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Nicola Hall - Revealing mRNA alternative splicing complexity in the human brain

London Calling 2019

Identifying the cellular pathways underlying psychiatric disorders has great potential to improve patient lives. Voltage-gated calcium channels (VGCCs), including CACNA1C, have been linked to the risk of bipolar disorder and schizophrenia, so are promising targets for new treatments. VGCCs are also important in the cardiovascular system, meaning treatments must be tissue-selective. We aim to identify brain-enriched CACNA1C isoforms as novel targets for new treatments. We used targeted long-range nanopore sequencing to characterise full length transcripts of CACNA1C in human brain and mouse brain, heart and aorta. In human we identified >250, and in mouse >190, mRNA isoforms, exons and splice junctions; including many predicted to modulate protein function. In mice, splicing was clearly tissue-specific, and we expect that this underlying principle of tissue-specific splicing will be conserved between mice and humans. Our data show that splicing of CACNA1C in human brain is far more diverse than is currently appreciated. This information will be critical to reveal pathophysiological mechanisms, and to identify brain-enriched VGCC isoforms that may be novel targets for new psychiatric treatments.

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In the second plenary talk of London Calling 2019, Nicola Hall (Department of Psychiatry, University of Oxford) began: "what's going on in your brain right now?". The activity going on that Nicola is interested in is: splicing. The study of splicing in the brain sheds light on how genes, and so the whole brain, is functioning, and helps in the investigation of psychiatric disorders.

Calcium signalling, Nicola explained, is essential for neurotransmission; Nicola showed the role of voltage-gated calcium channels in the tightly-regulated process of the passage of calcium ions through membranes. Voltage-gated calcium channels are important in the cardiovascular system, as well as in the brain. Loci within the gene CACNA1C, which encodes this protein, have been robustly linked to psychiatric disorders including bipolar disorder and schizophrenia. There are existing drugs which target the protein: Nicola quoted a study which found that treatment of patients with blockers reduced hospitalisation cases and self-harm.  CACNA1C produces multiple protein isoforms; differences in splicing impact the proteins produced, which in turn affect drug binding. To investigate the variation in the isoforms produced, Nicola stressed the importance of determining how different parts of the CACNA1C gene work together. To fully characterise splice isoforms of CACNA1C, the team decided to use long-range, targeted cDNA sequencing of full-length transcripts.

RNA was extracted from human brain samples, reverse transcribed and the long-read cDNA sequenced on the MinION device; 6 regions of the brain were sampled from each of the three tested. Data analysis was carried out via two pipelines. First, exon-level analysis was performed, in which reads were mapped to known and novel exons and their abundance compared. This analysis revealed that splicing in CACNA1C is "much more diverse than was previously known": 38 novel exons, 7 known, previously-annotated isoforms and 83 high-confidence novel isoforms were detected. 9 out 10 of the most abundant isoforms identified were novel, and 8 were predicted to encode functional channels.

Nicola showed a principal component analysis plot in which the isoforms detected in each region of the brain sampled were plotted. The results revealed that there is more diversity between splicing in different regions of the brain than there is between individuals, which is a promising result for future potential treatments, and suggests that splicing is regulated according to cell type and function.

Next, splice site-level analysis was performed, identifying canonical splice junctions and mapping reads to these. This method is able to detect small-scale variations, though quantitation is less reliable. This identified 195 high-confidence isoforms, of which only three had been previously annotated. The results again indicated that splicing diversity was much higher than expected in CACNA1C, and that more diversity is seen between different regions of the brain than between individuals. It also showed many "common themes" in splicing events, which frequently occurred in the same regions, though rarely in domains critical to structure, suggesting that the process is not completely random. The analysis was able to detect variations as small as a single amino acid codon, and some of the identified variations were quite abundant - one was seen in almost half of the samples tested.

Nicola then asked, what are the other sources of transcript variation? One source could be alternative start sites. 5'RACE (rapid amplification of cDNA ends) analysis identified two transcription start sites in the human brain: one known, one currently not annotated and predicted to encode a functional channel with a truncated N-terminus.

Nicola described how therapeutics would need to target brain-specific isoforms of CACNA1C to avoid off-target effects. To investigate this, the team are identifying brain-enriched isoforms that are absent in heart samples. As they do not currently have matched human heart and brain samples, their initial investigation uses samples from mice. Again, "huge diversity" was seen in splice isoforms, many of which were novel. Nicola showed data that demonstrated the clear distinction between isoforms present in the brain and cardiovascular system in mice, indicating that "splicing seems to be strongly tissue-specific." An alternative start site was also detected in the heart tissue. Isoforms are not conserved between humans and mice, so many splicing patterns were found in mice but not humans and vice versa; however, the team expect that the principle of tissue-specific splicing will be conserved.

Nicola concluded that the human CACNA1C gene encodes a wide variety of novel putative voltage gated calcium channels. In mice, the CACNA1C isoform profile differs between the brain and cardiovascular tissue, indicating the potential for identification of brain-enriched isoforms with different drug-binding capacity to cardiovascular isoforms which could represent future therapeutic targets. The team now plan to characterise the isoforms of CACNA1C in the brain and cardiovascular tissue of humans, and to carry out functional assays to see how the splicing observed affects protein function.

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