Human brain transcriptomic: towards understanding multiple system atrophy

Abstract

The human brain is a remarkably complex organ. It is a heterogeneous collection of billions of neurons and glial cells that are interconnected to form a finely tuned network capable of higher cognition. It is thought that the transcriptome may hold the key to understanding the complexity seen in the human brain. Next-generation sequencing allows the brain’s transcriptome to be probed at an unmatched resolution. This has uncovered a myriad of RNA elements, including RNA that does not code for protein, known as non-coding RNA (ncRNA). Originally, thought to be transcriptional noise, it is now appreciated that ncRNAs have numerous functional properties, with the ability to interact with DNA, other RNA molecules and proteins in different cellular compartments. It is thought that an increase in the number of ncRNAs being expressed throughout the brain, is a major driver of the increased intellectual capacity seen in humans and primates. The increase in the complexity of the human brain, also makes it prone to a number of different neurodegenerative and psychiatric diseases. These diseases are set to have dramatic economic and social impacts by the middle of the 21st century. To avert this looming epidemic an adequate understanding of the human brain is needed, so diagnostic tools and treatment targets can be developed. One such disorder is multiple system atrophy (MSA). MSA is a sporadic, rapidly progressing neurodegenerative disease. Currently no treatment exists and very little is known about the molecular basis of MSA. Before an understanding of the diseased brain can be reached an understanding of the healthy brain is necessary.

Here, the transcriptome of grey matter (GM) and white matter (WM) from the superior frontal gyrus (SFG) of the healthy prefrontal cortex (PFC) was analysed. This revealed pervasive transcription and highlighted the differences in the transcriptome profiles of distinct cortical structures throughout the brain. A number of protein-coding genes were expressed exclusively in GM or WM, including gamma-aminobutyric acid A receptor, beta 2 (GABRB2) and P21 Protein (Cdc42/Rac)-Activated Kinase 2 (PAK2), respectively. Further, an interesting phenomenon known as isoform switching was detected in genes such as the G protein-coupled receptor 123 (GPR123). It was also revealed that in the healthy frontal cortex long intervening non-coding RNAs (lincRNAs), a subclass of long non-coding RNAs (lncRNAs), appear to be important drivers of tissue differentiation.

To further establish the role of lincRNAs in the healthy human brain a comprehensive analysis of the oligodendrocyte maturation-associated lincRNA (OLMALINC) was carried out. It was found that OLMALINC is a recently evolved lincRNA with its highest expression levels in the human brain. OLMALINC was knocked down in human neurons and oligodendrocytes. Depletion of OLMALINC transcription revealed that it plays a role oligodendrocyte maturation. This study was one of the first functional characterisations of a lincRNA expressed in the human brain, and thus demonstrated the importance of the non-coding transcriptome throughout the human brain.

Next, the transcriptome of MSA brain tissue was profiled. This identified a handful of new genes that may be involved in the progression of the MSA. Genes that were differentially expressed between healthy and MSA tissue from the SFG included, alpha-1-hemoglobin (HBA1), alpha-2-hemoglobin (HBA2), beta-hemoglobin (HBB) and transthyretin (TTR). A number of differentially expressed lincRNAs between MSA GM and MSA WM were also identified. Very little is currently known about the genetic basis of MSA and this research article provides the most in-depth transcriptomic investigation of MSA to date. Due to its significant up-regulation in MSA WM linc00320 was selected for further analysis. While no conclusive link between linc00320 expression patterns and MSA was found, it was established that linc00320 was both human- and brain-specific. Interesting results concerning the biology of lincRNAs were also uncovered. While highly expressed in the human brain, no expression of linc00320 was detected in other human peripheral tissues. Further, no expression of linc00320 was detected in the following human cell lines; neurons, oligodendrocytes, adult astrocytes and fetal astrocytes. The inability to detect linc00320 expression in these homogeneous cell lines could be linked to the lack of cell-to-cell communication and tissue context of cell lines. Analysis of linc00320 splice variants demonstrated that alternative splicing can result in lincRNAs with different functional properties.

Together, using cutting edge sequencing techniques and bioinformatic tools this thesis provides a comprehensive transcriptional analysis of the unique transcriptome of the human brain, followed by the most detailed transcriptome analysis of the neurodegenerative disease MSA. The transcriptome wide profiling analyses are also coupled with comprehensive analyses of OLMALINC and linc00320, detailing their importance in human brain physiology.