Cross-disorder risk gene CACNA1C differentially modulates susceptibility to psychiatric disorders during development and adulthood

Single-nucleotide polymorphisms (SNPs) in CACNA1C, the α1C subunit of the voltage-gated L-type calcium channel Cav1.2, rank among the most consistent and replicable genetics findings in psychiatry and have been associated with schizophrenia, bipolar disorder and major depression. However, genetic variants of complex diseases often only confer a marginal increase in disease risk, which is additionally influenced by the environment. Here we show that embryonic deletion of Cacna1c in forebrain glutamatergic neurons promotes the manifestation of endophenotypes related to psychiatric disorders including cognitive decline, impaired synaptic plasticity, reduced sociability, hyperactivity and increased anxiety. Additional analyses revealed that depletion of Cacna1c during embryonic development also increases the susceptibility to chronic stress, which suggest that Cav1.2 interacts with the environment to shape disease vulnerability. Remarkably, this was not observed when Cacna1c was deleted in glutamatergic neurons during adulthood, where the later deletion even improved cognitive flexibility, strengthened synaptic plasticity and induced stress resilience. In a parallel gene × environment design in humans, we additionally demonstrate that SNPs in CACNA1C significantly interact with adverse life events to alter the risk to develop symptoms of psychiatric disorders. Overall, our results further validate Cacna1c as a cross-disorder risk gene in mice and humans, and additionally suggest a differential role for Cav1.2 during development and adulthood in shaping cognition, sociability, emotional behavior and stress susceptibility. This may prompt the consideration for pharmacological manipulation of Cav1.2 in neuropsychiatric disorders with developmental and/or stress-related origins.


INTRODUCTION
Major psychiatric disorders including schizophrenia (SCZ), bipolar disorder (BPD), major depressive disorder (MDD) and autism are moderately to highly heritable, and increasing evidence is suggesting a shared genetic etiology among them. [1][2][3][4][5][6] This might partially explain why many patients suffering from these illnesses display a substantial amount of overlapping symptoms. 7 One of the most consistent and robust genetic findings from genomewide association studies (GWAS) and meta-analyses of GWAS are associations of single-nucleotide polymorphisms (SNPs) in the α1 subunit (CACNA1C) of the voltage-gated L-type Ca 2+ channel (LTCC) Ca v 1.2 with SCZ and BPD, and to a lesser extent with MDD and autism. 1,[8][9][10][11][12][13][14][15][16] In support, candidate analysis studies have clearly indicated a shared genetic risk for CACNA1C across these disorders. 1,15,17 Further evidence comes from clinical studies, which have associated the primary disease-associated CACNA1C risk allele, rs1006737, with variations in human brain function and structure in patients, but also in healthy subjects. [18][19][20] Hence, the available GWAS and clinical data establish CACNA1C as a possible shared susceptibility factor which influences disease vulnerability for BPD, SCZ and MDD across current diagnostic boundaries. This is of considerable interest, in view of the fact that LTCCs have a pivotal role in modulating neuronal excitability, synaptic plasticity and gene expression. [21][22][23] However, the causality and mechanisms of how genetic alterations in CACNA1C affect the risk for an entire spectrum of psychiatric disorders remain largely unknown. Importantly, the associated CACNA1C variants by themselves only confer a marginal increase in disease risk. In addition to shared genetic risk factors, the environment represents an important contributor to the risk for many psychiatric disorders. In particular, adverse life events such as severe trauma and/or chronic stress represent strong risk factors not only for MDD but also for other psychiatric disorders including BPD and SCZ. [24][25][26] However, no study to date has examined whether the established risk gene CACNA1C interacts with established environmental risk factors to shape disease outcome.
Functional LTCCs are hetero-oligomeric complexes consisting of multiple subunits: α1, β, α2, δ and/or γ. The voltage sensor, selectivity filter, ion-conduction pore and binding site for all available calcium channel blockers is encoded by the α1 subunit. The LTCC family consists of four distinct members, Ca v 1.1-Ca v 1.4, with mainly Ca v 1.2 and Ca v 1.3 shown to have a prominent role in the brain, 19,22,23 and Ca v 1.2 accounting for~85% of the LTCCs. 27 Pharmacological agents, such as the LTCC-targeting dihydropyridines, have frequently been applied to assess the function of LTCCs in the central nervous system (CNS). However, this approach is limited by the fact that all LTCC antagonists available to date are not completely selective for either Ca v 1.2 or Ca v 1.3, and that most studies investigated acute rather than long-term effects. More sophisticated genetic approaches using transgenic mice have helped to address the selective functional roles of different LTCCs. 19,22,23 Constitutive deletion of Cacna1c was shown to result in embryonic lethality 28 and conditional inactivation of Ca v 1.2 in forebrain structures was repeatedly associated with impairments in cognitive function. [29][30][31][32] More recently, haploinsufficiency and forebrain-specific deletion of Cacna1c were also associated with anxiety-like behavior 33,34 and sleep disturbances, 35 two core endophenotypes of MDD and BPD. In addition, Cacna1c was shown to mediate survival of young hippocampal neurons 36 and brain-specific deletion of Cacna1c impairs discrete forms of hippocampal-dependent memory and neurogenesis within the dentate gyrus. 37 However, the underlying neuronal circuits that modulate the effects of CACNA1C on synaptic plasticity and behavior remain largely unknown.
In this study we aimed to investigate how CACNA1C modulates the risk to psychiatric disorders in dependence of environmental, developmental and circuit specific factors, using both genetic mouse models and human data.

MATERIALS AND METHODS
For a detailed description of the Materials and Methods, please refer to the Supplementary Information.

Chronic social defeat stress paradigm
The chronic social defeat stress (CSDS) paradigm is commonly applied to induce anxiety-and depression-related endophenotypes in mice, and was performed as previously described. [41][42][43] See also Supplementary Information.

Behavioral testing and study design
Locomotion and sociability were investigated with the open field (OF) test and the three chamber apparatus respectively, as previously described. 41,44 Anxiety-related behaviour was assessed in the light/dark box test as previously described. 45 Spatial memory learning and object recognition were investigated with the water cross maze (WCM) 46 and spatial object recognition task. The forced swim test (FST) was used to assess passive vs active stress-coping behavior (behavioral despair) and corticosterone levels in response to an acute stressor as previously described. 41 For details, refer to online Supplementary Information.
For the basal behavioral characterization of Cav1.2-Dev Glu-CKO (Figure 2), Cav1.2-Ad Glu-CKO ( Figure 2) and Cav1.2 CNS-CKO mice (Supplementary Figure S7), one batch of animals was used for each line with the following order of tests (OF, sociability, dark/light box test, FST and corticosterone assessment). A second and third batch of animals was used for the WCM test and long-term potentiation (LTP) recordings, respectively. In addition, a fourth batch of Cav1.2-Ad Glu-CKO mice ( Figure 4) was used for the CSDS paradigm with the following test order (sociability, OF, object recognition test, dark/light box, FST and subsequent corticosterone, adrenal gland and thymus weight assessment). The same applies to Cav1.2-Ad Glu-Het mice (Supplementary Figure S9). For the CSDS experiment in Cav1.2 Het mice ( Figure 3), two separate batches of animals were used. For batch 1, the OF and dark/light box test were performed. For batch 2, the following tests were performed in this order: sociability, object recognition test, FST and subsequent corticosterone, adrenal gland and thymus weight assessment.

Electrophysiology
The influence of Cacna1c deficiency on hippocampal LTP was conducted as previously described. 47 For details, please refer to online Supplementary Information.

Single and double in situ hybridization
Single and double in situ hybridization (ISH) was performed as previously described. 48 The Cacna1c probe was designed to target the loxP flanked

Study design (gene × environment interaction in humans)
Participants in this study belonged to a larger cohort (Grady Trauma Project) investigating the role of genetic and environmental factors in predicting outcomes to stressful life events. [49][50][51] Phenotypes and genotypes were available for a total of 4,023 individuals, predominantly African Americans belonging to a highly traumatized, urban population of low socioeconomic status. All procedures were approved by the institutional review boards of Emory University School of Medicine and Grady Memorial Hospital. Please see Supplementary Methods for further details and statistics.

Statistical analysis
Statistical analyses were performed using the commercially available software SPSS v16.0 (SPSS, Chicago, IL, USA) and GraphPad Prism v5.0 (GraphPad Software, La Jolla, CA, USA). The sample size was chosen such that with a type 1 error of 0.05 and a type 2 error of 0.2 the effect size should be at least 1.2-fold of the pooled s.d. All results are presented as mean ± s.e.m. Behavioral phenotypic differences between two genotypes were evaluated with Student's t-test (two-tailed). If data were not normally distributed, the non-parametric Mann-Whitney test was used. Time-dependent measures were assessed with multi-factorial analysis of variance (ANOVA) with repeated measures. For CSDS experiments, the effects of genotype and condition on all other behavioral and neuroendocrine parameters were assessed by two factorial ANOVA (twoway ANOVA). Whenever significant main or interaction effects were found by the ANOVAs, Bonferroni post hoc tests were carried out to locate simple effects. Statistical significance was defined as Po0.05. All data were tested for outlieres using the Grubb' test. Homogeneity of variance was tested using Bartlett's test. Animals were allocated to the experimental groups in CACNA1C modulates stress susceptibility N Dedic et al a semi-randomized manner and data analysis was performed blinded to the group allocation.

Forebrain Cacna1c is predominately expressed in glutamatergic neurons
Cacna1c is expressed throughout the mouse brain, including key limbic regions relevant for emotion and cognition such as the prefrontal cortex (Ctx), hippocampus (Hip) and amygdala ( Figure 1a). Double ISH against Cacna1c and markers of excitatory glutamatergic ( Deletion of Cacna1c from forebrain glutamatergic neurons during development and adulthood induces differential effects on anxiety and opposing effects on cognition In view of the results described above, we set out to genetically dissect the role of Cacna1c in glutamatergic neurons. For this, we crossed floxed Cacna1c mice to Nex-Cre mice, 38 in which Cremediated recombination is initiated during embryonic development (E11.5) in forebrain glutamatergic neurons. As observed from the ISHs and double ISHs, Cacna1c ablation in forebrain glutamatergic neurons (Cav1.2-Dev Glu-CKO ) was largely restricted to Vglut1-expressing cells, including the Ctx, CA1/2/3 of the Hip, and lateral and basolateral amygdala (Figures 2a and b, and Supplementary Figures S2). Next, we assessed behavioral endophenotypes known to be altered in animal models of MDD, BPD and SCZ, including locomotion/exploration in the OF test, anxiety in the dark/light box test, immobility in the FST, social behavior in the three-chamber test (sociability test), as well as spatial learning and memory in the WCM task. Similar to humans, deficits in cognition and social behavior are recapitulated by mouse models of numerous psychiatric disorders including SCZ, BPD and MDD. [52][53][54] Hyperactivity in the OF and FST are often associated with mania in mouse models of BPD, 53,55 whereas enhanced anxiety represents a core endophenotype of MDD and the depressive phase of BPD in rodents. 52 However, changes in locomotor activity are also commonly reported in animal models of SCZ and MDD. 52,54 Compared with their respective littermate Ctrls, Cav1.2-Dev Glu-CKO mice exhibited increased anxiety in the dark-light box test and decreased preference for the social counterpart in the sociability test (Figures 2c and d). In the FST, Cav1.2-Dev Glu-CKO mice exhibited increased active stress-coping behavior (reduced behavioral despair), evident by decreased immobility (Figure 2e). In addition, Cav1.2-Dev Glu-CKO mice displayed a pronounced hyperlocomotion in the OF test, which was not observed during the initial 5 min where novelty-induced anxiety is most prominent (Figure 2f). The OF was performed under low light conditions (15 lux) in order to minimize anxiety effects on locomotion, which likely explains the lack of effects on inner zone time ( Figure 2f). Hip-dependent spatial learning and memory performance was investigated in the WCM. 46 Compared with the regular Morris water-maze test, the simplicity of the WCM leads to short trial durations and therefore reduces the stress load compared to Morris water-maze training. In addition, using accuracy rather than speed as the main readout, allows for hippocampus-dependent strategies to be assessed from the first training day on. Cav1.2-Dev Glu-CKO mice displayed accuracy levels that barely surpassed the chance level of 50%, both during learning and relearning (Figure 2g), suggesting drastically impaired cognitive performance. In view of this strong effect on cognition, we additionally assessed hippocampal LTP at the Schaffer collateral-CA1 synapses. One hour after a 100 Hz tetanus stimulation, Cav1.2-Dev Glu-CKO showed a significant decrease in LTP compared with Ctrl mice (Figure 2h). Next we addressed whether the underlying neurobiological changes responsible for the behavioral alterations in Cav1.  (Figure 2o). Considering that deletion of forebrain Cacna1c was reported to impair long-term memory, 32 we re-exposed the animals to the WCM 30 days after relearning, but did not observe any differences between Cav1.2-Ad Glu-CKO and Ctrl mice (Supplementary Figure S6g). In view of our results that developmental inactivation of Cacna1c from glutamatergic neurons decreases NMDA receptor (NMDAR)-dependent synaptic plasticity and impairs spatial memory, we additionally assessed hippocampal LTP in Cav1.2-Ad Glu-CKO mice. Intriguingly, 1 h after a 100 Hz tetanus stimulation, Cav1.2-Ad Glu-CKO showed increased LTP compared with Ctrl mice (Figure 2p). Importantly, we observed that CNS-specific Cacna1c deletion (Cav1.2 CNS-CKO mice), which is initiated at E8, 56 results in similar behavioral alterations compared with Cav1.2-Dev Glu-CKO mice (Supplementary Figure S7). This further supports partially opposing roles for developmental and adult Cacna1c in emotion and cognition, and argues against the possibility that differences between Cav1.2-Dev Glu-CKO and Cav1.2-Ad Glu-CKO mice are due to the slight discrepancies in recombination patterns between the Nex-Cre and Camk2a-CreERT2. In addition, the observed behavioral changes were not caused by    Figures S6c,d).
Cacna1c differentially modulates susceptibility to CSDS during development and adulthood Chronic stress and/or trauma represent strong risk factors for a number of psychiatric disorders, including MDD, BPD and anxiety disorders such as post-traumatic stress disorder. [24][25][26]57 In view of the phenotype in Cav1.2-Dev Glu-CKO mice, we wondered whether developmental deletion of Cacna1c would increase the susceptibility to CSDS. We choose to investigate heterozygous Cacna1c animals (Cav1.2 Het , Figure 3a), considering that Cav1.2-Dev Glu-CKO mice already display strong behavioral deficits under baseline conditions, which might not be further aggravated by CSDS. Importantly, no gross behavioral abnormalities were previously reported for Cav1.2 Het mice. 33 Cav1.2 Ctrl and Cav1.2 Het mice were subjected to 3 weeks of CSDS. In accordance with other studies, 41,42 we were able to detect robust physiological and neuroendocrine changes evoked by CSDS, independent of genotype, demonstrating the efficacy of the paradigm (Supplementary Figures S8a-d). Chronically stressed Cav1.2 Het mice displayed drastically reduced locomotion throughout the entire test duration compared to both, non-stressed Cav1.2 Het and stressed Cav1.2 Ctrl mice ( Figure 3b). In addition, only chronically stressed Cav1.2 Het mice showed a significant reduction in the number of inner zone entries of the OF, indicating increased anxiety-related behavior even under lowlight test conditions (Figure 3c). Moreover, CSDS increased the latency to enter the lit compartment and decreased the time spent in the lit zone and number of lit entries to a much greater extent in Cav1.2 Het than in Cav1.2 Ctrl mice (Figures 3d-f), further supporting increased stress-induced anxiety in heterozygous Cacna1c mice. Sociability, immobility in the FST and spatial object recognition memory were not differentially affected by CSDS (Figures 3g-i), suggesting a specific impact of the interaction between Cacna1c and chronic stress on anxiety.
In view of the partially opposing effects of Cacna1c on anxiety and cognition during development and adulthood, we wondered whether stress vulnerability would also be differentially affected upon deletion of the calcium channel in adulthood. Physiological and neuroendocrine parameters were similarly altered by CSDS in Ctrl and Cav1.2-Ad Glu-CKO mice (Supplementary Figures 8e-h). In contrast to Cav1.2 Het mice, locomotion/exploration in the OF was only decreased in stressed Ctrl but not stressed Cav1.2-Ad Glu-CKO mice, with a similar trend in the number of inner zone entries (Figures 4a and b). In the dark/light box test, only Ctrl mice responded to CSDS with a significant reduction in time spent in the lit zone and number of entries, and a trend toward delayed latencies (Figure 4c), which implies enhanced resilience to CSDS in Cav1.2-Ad Glu-CKO mice. FST behavior, sociability and spatial object were similarly affected by CSDS in Ctrl and Cav1.2-Ad Glu-CKO mice (Figures 4d-f). Considering that we observed susceptibility to CSDS in heterozygous Ca v 1.2 mice, we additionally assessed whether a heterozygous deletion in glutamatergic neurons during adulthood (Cav1.2-Ad Glu-Het ) would be sufficient to induce stress resilience (Supplementary Figure S9). However, compared with Cav1.2-Ad Glu-CKO , Cav1.2-Ad Glu-Het mice only exhibited partial resilience to CSDS-induced anxiety in the dark/light box test (Supplementary Figure S9f). This points to a gene-dosage effect and suggests that complete absence of Cacna1c during adulthood, in forebrain glutamatergic neurons, is required to induce the extent of resilience to CSDS observed in Cav1.2-Ad Glu-CKO mice.
CACNA1C interacts with adult trauma to predict depression symptoms in humans A number of CACNA1C SNPs associated with BPD, SCZ and MDD were also shown to affect measures of anxiety, depression,

DISCUSSION
Our results demonstrate that spatial memory, hippocampal plasticity and anxiety-related behavior are differentially affected by the loss of Cacna1c from excitatory circuits depending on whether it is deleted during embryonic development or adulthood. Increased anxiety-related behavior as well as reduced social and cognitive performance, observed in Cav1.2-Dev Glu-CKO mice, are considered core endophenotypes of MDD and/or the depressive phase of BPD, 52,53 whereas increased locomotion and activity in the FST are often associated with mania in animal models of BPD. 53,55 Decreased immobility in the FST was previously reported for heterozygous mice by Dao et al., 33 which is in line with our results and earlier work demonstrating antidepressant-like effects for LTCC blockers. 27,61 However, studies using LTCC blockers have to be interpreted with caution, as these have been reported to induce nonspecific, aversive and stress-like behavioral effects possibly due to excessive blood pressure lowering caused by inhibition of Cav1.2 in the cardiovascular system. [62][63][64] Importantly, hyperlocomotion, altered social behavior, and learning and memory impairments, are also analogous to negative and cognitive symptoms of SCZ, a disorder believed to have neurodevelopmental origins. 24,54 Additional evidence also suggests that two missense mutations in CACNA1C predispose to autism, 65,66 another neurodevelopmental disorder characterized by impaired social interactions and altered cognition. In accordance with the observed cognitive impairments, Cav1.2-Dev Glu-CKO mice also displayed deficits in hippocampal LTP, which is considered the cellular correlate of learning and memory. 67 Moreover, we show that Cacna1c strongly interacts with the environment to shape anxiety-related behavior. Heterozygous deletion of Cacna1c, which is present throughout life starting at the earliest point of development (one-cell stage), significantly increased the susceptibility to CSDS and is likely to be mediated by Cacna1c absence in forebrain glutamatergic neurons. Although CACNA1C SNPs have not been consistently associated with anxiety in humans, altered anxiety-related behavior is considered a core endophenotype of MDD and BPD in animal models. 52,53,55 Different to our observations, a recent study showed that chronic unpredictable stress induces similar behavioral deficits in heterozygous Ca v 1.2 mice and their wild-type littermates when assessed 1-2 days post stress. Surprisingly, the stress effects persisted in wild-type mice when tested 5-7 days later but not in heterozygous knockout mice. 68 These discrepancies might be related to the different chronic stress paradigms. In contrast to a developmental inactivation, deletion of Cacna1c from forebrain glutamatergic neurons during adulthood blocked the adverse effects of CSDS on anxiety, enhanced LTP and improved hippocampus-dependent cognitive flexibility during the WCM, without affecting long-term memory. Our findings in Cav1.2-Dev Glu-CKO mice are supported by previous studies, showing that deletion of Cacna1c from pan-neuronal 37 or glutamatergic neurons during development 29 impairs synaptic plasticity and/or spatial learning, some of which might be attributed to deficits in adult neurogenesis. 37 However, it should be kept in mind that our LTP results cannot be entirely correlated with cognitive performance considering that we stimulated CA3-CA1 Schaffer collaterals, which leads to the potentiation of most CA3-CA1 synapses rather than those that would selectively be recruited during a learning and memory task. Electrophysiological and pharmacological data have indicated that LTP induction is mainly initiated through NMDAR-activity but can also include NMDARindependent and VGCC-dependent mechanisms. 69,70 Interestingly, the decline of some memory processes is attributed to the shift from NMDAR-dependent to VGCC-intracellular calcium storesdependent regulation of synaptic plasticity, especially during aging. [71][72][73] Along these lines, a recent study provides evidence for an association between increased hippocampal Cacna1c expression and age-related cognitive decline. 74 Although we did not assess aged animals, it can be speculated that ablation of Ca v 1.2 in adulthood partially blocks an evolving shift towards VGCC-and/or intracellular calcium store-dependent regulation of synaptic plasticity, hence facilitating NMDAR-dependent LTP. This potentially suggests that Hip-dependent spatial memory formation requires Ca v 1.2 during prenatal development, but not in adulthood where activation of Ca v 1.2 channels is even detrimental for LTP. Notably, calcium channel antagonists were shown to ameliorate age-related deficits in hippocampus-dependent memory, possibly mediated through facilitation of NMDAR-LTP or inhibition of long-term depression. 71,73,[75][76][77][78] On the other hand, specific inactivation of Cacna1c in the anterior cingulate between postnatal weeks 8-10, impaired observational fear learning without altering novel object recognition memory and classical fear conditioning, 30 highlighting the requirement for Ca v 1.2 during adulthood for specific cognitive functions. The fact that we observed no significant changes in spatial object memory performance under basal or stress conditions further implies that adult-specific Cacna1c deletion in excitatory circuits does not impact all cognitive domains. As observed in Cav1.2-Dev Glu-CKO mice, locomotion was also enhanced in Cav1.2-Ad Glu-CKO animals; however, social and active stress-coping behavior were only mildly affected, suggesting that these endophenotypes are partly regulated by forebrain-Ca v 1.2 during development and adulthood. In contrast, an earlier study demonstrated that forebrain-specific elimination of Cacna1c, using a non-inducible Camk2α-Cre, also results in increased anxiety-like behavior. 34 The discrepancy could potentially be attributed to different Cacna1c deletion time windows (p18 79 vs p91-97 in our study).
Although the Nex-Cre and Camk2α-CreER T2 drivers primarily target glutamatergic forebrain neurons, they do slightly differ in their recombination patters (for example,the Camk2α-driven CreER T2 is also expressed in parts of the thalamus, DG and central nucleus of the amygdala). However, the fact that developmental, CNS-specific Cacna1c knockout mice showed similar behavioral deficits as Cav1.2-Dev Glu-CKO mice, further supports opposing roles for Ca v 1.2 during development and adulthood. Nevertheless, we cannot rule out an important contribution of Ca v 1.2-expressing, GABAergic neurons in many of the disease-associated phenotypes. In fact, Cacna1c was reported to modulate processes related to drug addiction via GABAergic medium spiny neurons in the nucleus accumbens, 80,81 and promote susceptibility to social stress when deleted from this specific brain region. 82 It is important to point out that we only assessed a subset of behavioral tests relevant to MDD, BPD and SCZ in our knockout mice. Future studies evaluating anhedonia, psychostimulant-induced locomotion, sleep architecture and deficits in sensorimotor gating (prepulse inhibition deficits), will further refine the contribution of developmental vs adult Ca v 1.2 in psychiatric disorders.
Given our observations in mice, we further investigated if Cacna1c x stress interactions could also be found in human samples and checked for interaction effects of CACNA1C variants and adult trauma to predict current depressive symptoms As expected, the risk to develop depressive symptoms was increased with exposure to adult trauma in our study and this effect was moderated by loci within CACNA1C tagged by rs73248708 and rs116625684. Both tag SNPs were not in linkage disequilibrium with the main haplotypes associated with SCZ with genome-wide significance, but represent independent loci. The intron 1 SNP rs116625684 shows a weak, nominal association with SCZ in the Psychiatric Genomics Consortium data set (P = 0.021), with an odds ratio of 0.94 (http://www.med.unc.edu/pgc), suggesting a protective effect of the minor allele, which is also indicated by our results for the highest trauma group. The second SNP, rs73248708 is located in intron 3, which harbors the majority of CACNA1C SNPs associated with SCZ at genome-wide significance and also those linked to other disorders including BPD and MDD. 19 In fact, SNPs in this region were shown to lie in predicted enhancers capable of physically interacting with the CACNA1C transcriptional start site, and altering gene expression. 83 Although rs73248708 is not in linkage disequilibrium with the top GWAS SNPs, including rs1006737, it may independently moderate the function of this enhancer region. In fact, we could show that different sets of genetic variants may be involved in baseline transcriptional regulation vs. regulation following environmental impact, such as trauma exposure. 84 Replication in independent cohorts will be necessary to further validate these loci in gene × environment interactions. Importantly, it remains to be investigated whether the here identified SNPs have implications on protein levels or are of other functional relevance. So far, both decreased and increased Ca v 1.2 levels have been associated with the common risk allele variant rs1006737, [85][86][87] which might imply different regulatory functions across different brain regions and/or cell types. Whether the here identified SNPs, as well as previously associated CACAN1C risk alleles, can differentially affect gene expression in the brain upon perturbations such as severe stress and/or trauma will have to be assessed in the future. Considering the heterogeneity of MDD, our data raises the possibility that SNPs in CACNA1C might predict the risk for depression mainly in individuals with a history of trauma or severe stress. This could partially explain the nominal associations of CACAN1C with MDD in former genetic studies, which usually do not Ctrl for environmental factors. In addition, given our animal findings it would be interesting to examine for age × trauma interaction effects in the human sample. However, in the present cohort we did not have any information with regard to when exactly adult trauma took place and therefore cannot evaluate age effects.
There are some limitations of the interpretability of our findings, which should not be neglected. First, the Grady Trauma Project includes mostly African Americans. We cannot rule out that effects of CACNA1C variants are different in this specific population as compared with the European population where the effect of CACNA1C variants with regard to MDD were described first. Second, the GRADY sample itself is a highly traumatized cohort. It is possible that the described effects are only present in samples which show a high trauma burden and cannot be found in less traumatized cohorts. However, findings in this cohort can help to transit the results from mice to human data.
Although circuit-or cell type-specific absence of CACNA1C is not likely to occur in humans, epigenetic changes caused by environmental alterations may very well induce expression changes in specific tissues, cell types and/or entire brain circuits. In the clinical context this could imply that, individuals with genetic variants promoting reduced CACNA1C expression may either be at higher or lower risk to develop psychopathologies, depending on whether they experienced severe stress/trauma during development or adulthood. The pronounced effect of a prenatal Cacna1c depletion on emotional behavior and cognitive performance might account for its association with disorders such as SCZ and autism, which are increasingly believed to result from synaptic dysfunction during development. The fact that CACNA1C is also able to interact with the environment during adulthood, by modulating stress susceptibility, could explain its linkage to stressrelated disorder such as MDD and BPD. At the same time, Ca v 1.2 and Ca v 1.3 are expressed in midbrain dopaminergic neurons and several studies suggest a neuroprotective effects of LTCC blockers CACNA1C modulates stress susceptibility N Dedic et al in Parkinson's disease, 22,88 which further emphasizes the possibility that inhibition of Ca v 1.2 may be of therapeutic value in brain disorders that manifest at later stages in life. Concluding, we propose that the association of CACNA1C with multiple psychiatric disorders is related to its broad expression within key limbic regions and neuronal circuits relevant to emotion, motivation and cognition, and that alterations in CACNA1C gene expression during development and adulthood can result in diverging behavioral outcomes and differentially impact stress susceptibility.