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.

New drug targets in psychiatry: Neurobiological considerations in the genomics era.

After a period of withdrawal, pharmaceutical companies have begun to reinvest in neuropsychiatric disorders, due to improvements in our understanding of these disorders, stimulated in part by genomic studies. However, translating this information into disease insights and ultimately into tractable therapeutic targets is a major challenge. Here we consider how different sources of information might be integrated to guide this process. We review how an understanding of neurobiology has been used to advance therapeutic candidates identified in the pre-genomic era, using catechol-O-methyltransferase (COMT) as an exemplar. We then contrast with ZNF804A, the first genome-wide significant schizophrenia gene, and draw on some of the lessons that these and other examples provide. We highlight that, at least in the short term, the translation of potential targets for which there is orthogonal neurobiological support is likely to be more straightforward and productive than that those relying solely on genomic information. Although we focus here on information from genomic studies of schizophrenia, the points are broadly applicable across major psychiatric disorders and their symptoms.

Catechol-O-methyltransferase activity does not influence emotional processing in men.

BACKGROUND: Catechol-O-methyltransferase (COMT) regulates cortical dopaminergic transmission and prefrontal-dependent cognitive function. However, its role in other cognitive processes, including emotional processing, is relatively unexplored. We therefore investigated the separate and interactive influences of COMT inhibition and Val158Met (rs4680) genotype on performance on an emotional test battery. METHODS: We recruited 74 healthy men homozygous for the functional COMT Val158Met polymorphism. Volunteers were administered either a single 200 mg dose of the brain-penetrant COMT inhibitor tolcapone or placebo in a double-blind, randomised manner. Emotional processing was assessed using the emotional test battery, and mood was rated using visual analogue scales and the Profile of Mood States (POMS) questionnaire across the test day. RESULTS: There were no main or interactive effects of Val158Met genotype or tolcapone on any of the emotional processing measures or mood ratings. CONCLUSIONS: Our findings suggest that, at least in healthy adult men, COMT has little or no effect on emotional processing or mood. These findings contrast with several neuroimaging studies that suggest that COMT modulates neural activity during emotional processing. Thus, further studies are required to understand how COMT impacts on the relationship between behavioural output and neural activity during emotional processing. Nevertheless, our data suggest that novel COMT inhibitors under development for treating cognitive dysfunction are unlikely to have acute off target effects on emotional behaviours.

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.

Gabapentin and pregabalin in bipolar disorder, anxiety states, and insomnia: Systematic review, meta-analysis, and rationale.

The gabapentinoids, gabapentin, and pregabalin, target the α2δ subunits of voltage-gated calcium channels. Initially licensed for pain and seizures, they have become widely prescribed drugs. Many of these uses are off-label for psychiatric indications, and there is increasing concern about their safety, so it is particularly important to have good evidence to justify this usage. We conducted a systematic review and meta-analysis of the evidence for three of their common psychiatric uses: bipolar disorder, anxiety, and insomnia. Fifty-five double-blind randomised controlled trials (RCTs) and 15 open-label studies were identified. For bipolar disorder, four double-blind RCTs investigating gabapentin, and no double-blind RCTs investigating pregabalin, were identified. A quantitative synthesis could not be performed due to heterogeneity in the study population, design and outcome measures. Across the anxiety spectrum, a consistent but not universal effect in favour of gabapentinoids compared to placebo was seen (standardised mean difference [SMD] ranging between -2.25 and -0.25). Notably, pregabalin (SMD -0.55, 95% CI -0.92 to -0.18) and gabapentin (SMD -0.92, 95% CI -1.32 to -0.52) were more effective than placebo in reducing preoperative anxiety. In insomnia, results were inconclusive. We conclude that there is moderate evidence of the efficacy of gabapentinoids in anxiety states, but minimal evidence in bipolar disorder and insomnia and they should be used for these disorders only with strong justification. This recommendation applies despite the attractive pharmacological and genetic rationale for targeting voltage-gated calcium channels.

Kalirin as a Novel Treatment Target for Cognitive Dysfunction in Schizophrenia.

The cognitive dysfunction experienced by patients with schizophrenia represents a major unmet clinical need. We believe that enhancing synaptic function and plasticity by targeting kalirin may provide a novel means to remediate these symptoms. Karilin (a protein encoded by the KALRN gene) has multiple functional domains, including two Dbl homology (DH) guanine exchange factor (GEF) domains, which act to enhance the activity of the Rho family guanosine triphosphate (GTP)-ases. Here, we provide an overview of kalirin's roles in brain function and its therapeutic potential in schizophrenia. We outline how it mediates diverse effects via a suite of distinct isoforms that couple to members of the Rho GTPase family to regulate synapse formation and stabilisation, and how genomic and post-mortem data implicate it in schizophrenia. We then review the current state of knowledge about the influence of kalirin on brain function at a systems level, based largely on evidence from transgenic mouse models, which support its proposed role in regulating dendritic spine function and plasticity. We demonstrate that, whilst the GTPases are classically considered to be 'undruggable', targeting kalirin and other Rho GEFs provides a means to indirectly modulate their activity. Finally, we integrate across the information presented to assess the therapeutic potential of kalirin for schizophrenia and highlight the key outstanding questions required to advance it in this capacity; namely, the need for more information about the diversity and function of its isoforms, how these change across neurodevelopment, and how they affect brain function in vivo.

Targeting synaptic plasticity in schizophrenia: insights from genomic studies.

Patients with schizophrenia experience cognitive dysfunction and negative symptoms that do not respond to current drug treatments. Historical evidence is consistent with the hypothesis that these deficits are due, at least in part, to altered cortical synaptic plasticity (the ability of synapses to strengthen or weaken their activity), making this an attractive pathway for therapeutic intervention. However, while synaptic transmission and plasticity is well understood in model systems, it has been challenging to identify specific therapeutic targets for schizophrenia. New information is emerging from genomic findings, which converge on synaptic plasticity and provide a new window on the neurobiology of schizophrenia. Translating this information into therapeutic advances will require a multidisciplinary and collaborative approach.

Distinct roles for dopamine clearance mechanisms in regulating behavioral flexibility.

Dopamine plays a crucial role in adaptive behavior, and dysfunctional dopamine is implicated in multiple psychiatric conditions characterized by inflexible or inconsistent choices. However, the precise relationship between dopamine and flexible decision making remains unclear. One reason is that, while many studies have focused on the activity of dopamine neurons, efficient dopamine signaling also relies on clearance mechanisms, notably the dopamine transporter (DAT), which predominates in striatum, and catechol-O-methyltransferase (COMT), which predominates in cortex. The exact locus, extent, and timescale of the effects of DAT and COMT are uncertain. Moreover, there is limited data on how acute disruption of either mechanism affects flexible decision making strategies mediated by cortico-striatal networks. To address these issues, we combined pharmacological modulation of DAT and COMT with electrochemistry and behavior in mice. DAT blockade, but not COMT inhibition, regulated sub-second dopamine release in the nucleus accumbens core, but surprisingly neither clearance mechanism affected evoked release in prelimbic cortex. This was not due to a lack of sensitivity, as both amphetamine and atomoxetine changed the kinetics of sub-second release. In a multi-step decision making task where mice had to respond to reversals in either reward probabilities or the choice sequence to reach the goal, DAT blockade selectively impaired, and COMT inhibition improved, performance after reward reversals, but neither manipulation affected the adaptation of choices after action-state transition reversals. Together, our data suggest that DAT and COMT shape specific aspects of behavioral flexibility by regulating different aspects of the kinetics of striatal and cortical dopamine, respectively.

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.

Cellular calcium in bipolar disorder: systematic review and meta-analysis.

Calcium signalling has long been implicated in bipolar disorder, especially by reports of altered intracellular calcium ion concentrations ([Ca2+]). However, the evidence has not been appraised critically. We carried out a systematic review and meta-analysis of studies of cellular calcium indices in bipolar disorder. 2281 records were identified and 117 screened, of which 32 were eligible and 21 were suitable for meta-analyses. The latter each involved up to 642 patients and 404 control subjects. We found that basal free intracellular [Ca2+] is increased in bipolar disorder, both in platelets and in lymphocytes. The effect size is 0.55, with an estimated elevation of 29%. It is observed in medication-free patients. It is present in mania and bipolar depression, but data are equivocal for euthymia. Cells from bipolar disorder individuals also show an enhanced [Ca2+] response to stimulation with 5-HT or thrombin, by an estimated 25%, with an effect size of 0.63. In studies which included other diagnoses, intracellular basal [Ca2+] was higher in bipolar disorder than in unipolar depression, but not significantly different from schizophrenia. Functional parameters of cellular Ca2+ (e.g. calcium transients), and neuronal [Ca2+], have been much less investigated, and no firm conclusions can be drawn. In summary, there is a robust, medium effect size elevation of basal and stimulated free intracellular [Ca2+] in bipolar disorder. The results suggest altered calcium functioning in the disorder, and encourage further investigations into the underlying mechanisms, and the implications for pathophysiology and therapeutics.

Benchmarking pluripotent stem cell-derived organoid models.

Cerebral organoids are stem cell-derived, self-organizing three-dimensional cultures. Owing to the remarkable degree to which they recreate the cellular diversity observed in the human brain, they have attracted significant interest as a novel model system for research and drug development, as well as capturing the public imagination. However, many questions remain about the extent to which these cultures recapitulate neurodevelopment and the defining features of the human brain. To clarify the fidelity of human organoid models, Bhaduri and colleagues compared the molecular profile of brain organoid cells with that of primary cells from fetal brain. They observed that, whilst brain organoids broadly recapitulate the cellular profile of human brain, they lack the subtypes of cell classes seen in human brain. In addition, they showed marked expression of cellular stress markers, which could be reversed by transplanting organoid cells into neonatal mouse brain. The authors hypothesise that in vitro culture induces a cellular stress response and that it is this that impairs maturation. Thus, whilst their findings strike a note of caution in the use of organoids as a model for early human brain development, they lay a foundation for improving the accuracy of organoid models in the future.

Unraveling Mechanisms of Patient-Specific NRXN1 Mutations in Neuropsychiatric Diseases Using Human Induced Pluripotent Stem Cells.

Rare heterozygous deletions in the neurexin 1 (NRXN1) gene robustly increase an individual's risk of developing neurological and psychiatric disorders. However, the molecular bases by which different mutations result in different clinical presentations, with variable penetrance, are unknown. To better understand the molecular and cellular consequences of heterozygous NRXN1 mutations, Flaherty and colleagues studied how patient mutations influence the NRXN1 isoform repertoire and neuronal phenotypes using induced pluripotent stem (iPS) cells. Advancing from disease association to mechanistic insights, the authors provide insight into how patient mutations might impinge on neuronal function. This research highlights the value of iPS cells for elucidating otherwise elusive links between molecular and neuronal function. In addition, they provide further evidence of the importance of alternative splicing in the pathophysiology of neuropsychiatric diseases.

Correction: The genomic basis of mood instability: identification of 46 loci in 363,705 UK Biobank participants, genetic correlation with psychiatric disorders, and association with gene expression and function.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Voltage-gated calcium channel blockers for psychiatric disorders: genomic reappraisal.

We reappraise the psychiatric potential of calcium channel blockers (CCBs). First, voltage-gated calcium channels are risk genes for several disorders. Second, use of CCBs is associated with altered psychiatric risks and outcomes. Third, research shows there is an opportunity for brain-selective CCBs, which are better suited to psychiatric indications.

The genomic basis of mood instability: identification of 46 loci in 363,705 UK Biobank participants, genetic correlation with psychiatric disorders, and association with gene expression and function.

Genome-wide association studies (GWAS) of psychiatric phenotypes have tended to focus on categorical diagnoses, but to understand the biology of mental illness it may be more useful to study traits which cut across traditional boundaries. Here, we report the results of a GWAS of mood instability as a trait in a large population cohort (UK Biobank, n = 363,705). We also assess the clinical and biological relevance of the findings, including whether genetic associations show enrichment for nervous system pathways. Forty six unique loci associated with mood instability were identified with a SNP heritability estimate of 9%. Linkage Disequilibrium Score Regression (LDSR) analyses identified genetic correlations with Major Depressive Disorder (MDD), Bipolar Disorder (BD), Schizophrenia, anxiety, and Post Traumatic Stress Disorder (PTSD). Gene-level and gene set analyses identified 244 significant genes and 6 enriched gene sets. Tissue expression analysis of the SNP-level data found enrichment in multiple brain regions, and eQTL analyses highlighted an inversion on chromosome 17 plus two brain-specific eQTLs. In addition, we used a Phenotype Linkage Network (PLN) analysis and community analysis to assess for enrichment of nervous system gene sets using mouse orthologue databases. The PLN analysis found enrichment in nervous system PLNs for a community containing serotonin and melatonin receptors. In summary, this work has identified novel loci, tissues and gene sets contributing to mood instability. These findings may be relevant for the identification of novel trans-diagnostic drug targets and could help to inform future stratified medicine innovations in mental health.

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.