CACNA1C (CaV1.2) and other L-type calcium channels in the pathophysiology and treatment of psychiatric disorders: Advances from functional genomics and pharmacoepidemiology.

A role for voltage-gated calcium channels (VGCCs) in psychiatric disorders has long been postulated as part of a broader involvement of intracellular calcium signalling. However, the data were inconclusive and hard to interpret. We review three areas of research that have markedly advanced the field. First, there is now robust genomic evidence that common variants in VGCC subunit genes, notably CACNA1C which encodes the L-type calcium channel (LTCC) CaV1.2 subunit, are trans-diagnostically associated with psychiatric disorders including schizophrenia and bipolar disorder. Rare variants in these genes also contribute to the risk. Second, pharmacoepidemiological evidence supports the possibility that calcium channel blockers, which target LTCCs, might have beneficial effects on the onset or course of these disorders. This is especially true for calcium channel blockers that are brain penetrant. Third, long-range sequencing is revealing the repertoire of full-length LTCC transcript isoforms. Many novel and abundant CACNA1C isoforms have been identified in human and mouse brain, including some which are enriched compared to heart or aorta, and predicted to encode channels with differing functional and pharmacological properties. These isoforms may contribute to the molecular mechanisms of genetic association to psychiatric disorders. They may also enable development of therapeutic agents that can preferentially target brain LTCC isoforms and be of potential value for psychiatric indications.

Long read sequencing reveals novel isoforms and insights into splicing regulation during cell state changes.

BACKGROUND: Alternative splicing is a key mechanism underlying cellular differentiation and a driver of complexity in mammalian neuronal tissues. However, understanding of which isoforms are differentially used or expressed and how this affects cellular differentiation remains unclear. Long read sequencing allows full-length transcript recovery and quantification, enabling transcript-level analysis of alternative splicing processes and how these change with cell state. Here, we utilise Oxford Nanopore Technologies sequencing to produce a custom annotation of a well-studied human neuroblastoma cell line SH-SY5Y, and to characterise isoform expression and usage across differentiation. RESULTS: We identify many previously unannotated features, including a novel transcript of the voltage-gated calcium channel subunit gene, CACNA2D2. We show differential expression and usage of transcripts during differentiation identifying candidates for future research into state change regulation. CONCLUSIONS: Our work highlights the potential of long read sequencing to uncover previously unknown transcript diversity and mechanisms influencing alternative splicing.

Induced Pluripotent Stem Cells in Psychiatry: An Overview and Critical Perspective.

A key challenge in psychiatry research is the development of high-fidelity model systems that can be experimentally manipulated to explore and test pathophysiological mechanisms of illness. In this respect, the emerging capacity to derive neural cells and circuits from human induced pluripotent stem cells (iPSCs) has generated significant excitement. This review aims to provide a critical appraisal of the potential for iPSCs in illuminating pathophysiological mechanisms in the context of other available technical approaches. We discuss the selection of iPSC phenotypes relevant to psychiatry, the information that researchers can draw on to help guide these decisions, and how researchers choose between the use of 2-dimensional cultures and the use of more complex 3-dimensional model systems. We discuss the strengths and limitations of current models and the challenges and opportunities that they present. Finally, we discuss the potential of iPSC-based model systems for clarifying the mechanisms underlying genetic risk for psychiatry and the steps that will be needed to ensure that robust and reliable conclusions can be drawn. We argue that while iPSC-based models are ideally placed to study fundamental processes occurring within and between neural cells, they are often less well suited for case-control studies, given issues relating to statistical power and the challenges in identifying which cellular phenotypes are meaningful at the level of the whole individual. Our aim is to highlight the importance of considering the hypotheses of a given study to guide decisions about which, if any, iPSC-based system is most appropriate to address it.

Long read transcript profiling of ion channel splice isoforms.

Alternative splicing of RNA transcripts allows a single gene to generate multiple products and is a key means of generating functionally diverse voltage-gated ion channels. Splicing can be regulated according to cell type, cell state, and stage of development to produce a bespoke complement of protein isoforms. Characterizing the identities of full-length transcript isoforms is essential in order to fully understand a gene's expression and function. However, the repertoire of transcript isoforms is not well characterized for most genes. Long read nanopore sequencing allows full-length isoforms to be sequenced, therefore identifying full-length transcripts. Using this approach, we recently discovered that the human CACNA1C gene gives rise to a far greater repertoire of splice isoforms than previously appreciated. Here we provide a detailed overview of the technical approach we used to achieve this. The method described in this chapter combines long read nanopore sequencing with PCR targeting to selectively sequence transcripts of a specific gene of interest.

Long-read sequencing reveals the complex splicing profile of the psychiatric risk gene CACNA1C in human brain.

RNA splicing is a key mechanism linking genetic variation with psychiatric disorders. Splicing profiles are particularly diverse in brain and difficult to accurately identify and quantify. We developed a new approach to address this challenge, combining long-range PCR and nanopore sequencing with a novel bioinformatics pipeline. We identify the full-length coding transcripts of CACNA1C in human brain. CACNA1C is a psychiatric risk gene that encodes the voltage-gated calcium channel CaV1.2. We show that CACNA1C's transcript profile is substantially more complex than appreciated, identifying 38 novel exons and 241 novel transcripts. Importantly, many of the novel variants are abundant, and predicted to encode channels with altered function. The splicing profile varies between brain regions, especially in cerebellum. We demonstrate that human transcript diversity (and thereby protein isoform diversity) remains under-characterised, and provide a feasible and cost-effective methodology to address this. A detailed understanding of isoform diversity will be essential for the translation of psychiatric genomic findings into pathophysiological insights and novel psychopharmacological targets.