Rader, Stephen

Myelination is essential for proper conduction across all neural networks. Myelination in the central nervous system is performed by glial cells called oligodendrocytes. Mature, myelinating glia develop from progenitor cells through distinct differentiation steps, and gene expression markers have been established that are unique to oligodendrocyte lineage cells. This study aims to establish a reliable RT-qPCR protocol to quantify oligodendrocyte-specific gene expression according to the MIQE guidelines. Primers specific to the progenitor, immature, and myelinating stages of oligodendrocyte differentiation were validated, showing 90-100% efficiency. Reference gene primers were also validated, and their stability was determined to make recommendations for those to use as normalization factors. The expression pattern of oligodendrocyte-specific genes across stages of postnatal development was similar to previously defined RNA-seq profiles in isolated cell types. The validated RT-qPCR assays developed build a framework for future investigation on myelination during development and remyelination in disease.

Obesity is a disease that occurs when energy intake exceeds energy expenditure, concomitantly increasing risk of chronic diseases, including metabolic diseases such as diabetes. Research into therapeutics to correct dysregulations in energy balance is on the rise, and one notable neuropeptide being studied is pituitary adenylate cyclase-activating polypeptide (PACAP). PACAP has been shown to regulate thermogenesis, an energy burning process regulated by the sympathetic nervous system (SNS) in response to cold stress and overfeeding, but its role within the sympathetic nerves innervating and regulating energy metabolism in adipose tissues is not well understood. We hypothesize that PACAP is acting on PACAP receptors (PAC1, VPAC1, VPAC2) expressed in stellate ganglia innervating brown adipose tissue, the main thermogenic organ in mammals. We have established a reliable protocol for the isolation of two ganglia of the SNS (stellate and superior cervical) and provided recommendations of reference genes to use as internal controls for gene expression studies. For the first time, we confirmed PACAP receptor gene expression in the stellate ganglia, and saw sex-specific, differential gene expression based on housing temperature. We subsequently analyzed the expression of PAC1 splice variants in the stellate ganglia and our positive control tissues (adrenal gland and superior cervical ganglia), identifying at least two variants in these SNS tissues. This work adds to current literature on the study of thermogenesis and energy balance, and encourages future work characterizing G-protein coupled receptors (GPCRs) for their therapeutic application, enhancing our fundamental understanding of autonomic physiology in mammals.

The eukaryotic process of pre-mRNA splicing involves the removal of noncoding intron sequences and the fusion of the remaining protein-coding exon sequences. The splicing reaction is catalyzed by the spliceosome, a dynamic multi-megadalton ribonucleoprotein complex that, in humans, is composed of 5 small nuclear RNAs (snRNAs) and over 200 associated proteins acting on more that 200,000 introns present within 25,000 genes. The unicellular red alga Cyanidioschyzon merolae possesses a more tractable splicing environment, with only 4 snRNAs and 75 associated proteins interacting with 27 annotated introns found in 26 our of 5,331 genes. Intron-rich genomes can confer benefits to their host species such as improved gene expression, incredible proteomic diversity, and increased genetic stability. This raises the question of why intron-poor C. merolae has retained such a small number of introns and a dramatically reduced spliceosome. A comprehensive investigation into the precise role that introns play in C. merolae would require the systematic removal of introns and an analysis of the effects thereof. The ability to elucidate the role of splicing in C. merolae via genome-wide intron deletion, however, hinges on the feasibility of establishing the efficiently scalable CRISPR genome engineering tool in C. merolae. It also follows that such an endeavour would require an accurate picture of the intron landscape of C. merolae, and since the number of annotated introns in C. merolae is relatively small, it is especially vital to determine whether any introns are missing from the C. merolae annotation. To that end, a stable and inducible Cas9-expressing strain of C. merolae was successfully developed. Transcriptome analysis using RNA-seq data revealed the discovery of 11 novel introns and 1 misannotated intron, as well as the presence of alternative splicing in the form of alternative splice site usage.