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NIV ~R ITY

D - VIRO ME TAL ,' T DI E.'

F N RTH RN BRITI H OLUMBIA
June 2015

~·' Yueting

hao, 20 15

tra t
~th;lnPr

i ·ant hut the di\er~e mcchanisti · e\ents

ur) ( 1 Ilg) i a \\cll-knc \\n neurot

1' It\- ha\e -\et heen lulh

, soci ted \\ ith
e I I 1r d the me ·halll . Ill

l

r

1cllg neurc t

1 ' It

lucid · ted . In this study. we

partt ·ularl}

ih

r 1Ssihle roles

in

n ur deg ~neratl\ e d1 ea e.., ltke I Mktn on· dt..,ea e (I [ ) u~tng dopaminergic neuronal cells
and an n-human pnmate mt d l In the ·ell culture m )del. \\e cc mpared effects ){ Mel lg t
tho T indu cd h: 1-meth: 1-.f-phen: lr: ndtn1um ( 1f P ). a \\ell-e~tahli~hed drug that can
Parkins ni 'm-like : mpt m

indu

\ protctmlc appn ach \\as u~ed t identif) and analyl'e

Mellg afTe ted pr tein and their ht )]c g1cal function and associated pathwa;s in both the
cellular and marmo. et model .

)ur re ulh shO\\Cd that Mellg induced changes of

gen .. pr tein pr file are imilar to the effects as

l

1PP . l· \idence from proteomic results

·ugg t d M llg au

d neurc d generati\ e effecb not on!; as~ociatcd with PD but also other

neurodegenerati\

rder , uch a Huntington· . di ea e (} ID ).

di

and amyotrophic lateral

l;hcimer · s disease ( D).

ler i ( L ). \\'e also found brain regional pecific re ponse to

MeHg timuli. ba d on the protein profile affect d in the following order: cerebellum ->
occipital lobe (OL) > frontal lob
derivative m tabolic proc

(FL) of the c rebrum.

. ynaptic tran mi

ln the

ercbellum. carb h;drate

ion. cell dev lopment and calcium ·ignaling

are dominant functions and path'v\ay contributing to the motor deficit in Mel lg-treated
et. Mellg

marm

a

found to

electi I

target membrane pr t in

in the cerebellum

particularly in ·ynaptic membranes . MeHg af[i cted protein 1nv lved in energ; metaboli ·m
in both
the

L and F

f the cerebrum thr ugh different proteins and biochemical path\\ a) .·. In

L, protein

were enriched in functions of carbohydrate metabolic proce · . lipid

.ii

111

tab li pr

11 ular

111m

a id 111 tab li pr c , , . h 111

and regul ti n f b d\ Ouid l " I. In the
in" oh d in th
m tab li

l. diffi rentiall; e 1 re ed pre t ins 'v\ere main!

11 \ lc and ell di \ i. i n. gl; cer li p1d met b li pr

pro e.. . cellular ammc

pr te I) i . ·1he d) h me ·ta. 1

c.

, . . , ul fur

mpound

cid met b li ' proce"'"'· micr tubule-based pro ess. and

r \\ ater tran p 1rt and a ~ 1' iated path'v\a) s 1bscrved in 0 L

nd FL \\a fi und t 1 be the under!: mg mechani m f1r brain edema c b. cned in the Mel Ig
expo. ed mannc :ct.

\Ciprottn . u ·ha II 14(P,' P5)inthc ' erebellumand

PCl: in

L \\ "re e'hibited to he · re pr tein. in linking multifunction targeted b; Mel Ig. ·r his study

pr "ide
n ur t xt

a ne\\ per. pccti\ e upon under tandtng
dcfi it ·.

n urod generati\'e di

nd

. ugge:t

potential

mechani~ms

link .

bet'v\ecn

behind Mellg mediated
Mel lg

exposure

and

rder. in human

.iii

Table f

ntent

ppro al page ... ........... ................................................. ................................................. 1
b tract .. ... .......... ... ...................... .............................................................. .... ............. .. 11
Table of

ontent ........ .. ........ .. .............................................................. .. ................ ..... 1v

Li t f Table ............................................................................. ........... ...... .. .......... ... .. VII
Li t of Figur . ............................................................................................................. viii
lo ary .. ... ............................................................................................ ................ ..... . xi
ckno"'·ledgement .. ............ .................................................... ............. ....................... XII
hapter I. lntr ducti on .. .... ........................................................... ............ .................... 1
Rational e ......................... ...... ........ .................................................................................................. 3
Re earc h H yp th e i ...................................................................................................................... 4
Objective a nd

tru tur : ............................................................................................................... 4

Chapter II. L itera ture R ev ie"'· .. ... .............. .. ... .............................. ......... ... .. ... ..... .. ...... ... 6
1. Mercury in e nviro nm ent .......................................................................... ................................. 6
2. Toxic Effects a nd Mechani m of M eth ylm e rcury ................................................................. 9
2. 1 Targe t ites, )- mptom and pathologica l chan ge . ....................................................................... ... 9
2 2 Mec hani m of MeHg actio n ............................................................................................................. 11

3. Methylm ercury as a risk factor fo r neur·od ege nerative disea e ......................................... 23
4. MPP+ and its dopaminergic neurotoxicity ............................................................................. 27

Chapter Ill. Effects of Methylm ercu ry on Dopamine Relea e in MN9D Ne uron a l Cell
................ .................................................................................................................... 29

Abstract ........................................................................................................................................ 30
Introduction .................................................................................................................................. 31
Material a nd Method ................................................................................................................. 33
ell lin e and treatm ent ......................................................................................................................... 33
ell viability a ay .............................................................................................................................. 34
IIPL detec ti on o f DA and it · metabolites in MN9 0 ce ll cu ltu re medi um ......................................... 34
erni -reve r e transcript P R ................................................................................................................. 35
Immun obl ot .................................................................................................................................. ...... . 36
An aly i of MA -B ac ti vity ............................................................................................................. 36

Statis ti cal Analy is ....................................................................................................................... 37
Re ults ........................................................................................................................................... 37
. IV

Th t

I

90
it fM ll g nd PP t th e
9 0 ell
1n d pam111 e and 1t metabo lite re lea e fro m
f 1n Jtr e pos ure to cll g and PP on do pamm c )C lin g genes

of 1n 1tr

. po ure t

f Mell g and

Oi cu

PP on

. 37
37

................................ 38

38
n d pam 1n e C) l111 g pro te1n
-Bact I\ 1t_ ............................................................................... 38

ell g and MPP

ion ................................................................... .. ................................................................ 45

o nclu ion ................................................................................................................................... 48
ckno\\·ledge m nt .......... ....................... .. ............... .................................................................... 49

hapter IV .

ffect of Methylm rcuf!

n M 90 do pamin rgic n uron

IL : a

genomic and pr t omic analy i. .................................................................................. 50
Ab tract .......................................................... .. .... .. ... ... .. .............................................................. 51
lntr d ucti n ........................... .................. ..... ................................................................................ 52
Material and Meth d ............................................................. .................................................... 54
II ulture and hem1cal e p urcs .... .............. .. . . ...... ............ ............................................... 54
PO P R rra) a a) ................................................................................................................ 54
............................................................................................................... 55
Prot omi
nal) 1s
e tern blot anal) 1s .................................................................................................................. 57
Re ult ...................................................................................................................... . .................... 58
PO P R rra1 anal] 1 ....
58
The prote 111 abu ndance profiling. ................................................... ....................................................... 59
.. ............................................................................ 59
Path v. ay enrichm nt anal} i .....
Molecu lar function s and on·e lation anal} i ........................................................................ 60
Validatio n of proteomic re sult ................................................................................................................ 61
Discus ion ....................... .............................................. .. .................................................. ... ......... 71
Conclusion ........................................................ ... .... ... .. ... ..... .. .......................... ............................ 76

Chapter V: Proteomic Ana lys is of Cerebellum in Common Marmoset exposed to
Methylmercury ........ .................................................................................................... 77
Abstract ........................................................................ .. ........................ ............................ .......... 78
Introduction ..................................................................................................................... ............ . 79
Materials and Method ................................................................................................................ 80
Anim al and meth !mercury trea tm ent ................................................................................... ...... ..... 80
1-M / M ........ ..................................................................................................................... 81
Data anal ysis ....................................................................................................................................... . 83
We tern bl ot analy i ·························································································································· . 85
Result ........................................................................................................................................... 86
Protein id entifi cati on and ce llul ar co mponent di stributi on .......... ...................... ................................. 86
87
0 analy i of differenti all y ex pre ed protein in cerebe llum ...................................... ..... ...... ......
Pathway analysi .. ...................................................................... ................................. ......... ... .. ....... 89

v

eri fi at1 n f prot tn

1 n u tn g n alternat1 e method

........................................................... 89

Di cu i n ..................................................................................................................................... 98

hapter VI: Prot me pr fi ling r vea l r gi nal protein a lt ration in cerebrum of
common marmo t ( a llithri. jacchu e ·po, ed to m thylm rcury .......................... 103
b tract ...................................................................................................................................... 104
l n tr duction ................................................................................................................................ 105
Material and M th d . ............................................................................................................... 106
penmental an1malc; nd eth) lmer ur: dmtn1c;trat1on ................................................................ 106
amp le Pr parat1011 and Ma . pe trometrt
nal: c; 1c, .............................................................. 107
108
11 om ap1cn Databa e ear h nd uantltdtton
108
llular
mp nent D1 tnbut1 n
Data nal: 1 nd
c1p1tal l obe ................................... .. 109
Fun tional nrt hment nal: 1 for I rontal lobe and
110
Path\\ a: 1: nnchment nah 1 and Mapptn g
110
e tern 81 t nal)'il
R

ult ......................................................................................................................................... Ill
eth} Imer ur: -1ndu d Prot 111 bundan e ' han ges 111 th e hontal Lobe and 0 ip1tal Lobe of
Marmo t. . .
. .... ..... .. .. .... 111
nri hment Funct1 nal nal: i D1ffl rent1all} \pre sed Prote1n s fl r rLand C L .......................... 112
Functional nnotatton et\\Or" of FL and L of Marmose t b] lue
........................................... 113
K
1gnificant PathV\a)
114
Validation of Differential I] xpre ed Prote1n
114
.
.
D ISC U tOn •••••••••••••••••••••••••••••••••••••••••••••••••••••••• •••.••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 129
Co nclu sion .................................................................................................................................. 134

Chapter VII. Conclusions .......................................................................................... 136
Key findings of thi

tud y .......................................................................................................... 136

Contributions to knowledge and limitation ........................................................................... 141
Future research .......................................................................................................................... 142

Publications and Academic Activities during PhD. Study ......................................... 143
References ................................................................................................................. 144
Appendix 1: Establishing the Cerebellar Granule Neuron and Astrocytes Primary
Culture Model for Methylmercury Toxicity tudy .................................................... 162
Appendix II: Supplementary data ..................................................................... ........ 170
Appendix Ill: Authorship Statements for Published

hapter ................................. 185

.VI

t f Table
hapter Ill:

abl 1. L \' I of
in ub ti n V\ i th e I Ig r

P
and I I
( ~l
In M
cell culture medium after
P P .. C r ~ 4 h. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

hapter IV:

Table l . ignifi

nt han ged PI related ge nes in Mcl lg and MI p + treated M

cells 62

Tab! -· P related gene: pr tein. \\er " identifi ed in the tV\O ~~ays of P P ' R rray and
pr t me r fi I"' .................................................................................... 63
hapter V:

Table l . ignificant
( iolo gi al Pre ce. S) term ba, cd se ts or differentiall y e pressed
prot in in M ll g-treat d marmo et cerebellum ......................... ... .................... 90
able_. Enriched microR
and their target ge n · in th e ' functi n term of synapti
tran mi ion ......................................................................................... 91
Table . ompari on between protein quantification re ult u in g We t rn blot and
prote mic analy i .................................................................................92
C hapter VI :

Tablel. Functional
term (BP) differentiall y xpre d protein m Mel-Ig-treated
Frontal Lobe ...... .. .... ........................................... ............ . ..................... 115
Table2. Functional GO term (BP) of differentially expre ed protein in MeHg-treated
ccipital Lobe ............................... .. ..................................................... 11 7
Table3. K

G pathway identified in MeHg e po ed manno et frontal lob ............. 1 19

Table4. K

G pathway identifi d in Me l lg e po ed marmo et occipital lobe . . ......... 120

TableS. Proteomics re ult were confitmed by We tern blot in marmo et tV\O brain
region s .... ........... .......... ...... ... . ............... ............................................. 121

.VII

t f Fi ur
hapter III :

Figur 1.
inM

e-re p n
un c. 1 ( ) cth; !mer ·ur; ( ell g) and (B) MPP + toto i it
ell aftL:r _4 and 4 h L:\.P 1surc ...................................................... .40

igur -· T .. pi

I hr mat( gram.

fI

. I )I

' and I I

............................ ..... 4 1

1gure .
c-depcndL:nt efT~ ·t r (A) 1cll g and (I ) 1 I • on the c. pre ·si ns of Tl L
T and a- , ; n mR
anal; n .:d h; R r -P ' R .................................................. .42
igur 4.
e-rL:. p ns ~ ~ fTcct ( f (
cl lg and (B) MPP on the c. press ion s of TIL
and a- ) n pr tL:in anal;1 d h\ immun 1hlot ........................................... .4
Figure . Effe t · of

I Ig and

PP on M ( -B cti\ it; in M JD cells .................. .44

ha pter IV:

Figur 1.
lean pi t hov. the ge ne ex pr ion in 1.5 -fold change in Mel Ig 2.5 ~M and
5~M ( and B). MPP+40~M ( ) expo ed M 9 cell ....................................... 64
nrichment anal; i of K
Pathv..a; in (A) McH g l ~M. (B) M 1lg 5~M and
VID ..................................... 66
( )MPP+ 10~MtreatedM 9 c II analyzedh;
igure2.

igure3. (A) Heat map f common changed protein for ' 0 molecular functional
clas ification in MeHg and MPP treated MN9D cell . Prot ins abundance ratio
(treated/control) are compared bet\\oeen (B) MeHgl ~M /Con (I g2) and MPP +l O~M /C n
(log2) and ( ) MeHg 5~M and MPP + 10~M .................................................... 67
igure4 . MeHg induced all th prot in abundance change in MN9D ell . Protein
abundance wa di splayed a fold chang ........................................................ 68
FigureS . Protein abundance change induced by MeHg ba ed on molecular function
classification ......................................................................................... 69
igure6 . We tern blot verified ex pres ion of t\t\IO protein s (V ATI L and BAZ 1B) from
M analy i .................................................................................. . ........ 70
hapter V:

igure ] . A. I Ieatmap of pro tein abu ndance pattern in difTercntially expressed proteins.
B. ellul ar component di tribution fo r the to tal identified protein. (n=998. IPA mapped
I ) and differe nti all y e pre ed pro tei n (n- 10-) in marmo ·ct ccrebcllum ................. 93

.VIII

igur 2. n nn tati
pr t in in M

rk

nri hm nt ellul r

Figure .

f thr e fun ti nal gr up [! r differ ntiall
pre ed
t ereb llum ......... .. .. .. ... .. ........ .......... .... ...... 4
mp nent

nal) i u ing Babelomic · .............. .... 95

igur 4. The t p 1
n ni al pathv. ) identified h; th lngenuit Pathvva
nal sis
rt db their p_, alu , ................................................................ .. 9
Figure . We, t rn bl t 'crificd r ur pr tcin. identi fl d from M.' anal is in marm )Set
er b llum ........................ . ......................................... . ......................... 97
hapter V I:

igur 1.
MeHge

u ntifi ati n anal) , i. of identified pr tein s in frontal lohe and occipital lobe in
ed marn1 et .............................................................. . ...... ... 122

igur ~. II atmap f pr tein abundant hangc~ in differential!; c. pre ·sed pr tein s in FL
( ) and L ( ' ). nd ellular c mponcnL distribution for identified proteins in frontal
1 b (B) and ccipital I be, ( )......................................... ... ..................... 123
~ igur

3.
-nri hment depletion analy is of differential ex pre ed pr tein for frontal
I be and ccipital I b in MeHg-treated marmo. et. on!; the ignificant functional term
(P<0.05) are appear in bar chart ................................................................ 125
Figur 4. nnotation network f dif~ r ntiall y ex pre, ed proteins in FL (A) and L (B)
were vi ualized by lue
Clu pedia ............. .................. .. ........................ 126
FigureS. Validation of the proteomic r ult u ing Vv'B analy i of 4 protein in fr ntal
lobe and occipital lobe ................................................................................... 127
Figure6. Va opre in-regulated water reab orption pathwa; wa cia ified by K
pathway mapping ystem from uploaded 62 differentially ex pre ed prot in in FL .... 128

A ppendix 1:

Figure 1. Diagram of primary cerebellar astrocyt

i olation and purification .. . ......... 158

Figure2 . Diagram of cerebellar granule neuron and progenitor cell collection .......... 159
and
Nco-culture plating in a ratio of 1:6 for 7 da) · folio\\ ing
igure3. Diagram of
MeHg expo ure ................................................................................................................ 159
Figure4. Immunofluore cence of Cerebellum Astrocytcs (AS) and erehellar ranule
Neur n ( GN) co-c ulture for 10 day · ................................................................ 162

. IX

5. I
p tn t 1d
~ igur

nd m n
reb llum n ur n
f
taining f m n c r bellar a tr 1 te
4- mi ............................................................................ 16

igur .
ll viabilit; f c rebellar granule neur n and a tr
te in m n and
ultur ~ ll ~ing a .... h . p ur ( and 4 h . po. ure (B) t vari u
ncentration of
Mel Ig ............................................................................................... . 1 4

pp ndi

II: 'upplementary Data

hapt r
T ble 1.

_ identifi ed pr tein. datase t in

d Ig- trea tcd ce rebellum ............... 1 6

Li t f 1 2 difTercnti all; e'<pre..·ed protein: in Mell g- treated marmose t
abl
r bellum .......................................................................................... 166
bl

. Enri h d
i log i al Proce . term ba ed . cts of differenti all y c pressed
in MeHg-treat d marm et cerebellum ............................................... 169

Tab!
pr

4. ignifi ant
ellular 'o mp nent term and th e a soc iatcd differentiall y
ed pr t in in M Hg-tr at d marm et ce rebellum by · rill a ..................... 174

Figure l.Repre entati ve M /M
pec tra from ( ) DL ' 4 (P78 352) and ( ) ,' L ' 17A 7
(Q9P2 7) . .. ....................................................................................... 175
~ i gure

2. K G pathway enrichment anal y i of differ nti all y expres d protein 111
M Hg-treat d marmo et cer b llum u ing Web e talt. .... . ........... . ................... 177
Figure 3. alcium ignaling pathway was cia ified by KE G path~ ay m apping y tem
from uploaded 102 differentiall y ex pre ed protein ..... . ......... . ........................ 178
hapter VI :
Figure ].Representative MS/M pectra from PO (P02649) and DYNC1LI 2 (
in marmo set (A) occipital lobe and (B) frontal lobe ............................ 179

4 "~237)

.X

I

ary

1-m th l-4-ph n lp 'ridinium(M P+)
,4-dih dr , ph n) l

ti a i (I

lzh im r' di a e (

)

my tr phi lateral

P

')

l ' )

p 1ip pr t in ~ (
quap rin-4 (

P4)

Databa e [! r

nn tati n.

i uali;ati n. and Integrated I i. cover) (

ID)

i k large h m I g 4 ( L 4. P P 5)

pamm
pamm tran p rt r (
lutathion

T)

)

lutathi n per , ida e-1 ( PX 1)
Fr ntal lob ( L)
Homovanillic acid (HV )
Huntington· di ea

(HD)

Kyoto ncyclopedia of ene and

nome (KE

Liquid chromatography/electro pray i nization tandem m

pectrom try (HPL -E. 1-MS/M )

Mercury (Hg)
Methylmercury (MeHg)
Monoamine oxida e-B (MA -B)
N-m thyl-D-a partate (NMDA)
Occipiallobe (OL)
xidative pho phorylation ( XPHOS)
xygen pecies (RO )
Parkin on' di a e (PO)
Tyro ine hydroxyla e (TH)
Web e talt (W B-ba cd JCne e AnaLy i

oolkit)

a- ynuclein (a- yn)

.XI

ckn wled
Fir t falL I Vv uld lik t

pr"

me nt

m; p ial gratitude to my UJ er i. ing Pr fe or Lauric

h n. h nk ou [! r ) ur adv i c and guidan

thr ugh ut all f m d

t rate re car h and

th

vvriting. I apprc iated the pp I1unit) to \\Ork \\ith Lauri · t'v\ gr up in

th

m

r it; of

nthu ia ti

di

ttav\a. nd I am \Cf) gr teful t al l the m mhcrs in h th lab , C r our

u i n . and C r all the fun \\e had in the past fi e

thankful[! r Pr

NB ' and

r ' h 'v\ Lee .· m;

cars. I am de pi

)-:upen isc rand supeni ing

mmittee member,

C r b th hi academi ad\ i e and admini ·trati\e help . I \\C uld a] · like to thank my I hD.
mmitt e. Pr G

ezene I Iuber. Luke I larris and William

ippett for ·er mg as my

mmitt

m mb r. nd G r their Ia ·ting c c demi support and input .

he pa t

v r I y ar 'v\ r n t ea ) [! r me in both academia and perso nal Iife . I have

I arn d h v. to be m r ind pendent. and h 'v\ t : ta; ·tr ng vvill vvhcn facing iss ue in b th
acad mic and p r nal life . I am trul; thankful for my friend Licheng Pcng, r r her
kindne , nc urag m nt. [! r ur hared laugh and r; time. and travel memorie which I
'v\ill ne er [! rget . I'd al

like to give a h artfc lt. pecial thanks t my roommate . u an

hepard, my fri nd Lihua Liu and al o other friend . [! r their elfle

help and care during

my most difficult time of my I g injury .
I e p cially thank my family m mber : my mom. my dad. my brother and my i ter-in-law
for their ongoing upport and advic , thank you for alway standing by m . I would n t
have made it thi far without them . My mom ha alway been my be t li t ner while I wa
abroad in the pa t five year . he accompanied me for the mo t difficult and happie t time
1 e perienced.

Finally, thi re earch would not be completed without finan ial
aune

han'

Health,

anada Research

Research

funding from the B

Leader hip

hair in

nvironm ntal and

hair program and the Natural

unci! of anada, a well a the

upport from Profe or

c1ence

and

boriginal
ngineering

niv r ity [Northern British Columbia.

hank you everyone for your upport and a i tance. The kindne

and encouragement ) ou

have hown me has motivated and enabled me to complete thi the ts.

.XII

hapter I. Intr ducti n
h nerv u . ·. tem i. th main targ t ~ r man metal
m r ury. ar

111

m thylmer ur)

and lithium in the human h d; .
el lg) i. one

u h a I ad. aluminum. mangane. e.
mong them. mer ur

(Ji g). speciall y

f thL: m 1. t neur to . ic metaL ( ' larks n. 1987). and p c a

p t ntial ri k t h th ec I gi al and human health . Mellg i ~ a non-essential metal that has no
bioi gi al fun ti n in human bodie:. thu e'\ce. ~i\e Mel lg e. p ure cause adverse effects
(F rina t al.. _Q l_ ). Me I Ig to · i it) ha. been \\ell d cumented and can he traced back to the
nmg

utbreak · in Minamata Ba;. Japan during 1953-195 . A chemical

fa tory rei a d \\a t

containing Mellg (a b;pr )duct of acetaldehyde sy nthesi s) into

fir t M Hg p

Minamata Ba) . R ident around the ba; v.ho con . umed the fish and marine products from
the pollut d \\at r developed ·ymptom · ·uch a. tremor. ataxia. ·ens ry di sturbances.
dy arthria. and lo

of hearing (Clark ·on and

biop y re ult of Minamata di ea e patient

train. 200 : llarada. 1995 ). Pathol gical brain
ho\\ed

I vated mercury concentrations in the

cerebellum and cortice in the cerebrum. particular!) in the occipital lob

(Eto. 1997). , ince

then. numerou studie on Hg toxicity and mechani m of it actions have been conducted and
reviewed ( hapman and

han. 2000:

lark on. 1972: Clark on and Mago . 2006: Mergler et

al.. 2007: Mutter et al.. 2007). Th early tudie on the mechani m of 1Ig t xicit) tarted in
the 1950 . which identified Hg binding activit

with cell pla ma m mbran

and it

involvement in membrane tran port proce es. Later on during the 1960 and 1970 . the toxi
mechani m involved in di ruption of protein ·ynthe i . nucleic acid metaboli m. and the
cyto keleton were identified . After 1980. tudic revealed Hg induced di ·ruption to calcium
h meo tasi (Clark on. 1987). ' ince the 1990's new techniques and ski! I. de\ elopment in the

1

fi ld

f m 1 ular bi 1 g

gene

r pr tein

hav

in term

all Vv d re ar h r t

f th

me hani ti

high level

1..-

l~

0

a ti n of llg. , p i fi all

meth lmer ·ury

number of findin gs point t 1 the abilit

f Mel Ig to induce

f xid ti\ e tre, rca tiH~ H} g n ~pccies ( R .' ). disrupti n of mi rotubule and

intra ellular calcium h me ·t . i. ( t hison and I !arc. I 94: 'cc atclli et a!.. 2007 :
nd

pe ifi

it. high!; to,· i prop rt in rganisms (Faro et

(M ll g) ha be n e ' len i\ el re\ ie'Aed du

:R

identif ' and quantif th

eegal. _o 7) . P tential anti- 1 ·idant agents t
ati n \\ere

g n t xi it; and a

v.idel;

re1em

protect cellular fun tion from Mel lg

tudicd (Farina et al.. 20 11: Liu et al.. 20 14).

dditionally,

ciated glutamine glutamate c;cling alteration by Mel Ig has been

b rv din vari u e perimental tudie ( ' re. p -Lope! ct al.. 2007: Yin et al.. 2007) .

nvir nm ntal p 11 utant

uch a mercur; \\ere rep rted to c ntri but

neurod gen rative di a e (M nn t-T hudi

t

the eti logy of

t al.. 2006) . Mellg iLelf can trigger

neuropathological condition by affl cting multi-function in target brain region . Farina et al.
( arina et al.. 2012) de cribed the po ible mechani m for MeJig-induced neurodegeneration
which involved the binding of M Hg to

ulfhydryl group (thiol and eleno) of prot in

(including neurotransmitter receptors) and oth r non-protein thiol uch as glutathion . Thi
reaction evoke the redox tate change of thiol group on protein . ub equently I ading to
cell dy function.

MeHg can al o c ntribute to neurodegeneration through calcium

dy homeo tasis, neurotransmitter alteration and glutathione (

H) depl tion (Ca toldi et al..

2001 ). For example. MeHg inhibited dopamine y tern function in

ynapto orne of earh

postnatal rat (Drciem et al.. 2009). The e m chani tic characteri tic
featur

of several neurodegenerative di ea c

. uch a ·. P rkin ·on·

are al o common
Disea ·e ( PD).

myotrophic lateral sci rosi ( L ) and I Iuntington's di ·case (HD).

2

Rationale

n th ugh lini al
due t

mpt m

p t Mellg poi

f mer ur] neur

bani m cau:ed b; Mcllg i, till n t full

under t

el Ig.

path gene i

ffl ct /b havi ur and intrin ic m
m

r

h ni m

de · (Farina et a!.. . . . 0 I l ). the relation bet een these

nmg ept

f[i ct:

of

( articularly

it.

role ·

111

the

d. The
f

n ur deg n rative di ea e · need t) be clarified . I· urthermore. it ts unclear whether the
m I ular target · of Mellg in
pid 1111

tudi

gi

different

oft n pro\ ide

are

the

·ame.

Moreover,

mpletel; ab I i ·h Mel Ig t

ici ty (Farina et

mpar d t epid mi I gical . tud;. laboratory in e tigations (in vitro and in vivo)

larg ly fa ilitat

the under tanding

M Hg neuroto icity in dif[i rent
pattern of toxic outcom

From th

rcg1 n

nflicting results (Morken et al., 2005) and no
far to

ffl tiv treatment ha be n d vel pcd
al.. 2011 ).

brain

f beha\ ioral factor

and a. ociated me hani m

of

rgani m . and thi v.ill help to rev al the unpredictable

( a toldi tal.. 2008).

context of eco y tem health. thi

tud]

tem

from an environmental health

perspective. By u ing different re earch models (dopaminergic n uronal cell . non-human
primate common marmo et and primary culture ). thi
underlying

mechanisn1s

of

MeHg

toxicity

and

tudy v.a de igned to e plore the
the

potential

a ociation

\\ ith

neurodegeneration particularly Parkin on· di ea e. and to det n11ine key target protein, that
can be re pon ible for MeHg neuroto icity . We applied a y tem-ba ed "-omic "approach to
obtain high throughput gene/protein profile. thi allowed u to ob erve the integrated pi ture
f respon e to Mel Ig in both in \'ilro and in \'ivo models. The overall goal i to increa ·e our
under tanding of potential impacts of nvironmental to. ins. Md Ig. in human nen OLL '\stem
and it r lation to neurodegcnerative condition · uch as I D.
3

Re earch H pothe 1

Th fir t re

r h h; p the i t be t . ted in ' hapter I II and 1 vva · that Mc ll g an d crease

d pamine r lea

in a , imilar me h ni . m t that of 1-Meth; 1-4-Phen lp; ridini um (MPP +), a

kn vvn g nt 'O mm nl; u. ed t) induce path ge ne. i.· or PI , and the tvvo t . ins regulated in a
imil r manner in t ~rm:
ec nd part

r thi

tud) the pattern

r pr tein pr )file in a de paminergic neur nal cell line, M 9I . In the

stud) ( ' hapter

and ' hapter

I ),

a pro tee mic appr ach was used to

f differential!) e'<pre'). ed pr )tein~ in Mel lg-treated marmoset brain Ill

rder to determine the p tential regie nal di{Terence. .

') the open feature of "-omics"

nd part v.. uld be that some of the identified protein s
and th ira

ciated bi logi al function and path'Aays i: con ·i tent vvith th

e reported in the

lit rature , and ne'v\ pr tein. indentified that can . hed nevv light on the understanding of
MeHg neur t

icity . Th

identified enriched functi ns v..ould explain the

phenotype effect upon M Hg-treated marm

O bj ective and

bse rved

et.

tructure :

The principle objective of this project wa to explore the mechani m of MeHg intoxication
y tematicall y using an in ' 'ilro cell model and manno t in ri, ·o mod I and to inve tigate an
association with neurodegenerative di ea e

uch as, Parkin on· di ea e.

pecific objective

included:

1.

To inve tigate the effect of MeHg on a dopaminergic neuronal cell line. M 90
and compared that to 1-methyl-4-phenyl pyridini um ( MPP +). a \\ell-establi shed
ag nt associated with the pathogenesi . of PD .

4

tu

2.

T

tud

J

the ffe

( 'o!ltthrix

f Me ll g

ffect

th

Jocchu\

ell g

f

usmg

n a d paminergi

p sure in the erebellum
a

·h tgun

pre teomi '

ell line u mg a

M 9

f

mm n marm J

appr ach

with

t

liquid

hr matograJ h) c up led H ma s ~I e tn metr) .
4. T

mp re the , mprehcn, i \ e pr teo me exprcssi n of the marm )Set brain

r g1 n. (prefrontal c )I1e'\

)r the fr mtal lobe and the occipital lobe around the

al arine ·ul u: in cerebrum) expo~ed t Mel Ig using pr te mic techniques.

ntain

e\ en 'haptec and c ne appendix . ·1he introduction in ' hapter I includes

th ba kgr und. bjective and ·tructur . rationale and h; p thesi , of thi · re earch . ' hapter Il
i the literature re\'ie\\,. \\,hich

mpri e the general introduction of "mercury life cyc le".

M Hg in the environm nt and it

ocial determination of heath. Chapter I l al o include a

review of the recent d v lopment of toxic effect and mechani m of Mel Ig. particularly on
the CN . by illu trating ho\\, MeHg embodiment relate to it pathologic proce · and the
a ociation between these mechani tic proce e . Furthermore. the general review of
relationship between MeHg expo ure and neurodegen rative di ea e

uch a Parkinson·

disea e will be addres ed. Chapter III-VI comprise th

f the thesi . the 4

main body

chapters each containing a manu cript of research paper that aim to achieve each of the 4
objective described above. A g neral conclu ion along with the potential contribution and
pi tfall of this the i wil l be given in Chapter VII . Finally. a eparate chapter de cribing the
proce

of method deve lopment for a cereb li ar a trocyte and neuronal o-culture model i

included in Appe ndi l.

5

ha pt r II.

it ra tu re Review

1. Mercur an en ar nm nt

curot . in · are agent · that c n 'au c t 1 ic cffccL in luding p i. ning or dam ging on the
ntr l n n u . ; ·tem ( ' ,' ). I he:c \uhstan es <., uch a: mcrcur; (1 !g), mm gancsc (Mn) and
lead (Pb), al

, ' Ur in the natural en\ ir mment. 1hc ~c metal arc als

~1und in so lid waste,

h mica! v. a te .. hi I gi al and radic acti\ e materiab. as v.ell as industriaL municipa l and
agricultural \\a, te di:c harged intc \\ ~lter (.'iha ct al.. 200() . Mercur; is one of the mo st we ll
d

urn nted en\'ironmental p )llutant~ and i kn O\\ n to he hi gh!; t xic to v.ildlifc and human
lark

n et aL 20

). There are main!;

fc rm . of mcrcur; : elemental mercury or

qui k ilver (Hg 0 ), in rganic m r ur; or i nic mercury (Ji g· and I Ig2 · ) and organic mercury
uch a methylmercur; (M Hg) and thylmercur; ( ' arocci et al., 2014 ).

There are tv.o main ource of Hg emi ion : natural!; occurring emi sion and em is ion from
human activitie . Anthropogenic contribute to the majority of mercury mi s ion. Selin et al.
( elin

taL 20 10) u ed

-Chem. a global three-dimen sional atmo ph ric chemi tr;

transport model to estimate atmo pheric mercury depo ition from it emi ion ource 111

. .

This tudy reported 68°/o of Hg emi ion are from anth ropogenic ources ( 59° o from North
Ameri ca and 9°/o from out ide N011h America), 16o/o we re natural and the re t of the 16° o
we re from historical anthropogen ic Hg.

Mercury producti on, emi ion, atm o ph ri c tran port and di trih uti on in the en\'ironment are
a co mpl ex proces . Elemental merc ury (Hg0 ) or 1I g2 + in the atm osp here can be transported to
remote areas by wind , then depos ited into ocea n, ·oi l and land, and thi , represent · the

6

mi i n. b th natural and anthr p g mc
tn

rt 111 ana r bi

ba t ria. i ni

m r ury . Mel Ig nl r th
predat r fi h and

~

ur e

f I lg. F 11

ing I lg meth lati on

I lg an be c 11\ rted t Mellg. the mo t l

fonn

f

d chain thr ugh hi m gnifi ati n , and hiomagnifications 111

ther m nn

mammaL . J luman,

n ' Ume Mellg

nd vvildlife that

ntamin nt d cean or t rre. trial rgam . m \\ere \entuall affect d (f ri c 11 el aL 201 ).
In term

f mer ur) life ) cle bar cter.. mercury i able tc travel gl ball y carried by wind

and rain . Ther ~ r . m rcur) pc lluti n ism 1r, than a simple en\ ironmental health iss ue. The
in r a ing p ial i1 ti n and am unt
f[i ct

r re. earch ha\ e led to further efforts to under 'land the

f mer ur; in the n\ innment. and health impacts n Iiving 1rganisms.

Th mam our

f human expo. ure t 1 mercur; is fish and mammals' c nsumption. ' hina is

con ider d

f the large t ource. of anthropogenic I Ig emi ·sion s. ""hich is generated

n

mo tl; by c al combu ti n and m tal
ti u in

melting. Intriguingly. Mel Ig con entration in fish

hina are v ry lovv. even in the heavily Hg ontaminated area . The rea on may be

due to the hort aquatic food chain . and the fact that mo t of the fi h are pr duced b;
aquaculture. which are not favoured Hg bioaccumulation proce

according to Li · revie""

(Li et aL 201 0). Low fish Hg concentration ha re ulted in lo"" hair Hg level s in the fi h
eating population in

hina. Rice on the other hand \\Ia reported to contain high level of

Mellg from Hg polluted Qingzhen area and th Wan han mining area in

uizhou Province.

hina (Horvat el aL 2003 ). A signifi cant relationship \\a found bet\\-een the area v. here
there was high level of MeHg in the rice and hair MeHg level (r= 0.65. P< 0.001) (Li eta!..
201 0). Therefore, it appear

the source

g ographic location , and thi

for human e. po ure to Hg differ depending on

is as ociated v.ith particular characteri ' tic , of local

environment and Ii fe style.

7

h 11

In the

I l n

rr l ti n w
c p

ur

[i und

nutriti n

h rt tud . 'Aith 12

b t'A

d

p l un tur ted fatt

a id.

pr natal e. po ure \\ith
n t

find

maternal

Mcllg

and language d \ cl pmcnt 1f child. and PlJ F

(n-

II

ahc

(d

\\Crc [! und t be an impc rtant nutriti n fa t r t

c. p

urc

to

1el lg

rr 1111

fish

con. umption

tud ' indi ated

ial en\ ir nmental fact r ~ uch a. diet. especial!

fact r.

associat d

with

n ·hi ldrcn (ab 1ut ~O month~) (. ' train ct a!.. 2015 ). ·r his cohort

ut

Th

modify the

ell g and P. )- ·h 1111 t1r [ e\cl pmcntal Ind c . th ugh the rc ult did

n ur d \ elopmcnt

p pulati n (m·er g

a1r the p si ti

n 1c acid) in prcnat

n bl

fi h c n umpti n

moth r- hild

m

in high fish eating

. fi h meal \\Cck). are important in modif;ing Mcllg to ic effect.

h uld be

n ider d in ri k a.. c. sment and pol ic; determination . Recent
al

cmpha i7e

ep1g n tic-ba d

tudie

nvironmentally

n itiv gene in MeHg exp

genetic

pol; morphi sms

In

prediction

of

ed population . This is valuable in estimating

biomarker uncertainty and health outc me ( ee

iladri Ba u revie\\) (Basu et al.. 2014 ).

oodrich et al. ( oodrich t al.. 2011 ) found 5 p lymorphi ms ( '. ' TT 1 deletion. GSTP 1-1 05.
TPl-114.

5' and

PP 1 ') · TR) to be a ociated with mercury accumulation in urine

and hair levels in a population of dental profe ~ sional . indicating change of glutathione
yntheta e (

) and selenoproteins

the polymorphism modification of

EPP1 affect MeHg limination which r ulted from
S and S EPP 1 gene . The

re ul t

illu trate that

environmental factors are able to modify th epigenome and ingle nucleotid polymorphi ·m.
pigenetic tudies have dire t role to monitor biomarker and MeHg xpo ure source like
fi h con umption (Basu et al., 2014).

uch tudie will benefit m rcury ri k a e sment and

policy deci ion-making on improving management on

u ceptible population \\ith i\1eHg

po ure.

8

2. To ac

ffect and Mechani m of Meth lm r ury

2.1 arget ite

M Hg

th

ymptom and path I gical chan g
~

m t t '\I

rm

f m r ur) that hie a umulate. in aq mtic s

high e p ·ure in human · \\he

on~ume large amount~

and Ward) ( ' e catelli et al..

~0 I 0:

in rea ing le\ el

f I Ig and

.'anfcliu et al..

tcms,

au ·mg

1f fi sh and ea mammal s (,' himsha k
~ 00

). Man)' studies have shown

cl Ig in the hair and hlo d of people in cultures vvith high fi sh

n umption ( iez et al.. 20 9: Jochfeld. ~ 003: Pa~ ~o~ et aL 2008), for ~ ample, maternal
n umpti n f large

iI; fi ·h has been . h '" n , igni fie anti 1 correlated with total mercury in

n \\b rn ' hair in pani h ( iez et al.. 2009) .

M Hg t xicit)

extr m 1; damaging t

tage a it I ad to alteration of th
( randjean t aL 201 0).

the nen u

y tern m the early devel pmental

·tructure and functionality of the ncr

u

·y tern

\'en m l \\ do e , prenatal expo ure to MeHg can di srupt brain

d velopment in the fetu ( arpenter, 2001 ).

nimal tudie have hown that e po ure to low

dose of MeHg (0.02mg/kg/day for 7 week ) reduced birth weight of mou e pups, and
induced neurobehavioral defect

(locomotor activity, motor equilibrium impairment and

auditory dy function). The e anatomical and phy iological effect were matched by change
in lipid peroxidation, Na+/K+-A TPase and nitric o. ide

ignalling (Huang et aL 201 0).

Mat rna] nutrients change may alter the phy i logical et point and rai

the \'Uinerahilit; of

the off pring to the low level of MeHg in the environment ( Fo. et al.. 2013 ). Th re i.

lear

electivity of Mel Ig for pecific cell t pe and brain tructure (Sanfeliu et aL 2003 ). MeHg
preferentially accumulate

in a trocyte

di abling the capacity for a trocyte

to pro\'ide

metabo li and antioxidant upport to neuron . Thi inevitably leads to neuronal damag ~ and

9

th

a umul ti n

pr ~ rred

f

ll g 111 neur n (

hn r et aL 20 7). The me hani m

umulati n re n t yet full under t

of thi

d.

IIi t

h mi al r , ult from autop~ie of Minamata di s a. e p tients revealed that I Ig were

d p

ited in alm , t all the ti , ue and organ~ in human h d;. 1f ~hich kidney. brain. liver,

thyr id \\ler th m , t ·tained rgans ( hi g he ~ t le\ el: of I Ig accumulati n) ( Fto. 1997: ( kabe
and Takeuchi. 1
and

er brum

c rebrum

0) . In th ~ nen

U'> s_ ~tem. I Ig accumulati

r the a ute phase 111

n ~as highest in the cerebellum

1inamata disease case~. 'I he pathological changes of

·h wed that damage pred )minantl)

1ccurred in the calcarine regiOn , the

po t entral and pre entral g; ri. and the temp )raJ tran s\ erse gy ru s. The to icity in the
c r b llum \\a
purkinj

hara teri1ed b) I s · c f granu le neuron s hut a relative ~ell maintained

c II ( ~ to . 1997). indicating that granule neuron s are specific tartgeted cells for

Mel-lg toxicity. Brain ed rna and ub equent i chemic changes were observed in the early
tage of acute ca e . In chroni ca e . Hg accumulation ~a . lower in brain ti . ue than the
other organ ( kabe and Takeuchi. 1980). A neur logical study of 77 autopsy ca es from
Minamata di ea e found 80.5 o/o patient with

n ory impairment. 35.8°/o of patient had lo~er

e tr mity coordination. patients ~ith con triction of vi ual field
retrocochlear hearing lo

ccupied 28.8°/o. and

was in 15.3°/o ( chino tal.. 1995). Another evere occurrence of

MeHg poi oning occuned in Iraq in 1971-1972s with mortality rate of 459 ca e from 6530
record d ho pita! ca e (Bakir et al.. 1980). Patient exhibited

imilar ymptom (muscle

weakne s. ataxia. dy arthria and tremor, ev r ca e appear visual and hearing impairment)
found in Minamata di ea e. Notably, neuro logical deficits such a un teadine · and frequent
fa ll , early sign for Mel lg poisoning. were ob erved in the Iraqi patient . There are 3 major
fa tor reported to contribute to cause thi tragedy : people ~ ho consumed bread that \\as

10

tr

t d
c

ith M Hg

rding t thi

fungi id , br eding r ut fr m

hang

) mpt m and hi t path

v. r par II I v. ith the degree

2.2 Mechani m

al o th prin ipl targ t rgan

tud; ( Bakir et a!.. 198

ning ba d n th

ed m ther. nd prenatal e p

ur .

f Mel Ig

gi al alt ration . . The brain path 1 gical

r p is ming nd bloc d mercur le cl.

f McHg acti n

including Mel lg enter the hod) and c crt their t ), ic ciTe ts in 4 general pha e ·
a

rding t

arc ni and I arhicri. 1 8 ) and Philbert (Philbert ct al., 2000): ( 1)

Mar 111

d liv ry pha . entr)
re ept r/m tab li

)[ action; (2)

ph e. bind and rea t \\ith spe iiic cellular targets: ( ) to icodynamic

pha e, initiati n fbi
and functi n in

into orgam ·m and di trihution to the regton

h mica] hange : and (4) lead to the alteration of cellular structure
(clinical pha e).

ince the initial identification of l lg poi oning in 1956,

the clinical n urology and the effect of neur toxicit; of Mel lg have been v.idely studied.
How ver. the mechani m re p n ible [! r MeHg induced change in neurobehavioral effect
and function are till enthu ia tically debat d.

number of ·tudie have shov.n that Mel Ig

di rupt a number of cellular function including inhibition of prot in ynthe i . microtubule
di ruption. and increase of cyto olic and mitochondrial

vel

and di rupt of

neurotran mitter function in the brain, uch a catecholamine (Ro i. et al.. 1997: Fit anaki
and Aschner. 2005a: Johan on et al.. 2007:

anfeliu et al.. 2003 ). llere. I pre nt a mini

review on the mo t commonly proposed m chani m

[! r MeHg neurotoxic it) .

2. 2. 1 Oxidal ive stress and Ani ioxidanl defense

xidative tre s is a condition of fom1ation of total free radical in a cell that exceeded the
anti-ox idant capabi Iity that triggers variet of pathological condition ( Smcyne and Sme\ ne.
11

201 ). Tr nit in r a

gcn p 1e ( R , ) are

f r a tive

I ignall in g. nd ntio. id nt

ellul r pr

t th red . b ian

. I ligh

n

n/) m

ntrati n

in b d;

mm nl

e i t d in n rmal

uld rapid!

r m

e R

t

f R ,' may cau e o idati e modi f and

n rau. tad llir ct al.. ~0 1~ ). ' mentioned abo c.

damag

f macr m le ule

d li

ph c. llg enter. the ell. h) binding Jig mL pre. cnt in membrane. whi h include

f\'

1n

the first

thi I gr up. el n gr up. amino. phc sphak and car be x; I. Mcll g can bind t -, ,I I nd - 'e lI
ligand in th

) t ine amino acid resulting in the r m1ation of a Mel Ig

stcinc c mplc

i t a!.. ~ 14 : ' lark on. 19 7 . I hi · is al. o the major c, cretion route for Mel Ig. for

( ar

e. ample. Mellg i tran ported

th li\er canalicular membrane · into bile in the form of

H-Me g c mpl x ( utczak and Ballatori . 1994 ). , 'elen proteins ( ie .Glutathione p roxidasc

1.

PX 1) and non-pr t in abundant in -,, 1I (membrane tran sporter . Iipoic acid) are more

·u ceptible target
protein

f MeHg . .'elenopr tein

like

' PXI are also important anti xidative

again t MeHg toxicity. In addition. previous

tudy ha

sho-wn that Mel Ig

preferentially bind to high molecular w ight prot in (> 200Kda) uch a

·keletal mu cl

myo in heavy chain. which ha. been proved in tuna fi sh mu cle by metallomic approach
(Kutscher et al.. 2012).

xc ss1ve RO

generation 1s a well-document d mechanism of MeHg neuroto. icity

demon trated both in vitro (primary cerebellar granul neuron ) ( arafian and Verity. 1991)
and in vivo (rat cerebral cort

) tudie (Liu eta!.. 2014). R

i an important factor in redox

ignalling and maintain cellular oxygen homeo ta is. Imbalance bctv~een R
and anti-oxidative repair y tern can lead to <."'~ . idati ve

tre

production

(Shukla et al.. 2011 ). and

sub quent damage the cellu lar macromolecule and disturb signa l tran ·duction path\\ a) .·.
R

formation canal o ind irect! mediate glutamate cxcitoxicity. For example.

u ct al (Xu

12

~ rmati

L 2 12) h vv d M I Ig i gn i fi

t

idati

dam g

d h m

ta i

imbalan

vva

in mit

h ndria

111

rt . of rat. The auth r al

r bral

a re ult of R

n. lipid. pr tei n and D
dem n tr ted glutamate

m diat d inhibiti n of glutamine , nth ase.

' 2+

a

b n d a.· ne f th t .· ic mcchani m. ~ r Mel lg-indu ed R ._' generati n
f r t brain ·li e. and · ntributcd to the

hain effect of mit ch ndrial

dy fun ti n nd :idati\e . tre:· ( )ama et al.. 1994). Imp rtantly. R ,' i able t adversely
af~

t

llular ma r mole ule:

thr ugh o. idation . (

u h a I

dam g i an thcr m I cular me hani sm )b ·ened in Me l Ig nclll·ot

idati n

au ·ed

icity . C idative D A

may n gativel) afT ct n ur de\ ~lopment thr ugh a non-mutation mechani sm to alter gene
tran cripti n. fa ilitate formation of ·enobiotic-macromolccular adducts. and di rupt other
macromolecular target

uch a protein . lipid and ignal tran duction (Well s et al.. 201 0) .

repair and ap pto i are normal I) t'v\

re pon e. ~ llo'v\ ing DNA damage which arc

initiated by oxidative tre . It wa reported in murin embryonic fibrobla t · that 0
protein oxoguamne glyco yla
damage following MeHg

1 (

1)

protect cell

repa1 r

from D A double strand break

po ur . indicated D A repair y t m deficiency (

Gl -- null)

and inc rea ed the cellular en itivity to MeHg to icity ( ndovcik et al.. 2012 ).

lutathion (

H) is a critical part of cellular anti-oxidant defen e machinery and i required

to prot ct c lls from RO induced pero. idation of lipid membrane and D A damage induced
by MeHg (Gib on et al.. 2014 ). Anti-oxidative agent/drug can inc rea e G I I level
attenuated MeHg-induced n urotoxicity. For example. the antioxidant

and

(quercitrin and

quercetin) have been hown to provide neuron prote tive effect · against Me!Ig induced lipid
peroxidation and ROS formation in cortical brain slices of rats (Wagner et al.. 201 0) . Pretreatment

f tea polyphenol

( 1mmol/kg) rever ·ed Mellg caused GPX I reduction and

13

glut mat uptak di rupti n 10
f f1

n id Vvith

cl lg,

\\Cll

frat (Liu t al.. 2 14 ). M ri ctin, a member
mpl etely inhibited R

.

brain mit

ti on. In additi n.

G rmation

fa M ll g ; . t me

hondrial d · fun ti n (Fran

cllg ha. hi gh affinit y f r cysteine a. mcnti ned ab

ct al. ,

c. and

mpl c.· . tructure i simil ar to that f the ne utral amin acid .

r m thi a. p L methi mn e ma; al.

M ll g c. p ure b

gcn rati n and lipid

ugge. t th at anti -ox idati\ c agent. may . ervc a p t ntial treatment for

Me ll g int

methi nine .

rt

nti . idant pr p rti e ..

n indu ed b
.... 01 ). ~ h e e.· mpl

rebral

sef\ e a. potential treatm ent ~ r acute

mp titi n r r the bind ing . pot of th e Mel lg- ' ys tcin c compl ex (Roos ct

al.. _o 11 ).

2.2.2 Calcium hom eo tasi

Cal ium and cal ium ignallin g path'v\ a) pl ays an important role in Mcl-I g indu ced tox icity.
Alm o t all the id ntifi ed mechani m related to MeJ l g tox icit) 'v\ a initi ated or lead by oth r
fac tor to increa

f intrac llular calcium conce ntrati on ([ 'a 2 +]i ) (Roo · et al.. 201 2). There

are two main way that [ a2 ]i can be affected: one i the regul ati on of intrace llul ar Ca 2
tore, and another is pla ma membrane perm eability to

+

rupti on of either ource can

alter intrace llular calcium concentrati on (Atchi on and Hare, 1994 ). Intrace llular ca lcium
signaling i required to timul ate energy production by catalyz the key enzyme in Kreb
cycle which can directl y affect mitochondri al re pi ration fun cti on (Roo ct a!. . 20 12 ).
acted a a dri ving force in the mitochondri al matri x, and an in crea e o r cytoso li c Ca 2 •
ti mu lated a partate-glutamate exchange r ( l 25a 12, part or the malate-a. partate shuttl e).
thu s b o ting pyruvate upplicati on to ini ti al Kreb cyc le in co rti ca l neuron · (Li orente-Fo lch
et a!., 20 15 ).• akamoto et a!. ( akamoto et al.. 1996) demon. tratcd that ca lcium bl oc k. rs
(flun ari zine, nifedipine, ni cardipinc, and erapami l) attenuated the to.· ic effec ts (hod \ \\ ei ght
14

mpt m ) m rat indu e

and n ur

g1 al

gr nular

11 , al ium bl

k r Ounari7ine v.a

death fr m M I lg t xi it; .
b rv d the reducti n
ultur b) u ing

n ther , tud;

[ [ 'a- ]i and

· 'a 2 '

a2

nth~

.

' b

.

u mg pnmar

reb llar

h v.n t hav pr t ted fleet again t ce ll ·

ondu ted b;

]a: .

t a!. ( ' a o t a!., 2001)

h nne! hi ' ker (ben/ami!). and inhibitor (thap ·igargin

f ' a 2 •-

shO\\ed that Me l lg induced R S [! rmation that

a 2 • m hili/ali n fr 1m endoplasmic reti ulum store and e tracellular

ther h nd. an earlier stud) puhli . hed h) ( ;ama et al. (Oyama et al., 1994)

r p rt d that MeHg (I 0)1
enhancement

M ll g.

cl Ig ' ) l<. t ·icit; in rat cerebell r granule neuron

P e Ill nd pia mic reti ' ulum . I he) al
\\>a indep nd nt c f

b

) induced increasing le\el~ of [ 'a 2 ]i V\hich resulted in the

f R , g n rati n in isolated cerebellar neurons )f rats . .'imilarly. another

tudy dem n trat d in rat tri tal ·ynapt

ome · that RO ,' formation V\a a downstream event

of mit chondrial dy function indue d h) Mellg V\hich ""a dependent on an increas
mitochondrial calcium level ( reiem and
whether [Ca 2+]i o erload and R

of

eegal. 2007) . Therefore. it i . till d batable

generation indue d h; MeHg are a cau. e-effect or an

independent event . However. there i little doubt that r gulation of intracellular calcium
level i a critical factor in the n urotoxicity outcome indue d by M Hg. In addition. it wa
hown in

57BL/6J mice orally exposed to MeHg ( 1 or 5mg/kg) for 11 day led to R

formation. [Ca 2 +]i alteration. decrea e mitochondrial membrane potential and r localization
of cytochrome c (Bell um et al.. 2007). The e events did not accompany a ignificant n uronal
ce ll (mi c cerebellar granu l ce ll ) death. indicating that ubcellular biochemical chang ,
occurred without lethal cell inj ury in hort term e. posure to Meiig in mice (Bellum et al..
2007). Though Me l lg may not direc tl y lead to neurona l death, inhibitio n c r glutamate uptake
may cau e ce ll energy d fi cit ( ehinde r et al.. 1996) as a resu lt of mitochondrial d) sfunction,
ca lcium dy homeostati

and reduced glutathi one leve l.

bnormal calcium ·ignalling or
15

al i urn i n d h n1

ultim tel

tgn !ling r athv,a; i
~ napti

1 ad t

fl

f bi

n rg ti

1n

mit

hondrial

tn\ h d in man; impc 11ant neur nal function such a

pia tici t). the g~nerati n of brain rh) thms and asso iated learning, mem r and

c gniti n ( erridg . _o I ). Ph :ph lipa~e ' is knov.n a. initial fact( r of calcium signalling
thr ugh

ti\ ati n of JP~ In ~ it I tri pho ph ate). a sec md messenger. i capab le of regulate

n entration betv.een e'tra and intracellular calcium store ( ' hang, . . . 011 : Venkatachalam et
aL '

2) . Mellg ( ~M) \\as

r und to initiate calcium signalling by activation of

Pho ph lipa e ' and phosph lipase
111

m u

2 \\hich re . ulted in the release of interleukin-6 (IL-6)

c r bra! glia ( 'hang. 2011 ). Ba ·ed on the

bsef\ation of Mc11g increasing of

[ a2 Ji in pinal motor n uron culture . Ramanathan ct al. (Ramanathan and Atchi son. 2011)
+

rep rt d that M Hg-induc d calcium alteration v.a. related v..ith the glutamate receptors
mediated pathway .

pplicati n of NM A receptor blockers ( AP-5 or MK- 01) ignificantly

reduced MeHg-induc d calcium influx . In
intracellular

..,

a~

ummary. the con equence

level is an important mechani m for th

downstream pathway that lead to neurodegeneration or cell death.

of increa ing

activati n/inhibition of
alcium i al o the key

ion in linking the proce s of synaptic transmission and neurotran mitt r relea .

2. 2. 3 Synapl ic transmission and Neuro/ransmi//er release

Another well-documented mechani m for MeHg to. ic action
neurotransmitter (NT) r lea e in the proce

the alteration of the

of synaptic transmi ssion .

xon terminals are

distal termination of a on that functioning a storage and release of Ts . rhi · proces: i · aid
by opening of vo ltage-gated Ca 2-t channel · when depolari;:ation occurred in the pres) naptic

16

m mbran

f [

n

a ad

lin
a ~+ ]i

t L l

1 . B th intr - and e tr

in n n e t rmin 1 • nd the

ignal

f fu i n f ;napti \e i le t r lea c

t hi . n. 19

n n

mpetiti\e m nncr t inhibit ' a2 mflu

f al ium th n an evoke a . ignalling
·1 b; e. c;t s1

el Ig \\

he human brain
ynap e. The

t s napti

left (I enn

~ 11 v.ing an acti 1n p tcntiaL but incrca e the

I [ a2 ]i in nene terminal (I nn) and

n ur tran mitter

l ium c ntribut t the high

r 'P rtcd t blc k ' a 2 ' channeL in the ncr c terminal 111 a

nd

n ntrati n

).

cllular

tchi ·on. 1996) . ·1 h~ ma111 target d

f Mel lg arc glutamate ( ' ]u). d paminc (I

). and cholinesterase ( 'hE).

ntain · apprc '\imatel; 10 11 neur )n . . and each neuron has ab )Ut 7000
. ;nap · ~

are the · mnccti\c stru ·turc that allov. s "cross talk" between

n ur n ( adiq et a!.. 2012 ). It ha · b~en ·ho\'- n that Mel Ig altered the s; naptic transmi ssion
by changing either th

ndu ti n nene action potential or directly inhibiting synaptic

tran mi i n. or both ( tchi
acid receptor in
non-NMD

nand Hare. 1 94 ). Mel Ig particularly inhibits e, citatory amino

. Bl eking

f ex itatory amino acid receptor of

MDA receptor and

recept r po tponed th increa e of [ 'a 2 +]i mediated by Mel Ig (Ramanathan and

Atchison. 2011 ). The amino acid L-glutamate a
mammalian

the major e citat ry

ignal

in the

. plays a critical role in neuron development including ynap e induction.

elimination, cell differentiation and death. Glutamate i con tantly ecret d from the cells but
removed quickly from extrac lluJar fluid. and there are sev raJ thou and-fold gradi nt
difference aero

plasma membrane . In normal condition. only a mall amount glutamate i

ke pt in ex tracellular pace (Danbolt. 200 1). Therefo re. it i importa nt to maintain a ll1\\er
level of glutamate in the e tracellul ar fluid .

lutamate uptake i the primary mechani m for

maint nance of glutamate co ncentrati on

whi ch pro tec t

the brain from glutamate

excitotox icity. There are a number of report prov ing that the inhibition of glutamate uptake
an important mechani sm

f Mel lg- induced exc itoto:icity. }~ or example . .luare/ et al
17

t aL 2
front l c rt

2 . u d mi r dial

f rat

p

d t

Anoth r tud; r ported Mel Ig at
uptake 111 a

b er e glutamate fluctuati n in the

M I Ig and [! und an remark ble increa e

glutamat c 11 e11trati n. It \Na al
inhibiti 11 of glutamate uptak

i t hniqu t

f e tra ellular

rep rted that Ia tational e p sur t M I Jg caused an

1n cerebellar . lice,

f mou e pup ( Manfr i et a!.. 2 04 ).
icle glutamate

n entrati n: bet\\-een 2-1 011M inhibited

ncentrati n dependent manner. the authc r~ als

dem 11strated the failure of

glutamate upt kc \\a re ulted from inhibition f the proton gradient and I J+- 'I Pa e activity
inrat") naptic\~

i le(P rciunculactal..200 ).

Cdutamate re ept r ig11alli11g v..a f und t be an thcr ke) target for Mel Ig.
(u-ami 11o- -h) dr . ) -5-meth) 1-4-i oxaz lepropionic acid recept r) and

MPA receptors

M A recept r ( -

mdh) 1-d-a partat r cept r ) are glutamate receptor that play vital roles for modulation of
. naptic tra11 mi 1011. y11aptic pia ticit; in
Leveille et al.. 2008:

' (Danbolt. 2001: Ha himoto et a!.. 2001:

ku and Huga11ir. 2013: ' hepherd and Huganir. 2007). motor learning

and coordination (Ha himoto t al.. 2001:

ku and H uganir. 2013 ). Activation of

MD

receptor was reported a a mechanism of MeHg-induc d excitator neurotoxicity. An in ' 'ivo
c periment studying effect of MeHg on brain developm nt and vulnerability to NMDA
r~c.eptors (Miyamoto et aL 2001) found that the mo t

ever neuron al damage occurr d in

the occ ipital cortex of P 16 rat . and the MeHg-induced neurodegeneration \\a
attenuated by an NMDA receptor antagoni t. MK-80 1.

·ignificantl)

dount e and Chan r ported in an in

nlro study that exposure of MeHg 0.25 and ljlM to human

H-SY5Y cells for 4h re ulted in

an increa e of NMDA receptor level and ub equ ntl; cau ed cell death ( dount e and

han.

2008). Ba u et al. (Ba u et al., 2007) reported a decrease in NMDA receptor leveL in \\ ild
mink brain exposed to 0-2ppm Me ll g in dietary . The author. suggc ·ted that the decrea ed

18

M

r

r ult

pt r

indi at d ab vc. a tr
thu

f

mp n at r

m

hani m [! r limiting Me ll g mediat d

; tc arc more su cpti blc t

cl lg a cumulate than neurons, and

r pre ent an imp 11ant target fc r Mcl lg tox ici t; ( . chn r et al.. ... 007 ). Besides

glutamat r uptake. the a. tr c; tic glutamate tran sp rtcr is an ther regulated mechani ·m t
balan c glutamate home sta ·is and the le\el of extracellular c. citoto 1c amm J acid .
Jut mat a partate tran. p rt r ( i L , ' I) and glutamate tran sporter- ] ( :iL'I- I) are primary
glutamat

tran port ~r. that are found prcdc minantl; in astrocytcs (Mutku · ct al., 2005).

MeHg \\a

h \\n t inhibit the tran port of c;stine and c;stcine in astrocytes (A ·chner ct al.,

2007). MeHg \\a

neur n

al o rep rted to affect s dium tran sportation bet\\cen astrocyte and

through in hi bi ti n

f a tr cyti

cy. tine uptake on the ·ystcm X( A ' ) (cysti nc

tran port y t m in a trocyte ) ( II n et al.. 200 1b). Treatment of ' II -K 1 cells with Mel Ig
ignificantly increa ed the xpre

n of

LA T tran porter and decrea ed cxprc s1on of

LT-1 in protein level (Mutku et al.. 2005). Another stud; al o reported that the mR A

level

of two

lu transporter .

LA T and

LT -1. \\-ere down-regulated in the cerebral

cortex of rat treated with MeHg (Liu tal.. 2013 ).

In addition to

lu, other NT such a cholinergic and the dopaminergic y t m \\-ere al o

reported to be affected by MeHg. Musc le chol ine tera

activity was hown to be inhibited in

H. malaharicus fo llowing low do e of M Hg e po ure in a fi ·h model ( lve Co ta et al..
2007). However, Ba u et al. (Bas u et a!.. 2006) reported no significant e ffect of Mellg on

bl ood cho line tera c ac tivity in capti ve mink, but hi gh ' hE activit) \\a detected in th e
occipital co rtex and basa l ga ngli a of mink brain , also they ob erved a paralleled increased
mu carini c cholin rgic rece ptor leve l. The c studies indica ted a potential di ·ruption of
19

h lin rg1 n ur tr n mi
m

iII b

)11

pti

tran mi

1

n

u h a

f~

t

fMeHg nth dopamin rg1c

el Ig . emingl) targeted multiple fa

di e

tr n mt. 10n pr

he

ti n enti tied "M th lmer ur

d In the

addr

n ur d g ner ti\

n regul ted b Me ll g.

e ..

ri k factor

a
In

~ r

the glutamate

ther pr ce .. es affected b; Mel lg invol ed 1n synaptic

neuronal p tential and the S)naptic do~n tream . ignal ling ar

furth r addre cd a. it i be) ond the

C

n t

pe of thi . re\ leV. .

. . . . -1 Effects on C\to.\keleton microtuhuln am/ Other.\

Mi r tubule i

ne of th" thr ~e mmn comp ment ,

ther part

Jude mi rofilament (made or pr tein actin) and intermediate filaments .

111

Micr tubule pia; a \ita! r lc in mit

1 · pro

r the cyt )Skeleton in eukaryotic cell ·,

e ·. including mitotic :pindle in dividing cells,

cell cycle arre t at M pha e and Iat ap pto i ( M IIi ned

and ' ajate, 2003 ). ' yto ·keletal

protein are required for cell movement. mitotic ·pindle fom1ation. organelle and ve icle
tran port chromo omal

gregati n. cell ignaling and cell divi ·i n. thu any impairment f

microtubule would affect th

a ociat d function (Cre po-Lopez et al.. 2009) .

ffects of

Hg pecies on microtubule n twork ~a introduced in the 1970 . mercury preferabl; bind t
protein that form the microtubul

. tubulin and kine in . Microtubule di ruption ~a reported

in a number of MeHg expo ed experimental model . Berget al. (Berget al.. 201 0) conducted
a proteomic

tudy where they identified the group function of tubulin/di ruption or

microtubule in Mel Ig e po ed Atlantic cod brain. An in \'ilro study u ·ing di !Terentia ted N2
neurobla toma cells showed that MeHg perturbed tubulin tyro ·ination. the earl; neuronal
biomarker of mercury toxicity, implicating the neurite outgro'v\th 'v\as inhibited (La\\ ton et al..
2007). Me l Ig i known to di rupt cell c cle through microtubulcs hccau~c the mitotic spindle

tructure comprises the most abundant microtuhulcs proteins. In neuronal (mouse
20

n ur bl
and
1111

2/

t rna) nd n nn ur nal ( P L.. c 11 , M 1Ig tr atment I d t
pt

ub equ nt

ted

ell

arre ted

) le p rturbati n thr ugh int rfcring with

an imp rtant v nt in th development of ap pt , i in b th ell line , (Miura

r tubul

t al .,

i , indi

pha.

).

icr tubul e

rc

riti al fil ament us stru ture

t

reg ul ate the

nset of

ap pt

, and th p5 independ ent path\\ a): \\ a. in\ olved in the Mel lg m di aled ap pt

(Miura

t a!..

mi grat r; pr

).

sho wn t

el lg- indu ed mi crotubul e dis rupti n v., as a!

nd di . turb a'\ nal elongatio n in th e cultured embr

inhibit

nal carcin rna cell s,

nd the e path I gi al h nge are important t ) un de rstand )f Mel Ig induced tru turc crash
f

r b llum and

ther brain regi n in the deve l pm ent of utero and postn atal tage

(Philb rt t a!.. ~ 0 0 ). ince M Hg hi gh! ; rea t Vv ith sulfh ydr; l gro up in cy tein e re idues of
ce llular mi crotubul pr tein (Philb rt t al. . 2000) . Mel Ig can potenti all y inhibit mi crotubul e
polymeri zati on, and thi effe t i a um ed thr ugh binding to the free sulfh dry! group at th e
end and on th e urface of mi crotubule : thi ha bee n prove n in ''ilro e perim ent (V oge l et al. ,
1985 ).

In recent year , the role of glial cell e pec iall ; a trocyte . in maintainin g functions of
neurons have been bett r defin ed. They not onl y pro\ ide ph y ical structure to

upport

neuron , but also functi onall y interact with n uron or non-neuro n ce ll

in ph y ical and

di seas conditions, which include neuronal ynchroni za ti on, ynapti c tran. mi

ion regul ati on

and controlling blood flow (Volterra and Meldole i, 2005) .

li a ce ll infl amm ator; ignaling

respon e to MeH g i a n wl y empha ized mechani m in re pon e to M 1-lg neuroto. icit) . In
attempt to te t Mel lg on the re pon e of pro-inflamm atory cytokine from gli a cell s. B a ~' ' Ctt
et al. (Ba sctt et a!., 201 2) found Mel lg dec rca ed IL-6 relea 'e in a do. e depend ent manner in
microglia/a tro yte culture with pre-stimulation with toll -like rece ptor li gand . AI ' O. the ratio

21

f mi r gli
11.

m difi

nd
h

tr

111

r ult

indi at

that

eH g

inflammat f) r

' I 7.

L4.

b an 1mp rtant fa t r affe ting l L-6

an a [! ct micr glia

ti nth int ra ti nb tvv en m1 cr gli astr

h m kine

ell

'

fun ti n thr ugh
lTilCf arra

. In an her

t

' ' L9 and

ti on \\ ere f und tc be , ignifi

L 1.... ) that were a

tud ,

ciatcd with

ntl ) in ' fCa cd (.... - [! Jd hangc ) in re ponsc

erebcllum ( I Omg kg da; for 10 da , ) (II an g ct al. . 20 II) . In

eH g timuli 111 m u

t

[! und t

ulture

dditi n. gap jun ti n hannel , a

nncc ti n . tru turc bet-w een a. tr cytes, play · a vital role

in n ur -gli int racti n (R ua h t al. . ~000) . ' nn cx in protein c nditi on , arc important in
th

[! rmati

n

f gap JUn ti n

111

n ur n-gli a co mmuni ca ti ns.

dj ace nt astr cytes

JS

connected thr ugh gap juncti on hannel ( ' J ' ).e.g .. '. 4 and hemi chann cls t regul ate A TP
relea

and glutamate tran mi i n ~ 11 wing intrace llul ar calcium elevati on to respond

neur nal glutamat r I a in g (Maragaki and R th tein . 2006:
ther i little r earch to inve ti gate th e rol e of

J ' fun cti on

r Il ana et al. , 2009) . ,'

n mercury neuron tox icity

(Kawa aki et al.. 2009 : Kothmann t al.. 2009). The arli er publi cati on
inhibited the function of

J

far.

howed th at MeHg

both in cultur d rat o teo bl a t-like cell (. chirrm acher et al..

1998) and rat pro imal tubular cell

(Yo hid a

t al.. 1998 ). Mercury chl oride wa al o

reported to affect junctional coupling in epitheli al canine kidney cell ( leo et al.. 2002).

Thi

literature revtew hi ghli ghted a number of sing! mec hani m

re ponsible of Mell g

neurotoxicity. It is important to note that there are many potenti al known and unkno\\ n interconnections b tween each of these m chani sms. For exampl e. both the event of ox idat ive
tre

and glutamate dy homeosta i were m chani m invo lved in M ll g tox ic it) ob en ed

in rat c rebral corte , and the potential
al

provoke m rc RO

f

lu dy homeo tas i cau cd Ca 2+ overl oad could

formation (Liu et al. , ~ OL ). Mell g cxpo ·urc induced

.· idati\e

22

H d pl ti n, R

~ rm

glut mate tr n p rt r and

" rl ad

t\.\ 0

e

dam g (

2 14 ).
ffl ted

h

nn

h th fll1

t d pr

ti n and the

it

m 111 metal

wa m diated b

p th a

f R

'

( eng

rt

f intr el lular calcium in the rat

t a!.,

gcnerati n and glutam tc d h meo ta i

ellg-indu ed neur t . icit; ( , chn r taL 2007).

3 . Meth y lmercur a a n k fa t r f r ne urod e

h

it

indu ing n ur heha\ i )ral

m th lm r ur; and le d ( 'arp nt --r. ~00 1).
), p rkin

ne rative di ea e

hanges after dcvel pmental c p

cure degenerative di · rd r

uch a

( rr ) and am) tr phic lateral ·cler

n' . di ea

ure arc

lzheimer'
(AL, ) affect

v raJ million pe pie V\ rldV\ide (Migli re and 'oppcdc. 2009). The inheritance of m st of
n urod g nerativ di · a

rar ly [! II "' Mendelian laVv but in ·tead reflect a more complex

interacti n V\ith multi pi predi p

ing gene and en vir nm ntal factor · (

va t majority of AD. PD and

L

contribution of comple

occur a

p radi

chner, 2009). The

D rm , likely re ulting from th

int raction betwe n gen tic and nvironmental pollutants uch a

pe ticide and h avy metal . Interacti n between gene and environmental factor is crucial
in modulating the vulnerability to PD ( L' Epi copo et a!., 201 0) . Th alteration of tubulin
tructur , mitochondrial dysfunction and sub equent biological change in lipid, protein and

DNA peroxidation are common change ob erved in AD (Carocci

t al..

2014) and PD

patients. These pathological changes are al o often ob erved in MeHg induced neurotoxicit)
in different exp rimental model .

PD i a commonly occurring neurodegenerati c di order characterized b.}

elective lo, · of

dopaminergic neuron and progres ive neurodegencrativc movement di ·order that produces
mu cular rigidity, bradykinc ia, tremor of re ·ting limbs and loss of po ·turn! balance

23

( bdulwahid
pre

rif nd

I nt and d

hm d Kh n. 2 10: Jiang t 1.. 2

in th br in.

nigra. v ntral t gm ntal ar ~a

1·

ncur n main!

nthc i7 d

t v.idel; . tudied s;~tem · in the brain . ince iL di sc

7 : J nc. and

in th sub tantia

ncur I gical and en do rinol gical diseas s such

hi7 phrcnia. and I Iuntingt n · s, d i ca. e ( ' ui et a!.. ~006 ).

unn tt. _

pinephrin ,

nd h; p thalamu. ( i oullon. 2002). The dy. function of

d paminergic rclat d pathv\ a) . c n pr du

am ng them

) i the m st

pamine (

um nt d ate h lamin neur tran mitter am ng th h rm ne

n r pin phrin . and

a

7 .

neurons ha c been
cry (Bjorklund and

iller. _00 :Yo ung et al.. 2011 ). In addition. dopamine rc carch

ha b n unique \\ithin the neur . cience in a v.a; that is ahlc to bridge basic science and
clinical pra ti

(Bj rklund and

unnetL 2

7b). ince I

is involved in many important

a pect of behavi r. uch a m tor controL m od regulation. cognitive ta k . addicti n, Jeep.
puni hment and re\1\- ard ( i oull n. 2002) ( Li
neurotran mi i n m diat

and Roeper. 2008 ). D paminergic

through everal linked pr ce es. including sy nthe i . rclea e.

torage. r ceptor activation. uptake and metaboli m. Tyro inc hydroxyla

(TH). an enzyme

that limit DA ynthe i . convert L-tyro ine to L-

P . \1\-hich i then decarboxylated to

DA. Then DA is tran ported into

through the ve icular monoamine

ynaptic ve icle

tran porter (VMA T). with presynaptic action potentiaL

r leased from the cell.

nee

relea ed, DA activates a variety of po t ynaptic receptor \1\- hich ar Iinked to different c II
signaling pathway . DA transmi sion is inactivated by re-uptake the of D

tran porter (DAT)

into the pre ynaptic neuron where it i metabolized by catechol-0-methyltran ·fera e ( ' MT)
and monoamine oxida e (M 0) (.lone and Miller, 2008). Environmental neurotoxicant · are
able to depre

or enhance dopaminergic n urotran mitter by impacting th

proce , leading to pathological alteration
add iction (Jon

and Mi ller, 2008).

in DA-assoc iated disorder

D

cycling

such a

PD and

nd rslanding the molecular mechani sm or hO\\ those
24

nz m

k

m th
nt

n ur

d pamm

and d pamm rg t

lib

'Will fa iii tate identifi ati n or d , 1gn

f n

a ad

path a

re pond to

I gical targe t to

ph arm

r lated to dopamine neur tran mi tter di order.

pr te t nd treat di e

lth ugh the eti 1 g; and path )gene, i.

f P

1• p

rl; under. tood. interacti n between

g ne · nd ~n\ ir nmental fa tc r. haH: been impli a ted a. critical fact rs 111 m dulating the
vulnerabilit; t P
p llutant

t aL. 2000 ).. ' e\ era! ·tudi s ha\ e rerorted that en vir nmental

u h as pe. ticide: and hea\; metal

( j rklund, 1

al

(I Ieller

M i h lk et al.. 200 : R

been h 'Wn t link t a\ ariet;
lzheim r'

di ea e and Parkin

f PO

s ct a!.. 2006h ). r, posure to heavy metal ha

related di ea ·es . uch as

ni m (J n

ttenti n

and Miller. 2008) .

eficit

isorder.

'hr nic e p ·ur

of

g n rati n ha h en pr po ed to contri hute to the neurodegenerati n

rot n
f

r0

pia; a ke; role in the pathogenesis

neuron

ander

and

r enamyre. 20 l 3 ). ' harle. et al. ( ' harle

et al.. 2006)

analyzed data collected from L049 men aged het'Ween 7 l to 93 and D und a igni ficant
a ociation between occupational e po ure to pe ticide . olvent . metal , mangane . and
mercury (during their middle age) and 14 movement abnormalitie . It wa found that high r
exposure to any metal , and

p cifically mercury, wa a ociated with abnormal fa ial

ex pres ion. Dantzig (Dantzig, 2006) tudi d 14 patient with Parkin on' di ea e, and found
detectable amou nt of blood Hg in 13 patient who a! o had

rover'

di

a e (tran ient

acantholytic dermato i ) and proposed that llg cou ld be involved in the etiolog; or PD and
rover's di ease.

A di cu ed earl i r. the syndrome cau d by Me ll g poi ·oning is called Minamata di ·ea:e
( eccat IIi et al. , 20 10). The early ign or congenita l Mellg poi ·oning can he mistal-.en for
other di ea c and over-looked, especia ll y in mild cases ( randjean et aL 20 I 0) . l--or
25

amp le, th

ndr m

pi

h p

the ta ,

.
mt a

t a!.. 1

d

h aring (

ntr 1 tud; .

c e
t

.

111

ith r m ngan

pr du

), v. hi h bar m n)

r c pper \\ a. as

iatcd 'v\ith P . 1 he

ppcr. v.. a. a.

iated 'v\ith P

cupati nal e po ur
als

[t und

howe

r,

indi ate that

f the

th t dual

ccupational

r nn al n \\ a: n t related t th e n. et f P

arl ) ex pc ure t

chemi cals in creased th e

metab lie ;ndr m lik di ea c and c th er health i ue in later life (F
ampl e i

f

imi I ri tic. t P . In a p pulation -ba cd

rell ct al ( J rcll ct al.. 19 9) ~ und that hr 111

p ur t 1r n, m r un

doe

d in adult during the Minamata and Iraq

n r di turban c , tr m r , d arthria, and lo

f le d. 1r )n and

mbin ti n

vid n

d b Mel Ig b r

u ccptibility to
ct al. . 201 ). An

p ur t meth; lazo. ymcth anol (a ncuronto in ) from th e ource of fo od

or m dical treatm nt potenti all y contributed to th e eti ology of AL,, and PO dementia
compl e

ndrome in region pecifi c p pul ati n ( ' uam. Kii and W st Papua). ' urrent

know! dg indicat

that th re are van ou ri. k fac tor leadin g to the dev lopmcnt of PO

phenotype. which e pre sed in th in cr a ed odd rati o for PO in epid m1

g1c tu die and

characteri tic ofth e ph notype produced in experimental model ( 'ory- , I chta t al. , 2005) .
Wh ther MeHg expo ure i a ri sk factor in th e form ati on of PD phenotype i

till not kn own.

However. th ere are known ynergisti c effe ct of vari ou to in that can re ult in co nditi on
like PD. For example. oxidati ve tre

is an important factor o curr d in the proce

neurod egen rati on ( hukla et al. , 2011 ). Many of the MeHg induced change

f

uch a

oxidati ve stre

and di turbance of neurotran smitter functi on have al o been impli ca t d a

major factor

in PD . Moreover, th ere ar

likely other overl apping and/or unknO\\n

mechani ms in the etiology of PD . Re earch on the e. pl orati on of envi ro nm ental toxicant ·
uch as M I lg e po urc and relevant di order ha e trcmcnd ou · impli ca ti o n ~ in prevention

26

and lini al

gn

m hani m

fM

MPP

ti

( ang n

i a m t b lit

pr du t

id n

f MPl P. a \\ell kn 'Wn to icant that el cti ely de troy

d pamin rg1

neur n in the uh. tantial ni gra. v.hi h is haractcri ti

.... 01

. MPT

\\a identifi d b) a

n 'vV

nth ti

th m hani ti
drug in anti- P
and th n entry

~ th

(llarc et al. ,

imilar

hrc nic . ign · of Parkin s n ·

di ca c

n. MPTP MPP ' \\a. \\ide!] used a parkin on m mimic m
u1 t al., 20 I 1: Zhang et al.. 2011) and devel pment of new

tud; of

tal.. 20 l 0: ' a

(

of P

identl; in 19 ~ fr m pc pic \\h addi ted in admini ster a

mpound \\hi h c n induce

(Lang t n tal.. 19 . ., ). in

need d to larif

ll through

tal.. 201 0) . MPP + i · c nvert d from MPTP by MA ,

T. which particularly high e pre · ed in the cytopla m and

membrane of ub tantia nigra par ( torch t al.. 2004 ). MPP ' i d liv r d from cytopla m
into the mitochondrial matrix by the gradient betwe n inner m mbrane . and then combine
with NADH dehydrogena e of complex I to top electron tran port. I ading to
The ub equent effect of the energy d ficit cau

TP depletion .

of extracellular DA level in

triatum, and further promote the generation of R S. uch a hydro yl radical ( · H) ( ing r
and Ramsay. 1990). Therefore, the important manife tation of MPP+ excert it neurotoxicity
is mediated through inhiting respiratory chain compl

I in mintochondrial. Intraventricular

infu ion of MPP on rat ha been shown to reduce triatal DA level and progr

ive lo t of

TH-positive neurons. Mitochondrial appear ~ woll n. and conden ed material \\ re found in
DA neurons (Yazdani tal., 2006). Mor over. execs ive production of free radical in rea ·e
memberane lipid p ro idation thu ace leratc the vulnerability of dopminergic cell death
( bala, 2002) a wel l a

ther neuro logical ympotom 'Which featured by PO patient .
27

High-throughput

cr emng m th d

ha e dem n, tr ted that multiple

In

ed int

MPP

indu ed d p mincrgi

indu

d gen

hang

in th

ateg ric

ample, MPP + in 40 ~M

n ur

f ap pt

ignaling pathwa

. . id ti ve tr s. and ir n binding

n

ell . a midbrain d rived d pamincrgic ncur nal cell line (Wang ct aL 2007).
ha

g n

m d pam in rgic

hang

p pt

and

mit

t al ( ' ha

et a!. ~ 15) :tud\.

imilarl , in

h ndrial

PP + 'A a

ignaling tran ·du ti n. tran , porter

d grad

y tem. v.

al

and iron transport.

d\ ·functi n. I paminergic signaling transduction

d m n tr ted a th m t affl ct d function affected b; MPP +.
MPP n ur t xi it; . . u h

hown triggering signifi ant

was

ther mechani ms related to

· ubiqui tin pro tea · me . ; tern (UP ,' ). an intraccll ular protein
h 'An a · ciation v.. ith MPP induced d pamincrgic n urotoxicity

(Lim. 2007). It ha be n r p rt d that Puerarin. an anti-oxidative c mponent extracted from
Pueraria I bata and

h'A i Pu raria th m

ni i Benth. v..as able t

protect , , 11-SYS Y from

MPP+ ( 1mM) elicited c ll death through r gulation of UP ' functi n ( ' heng et al.. 2009) .
Mor over. MPP e hi bit d neuroto icit; wa reported to be related to the metal m taboli . m
in idiopathic Parkin on· di ea e (Hare et al.. 2013 ). In a population ba d ca e-control tud).
Gorell et aL ( orell et aL 1997) howed that occupational expo ure (more than 20 year ) of
lead-copper. lead-iron and iron-copper ignificantly as ociated 'Aith PD than any of the e
metal alone, indicating the e metal may erv a n k factors alone or together to facilitate
the d velopmcnt of PD. However. th

mechani m underlying the e effect remam to be

ill ustrated.

28

hapter III.

ffect

f M thylmercur
eur nal

n D pamin Relea e in MN9D
ell

ame and lffiliation of 1111lor :

Yu ting ha 3 ,

ing Man ' hanh *

ur ·c and I·n\ir nm ntal ,' tudie. ,
Univ r it) Wa). Prin e ' e rge. B ' . ' anada
a unb

of
....

rthern Briti h

4Z9

cpartment f Bi I gy. nivcr ity of )ttawa,
1 6
Fmail : lauri .chan au ttaV\a.ca

0 Mari e ' uri c,

29

b tract

len

f Parkin. on' di ea e

ur t etnir nment l p llutant , but them chani m

f path gen si are till

_. pid mi l gi al evid n
(P ) nd

p

un lear.

he bj

d pamin rgi

tiv

ha, h \\n a

f thi . tud

L

iati n b tV\ en pre

t

tn\

• tigate effect.

and c mpare that t

n ur n l ell line.

f meth lm r ur (M ll g) on a
1-meth l-4-phen lpyridinium

(MPP , a \\ell-e ta li hed agent a..

iatcd \\ith pathogene i of PI . M

e p

(10-400~tM) ~ r 24 or 4

d to Mellg (1-10)-lM) and MPP

h.

D elL were

ur results . howed that

M Hg indu ed ell death v. ith b th time and de e dependently . M J lg al o dccrea ed the
r I a

of d pamine (

(H

) imilar t th effe t

th

ame tim . both MeHg and MPP .. de rea ed the ynthc i of tyr

). .4-dihydr x; phcn; !acetic acid (D P

' ).and hom

anillic acid

f MPP . . There V\a an incrca. e in I ( PA ' I IV AID

and dopamine tran p rter (D T) at the mR

and pr t in level .

ratio . At

inc hydroxyla e (Ttl )
Xpre

I

n

f the U.-

ynuclein (a- yn). a hallmark neuropathological indicat r of PD. wa al o up-regulat d at
the mR A level but not at th
oxida e B activity wa
The e finding

protein level after MeHg and MPP + do ing. Monoamine

up pre ed in all MeHg treatment and MPP ( 1).lM )-treated c II .

suggest that MeHg can disrupt th

ynthe i . the uptake of D

and th

metaboli m as well as alter the biology of a- yn imilar to MPP . Expo ure to MeHg rna;
potentially be a ri k factor for the development of PD.

Key word : Heavy metaL Neuroto icity; MP P+; Parkin on· di ea ·

30

Introduction
Parkin

n di ea

milli n

fp

hav r

gni7 d a p

(J n

and

(P ) i

f th m

pie v. rldv. ide. The
iti\e

t c mm n neur d g n rati

·a t au ·e of P

e er. rec nt

n. 197 ~).

rk

ccurren e of PO
metal · have

ciated '' ith the path gene is f PI (R )OS, ..... 00 : Michalke, 2009).

m thylm r ur] (M Hg).

and Mag

is , till unclear. I I

rr lati n bctv.cen indu. trializati )n and the

a w 11-knO\\ en' ir nmental neur t

M rcur ·

di ea e that affect

iller. _ 0 ). r n' ir nm ntal p )llutants :u h as pesticide and hea

b n r p rt d t be a

( lark

n

re m re t

ntral n rv u

icant. The orgamc

1c to li,ing organi ms than it

) tem i the mam target of rgam

[! rm '.

such a

1n rgamc form
mercury ( ' lark

n

. 2 06). M r ury an b accumulated in the c rte , ·triatum. hipp

ampu , brain

ord . The m t comm n clini al neur I gi al

ympt m of

t m, cerebellum. and pinal

MeHg p i oning ob erved in adult in Japan and Iraq epi
, en ory di turbances. trem r . dy arthria. and lo
Par the ia i u ually the fir t neurofunctional
2006 ). Pathological re ult
central nervou

de were hypoesthesia. ata 1a.

of hearing ( inomiya et al.. 1995 ).

ymptom ob erved ( lark on and Magos.

in the victim of Minamata our break al o indicated that the

ystem wa th primary it of MeHg poi oning ( ' lark on and Mago . 2006 ).

The motor dysfunction

caused by MeHg e po ure ar

imilar to th

ymptoms of

Parkin n · disease.

The bi ological mechani m of MeHg on central nerv u

y tem are ti ll unc lear.

ne of the

e tabli hed mechani m by whi ch MeHg can e ' ert to. icity i th roug h the di . ruption of
neur tran mitter fun cti on (Fitsanaki

and A chner. 2005 b). Dopamin e (OA) i the most

preva lent catec holamine neurotran mitter co mpared to epinephrine and norepinephrine in the

31

br in.
nd

h
rin

di ea

di rupti n
gi

l di a

f d pamtn rg1

u h

r !at d path a

P rkin
m

( u i et aL .... 0 . Th re i

n

111

1 ' eL (Far

P L ... , th intr - ellul ar dopamine
M Hg (

tt ri ct al.. _

meth 1- -a artate ( M

au

neur 1 gi al

nd

hiz phrenia, and I luntingt n'

di

vid n e f Mellg affe ting th relea

mpl . inje ti n f MeHg int the tri tum in fe ly m
dep nd nt in rca

n

.F r

f

in g rat indue d a concentrati n-

ct a!.. ~002 ). In th rat phc cr m c t rna cell I in

ntent v. a. d ·c-dcpend cntl ] decrea cd after

po ure t

). Mel Ig can aL o medi ate its nc ur t . icit) by interferin g with
) or d pamine rgi rccept r. .

egati e co rr lati on were ~ und

betw n t talll g and d paminergic

2 rece ptor den. ity in v.. ild mink (Ba u ct al. , 2005a) and

wild ri ver tt r ( a u et al. , 2 05b ).

I . Mef Ig wa. reported to in crea e NMDA recept r ·

both in vitro and in ,.i,·o (Mi yam t et al. . 200 1:

d unt

and

han, 20 8) . The e re ulL

ugge ted Me g c uld di rupt d pamin ergic path v..ay in the central ner ou

1-meth yl-4-phen lp ridinium ion MPP+ i

a we ll

anatomi caL behavioraL and biochemical change

y tern .

tudi ed comp und th at can induce

im ilar to th o e ee n in Parkin on' di ea e,

whi ch i characterized by ni gro tri atal dopamin ergic n uro-d ge n rati n (Heikkil a et al. .
1984 ~ Yuan et aL 2008). MPP ha bee n hovvn to cau e a lo

of th e stri ata l nerve termi nal

a indicated by decrease in tyro ine hydroxylase (TH ) and dopamin e tran porter (OAT) a
well a dopamine and its metabolites leve l (Nakai et al. , 2003 ). a- ynuclein (a- yn ). a
presynapti c protein closely related to DA regulati on (l· ountain and R., 2007: Fountaine and
Wade-Matiin s, 2007: Herm an on et a!.. 200 ~ Zhou et al. . 2006) i a neuropathological
hallmark of PD . Pro hibiti ve ac ti vity of DA ha

bee n foun d to be high !) rela ted to

acc umul ati on of th e a- , yn protofi bri I (C onway et al. , _oo I ). M pp+ has bee n ho\\ n to cause
an increa e in a-Sy n aggrega ti on (Jeth va et al. , _o 1 I). In addition, Mo n amine oxidase-B

32

(M

1

a mit

h ndrial

nz m

MPP ~ (

udim and

akhl ..... 0 ). M

that has b en

iatcd

ts po i ti

inhibit r ha. be n d e ~ c l pcd in th e treatm ent of P

and M
2

.

rt MPTP 1-meth '1-4-ph n l- L2.. -t trah dr p rid inc) to th a tive

n

d m n tr t d t
m tab lit

f M

f th

n

i th PO,

(Y udim and Ri dcrcr.

4) .

Th mam bj cti\'e f thi
it me hani m

mpan , n to MPr ·. ~· c meas ured th e rclca. e f

111

with MeHg and M P
of ynth
h dr

izin g

yla

·tud ) i. t in\ c. ti ga tc th e cflcc t of M ll g n

+

•

regul ation and
and its metabo lites

·p ure u ing a neuronal ce ll Iinc M 9 . whi ch have th e properti es

at ch I min e and ex prc. in g ncur n- ·pcc ifi c mark er

uch as tyro ·in

(Herm an n. _00 ). We hvp th e iz th at Mellg can dec rea ·c th e relca c of

by: 1) d cr a in g the
limiting nzyme for D

A

ynthe i 'v\ith dee r a e in l] r in c hydr xy la ·e (T ll ). th e ratei : 2) dec rea in g th e DA re- uptake through th e decrea e

ynth

f

dopamine tran porter (D T). a tran m mbrane protein th at m di ate th e r -uptak of DA;
and 3) reducin g DA metabolite by inhibition MA -B ac ti vity. W will al o in e ti gate the
effect of MeHg on the synthe i of a- yn in the M 9D cell .

Material and Methods

Cell line and treatments

MN9D cell s ( hoi et aL

199 1) were main tained in DM M ( igma. M . U

uppl emented with 1Oo/o (v/v) fetal bovin
100!-lg/mL streptomyc in ( ibco,
2 at 37°

erum , 3. 7g/L Nal-JC

1.

)

1OOU /mL penici ll in. and

nt. , Canada) in an incubator 'v\ ith an atmosphere or 5° 0

ell s were gr wn in poly-0 -ly ine coa ted lla ·k (BD. NJ, U,, ). MN9D celL

w re e p cd to different d c of Mel lg or MPP +. Stoc k so lut ions ( 1mM MeHg or 1OmM
33

MPP )

er

pr par d in \\at r. Medi

btain d fr m

]fa

ar

wa r pi

) and

d

er

48h. Me ll g

nd MPP

1

wer

igm - ldrich ( nt. ' anada) .

ell viabi lity a ay

n M

a

a h \\ 11

) \\a
f

arricd

-v. 11 plate

n entrati n bet\\

all

i r ~4h he~ r

bout

adding the neurot

x ]O

c lL

ere

lated in

1n Mellg or MPP + at

n I ~tM t l 0 ~tM, r l O~tM t 400 ~M, r ·pe ti vel . , ample treatment

ndu t d \\ith fi\
in triplicate .

ut t1 '\aluatc ell \iahilit)' .

rcpli ate in each , periment and each e, periment wa

onductcd

ntr I and blank amp! \\ere in luded in all ample plate . Treated ce ll s were

r , oluhiliz d MTT ~ nnazan produ t wa

ed t gr \\ [! r _4h and 4 h. The am unt

determined by p troph t m tf) at

95 nm . ' ell viability \\a e pre ed a percentage of

the contr I gr up.

HPLC detection of DA and it metabolite in MN9D cell culture medium

After 24h incubation with MeHg or MPP +. 200~1 aliquot of m dia from 1 x 10 7 ce ll w re
mixed 1:1 with ice-cold 0.5M metaphosphoric acid, and filtered through a 0.22~M centrifuge
tube filter ( orning. NY, U A) at 15,000 rpm 15 min at 4° . The flovv parts \\er collected
and 20~L

ample was then loaded. 0

, 3A-dihydroxyph nylacetic acid (0 P

homovanillic acid (HV A) were analyz d by
detector (pul ed Water 464, Millipore, M ,
usmg a

IL

N HPL

) and

y tern with electrochemical

). The isocratic

paration was perf rmed

18 rever e-p ha e column ( 150 x 4.6 mm, 4~M: Phenomenex. ' . U A). The

mobil e phase was made up of a 0.1 M acetate, 0.1 M citrate butTer at pl-14. cc ntaining
228 mg/L _, OTA tetra odium

alt. 200mg/ L sodium octyl sulfate and 10°o methanol a

de cribed previously (Vicente-Torr s et al., 2002). The OA, DOP

' and IIV

release le\ el
34

ln

11

1 ul t d ba d n a m1

!uti n.

nl

th r\\-1

tra n

ript P R

f
( nt.

m nti n d.

a

rding t

n

n c n ntrati n

II reagent \\-er purcha ed from

igma- ldrich

anada).

em1 -rev er

V\a i )lated fr m ·e ll , mplc, arter 4 h of e. p sure. vvith th u. e f

tal R
R

d tandard ur

a 1 Plu Mini

it ( tagen.

Ou r m ter ln\·itr gen.

c nccntration V\a. mea ·urcd in a

nt.. 'anada) . R

nt. 'anada).

11g of i ·olatcd total R

u ing th T qman Rev r c Tr n. cription Reag nt · kit ( pplied
a final\' lum

lumn
ubit

V\a. rever. ed tran cri bed
io y ·tem ,

nt, anada) in

f I 00 111 follov, ing the manufacturer's in tructions. Primers (Liu et al.. 2008;

Zhang tal.. ~0 I Oa) u ed were

T (forvvard 5' -T ' '1 GGT ' ATTC:iT'I 'T ' 'TC- 3'.
-...,, ). 1 H (forward 5'-

A

T

5'-T

ynuclein (forward 5'-T
A T
revrse5'-

T

T
ATAGA

T T

' GT

TC A A 'TT ACA A-3'),a-3'. rever e 5'-

-3'). GAPDH (forward 5'-TTGTTACCA C GG
T TTTA

CG-3'.

G-3')a aninternalcontrol.

ycling condition wer 95 ° for 5 min. 0 cycle of 95 °

for 30 ec. 56° - 60°

for 30 ec.

72 ° for 30 sec and follow d by a 5 min e ten ion tep at 72 ° . The product w reanalyzed
u ing agaro e gel electrophoresi . Band inten itie w redetermined den itom trically u ing
ene Tools oftware ( yngen . MD, U A). and the r ult ar

hov.n a r lative e. pr

1011

of ratio of target gene to internal c ntrol.

35

lmmun blot

fter 4 h f

p

ur

elL \\ere h n

ktail

nd

dium

rth \anadate (,' anta

h m gent7 d b) · Jni ati n and
·on entrati n \\a
pr tein

dermin ed b) Bradfl rd as. a)

e membrane ·.

1; i buffl r kit vvi th PM. F, pro tea. e
). The cell

ru1.

ere then

entrifugcd at I ~.O OOrpm for _0 mm at 4° ' . Pr te in

(_ -60~tg) \\ere . cparatcd

nitro ellul

t d in Rl

Bi -Rad. ll er ulcs,

' ).

liqou ls

n I 0-15° o a r) !amide ge ls and tran ferred

1emhr ne: \\ere hi< eked \\ith 3° o BS

sc lution

111

f
nto

TBST

.·olution befl re incuh tim \\ ith the fl liO\\ ing antihodic. : 'Ill ( ' hem icon. l :500), OAT
( hem icon I : ~
loading

). and u-.') n ( ' hemic< n. 1: 0 ). ' P I I ( ,' anta

ntrol. Pr tein hand \\ere dete ted h) a

quantifi d b) 1ene T ol anal)

ru7 , I: 00) was used a a

.3'-Diaminohenzidine (0/\B) kit, and

ft\\are (, ;ngenc. MI . US ).

A naly i of MAO-B activity

ell were collected aft r 48h treatm nt. V\a h d and re- u pended in c ld
mM K L 120 mM NaCL 50 mM

al--bP

4. pH 7.4 ). then

a/K buffer (5

onicated . , upernatant were

removed for protein concentration analy i (Bradford a . ay) . MA -B activity was mea ured
a de cribed earlier with modification ( tam ler et al.. 2005~ Zhou and Panchuk-Vo lo hina.

1997). Briefly, the reacti on tarted when th M 0-

inh ibi tor clorgyline ( 1mM) V\a

incubated with ampl e fo r 30 mi n at room temp rature, then 100111 of work ing olution
contain ing Am plex Red (20 mM ), hor e radi sh pero, ida e typ IV (200

/mL). tyramine

( 100 mM) and Na/K buffe r we re add ed into the cell ample . Incubation. \\ re continu d for
a minimum of 30 min . pecifi c flu ore ce nc of re orufin , the oxidati on produc t of Ample.·
Red reage nt, wa m a ured u in g a Multilabel Mi roplatc Reader (Bioscan Inc ., Wa ·hington

36

i th , itati n at

Onm and m1

rufin pr du t wa

e a pm 1/mg pr t in/minut .

pr

tati tical

nal

II d t

pre entcd a me n

ar

1

analy i V\ith ne-V\ay
r t-te t
(**)

n det cti n at 9 nm. R

r tV\ gr up

' .

. ,' tati . tical

. ignific nee

as obtained b

te ·t. [! II v.ed b) L,' 0 int r-group
mpan

n u ing ,' P,',' I .0 ,' ftV\arc .

mpan

vanance

n post ho test ,

aluc f P<0.05 (*), ? <0.01

r P< . 01 ( *** v. a r garded a . igni fi ant.

Re ult
T he to icity of Me H g and M PP to th e M 9 0 ce ll v iabi li ty

MeHg d crea d c ll viabilit at con

ntrati n of 5~-tM, 7 . 5~-tM, and 10~-tM in 24 and 48h

( ig. 1 ). The

~ 8~-tM in 4

50 wa

e timated to b

h. In contra t MPP + do ing re ulted in a

dec rea e of cell viability at all concentration ( 10~-tM -400~-tM) for both 24h and 48h (Fig . 1B) .

Changes in dopamin e and it metabolites relea e f rom MN9 D cell

Typical HPL

chromatogram of the culture m dia after the MN9D cell v..ere xpo ed to

MeHg and MPP are hown in Fig 2. The quantitie of e. tracellular DA produced b) the
MN9D cell declined with increa ing do es of MeHg (Table L (F(3,8)=, .607, ? =0.065) . The
lowest DA relea e was 0 . 1 2 ~-tM when do d with 10~-tM MeHg for 24h compared to 0., 4 ~tM
in the co ntrol. MPP+ exerted the stron ge t effec t at 50~tM resulting in 0.04~tM (P 0.0 I) on
DA release in the media.

here were decrea e tendencies in D PAC and I I A release from

ce ll media treated with Mel Ig and MPP+. The DO PAC llV AIDA ratio increased slight!)

37

ith M Hg

p

ur

.P

F . ) 0.

)

nd

ignifi anti

wi th MP P+ e p ure

P<O. 1).

ffect of in vitro e po, ure to M eH g and MPP

gel of Rf-P ' R result · and the quantified den ·it

ann d agar
T mR

fthc bands arc hown in

lc\cl in 1-7 . ')~M Mcl lg treatment did n t differ fr)m that in normal

ell

h 'v\e\er. ·ignifi

level

111

c 11

n dopamin e y cling gene

nt rcdu tion appeared in the d se

f 1O~M (Fig.

). Til mR A

do ed \\ith Me l lg ( 1-1 O~tM) remamcd unchanged . In comparison. u- ,' yn

ho'v\ d an increa ·ing trend in a do e-depcndent manner 'v\ith both Me l Ig and MPP +
tr atm nt ( ig . .., ).
and THe, pr

d

-dependent dO\\n-regulation trend 'v\a al o ob ervcd ~ r DJ\T

ion 'v\ ith MPP .. expo ure (Fig. 3 B).

Effects of in vitro ex po ure to M eH g and MPP+ on dopamin e cy clin g protein

After 48h of expo ure. pr t in lev I

f both TH and OAT were decrea ed ignificantly in

MND9 cell do ed with 511M and 7.5~LM MeHg (Fig. 4A) . The arne trend app ar in MPP -'
treated group

and the notable difference 'v\a

effectiv do se reported in previou

induced at a do e of 50!-lM. a similarly

tudy ( pittau et al.. 2012) (Fig. 4B) . u- , yn expr · 10n

level were reduced in the cells with either MeHg or MPP exposur

(Fig. 4A and B).

Effects of M eH g and MPP+ on M A O-B activ ity

The en ymatic a say howed M

-B activitie wcr

uppre ed ignificantly in all MeHg

treatments (0.111M , 0.5!-lM. I 11M and 5 ~LM ). with 18-fold

dccn~a ·c in eel L do ed v~ ith 5 ~L

1

Mell g (P<O.OO 1). The MA -B activity wa, lower in cells treated \\ ith 1~LM MPP \\ hil e
higher at high do e ( 10011M) expo urcs (Fig . 5).
38

p

ell ulture m dium after
and I I
in ubati n \\lith M ll g r MPP+ [! r .... h

D

ncentration

M Hg

MPP

0

+HVA/DA

DOP

HV

DOP
1 00

0.65

0.25

0.20

4. 1

1.7 8

.27

. 16

0.

0.5-

0.2 6

0.2

5.

2.55

15

0 10

0.8

0.5

0. 1

0 16

8.5

6. 5

10

0. 12

0.05

0. 0

0 0

0 08

0. 10

9.08

6.7 8

0

0. 4

0. 1

1.00

0 5

25

0 20

.1

1.78

50

. 4

0.02 ..

0.69

01

D

are mean ±

25.49 1 .5 I·•

. n t de tec tab le. ** P<O.O 1 \ '.\ co ntr
ac id: }{
. h mo\·anill ic acid .

39

120

A
......

48h

24h

100

--

0

e:..
.~
.0
(11

·:;
Q)

u

80
60

I* '

40

t I

t

20
0
0

1

0.5

0.1

5

7.5

10

MeHg ( ~M )

120

8

~
e:..

.£
.0

..

80
60

-

40

Q)

48h

100

(11

·:;

24h

..

~

....
k

...

.

·~

"'*

....w

200

400

(..)

20
0
0

10

50

100

MPP ·( ~M )

Figure} . Do e-respon e curve of (A) Methyl mercury (McH g) and (B) MPP + cytoto. icit) Ill
MN9 0 cell s after 24 and 48h e po ure .
descr ibed in ecti on 2.2 Values a r mean ±
control)

e ll v iability wa mea ured b) MTT a a; a
.0 (n=3-6) . (P<O.OS, ** P<0.01, ***P<0.001 rs.

40

80

A

-

70
~

50

c

40

c

30

..,

1 JIM M H(

60

c

~

-

0.2 JIM t till I ( I d

A

41

,.,
0)

(/)

20
10
0
2

6

4

10

8

12

Retention time (min)

8

80
-

70

0 fJM MeH
15 pM MeHq

-

5 pM MeHq
50 pM MPP+

60
A

50

,.,
c0)
(/)

40
30
20
10
0
2

4

6

8

10

12

Retention time (min)

Figure2. Typica l chromatogram of D , DOPAC and HV A. (A) 0.21JM tandard and cell
culture medium from I 11M Mellg incubation analyzed by HPLC : (B) Cell culture medium
sample from difii rent concentrati on in Mel Ig and MPP + for 24h analyzed by I-IPL .

41

1.4

A

TH
I

---- ----------__ ............
~-----.

MeHg 0

8

I

1

5

7 .5

1 05 -1
~

OAT

c

:J

~

a-Syn

"'....
;::

07

• TH
•OAT

.D

....

<t

0 35

a-Syn

GAPDH
0

10 (~)

0

1

10

5
7.5
Me Hg ( ~M )

1.6

..

TH
......

c

DAT

....

:J

~

...."'....
a-Syn

12 '

0.8

u

u

'"'

• TH

....

u

OAT

.D

....

<t
a-Syn

GAPDH
0

MPP• 0

10

50

200

400 (~)

0

10

50

200

400

MPP · ( ~M )

Figure3 . Dose-dep ndent effect of ( ) M Hg and (B) MPP+ on th e pre

ion of TH. OAT

and a-Syn mRNA analyzed by RT-PCR. Bar graph ho~ing that 0-1 011M M Hg e po ure
on MN9D cells in 48h. Th mRN level wer hown ba ed on one repre entative data from
three separate e periment . Each band wa quantified by d n itometry and normalized to that
of APDH . *P<O.OL ***P<O.OOl vs control.

42

16

A

•OAT • a-Syn

TH
OAT

~c 1 2 j

:::J

>-

ro o 8
~

.::

a-Syn

.0

< 04

GAPDH
0

MeHg 0

1

5

7.5

10

0

(~)

1

24

8

I

TH
~

18

-,

r

t

5
MeHg ( ~M )

75

10

TH • OAT • a-Syn

I

J

c

OAT

:::J

~ 1.2 .J
~

a-Syn

~

.0

< 0.6

.J

GAPDH
0

MPP• 0

10

50

200

400

(~)

0

10

50

200

400

M PP+( ~ M)

Figure4. Dose-re pon e effect of (A) MeHg and (8) MPP + on th e pre ion of TIL DA T
and a- yn protein analyz d by immunoblot. MN9D cell were exposed t MeHg or MPP + at
diffi rent concentration for 48h. The total homogenate wer prepared from M 9D c II .
Data how a typical e periment performed in triplicat . ach band wa quantified b;
den itometry and nonnalized to that of GAPDH. Value are mean ±
independent xperim nts. *P<O.OS, ** P<O.O L *"'* P<O.OO 1 rs control.

.D from three

43

_...

c:

5

•MeHg

-eEEn 4
0

E

a.

D MPP+
,..
~

T
J.

3

~

i:'

>
......
(.)

~

CD
0

2
~-

1

-=~

I

~

~

~ igure5 .

e po

0

ffect of MeHg and MPP ~ on M

-B acti\ it} in MN9D cell . M 9

cell

were

d to a dif:D rent range of M Hg and MPP ~ [! r 48h . A one tep nuorom tric method

wa u d t determine M
-B activit} . Re rutin pr duct wa e pre ed a
protein/minute.
perim nt wer run in triplicate . "**P<O.OOl 1'S control.

pmol/mg

44

Di cu
.

.

h d ammergt
MPP indu
th t the

~

11 line

d n ur
"o

ha. been . ho\\ n t l e an ffecti v model [! r the stud of

·icit; in P

Kim et al.. ~0 11: Wang et aL ~0 08 ). It ha b en shown
ells i

f MPP f r 1 9I

yt t xicit) in

O~t

1. and at 40~M. MPP + induced ignificant
). In thi s stud) \\e se lected 0-.... 00~M MPP t

elL (llu:sametal..l

ur p

iti\e

ntrol \\hi ·h au. ed 40°o cell mortalit; (hgl

). In the MTT a ·ay. we found

that th E

"0

f Mel Ig f r M 9I cells ,,a. around 8 ~tM .

ur re sults sh wed that Me l Ig

d r a ed

II pr t in '\pre .. ic n at ~ ~l

a

Th

effect"' r

benedatd

mparable t th c nditi n
in MN9
th

and decreased the pre tei n level )f I

e belov. thei· ·~o .inthe80°osurvivedcells(Fig.l
f th

f th

comparabl

d pamine cycling protein and D

)that

PP + treated c II . rhu . ur results howing effect

c 11 tr at d \1\ith the . perimental d

under ·tanding

·1 at 7.5 ~M .

range r r Mel Jg and MPP + are relevant in

ffi ct of th

e t\1\

c mpound . on e pre sian

f

content .

Thi i the fir t report that how M Hg may har
of MPP in dopaminergic cell . Our r ult

imilar path logical mechani m a tho e

hoVved MN9D cells exposur to M Hg re ulted

in a decrea ed relea e of DA and DA metabolite (D P

and HV ) in a imilar manner a

that of MPP+. HV A leve l were more sen itively affected by MeHg compared to D P
the present tudy. which could e plain in part the lower media I v 1 of D

' in

due to HV

being its primary extrac llular metabolic product (El worth and Roth . 1997). imilar r ult
were reported in an in vitro (Wang et aL 2008) and an in ''h'o tudy (Xu et al.. 2005 ) \1\here
they reported MPP+ or MPTP cou ld induce a decrea ing re ponse of D . D P
from MN9D cell and m use triatum.

and I IV

n th other hand. some in ,.i,·o tudie illu ·trated a

con entrati on dep nde nt in rease of e trace llu lar D

and it metabolites in free!) mo\'mg
45

triatum u ing a mi r dial
200 : ar

p

t al.. 2 07:

t hnique timulat d b M ll g and MPP+ ( ar

ang et al.. 2

f

rna

H

rati a an inde. h
~a

2005 ). Th rati

ar

). The e re ~ ult

d p nding

~ith

c n idered a a marker f

ncurrent dee r ~ase in I

the

( ab.1), indi atin g that th e rate
m tab lit

p

m d L

n /or

nditi n .

been u ed t a . e. d paminergic a ti ity (Lee et al..

a hi gh r turn " r (Thiffa ult et aL 2000) . We
rati

ugge t that effe t of MeHg on the

di ffl r n t

n

l al. ,

turn ver with a hi gher rati repre entin g
h. erved an increa e in

p

I IV

A

relea e after incubate ~ith Mel Ig and MPP + 24 h

r dec rea ·e of

relea e was fa ·ter than th at of it

and 1I

Th li fl c cle of d pamtn rgic neurotran mt
uptake and m tab li m.
conditi on , r lea ed D

hi

dynami c pr c

n inclu d OJ\ ynth e. i , relea · , t rage, redetermin e

OA de tin ati n. In norm al

interact 'vV ith a vari ety of po t ynapti c rec ptor , then ac ti vate

different cell ignalin g pathway (Jone and Mill er. 200 8), re ult in di ffl rent b hav ior effect .
To d tem1ine whether the decrea e in culture medi a level of D

and it metabolite 'vV ere

associated with M Hg-induc d alterati on in dopaminergic neur tran mi

ion, key protein

involved in the li fe cycle of dopamine includin g tyrosin hydroxyla e (TH). DA tran porter
(DAT) and monoamine oxidase-B (M

-B) , we re mea ured to examin the D

ynth e i ,

re-uptake and metabolite, respecti vely. The e pr teins have pr viou ly heen reported to be
expre ed in MN9D dopamin ergic neuronal cell line (L 'vV er et al., 200 8).

ur r ul ts

howed that DAT and 1I decreased with increa in g MeHg co nce ntrati on at both the mR
and protein levels (Fi g.

and 4 ). Thi agr es with the r suit oh erved by Faro e/ a/. (Faro et

al., 2002) that OJ\ uptake wa inhibited by trea ting Mell g in 1'h 'o. Mice expressi ng ltn\er
leve l of DAT was al o proved to ca u e reducti on or intrace llular I

stor --s (Egana ~t al..

46

2 0

r p n e
~-tM,

ffl t

vva

7. ~-tM and 1 1-l

n th

f Til in M

imat

-fl Ids de rea e in the

M llg, ~ith an ppr

p

d t

~-tM

tr atm nt gr up

mpared t th

ntrol group ( ig. 4 ).

M Hg

p ur vvill ( 1

u e the uppr

r that\

tra ellul r en vir nm nt nd ( ~ de rea, ed e. pre ion

th

11

pr

ur re ult

ugge t th t

uld inhibit 0

re-upt ke fr m

f Tl I that

uld decrease the

f

Tran

ripti nal pr filing identified an incrca ·c in u- ,' ]n cxpres, 1011 with do e dependent

mann r by Mellg (Fig. ). a- , ; n aggregati n ~a · rep rted to be highly related to PD .
Incr a ing pr t in
(Pal

ncentrati n v. a

ne fl rm of a-Synuclein aggregation process

log u et al., 200 ). Tran fecti n ~ith mutant u- ,' ;n led to u-.'yn aggregation in MN9D

cell (Wu et al.. 2011 ). Th increa ·ed u- ;n gene expre i n timulated by MPP + or MPTP
was con i tent with m t rep rt (Vila et al., 2000) but diffl r nt from the re ult , f Xu eta/.
(Xu et al.. 2005; 2005 ) and KUhn et a/ (KUhn t al.. 2003 ). They ob er ed a decrea e of uyn gen expre ion treated with MPTP 24h in \'iro. The slight r duction in a-, yn protein
expres ion ( ig.4) wa

diffl rent from it

mRNA level vvhich may be the re ult of the

relatively hort duration of incubation (48h) or the po t-tran lational modification of a- . yn in
re ponse to high do e of MeHg and MPP+. Recent report have identified that a- yn i
involved in regulation of TH and DA T trafficking. Overe. pres ing a-, yn ould dec rea e D
ynthesis in MN90 cell (Tehranian tal. , 2006). By reducing the activit; ofTH (Perez tal..
2002) and forming comple e with D T (Wer inger et aL 200., ). a- yn could regulate D
bio ynthe i and DA T amount in th pia ma membrane .

Monoamine

oxida e

(MA )

neurontran mitter , uch a

involved

erotonin and D

111

metaboli ' m

of

(Chakrabarti et al., 1998).

man)

monoamm

ur result shO\\ed
47

that M

-B acti iti

wa

r inhibit d after

ith pre iou

acti it

report that MeHg ha

in different rat brain

(Beyr ut

t aL 2006:

po ur t dif[i rent do e of M Hg.

napto

n d m n trated t

b

his re ult

reduce M 0

m s and impacted embr o brain development

hakrabarti et aL 1998) .

loVver M

a ti ity i a ociated with

human bl od-Hg c n

ntrati n ( tam! r et al.. 2006) .

ho

-B activity. but th trend wa rever ed at the higher do e of MPP t at

d a r du ing M

100)J.M (P <0.01. Fig. ). Therefore. ef:fl t
the do e. The effect f the d
high M

ell tr ated with 1 )J.M MPP+ also

f MPP + on M

-B activity vary dep nding on

f MPP + ( 1OO)J.M) i more c n i tent with the hyp thesi that

-B activity c uld mediate neuroto icity in P

model by increasing the metabolic

rate of D . There i al o report that patients with idi pathic PD have an increased MA -B
activit

in platelets (Bonucc lli et aL 1990). Th

difference in effects between MeHg and

MPP+ on MAO-B activity can be attributed to the trong affinity of MeHg to thiol group. e.g.
cysteine residue

in protein. A

MAO wa reported to be inactivated by methylmercuric

iodide (Gomes et aL 1976). MeHg may bind directly to cysteine residues of MAO enzyme
and inhibit its activity.

Conclusions

Our results show that MeHg. at least in high concentration, can exert similar effect

on

MN9D cells that are comparable to those by MPP+. MeHg can decrease the release of DA by
decreasing the DA synthesis with decrease in TH. and by decreasing the DA re-uptake
through the decrease of DAT. However. MeHg decreased MAO-B activitie while MPP+ in
high dose treatment showed an increa e. This comparative tudy improve our under tanding
concerning DA a sociated gene/protein and metaboli m changes underlying the MeHg

48

indue d path I gical alteration .
b a ri k factor forth de

ur r ult

ugge t that e p ur t M Hg may potentiall y

1 pm nt f P .

Acknowledgement

W thank

r.

uanji Wu ~ r hi e, pert HPL ' techniqu a i tan

49

Chapter IV. Effect of Methylmercury on MN9D dopaminergic neuron
cell : a genomic and proteomic analy i
N ame and Affiliation of utltor :
Yueting haoa,

ani 1 tg y h, Zhibin

ingh, Ryan Mail lou L, I ling Man

hanc*

aProgram f atural Re ource and Envir nmental tudies, niversit of orthem British
olumbia, Prin
e rg . Briti h olumbia. anada V2N 4Z9
mail shaoy@unbc.ca
h ttawa In titute f
tern
iol gy. niver it of ttawa, ttawa, Ontario,
8M5
mail : dfig y ra u tta'Aa.ca : Zhibin ing. Email: ningzhibin(a,gmail.com
c

entre for
dvanced Re earch in Envir nmental
niver it of Ottawa, ttawa. ntari . 'anada K 1 6

anada K 1H

enomics, Department of iology,
mail : rmailloux ~ uottawa.ca

*c entre for dvanc d R earch in nvironmental en mic , Department of Biology,
ni r ity of Ottawa. ttawa. ntario , an ada K 1N 6N 5
orresponding author, Emai 1:
laurie .chan(ci{uottawa.ca

50

Ab tract

. . p ure to n ir nmental hemical ha been impli at d a risk fact r for th development
f n ur degenerative di ea e.

ur prevt u

e. p ure can di rupt ) nthe. i . th

tudy

hov.ed that methylmercury (MeHg)

uptake and the metaboli m

f dopamine similar to I-

meth 1-4-phen ·Ipyridinium (MPP_._ ). The objective of thi study wa to inve tigate the effect
of M Hg , po ure
cell

w r

n gen and protein profiles in a d paminergic M 9

treated -with MeHg ( 1

p ptid

w r

Parkin

n' di ea e (P )

JlM) and MPP + (I 0

e. tracted fr m different treat d c II

ionization tandem ma

cell line . MN9D

0!-lM) for 48 h urs. RNA and

and analy7ed using a real-time P R

rray and high-performance liquid chromatography/el ctro pray
pectrometry (HPL ' -E I-M /MS). PCR array result showed that

16 out of the 84 PD related genes ( 19°/ o) were ignificantly changed in the MeHg 2.51-lM
treated cells. and 33 genes (39o/o) were changed in the

5~-LM

MeHg treated cell . In

comparison, MPP+ treatment resulted in significant changes in 22 gene (25%). A total of 15
common genes were altered by both MeHg and MPP +. and dopaminergic

ignaling

transduction was the most affected pathway. Proteomic analysis identified a total of 2496
proteins of which 188 proteins were differentially changed by MeHg 111M treatment and 233
proteins were changed by MeHg 2.5!-lM treatment. MPP+ treatment resulted in 395 protein
changes. A total of 61 common proteins were changed by both MeHg and MPP + treatment.
Altered proteins were mainly involved in energetic generation related metaboli m pathway
(propanoate metabolism, pyruvate metabolism and fatty acid metaboli m). oxidative
phosphorylation, protea orne, PD and other neurodegencrative di order . A total of 7
genes/proteins including Ube213 ( biquitin-conjugating enzyme E2 L3) and Th ( fyrosine 3monooxygenase) were changed imilar by both the genomic and proteomic anal) sis. These
51

re ult

ugg

t that M eHg a nd MP

e ffl t in th path gene i of P

Ke w rd : M eth ; !me r Uf).

+

har

man

imilar ignaling pathwa

and endp int

and o ther neur degenerati e di ea e .

eurot xic i t;. ' ene e. pre

ion. Pro teo me. Parkin on 's d i ease

Introduction

Parkin

n · di

a e ( P ) i.· chara teri7ed b.: the pr gressive los

the ub tanti a nigra par · compac ta ( hinta and
till uncl ear: h ~ever. there

nder en. 2005). T he e ac t cause o f PD is

evidence tha t dy functio na l m itoc hondri a l meta b Ji sm ,

unc ntr li ed r a ti ve oxyge n ·pec1e

pr d ucti n. and d i rupti n of ubiq uitin pro tea om e

t m pl ay ce ntral ro le in the pathogene ·i of PD (H
d feet have al

f do paminergic ne uro ns in

t al. , 20 12). In addi tio n, ge neti c

been hown a potentia l ri k factor via the mutations or po lymo rphi sm of

Snca. Pinkl . Lrrk2. Park ~. Htra2 and Park2 (the gene th at en cod s Park in) (Bogaerts et a!.,
2008).

tudi e has hown a po iti ve corre lation b tween indu tri ali zati on and th e occurrence

of PD (G itl er, 2009: Jones and M ill er. 2008 ). Environmental po lluta nt

uch as pesti cid s

and heavy 1netals have been reported to be ri sk facto rs lead ing to the path ogenesi of PD
(Michalke et al., 2009: R oos et al., 2006a).

Methylmercury (MeH g) is one of the m ost tox ic fo rn1s of m erc ury (K eyvan hokooh et a] ..
2009). M eH g causes damages to the nervous ystem in t he early deve lopmental tage as it
a lters both the structure and fun cti onality of the ne rvo us system (G randjean et a!., 201 0). It
was showed that possibl e mechani sm s of M el lg neurotoxic ity in c lude inh ibition of glutamate
uptake, increased reacti ve oxygen species ge neration, ind uction of oxidative damage.
depl eti on of glutathi o ne (G SH ), increase of cyto oli c and mitoc hondr ial Ca 2+ levels, and
mitocho ndrial dy fun cti o n (Farina et aL 2011 : Fo nnum and Lock, 2004:

m·afian and Veritv.

52

1991; Wagn r

tal.. 2010).

number

microtubule di rupti n, ubiquitin-pr tea
c

f tudi
me

rep rted that M Hg could al o induce
tern p Iiurbation and dy regulation of c 11

le ( r p -L pez et aL 200 ) ( eccatelli et al., 2007:

fD t

20 10) . Th
o idati e tre

f MeHg

reiem and

egaL 2007: Yu et aL

n di rupti n of n rmal neur nal functi n, induction of

and damage. and impairment of mitoch ndrial function uggest that it c uld

a] o be a p tential cau ativ agent for development of Parkin on ·s di eas (Ho et al., 20 12).

an
tetrah dropyridin

active

metab lie

product

(MPTP) . MPP ")MP P ha

inhibitor of c mplex I of th

of

n uroto in

1-methyl-4-phenyl-12J.6-

been widely d monstrated to be a se lective

mit chondrial electr n tran port chain and can induce a

e to PD in both cellular and animal mod

( berhardt and , chulz, 2003~ Petit-

Paitel et al.. 2009) . Energy depletion and oxidative tr

are well documented to be the main

ndrome cl

contributors to neuronal death in the substantia nigra following MPP+ admini tration
(Mizuno et aL 1998). Dy functional ubiquitination and proteasomal-mediated protein
degradation have been demonstrated to be common features in PD patients (Fishman-Jacob
et aL 2009: Zhou et al.. 2005). In addition. it has been revealed that gene involved in cell
cycle,

vesicle

trafficking.

synaptic

transmission.

dopamine

metabolism,

and

cell

adhesion!cytoskeleton display sharp changes in expression in sporadic PD cases (Grunblatt et
al., 2004: Hauser et al., 2005: Moran et al., 2006: Zhang et aL 2005).

High-throughput test such as array technologies and proteomics are powerful tools that can
be used for sys tematic analysis of cell ular events and interpretation biological pathway
affected by environmental chemicals (Dowd. 20 12: Jennings, 2014 ). In our previou study.
we showed that MeHg ex hi bi ted toxic effects simi lar to MPP+ re ulting in a decrease in
tyrosi ne hyd roxy lase (TH) and dopamine transporter, as well a dopamine and its metabolites
53

uch a
111

e tigat

M n amm

id

M 9

c ll . The purp

of the

ffect

111

the under! ing me bani m

m g n

and pr t m , pr
fP

cau ing path

f thi

study i

to

of MeHg u mg a gen m1c and

ll line . W h pothe ized that the profile of

appr a h in a d paminergi c M
hang

e

1on. indue d b; Mcllg

imilar to th

c of MPP+

.

Material and Method

ell culture and chemical e po ure

MN90 cell ( h i et aL 1991) Vvere maintained in OM ~ M (, igma, M ) upplementcd with

10°/o ( /v) fetal bovine

treptomycin (Life Technologi

So/o 02 at "'7°C

aH

. 1OOU/mL penicillin. and 1OO!lglmL

urlingt n.

) in an incubator with an atmosphere of

erum. 3.7g/L
Inc..

ell were grown in poly-0-ly in coated fia k (BD lnc, Mi

MeHg and MPP+ compounds were obtained from Alfa

e ar. MA and

i auga, ON) .

igma (Mi

is auga,

ON). Sodium Butyrate ( igma. Missi sauga. ON) with 1mM was u ed for cell differentiation
for 5-8 day before MeHg or MPP+ exposure .

PD PCR A rray ass ay
The mouse RT 2 ProfilerTM PCR array (Qiagen. Toronto. ON) wa u ed for thi study. Thi
array contains 84 key genes directly or potentially involved in Parkin on's disea e. and also 5
house-keeping genes were included as reference genes in the same array plate. In addition.
thi array comprises one well for mea ure genome contamination. 3 well of po itive PCR
control (PP ) and 3 we ll of RTC (reverse tran cription control. reaction without rever e
transcript enzyme) .

54

Triplicat

11 amp!

re

p

d t M Hg and MPP + (MeH g 2. 5!-LM, M Hg 5!-LM and

MPP+ 40!-LM) and

ntrol.

h incubation, t tal R

e tra t d b

1agen R ea

Mini Kit [! II wing manufacturer'

u mg

a

qual it

e timated

rm

pectroph t m
and quantit ~.
R

b)

h rati of

runmng

[! m1ald

hyde

kit for

denaturing

260/2 0 for each R

i . RT 2 YBR

were

in truction . RNA

g L and

Nan Drop
purity

ample mu st be reached above 1.8. The
RT 2 RN

' P 'R

). R v r e-tr n cripti on reacti on Vvere c ndu ctcd u in g
nth

[! ur group

Fi h r c1 ntific. Waltham, M ) wa used to te t R

quality wa furth r confirm d u mg a m u

T rant

fr m th

rray ( iagen,

iagen RT 2 Fir t trand

ree n qP R rn a termi x ( iage n, Toronto,

), water

and cD

aliquot ampl e from fo ur gr up Vv ere loading to P ' R Array pl ate, and run in

Bio-Rad

FX96 real-tim e P R in trum nt. Running conditi on was appli ed accordin g to th e

array in tructi on. The a erag expre i n of two hou ek eping genes (Gapdh and I Isp90ab 1)
were u ed for normali zati on. The !'J.!'J. t method wa

u ed to calcul ate th e fold-change

difference between treatment and control group . Gene differenti ated expre sion wa tested
by stud ent' s I test between each treatment and control group .

Data were expr sed as

mean±SD, and fold change with p <O.OS was co nsidered significant difference. Ther fore. a
total of 12 PD PCR Array were appli ed in thi s study.

Proteo mic A nalys is

Sample prepara! ion and l-IP LC-ESI-MSIMS

For proteomi c stud y, MN9D cell s were expo ed to different do es of MeHg (O!JM, 1~L M. and
5!-LM) and MPP+ (} O~tM ) for 48h . After 48h, ce ll were wa hed with cold PB S and harve ted
by sc rapping. The total proteins were ex trac ted fro m the ce ll · using RIP A lysi

butTer

55

c ntaining PM F, pr tea
Biot chn log

In .,

inhibitor

alia .

).

c ntrifug d at 12.000rpm ~ r
impro

d Lowr

pr cedure of in

co ktail and

m th d u mg

.

ribed b;

Protein

ha

et al. (, ha

e tra t w re pr cipi tated. dige ted. and dried
fra tionation by five t p
analyzed u ing an

anadate ( anta

ruz

onication and

Pr tein c ncentration wa determined by an
ay kit (Bio-Rad, Mis i auga,

luti n dige tion and Rever ed-Pha e

wer p r[i nned a de

xih

ere th n h m g nized by

he e tract

0 min at 4 o

dium

). The

iquid

hromatography (RP-L )

et aL 2015) .

riefly. protein in the cell

ample

ere

ubjected to StageTip

X

f pH elution (pH .0. 4.0. 6.0. .0. 10.0). Digested peptides were

gil nt 1100 capillary HP

A) coupled with L T -Orbitrap rna

1 tem ( gi I nt Technologies.

pectr m ter (Thermo

anta

lara,

lectron. Waltham, MA).

equipped with a nano-el ctro pray interface operated in positive ion mode. All data were
recorded with Xcalibur software (Thermo Fi her cientific. an Jo e,

A).

Database search and Bioinformatic analysis

All raw files were processed and analyzed with software MaxQuant (Version 1.3.0.5) against
Swissprot protein database of mouse (20 12, July Release). including commonly observed
contmninants. The criteria for protein quantitation and identification were perfom1ed as
previously reported (Shao et aL 20 15). All the identifications from contaminants and
reversed database were removed using Persues software (Version 1.3.0.4). Abundance ratios
of proteins were calculated from the label-free quantitation of each group in the format of the
fo ld changes. Proteins with two-fold changes of treated/control ratio were filtered for the
subsequent data analysis. Heat map functional classification, correlation analysi . and the rav~
ratio of LFQ treated/contro l were transformed to logarithm (base 2) cale.

56

ld ntifi ation
re

urc

f

nrich d pathwa

.7 ( ataba

:D r

wa

nnotation,

c nduct d b

earching DAVID bioinfonnatics

i uali zati n, and Integrated

t aL 2009) . The 2-[i ld dif:D r ntiall

e pre ed protein

id ntificati n w re uploaded int

:Dr K

musculu,

pec1e

ann tati n databa

wa

elected a

VI

i covery)(Huan g da

from M Hg and MPP+ M

bi log ical pathway analy is.

backgr und ge ne

et. K ~

. M difi ed Fi h r e ac t te t wa

was

he Mus

e lected as re:D rene

adju ted fo r multipl e test usm g

Benjamini and B n:D rroni pr cedur

T he to ta l id e ntifi ed prote ins and 2 fo ld -cha nged

prot in

ftv.,are to perforn1 compan sons. Identification of

w r

upl oad d int

IP

mol cular functi on (MF ) in G ne O nco logy (

) was onduct d u ing Pa nther c las ificatio n

sy tern (Mi et aL 201 3 ), and the uncla ifi ed prote in
niprot protein knowledge ba e (

we re

earched and de fin ed usin g

n ortium . 20 15) . Fo r prote in w ith multipl e MF , the be t

two known function were a igned fo r each category. Prote in rema in uncl assifi ed in both
Panther and

niprot tool

were a signed to th e category in "other ". F urtherm ore, the

abundance change patterns of selected protein were di spl ayed as heat maps using Per ues
software, and proteins with similar MF w ere further grouped . Correlation ana lys i for all th e
identified proteins from M eH g and MPP+ treated cell wa conducted by u ing

PS

16.0

(SP SS Inc., Chicago, IL).

W estern blot analys is

MN9D cell s were harvested, lysed and quantified as described as in MS (mass pectrometry)
sampl e preparation. Total prote in s extract with 6!J.g we re eparated on 10°/o ae ry lam ide gel
before transfer of prote ins onto PVDF membranes by usi ng co nve nti onal vvet tran -b lot unit
(Bio-Rad, Mi ssissauga, ON ). M embranes we re bl ocked w ith So/o ki m milk in TB T (Tri Buffered Saline and Twee n 20) so lution for one hour befo re incu bation with the primary
57

napti

v

i le m mbran

kina e B Z1B (

Z1

protein

) antib die .

ft r wa bing

1 ading contr 1.

~

oluti n. the membrane

time

-1 h mol g-lik

(

T1 L) and

yro in -protein

P II (g at p 1 cl nal antib d ) was used as a
with TB

(Tri -Buffered

aline and Tween 20)

re incubated ~ith appr priate second antib dies. Prot in bands

w re d te ted by

hemido

!mag J oftwar .

11 antibodie were purcha ed from , anta ' ru7 Biotechnology Inc . . anta

ruz.

and - beam.

imaging y tern ( i -Rad. Hercules.

). and quantified by

ambridge. M .

Re ult

PD PCR A rray analy is

Volcano plot

how d that out of the 84 PD related gene included in the P R Array, 16

gene were ignificantly changed with 2.5 ~-tM MeHg treatment (Fig. 1A). and 33 gene were
ignificantly changed in MeHg ( 5 ~-tM) exposed MN9D cells (Fig. I B). In MPP + treatement
( 40~-tM) group. 21 genes were changed significantly (Fig. I C). Among all the statistically
changed genes, a total of 15 genes were found to be common between MeHg and MPP+.
They are mainly distributed in the 10 functions associated with PD: dopaminergic signal
transduction pathway (Ddc. Park2. Park7. Slc6a3.

nca and Th). Transporters and ion

transport (Atp2b2, Psen2. Slc 18a2, Slc6a3 and Snca). mitochondria (Park7. Snca and Th),
pro-apoptosis and anti-apoptosis (Tcf712 and Snca). parkin substrate and complex (Atxn3 and
Park7), cytoskeletal organization (Mapt and Park2). ubiquitination (Park2). ynaptic v sicle
(Th), cell adhesion (Nfasc), and MAP kinase (Fgfl 3) (TabS.l ).

58

The protein abundance profilin g

In thi

tud;. vv u ed label-free quantitation (Luber taL .... 01 ) meth d t mea ure relative

pr tein abundan e in M I Ig nd MPP + treated M 90 cells.

total of 185 , 2005, 2061 and

19 0 pr tein were identified fr m non-treatment. Mellg I ~M . Mellg5~M. and MPP +1O~M
gr up after filtrati n of the n )n-detccted \alucs.
th

e pr tein

protein (IP

....-fold change of treated/control ratio of

v. re filtered manual!;. and table ,' 1 illustrates all the 2-fold

changed

mapped) in Mel lg and MPr · groups v.. ith gene entry name, fold changes. cell
total )f 16 changed pr tein . were found in the Mel Jg and

I cation and protein t) pc .

MPP + treat d M 9

cell v.hich v.erc u ·cd for the[! llov.ing anal yses.

Path w ay enri chm ent analy i

To get an overviev.- of th biological function and pathv.-ay induced by the two toxicant ·. a
pathway enrichment analy i vva conduct d u ing D VID online

urcc. The results showed

that pathway in cyto keleton. amino acid metaboli m. glutathione metaboli . m. and
neurodegenerative di ease

(PD. AL . AZ. HD) are

pecific enriched in MeHg treated

MN9D cells (Fig.2A and 28) . Hungtinton · · di ea e wa the most afD cted categoric which
contained eight protein

in MeHg 1~M (P=O.Ol 0) and ten protein

(P=2.99E-06) treated cells. Notable gene

in the MeHg S~M

involved in G H metaboli m included

R.

H. ADPJ-I

G6PDX, IDH 1, G LM and HNRPLL that are required for the bio ynth

f

production, and the reduction of glutathione di ulfide (

H. While. MPP +

) back to

treated MN9D cells was howed to be enriched (P= 1.61 E-18) in spliceo ome path\\ay which
included 28 proteins ( ~ ig.2 ). Protein in propanoate metabolism. p) ru,·ate metaboli ·m. fatt\

59

idativ

acid m taboli m,

ph

phor lati n, prot a m . and Parkin

n'

di ea e were

chang d b both MeH g and MPP treatm nt ( ig.2 ).

GO Molecular function and

We c mpar d the pr tc m
M Hg in MN9
tr ated cell ,

orrelation analy i

hange

( 2- ~ ld

f

ell . When c mparing the prole me pr filin g betwee n MPP + and Mel lg
1 pr tein V\ r comm nl ; changed. The e prot ins were expressed similar

manner f up and d V\n-regul ati n ( 1g.
main!

) of MPP + and Mell g, a well a 2 d

involv d in

). The main MF of th

id r ducta e activ ity. cyt

common proteins are

keleton. tran fera e acti vity, nucl eic acid

binding and pi icin g. C) to k leton protein , rec ptor and signal tran ducti on.
acti vity with eight pr tein (

ADM.

xid orcducta e

L H7 A 1. LBR. ND FS 1, PMPCA, PR X4 , SDHC

and V AT1 L), i the mo t numbered molecul ar fun cti on hared between two tox ins (Fi g.3A).
onsistent with the above observations, the correlation analys is gave a coeffici ent of 0.504
and 0.525 with both P valu e < 0. 0001 when comparing protein abundance rati o of MeHg
1~M with MPP+ 1O~M . and MeHg 5 ~M with MPP+ 1O~M . re pecti vely (F ig.3B and Fig.3C).

Using our experimental paradigm, we also identifi ed change in the expre sion of speci fi c
proteins between two different dose of MeHg, ( 1 ~M and 5 ~M ) . MeHg treatm ent ( 1~M and
5 ~M MeHg) resulted in alteration in expression of 90 protein

compared to th e co ntrol. A

total of 68 and 69 proteins were found up-regulated in MeHg 1~tM and 5 ~M expo ed MN9D
ce ll s~ 22 and 2 1 proteins were down-regulated in

1~M and 5 ~tM MeHg treatments. The tV\o

do ses of MeHg induced a dose-re pon e effect for the identifi ed protein (F ig.4 ). Protei n
with 2-fold change were furth er di splayed a hea t map in Fi g.5. The molec ul ar f'unction of

60

Table 1. Significant changed PO related genes in MeHg and MPP+ treated MN90 cells

Gene description

MeHg l 1-1M (Gene symbol)

MeHg S1-1M (Gene symbo l)

Dopaminergic
signal transduction

Ode (0 .68), Snca (0 .73)

Transporters and
Ion transport

Gria3 (0.62), Kcnj6 ( 1.3 7),
Sic 18a2 (1.27), S I OOb ( 1.3),
Snca (0.73), Sytll ( 1.35)

Mitochondria

Snca (0.73), Uchll ( 1.32)

Pro-Apoptosis and
Anti-Apoptosis
Parkin substrate
and Complex
Cytoskeletal
Organization

Casp9 (0.79), Snca (0.73),
Tcf712 (0. 78). Ubc (I .43)

Atp2b2 (0.45), Sic 18a2 ( 1.43 ), Slc6a3
(0.33 ), Snca (0.68), Gria3 (0 .3 7), S I OOb
(0 .63), Slit I (0.07). Sytl (0.43)
Park7 (0 .58), Snca (0 .68). Th (0.49), Casp7
(0.58). Vamp! (0.46)
Tcf712 (0.47), Snca (0 .68). Ape (0 .72). Bdnf
(2 .3 8), Casp9 (0 .5 I ), Ubc (3 .05)

Sytll (1.35)

At:\.113 (0 .58). Park 7 (0 .58), At:\.112 (0 .5)

Ubiquitination

Ubc(l.43), Uchll (1 .32)

Cell Adhesion
Synaptic Vesicles
MAP Kinase
Inflammati on
Others

Fn I (0 .63)
Sytl I ( 1.35)

Ode (0.33), Park2 (0 .63), Park7 (0 .58),
Slc6a3 (0 .33). Snca (0 .68), Th (0.49)

Mapt (0 .54 ). Park2 (0 .63 ). Ape (0 .72)

Fn I (0 .63)

Park2 (0.63). Ubc (3 .05), Ube2k (0 .66),
Ube213(0 .55)
Nfasc (0 .64), Ape (0 .72). Fn I (0.34)
Th (0.49), Sy11 (0 .43)
Fgfl3 (0 .51 ), Ape (0 .72)
F11 I (0.34)
Chgb (0 .62), Olk I (0.43). Rtn I (0.48)

MPP+ 401-!M (Gene symbol)

Ode (0 .63), Park2 (2 . 17). Park7 ( 1.29),
Slc6a3 (2 .58}, Snca ( 1.61 ), Th (0.68),
Ord2 ( 1.73). Nr4a2 ( 1.72)
Atp2b2 ( 1.61 ), Sic 18a2 ( 1.45), Slc6a3
(2 .58). Snca ( 1.61 ), Drd2 ( 1.73)
Park 7 (I .29), Snca ( 1.61 ), Th (0 .68)
Tcf712 ( 1.68). Snca ( 1.61 ), Nr4a2 ( 1.72),

Pse112 ( 1.67). Casp3 ( 1.26)
Atxn J ( 1.28). Park 7 ( 1.29). Gpr37 (2.40),
stub I (I ,29)
Mapt (I .51). Park2 (2 . 17)
Park2 (2 . 17). stub I ( 1,29)
Nfasc ( 1.54), NrxnJ ( 1.64)
Th (0.68)
FgflJ ( 1.58)
Ncoal (1.45)

Note: the numbers in bracket indicate the fold change of each of gene.

62

Table2. P

r lat d g n

y mbol

txn2
Baspl
a p3
dc42
hgb
H pa4
Mapt
Nefl
N f
Park7
Ppid
P rd x2
ept5

Skp la
lc25a4
Srsf7

Stub]
Th

Ubc
Ube2 i
Ube2k
U be2 13

Uchl 1
Vdac3
Ywhaz

/pr tein

ere id ntifi d in the two a ay of P
pr teom pr fil e

Proteomic

Protein Na me

Brain ac id olubl protein 1
a pa ell di vi ion ontrol prot in 42
h molog
e retogranin-1
Heat h ck 70 kD a pro te in 4
Microtubule-a oc iat d prote in
euroftl ament li ght p I) pept ide
e icle-fu in g ATPa e
Pr teinDJ - 1
Peptidyl-prol I ci -tran
1 omera
D
Peroxiredox in-2
eptin-5
-ph a e kin ase-a oc iated protein
1

ADP/ATP tran toea e 1
Serine/arginin e-ri ch spli cing
factor 7
STIP1 homology and U boxcontainin g protein 1
Tyros in e 3-monooxygenase
Ubiquitin-40S ribo omal protein
S27a
SU MO-co nju gatin g enzy me
UBC9
Ubiquitin-conjugating enzyme E2
K
Ubiquitin-conjugatin g enzy me E2
L3

Ub iquiti n carboxyl-term inal
hyd rolase isozy me L 1
Voltage-dependent ani onse lecti ve chann el protein 3
14-3-3 protein zeta/delta

P R Arra and

0.85
1.1 4

1.18
1.1 7
1.26

0.85
0.84
0.92

0.88

0.76

1.22

1.30

1.08

0.62
1.24
0.54
1.00
0.83
0.58

1.20
1.1 9
1. 5 1
1.05

1.1 6
0.85
0.73

0.87
0.95

1.32

4.89

7.3 1

1.29

I .2 1

1.33

0.93

0.99

1.16

1. 50

1.06
0.82

0.92
1.08

I .I 0

0.98

0.77
N/A

1. 1 I

- I .06

0.81

0.9 1

0.88

1.32

0.69

0.7 1

0.76

0.78

0.75

0.95

0.87

1.29

1.24

N/A

0.49

0.68

0.93

0.66

3.05

1.22

0.74

0.6 1

0. 83

l. I I

1.21

1.60

0.66

1.07

0.87

0.26

0.55

0. 98

0.64

0.04

1. 12

1.10

0.78

0. 8 1

0.92

1.05

I. 15

1.03

1.0 I

0.9 1

1.07

0.85

1.64

1.16

I .I6

63

A 1 E-04
1 E-03

~
ro

1 E 02

>

a.
1 E-0 1

7

5

l
log 2 Fold Differen c e (MeHg51AM/Control)

Log2 Fold Differen c e (MeHg2.51JM /C ontrol )

c

1 E-05

1 E-04

Figure 1. Vo lcano pl ot

1 E-03

ex pre sio n in 1.5 -foJd chan ge in M eH g
2.5 ~M and S ~M (A and B), MPP+ 40~M
(C) expo ed MN 9D ce ll s. T he blue Jines
indicate the thre hold fo r th e P value of

<1.>

:J

ro

>

how the gene

1 E-02

a.

the /-test.

1 E-01

1 E+OO
-7

-5

-3

-1

3

5

7

9

Log2 Fold Differen ce (MPP+401JM/Control)

64

Fatty ac1d metabolism

1 7%

Alan1ne . aspartate and glutamate metabolism

17%

A

Proteasome

17%
2 2rYo *

Pyruvate metabolism

2 2% *

Propanoate metabolism

Alzheimer's disease

28%

Ox 1dat1ve phosphorylation

28%

Insulin signaling pathway

34%*

Parkinson 's disease

3 4% *
3 9%,

Regulation of actin cytoskeleton

3 gof<, *

Pur1ne metabolism

4 5% *

Huntington 's d1sease

0

2

Pyruvate metabolism

1.3%

Cit rate cycle (TCA cycle)

1.3%

B

Fatty acid metabolism

1 8% *

Propanoate metabolism

1.8% *

Amyot rophic lateral sclerosis (ALS)

2 2% *

Glutat hione metabolism

2.2% *

Valine, leucine and isoleucine degradation

2.2% *

Ribosome

3.6% *

Parkinson's disease

4.5% *

Alzhe imer's disease

4.9% *

Oxidati ve phosp horylat ion
Hunt ington's d isease

10

8

4
6
#Protein s/Term

4.9% *
_.,",--'II

0

~"f-./""'~-~

:~~,..,..~~~T'!""'>>~~·~"'~)J"~"-~""!o!'!''j

.

3

'

.

12
6
9
# Protein s/Term

6 3% *

15

18

65

Fatty ac1d metabolism

08%

Pyruvate metabolism

08%

c

Propanoate metabolism

1 1% *

Parkinson 's d1sease

13%

Ox1dat1ve phosphorylation

1 3%

Arginme and prol1ne metabolism

1 3%.

Proteasome

1 3%.

Pynm1d1ne metabolism

16%

Ubiquitin mediated proteol ysis

1 8%

Cell cyc le

2 1% *

Ribosome

3 2% *

Spliceosome

7 4% *

0

6

18
24
12
#Protein s /Term

30

36

Figure2. nrichment analy i of KE G Pathwa; in (A) MeHg 111M. (B) MeHg S11M and
(C) MPP+l 011M treated MN9D cells analyzed by D VID . Th elected 12 enriched
categories were applied in the figure .

66

l
oxidoreductase
activity

B
4 .0

BRIX l

structural
const itu en t of
cytoskel eton

DY N LL2
STOM
T U BB3
T U BB 4A
1\PRT

;;;
QJ

iii

c..
n

........
0

::I

0

O'Q
N

0

:::1.
.....

0'1

....

CD

~

transf erase activity

:::E -2 0

-40

r- hel icase activity

1

-40

-2 0

1

r

0.0

2.0

r

4 .0

6.0

MPP+10~M/ Con (log2)

~

SLTM
PI\BPC 4

nucleic acid binding

c

r- RNA splicing fa ctor activity

RB M 28
RB M 39
STX12
TFG

receptor and signal tran sducer
activity

IT PI\
SMC4

FI\F1
GI\T 1\4
N u ll2
M EPCE
M TC H 2
N CI\PG
OSB PL1A
PLRG1
RN H1
SSSC/\1

c

::t

XRCCl
EIF3H
SDPR

GL01
51001\6
PS M G1
I\P1 61
I\P2 M 1
ARPP19

..

:::E

DDXl
DDX18
DDX191\
DHX30
H2AFY
HIST1H1D

I\BCF2
M E M 01
NAPl L4
N UP50
TI M M BA
EIF2B4

:.

!2 0 0

AS3MT
BI\ZlB
PIGK
SDF2L 1
TBL2
M I\P2K1

-I

\

cg; 2.
g

~ hydrolase activity
....J

prote in binding
transporter activity
translation initiation factor activit)/
lyase activity
calcium ion binding
chaperon

l

4 oI

~ 201
c

80
~

i-"iI

...

-4 .0

I
-4 .0

others

I
-2.0

I
0.0

I

2.0

4 .0

MPP+10~M /Con (log2 )

Figure3 . (A) Heat map of common changed proteins for GO molecular functional
classification in MeHg and MPP+ treated MN9D cells. Protein abundance ratio
(treated/contro l) are compared between (B) MeHgl1-1M/Con (log2) and MPP +l 01-1M/Con
(log2) (Spearman correlation coefficient rs=0.504 with a p-va1u <0.0001) and (C) MeHg
S1-1M and MPP+101-1M (Spearman correlation coefficient r.,=0.525 with p-value <0.000 1).

67

liJM MeHg

• 51JM Mc Hg

u

.

~

0

~

£
"C

:2
0

,~ ~- ~ _,.~ "' . ,.. ~ ~-0 ~~ ,(:- "' ~ <!'.. ~ ~- 4!' ,:)>' ..~ .::-~ r,"> <" ~ ~ ~ ,., •• ..1- ~., <!> .,~ ..... #' ~.. (' .? ..,, ,~ " .t "' .,~ ~· ~· ~.. .t! ¢" *' •" ...~ y ._, .p .;,' ._, "' ,r ~~ ~.,. "./':
,..::' ._.." .;;~ v ~ • <!'+ ~- ~~ ~ ~ .,.~' ;~- ~ - "-"0~ "_,.,. '' ,..JJ <Yft(J ,:.o·_.f/o 6,..., ~~i' .:; "'<I • <~' ~-' <rA~' $' ._(" ~<f' ,..- ~<1- _..J-~'.1' .f ~" a-$' .l' t ~~ <i' ,..:t_
,. .i" ,~ ,.rt ;

o"'

v

.,:-

" o~-·

,..Y

' <'l

q

c.;

~

q

q

"

o''

1

'l"

.Y

~

~.,

5'-<i>+'',--'"'.,~;..~·~~:f~J?.;'l>~iifJI.fl."'~~~~"J.<fl."''

~0

cf

_,

_,

Figure4. MeHg induced all the protein abundance changes in MN9D cells. Protein abundance was displayed as fold changes.

68

ATPSO
CTSZ
DOX17
GNL1
GTPBP4
OLA1

hydrolase activity

PPP1CB
PSPH
SEC231P
CKB
ONMT1

-

NAA25

transferase

PBK
PIGK

activity

PPAT
PYGB

0

CERS2

N

H2AFV
SBOS

}

ACAOL
IDH1

} oxidoreductase activity

O"Q

nucleic acid
binding

SRSF10
KTN1
OSBPL 1A

structural constituent of cytoskeleton
ligase activity
RNA splicing factor activity
kinesin binding
phospholipid binding

CNIH4
PCNP

others

DYNLRB1
MTHF01L

}

WDR12

FigureS. Protein abundance changes induced by MeHg ba ed on molecular function
classification. Protein abundance was disp layed as fold chang in log2 cale. Oth rs
indicate an undetermined molecular functiona l category.

69

250

B

*
200

A
"'"'

~

V Tl

150

OJ.

c

...

\ 1\l l L

.::

PO H

:;;;

BAZ I B

100

0

:....

B ZIB

APOH

50

0

( o nt rol

l ~l \1\l e ll g

SJ.! '\1\I ellg

IOJ.~MMPP +

Figur 6. We tern bl t verified expre . ion of t'AO proteins (V ATl L and BAZ1 B) from
M analy i . . r pre entative blot from t'AO protein . B. quantifying data were obtained
through three individual run with or without Mel-Ig and MPP + treatment for VA Tl L and
BAZlB.

70

Di cu

wn

nal

of th impa t f M Hg r MPP + n protein or gene e pre sion patt rn in the

c nte t f n ur to icity ha be n rep rted in the pre 1ou

tudie (Kc

an h k oh et al.,

200 ) ( endrell et al.. .... 0 10: Xie et aL ~0 11 ). This i the fir t rep 1i to usc both genomic
and pr teomic ppr a h to tud; the effec ts of MeHg and it relation hip with PD u ing a
d paminergi cell line . lJ ing a known PI mimic chemical MPP + as a positive control,
th

comparati\·

r ult

hov, that Mel Ig and MPP • to icity affected many similar

function and pathway

In our P R anay re ult . V\

c nfirrned that MPP + . ignific antl y changed 22 genes that

have been invo lved in P

pathogene i . With eight proteins changed. dopaminergic

ignal transduction i

the most biological pathway affected by MPP +. It is well

documented that dopaminergic signaling i compromi ed during the pathogene i of PD
(Girault and Greengard, 2004 ). It i also known that MeH g affect neurotran mitter release
including DA (Faro et al.. 2003 ). A total of 15 g nes were found to b common between
MeHg and MPP+ treated groups. and 6 of them (40o/o) were functioned in dopaminergic
signal transduction pathway (Ddc. Park2. Park7, Slc6a3. Snca and Th). Ddc i an enzyme
to catalyze syntheses of dopamine and serotonin (Hwu et al.. 20 13). MPP+ in low do e of
1OOnM was shown to inhibit Ddc activity in PC 12h cells (Naoi et a!., 1988 ). Our re ults
showed that Ddc was down-regulated in both MeHg and MPP+ treated group , indicated
that MeHg and MPP+ have overlapping toxicity in term of altering gene expre sion
patterns in dopaminergic ignaling of PD. Other functions shared by the t\\O toxicants
included

tran portation,

mitochondria,

apoptosis,

cytoskeleton

organization

and

ubiquitination . These functions are the well accepted mechani sms as ociat d \\ ith PD
71

pathogene i

( run blatt et aL 2004 ). We noti

ignificant hanged gene w re

pre

d

that a numb r of c mm n

hared

ppo it b tween the tw to in , for e ample,

two gene ( t n

and Park7) in parkin ub trate and comp le . and D ur gene (Park2,

Park?,

n a) in d paminergic ignal tran duction were d wn-regulated with

lc6a and

M Hg 5 )lM treatm nt. but up-regulat d in the MPP + 40 )lM treated cell ( ab. 1). This
impli

MeHg bared imilar path\\a.

in term

\\ith MPP + but may function in different manner

of targeting prot in . In additi n. function in tran portation including gene

fun tionalized a tran porter and i n tran port \\ere the most affected category by MeHg,
thi

re ult support

tran portation di rupti n 1

ne

f the imp rtant mechani sm

of

MeHg neuroto icity ( lien et al.. 2001 a).

In compared of PO P R array. proteomic appr ach provides a global proteome change of
the two toxicant in MN9D cell . We ob erved 13 prot in (2-fold changes) in MPP +
group were overlapped with the changed proteins in MPTP induced PD mou se brain
(Zhang et aL 201 Ob ). The different numbers of dysregulated protein between our study
and Zhang et al study may cause by differences in experimental paradigm . We u ed cell
lines as subjects . and they used mou e brain tissue . Pathway in propanoate metabolism,
pyruvate metaboli sm, fatty acid metabolism. ribosome. proteaso me. neurodegenerative
disorders, and oxidative phosphorylation were showed enrichment in both MeHg and
MPP+ treated MN9D cells (Fig.2). The catabolism of propanoat i involved in succinate
and fatty acid synthesis (Wayne A. Fenton .. 2001 ). Metabolic alteration such a pyruvate
metabolism. mitochondrial fatty acids o idation is critical

proces

111

triggeri ng

mitochondrial impairment (Parra et al., 2001 ). The resu lts of path\\lay analysi suggest an
energy d ficit occurred in Mel l g and MPP + exposed MN9D cells. and support

72

mit

hondrial d

functi n i an imp 1iant m chani m in P
al

P=2.29 -05) wa

D un

that pathwa) in

tr ngl · afD ted b

M Hg5~-tM with 11 pr t in

F 4,

10,

In mitochondria,
bio ynthe i

[

TP5 .

idati e ph

phorylation ( XPH

MPP + and MeHg e po ure

. pre wn

TP5L,

path ge nesis and MeH g

altered ( DlJF B L N
TP J, ' X5 .

X

pecially with

F, L NO

2,

, and SOil ) (Fig.2).

idative pho phorylation i. required to couple nutrient xidation to the
TP, the uni\er a! energy currency (KVvok et aL 2010) . These findings

indicated that MPP+ and Mellg can trigger the obstruction of o idative phosphorylation
pr ce

by altering the expres ion of ke; protein required for the assemb ly and function

of the re piratory chain in mit chondrial. which further confirmed our ob ervati on of
ene rgy d ficit occurred in both two toxicant treated M 90 ce ll s. [n line with pathway
analysis, a previous report showed that MeHg inhibits
reactive oxygen species (ROS) (Mazzio and

XPHOS and increase ce llul ar

oliman. 20 12). and treatment with

glutathione pero ida e mimetic (eb elen) rever es MeHg-induced neurotoxicity (Yin et
aL 2011 ). Except OXPHO

pathway. we ob erved that MeHg 5~-tM al o induced the

alteration of 5 proteins (G R. G6PDX. IDH 1. GCLM and HNRPLL) in glutathione
metabolism. The protein alterations in GSH metabolism indicate the di abi lity to protect
oxidative damage including its rol e in rendering toxic soft ac id metal

like MeHg

nontoxic in mitochondrial (Ballatori, 1994 ).

Intri guin gly. except PO , we found that M eHg also targeted proteins as ocia ted \vith other
neurod egenerative di so rders such as Huntington disease. A lzheimer disease (AD) and
ALS. Actually, Huntington di sease was the most signifi cant enriched pathway aiTected
by M eHg, with 8 proteins in 1 ~-tM and 10 proteins in 5~tM Mellg (Fig. 2A and B) . MeHg

73

ha be n i1np li ated to indu e neur deg n rati
a

(Zahir t aL 2 06) and

L

(J hn

n and

MeH g har

man

imilar biol gical pathwa

MeHg d

af[i cted

imilar gene /protein

pecificall

in P .

euronal accumulati n

a

tchi on. 2009).

ur re ults indi ate

a in other neurodeg nerativ
MPP+ in Parkins n'

f M Hg rna

di ease.

disease but no t

be a predi po ing fact r [i r

vera! neur degenerative di ea e .

m I cular fun tion

f M Hg and MPP .. in heatmap exhibited ' imilar up and down-

regulated common protein. ( 61) bet\\.een the tv...o to ins (Fig. A), with 8 proteins
(A ADM. AL H7

1. LBR.

D F 1.

identifi d. o idoreducta e activity wa

PMP A.

PRDX4. SDJIC and

V ATl L)

the mo t numbered molecular function

hared

between two toxins. which confirmed pathway in OXPH S i a critical process shared by
MeHg and MPP+. V·/ e tern blot

howed that expre ions of V ATI L induced by two

toxicants agree with prot omic prediction (Fig.6). In addition, we ob erved a dose
response effect of MeHg 111M and S11M in term
MN9D cells (Fig.4). The molecular function

of regulation protein expres ion

of those dysregulated protein

in

in MeHg

treatments, mainly involved in hydrolase activity and tran ferase activity may reflect the
specific mechanisms for MeHg neurotoxicity that different from MPP+ (Fig.5).

Consistent w ith the above observations, a positive correlation of protein abundance ratios
was observed between MeH g I 11M and MPP+ (rs=0.504) and MeHg 5~LM with
MPP+l 011M (rs=0.525) (Fig.3B and 3C). Thi re ult futiher confirms the identification of
changed prote in s ind uced by two tox in s wa corr lated. and MeHg in high dose present
more correlati on with M PP 1 than Mel Ig in low dose.

74

c mbined th data f P

inall ,

pr tein /g ne in tw

t

P 'R

rra and prote mic profil . We found

v n

m e pre ed imilar manner in b th pr teomic and P R array

a . a (Tab.2). The ubiquitin-prot a orne play an 1mp rtant r le in mitochondrial protein
d gradati n (lie
c njugating

and Rutter, 2011: Margineantu et aL 2007) .

n7ym

E~ L

) i an ubiquitin liga e en?yme to mediate PD related genes

Parkin (Park2) degradati n
kn ckdown of Ub 21

f mit chondria organelle

enzyme lead t

pre ent

tudy. Ube213 were

and mitochondrial protein,

ignifi ant decrea e of autophagic clearance of

depolariz d mit ch ndria ( ei ler et aL 2014 ). thu block
In th

be213 ( biquitin-

f mitochondrial metaboli m.

ignificantly d wn-regulated in MeHg and MPP +

treated M 9D cell in b th tran cript and protein level (Tab.2) . Which uggest a critical
role of Ube213, similar to MPP+. in MeHg mediated mitochondrial dysfunction. , imilarly,
a ignificant pathway of protea orne was identified in both Mellg ( P= 0.024) and MPP +
(P=0.031) treated MN9D cells (Fig. I A and

). A dysfunctional proteosomal degradation

pathway has been implicated in the neurodegenerative proce

of PD and mercury

neurotoxicity (Li and Guo. 2009: Lim and Tan. 2007: Yu et a!., 201 0) . The e finding
suggest that ubiquitin-proteasome system were common target affected in both MeHg
and MPP+ treated MN9D cells. Moreover. tyro ine hydroxylase, an enzyme to limit
dopamine synthesis, were down-regulated in both MPP+ and MeHg-treated cell . This i
also consistent with our previous result that Th expre sion in mRNA level was decrea ed
with the dose increasing of MeHg (chapter I II), which further confirmed Th as an
important target enzyme, i inhibited dopamine synthesis process in both two toxicants .
Taken together, these re ult

upport our hypothesis that MeHg share many similar

biological function and pathway in the pathogene -is of PD .

75

onclu 100

MeHg har

man

wh re th

tw

t ,m

affected e. pre

1

with MPP + in the path gen s1 of P
n

f

pr teins in vol

d

In

o idative

H homeo ta

propanoate metaboli ·m. and p ruvate metaboli m

which c ntribute to energy defi cit.

opaminergic . ignal tran ducti on and mitoc hondri al

ph

ph r Jati n.

imilar ignaling pathwa

d functi n w re addr

d in tran ripti on leve l in both MeHg and MPP +e po ed cell .

Whil pr t in change abundance for M J-I g uch as hydro la e acti vity may r present th e
di tinct tox ic mec hani m of neuroto. ici ty ~ r M Hg. MeHg not only co rrelated with
MPP+ neuroto icity but may in vo lve in th e dev 1 pment of
di order

uch a Huntington di ea. e.

ur re ult

th er neurodegenerati ve

h v-. th at MeHg e posure can induce

hifts in protein/gene e pre sion pattern that are akin to change observed in a ce ll
culture imulati on of Parkin on· s di ea e. The current prote mi c and PCR arrays data
are compl ementary by providing the di ffe rent sets of regul ated protein /genes for MeHg
and MPP+.

76

Chapter V: Proteomic

naly i of

ereb Hum in

ommon Marmo et

e po ed to Methylmercury
N ame and Affiliation of A ut!t or :

Yueting ha *, Megumi Yamam tot. Daniel Fige; t, Zhibin Ningt, Bing Man

han ~

ur e and ~ nvir nmenta l , tudic Program, University of Northern Briti sh
olumbia, Prin e eorge, Briti h 'olumbia. 'anada V2 4Z9
mail : haoy(a{unbc .ca

* atural R

epartment of a ic Medi al c1encc,
Minamata, Kumamoto. Japan 67-000

t

ati nal Jn titutc for Minamata Di sease,
~ mail : yamamot ra,nimd .go .jp

ttawa In titute of y tem Bi l g). niversity f Ottawa, ttawa.
8M5
~ mail :
aniel
Figey
dfigey a,uottawa.ca ;
K 1H
ningzhibin CZ: gmail.com

t

ntario. Canada
Zhibin
Ning

~ entre for

dvanced Re arch in Environmental Genomics. Department of Biology,
mail :
Univer ity of ttawa. 30Marie Curie. Ottawa. ntario. Canada KlN 6N5
laurie.chan ~uottawa.ca

77

Ab tract

The cereb llum i kn wn

th maJ r target region

but the mechani m ar

till not full) under t

f m th lmer ur (Mellg) to ici ty,

d. We

tudied the effects

f Mel Ig

e p ure in the erebellum of comm n marmo et ( 'u/lithrix iacchus) using a shotgun
pr te mic appr a h with liquid chr matorgraph) coupled to ma
of 1000 pr t in

'v\ere identified. and 102 pr tcin

e pre ed in the

er bellum of common marm

M Hg/kg b dy w ight ~ r t\\

'v\ere

spectrometry. A total

ignificantly dif~ r ntially

ct 'v\ith orally dosed Me l lg ( 1.5 mg

v. eek ) c mpared to th

e of th control group. Functional

enrichment analy i and path'v\ay predictions show d that the differentially expre sed
protein

were involved

111

carbohydrat

d rivative metabolic procc . ion transport

including ynaptic tran mi Ion. cell development and calcium signaling pathway and
cellular component enrichment analy i

ho'v\ed that the] were mainly distributed in

pla rna membrane. excitatory ynap e and ynaptic membrane . These results indicate that
synaptic transmission and calcium ignaling pathways are the core functions affected by
MeHg. We found a total of 21 novel proteins affected by MeHg in ynaptic tran n1i sion
and calcium signaling pathways. DLG4 (PSD-95) and MIR-19A/MIR-19B were found to
be potential key targets leading to the multiple effects of MeHg neurotoxicity. These
comprehensive results show the global effect

of MeHg on cellular function

and

pathway leading to neurological deficits in common manna et.

Key word s: Methylmercury, proteomics, neurotoxicity. synaptic tran mi

ion. calcium

signaling pathway, primate

78

Introduction

M th lm rcur

(MeHg) i

cer b llum i

ne

a wid 1

kn

n

f the maj r target region

nvironmental neurot

icant and the

again t M Hg-e p

( lark on and

ur

Mago , 200 ). The mo t well-knov.,n MeHg poi oning incident happened in Minamata,
Japan in the 1950 wh re Minamata di ea. e patient
cereb liar dy functi n ( ' lark
in th
di ea e.

ermi and it

uffered from ata ia a a r

n et aL 2 0 ). The central type

f atrophy was

ult of
b erved

urr unding region in cerebellum in pr l nged ca e of Minamata

erebellar impairment v.erc featured by the l

integrated purkinje cell (Takeuchi and Eto. ] 999) .

of granule cell and relatively

large amount of mercury was found

in the remaining nerv c II . glia cell and phag cyte in the le ioned granular layer of
Minamata di ea

ca es (Okabe and Takeuchi. 1980).

Numerous experimental

tudie

have been publi hed on the molecular mechani ms of

MeHg neurotoxicity (Farina et al.. 2011 ). However. most of these studies focu ed on the
effects of MeHg on a specific mechanism or pathway, and very few studie report the
systemic effects in the brain. Omics technologies such as proteomics provide a poVverful
tool to study cellular function in response to environmental stimulus at the biological
level (Dowd, 2012; Jennings. 2014). In a recent proteomic study. Kong eta! .. found a
deficit of metabolic statu in rat somatosensory corte

with chronic exposure of MeHg

(40flg/kg/day) (Kong et al., 2013 ). Another proteon1ic study for MeHg toxicity reported
that non-phosphorylated cofilin, an important protein to regulate actin dynamics. \\as
significantly increased with 60nM MeHg e posure for 10 days in primary culture of
cerebel lar granule neuron (Yendrell et al.. 201 0). Rodents and primary cell culture \\ere
used a tested models in these studie .
79

on-human primate ha e a c mm n ance tr to human being, and in many a pect they
har

man

imilaritie

ith human than rodent . uch a

brain organization and c mple

neuroanat my, intricacy of

ognitiv capabilitie . Therefl re, primat

are r garded

a th be t model for the under tanding

f m le ular mechani m for human particularly

inn ur d g n rativ di ea e ( apitani

and Emborg. 2008).

of the m

t utiliz d n n-human primate specie

mmon marmo et i one

in biomedical research offering unique

b havi raL neuroanat mica!. and neurobiochemcial simi laritie
repo1ied that comm n marn1

et

ar

to human . It ha been

particularly appropriate model

for Mel Ig

icity tudie ver u other primate model . a the pathological changes in target
brain regi n

(cerebellum. cerebrum and peripheral nerves) induced by MeHg were

pre en ted in common marmoset ( to et al.. 2002; _. to et al.. 2001) but not in other type of
monkey

uch a rh su monkey ( hav. tal.. 1975 ).

The objective of this study is to use a hot-gun proteomic approach to tudy the effect of
MeHg on protein expression and their consequent change

in biological function

through functional and pathway analysis in the cerebellum of common marmoset.

Materials and Meth ods

A nim als and methylmercury trea tm ent

Experimental

conditions concermng MeHg-exposure

in

common

marmoset wa

previously described (Yamamoto et al.. 2012) . Briefly, six fl male common man11oset
were administered MeHg chloride orally (1 .5 mg Hg/kg body weight) or vvith a vehicle
so lution (containing 7.5 mM

-cysteine) daily for two vvceks follovvcd by tv.o v.eek

without treatment (n=3 in each group). The manno et were betvvecn two and three year
80

di

ct d aft r n

0°
R

re betw en 250g and

eight

ld and their bod

mmittee at the

11 animal pr cedure
ati nal In titut

were appro ed by the Animal

for Minamata Di sease. and all procedures

uide [! r the ' are and LJ e of Laborat ry

conD nned t the
Lab ratory

he cereb llum regi n were

the ia and were imm diat 1 fr z n in liquid nitr g n and tored at -

[i r pr te mic analy i

ear h

Og.

nimal by the Institute of

nimal Re ource ..

HPLC-E 1-M

fM '

In solution degistion and strong cationic exchange (SC)() fi·aclionation

erebellum

ample

di

ected from mam1o et treated with Mel-Ig and v hicle were

thawed, homogenized in 100 mM

mmonia bicarbonate (AB ), 8M Urea, 50 mM

Dithiothreitol (DTT). protease inhibitor. 1 mM Ethylenediaminetetraacetic acid, and
lysed with sonication. The ampl

were centrifuged at 16.000 g for 10 minutes and the

protein concentration in the supernatant of each sample was determined by modified
DC rM Protein Assay (Bio-Rad, Mississauga, ON) .

Protein extracts from each ample ( 1OO)J.g) were precipitated by cold acetone overnight.
A mixture of 8M Urea with SOmM ABC was used to recon titute protein pellet.
Reduction and alky lation were performed by addition of DTT to a final concentration of
20mM at 56°

for 30min followed by 60mM iodoacetamide at room temperature for

another 30min. Samples were then digested with tryp in at a protein-enzyme ratio of 20:1
at 37°

overnight with gentle baking. The dige ted peptides vvere acidified"' ith formic

acid (FA, O.So/o (v/v)), and fractionated by

tageTip SCX into fiv

fraction b; tive pH

steps (p i l3 .0, 4.0, 6.0, 8.0, 10.0). The peptide vvere then desalted on a Sep-Pak column
81

aug a,

(Wat r, Mi
an J

) and ly philiz d u ing a peed

ac (Thermo

h r c1 ntific,

).

Mas . . pee/rom J/ric analysi.

gilent 1100 apilar)-liPL
with

- rbitrap ma

y tcm ( gilent Technologic , , anta
pectr meter (Thermo Fi her , cientific,

lara,

) oupled

an Jo se, C ) was

u ed to anal ze the peptide mixture . The olvent sy tcm contained buffer A (0.1 °/o FA
in water) and buffer

(0.1 o/o F

in acetonitrile) . Each peptide ample was acidified with

20j..d 0.5o/o (v/v) formic acid and 8).11 of \\hich v,a loaded on a fused silica analytical
column (75 ).lm I. D. x 100 mm) packed in-hou
: Dr. Mai ch

Vvith 3)-lm ReproSil-Pur C 18 beads ( 100

mbH. Amm rbuch. Germany) . eparation of peptide wa performed at a

flow of 200nL/min u ing a gradient elution from 5 30°/o buffer B in 2 hour .

LTQ-Orbitrap mass spectrometer was equipped with a nano-electrospray interface,
operating in positive ion mode . The spray voltage wa set to 2.0 kV and th t mperature
of heated capillary was 200 °C. One full MS scan from 300 to 1700 m/z was performed
followed by data-dependent MS/MS scan of the 5 mo t intense ions. Dynamic exclusion
was set to repeat count of 1 in 30s, and exclusion duration of 90s. For the full M

scan,

the scan range was defined at m/z 400 with R = 60,000 in Orbitrap analyzer, and the LTQ
analyzer was applied for subsequent MS/MS analysis. To achieve an impr ved mass
accuracy, a real time internal calibration was performed by locking the ma s background
ion at 445.120025 in the Orbitrap mass analyzer.

82

Data analy i

he p ak li t fr m ra
and earch d again t
comm nl

file w reg nerat d with
wi

ftware Ma

Prot pr tein databa e f human ( er i n 20120711 ), including
ntaminant . The foll wing criteria were applied: cy teine

ob erved

arbamid m th lati n \\a

et a

(+ 15 .99492

-ten11inal acet; lati n vvere , et a

a) and pr tein

nzyme pecificity wa
nl

two mi

1on rna

a fi ed m dificati n: the methionine o idation
ariable modification .

et to tr]p m, n t all \\ing ~ r clea age N-terminal t

ing cl avage of tryp ·in v.er

toleranc

uant (Ver ion l. .0.5)

alloV~ed.

For M /M ,

were 7ppm, and fragm nt ion rna

proline.

pectra, the precur or

tolerance wa set to 0 .8 Da.

Razor and unique peptide \\ere applied for quantitation. The false di scovery rate was
limited to 0.01 on both protein and peptide I vel. A minimum

equence length of six

amino acid wa required for peptide identification.

Protein ident({tcation and cellular component distrihution

For protein identification. if the identified peptide sequence of one protein was equal to
or contained in another protein's peptide seL these two proteins were group d together by
MaxQuant and reported as one protein group. For quantitation, a minimum of 2 spectra
counts is needed . All the identification from contaminants and reversed databa

were

removed by Persues software (Version 1.3 .0.4 ). Abundance ratio s of protein were based
on the label-free quantitation of each group in the format of fold change . The calculation
of differentialJ y ex pres ed proteins was performed u ing Mann-Whitney U test v'v ith
SPS

16 (S P S Inc.,

hicago, IL) . The differentially expressed proteins in each triplicat

83

ampl

f th

n i tenc

ontrol and M Hg-treated gr up
f ampl

nal

i

lu t red into a heatmap and th

within ea h f gr up wa te ted u ing Per ue

ellular component attribute
Pathwa

er

(IP )

f a ll th e id entifi d pr te in s w ere clas ificd u ing Ingenuity
ft\\ are ( iage n. Red\\ ood.

). Th

di tri buti n b twe n ll identifi d pro tein a nd th e di ffe renti a ll
compared . Th

oftware.

di tributi on \\ a

cellular compon ent
pre ed proteins were

: pre ed a the percentage of pr tcin as

iated w ith

ach c ll comp n nt aga in t the di stributi o n of the to ta l id entifi d prot ins. A Fi sher'
e act t t wa
dif:D rentia ll y

p e r~

nned to te. t the diffe rence between the di stributi o ns o f th e
d pro te in a nd tho

o f the to tal id e ntifi ed prote in s.

Functional gro up mapping G O enrichment dep letion analys is

The functional enrichment anal ysi

of the 102 differenti a ll y changed prote in

w as

conducted using Cytoscape ClueG

plug- in (Bindea et al.. 2009) against th e id entifi ed

proteome. i.e . th e 1000 proteins. Th e fun ctional class ificati on was based on the Gene
Oncology (GO) bio logical proce

. A two-tail ed hypergeometri c stati sti c te t wa

conducted to determine the mo st disturbed biological function s re u]t

fro m the

di fferenti ally ex pressed prote ins. Bonferroni m ethod was used to correct the pro bability
value. and the GO term restricti ons (# of ge ne/term > 18 and o/o of term ge ne 2:. 15o/o) were
appli ed to reduce the related term redund ancy. Onl y

0 te m1 s w ith P va lue Ie , than

0.05 were accepted a nd prese nted . Cytoscape Cl ue Pedi a plug- in (Bindea et a!.. 20 13) wa
used to visuali ze the known interactions (from STRING 9.0) betwee n the GO term s and
the associated prote in s.

84

Two nlin

b

t aL 2009),

ere u ed D r

than 0.05

r er oftware,

a u ed a

ab I m1

4 .2 (M dina t al.. 20 10) and

ellular c mp nent nrichment anal

ori lla ( den

P value les

ut ff.

In rder t explore the p tential regulated tran cri ption factor o [ Me l-Ig on ion tran port,
Web

talt (WE -ba ed

Ene ._ eT

identify enrichment of miR

naLy i Toolkit) (Wang et aL 2013) wa used t

targe ted ge nes in the GO term of ynaptic tran mi

1 n.

Pathway enrichment anu/ysi.\

Pathway enrichm nt analy i on the differentially e pressed protein
u ing IP

and Web

talt onlin

ource .

were conducted

li t of 102 differentially expressed protein

in marmo et cerebellum wa uploaded into the IPA database.
identified and were eligible for generating Canonical

ne hundred proteins were

ignaling pathways . Web e talt

online source was also u ed for pathway analy is which provided the direct connection to
biological pathway

from Kyoto Encyclopedia of

Hypergeometric test was u ed to calculat

ene and Genomes (KEGG ).

P value from the differentially expressed

proteins against the identified 1000 proteome . Bonferroni method wa applied to correct
the P-value for KEGG pathway analysis.

Western blot analys is

To validate the results from the proteomic analysi , four proteins, Disks large homolog 4
(DLG4), Sodium/calcium exchanger 1 (SLC8A 1) Vesicular glutamate transporter 1
( L l 7A7) and T-complex protein 1 subunit zeta (C T6A) were quantified u ing
western blot. Protein extraction and concentration determination were the same as

85

rib d in

tion 2. 1. Pr t in ampl

(1 0-401-lg)

ere 1 ad d and eparated by 10-12°/o

). Membrane

er block d for lhr with 5o/o kim

aline +Tw en-20), and incubated with

.
.
anou pnmary

antib di

50. ab 1825 ). , L 17 7 ( 1: 00. ab 104 98), and

L ' 8 1 ( 1:800,

ab 1779 2) ( beam.

ambridge, M ), and

T6

Biot chnolog

anta

overn ight.

milk in

B

(Tri Buffered

Inc.

ruz.

' ) at 4°

( I :200, sc-1 896) ( anta

ruz

fter wa hing by TB T,

membrane wer

incubated \\ith llor radi h peroxidase-conjugated (liRP) eco ndary

antibodi

ruz

( an ta

iotechnolog; In .

anta Cruz. C ). goat anti-rabbit lgG-1 IRP

( 1:12000. c-2004) or mou e anti-goat Ig -HRP ( 1:12000. sc-235 4) for 1 hr at room
temperatur . The blot

were

tained b;

Iarity We tern ECL Sub trate (Bio-Rad,

Mi i auga, ON). and target band vvere vi ualized by using Chemidoc imaging system
(B io-Rad , Hercul es, CA). The target band wa quantifi d by using image J software and
the density of each band wa normalized against GAPDH .

Results

Protein identification and cellular component distribution

Proteomic analysis across the 6 cerebellum sample in control and the MeHg-treated
groups resulted in the identification of 2563 proteins (TabS.l ). Representative MS/MS
spectra for two proteins DLG4 (P78 52, Di sks large homolog 4) and L 17A 7 (Q9P2U7.
Vesicular glutamate tran sporter 1) are shown in FigureS .1. Only proteins identified in all
6 samples in control and MeHg- treated mamoset cerebellum were included in our
analysis, which resulted in 1000 proteins.

total of 102 proteins -were found to be

86

differ ntiall

e pr

d (

d crea ed and 16 increa ed) in the M Hg tr ated ample

c m ared t th c ntr l (Tab, .2 ).

th

f di fferenti all y changed protein

abundance chang

between the

ig. 1

h w

contr

and M Hg-tr ated manno. et cer bellum ampl e . Th tripli cate sampl e in th e

c ntr 1 gr up and the the MeHg treatmen t group ~e re clearl y eparated and a good
y in the pr tei n patt rn wa. fou nd ~ ithi n each gr up . Re ults of IPA gene
ont log

(

) analy i

f c llular compo nent are

abundance f id ntifi d pro tein wa a.
foll owin g d

ho\\. n in Fig. ! B. The relative

ciated \\. ith vario us cellul ar co mponents in th e

endin g ord er: cyto pla m (62o/o) > nucl eu ( 16o/o) > plasma membrane

(1 5o/o) > extrace llular space (3o/o).

organell es. The relativ

The remainin g 4°/o w re a oc iated with oth er

percentage of di fferenti all y ex pres ed proteins di stribution

fo llowed the ord er of cytopla m (55o/o) > pl asma membrane (27o/o) > nucl eu (14°/o) >
extracellular space (1 o/o), and other organell es co mprising 3o/o. There was a signifi cant
difference in organell e di tributi on between all th e identifi ed proteins and th e
differentially expressed protei ns conducted by Fisher's exact te t (P=0.046).

GO an alys is of differentially ex pressed proteins in cerebellum

The functional enri chm ent analysis results showed that 62 out of the 102 differentially
expressed proteins were included in 24 significant GO terms (P<0.05) (Tabl e 1). All the
proteins associated with each of th ese 24 signifi ca nt GO term s are hown in Table
These signifi cant GO term s we re grouped into three groups and th

.1.

key biological

fun cti ons of these groups includ e: 1) ca rbohydrate derivative meta bolic proce s and
purine as oc iated nucleotid e metaboli c process; 2) ion tra nsport inc luding vesicle-

87

m diat d tran p rt and ynapti
m rph gen

i in ol

tran mi wn: and

) cell de el pment a well a

d in n ur n differentiation. Ba d

functi nal annotati n net vvork analy i clu tered the
different bi

gical function group

differential!

e. pre ed protein

(Fig. 2 ). It

11

n the pr tein imilarity, the

ignificant
, hovv n

tenn

into three

i ually that 1110 t of the

are a ociatcd vvith the carbohydrate derivativ

m tabolic proce , and purine and ribonu leotide m tab lie proce se . ~o re proteins uch
a

D

4.

TP2 2 and PL

1 \\ere found to be important m linking biological

functi n m arbohydrate deri\'ative metabolic proces . synaptic transmi s10n and cell
development.

Web e talt miR A enrichment analy i res ult showed th e top 10 enriched miRNA
targetting the 20 proteins included in the GO function term of ynaptic tran mi ssion
(Table 2). MIR-19A and MIR-19B were the only significant miRNA that targeted five
down-regulated protein (NAPB, PLCBl, SYTl, BSN, ATP2B2 and NAPB) in th
process of synaptic tran mission (Table 2).

Results of the GO cellular component enrichment analysi

showed the differentiall y

expressed proteins was significantly enriched in pia rna membrane (46.1 o/o). pia rna
membrane part (33.3o/o). membrane fraction (21.6%) and cell junction (15.7o/o)
(Babelomics, Fisher's exact test, P<O.OS) (Fig.3 ). Results from the Gorilla analysis also
showed similar results that plasma membrane part (a chi ld term under plasma membrane).
excitatory synapse and synaptic membrane were the mo st enriched cellular component
(TabS.4) .

88

Pathway analy i

Path a anal
top 16

i u ing IP

identifi d a t tal

f 72 ignificant

anonical pathway are hown in Figure 4.

ignaling were id ntified a
an nical pathway included

an nical pathway . The

alcium transp rt 1 and calcium

the t p and the 4 111 ranked pathway .
protein

ther important

ignaling mediated by Tubby, remodelling of

epith lial adh rent , glutamat rgic ~ ynap e and gap juncti n (Fig.4 ). Results of KI~
Pathway pr dicti n u ing Web ' e ~ talt t ol al o ho\\-ed the calcium ignaling pathway a
th

nly ignificant path\\- a; that included I 0 f the l 02 differentially ex pre ed proteins

from M Hg treatm nt (Fig . 1-2) .

Ve rification of protein express ion us in g a n alternative m eth od

Results of We tern blot are hown in Figure 5.

hese protein were chosen as they are

important in ion tran port/ ynaptic transmi ssio n (SLC 17 A 7), and calcium pathway
(SLC8A 1). DLG4 was the common protein involved in all the significant term s
(carbohydrate derivative metabolic process, ion transportation and cell development a
well as cell morphogenesis in neuron differentiation) in MeHg-treated cerebellum.
CCT6A, a protein that was not differentially expressed was also elected as a negative
control for validation. There was a decrease expression of DLG4, and
significant change in

L 17 A 7 and no

T6A. These results agree with the MS result . H wever. while

SLC8A 1 was shown to be significantly decreased in the MS results, the we tern blot
resu lts showed a decrease trend but the resu lt wa not significant (Table ., ).

89

Tabl 1. ignificant

( i logical Pr

term ba d et

f differentially

pre ed

pr t in in M Hg-treat d m rm et cerebellum

Group

ro up 2

Grou p 3

GOlD

:00 55 0 6
:000 75
G0 :00091 17
: 1901 565
:00 7252 1
G :0006163
:0019693
G0 :0009 25 9
G0:00091 50
G : 19011 36
G0 :0006811
G0 :00 5 1049
GO:OO 16192
G0 :004690 3
G0 :0035 637
G0: 001922 6
G0 :000 7268
G0: 0048468
G :0032990
G0:0000904
G0 :0048858
G0 :0048667

rm

arb h; drate deri' ati' c metaboli c proce
rgan oph o phat metaboli c pr ce .
nu I ba c-co ntainin g mall mo lec ul e metaboli c
proc
nu cleo id e ph o ph ate metab li e proces
nu I otid m tabo li c pr ce
orga nonitr ge n compound ca tab li e proce s
purin -co ntainin g ompound mctaboli proces
purin e nucleotid e metaboli c proce
rib o e ph o ph ate metabolic proce s
ribonucleotide metabo li c proce
purine ribonucleotid metaboli c proce
carbohydrate deri va ti cataboli c proce
ion tran port
regulati on of tran port
ve icle-m edi ated tran port
secreti on
multi ce llul ar orga ni mal signalin g
tran mi sian of nerve impul e
synapti c transmi ss ion
ce ll deve lopm ent
ce ll part morph oge nes is
ce ll morph ogenes i in vo lved in di fferenti ati on
ce ll proj ecti on morph oge nes is
ce ll morph ogenes is in vo lved in neuron
differentiati on

A ociated
Protein # ( 0/o)

Term P Value
orr.
Bonferroni

22 (1 5.7)
25 (I 5.9)

7.86 £- 109
9.54 .. - 100

22 ( 15. 7)

. 14£-96

22 (1 7.2)
22 ( 17.2 )
18(1 5.8)
20 ( 18 .0)
20(18 .3)
20 ( 19 .2)
20 (19 .2)
20( 19 .4)
18 (22. 0)
2 1 ( 15. 8)
2 1 ( 15.3)
23 ( 15. 1)
19 ( 17. 1)
24( 16.7)
22( 15.9)
20( 15.5)
28 (15 .2)
19 ( 16. 1)
18 (15 .8)
19( 16.4)

8.5 9 - -95
1.57£-94
1.86 £-65
I .48£-52
1.46£-42
4.29 -3 5
2.92£-34
6.73 -32
3. 12£-2 1
5.95£- 105
3.80 - 104
4.49 - 10 1
2.22 -99
I .88 -6 8
3.24 -56
1.37£-34
3.91 -103
3.85 -50
I .24 -46
4.49 -46

18(17 .0)

2.44£-22

90

Tablc2. nri h d rni r R

and th ir targ t g n

fun ti n t rm f

in th

naptic

tran mi i n

mi croR

h a

TT

ha
h a

1358
h a_
TT ,MIR-17-S P.M IR20 .MlR-1 06 .M IR-1 068.M IR208,MIR-519D
GGG,MIR-1258.MIR-

125
h a

Ge ne ymbol

,MIR-19 .M IR-19
.MIR-15

h a CT

#o f Ge ne

P-va lu e (corr.
Bonferro ni )

0.0297
4
4

4

3

A TGT .MIR-128 ,M IR-

P25. PL

YTI,

TN Pl. PL BL 8
K J10. PL ' 81, SL 17/\7.
TP282
' A , L ' 17A7. 8
.'L 6 L

1288

I.

A , SYT1

0.069
0.1122
0.] 287

0.24 75
0.3201

h a TTTTG G.MIR-373

3

DLG4. RA 11A. L 17A7

0.4059

h a_TGCACTT,MIR-519C,MIR519B.M IR-519A

3

KCN.J I 0. PLCB I. SYT I

0.5049

h a AAGCAAT,MIR-137

3

PLCBI, YT1. 8 N

0.5049

hsa CACTGCC,M IR-34A, MIR34 ,M IR-449

3

CNTNA PL PLCBL , YT1

0.5049

91

Tabl

01npan

n pr t in qu antificati n re ult using W tern blot and
pr teomi

niqu e
ene y mbol

Pro tein

anal

Fold

hange (T rea t/ o ntro l)
wi s-Pro t ID

a rn e
P e ptid e

W es ter n bl o t

HP

...
")

0. 6

0.

P78 52

10

0.47

0.61

9P2 7

9

0.26

0.75

P324 18

21

0.62

0.68

P40227

v icular glutamate
17 7
tran p rter 1
dium/ al iurn
L 8 I

exchange r 1
T-cornpl e protein 1
T6A
ubunit zeta

92

I

A

I

~

"'I
<.0

nn

~

"'I

<.0

w

~

"'
I

<.0

()
0

:::>

w

I
()
0

:::>

()
0

=.

B
70
• Identi fied proteome

60

-

~

• Differential expression

50

~ 40

~

~ 30
~

~ 20
10
0
Cytoplasm

Plasma
Membrane

Nu cleus

Extracellu lar
Space

Other

F igure 1. A. H eatmap of prote in a bund ance pattern in di ffe renti all y ex pressed protein s. B.
ellular component di stributi on fo r the tota l identifi ed protei ns (n=99 8, IPA mapped ID )
and diffe renti ally expre ed protein s (n= l 02) in marm o et cerebe ll um . There was a
significant diffe rence in the di stribution of subce llul ar locali zation between total
id entifi ed proteins and signifi cantl y changed prote ins (F isher's exact test P =0.046).

93

f8X02
Bt VRB
PSMC3
SR
VCP

car

GNB5

2

A

hydrate de nva ~ve calaboltc
process

}11trogen compound ca[?'
procass
punfllH:Ontalntng comP9unQ_
_,....--metabolic Prt>ClY

•

SPTANI

RUVBL2
C,MPS

(
GBAS

nbose
ONMI

cell morphogenesiS Involved 1n
neuron d1fferenhat10n

PHGOii

carbohydrate
.AP
derivative metabolic
nucteotrde metabolic process
process
...,e metabolic procass
• ...At.
PIP4K2V
IIRF~

I

•

L1CAM

c II pari morphogenesiS

cell prOJeChon morphogenesrs
cell morphogenesiS rnvolved 1n
ATP6V10
GNA.ORASI
d1fferentratron
SPTBN2
OLG4
•
ANK2
PAKI SRCINI
~ ·K3A
Stde ohosphate metabolic
RAB3A
NTNAP ' NRCAM
procass
111

•

punne nucteotrde metabolic p

puMe nbonucleol!de ~
VPS4A

A P2B2

cell development

PLCB1

ATP6V1H

ANKI

•

LGII

HANK1

co TB

organophosphate metabohc proce~
nbonu

~de metabolic proces:s-

GNAS

•

KCNJIO

GLIJ0 1

•

nucteobase-contarnrng small
molecule metabolic process

lC8At

f)

•

BSN

SUB !

•
SCNlA

synaphc transmiSSIOn

ion transport

LRPI
PPIP

SNAP2~

r t\2
SYT'

NAPB

Ar ::Z112 AMPH
S(P
$

CAM~ 2A

•

SlC17A7
•
SLCGAI

Figure2. An annotation network of three functional group for differentially expressed
proteins m MeHg-treated marmoset cerebellum. The network was generated by
ClueGO+Cluepedia, the GO "Biological Process" was selected, GO term connection
restriction (Kappa score2:0.3 ). Colour nodes represent the functional groups of GO term,
and the size of node indicates the significance of the terms. The identified changed
proteins associated with each term are high lighted in red .

94

• Identified proteome

Cell junction

• Differential expression

*

Plasma membrane part

Plasma membrane

*

Membrane fraction

*

0

10

20

30

40

50

Percentage (%)

Figure3. G enrichment cellular components analysi u ing Babelomics. The a teri ks
indicate ignificant enrichment (P<O.OS) of differentially expres ed protein against
identified proteome. The percentage repre ents the ratio of po itive genes annotated in
blue bar (differentially ex pres ed protein ) and red bar (identified proteome) compared to
the sum of negative and positive gene size in the corresponding term .

95

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""

01

r.71

Figure4. The top 16 Canonical pathways identified by the Ingenuity Pathway Analysis software sorted by their P-values. The ratio
presented is defined as the number of the differentially expressed proteins found in the MeHg treated marmoset cerebellum over the
total number of proteins involved in each ofthe pathway .

96

8

A
Control

MeHg

:X:

c

a..

0.

<

Q

DLG4

-

0.

~

0.4

0

....1

c

• •
••
•

0.6
X
0
~

<
!2 0.4

.....

•
•

0.2

...<
0
......
w

• •
• ••

0.2

SLC17A7
0.

CCT6A

0.0
Con trol

SLC8A1

M Hg

X
0

~

~

<

!2

...<
........ 1.
0

......
<D

ConiJol

MHg

,.,.

2.
X
0

< 1.

GAPDH

•
•
•
••
•

••

•

••

0.

0.

•
•
•
•
••

!2

...< 0.

1-

0
0

0.

0.
0.

Control

MeHg

FigureS. W e t m bl ot ve rifi d fo ur protein identi fie d fr m M

Control

MeHg

analy i in marm o et

cereb llum . A . r pre entati ve bl ot we re e l cted from fo ur prote in , and the intcn ity of
each band wa qu antifie d by den itom etry. Pl ot o n B how each valu o bta in ed from
intensity of indi idual band before and after MeHg treatm ent in cere bellum for those fo ur
proteins (n=6, 2 rep! icate I ampl e) .

97

Di cu

hi

wn

i the fir t tud

pr t orne change
tr at d m aim

u m g n n-human primate a a m ode l to inv

f M e Hg ne ur

et m nkey

ti gate the g lobal -

ic it in th cerebellum o f co mm on marm

e l. MeH g

h wed li ght akine ia and drunk -kind o f m tor dy function

mpt m a (Yamam o to et a!.. 20 1 ~) . M ell g trea tment in crea d th e t tal concentrati n
f t tal m r ur) (H g) in the c rebe llum of the three M eHg- treated m am oset m onkey to
2 .5, 2 . and 2 1.1 ~ g/g re pecti ve l;, c mpar d t 0 ~ gig in th contro l (Yamamoto et a!.,
201 2). Th

f_G ct o n motor fun ti on are imil ar t th

e

b 'erved in hum ans e posed

t hi gh level of M eH g ( lark on and M agos, 2006; F rum kin et al. , 200 1) sugge tin g that
marmo et m onkey i a good mode l fo r hum an MeH g to ic logy tud y. In thi

tud y, we

used hum an databa e earchin g fo r prot in ide nti fica ti on fro m M /M S data. T he ma in
reason are the simil ari ty of n uroanatomy and behav ior of comm on m arm ose t to hum ans,
and the gene ontology fo r m armo et database was not full y built at the tim e thi

tud y

was co nducted . M oreover, o ur result of Western blot using hum an ori gina ted antibodi e
were found to be applicabl e fo r m armoset bra in sampl es . Thi

a] o sugge t

the

hom o logy of genom e sequence between the two species .

M eH g treatment resulted m a decreased expressiOn of 86 proteins and an in creas d
ex pressiOn of 16 protein

m the cerebellum . The number

of di ffe rentia ll y expre sed

protein are similar to those reported by Zhang et al. stud ying effec ts of MPT P on rat
cerebellum (Zhang et a!., 201 Ob) . The ce llular compo nent di stributi o n ana ly i
that M eHg treatm ent did not just cau e globa l no n- pec ifi c effec t

howed

o n a ll ce llul ar

compon ents but had specifi c effec t on the prote in s in the cere be ll ar pl as ma memb ranes

98

( ~ ig.l

). Re ult

f th

ellular component

nrichm nt anal

i al o

h w d that

M Hg af-D ct d pr t in main! 1 cated in the la ma membran (part), m re p
nrich d in

nap e,

MeHg treatment ~a

~ und

deri ati e metab lie proc
differential!

naptic membrane and c ll juncti n (Fig. and Tab .4) .

to affect three mam functi n

11

including carbohydrate

. 1 n tran port and cell devel pment (Tab . I).

e pre ed protein

metabolic proce

f th

ifically

\\er

e pr tein

total of 22

found involving in carbohydrate derivative
~ere d

wn-regulated except for FBX 02 , GNAQ

and VCP , indi ating MeHg n gatively affect d carb hydrate m tabolic pr ce s which
wa the main

urce

fen rg)

upply . , imilar effects on carb hydrate metaboli m were

al o reported in ur arli r tudy in

mato

n ory cortex of rat with a 1 w do e of MeHg

treatment (Kong et al.. 2013 ).

Among all the ion transport functions,

ynaptic tran mi ssion was the mo st important

function affected which is similar to the observation in MeHg expo ed rat cerebellum
(Radonjic et al.. 201 3) . Twenty differentially changed protein

were a ociated with

synaptic transmission (TabS.3 ). and four of them (ATP2B2 , CAMK2 A, SLC17 A 7 and

SYP) had previously been reported to be related to mercury toxicity (Fujimura et al..
2012; Reddy et al., 1988; Yoshida et al.. 201 1) . Furthermore, the 20 proteins as ociated
with synaptic transm ission are mainly involved in the neurotransmitter regulation ( BSN,

DLG4, LGll , NAPB , RABllB , RAB3A and SLC6A l ), glutamate secretion and
glutamate receptor signaling pathway (DLG4, PLCBl , SHANKl , SLC 17A7, SY P and

SYTl ), synapse calc ium-depende nt bi nding (ATP2B2 , SNAP25 and SYT l ) and other '
(AMPH , CNTNAPl , ITPKA, KCNJ10, NCAN). The identification of cellular

99

mp n nt t rm

nap e and

f

that MeHg impa t th

n ur tran mi i n b

ugge ted

naptic membrane (Tab .4) al
citator

ynap

tran mi ion

naptic m mbrane . The e re ult are c n i tent with the previou

tudie

that

am in acid glutamate imbalance inn urons wa on

citatot

me hani m in MeHg

MeHg effect
(Radonj i

n brain

·p

di rupti n protein

m

f them

t impotiant

ur (Yin et aL .... 009): ( arina et al., 20 11 ).

tructure and

t a!.. 201 ). W al

ell m rph logy ha e been

ten ' ively r port d

found igni ficant effec t on prot in a ociatcd with c II

de elopment cell part m rph g n

i , cell m rphogen

i involved in differentiati n,

cell proje ti n morph gene i and cell morph gene i (group 3, Tab. I) . The e re ult
demon trate the global effect

f MeHg on cell development neuronal morphology

feature and n uronal differentiation in the cere bell urn .

alcium homeo tasis di sruption ha

been well documented as one of the core

mechanisms of MeHg toxicity (Limke et al.. 2004: Yallapragada et al., 1996). Calcium is
involved in almost all types of the synapses plasticity regarding motor learning proce s in
cerebellum (Lamont and Weber. 2012). Our re ults showed calcium ignaling pathway
was the top pathway affected by MeHg as predicted by both IPA (Fig.4) and WebGestalt
tools (FigS.2). Also, when a more broad-term examination using #gene/term <5 with
term fusion was applied for GO functional analysi , calcium ion transport and cellular
calcium ion homeosta is were observed in the 60 significant GO term

(TableS.3 ).

Moreover, a total of 10 (ATP2A2, ATP2B2, ATP2Bl, CAMK2A, GNAS, GNAQ,

ITPKA, PLCBl , SLC8Al and SLC8A2) of th
K GG

calcium

signaling

pathway

102 changed protein were mapped in

(Fig .3).

Out

of

which,

five

protein ,

100

TP2Bl/ATP2B2 (Burl ndo t a!., 2004),
(Yo hida t al., 2011 ),
re lat t

mercur

TP2 2 ( a o et al., 2001 ),

L SAl ( urieri et aL 2011 ), ha

MK2A

been pr viously reported to

t , icity. Th r ~ r . the c re ult confinn d that di rupti n

f calcium

path a i a ke mecahni m [! r Mcllg neur to icity .

mong th

102 differ ntially changed pr tein . DLG4. also kn \ n a P , D-95. i a core

protein that afD ted multipl

fun tion

bioi gical functional term . Thi
h wn by both L -M /M

a

it \\as inv 1\ed in all of the 22 significant

;naptic maker \\US down-regulated (about

-folds) as

and \\e tern blot re ults (Tab.3 ). DL 4 ha been ·hown to

play a maj r role in regulating the interaction bet\.\een po t-synaptic receptor particularly
ionotropic glutamate re eptor and a ociatcd
Porras et al.. 2012).

ignaling mol cules (Nash et al.. 2005~

nother key target wa MIR-19A/MIR-19B as they were shown to

target 5 down-regulated protein (N APB , PLCB 1, SYTl , BSN, A TP2B2 and NA PB )
involved in synaptic transmi sion (Tab.2). MiR-19 family plays an important role in
regulation of key gene in the neurodegenerative di ea e and variety of cancer proces .
For example. MIR-19 is a co-regulator to regulat the ataxin 1. a gene that contribute to
the progress of spinocerebellar ataxia type 1 (Lee et a!., 2008). The effects of MeHg on
miRNA warrant further investigation .

In summary, usmg a prin1ate model. we have shown the effect

of MeHg on th

dysfunction of motor function was mainly caused by disruption of carbohydrate
derivative

metabolic proces . synaptic

transmission

process

particularly

calcium

signaling pathway, and cell morphogene is. We r ported a total of 21 novel protein
affected by Mel-lg in

ynaptic transmi sion (Tab . ., ) and calcium associated path\.\ a)'

101

( 1g . ). DLG4

nap tic tran mi

nd MIR-19 /MIR-19
1

n f MeHg n ur t

contribute t the impr

re [! und a th

k

targ ts invol ved in

it; . Furth r in e ti gati on of th ir function wi ll

ed und er tandin g f the path ge ne 1 of Mel Ig neur to

ity in

human .

102

hapter VI: Proteome profiling reveal regional protein alteration in
cerebrum of common marmo et (Cal/ithrixja cchus ) e po ed to
meth y lmercury
a me and Affiliation of

utlt or :

Yu ting ha *. Megumi Yamam t +.

lumbia.

aniel Figcy ! _ Zhibin

ingt, !ling Man

han ~

and nvironmental ,' tudie Program. University [ orthern Briti h
eorg . Briti h olumbia. anada V2 4Z9 ~ mail: shaoy(a{unbc .ca

partn1 nt of a i Medi al cience. ati nal In. titute for Minamata
Minamata. Kumam to. Japan 867-0008 Email : yamam to rcL/nimd.go.jp

t

1 ea e,

ttawa In titute of
tern iol g;. niver ity of ttawa. ttawa. ntari , Canada K 1H
8M5 ~ mail : Daniel Figey dfig y a lu ttav,.a.ca; Zhibin ing ningzhibin(a:gmail.com

!

~ Centre for Advanced Re earch in Environmental

niver ity of ttawa. OMarie
mail :laurie.chan'c(uottawa.ca

enomic , Department of Biology,
urie. Ottav,.a. Ontario. Canada K 1 6N5

103

Ab tract
Meth lmercur
rtic
r gwn
e pr

along deep ul i and fi ur
till unclear. Thi
ion in t\\

cipital 1 b

lecti ely damage the cal anne and precentral

(MeH g) i kn wn t

reg1on

The m lecu lar

ent of Mel lg le ion

111

the e

tudy aim to identify and analyze the differential pr teome
f th cerebrum (pr fr ntal c rtex of the frontal lobe and the

around th e ca lcarine

ul cus in ce rebrum ) o f th e marm

t ( Callithrix

j acchus) treated Vvith McHg ( 1. mg/kg body Vve ight ~ r 14 days) using a shot-gun

prot omic appr ach.

t tal of 1045 and 1062 pro t in Vv ere identifi ed in th e frontal lobe

(FL) and occipital lob ( L), of whi ch. 62 and 89 protein were found
changed in FL and

L re pecti ve ly.

that the lipid metabolic proce

ignifi cantl y

un cti onal enri chm ent/depl eti on analys is showed

and prot olys is occ uned in both two lobes. Differenti all y

e pres ed proteins in FL are pecificall y enri ched in th e fun cti on of cell cycle and cell
division, sul fur compound metaboli c proce s, microtubul e- ba ed process and glycerolipid
metabolic process. In compari son, protein were enri ched in th e fun ctions of transport,
carbohydrate metabolic process, chemi cal caused homeo tas is and regul ati on of body
fluid levels in OL . Pathway analysi

predi cted that vaso press in -regul ated water

reabsorption was di sturbed in MeHg-treated FL. Di srupti on of energy production were
shown by changes in pyrimidine metaboli sm and pyruvate metabolism in FL, and
butan ate metaboli sm in OL. Our res ults showed that MeHg induced regiona l spec ific
protein changes in FL and OL but with similar endpoint effects such a energy dimi ni h
and edema. The result of water transport dysfunction upport the phenotype effects of
edema observed in the marmoset brain. APO

and G PX 1 were fo un d to b key target

protein dedi catin g Mell g multiple functi ons in OL. This is the fi r t report of the \\hole
104

prot

m

hang

f primate cerebrum ~ r MeHg t

the und r tanding f m lecular ba i

Key word : Meth lm rcur , Prot

f MeHg int

m1c ,

erebrum,

icit

tudy, and th r

ults improve

ati n in human.

curoto icit ,

ammon marmoset

Introduction

Mercur a a global envir nmental to. icant po ed a potential health ri sk for wild-life and
human

( ri coil et al.. 201 ). Mercury ha

been used for

ecd fungicide and as an

indu trial byprodu t (acetaldehyde cataly t ) in the Ia t century. Fi h and marine mammal
con umpti n are th main
2003).

inc

th

urce

f mercur) expo urc for human ( ' larkso n and

train,

fir t outbreak of m rcury p i oning in Minamata, 1956s, mechani sms

for mercury to icity effects in different pecie have been widely studied (Farina et al.,
2011 ).

erebellum and cerebrum are the main target

for MeHg neurotoxicity. The

typical symptoms ofMeHg in Minamata di ease were ataxic gait low movement as well
as vision deficit (Eto, 1997). The autopsy re ults of Minamata di sea e cases hawed that
in acute phase, the level of mercury wa

found highest in cerebrum and cerebellum

compared to the other regions in nervous system (Okabe and Takeuchi, 1980) .
Methylmercury induced brain morphology changes, including neuron lo s , particularly
occurred in the calcarine cortex and cerebellar cortex (0 hmichen, 2006; Takeuchi T.
1979). Brain edema happened in the perivascular space and neuron swo llen in th
cerebral cortex in acute case (Eto, 1997).

erebrum in frontal lobe and occipital lobe

have been documented as sensiti ve bra in regions for mercury accumulation in different
spec ies, such as rin ged seal (Krey et a l. , 20 14 ), be luga whales ( 0 tertag e/ a/., 20 13 ),
otters (Do rnbos el a/. , 20 13) and co mmo n mam1o et (Yamamoto el a/., 2012).

105

De it the num r u

tudi

reporting neurot

icit

mammal , ther i little in:fl rn1ation on the c mpari
brain regi n
t maticall

f MeH g in dif:fl rent pec1

of

n m chanism f M llg in differ nt

pecifi ally. Thi i partly du t a lack fa relevant model and tool to
tud , the global mechani m

f mercury to icit in the

regwns.

ue to

the imilar character with human being in anatomy and beha vior, non-human primate
uch a c mm n marm

t e. hibit m

rc advantage than the other primate , and rodent

p c1e in biomedic al re earch e, pec iall ; in neuro cicnce and tox icological screenin g
(Ki hi et a!.. 20 14:
u

kan el of , 20 12 ). Th

bjecti e of thi s stud y wa to explore the

of common marm o et a , a model to tud ] the mec hani sm of Mel-l g sub-acute

toxicity. The pecific bj cti v wa to id enti fy and co mpare th e global proteo me changes
of frontal lobe ( L ) and occipital lob

(

L ) of th e ce rebrum induced by McHg ex po ure.

Us ing bioinfonnatic analys i includin g bi ological cellul ar fun cti on, pathway and protein
network anal ysis. the mechani sms und erl yin g th e phenotypic change induced by MeHg
were investigated. Western blot was u ed to validate the important protein s re ult from
proteomic prediction.

Material and Methods

Ex perim ental animals and Methy lm e rcury A dministration

Experim ental co nditi ons co ncernmg MeHg ex pos ure

tn

commo n 1narmo et was

previously described (Yamamoto el aL 20 12). Briefl y, s1x co mm on marm oset we re
di vided into MeHg-treated and vehi cle groups. Methylm erc ury chl ori de ( 1.5mg/kg bod)
we ight) was admini strated using an indwe ll ing infant reeding tu be ~ ith 3 marmo et for
14 days, foll owing 14 days without treatm ent . The co ntro l group \\as given the ame

106

f

c ncentration
appr

d b

the

-c

t 111 (7.5M a MeHg-tr ated gr up.

nimal Re

ar h

uide for the

nimal

b

th

In titute of Lab ratory

hi I

and MeHg-treat d group

are and

e of Laboratory

L and

regwn

111

ected after anesthesia. Di

ected ti

ue

nimal Re ource . The

\"vere di

were

for Minamata

ati nal In titut

mmitt e at th

i ea e, and all pr c dure c nform d to th

11 animal pr cedur

ample were immediately fr zen in liquid nitr gen and stored at -80 °C for proteomic
analy i

ample Preparation and Ma

FL and

S pectrom etric

naly i

L ample in triplicate from vehicle and Mel Ig-admini trated groups were used

for liquid chromatography-tandem ma

pectrometry (L -M. IM. ) analy i . Tissue

preparation and protein quantification u d the

ame methodology and pr cedu re as

described previou ly ( hao et a/., 2015 ). Briefly, proteins extracts ( 10011g) w re
precipitated, reduced and alkylated, followed by tryp in digestion. The digested peptides
were acidified by formic acid, and fractionated by

tageTip strong cationic exchange

fractionation into five fractions by five pH steps (pH3.0, 4.0, 6.0, 8.0, 10.0). Lastly, the
peptides were desalted on Sep-Pak column (Waters Corp., Milford, MA) and lyophilized
in a SpeedVac (ThermoFisher Sc ientific, San Jose, CA).

An Agilent 1100 capillary-

HPLC system (Agilent Technologies, Santa Clara, CA) coup led with L TQ-Orbitrap mass
spectrometer (Thermo Fisher Scientific, San Jose, C A) was u ed to analyze the peptide
mixtures.

107

Homo

The
d

apaen Databa e earch and Quantitation

arne human databa

e r h and pr tein identificati n procedure were u ed a

cribed pre iou 1 ( hao et al., 2 15). The peak I i t fr m raw fi le wer

wi th

ftware Ma.

uant ( er ion 1.. 0.5 ) and

general d

earched against , wissProt pr tein

databa

f human (ver i n .... 0 120711 )_ including comm nly b cr ed ontaminants. For

M /M

pectra. the precur

t 1 ranc wa

r ion mass tolerance \\ere 7ppm. and fragment i n mass

t t 0 . Da. Raz rand unique peptide Y\ere applied for quantitation . The

fal e di cov ry rate v.a limited to 0.01 on b th pr tein and peptide level. A minimum
equenc length of ix amino acid wa requir d for peptide identification .

For protein identificati n. if the id ntified p ptide sequence of one protein was equal to
or contained in another protein· peptide et. the . e tVvo proteins were grouped together by
MaxQuant and reported as one protein group. For quantitation. a minimum of 2 spectra
count i needed .

Data An alys is and Cellular Compon ent Distributi o n

All the contaminant and reverse data were removed. and then the identified protein were
converted to

niprot gene ID by Persues software (Version 1.3.0.4).

bundance ratio of

proteins were based on the label-free q uantitation of each group in the format of the fo ld
changes. The calc ul ati on of differently expressed proteins was performed u ing MannWhitney U test w ith SP SS 16.0 ( P

Inc., C hicago, IL) . Differentiall y expressed

prote ins in each of co ntrol and M eHg- treated group were furt her clustered into heat map
to test the gro up va ri ance using Persues software.

108

ellular c mp n nt d i tributi n wa cla ifi d b u ing lng nuit Path a
ft are (Qiag n, R dw

nalysi (IP )

d.

mpari on of total id ntified pr tein and differ ntiall e pre cd pr tein was given by
th p r ntag
e t

f ach ub-c llular locati on aga inst it total number . A Fi her'

_. act

a c nduct d to te t the rganell e di tributi on difference betwe n t tal proteins

and di fferentiall y e. pr , ed pro tein in the tV\ lobe of ce rebrum .
GO Functional Enrichment

naly i for Frontal Lobe and Occipital Lobe

To identify th

v r-r pr entati ve functi onal catego ri e

anal y i

(g ne ont logy) bi ological fun ction wa co ndu cted usmg 'ytoscape

for

111

FL and

L, enri chm ent

lueGO plug- in (Bindea el a/. 2009) . The di ffe renti all y ex pre s d protein wer te ted
in th e ba e of the reference of human G

(bi ological process) annotati on again t the

proteome identifi ed in FL (1 045) and

L ( 1062). A two-sid

hypergeom tri c

(Enrichment/Depletion) test wa used to calcul ate the enri ched catego ri es. and Bonfe rroni
test was applied to correct the P value. Onl y P <0.05 was co nsidered as ignifi cant terms.
To reduce th e redundancy of the GO terms. we applied the restri ct parameter in OL (# of
genes as 9, and min °/o as 10).
Hierarchy groupmg was undertaken to clust r the functio nal tenn based on the
perspecti ve of their parent tenns. We defined the min # co mm on parent a 3 and max #
as 4 for the mapped clu sters that representing from the total associated gene with the
term . To visualize the bi ological relevance of related proteins in FL and OL. a functional
annotati on network was generated based on the gene simil arity (Kappa core wa set as

109

0. ) D r

h of the t o 1 b

the kn wn int racti n (fr m

Pathway E nrichment

furth er addre

lueP dia (Bindea e/ a!. , 20 1 ) wa applied to vi ualize
RIN

the identifi d biological functi on affec ted by M Hg, the diffe rentiall y

u ing Webge talt (WEB-ba ed

reD r nc

ciat d gene .

naly i and Mappin

pre ed pr tein in FL (6-) and

linked K

9.0) b twe n a

L ( 9) V\Cre further appli ed for path way predi cti n
~ n e , eT

naL

is Too lkit) (Wang et ul. . 201 3 )which

path'vVay databa e. The id entified proteo m in FL and OL were u ed a
background D r th

enriched KE

(Kyo to Encyc lopedia of

ene and

enome ) path'vV ay term predi ctio n. I lypergeometri c te t was used to calcul ate P values
fr m th diffe renti all y xpre
were included in KEG

d prot in ~ th e ize of GO et equal or above 2 genes

pathway analy i . Bonfe rroni method was appli ed to co rrect th e

P value. Protein invo lved in int rested pathway were further mapped into onlin e K GG
pathway mapping sy tern (http ://www.genome. jp/kegg/).
Wes tern Blot A nalys is

To verify the quantitative changes of the proteomics data. fo ur protei ns were elected
including Di sks large homolog 4 (DLG4), Cytopl asmi c dynein I li ght intermedi ate chain
2 (DYNC l LI 2), Glutathi one pero idase 1 (GPX l ) and Aq uaporin -4 (AQP4) to be
analyzed by We tern bl t. We select these 4 proteins because th ey arc important protei n
in linking multi functi ons (DYN 1LI2 and GPX 1) and key protein in interpretati on brain
edema (DYN 1LI2 and AQP4) in th e region of FL and OL. In additi on. wea l o \\ant to
verify the ex pre sion of non-differential ly ex pressed pro teins such a DLG 4. Briefl y.
protein

10-40!-!g we re loaded into I 0- 12o/o SDS-PA 1E pre-cast ge l , (Bi o-Rad.

110

Mi

) Dr

aug a,
tank bl tting

paration, then tran ferred into PV

tern (Bio-Rad. Mi

with So/o fat-free milk in T

T ( n

m mbrane by u mg

auga,

).

he membran

were blocked

Buf[i red

alin

+Tween-20) for 1h ur. and

appropriate primar anti bodie were incubated v. i th membrane in So/o TB
(4° ).

overnight

fter probing v.ith hor eradi h per . idase-co njugated econdary antibodies ( anta

ruz Biotechnology Inc,

anta ' ruz,

). membrane v.ere wa hed and target band

wer d teet d u ing enhanced chemilumine cence clarity reage nt (Bio-Rad, Mi ssi sauga,
), and

i ualized by ' hemi-doc ( io-Rad, Hercule ,

of band inten iti

wa performed u. ing Image J oft ware . All the primary antibodie

were purcha d from
Inc. (Santa

ruz.

A) Imager. The quantification

beam ( beam. Cambridge. MA) and , anta Cruz Biotechnology

A): DL 4 ( 1: 50. ab 18258 ).

(1 :300, sc-66 866), AQP4 (1 :400.

PX 1 ( 1:1000, ab l 08427), DYN 1LI 2

c-9888) and econdary antibodie were from , anta

Cruz Biotechnology. The equences of antigen amino acid for tho e human originat d
primary antibodies were 2: 90o/o similarity with common marmo et.

Res ults
Methy lm ercury-indu ced Protein A bun dance C hanges an th e F rontal Lo be and
Occipital Lobe of Ma rm oset

A total of 1045 and 1062 proteins in triplicate runs were identified from frontal lob and
occipital lobe, among them 168 and 185 protein were uniquel y expres ed in FL and OL
respective ly. Typical MS/MS spectra for DYN l LI2 (04323 7. Cytopla mic dyne in 1
light intermediate chain 2) in FL and APOE (P02649.

polipoprotein E) in OL were

presented in Figure S 1. Man-Whitney U test showed that 62 proteins (28 up-regulation

111

and

4 d - n-regulation ) in
re differ ntiall

ere commonly e pr
Heat map from
diH rentiall

and

pr

L and 89 ( 18 up-regulati n and 71 down-r gulation ) in
ed.

d betwee n tVv regi n (Fig. I ).
L hov.- ed th

prot in

con i terr e

of protein abundance change of

e. pre ed protein . and a clear eparati on wa ob erved between Mel Ig-

treated and untreated ampl e (F ig 2
L and

pr tein ( YN ' 1 I2. HI Tl H2BH and P )

nl

and ' ).

are hov,:n in F i g~ (B and
wer

i tri buti on of

cellular component in

). In FL the total and di fferenti all y ex pressed

di tributed in the fo ll ov.- ing order: cyt pl asm (63o/o. 70o/o), pi a ma

membrane ( 17o/o. 11 ° o). nucle us ( l ., o/o. 1.,o/o). ex tracellul ar space (3°/o, 3%) and the
remaining other organell e (4°/o. 3°/o). In

L. th e relevance percentage for ub -cellular

location fo r total protein and diffe renti all y expre sed pr teins are cytopl as m (63%, 66o/o),
plasma membrane ( 17o/o. 17°/o). nucleu ( 12o/o, 1Oo/o), extrace ll ular pace (3o/o, 5o/o). and
other organelles taking 5% and 2o/o re pectively. There are no ignifi cant diffe rence of
organelle di stribution between the identified proteins and th e di ffe renti all y expressed
proteins in FL and 0 L.
GO Enrichm ent Functional Analysis Differentially Expressed Protein s for F L and OL

An enriched functional analysis fo r FL and OL were conducted usin g ClueGO software.
In FL. the analysis indi cated fi ve signi ficant hi erarchy groups and non-grouped term
(F ig.3A) with 39 proteins and 24 term s were included (Tab 1). The hi erarchy terms 'Were
clustered manuall y into 5 parent term s: 1) Ce ll cyc le and Ce ll di vi ion; 2) Cel luar
metabo li c process; 3) Proteoly is; 4) mi crotubu le- based procc s: 5) Mult i-organism
process including modifi cati on of morphology or phys iology of other organism . The non-

112

gr uped functi n t nn includ d pr tein ubiquitinati n (P=5.24 - 14) and developmental
gr wth (P- 1.45 -- -04 ).
in all 24 tenn

f

t rm

in 19

. In

ell divi i n wa the m

t ignificant t rm ( .5 6 -1 4) id ntifi ed

. 55 out f 9 differential] e pre

in re pon e t

Mel Ig expo ure. These 19

d proteins were includ d
term

were manually

clu t r d int 4 parent group and 5 non-gr uped tenn s (Fi g. B): l) llomeo tatic process;
2)

rganic

ulfur

ub tanc e me tab li e pr cc ;

mp und metabolic proce

) Tran port; 4) ' ell ul ar metabolic process;

(P- 0.0 8) and glycerolipid metabolic pr cess

(P=7. 1 ~ -05 ) \\1 re in luded in cellul ar metaboli c process. The non -grouped term
compri ed intracellul ar

ignal transducti n. regul ati on of multi cellular organismal

development n gative r gul ati on of re pon e to timulu s. proteo ly i · and regul ation of
body fluid level . Biological proce

in intracellul ar signal transdu ction co ntain ed th e

hi ghe t proteins number ( 19) again t it associ ated protein (Tab2). Lipid as. ociated
metabolic proce s and proteo lys is were the functi ons that we re co mmonl y affec ted by
MeHg in both the FL and OL.
Functional Annotation Network of FL and OL of Marm oset by C lu eGO

The functional annotation network provided the visuali zati on of th e functi onal conn ecti on
and associated proteins between group term s. A total of 3 7 GO term connec ti ons 'W re
found in the FL ( ig.3A) and 12 term connections in OL clustered network (F ig.3 B). due
to the restriction of parameter set in OL. Each of the e cluster represe nt di tTerent
biological functi ons based on their protein sim ilarity. Cytopl a mi c dynei n 1 light
intermediate chain I (DYN 1L ll ) in FL and Apolipoprotein E (A POE) in OL net\\Ork
were shown as the co re protein to co nnec t mu lt ip le ce ll ular bi ological processes .

113

KEGG

ignificant Pathway
pathway predicti n refl ected the functi nal ef~ t of differentially

here ult of K
pr

ed protein on p cific ce llular pathwa

1 gical function anal y i . the re ult

orre ponding t

M H g-tr at d

L

re pond to mer ur

h wed

va. pre in-rcgulat d

water

timuli in

f K.

reab orpti n,

L and

pathway in
pyrimidin

metaboli m. pyruvate m tab li m and neurodegenerative di ord er such as Parkinson ·
di ea e and Huntin gt n ' di ea e (Tab.3 ), th ough no signifi cance bowed in adjustm e nt P
alue . Pathway in va o pr
62 differential!) e pr

in-r gulated \\ ater reab o rption inc lud d 4 pr te ins out of the

ed protein in FL (Fi g. 6 ). Fo r th e

identifi ed the impo rtant pathway

L. KE

.J pathways anal ysis

uch a tryptoph an meta boli sm , butanoate m etaboli m ,

valine, le ucine and isol eucine d gradati on. meta bo li c path ways and ti ght juncti o n (Tab.4 ).
Pathw ays associated with energy producti on such as pyruvate m etabo li sm and butanoate
m etaboli m , and neurod egenerati ve di sea es (Parkin so n '

di ea e and Huntingto n 's

di sease) were found in common in the two lobe of cerebrum .

Va lid ation of Differentially Ex pressed Protein s

W e tern blot was conducted to veri fy the four screened protein from the r ult of m a
spectrom etry. As expected, the express ions of DLG4 and DYNC 1 LI2 from MeH g-tr ated
L were up-regul ated (Fi g.5 ), and GPX 1 was significantly down-regul ated w ith 2.6 fo ld

(P<O.Ol) in OL region (Table] ). However, the ex pre sia n of AQP4 in OL intends to be
in creased, but was not stati sti call y signifi cant. Ge nera ll y, We tern blot resul t were in
line with the proteomi cs ana lysis.

114

Table 1. Functional GO terms (BP) differentially expressed proteins in MeHg-treated Frontal Lobe

GOlD

GO Tenn

Associated
Proteins #
(o/o)

Term Pvalue Corr.
Bonferroni

Associated Genes Found

G0:0000075

cell cycle checkpoint

4(16.7)

1.09E-02

G0:0022402

cell cycle process

14(11.1)

1.06E-6 7

G0:0044770

cell cycle phase transition

7(12.3)

1.08E-06

G0:0051301

cell division

8(11.1)

".56E-14

G0:0044772

mitotic cell cycle phase transition

7(12.5)

2.44E-06

4 ( 16.0)

4 .37E-02

DYNCI LIL PSMAL PSMB6. PSMC4

4 (16.0)

4.45E-02

DYNCI LIL PSMAL PSMB6. PSMC4

4(12.5)

2.07E-05

DYNC 1 LI I. PSMA 1. PSMB6. PSMC4

4 ( 16.0)

2.79E-02

DYNC 1LI I. PSTv1A 1. PSMB6. PSMC4

7(13.0)

1.07E-03

7 ( 13 .0)

5.01E-05

7 (13.0)

5.96E-O~

7 (13.0)

1.97E-04

GO: 1901987
GO: 1901990
G0:0007346
G0:0010948
G0:0019048
G0:0035821
G0:0044003
G0:0051817

regulation of cell cycle phase
transition
regulation of mitotic cell cycle phase
transition
regulation of mitotic cell cycle
negative regulation of cell cycle
process
modulation
by virus of host
morphology or ph ys iology
modification of morphology or
physiology of other organism
modification by symbiont of host
morphology or physiology
modification of morphology or
physiology
of other
organism

DYNC1 LJL PSMAL PSMB6. PSMC4
ANK3. ANXAI 1. CHMPIA. DYNClLIL MAP4.
PPP 1CB. PRKAR2B. PSMA 1. PSMB6. PSMC4,
RABIIA. RAB35. TUBB2A. TUBB4A
DYNC I LIL PPPl CB. PRKAR2B. PSMAL
PSMB6. PSMC4. TUBB4A
ANK3. ANXAI L CHMPlA. DYNClLIL MAP4.
PPP1 CB. RAB II A. RAB35
DYNC I LI 1. PPP 1CB. PRKAR2B. PSMA 1.
PSMB6. PSMC4. TUBB4A

DYNC1LI1. E IF~H.
SLC25A5. VIM
DYNCl LIL EIF-lH.
SLC25A5. VIM
DYNCl Lll. E IF4H.
SLC25A5. VIM
DYNClLIL EIF4H.
SLC25A5. VIM

KLCI.

PPIA.

PSMB6.

KLCL

PPIA.

PSMB6,

KLCI.

PPIA.

PSMB6.

KLCl.

PPIA.

PSMB6.

115

Table2. Functional GO terms (BP) of differentially expressed proteins in MeHg-treated Occipital Lobe

GOlD

GO Term

Associated
Protein # (% )

Term P-value
Corr.
Bonferroni

G0:0019725

cellular homeostasis

12 ( 14.0)

9.66£-22

G0:0042592

homeostatic process

14 (10 .8)

7.42E-1 06

G0:0048878

chemical homeostasis

10(11.0)

1.25E-48

G0 :1901135

carbohydrate
metabolic process

15(10.0)

1.29E-1 07

G0:0006629

lipid metabolic process

11 (11.1)

6. 14E-112

10(12.8)

1. 94E-l 04

9(11.4)

1.79E-29

G0 :0044255
G0 :0044723

derivative

cellular
lipid
metabolic
process
single-organism carbohydrate
metabolic process

G0:0051049

regulation of transport

16 (10.3 )

1. 17E-99

G0:0051050

positi ve regulation of transport

10(13 .3)

8.91E-19

G0:0006897

endocytosis

13 (15 .9)

1.57E-16

Associated Proteins Found
ADDL APOE. GLRX. GPXL HP. IMMT,
OXCTL PICALM. PPTl. PRKCB. PRNP. TXNLl
ADD!. APOE. AQP4. GLRX, GPXL HP, IMMT,
LAMTORl. OXCTL PICALM, PPTL PRKCB,
PRNP. TXNLl
A PO E. AQP4, HP. IMMT. LAMTORL OXCTL
PI CALM. PPTI. PRKCB. PRNP
AK 1. AK5. ALDH5A l, APOE. ARL8B. A TP5F 1.
CMAS. DGKB. DNMl. GANAS. G UCY1A2,
NGEF. OGT. RAB18, RAB5A
ALDf-15A l. APOE. CMAS. DLAT. GPXL
LAMTORl. LRPI. OXCTl. PC. PPTL TECR
ALDH5Al. APOE. CMAS. DLAT, GPXL
LAtviTORl. LRP I. OXCTL PPTL TECR
AGL. ALDH5A l. CMAS. DLA T. GANAB.
OGDHL. OGT. PC. PICALM
ADDL APOE. CDH13. GFAP. LAMTORl. LRPL
MAOB. NCS 1. OXCTl. PI CALM. PPTI. PRKCB,
RAB5A. SCAMPS. SNAP25. WDRl
ADDl. APOE. LRPL NCS 1. OXCTL PICALM.
PPTl. RAB5A. SCAMPS. WDRl
ADDL AP2A2. APOE. CDH13. CLINTL DNML
LRPL
PICALM. PPTL
RAB18.
RAB5A.

117

G0:0006520

cellular amino acid metabolic
process

9(11.7)

6.07E-16

G0:0006163

punne nucleotide
process

12 (10.3)

2.07E-36

G0:0055086

nucleobase-containing smal I
molecule metabolic process

15 (10.1)

8.07647E-96

G0:0072521

purine-containing
metabolic process

12 (10.2)

1.07806E-42

G0:0035556

intracellular
transduction

19(10.2)

2.03E-111

11(11.2)

3.37E-112

10(12.0)

9.16E-l06

G0:2000026
G0:0048585

metabolic

compound

signal

regulation of multicellular
organismal development
negative
regulation
of
response to stimulus

G0:0050878

regulation of body fluid level s

9(10.2)

1. 79E-29

G0:0006508

proteolysis

9(12.3)

9.7408E-112

SCAMPS, WDRl
ALDH1LL ALDHSAL ALDH6AL ASRGLl,
GPXl, PRNP. PSMB 1, PSMB4, V ARS
AKL AK5, APOE, ARL8B, ATPSFL DGKB,
DNML GPXl. GUCY1A2, NGEF. RAB18,
RABSA
AKL AK5, ALDH6AL APOE, ARL8B, ATPSFL
DGKB, DNML GLRX, GPXL GUCY1A2, NGEF.
PICALM, RAB 18. RABSA
AK1. AK5. APOE. ARL8B. ATPSFL DGKB,
DNML GPXI. GUCY1A2. NGEF, RAB18,
RABSA
APOE,
ARL8B.
CDHI3,
DGKB,
GPXL
GUCYIA2, LAMTORl. MRAS. NCSL NGEF,
OGT. PI CALM, PRKCB. PRNP, PSMB L PSMB4,
RAB18. RABSA, \VORl
ADD!. APOE. DGKB. GFAP, GPXL NCSL
NGEF, OGT, PRKCB, PURB, WDRl
AP2A2, APOE, CTNNA2, GPXL HP, LRPL
PRKCB. PRNP. PSMB4. WDR1
APOE. AQP4. ATP1B3. DGKB. GUCYIA2.
PIC ALM, PRKCB. RABSA. WDR 1
APOE. ASRGLL GPXl. HP, LAP3. OGT.
PITRMl. PSMB L PSMB4

118

Table3. KEGG pathway identified in MeHg exposed marmoset frontal lobe

KEGG ID

Pathway Name

Associate
d Proteins
# (%)

ID:04962

Vasopressin-regulated water
reabsorption

4 (23.5)

ID:04910

Insulin signaling pathway

4(21.1)

ID:00240
ID:05130
ID:05012
ID:04540
ID:04270

Pyrimidine metabolism

2 (40)
2 (8)
2 ( 4.4)

Gene Symbol

P-value

6

P-value

NSF. DYNC I Lll. DYNC 1LJ2.
and RAB 11 A
PPP1 CB. PRKAR28. PYGM and
FASN
TXNRD I and CMPK 1
TUBB4A and Tl f882A

0.4438

NDUFA8 and SLC25A5

1

1

0 .1663

1

3 (12.5)
2(11.1)

TUBB4A. GNAQ and rUBB2A
PPP I CB and GNAQ

ID:00620
ID:0451 0

Pathogenic Escherichia coli infection
Parkinson's disease
Gap junction
Vascular smooth muscle contraction
Pyruvate metabolism
Focal adhesion

3 (15.8)
2 (7. 7)

ID:05016

Huntington's disease

4 ( 6. 7)

ACYP2. PC and PKM
PPPlCB and TLNl
GNAQ. NDUFA8. SLC25A5 and
GRIN28

0.015

0.345

0.0223

0.5129

0 .0309

0 .710

0.2903
0.098
0.4644

1

0.4839

1

119

Table4. KEGG pathway identified in MeHg exposed marmoset occipital lobe

KEGGID

Associated
Proteins #

Pathway Name

Gene Symbol

P-value

MAOB and OGDHL
ALDH5A 1 and OXCT1

0.1421
0.173

I:!.P-valu

(o/o)

ID:00380
ID:00650
ID:00280

Tryptophan metabolism
Butanoate metabolism
Valine, leucine and
degradation

2 (25)
2 (22.2)
isoleucine

2(15.4)

ID:OllOO

Metabobc pathways

16 (9.6)

ID:04530
10:04670
ID:04145
ID:0501 6
ID:04666
ID:04972

Tight junction
Leukocyte transendothelial migration
Phagosome
Huntington's disease
Fe gamma R-mediated phagocytosis
Pancreatic secretion

3(10.7)
2 (10.5)
3 (8.8)
3 (4.8)
2(8.3)
2 (8.3)

ALDH6A 1 and OXCTl
AK5, AKL AGL, ATP5fL
ALDH5A LALDII6A 1. CMAS,
DGKB. DLAT. GANAB. MAOB.
LAP3. NNT, OGDIIL PC and
PPTI
MRAS. PRKCB and CTNNA_
CTNNA2 and PRKCB
DYNCI Ll2. RAB5A and TUBA8
AP2A2. GPX1 and ATPSF l
DNM 1 and PRKCB
A TP 1B3 and PRKCB

0.3028

1

0.3223

1

0.428
0.4882
0.5615

1

1
1

1
1
1
1
1

120

TableS . Proteomics results were confirmed by Western blot in marmoset two brain regions

Unique
Swiss-Prot ID

Gene Symbol

DLG4

Brain

Protein N arne
Pep tides

P78352

Fold Change
HPLC-ESI-

Region

MS/MS

Western Blot

26

1.12

1.73

FL

intermediate chain 2

9

2.69

1.29

FL

Disks large homolog 4
Cytoplasmic dynein 1 light

043237

DYNClLI2

P07203

GPXl

Glutathione peroxidase 1

9

0.42

0.39

OL

P55087

AQP4

Aquaporin-4

5

2.11

1.28

OL

121

•••

••
•
•
•••
:
••
••
••
•

••
••••

••••••••

59

••• •••••••

••••
••
••
••
:
••
••
•
•
••••

••••••••• ••

•••

•

.3.

•
•••
••
:
••
••
•

•

86

••
••

• ••
•••
••
••
••
•
:
••
•
••
•
••
•••

••• •••••••• •••

Figure I. Quantification analysis of identified protein in frontal lobe and occipital lobe in
MeHg exposed marmoset. Numbers and PPI network of total identified proteins in upper
panel and differentially expressed (statistical difference) protein in lower panel. A total
of 1230 proteins were identified, and 877 overlapping protein were detected from the
frontal lobe and occipital lobe in triplicate runs (upper panel). 148 protein are
statistically different and only 3 protein shared between the two regions .

122

8 80
70

• Total identified proteins

60

• Significant changed proteins

;,!!

!!..50
Q)
Cl

~
40
c:
Q)
~ 30

Q)

Cl.

20
10
0
Cytoplasm

Plasma
Membrane

Nucleus

Extracellular
Space

Other

0 10
• Total identified proteins

60 4

• Slgmficant changed proteins
"0' 50
0

~
40 ~
nl
i:

...~ 30
~

Cl.

20 ~
10 ~
0
Cytoplasm

Plasma
Membrane

Nucleus

Extracellular
Space

Other

Figure2. Heatmap of protein abundant changes in differentially expressed proteins in FL
(A) and OL (C), and cellular components distribution for identified proteins in frontal
lobes (B) and occipital lobes (D). There was no significant difference in the distribution
of subcellular localization between total identified proteins and changed protein s in FL
and OL.
Note: IPA mapped total identified proteins m FL, n= l 039. differentially e. pre sed
protein n= 62; IPA mapped total identified proteins in OL, n= 1059, diff rentially
expressed protein n= 89

123

developmental growth
protern ubtqurtlnatron

12 1%

mrcrotubule-based process
microtubule cytoskeleton orgamzatron

11 1%

proteasome-medrated ubrqurtrn-<Jependent protern catabolic process
P ro t eo ly s1s
....__ _ _ _ _ _ _ _ _ _ _ proteasomal protern catabolic process

12 5%

glyceroliprd metabolic process
ur compound metabolic process
cofactor metabolic process

12 5%

A
.
tub
b
..,
M lcro
u 1e- ase~ process

.

L

Cellular metabolic
process

L

11 4%
30%
11 8%

c:

200%
54%

cellular amrno acrd metabolic process

Multi-organism
process

1%

r - - - - - - - - - - - - - - - - - - - - - - - - - rnteractron wrth host

115%

modrfrcatron of morphology or physrology of other organrsm rnvolved rn symbrohc rnteractmn
modrfrcatron by symbront of host morphology or physrology
modrfrcatron of morphology or physrology of other organrsm
L - - - - - - - - - - - - - modulatron by vrrus of host morphology or physiOlogy
r - negative regulatron of cell cycle process
regulatron of mrtotrc cell cycle
regulatJon of mrtotrc cell cycle phase transrtron
regulatron of cell cycle phase transrtron
Cell cycle and
mrtotrc cell cycle phase transrtron
Cell division
cell cycle phase transrtron

13%
13%
13%
13%
16 0%
12 5%
16 0'}
16 o•,.
12 5%
12 3%

cell diVISIOn
cell cycle checkpornt
L - - - - - - - - - - cell cycle process

11 1%
16 7%
111%

0

2

4

6

8

10

12

16

14

#Proteins 1 Term

124

8

proteolysis
regulation of body flu1d levels

12 3%
10 2%

negat1ve regulation of response to st1mulus

Cellular metabolic
process

L

12 0%

regulation of multicellular orgamsmal development

11 2%

Intracellular s1gnal transduction

10 0%
10 2%

cellula' ammo ac<d metabolic pmcess
purrne-contarnrng compound metabolic process
punne nucleotide metabolic process

11 7%
10 2%

nucleobase-conta1n111g small molecule metabolic process

I

10 3%

posrt1ve regulation or transport
endocytosis

101%

Transport L _

15 9%

regulat1on of transport

Organic.
c
substance
metabolic process

13 3%
10 3%

mgle-orgamsm carbohydrate metabolic process
cellular hp1d metabolic process

11 4%

hp1d metabolic process
carbohydrate denvat1ve metabolic process

Homeostatic
pro cess

I

12 8%
11 1%

chem1cal homeostasis
cellular homeostasrs

11 0%

'

14 0%

L_ homeostatic process

10 8%

0

2

4

6

8

0

12

14

16

18

20

#P rotein s I Term

Figure3. GO Enrichment/depletion analysis of differential expressed proteins for frontal lobe and occipital lobe in MeHg-treated
marmoset only the significant functional terms (P<O.OS) are appear in bar charts. The functional terms were clustered manually
into each of parent term based on QuickGO web-based too l (Binns et a!.. 2009) . A. Functional groups of differentially expressed
proteins specific for MeHg induced fronta l lobe. B. Functional groups of differentially expressed proteins specific for MeHg
induced occipital lobe .

125

8

A

purine nucleotide
metabolic process
M

gtycerolipid
metabolic process
cellular amino actd
metabolic process

p uri ne-e onta1 nlng
compound
metabolic process

developmental
growth

,
)

cellular amino ac1d
metabolic orocess

proteolysiS

carbohydrate
derivative metabolic
process
nucleobase-contalmng lntJacellular signal
small molecule
transducbon

regulation of
multicellular
orgamsmal
development

metabolic process

interact ion wrth host

...,

1'ti!Jk

negative regulation
-,= , ~"'
of cell cycle
process

~..t.-1.0J(J

•

.JeQ.'l=~ms.

microtubule
protein
cytoskeletorPbiquttlnation
organiZation

endocytosis
regulabon of body
negative regulation
fluid levels
of response to
stimulus

..:Q..f.Ji

regulation of mitotic
cell cycle

Figure4. Anno tation netwo rk of di ffe renti all y expressed proteins in FL (A) and OL (B) were vis ualized by ClueGO+Cluepedia.
Co lor nodes indicate functi onal group terms connected based on the ontology' s hierarchies. and gray nodes are terms not grouped.
The size of the nodes indi cates the term significant (the bigger the more significant ).

126

B

25
12

A

*

:r

••
••
•

~ 09

Co ntrol

<

Me Hg

~

~

Cl

06

~

OLG 4

0

03

OYN C1LI2

00

GPX l

:r

•••
•
••

•

<

~

0

a..

1 35

<
~

~

15

N

:J
<J

z

10

>0

180

:r

GAPOH

a..

*
20

05

0 90

:r

•

a..

••

Cl

0

a..

•

•

0 45

•

•
•

•
••

•

08

~
~
a..

••

04

0

<

•

0.6

c(

X

•

00

**
AQP4

0

02

••
••

••••

00

0 00
Control

MeHg

Control

Me Hg

FigureS. Validation of the prot om1c re ult u ing WB analy is of 4 proteins in frontal
lobe and occipital lobe .
. Representative blot of 4 protein from control and MeHgtreated marmo et occipital lobes. B. emi-quantitative mea urement of target proteins as
indicated by 6-7 WB analysi . Target proteins were first normalized by against their
corresponding housekeeping proteins-GAPDH (n= 6. 2 replicates/sample) .

127

VASOPRESSIN REGULATED WATER REABSORPTION

T~ght June n

)

---!)
.,0

Renal in ten titiwn
flu..il (b d)

Co Bet~ duct p rinc~ al cell

urinary lumen
F-acb.n
A~

Basolaterel
membrane

derolyme117.11.

membrane

V2R

AVP

Mlcrotubule

AC
E.xoc ytos IS

~

I AQP2 I \
l
\
Rec

hng\
l

-------~-------,-- ~

\

l
\

I AQP2 I

I AQP2 I --..

Unnary excrebon

/

I

Degradabon

I Reb5 I
I AQP2 I

I
I
I
I
I

- - • Water homeostasiS
0

H;P

I

+

Earl endosome
Tight junction

I AQP3 I

04962 7121/10
(c) Kanelusa Laboretones

Figure6 . Vasopress in-regulated water reab orption pathway was clas ified by K G
pathway mapping system from upload d 62 differentially expressed proteins in FL. Protein
hi ghli ght with red were the one mapped in Vasopre in-regulated \\later reabsorption path\\a).

128

Di cu ton
er brum, e p iall in fr ntal 1 b (F ) and ccipital 1 b
n

f th main targ t r gi n

(cal arine and

(

) ha been h wn to b

f M Hg neur to i ity ( t . 1997) . With tw d ep fi sure

ylvian) in cer brum. marmo et ha e more

imilar neuroanat my to

human than rodent Vvhich lack de p fi ure. in the erebrum . Therefore , mann
good model [! r n uro
human databa

[! r pr

1 n

et i a

e tudy relevant to human (Eto e/ a!. 2001 ). We searched

tein id ntifi ati n from marmo et M , /M

data, and us d human

originat d antib die in We tern bl t for validation . There ults (Fig.5) further proved the
genom

imilarity

f the two

pecie . and the

trategie

vve used in thi

appropriat . MeHg treatment cau d m r ury accumulati n in FL and

tudy were

L. The average

concentration of total m rcury in OL (31.41Jg/g) wa higher than FL (26.71Jg/g) in the
MeHg treated marmo et (Yama1noto et al.. 2012).The e results agree with other studies
howing that OL were more usceptible to MeHg effects compared to other cortical
regions in Minamata disease patient

(Okabe and Takeuchi 1980) and in common

marmoset (Eto et al., 2001 ).

The global proteome of FL and OL in the cerebrum of the MeHg expo ed manno et wa
examined.

A total of 877 proteins were identified in both FL and OL regions. The

number of the identified proteins was similar to those reported by We seling et al
(Wesseli ng el a!., 20 13) using similar label-free LC-MS methods in rat frontal cortex .
Out of the 151 differentially expres ed protein identified in FL and OL, only 3 proteins
(DYNCI LI2, HISTI H2BH and PC) were common! af[i cted in both region (Fig.1).

Thi s indi cated that the effec ts of Me l fg on pro tein abundance change Vvere regionally

129

p

iii in the t

f c rebrum. hi ob rvation i further upported by the re ults

lob

of the h at map anal

1

that h wed hi gh d gre

f con i tenc

of protein e pre ion

ithin th tr atment and c ntr 1 group but a clear di fferen c in protein pattern betwe n
th t

gr up

protein in th e

and

). Ther were more differ ntially xpressed

L ( 6) than in the L (5 9).

he majorit pr tein were di tributed in the

and

111

cyt pia min both L and
location betw n th

( ig.2

L ( ig.2

and

her wa al

differenti ally e. pre ed protein

prot in in the t\\ region . T he e re ult
cellular organ ll e within th FL and

ur re ult

).

no difference in subcellul ar

and that

f the t tal id entifi ed

ugge t that Mel lg did not target parti cul ar sub -

L of ce r brum .

how that the effect of M J-I g in th e FL we re not very specifi c and were

medi ated primaril y through the di rupti on of c II di vi ion, decrease in key anti ox id ant
protein , and interference in the proce ses of energy generati on. These are refl ected by
the protein affected by MeHg in the FL are mainl y in vo lved in cell cyc le and cell
di vision, microtubul e-based process, cellul ar metaboli c proces in cludin g cellul ar amin o
acid metabolic process and sulfur compound metaboli c proce , proteolys is, and growth
development (Fig.3A). The e findin gs are similar to those reported in a previo us
proteomics stud y (Keyvanshokooh et a/.. 2009) that proteins in ce ll metabolism, cell
division and signal transducti on were affected in juvenil e belu ga brain with 0.8ppm
MeHg exposure for 70 day . Among the significant changed functions, su lfur compound
metaboli c process acco unted fo r the highest percentage (20o/o) of proteins compared to
other functions in FL (Fig3 .A). As MeHg is known to react particularl y with ulfhydryl
group (-S H) and

elenohydryl group to form MeHg amino ac id or protein complex

( arocci et a/. , 2014 ), the signifi cant change in protei ns invo lved in ulfur compound

130

m tab lie
m

hani tic
tr

idati
elen

nz m

M H g.

b
e p

(BPNTl ,

pr c

id en e [i r u h pr
b d

h

e

N,

P , TXNRDl ) pr

I o. the mi r tubul

ba ed proce

u h a T

wa

ided

the

ugge t th at Mel Ig can au e

re ul t al

RDl , a major antio idant

ll c cle and ce ll di i i n wa the large t ategor

f ce ll di vi i n in mit

ob

N

rea ing ulfl1 dr I prote in

ure ( abl e I ).

microtubul e

MTHFDl ,

f function affected

identifi ed

n itive to Mell g

mi cr tubul e i, imp rta nt c. to ke lento n tructure in manipula tion
i . M ll g \\a

like ly d i rupting ce ll di vi i n pr ce s thro ugh

d p lym eri zati o n (C lark on. 199 : Miura et al., 1999 ). M o reove r, we

rved functi onal enrichment in th e glyce r lipid metab li e process a oc iated w ith 4

di f£ r nti all y chang d pr te in

(A LY , BP NT 1, F S , PPPl

ACLY and FASN are key enzy me
parti ularl

B ) in the FL (Tab 1 ).

fo r lipogene i and chole terogenesis w hich are

important for the ge nerati on of acetyl- oA for ace tylcholine

ynth esis

(Beigneux e/ al. . 2004 ), and de novo fatty acid synthe is (Kn o bloch e/ a/. . 20 13 ). A
acetyl- oA is the core enzym e connectin g fatty acid m etabo li sm enter into Krebs cyc le
(Edmund s e/ a!. . 2014 ). M eH g can target the processes of energy generati o n and interrupt
nutrition support in FL of mannoset brain . In parall eled w ith the biological functi on
analy is. we al so identifi ed specific path ways in pyrin1idine m etabo li m and pyru vate
metaboli sm in FL (Tab.3 ), which act as the essential energy source in Kreb

cycl e

(Prasad e/ a/. , 201 1; Schroder e/ a!. . 200 5).

In comparison, th e effect of M eHg on the OL protein s were more x ten ive but ap peared
to target some key prote in s uch a apolipoprotein

(APOE ), G luta thi o ne Perox idase- ]

(GPXl ) and Aquaporin 4 (AQP4). MeH g wa o bse rved impa irin g the function in li pid
metabo li c process (A LDHSA l , APO E, CMAS, D LAT, GPX l , LAMTORI, LRPl,

131

OX Tl , P , PP 1,

E R) and

llular lipid metab li

MA , D AT, GP 1 L MTORl
tal.(

ndre ll et a/ .. 200 7 ) a l

nz m

in the

e p

nthe i

of neura l lipid . wa

impa irment

s ( LDHS 1,

PO ,

Tl, PPTl , TE R) ( ab2). Vendr ll

rep rt d that -ketoacid -

d to 60nM M eHg ~ r 10 da

In dditional t

RPl , 0

proc

enz m

tran ferase L a k y

d crea cd in cer be liar g ranule cell

.

f lipid meta b li m . encrg

gen rati o n wa di rupted in th e

a evide nced by functi n alterati n of carb hydrate deri vati ve m etabo li c process and
ing l - rgani m

arbohydrate m etabo li c proc

. M oreo er, the o b erved change

in

butanoate metabo li m (Tab.4 ), an important pathway und e r carbohydrate m etabo li sm
provide further evid ence that M eH g indirec tl y influ enced th e energy meta bo li sm . T h se
results agree w ith our previou prot omi c tudy (K ong et a/.. 20 13) bowin g th at 25o/o of
their protein

changed in the m eta boli c of carbo hydrates and energy generati on

associated m eta boli m in the om atosensory cortexes of rats dosed wi th M eHg.

Our results also confirm that APOE is a key protein in mercury neurotoxicity (Godfrey e /

a!. , 2003).

APOE was fo und to be the mo t intense interacti on hub in O L n etwork

(Fig.4B) w ith 3.7-fo]d

increase. A PO

is a famil y member of apolipoprotein

functioning as regulate lipid transport in the bod y (Hatters e/ a/. , 2006 ).

n epid mio logy

stud y showed a significant correlati on between APOE 4 genotyping and 465 pati ent
diagnosed with chronic mercury to icily (Woj cik el a/. . 2006 ). APO ~ and other a ltered
proteins in lipid assoc iated categorie

in OL indi cated that different pro tein s and

separated biochemical processes in the two region contribute to the en rgy uppl y deficit.
These type of prote ins and associated function s ugge ted that chan ge in energy derived

132

carb h drate and lipid metab li m are a c re featur of M Hg neur to icity in both F
and

L.

B id

carb h drat

and lipid metabolic pr c

e , Me l Ig al

ar~ ct

transport,

intracellular

ignal tran duction, prot oly i , multicellular organismal development,

cellular amin

a id and nucle tide metabolic proce

were r pot1ed previou 1; t
K

be related t

th

L.

PX1

1

11 of the e function

lutathi ne Per xidase-1 (G PXl ) was

[the total identified 19 functional term

a family member of elenoprotein

hydrogen pero ide .
We tern blot)

ut

L.

Mel Ig into ication (Allen et al., 2001 a;

an hoko h t al.. 2009: Kong et a!., 2013 ).

invol ed in 12 MeHg affected functi n

in the

111

which catalyze the reduction of

he down-regulation of GPXl (2.5-folds in both LC-M /MS and

ugge ted glutathion

depletion occurred in M Hg affected multiple

functions, and the decrea e in anti-oxidant capacity could also be one of th

of MeHg

toxicity in the OL.

We observed the brain edema occurred in MeHg treated marmoset (Yamamoto et al.,
2012). The identification of water transport related pathway in the vasopressin-regulated
water reabsorption in the FL (Tab.3) provides the biological pathway evidence to the
observed effects of the brain edema. Vasopression regulate water reabsorption by
secreting A VP (antidiuretic hormone vasopressin) through neurons projection to regulate
water reabsorption in kidney (Boone and Deen, 2008). This pathway is one of the main
ystems to maintain water homeostasis in our body. A total of 4 differentially expressed
prote ins (DYNClLil , DYNC1LI2, NSF, RABll A) can be mapp d in pathwa; of
Vasopre sion-regulated water reabsorption (Fig.6 ).

In contrast, we found a ignificant

133

alter d functi n in regulati n of b d

AQP4, A TPl 83, DGKB, G
( ab2).

In th

previ u

tudy,

al

rep rted t

be clo el

ith 9 protein

r p rted the
p

relat d t

ver-

d marm set

blot analy i (Tab.
te t . The re ult

QP4 \\a aL

olved (APOE,

pre ion of

L

QP4 and it

(Yamam t et aJ., 20 12).

the reablorption of

m r gulati n and r gulati n of brain edema (King and
ignificant up-regulation

111

M, PRK B, R BSA, WDRl ) in th

Y1A2, PI

r lati n hip with brain edema in MeHgP4 wa

fluid 1 el

erebr

pinal 0 uid ,

gre, 1996). In this stud y,

b erved in MS results, th ugh not in We tern

and Fig. ). and thi may due to technical variation between the two
f

P4 activati n indicat d that MeHg can increase permeability of

AQP4 channel to enhance brain wat r flu ,
with other 8 protein

resulting in brain edema. 1 aken together

identified in functi nal group of regulation of body f1 uid levels

itnplied a water transport dy hom o ta i occurred in the

L of Mel-Ig- treated mamoset.

These re ults offered the mechani tic explanations of observed edema in both FL and OL
of the cerebrum in the MeHg exposed manno et .

Conclusion

This is the first report of whole proteome alteration in primate cerebrum region

with

MeHg exposure. Occipital lobe was more susceptible to MeHg effects than the frontal
lobe. We demonstrated the importance of energy diminish (carbohydrate and lipid
metabolic processes), anti-oxidant depletion, and dysfunctions of water transport as a
conseq uence of MeHg exposure. The two brain regions showed imilar endpoint effect in
energy metabolism but acting through different proteins and biological pathways .
Disruptions of regulation of body fluid levels including pathway in vasopression-

134

r gulat d

ater reab

brain edema.

rpti n are th Imp rtant mechani m f MeHg cau ing marmoset

PO E wa the

re pr t in linking multi functi n in Meilg-treated occipita!

lob . Th id ntifi d bi I gical functi n and pathwa
f MeHg-indu

pr vide the po sible 1nechani m

d neur nal dcfici t in frontal! be and ccipi tal lob .

Acknowledgement

W appr ciat Fr d li ma for hi profc

nal advice and help in bioinformatics analysis .

135

hapter VII.

Ba ic life

c1en

r

ar h i

t

onclu ion

d t rmme the prec1 e

tep

f the compl

c scade and

p t ntiall target m le ul and bi logical pathv.ay involved in different functi n or di ea es.
The g al i to devel p n v l therapj t target these proc
under tanding
important

f the neur

e or promote health.

hemical and fun ti nal circuitry deficits 1

trategy particularly for neur degenerati e di ea e

eviden e h v.ing that envir nmcntal p llutant
can be linked with neur degenerative di
interaction

se and impr

an

( olde, 2009) . There are

uch a methylmercury (MeHg) potentially

a e . H wever, very I ittle i

known of the

f to i ant-di ea e uch a M I-Ig and PD . The entry and di tribution of MeHg in

organi m i a com pi , proc
of MeHg poi oning, it i

. In pite of progre

in under tanding of the initiating factor

till no cure so far to remove mercury/MeHg even in the early stage

of mercury poi oning (Farina et al., 2011 ). How to prevent MeHg-caused neuron dysfunction
as well as the sub equent cell death i the key point to treat or prevent the MeHg-cau ed
pathological progress. In this respect. a strategy which aims at systematic identifying
sensitive molecular targets of MeHg as well as the association with neurodeg nerative
disease posed a critical role in prevention and treatment of mercury poisoning and other type
of neurodegenerative conditions.

Key findings of thi

tudy

Thi s the is has two aims: o ne was to investi gate the assoc iation of MeHg toxicity with PD .
MPP+, a well -known neurotox icant that can induce PO like symptoms i used a the positive
contro l. T he second a im was to systemati call y tudy the protein profi le in respon e to Mel Ig,

136

a

ell a identifi ati n th
planati n fl r

Th

d ffl ct indu ed b M I lg.

P 1 manu cript pr

nted 1n

g ne /pr tein for d pamin

hapter III

nz m

r gulat d by th
content .

nz 'm

re p n ible ~ r D

e, p

he e re ~ ult

ure

h w d th

c mparative changes of key

induced b MeH g and MPP ~ using a dopaminergic

) c cl

n ur n cell model. The rat -limiting
DAT, and

in bi 1 gical fun lion and pathway to seek

ub equ nt chang

T I I [! r

nthesis

metaboli m M

, dopamine transporter

-B were

ignificantly down-

f both toxicant , re ulting in a decrea e in extracellular DA

ugge ted MeHg c uld affect many c mm n key gene /protein in

regulation dopamine dynamic

imilar t

MPP +. To further explore the underlying

mechani m of the effl ct of MeHg and MPP +, a gen mic and proteomic approach was used
to study their effects on key PD gene and on the total proteome u ing the same MN9D cell
model in the 2nd manu cript pre ented in

hapter IV . A total of 6 genes including TH in

dopaminergic transduction signaling pathway were found to be affected by both MeHg and
MPP+ treated MN9D cells. This result further confirmed our ob ervations that the two
toxicants can induce similar pathophysiological vulnerability to DA and dopaminergic
associated signaling. Proteins in energy metabolism including propanoate metaboli sm,
pyruvate metabolism and fatty acid metabolism, oxidative pho phorylation were altered with
treatment of MeHg and MPP+. The disruptions of these cellular functions in the dominergic
celJs can be the underlying mechanism of PD . In addition, we also found that MeHg
interrupted pathways involved in other neurodegenerative diseases including AD, ALS and
especially in HD. Resu lts from these two chapters clearly sho\\- that MeHg can b a potential
risk factor for PD by affecting imilar biochemical pathway and endpoint effect

uch a

137

en rg

d fi it that ar

affl t d b

MPP +. M re

er, MeHg rna

al

involve m other

n ur d g nerati e condition .
Th

yd

and 4 111 manu ript pre ented in

c mpr hen i

hapt r

and VI h wed for the first time the

prote me change 1n di f[i rent brain rcg1 n (cere bell urn , fr ntal lobe and

cipital l be) f marm . t e. po ed t McHg. Th re wa a clear regi nal difference in the
number and profil e
chang

in th

identified in th

f pr tc in

affec ted b; MeHg leadin g to th e differenti al functional

thre brain region . Ba ed on the number of differ ntiall y ex pre ed protein
r g10 n , w

h V\ ed th e r lative vuln erabl e sites in res ponse to Mell g

timuli in the following order: er bellum ( 102) >

ccipital lobe (89) > frontal lobe (62).

Moreo er, MeHg affect d protein as ociated with th e most di ver e cellular fun ction s and
number of biological pathway in th e cerebellum and occipital lobe. Th se results support
previous observati on that cerebellum and occipital lobe were the primary target of MeHg
(Eto, 1997).

alcium signalin g and ion transport espec iall y ynaptic transmi ssion were

identified as the main affected pathway and function in the cerebellum of MeHg expo ed
marmoset. Corresponding to the biological functi on alterati on , MeHg wa fo und mainl y to
target organelle di stributed in pl asma membranes e peciall y in synaptic membrane in
cerebellum . One of the most important findin g of our tud y is the identifi cati on of DLG4
and MIR-19A/MIR-19B as the key targets leading to multipl e effect of MeHg in the
cerebellum. In the frontal and occipital lobes of the cerebrum of MeHg treated marmo et
APOE was identifi ed as a key affected protein that serve as a hub linkin g mul ti-functio n .
Thi s direct cause-effect relationship provide e cell ent mechani tic upport for the previou ~
reported correlati on between the APO 4 genotype and pati ent with chroni c mercury toxicity
(Wojcik et al. , 2006), and furth er support the proposed usc of APOE a a potentia l clinical

138

icit

bi n1arker [! r n1 r ur

ith

and in

an u neur degenerati e di ea
tudi e

ith P , we al o id ntifi d interaction of
he pr teomic re ult of both the in vitr

sh wed tha t M H g indu ced
a

Parkin

n 's

di ea e,

111

pr teins ass ciated with
and

ugge ting that M H g can be a ri k fac tor in the deve lopment of patho gen

is of

di ea

uch

hange

di ea e

neur degen rati e
lzh imer '

et aL 200 ). In a ditional to the evidence

that MeH g i a ociated

upp rting ur h p th
M Hg

( odfr

Huntin gton 's

van u n urodegene rati ve co nditi n .

Th

ultimate g a l of mechani ti c

tud y i

t

id entify the molecul a r, bi ochemi cal or

ph

iol ogical change that can ex pl ain the fun cti o na l or behav ioral changes o b erved . In thi s

study, we demon trat d the foll owing mech ani m s: i ) Me l lg impaired energy ge neration in
m armoset brain by affecting key prote in invo l ed in carbohydrate deri ved metaboli m and
lipid metaboli sm in all three region of marm o et brain ; i i) ignificant alterati ons of syna ptic
tran mi ion, carboh ydrate derived m ta bolic process and neuro nal mo rpho logy in the
cerebellum may explain the motor dy function sign presented in MeH g-d o ed mam1 o et: iii )
M eH g caused brain edema by altering the key protein A QP4 as well a its associated water
tran sport pathway.

In sun1mary, our results suggest that M eH g can be a risk factor for multiple neurological
disorders (PD , HD , AD, ASL) . The present res ults (Chap Ill and IV) speci all y foc u ed on the
compari sion of biological pathway

and molec ul ar changes o f MeHg and MPP + in the

context of PD pathogenes i , whi ch not onl y provided the ev idence in the a sociat ion of
MeHg with PD but also gave the additiona l hints for other dopaminergic r lated di order .
such as I ID , schi zophreni a and dru g addiction . furth erm o re, the re ult of C hap ter V and VI
demon strated a compreh nsive overview of the mo lec ul ar change and targets across the
139

c r b llum,
target

L, and

L r gion m M Hg e p

uld p t ntiall

bee m

g

d

ph rmarc 1 gi al target :D r tr atment

d marm

chapter ,
tran mi

4 and

P

id entified mo lecul ar

andida tc :D r de el ping biomarker in diagn
f Me ll g int

m clini c per pecti v

or

icati n. Particularl y, the e tudi e are

1mp rtant in prov iding th ba i .G r the d ve l pm nt of pre
mi tigate the e ad ve r e effl ct

et. The

nti ve and treatment trategie to
e ampled candidates in previous

ar c r protein in linkin g and m aintaining fun cti ons of synaptic

n and lipid m eta bo li m in the phy ical conditi n. ignifi cant changes of these two

protein c uld be pr mi ing m ark er. or a the candid ate targe t in predi ction/ treatm ent for
M eHg p i 10nmg. M reover, the "omi c " appr ac h wa pr ved as a re li abl e and powerful
method to id enti fy k y targe t prot in . and provi ded a n ve l way to 1uc idate th e r gionalspecifi c bi ologica l fun cti on and pathway a oc iated w ith M eHg n urotox ic ity. T herefore,

L -M S/MS i apprope rate t chnique and t st me thodo logy to evalu ate th e effects of M e H g
exposure on hum an nervo us

y tern. Finally, results of thi s stud y provid e th e indirect

guid ance to policy deci sion -makin g in the deve lopment of regulati on fo r MeHg emi ss ions
and exposure advisori es to protect the health of sen itive popul ati ons. Hg is a glo ba l i ue
encountered in various countries including Canada. In term s of potential tox ic effects of Hg
in the ai r, food, w ild life and human health, there is still far too littl e to know the e act toxic
m echanisms of MeH g p arti c ular on hum ans, the top chefs in the food chain. T he present data
provides the dose effects of M eH g that co ul d be reffe red for safety evalua tio n, and for further
fac ilitate o ur understandi ng of the pathogenesi changes of MeHg toxicity from laboratory
anim als to peopl e. Partic ulary, the primate m ode l used in th is study provided the more direct
proofs fo r predi cti ons of I lg on human hea lath effec ts. OveralL this study contribute to a
better understandin g of magnitud e in the uncertain mecha ni tic questions of MeH g
ne urotox ic ity in humans.

140

ontribution to knowledge and limitation
ntribution :

•

T

the c n id erati n of M eHg a

neur 1 g i al di
e p
•

rd er . and identi fy ing on the p

ri k fac tor for PD and oth er
ible linkag

between M eHg

ure and n ur deg nerati v di ea e .

To the ide ntifi ati n

f the electi ve effec t of M eH g on pl asm a m embran pr t m s,

particul arl y n prot in
•

a co ntribut r

f ; napti membrane in cerebe llum.

To pro id e furth er in ight into the deve lopment of k y prote ins as bi om arker for
predicti on and treatm ent fo r M eH g int x icati on . T he bi om arker co uld be useful for
future hum an ri k a e m ent and oth er epid e mi o logy studi es.

•

To promote the evidenc e- bas d p li cy makin g in translati on to nv ir nm enta l-heath
managem ent and po licy proce

into protecti on of human from env ironmental and

man-mad e expo sure of M eH g.

Limitations:

o

The question po ed in the Chapter III and IV is wheth er M eH g expo ure and PD
share similar etiopathology . In the normal bi olog ical co nditi on. co mpensatory
processes occur first before damage manifestati ons ( lark on et aL 2007).

hort te rm

and high concentration of M eHg ex pos ure can produ ce acute toxic effect wherea
long term low do se e posure may induce co m pen a to ry respon e
different net tox ic effects.

\\ hich ca use

In thi s tud y, the ce ll culture dos ing ex periments we re

de igned to have a do in g period of 24h -72h and the marmoset ex perim nt \\ a a lso
de igned to stud y the

hort te rm acute effects. Th ere fore, our result , m a) not
141

n ce aril

b refl ecti e

f the 1 ng term 1

do e ituation e perienced by the fi sh

n urn r .
o

Ther are limita ti n in th u e fbi informati c t identify pathway a
chang d pr t in . W e id entifi ed the indi idua l path wa
till la k

f v iden e and kn ovv l dge t

ci ated with

in each of bra in regi n , but

und er tand the ca cade ef-[1 ct /pa ttern and

r lati n hip betwee n all th affec ted pa th wa

. urth r studi e are n eded to confirm

the do e-re p n e effec t vvith a mo re e tab!i hed m e thod .
o

Th

predict d fun cti on and path way

multipl e do e . longe r term anima l d

need to be furth er ve rifi ed by additi onal

ing e pe riment .

Future research

1) The

creemng re ults of th

pre ent proteomi c wo rk prov id e the important bi o logical

pathway and proteins in linking M eHg-affected multi -functi o ns. T he prec ise fun cti ons of
key proteins and regul ated factor id entifi ed including DLG 4 and MIR - 19A/B in cerebe llum
and APO E in occipital lo be w ill need to be further clari fie d .

2) This study identified the connection of M eH g-neurodegenerati ve di sease

thro ugh

pathway enrichment analys is. The detailed underl ying mechani sm s of interacti on of M eH g
w ith these ne urodegenerati ve co nditio ns particul arl y in HD , AD and ALS need to be further
investi gated .

142

Publication and

cademic

ctivitie during PhD.

tud y

In Journal
hao Y, Yam am t M,
mmon Marm

hao Y ,

c 11 .

igcy
ing Z. han I IM. Pr te mic naly i of ere bellum in
et -- xpo ed t Methylmercury . To ic I ci ( ford). 2015, 146(1 ): 43-51

han HM . g ct
[ methylmercur on dopamine relea e in MN9D neuronal
ic I M h M th d ..... 0 15 25( ): 7-44

ing Z. Ryan M . han l-IM . -:ffects of Methylmercury on M 9D
hao Y. 1ge
d paminergic n ur n ell : a genomic and pr teomic ana lysi . Manu script accepted by
c1en . ept. 2015. ublicati n in pr ccs
Journal fT

Shao Y . Yamamoto M. 1gey
ing Z. han HM . Proteome pr filing reveal s regional
protein alteration in cerebrum f c mm n marm et ( Callithrix jucchus) exposed to
methylm rcury . Manu cript ubmitted to rchiv ofT xicology
Shao Y and han HM . Mini-review: Mechani sm
update. Manu cript in preparation

of Methylmercury neurotoxicity

an

Academic A ctivities

'r

Workshop in Pathway and Network Analy is of -omic Data Toronto. June 1-3. 2015

r

Proteomic Analysi of erebellum of Common Marmoset Treated with Methylmercury.
Poster presentation in SOT's 54th Annual Meeting. San Diego. U A. March 24, 2015

r

Effects of Methylmercury on MN9D Dopaminergic Neuron ells: A Proteomic and
Genomic Analysis. Po ter presentation in Society of Toxicology of anada ( TC). 46th
Annual Symposium. Ottawa. Dec 12.2014.

r

Methylmercury Induced Regional-specific Protein Change in the Marmoset Monk ;
Brain. Poster pre entation in the Global Biotechnology Congress. Bo ton. U
. Jun 18.
2014 .

.r

Effects of Methylmercury on Dopamine Relea e in MN9D Neuronal Cell . Po ter
presentation in the 1Oth International onference on Mercury a Global Pollutant
(I M P). H a lifax , anada. Ju ly 20, 2011.

143

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161

Appendix 1: E tabli hing the
Primary

erebellar Granule Neuron and A trocyte

ulture Model for Methylmercury Toxicity

tudy

Background

r wth factor

(F ' ,

F and B

F) have be n

hoVvn t

alter tatu

n gap junction

intra ellular ( J ) communi ation betw en a trocytcs and neur n. There i little rc earch to
in e tigat the role

J

f

functi n on mercury neuron to icity.

erebel lum is known as a

target regi n for mercury to , icit . The objective of this study is to develop primary
erebellar

trocyte (

) and ' erebellar

m del for furth r tudy of th
Junction Channel
Until now, w
Cerebellar

ranule Neurons (

groVv1.h factor F F2 ( Fi br blast

N) -mono and co-cultures
rowth Factor 2) on

ap

onne in4.., ( x4 ) communication in respond to Methylmercury (McH g) .

have

ucce fully e tabli hed th

ranule Neuron

co-culture

(Fig.4)

model

f mou e

r bellar Astrocytes,

and mono culture (Fig.5 ), which were

e timated by ingle IF (immunofluorescence) and double IF staining a say for MAP-2 (for
neurons) and GF P proteins (for astrocytes).

erebellar A

and

GN mono and co-cultures

were expo ed to MeHg (0-20!-lM) for 24h or 48h. The results of the cell viability assay
demonstrate that MeHg induced a time and dose dependent decrease of the cerebellar A
GGN in both mono and co-cultures (Fig.6 ). The mono-cultures are more sensitiv
lower doses of MeHg than the co-culture. These results indicate that cer be liar A

and

to the

and CGN

cu ltures are appropriate cell models to study MeHg toxicity. The primary cell viability a ay
provides the range of concentration to be used to further study the effects of FGF -2 on the
regulation of Cx43 in the cerebell ar AS and CGN co-culture.
T he protocol of c -culture AS and CGN modeL results of IF staining and cell viability a sa;
in AS and

GN cultures are present as fo llows. The animal experimental de ign and

m ethodo logy were app roved by th e U ni ve rs ity of Ottawa Animal Care and U e committee
(J une, 20 12).

162

Protocol of Co-culture Astrocytes and Ce rebella Granule Neuron

Astrocytes preparation was based on Marek eta!. paper
(Marek et al., 2008) with modification. Cerebellar Granule
Neuron (CON) culture protocol was referred from Hae Young
Lee etal. video article (Lee et al., 2009). Below is the
procedure of preparation co-culture of CGN and AS.
Primary astrocyte cultures were collected from the cerebella
of post-natal day 4-6 mice.
1)
Remove meninges and mince cerebellar ti ssues,
incubate in rocking water bath/ or MACSmix™ Tube Rotator
at 3 7°C for 30 minutes in 1OmL HBBS + 300jlg/mL Dnase I
and 0.25% trypsin (I0011L)

6)
Add 1OuL of Anti-ACSA-2 Micro Bead per 10011L
buffer. incubate for 30min at 4°C with gentle mixing every
5min
7)
Wash once with separation buffer: re-suspend m
500j1L buffer I 1X 108 cells
8)
Prepare MS columns by adding 5mL of buffer, wash
with buffer 3mL x 3

""

Post-natal day 4-6
Balb-c m1ce

2)
Triturate digested cells with 0.25% FBS, wash and
spin (300 x g for 1Omin)

Cerebellum dissection
and trypsm
diSSOCiatiOn

Smgle cell
suspension

M1crobead
(Ant1-Giast)

3)
Re-suspend pellet in HBBS and pass through 70~tm
nylon mesh screen. wash and spin

~

Astrocyte

4)
Re-suspend dislodged cells in 1OmL of MACS
separation buffer. pass suspension through a 40!-lm sc reener
into a 15mL tube

Purified cerebellar
astrocytes

5)
Spin (300 x g for 5min at 4°C). wash with buffer and
re-suspend at 1x 10 7 cells / 1OOuL buffer

Figure 1. Diagram of primary cerebellar astrocytes isolation
and purification

Elut1on on MS
magnet1c column .

Ant1-GLAST
Microbead Kit

163

9)
Place column in fresh 15ml tube and add cells, use
plunger to flush with 5mL buffer
Spin 15ml tube and dilute astrocytes enriched
10)
suspension with MEM medium to obtain the desired density
(l x l0 5/w in a 12-well plate. ~ 100,000 cells/mL, depending on
the time required to reach confluence)
Place AS enriched cultures into C02 incubator for 2
11)
weeks. medium was changed twice per week

16)
If culture remained beyond 7 days, add 500 ~1
SFM/MEM+ once per week of incubation thereafter to
compen sate for evaporation, resume incubation.

Cerebellum
d1ssect1on

Post-natal day 4-6
Balb-c mice

12)
When glial cells reached confluence and form a
uniform monolayer on the cover slips, add FUDR so lution
(ex.12.5 1-11 FUDR per petri dish)
2-weeks old purified AS ( +) were pre-seeded 1-2 days before
CGN and progenitor cells collection.

13)
After collection of CGN, count cell density with a
hemacytometer and trypan blue and add SFM to reach the
desired cell density (100,000 to 350,000 cells/mL). For 12
well plates. plate 2.5-3.5 x l 0 5 cells/well
14)
For cerebellar CGN and AS co-culture, seed CGN (5 6 folds density as AS) on top of pre-plated monolayer of
astrocytes (2 weeks old) with SFM/MEM+medium.
Add cytosine arabinoside to a final concentration of
15)
51-!M in SFM/MEM+ medium to reduce the proliferation of
glial cells

•

~

J;><

Papam Dissociation
System .

b-1

Enri ched
cere bellar
neurons.

Neurons collected
m culture
supernatant

Suspension plated on
POL coated plate .

Figure2. Diagram of cerebellar granule neuron and progenitor
cells collection

2-3 days
1n-v1tro

Plate
cerebellar
astrocytes

7 days
m-v1tro

Plate CGN on
astrocyte monolayer
(80% confluency)

Co-culture.

Figure3. Diagram of AS and CGN co-culture plating in a ratio
of l :6 for 7 days following MeHg exposure

164

Li t of chemical and reagent :

HB

,n

alcium, n Magne ium, n Phenol Red (In itrogen, 14175-095)

Minimum

ential Medium (M M) (In itr gen. 110 50 0)

ur ba al

-Medium (lnvitr gen. 12 49-015)

Penicillin- tr pt mycin/ P

luti n (lnvitr gen. 15140-122)

B-27 erum re

upplement (50. ) (lnvitr gen. 17504-044)

Tr pin- ~ DT

Jution (Invitrogen. R-001-100)

H at-Inactivated F

(Invitrogen, 1~4 4028)

lutaM X (lnvitrog n,

050061)

MA

mi TM Tube Rotator (Miltenyi Biot , 130-090-753)

MA

B A tock olution (Miltenyi Bi tee. 130-091 -376)

Anti-AC

-2 MicroBead, Mou e (Milten i Biotec, 130-097-6 78)

orning 15m1 graduated plastic tube PET (Fisher Scientific, 05-538-51 A)
Cell Strainer (Fi her cientific. 40~-tm 22-363-547: 70~-tm 22-363-548)
Characterized FBS SOOml (Fisher Scientific, H3039603Hl)
lOOmM sodium pyruvate solution (Sigma-Aldrich, 8636-1 OOml)
Cytosine ~-D-arabinofuranoside (Sigma-Aldrich, C 1768-1 OOmg)
45o/o (w/v) D-glucose (Sigma-Aldrich, G8769-l OOml)
MITO + Serum extender ( orning, 355006)

165

Prepare culture media and

lution:

erum-free medium ( FM) and 10% FB medium
4 ml of

ur ba al

500 !J.l 1OO x

-Medium

lutaM

500 !J.l 1OO x P ni illin- tr pt myc m
6.25 !J.l 2 M K 1 (final ... 50 !J.M).
plit in 2 aliqu t f9 ml and 4 ml. T pr pare 10°/o FB medium, add lml fh eatinacti ated
to the 9 ml aliqu t. T prepare FM, add 800 !J.I of th e erum -free
uppl m nt -27 t the 40 ml aliqu t. Filt r teriliz d and tore at 4° for a rna imum of 2
w ek .
MEM+ (MEM/1 0% FBS medium 1OOml) * an be t red at 37°( ~ r 1week
87 .6ml
nditi ned M ~ M ( vlinimal e enti a! medi a with Earl salt and without Lglutamine) + lml P (100 )
1. 33 ml

45 o/o (w/v) D-gluco e

lml

1OOmM sodium pyruvate oluti n
MITO (5ml H2

in 1 vial of MIT + erum E tender)

Filtered using a 0.22 !J.M nylon mesh bottle filt er
lOml

FB

SFM + MEM( +) solution
66ml

SFM

33 ml

conditioned MEM+

FUDR solution *Prepare lml aliquots and store up to 1 year at -20°(
203 m! MEM (with Earl e's salts, without L-glutamine and phenol red)
1OOmg 5-flu oro-2' -deoxy uridine
198mg uridine
Filter using a 0.2!J.M nylon mes h bottl e filter
Cytosine arabinoside * tore up to 6 month s at - 20oC.
To mak e 2mM
water.

ytosine arabinos ide, di sso lve 2mg c tosi nc-l-f3-D-arabino-furano ide in 5ml

166

Figure4. Immunofl uorescence of Cerebellum Astrocytes (AS) and Cerebellar
ranule
Neurons (CGN) co-culture for 10 day . Astrocytes are tained with GFAP, repr ented in
green (A), and neurons are stained with MAP-2, repre ented in red (B). Nucleus stained with
DAPI represented in blue (C). D is a merged image of AS and CGN in co-culture, E i a
phase contract image of AS+ GN co-culture (A-E, object ive lens 40 x), and F is AS and
GN co-culture (objective lens 1Ox).

167

A

B

c

D

FigureS. Immunofluorescence of mono cerebellar a trocytes and mono cerebellar granule
neurons of postnatal day 4-6 mice. Immunocytochemi try again t MAP-2 with DAPI (A) and
without DAPI (B) staining in

GN enriched cultures (1 Ox). Merged

FAP and D Pl

staining (C), and GF AP ingle taining (D) in i olated cerebellum astrocytes ( 1Ox). Boxe
indicated the expression of mono CGN and a trocytes under 40 x obj ctive len .

168

A

-

CGN 24h

Co-culture 24h

A 24h

140
120
100
0

80

!:

c
0

60

~

40

u

20
0
-20
M e H g( ~M )

B

--- CGN 48h

Co-eulture 48h

A

48h

120
100
80

-...
0

c

60

0

u

~

40
20
0
-20
MeHg ( ~M )

Figure6. Ce ll viability of cerebellar granule neurons and astroc ytes in mono and co-cu lture
fo ll owing a 24h exposure (A) and 48h expos ure (B) to various concentrations of MeH g.

169

Appendi II:

upplementary data

hapter V:

Table

1.

25 6 id ntified pr tein

Tabl

2. Li t [ 102 di f~ renti all y e pre ed protei n in MeHg-treated manno t cer bellum

Gen e ymb ol

Protein Na m e

14o rfl 59
y

I

RY B
PP IA
K NJI O
PH GDH
- IF2 2
081
LUDl
FBX02
TUBB
GNAQ
CYF lP2
CKB
VCP
TU BAl A
CCT3
ATP2A2
RAB J A
SPTAN1
PSMC 3
ATP6VlH
DNM I
SNAP25
RABIIA
NTNAPI
IMMT
HBB

T rea t/ ontrol (log2)
I. 75

lycoge nin - 1
lph a-c ry tall in
hain
Peptid) 1-prol) I ci -tran i omera e
P- en iti ve in v.a rd rectifi er p ta ium chann el I0
D-3-ph o ph oglyce rate dehyd r ge na e
Eukar oti c translat i n initi ati on fac tor 2 ub unit 2
CD81 anti ge n
Glutamate deh drogena e
F- bo onl y protein 2
Tubulin beta chain
Guanin e nu cleotide- binding protein G( q) ubunit
alph a
Cytopl a mi c FMR !-interactin g protein 2
Creatine kinase 8 -ty pe
Tran iti onal end opl as mi c reti culum ATPa e
Tubulin alph a- ! A chain
T-compl ex protein 1 subunit ga mma
arco pl as mi c/endopla mi c reti culum ca lcium
ATPase 2
Ras- re lated protein Rab-3 A
Spectrin alpha chain , non-e rythrocyti c I
26S protease regulatory ubunit 6A
V-type protonATPa e ubunitH
Dynamin - 1
Synaptosomal-a soc iated protein 25
Ra -related protein Rab- 1 I A;Ra -related protein
Rab- 11 B
ontactin-as oc iated protein I
Mitochondrial inn r membrane protein
Hemog lobin ubunit betaJ lemoglobin subun it delta

1.27
1.22
1. 10
1.09
0.86
0.84
0.75
0.7 0
0.60
0.55
0.49
0.45
0.42
0.3 1
0.2 1
-0.30
-0.3 I
-0.3 6
-0.42
-0.43
-0.45
-0.47
-0.47
-0.50
-0.54
-0.55
-0.60
170

VP 29

H
p
yp

PB
R 4
PIP4 K2B
XR 5
TP2BI
EPT9
R
M
M PR 3
R VBLI
AMPH
AT P6V ID
R YBL2
GBA
YTI

TLN2
NONO
CA MK2A
RPL26
SLC6A I
SGTA
HOM ER3
AN K2
IF3A
HUW I
LTB
ATP28 2
B N
SL 17A7
SU B I
/\ P I
MRPS3 6

acu Jar protein rtin g-a ciated prot in 29
Beta-addu in
erin e/thre nin e- pr t in kin a e P K I
p trin beta chain , non- th rocyt ic 2
5-o. pr I in a e
ual pec ificity pr tein pho. ph ata e
ynaptophy in
B ta- olubl
, F attac hm ent pro tei n
Protein DR 4
Ph o ph atid y I in ito! 5- ph o ph ate 4-kin a c ty pe-2
b ta
-ra; repatr cro -c mplementin g pro tein 5
Pi a ma m mbrane ca lcium -tran port in g A TPa c I
eptin -9
ur nal c II ad h ion molec ul e
Mi crotu bul e-a ocia ted pro tein RP/[: 8 fa mil y
me mbe r 3
RmB -lik I
Amphiph y in
V-type proton AT Pa ·e ubunit D
Ru vB-like 2
Protein ip nap homolog 2
ynaptotagmin - I
Tal in -2
No n-POU domain -containin g octamer-b in ding
protein
Cal cium/ca lm odulin -depend ent protei n kinase ty pe
II subunit alpha
60S ribo omal protei n L26
Sodium- and chl orid e-dependent GA BA transporter

-0.6
-0.6
-0.68
-0.7 1
-0.74
-0.75
-0.76
-0.7 8
-0.7 8

I

-0.94

Small glutamin e-ri ch tetratr ico peptide repeatcontainin g protein alph a
Homer protein homolog 3
Ankyrin-2
Eukaryo ti c translati on initiati on fac tor 3 ubun it A
E3 ubiquitin -pro tein l iga HUWE I
Clathrin li ght chain B
Pl as ma membrane ca lcium -tra nsporti ng ATPa e 2
Protein bassoo n
Ves icular glutamate transporter I
Acti va ted RN A polymerase II transcri ptio nal
coacti va tor p 15
Adenylyl cyc la e-a oc iated pro tei n I
28S riboso mal protein SJ6, mitoc hondria l

-0.7 8
-0.79
-0.8 I
-0.82
-0. 84
-0. 84
-0. 87
-0. 87
-0. 88
-0. 88
-0. 88
-0.89
-0.90
-0.91
-0.91
-0.92

-0.97
-0.98
-0 .99
-1.00
-1 .05
-1.06
-I .07
-1.09
-1 .09
-I .I 0
-I . I 4

-I . 14

171

85
PrLZ
Kl

p 4
A

P K2
PITRM 1
P2
VRB

L ll
OL 4
RF5
G K3
LGA L L
RCI 1
N2 A
MBL
AMKK2
HNRPOL
Ll CAM
SNRNP70
LRPl
SLC8Al
CS PG 3 variant
protein
C2C 0 5
RBM1 4
LSAMP
SHANKl

JTPKA
GMP S
OMXL2
MAPRE2
PL 81

uan1n nu I tid -binding pr t 111 ubunit b ta-5
Tum r pr tein 0 52
nk rin- 1
acuolar pr tein rtin g-a oc iat d protein 4
uanine nucl otide- binding protein ( ) ubunit
a lph a i o:D rm hort
[P) rll\ ate d h) dr gena e [lipoamid e] ] kin a e
i 7 me 2. mitochondri a I
Pre equen protea e. mitoc hondri al
Mitofu in -2
LIT-R
Rh o - Pase-ac ti atin g protein 2
Fl a\ in redu cta
OPH )
dium/ca lcium e;\.c hange r 2
EL
-I i" prot in I
eu ine-ri ch gli oma- in ac ti\ ated protein
Oi k large homo I g 4
OP-rib ylati on fac tor 5
lycoge n ynth a e "in a e-3 alph a
alectin -relat d protein
R kin a e ignalin g inhi bitor I
odium chann el protein t) pe 2 ubunit alpha
Carboxymethylenebut no li da e homolog
alcium /ca lmodulin -dependent protein kin a e ki na e

- 1.1 7
- 1.18
- 1. 18
- 1.19

2

-1 .74
-1.74
- 1.74
- 1.93

Heteroge neo u nuclea r ri bonucl oprotein 0- like
eural ce ll adhe ion molecul e L I
Ul small nu clea r ribonu cleo prote in 70 kDa
Lo\\ -density lipoprotein rece ptor-related prote in 1
5 15 kOa ubunit
Sodium/ca lcium exc hanger I
Neurocan core protein
C2 domain-containin g protein 5
RNA -binding protein 14
Limbi c system-assoc iated membrane protein
H3 and multipl e ankyrin repeat domai n protein I
In os itol-tri sphos phate 3- ki nase A
GMP synthase [glutamine- hydro lyzing]
Om X- 1ike protein 2
Mi crotubu le-as oc iated protein RP/EB fami I)
member 2
1-pho ph atidylin o itol 4.5-b i pho ph ate
ph o ph odi e tera e beta- I

- 1. 19
- 1.25
- 1.26
- 1.29
- I .3 9

- 1.40
- 1.42
- 1.42
- 1.4 8
- 1.49
- 1.49
- 1.50
-I .5 I

-1.59
- 1.60
- 1.70

-1 .93
-1 .95
-2 .0 I
-2.05
-2 .06
-2 .07
-2 .31
-2.43
-2.46
-2.57
-3. 14
-3.33

172

TableS3. Enriched GO Biological Process term based sets of differentially expressed proteins in MeHg-treated marmoset cerebellum

GOlD
GO:OOOI50 8
G0:0001558
GO:OOOI701

GO Term
regulatio n of acti on
potential
regulation of cell
growth
in utero embryonic
development

Nr.
Genes

o;o
Associated
Genes

Term P-value Corr.
Bonferroni

Associated Genes Found

5

21.7

2.75E-03

ANK2, CNTNAPI, GNAQ. KCNJ 10, SCN2A

5

15.2

1.35E-IO

CRY AB, GSKJA, LGII. NRCAM, RAB II A

5

17.9

4.07E-15

EIF2S2, LICAM. MFN2. PSMCJ, SLC8AI

G0:0006184

GTP catabolic process

10

21.3

9.33E-09

G0:0006811

ion transport

21

15.8

I. 71 E-1 04

G0:0006816

calcium ion transport
cellular calcium ion
homeostasis

5

15.2

2.53 E-08

ARF5. DLG4. DNM I. GNAQ. GNAS. GNB5. PLCB I,
RAB II A, RABJA. SRGAP2
ANKI. ANK2, ATP2A2, ATP2BL ATP282, ATP6VIO,
ATP6V I H. CAMK2A. DLG4, HBB. KCNJ 10, RABJA.
SCN2A, SHANK I. SLCI7A7. SLC6AI, SLC8AI,
LC8A2 . SNAP25. SYTL VPS4A
ANK2. ATP2A2. ATP2B2. CAMK2A, SLC8A I

6

17.1

6.77E-09

ANK2, ATP2A2. ATP2B2. DLG4. IMMT, SLC8A I

G0:0006874
G0:0006887

exocytosis

12

21.8

3.38E-05

G0:0007268

syna ptic tran smission

20

15.5

3.93E-34

G0:0007409

axonogenes1s

15

16.0

8.46E-19

G0:0007411

axon guidance

13

20.0

1.76E-08

G0:0007611

learning or memory

6

19.4

6.49E-03

G0:0009117

nucleotide metabolic
process

''

17.2

4.52E-94

L. ' -

ANKL CAPI, DLG-t NAPB. PAKI, PLCBI. PPIA.
RABJA. SNAP25. SRCIN I, SUB I. SYT I
AMPH . ATP2B2. BSN. CAMK2A. C NTNAPI , DLG_.,
ITPKA , KCNJIO , LGII , NAPB, NCAN, PLCBJ ,
RABIIA , RABJA , SHANKt , SLCI7A7, SLC6AI ,
SNAP25, SYP. SYTJ
ANKL ANK2. CAPL CNTNAPI, DLG4. LICAM. LGII,
NCAN, NRCAM. PAKI. RABIIA. RABJA. SPTANI.
SPTBN2. SRGAP2
ANKI. ANK2. CAPI. CNTNAPI. DLG4, LICAM. LGII,
NCAN, NRCAM. PAKI, SPTANI. SPTBN2. SRGAP2
AMPH. DLG4. PLCBI. SHANKI. SLC17A7. SLC6AI
ARF5. ATP2B2, ATP6V I H, CAP I. DLG4, DNM I, GBAS,
GLUD I, GMPS. GNAQ. GNAS. GNB5. GSKJA, PHGDH,
PLCB I. PSMCJ. RAB II A. RABJA, RUVBL2, SRGAP2.
VCP, VPS4A

173

G0 :0009 150

pur ine ribo nucleotide
metabo lic process

20

19 .4

1.93£-31

ARFS, ATP2S2, A TP6V I H, CA P l, DLG4, DNM I, GS AS,
GMPS. GNAQ. GNAS, GN S S, GS K3A, PLCS I, PSMC3,
RAS I I A, RAS 3A, RUV S L2, SRGA P2, VCP, VPS4A

G0 :0009 152

purine ribonucleotide
bi osynthetic process

5

15.2

3.7 8£-05

CA P !, GS AS, GM PS, GNAS. GS K3A

G0 :0009 154

purine ribo nucleotide
catabo lic proce ss

15

20 .5

2.02£-11

G0 :0009 166

nucleotide catabolic
process

16

20 .3

1.41 E-15

G0:0009205

purine ribo nucleos ide
triph osphate metabo lic
process

16

18 .8

2.94£-12

G0:0009790

e mb ryo deve lo pme nt

10

18.9

9 .62£-120

G0:0015711

o rganic a ni on tran spo rt

7

21.9

8.43£-11

G0:0016192

vesic le-medi ated
transport

23

15.1

1.29E-1 00

G0:0016570

histone modificat ion

5

29.4

2 .7 1E-1 0

G0:0019226

transmission of ne rve
impulse

22

15.9

9.30£-56

G0:0019637

organophos phate
metabolic process

25

15.9

2.74E-99

G0:0023061

signal release

9

15.5

3.05£-09

G0:0030097

hemopoiesis
regulation of cel lular
catabolic process
secretion by ce ll

6

15. 8

1.35£-35

8

17.0

6.21 E-26

17

16.5

1.17£-54

G0:0031329
G0:0032940

ARFS, ATP6V I H. DLG4. DN M I, GNAQ, GNAS, GN BS,
PLCS I, PSMC3. RAS II A RAS3A, RUV S L2, SRGA P2,
VCP. VPS4A
ARFS, ATP6V I H. DLG4. DNM I, GNAQ, GNAS, GN BS,
GS K3A, PLCS I, PSMC3, RAS II A, RAS3A, RUV BL2,
SRGAP2. VCP. VPS4A
ARFS, ATP6V I H. DLG4, DNM I, GSAS, GNAQ, GNAS,
GNSS. PLCB I, PSMC3, RAS II A, RAB3A, RUVBL2,
SRGAP2, VCP, VPS4A
ATP282, EJF2S2, GNAQ, GNAS, LICAM, MFN2,
PHGDH. PLCB I. PSMC3, SLC8A I
HBS. KCNJIO. RAS3A, SLCI7A7. SLC6Al, SNAP25,
SYTI
AMPH, ANK I, ARFS, ATP6V I H, CAP I, CL TB, CYFIP2,
DLG4. DNM L LRP I, NAPS, PAK I, PLCB I, PPIA,
RASIIA. RAB3A. SNAP25. SPTSN2. SRCINI, SUB I,
SYP. SYT I, VPS4A
HUWE I, PAK I, RSM 14, RUVSL I. RUVBL2
AMPH. ATP2S2, BSN. CAMK2A, CNTNAPI, DLG4,
ITPKA, KCNJ 10, LGII, NAPS, NCAN, N RCAM. PLCB I,
RASIIA RAB3A. SCN2A. SHANKl, SLCI7A .,
SLC6A I. SNAP25, SYP, SYTI
ARFS. ATP2S2, ATP6VIH, CAP!, CD81. DLG4, DNMJ,
GSAS, GLUD I. GMPS, GNAQ. GNAS, GNBS, GSK3A
lTPKA, PHGOH, PIP4K28, PLCB I, PSMC3, RA B IJA,
RAS3A. RUVBL2. SRGAP2, YCP, VPS4A
CA MK2A. GLUDI, GNAS, NAPS. RAB3A. SLC17A.,
SLC6AL SNAP25, SYTI
ADD2, ANKJ, GNAS, LICAM, TPD52, XRCCS
DLG4. GNAQ, GNAS, GSK3A, PLCB I, RBMI4,
SRGAP2. VCP
AN K I, CAMK2A, CAP!. DLG4, GLUDI, GNAS. NAPB,

174

PAKI, PLCBI, PPIA, RAB3A, SLCI7A7, SLC6AI.
SNAP25, SRCIN I, SUB I, SYTl

G0:0032970

regulation of actin
filament-based process

7

17.9

1.98E-05

G0:0035637

multicellular
organismal signaling

24

16.7

5.39E-68

G0:0040008

regulation of growth

8

17.4

2.08E-29

G0:0042060

wound healing

12

15.4

5.67E-39

9

19 .6

1.21 E-08

7

20.0

3 . 13E-35

5

27.8

1.02E-02

G0 :0042391
G0:0043009
G0 :0043547
G0:0044057
G0:0044708
G0:0045936

regulation of membrane
potential
chordate embryonic
development
positive regulation of
GTPase activity
regulation of system
process
s ingle-organism
behavior
negative regulation of
phosphate metabolic
process

ADD2. ANK2, LRPI, PAKI, SHANK!, SPTANI, SPTBN2
AMPH. ANK2. ATP2B2, BSN. CAMK2A, CNTNAPI,
DLG4, ITPKA, KCNJ I 0, LGI I, NAPB, NCAN, NRCAM,
PLCBI, RABIIA, RAB3A , SCN2A, SHANK!, SLC17A .,
SLC6A I, SLC8A I, SNAP25, SYP, SYT I
CRYAB. GSK3A. LGII, NRCAM, PLCBI, RABIIA,
RUVBLI, RUVBL2
ATP2A2. ATP2B I, ATP2B2, CAP I, GNAQ, GNAS, HBB,
LICAM , MFN2. PAKI. PPIA, SLC8A2
ANK2, ATP2A2 . CNTNAPI, DLG4 , GNAQ, KCNJIO ,
SCN2A, SHANK!, SLCI7A
EIF2S2, GNAS, LICAM, MFN2 , PHGDH, PSMC3 ,
SLC8AI
DLG4. GNAQ. GNAS, PLCB I, SRGAP2

15

19.7

1.42E-16

8

16 .3

6 .08E-I 0

ANK2, ATP2A2. ATP2B2, CAMK2A, DLG4. GSK3A,
ITPKA, KCNJIO, LGII, NAPB, RABIIA, RAB3A .
SLC6A I. SLC8A I. SYP
AMPH. DLG4. KCNJ I0, PLCB I, SHANK I, SLC 17 A
SLC6A I, SPTBN2

5

15 .6

1.69E-12

DUSPJ. GNAQ. GSKJA, LRP l, SRCIN I

G0:0046128

purine ribonucleoside
metabolic process

17

18 .3

6 .3 0E-18

G0:0046130

purine ribonucleos ide
catabolic process

15

20.5

1.16E-IO

G0:0046903

sec reti o n

19

17.1

6 .39E-99

G0:0048468

cell devel opment

28

15.2

1. 12E-102

ARFS. ATP6V l H. DLG4. DNM l. GBAS, GMPS, GNAQ,
GNAS, GNBS, PLCB I. PSMC3. RAB II A, RAB3A,
RUVBL2, SRGAP2, VCP , VPS4A
ARFS. ATP6VIH. DLG4. DNMI. GNAQ, GNAS, GNBS,
PLCB I. PSMCJ , RAB ll A. RABJA. RUVBL2, SRGAP2,
VCP. VPS4A
ANK I. ATP2B2. CAMK2A. CAP I, DLG4, GLUD I,
GNAS. NAPB, PAKI, PLCBl. PPIA, RAB3A, SLCI7A
SLC6A I. SNAP25, SRCIN I, SUB l, SYT I. TPD52
ANKI. ANK2, ATP2B2. CAPL CNTNAPL CRYAB.
DLG4. GNAQ, GSK3A. KCNJIO , LICAM, LGII,

175

MAPRE3, NCAN, NRCAM, PAKI, PHGDH, PLCBl,
RAB I I A, RAB3A, SHANK I, SLC8A I, SPTAN l,
SPTBN2 , SRCIN I, SRGAP2, SUB I, XRCCS
ANKI, ANK2, ATP282, CAP!, CNTNAPI, DLG4,
LICAM, LGII , NCAN, NRCAM, PAKI, RABIIA,
RAB3A, SHANK I, SPTAN I, SPTBN2, SRCIN I, SRGAP2
ANKI, ANK2, CAP!, CNTNAPL DLG4, LICAM, LGII,
NCAN, NRCAM, PAKI, RABIIA, RAB3A , SHANKl,
SPTAN I, SPTBN2 , SRCIN l, SRGAP2
ANKI. ANK2, ATP6VID, CAP!, CNTNAPl, DLG4,
LICAM, LGII. MAPREJ, NCAN, NRCAM, PAKI,
RAB II A, RABJA , SHANK I, SPTAN I, SPTBN2,
SRCIN L SRGAP2
ANK2 , ATP2A2 , ATP2B2. ATP6V I 0, ATP6V I H, CKB,
DLG4, IMMT, KCNJ 10, PDK2 , SLC8AI
CAMK2A, GLUD L GNAS, NAPB, PLCB I, PPIA,
RABJA , SLC6A I, SNAP25. SRCIN L SYT I
ANK2, ATP282 , CAMK2A, CRYAB, DLG4, GLUDI ,
GNAQ. GNAS. GSKJA, LRP I, NAPB , PLCB I, PPIA,
RAB II A. RABJA. SHANK I, SLC6A I, SLC8A I, SNAP25 ,
SRCIN I. SYT I

G0:0048667

cell morphogenesis
involved in neuron
differentiation

18

17.0

7.02E-22

G0:0048812

neuron projection
morphogenesis

17

15.7

4.29E-24

G0:0048858

cell projection
morphogenesis

19

16.4

1.29E-45

G0:0050801

ion homeostasis

II

15.9

I .0 I E-25

G0:0051 046

regulation of secretion

II

21.6

7.08E-17

G0:0051 049

regulation of transport

21

15 .3

1.09E-I 03

G0:0051276

chromosome
organization

6

15 .0

3 .22E-83

G0:0051640

organelle localization

9

17.6

6 .67E-05

10

15.9

3.14E-22

6

18 .2

2 .63E-21

CAMK2A, CAP I, GSK3A. LICAM, PLCBI, TUBAIA

6

15.4

3A5E-09

ATP6VID, ATP6VIH. GNAS. GSK3A, PAKL PDK2

7

23.3

2.31E-14

CAPL DLG4, GNAQ, GNAS. GSKJA PLCBL SRGAP2

11

21.6

2.68E-09

ARF5, DLG4, DNMl, GMPS. GNAQ, GNAS, GNB5,
PLCB I, RAB I I A, RABJA , SRGAP2

G0:0055082
G0:0071345
G0:0071375

G0:1900542
GO: 1901068

cellular chemical
homeostasis
cellular response to
cytokine stimllllus
cellular response to
peptide hormone
stimulus
regulation of purine
nucleotide metabolic
process
guanosine-containing
compound metabolic

HUWEI. PAKI. RBMI4 , RUVBLI, RUVBL2, XRCC5
AMPH , DLG4. MFN2, NAPB, RABIIA, RAB3A ,
SNAP25, SYP. SYTI
ANIC , ATP2A2, ATP282, ATP6V I D. ATP6V I H. CKB,
DLG4 , IMMT, L ICAM. SLC8A I

176

process
GO: 1901135

carbohydrate derivative
metabolic process

22

15 .7

2 .26E-I 08

G0 : 1901136

carbohydrate derivative
catabolic process

18

22.0

8 .9 7 E-2 1

8

15 . 1

1.05 E-48

18

15 .8

5. 3 5 E-6 5

7

15. 9

3. 03 E-22

G0 : 1901137
GO: 1901565
G0 :2000145

carbohydrate derivative
bi osynthetic process
organon itrogen
compound catabolic
process
regulation of cell
motility

ARF5, ATP28 2, ATP6VIH , CAP!. DLG4, DNMI ,
FBX02, GBAS, GMPS. GNAQ, GNAS, GNB5 , GSK3A,
NCAN . PLCB I. PSMC J. RAB II A, RAB J A. RUVBL2 ,
SRGAP2, VCP, VPS4A
ARF5. ATP6VIH . DLG4 . DNMI . FBX02. GNAQ, GNAS.
GNB 5. GSKJ A. NCAN . PLCB I, PSM CJ, RAB II A,
RAB J A. RUVBL2. SRGAP2. VCP, VPS4A
CAP I. GBA S. GMPS. GNAS, GSK 3A, NCAN , PLCB I.
VCP
ARF5, AT P6Vl H, BLVRB, DLG4 , DNM I, GL UDI ,
GNAQ, GNA S, GN8 5, NCAN , PLCB I, PSMC3, RAB II A,
RAB J A. RUVBL2. SRGAP2, VCP, VPS4A
LRP I. PAK I. PLC BI , RBM I4, SLC8AI , SRCINI ,
SRGA P2

177

TableS4. Significant GO Cellular Component terms and the associated differentially expressed proteins
in MeHg-treated marmoset cerebellum by GOrilla
GOlD

6

GO Term

P-value'

Protein
Count

Associated Proteins

G0:0044459

plasma membrane
part

3.60E-03

34

SYP. SPTAN I. ATP2B I. DLG4 . ATP2B2. SCN2A. ATP2A2. LRP I. SLC8A I.
SYTI. CNTNAPI. CAMK2A . SNAP25. C2CD5. HOMERJ . CLTB . SHANKI ,
CD81 . GBAS. NDRG4 . AMPH . GNAS. SRCIN I. KCNJ I 0, SRGAP2. GNAQ.
SPTBN2. L ICAM. PAK I. DUSP J . SLC6A I. ANK2 . ANK I. NRC AM

G0:0060076

excitatory synapse

2.68E-03

6

SYP. DLG4. BSN . SHANK I. SYT I. SLC 17A

G0:0097060

synaptic membrane

1.62E-02

12

SNAP25 . SYP. Ll CAM . HOMERJ . DLG4 . SRCIN I. SHANK I. ANK2. SYT I.
SRGAP2. ANK I. CAMK2A

P-value corrected using the Benjamini and Hochberg method

178

A
DLG4 (Disks large homolog 4) : P78352

LQAAHLHPIAIFIRPR
B~ Ions

L

0

A A

Y~ Ions

R
P
B Ions 1 - L - t

H

R

L

H

P -l

-1-t-F

F -f

+A+-

H

A +-A-+

R

P-l

t A-l A ~ 0--1
p
H

H

Y Ions J - R ---+-P --1-R --+-- --t--F - t

A-+--1-

A

1-f

P

-F-

-H-

L-

1 -i- R - - ! - - P
H-

A

A

0

+

"'
Sequence

LOAAHLHPIAIFIRPR

.. .
I

+

~
c
a.•

"'
c

,f...

""

.,.
"....

"'
')
~
~,

""
"

'I

0

100

~00

C"l

,,

00

X.

.....

,...,

300

r

,_

'

400

500

600

700

BOO

900

0

1000

1100

1 ~00

1 300

1400

1500

1600

MIZ

179

8
SLC17 A7 (Vesicular glutamate transporter 1 ): Q9P2U7

ILQGLVEGVTYPACHGIWSK
8::' Ions H

L

Q

Y:' Ions 1-V.

s

W

8 Ions

I

GI-L

V

E Gi-V T

HGI-H

C

H

V~G~

v

L-t-0
G
S
W-----!-1

Y Ions f-V

P ~A

Y

C -!AI ~+

E

l

H

G

p

A

p

y

T

G-+ V
-C

A

H

-Y-+-T

G

1-t--W-

E-t-V-!-L

G

0

100

Sequence
MH+

90

ILOGLVEGVfYPACHGIWSV

::'::'::'8

53

Charge= 3+
Score= 1 ::'1 '21

80

"

.[

.

..,.+

0

100

::'00

300

400

500

600

700

BOO

900

1000

1100

1 .00 1 300

1400

1500 1600

1700

1 BOO

M/Z

igure l.Repre entative M /M

spectra from (A) DLG4 (P78352) and (8) SLC17A7

(Q9P2U7) in marmo et cerebellum

180

Cell adhesion molecules (CAMs)
Chagas disease (American trypanosomiasis)
Vascular smooth muscle contraction
Wnt signaling pathway
Dilated cardiomyopathy

l ATP2A2, ATP2B2,

l ATP2B 1, CAMK2A,

Malaria

i GNAS, GNAQ, ITPKA,
i PLCBl , SLC8A2 and
i SLC8Al

Alzheimer's disease
Gap junction

L- - - - - - - - - - - - - - - - - - - - - •

Pancreatic secretion
Calcium signaling pathway

------------------------

t

-------*

0

2

4

6

8

10

# of Genes/ Term

Figure 2. KEGG pathway enrichment analysis of differentially expres ed proteins tn
MeHg-treated mannoset cerebellum using WebGestalt

181

12

r

CALCIUM SIGNALING PATHWAY 1

SLC8A1 and SLC8A2

Nt

1

I
I
J MLCK J

j-

Con~bon

[:1lliU f --• Metabolism

Newotrucsl!Utlet,

au!¥:old

Newotrucsnul"'et

h:mrone,
aul¥:old

Grow1h fact>t

ITPKA
NAADP

SIP

'·- - - - - - - - - - - - - - - - - _. ExocytJsJS
Sembon

04J20 4115114

(c) Kanelusa l.!boratooes

FigureS3 . Calcium s ignaling pathway was classified by K EGG pathway ma pping system
from uploaded 102 differentially expressed proteins. Protein s with red labels were the
one mapped in calcium signaling pathway.

182

hapter VI:

A
APEO (Apolipoprotein E): P02649
8 Ions f - -

L

R

Y Ions t--- - R -

v

-!-A-+

I

+A+G
Al
A-+---l ~

0

- - 1 - A - t - G - t - A - + - - - 0 - - + ---Y-----1r---V- -t -

tr.)

>

100Sequence= RLAVYQAGAR
MH+ = 110 4 6~63

90-

L£ 1

.....

Charge= :? +

(X)

""

(J)

80-

Score= 1 :?6 07

en

.....

Expectvalue = 0 01 4 035
70r-

0 ;:
<.C '
(..,

601-

r""

cr
M

~

....

VI

M

~501-

C)

J

c

(IJ

0

....

("'")

-

L['

r '

401-

--;:;:;
I

'""

~

r 1

~

=

>-

301-

<o

r1

- -

.r.
.._

r -,

',...
If

<.C•

a:

,...""'

,_r--~ ,~

'r""

._. "l""f' (' ~

'"-

~

...

co

-r ~ a
~ ..,.

r'

::2
-....

,..

-

~

~

~01-

I

+I.,-

i'

+ --;;;:;
.q
;f_ .:....
...,-

.
"
=

0'

M

""'

oq
<L

, r'

....

"-' 0
CD

<L
D

..,. ''
,
,'"," T
C)

(Y"

,_

L[

CD

f.C,

;::..,

'"' '

' ,.,

£

'L

, , 1.£
~ .0

~-

.

.'

'

..,

:. ,._

0"1
~

I

;:;-'

'
-,

1=-

~
~

~

~

101-

0

I

0

100

I

I

I

:?00

300

I

400

I

I

I

'500

600

700

I

I

I
I

BOO

I

I

900

I

1

1000

M/Z

183

8
DYNC1LI2 (Cytoplasm ic dynein llight intermediate chain 2) : 043237
B~ Ions rD-tA+-V

F-+-1-+-P+A~GI-W-t-D

Y~ Ions 1-V -I-V -t-E

N

D-t-

B Ions f - D -+-A-tF
Y Ions f----V.
V. -+--E

1 ~-f-E

-lGtA-t P +-

V-l

F t-V-tA1
-t+-P-t-A-+- G
W
D---!- I,J - - 1 - E - - - 1 - - V -----1
N---!- 0
W-+- G -+-A-+- P - + -1-+--- F ----+--V - ! -A -l
+-

0>

>

100
Sequence= OAVFIPAGWDNHV
MH+ = 1 589 7966

90

Charge=~+

Score= 1 ~5 03

80

Expectvalue = 0 00 4 9~13

")

,...,
~

0

1 00

~00

300

(~J

0

~

(T'}
(T'}

..,.

400

+

C"'J

~
y

C'

+ ..,.
T >.C

500

...c
T

+
+

600

700

800

900

1000

11 00

1 ~00

1300

1400

1500

M/Z

Figure l .Repre entative MS /MS pectra from (A)
lobe and (B) DYN 1LI2 (04323 7) in frontal lobe

PO

(P02649) in marmoset occipital

184

Appendix III: Author hip

tatement for Publi bed

hapter

Statement of Authorship (Chapter III)
The manu cript in hapter III titled on " ffect of Methylmercury on Dopamine Release in
MN9D Neuronal ell "i author d by Yueting hao and lling Man han.
Dr. Laurie han provid d guidanc in the overall tudy design of the manu cript. Yueting
hao design and conducted the experiments, performed the data analysis and drafted the
manu cript. Dr. Laurie han pro id d the comments based on hi expertise knowledge in
toxicology. Yueting hao updated the manuscript based on Laurie's feedback, and Dr. Laurie
Chan made a fmal approval of this article and submitted it to the publisher.

Yueting Shao

Hing Man Chan

185

Statement of

ut ho ., hip ( bapter

Th manu ript in hapter \ titled on 'Pro\ omic
eth) lmer ury" is uth re,d
1annosc c ·posed t
an han.
D ·el ·i ey . lhibin

)

naJ..·sis of Cerebellum 1
ammon
'uetin
hao, tc mi Yamamoto,

stud cerebellar prote me in meUl)lmercun.
Yuetin
bao developed the conception
xpo d common mann se and utline of e ·periment nHcr initial al ·· n" "'1th her upcrvi or
Dr. u ic Chan C mmon manna t samples " r collec ed b... r. 1cgu i Yamamoto i
~auonal In itute fo . 1inamata Dise se, Japan. Dr Zhibin . ing in Onawa In tHute o
) ~1em Biolo
ondu ted LC- 1 , f ru 1) Js and pru\'lded lbc advice for u ing Per ·u
hao performed data analy 1 and conducted the validation say. Dr
ftw . Yu tin
Damel fige.: ( nawa In titute of y ems Biology provided he critical comment on
analyzing method Wld data interpretation. and 'ueting h updated th r suits based o
Daniel Figeys· omments. and Titten thi manuscript. I urie Chan provided tlu: irnportan
revisi n or thi raft. and Yueting hao re-organized the dr t based on Launc 'han'
feedback. ThL draft was then reviewed by other co-au tho : D . Megurni Yamamoto. Daniel
I i y an Zhibin ing. Yueting ·ha integrated the commcn from the co-authors. upd ted
manu ript and finall) approv d y Lauri\: Chan for ubrni sion.

ueting hao

I aniel Figey

llin

1e um1 'amam t

/hi bin

an h n

186