Dysfunction of cAMP–Protein Kinase A–Calcium Signaling Axis in Striatal Medium Spiny Neurons: A Role in Schizophrenia and Huntington’s Disease Neuropathology

Background Striatal medium spiny neurons (MSNs) are preferentially lost in Huntington’s disease. Genomic studies also implicate a direct role for MSNs in schizophrenia, a psychiatric disorder known to involve cortical neuron dysfunction. It remains unknown whether the two diseases share similar MSN pathogenesis or if neuronal deficits can be attributed to cell type–dependent biological pathways. Transcription factor BCL11B, which is expressed by all MSNs and deep layer cortical neurons, was recently proposed to drive selective neurodegeneration in Huntington’s disease and identified as a candidate risk gene in schizophrenia. Methods Using human stem cell–derived neurons lacking BCL11B as a model, we investigated cellular pathology in MSNs and cortical neurons in the context of these disorders. Integrative analyses between differentially expressed transcripts and published genome-wide association study datasets identified cell type–specific disease-related phenotypes. Results We uncover a role for BCL11B in calcium homeostasis in both neuronal types, while deficits in mitochondrial function and PKA (protein kinase A)–dependent calcium transients are detected only in MSNs. Moreover, BCL11B-deficient MSNs display abnormal responses to glutamate and fail to integrate dopaminergic and glutamatergic stimulation, a key feature of striatal neurons in vivo. Gene enrichment analysis reveals overrepresentation of disorder risk genes among BCL11B-regulated pathways, primarily relating to cAMP-PKA-calcium signaling axis and synaptic signaling. Conclusions Our study indicates that Huntington’s disease and schizophrenia are likely to share neuronal pathophysiology where dysregulation of intracellular calcium homeostasis is found in both striatal and cortical neurons. In contrast, reduction in PKA signaling and abnormal dopamine/glutamate receptor signaling is largely specific to MSNs.

Inhibitory GABA (gamma-aminobutyric acid)-releasing medium spiny neurons (MSNs) are the principal projection neurons of the basal ganglia, receiving inputs from both cortical glutamatergic neurons and midbrain dopaminergic (mDA) neurons. MSNs are critically involved in a variety of essential functions including voluntary motor control, habit learning, and reward processing, and dysfunction followed by loss of this neuron population underlies Huntington's disease (HD). A growing body of evidence identifies loss of function of transcription factor BCL11B (also known as CTIP2) as a driving force behind selective neuron degeneration in HD.
BCL11B is expressed by all MSNs and is required for transcriptional regulation of striatal genes, patch-matrix organization, spatial learning, and working memory (1)(2)(3)(4). BCL11B is also highly expressed by cortical layer V/VI neurons, where it plays a role in corticospinal motor neuron fate specification and axon development (5,6). BCL11B protein level is reduced in both human and rodent mutant huntingtin (mHTT)expressing cells, resulting in mitochondrial deficits prior to the onset of MSN death (7)(8)(9)(10). We have recently demonstrated that human BCL11B-deficient and HD MSNs share dysregulated gene expression and present with deficits in signature striatal protein phosphorylation, DARPP32, and GLUR1 (11). In addition to the striatum, BCL11B-expressing neurons in other brain regions are affected in the later stages of HD, such as the cortex, hippocampus, and hypothalamus (8). Thus, these findings point to an important role for BCL11B in HD pathogenesis.
Loss-of-function mutations in the BCL11B gene have also been identified to cause immunodeficiency and neurodevelopmental delay with speech impairment and intellectual disability (12). Furthermore, several studies have recently demonstrated genome-wide significant enrichment of polymorphisms increasing risk for schizophrenia (SCZ) in the BCL11B gene (13)(14)(15)(16)(17). It has been widely accepted that pathogenesis in neurodevelopmental and psychiatric disorders is driven by cortical interneuron and glutamatergic neuron dysfunction (18), but novel evidence suggests that MSNs also play an important distinct role. Several gene enrichment studies integrating single-cell transcriptomics and large psychiatric genome-wide association study datasets revealed that psychiatric risk variants were highly enriched in genes expressed by MSNs that differ from genes expressed by cortical neurons (17,(19)(20)(21). Considering that polymorphisms affect both pan-neuronal and subtype-specific neuronal genes, it is important and necessary to investigate whether resulting cellular pathology is distinct or shared between striatal and cortical cell populations. It also remains to be determined as to what extent HD and SCZ neuropathology may overlap due to disrupted function of BCL11B, and if the same or different biological pathways are at play in these conditions. Together, these findings lead to the hypothesis that BCL11B regulates important signaling processes in MSNs and cortical neurons, whether shared or distinct, that may be particularly vulnerable to psychiatric disorder risk variants and HD pathogenesis.
Using BCL11B-deficient human embryonic stem cells as a model to address the above questions, we demonstrate here a role for BCL11B in mitochondrial function, calcium signaling, and dopamine/glutamate signal processing predominantly in MSNs, with much milder deficits observed in cortical neurons. Our study reveals significant enrichment of psychiatric disorder risk genes in BCL11B-regulated signaling, including cAMP-dependent protein kinase A (PKA) and DARPP32 signaling, specifically in MSNs, and calcium and glutamatergic synaptic signaling in both neuronal types. Similar biological pathways are identified in HD, suggesting a shared role for BCL11B in the pathophysiology of HD and SCZ. We therefore prioritize promising targets for further mechanistic investigations and development of new therapeutics in these conditions.

Mitochondrial Assay
The cell-permeant mitochondrial membrane potential sensor JC-1 (Thermo Fisher Scientific) was added directly to live cells at a final concentration of 1 mg/mL for 30 minutes at 37 C.
Where indicated cells were preincubated with the nitric oxide donor SNAP (1000 mM; Tocris) for 24 hours. Samples were analyzed on a flow cytometer according to the manufacturer's protocol.

Electrophysiology
From 20 days of differentiation (days in vitro [DIV]), MSN progenitors were cultured in astrocyte-conditioned medium supplemented with maturation factors according to an established protocol (26). Whole-cell patch-clamp recordings were acquired from control and BCL11B KO #4 MSNs at 40 DIV in aCSF at room temperature. SKF-81297 (10 mM) was added to aCSF where specified, while DL-glutamic acid (200 mM) was focally applied (30 ms) to the patched cell and the evoked current response recorded. Data were analyzed with Clampfit software (Molecular Devices) and exported to and plotted using Origin (OriginLab).

Statistical Analysis
For normally distributed data as determined by the Shapiro-Wilk test, we performed either two-tailed Student t test or two-way analysis of variance followed by post hoc Bonferroni correction for multiple comparisons. Alternatively, data were subjected to nonparametric Mann-Whitney U test or Kruskal-Wallis test followed by post hoc Bonferroni test.

BCL11B-Deficient MSNs Display Mitochondrial Deficits
We showed previously that BCL11B KO MSNs exhibited increased oxidative stress-dependent cell death (11). Because mitochondria are known to play an active role in this complex cascade of events in various models of neurodegenerative disorders (29), we set out to investigate whether loss of BCL11B would compromise mitochondrial health in MSNs, cortical neurons, and mDA neurons. Mitochondrial membrane potential sensor JC-1 was used to measure metabolic activity, a key indicator of mitochondrial health ( Figure S2A) (30). Mitochondria in BCL11B KO MSNs were found to be significantly more depolarized than in control cells at 40 DIV, indicating decreased metabolic activity in these neurons ( Figure 1B).
To investigate whether this impairment in BCL11B KO MSNs would render them more susceptible to oxidative stress, cells were treated with the nitric oxide donor SNAP, which was previously shown to generate reactive oxygen species (31). This induced a marked depolarization of the mitochondrial membrane in both groups, with a twofold greater amplitude in BCL11B KO cultures ( Figure 1B, Figure S2B). We ascertained . All line graphs and dot plots depict mean 6 SEM for each genotype. Box-and-whisker plots depict data for each genotype (center line, median; 1, mean; box limits, upper and lower quartiles; whiskers, 2.5 and 97.5 percentiles). Means for individual clones are indicated by red-shaded circles next to BCL11B KO data. See also Figures S1 and S2. BDNF, brain-derived neurotrophic factor; CTX, cortical glutamatergic; KCI, potassium chloride; KO, knockout; LDN, LDN-193189; mDA, midbrain dopaminergic; MSNs, medium spiny neurons; n.s., not significant; SB, SB431542; SHH, sonic hedgehog; Tg, thapsigargin.

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Biological Psychiatry: Global Open Science July 2023; 3:418-429 www.sobp.org/GOS that the observed mitochondrial deficit was BCL11Bdependent in MSNs by repeating this assay in striatal progenitors prior to BCL11B expression and detecting no deficit ( Figure S2C). We next investigated whether these mitochondrial deficits were specific to MSNs by performing the above experiment in cortical and mDA cultures. Although exposure to SNAP had a significant depolarizing effect in BCL11B KO but not control cortical neurons after Bonferroni correction, there were no differences between the genotypes in either treatment group ( Figure 1C). In addition, no differences were detected due to loss of BCL11B in mDA neurons, with both groups responding to SNAP similarly ( Figure 1D). Taken together, our data suggest that BCL11B is required for regulation of mitochondrial membrane potential and has a protective role against oxidative stress, most prominently in MSNs and, to a lesser extent, in cortical neuron cultures.

BCL11B KO Neurons Present With Abnormal Intracellular Ca 21 Regulation
Mitochondrial dysfunction in neurons has previously been attributed to high intracellular Ca 21 levels, particularly in the context of neurologic disorders (32). Given the impairments in mitochondrial health in BCL11B KO neurons, we hypothesized that loss of BCL11B would result in deficits in intracellular Ca 21 signaling and therefore performed calcium imaging in BCL11B KO and control neuronal cultures at 50 DIV. To investigate the effect of BCL11B loss on the regulation of Ca 21 levels, intracellular Ca 21 was depleted using a Ca 21free aCSF solution and store-operated channel-mediated Ca 21 entry was induced by application of the sarco/endoplasmic reticulum Ca 21 ATPase inhibitor thapsigargin (33,34). Significantly larger store-operated channel-mediated Ca 21 signals, suggesting greater Ca 21 influx, were detected in both BCL11B KO MSNs and cortical, but not mDA, neurons compared with their respective control cells ( Figure 1E-G). Consistent with this finding, markedly larger Ca 21 signals in both BCL11B KO MSNs and cortical, but not mDA, neurons were also observed in response to depolarization of the plasma membrane by external potassium chloride ( Figure 1H-J). These data indicate that BCL11B-dependent abnormal regulation of intracellular Ca 21 may underlie the depolarized mitochondrial membrane potential and increased vulnerability to oxidative stress in MSNs and cortical neurons described above.   Figure S3A). In MSN cultures, smaller and less frequent DCa 21 were detected in BCL11B KO neurons compared with control cells (Figure 2A). Indeed, this was reflected in a 36% decrease in DCa 21 amplitude and a 33% increase in inter-DCa 21 interval in BCL11B KO MSNs ( Figure 2B). This phenotype was specific to MSNs, because no deficits were detected in BCL11B KO cortical and mDA neurons, either in DCa 21 amplitude or intervals ( Figure 2B). We next sought to gain mechanistic insight into the reduction in spontaneous DCa 21 in BCL11B KO MSNs. Crosstalk between PKA and dopamine signaling pathways plays an important role in calcium signaling and DARPP32 phosphorylation in MSNs (35,36). Given reduced levels of DARPP32-Thr34 phosphorylation and PKA signaling in BCL11B KO MSNs identified previously (11), we hypothesized that activating the PKA pathway would rescue DCa 21 deficits in these neurons. To this end, we compared the effects of drugs known to modulate PKA/DRD1-mediated calcium signaling in the striatum, including CDK5 inhibitor roscovitine, DRD1/DRD5 agonist SKF-81297, and cAMP analog 8-bromo- All line graphs and dot plots depict mean 6 SEM for each genotype. Box-andwhisker plots depict data for each genotype (center line, median; 1, mean; box limits, upper and lower quartiles; whiskers, 2.5 and 97.5 percentiles). Means for individual clones are indicated by red-shaded circles next to BCL11B KO data. See also Figure S4. AP, action potential; Ctrl, control; CTX, cortical glutamatergic; KO, knockout; mDA, midbrain dopamine; MSNs, medium spiny neurons; n.s., not significant; RMP, resting membrane potential.  Figure 2C, Figure S3B). Together, these findings suggest that loss of BCL11B disrupts PKA-dependent calcium signaling in MSNs, a deficit in spontaneous DCa 21 that is not observed in either cortical or mDA neurons.

Abolished Dopaminergic Modulation of Excitatory Signaling and Impaired Glutamate-Evoked Responses in BCL11B KO MSNs
Glutamatergic transmission in MSNs is known to be enhanced by dopamine acting on postsynaptic DRD1 (38). Considering abnormal dopaminergic and glutamatergic synaptic signaling in BCL11B-deficient MSNs suggested in a prior study (11), we next inspected physiological implications of this by performing patch-clamp electrophysiology in MSN cultures at 40 DIV. While both control and BCL11B KO neurons displayed similar basic membrane properties, firing frequency was markedly higher in BCL11B KO MSNs ( Figure 3A, B). Glutamate-evoked currents were not significantly different between BCL11B KO and control cells at baseline; however, only control MSNs showed a significant increase in the current amplitude following treatment with DRD1/DRD5 agonist ( Figure 3C, Figure S4A). Intracellular Ca 21 is delicately balanced and plays an indisputable role in determining neuronal excitability. Therefore, we next tested the effect of BCL11B loss on neuron response to excitatory stimulation by measuring DCa 21 evoked by glutamate pulses in MSNs, cortical neurons, and mDA neurons ( Figure 3D-F). BCL11B KO MSNs initially exhibited larger glutamate-evoked DCa 21 but desensitized faster than control MSNs, a sign of excitotoxicity ( Figure 3D). Indeed, significantly greater increases in intracellular Ca 21 levels over time together with fewer glutamate-evoked responses were observed in BCL11B KO MSNs compared with control cells. This phenotype was mostly specific to MSNs because only a  Table S2). (B) BCL11B target genes within shortlisted core biological processes affected by the loss of BCL11B in MSN and CTX neurons. Next to gene name, color icons indicate significant association (differential expression or identified risk variant) with HD (pink), SCZ (green), NDD (blue), and ASD (yellow). (C) Exclusion of BCL11B target genes from differentially expressed gene list results in several pathways in MSNs becoming either drastically less significant or not significant at all, including cAMP-PKA-calcium signaling axis pathways and dopamine synapse signaling, while having almost no effect on signaling pathways in CTX neurons. (D) BCL11B-regulated signaling abnormalities in MSNs and CTX neurons are shared by HD neurons (full gene set lists are presented in Table S4). In pathway graphs (A, D), dot size corresponds to gene set size, dot color corresponds to 2log 10 (pval), where p_adj , .05 dot is framed inside a black circle. See also Figures S5 and S6 and Tables S1-S4. ASD, autism spectrum disorder; BDNF, brain-derived neurotrophic factor; CTX, cortical glutamatergic; DA, dopamine; excl., exclusion; GABA, gamma-aminobutyric acid; HD, Huntington's disease; incl., inclusion; mHTT, mutant huntingtin; MSNs, medium spiny neurons; NDD, neurodevelopmental disorder; p_adj, p adjusted; PKA, protein kinase A; SCZ, schizophrenia.  Figure 3E), while no differences were seen in mDA neurons due to loss of BCL11B ( Figure 3F).

BCL11B Role in SCZ and HD Striatal and Cortical Neurons
A key measurement of neuronal functional maturity is the development of complex morphological features such as dendritic branching. Analysis of neuron morphology revealed no striking differences between control and BCL11B KO MSNs ( Figure S4B-E). In conclusion, although BCL11B-deficient MSNs were largely comparable to control cells in membrane properties and morphology, they presented with significantly impaired responsiveness to physiological stimuli, including glutamate-evoked Ca 21 signaling and DRD1-mediated modulation of glutamate-evoked currents, a feature characteristic of MSNs in vivo (38).

cAMP-PKA-Calcium Signaling Axis Is Driven by BCL11B-Dependent Transcription Programs
To investigate molecular mechanisms and pathways leading to pathological changes in BCL11B KO MSNs and cortical neurons, we performed whole-transcriptome RNA sequencing analysis at different stages of differentiation (Table S1). To elucidate specific biological processes regulated by BCL11B, we performed Ingenuity Pathway Analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis of proteincoding differentially expressed genes (DEGs) (false discovery rate p adj , .01). Genes regulating calcium signaling, mitochondrial function, and oxidative phosphorylation were found to be significantly altered in both neuronal types ( Figure 4A, Table S2). Moreover, DA-DARPP32 feedback in cAMP signaling deficits was specific to the MSN population, while PKA and cAMP pathway dysregulation was stronger in MSNs but also present in cortical neurons. Furthermore, synaptic genes regulating dopamine, glutamate, and GABA neurotransmitters were significantly altered in MSNs from as early as 20 and 30 DIV. In contrast, only glutamatergic and GABAergic synapse signaling was affected in cortical neurons. In line with previously identified roles for BCL11B in the brain (3,4), we also observed significant dysregulation of BDNF (brain-derived neurotrophic factor) signaling specifically in MSNs, as well as abnormal learning and memory signaling in both neuronal types.
To further elucidate the role of BCL11B in the identified pathogenic pathways, we asked whether the enriched pathways are driven by BCL11B target genes ( Figure 4B, Figure S5). Once BCL11B target genes were excluded from the DEG list, several pathways in MSNs became either drastically less significant or not significant at all, including cAMP-PKAcalcium signaling axis pathways and dopamine synapse signaling ( Figure 4C, Table S2). While BCL11B target genes appeared to play a weak role in cAMP-PKA signaling in cortical neurons, their exclusion did not affect calcium or dopamine synapse signaling in these cells. Transcriptomic data corroborates cellular pathologies identified in knockout neurons, suggesting that BCL11B plays a role in mitochondrial and intracellular Ca 21 signaling in both neuronal types, while its MSN-specific role converges on a few select pathways primarily regulating the cAMP-PKA-calcium signaling axis and downstream glutamate/DA-DARPP32 neurotransmission.

BCL11B-Regulated Signaling Abnormalities Are Shared by HD Neurons
BCL11B hypofunction has previously been demonstrated to play a role in MSN degeneration in HD by regulating  Table S7). In graphs, dot size corresponds to gene set size, dot color corresponds to 2log 10 (pval), where p_adj , .05 dot is framed inside a black circle. See also Figures S5 and  S6 and Tables S5-S7. ASD, autism spectrum disorder; CTX, cortical glutamatergic; DA, dopamine; GABA, gamma-aminobutyric acid; KO, knockout; MSNs, medium spiny neurons; NDD; neurodevelopmental disorder; p_adj, p adjusted; PKA, protein kinase A; SCZ, schizophrenia.

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Biological Psychiatry: Global Open Science July 2023; 3:418-429 www.sobp.org/GOS Biological Psychiatry: GOS mitochondrial signaling and protein phosphorylation (8,11). We therefore investigated potential overlap between BCL11B-and mHTT-mediated transcriptomic changes by comparing our DEGs in both neuronal types with four publicly available datasets from human and mouse HD models ( Figure S6) (7,10,39,40). Gene set enrichment analysis indeed revealed that all MSN and cortical DEGs were significantly enriched for mHTT-regulated genes (Table S3). Further investigation of concordantly dysregulated genes between BCL11B KO and HD models confirmed deficits in DA-DARPP32 signaling, cAMP-PKA-calcium signaling axis, synaptic signaling, oxidative phosphorylation, and mitochondrial function predominantly in MSNs and to a lesser extent in cortical neurons ( Figure 4D, Table S4). Multiple BCL11B target genes in these pathways have been previously shown to be affected in HD ( Figure 4B). These results provide further support for a regulatory role for BCL11B in the cAMP-PKA-calcium signaling axis and downstream DA-DARPP32 neurotransmission events, processes that are particularly vulnerable to mHTT.

A Role for cAMP-PKA-Calcium Signaling Axis in the Neuropathology of Psychiatric Disorders
A direct role for MSNs in psychiatric disease pathogenesis has been suggested by recent gene enrichment studies (19)(20)(21). Moreover, independent studies have fine-mapped and prioritized the BCL11B gene among a few others as a candidate causal SCZ risk gene (17,20,41). Considering these reports, we wonder whether the BCL11B-dependent neuronal phenotypes described in this study may contribute to pathogenesis in psychiatric conditions. To gain support of this hypothesis, we investigated BCL11B-regulated genes in MSNs and cortical neurons, with particular interest in the signaling pathways responsible for the observed convergent and MSN-specific deficits, and their enrichment for risk variants in SCZ, neurodevelopmental disorder (NDD), and autism spectrum disorder (ASD). Neurologic disease gene sets were collated by integrating findings from transcriptomics, genome-wide association study datasets, and other functional genomics studies of SCZ (13,14,20,21,(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54), NDD (51,53,55), and ASD (46,53,56) ( Figure S6, Table S5). In line with neurodevelopmental components reported in SCZ and ASD, an overlap of about one third of genes was detected between these two conditions and the NDD gene set. Gene set enrichment analysis revealed that BCL11B KO MSN DEGs were enriched for SCZ and NDD risk genes but not ASD risk genes at all stages of differentiation, while CTX30 DEGs were enriched for all disease risk genes ( Figure 5A, Table S6). In MSN cultures, BCL11B-dependent enrichment of DA-DARPP32 and cAMP-PKA-calcium signaling functional terms was much stronger and appeared earlier for SCZ than NDD risk genes ( Figure 5B, Table S7). Furthermore, SCZ risk variants were enriched in synaptic signaling affecting all three neurotransmitters, while NDD risk genes were present only in glutamatergic and GABAergic synaptic pathways. In cortical cultures, dysregulated pathways were more enriched for SCZ and ASD than NDD risk variants. Most BCL11B target genes in these pathways have been previously implicated in at least one of these three neurologic disorders ( Figure 4B). Together, these findings suggest that synaptic signaling and cAMP-PKA-calcium signaling axis dysregulation may contribute to SCZ neuropathology in the striatum. In contrast, in cortical neurons, some of these BCL11B-dependent phenotypes, although affected less severely, may contribute to both SCZ and ASD pathophysiology.

DISCUSSION
In this study, we provide evidence supporting a role for corticostriatal transcription factor BCL11B predominantly in HD and SCZ than in NDD and ASD neuropathology. We further strengthen the hypothesis that MSN dysfunction may contribute separately from cortical neuron pathology to psychiatric disease development. Such neuron subtype and multiple disorder cross-examination allows the opportunity to study gene expression patterns and determine convergent versus distinct BCL11B-regulated mechanisms contributing to pathogenesis of different diseases. Indeed, our gene enrichment analysis and functional assays reveals a limited number of core biological processes affected by the loss of BCL11B that are highly specific to the MSN population, such as the cAMP-PKA-calcium signaling axis, DA-DARPP32 signaling, glutamate-evoked calcium signaling, and mitochondrial health ( Figure 6). Intracellular Ca 21 signaling deficit is the only phenotype common between MSNs and cortical neurons, while other calcium and mitochondrial signaling deficits are only mildly present in cortical neurons. Transcriptomic analysis suggests that identified BCL11Bdependent biological processes are mainly concordant between HD and SCZ in MSNs, while cortical neuron pathways were also enriched for ASD risk variants. Furthermore, our study predicts involvement of BCL11B target genes in regulating these pathways, with many genes identified either as risk factors for or differentially expressed in psychiatric disorders.
Transgenic expression of Bcl11b in STHdh Q111 HD cells (that exhibit reduced levels of BCL11B) partially rescued mHTT-induced defects in mitochondrial metabolic activity (8). This suggests that restoring or increasing BCL11B levels can reverse at least some of the mHTT-driven impairments. Mitochondrial dysfunction and elevated intracellular Ca 21 levels are highly interdependent phenotypes in neurodegenerative diseases (32). Similar to HD cells, BCL11B-deficient neurons present with mitochondrial deficits, vulnerability to oxidative stress, and abnormal regulation of intracellular Ca 21  A connection between disturbed calcium signaling, excitatory stimulation, and MSN apoptosis has been established in HD models (34,(71)(72)(73)(74)(75). We demonstrate that glutamate induces elevated Ca 21 responses in BCL11B KO MSNs, a feature of excitotoxicity, which suggests that BCL11B hypofunction might be the cause of aberrant calcium signaling and MSN apoptosis observed in HD. Although BCL11B knockout appears to enhance intrinsic excitability of MSNs, it simultaneously impairs dopaminergic modulation of glutamatemediated excitation. Together, these results provide strong evidence of a central role for BCL11B in regulating calcium homeostasis in MSNs and, to some extent, in cortical neurons, the disruption of which in BCL11B-deficient and HD MSNs induces Ca 21 overload, leading to excitotoxicity MSNs and CTX neurons and their likely contribution to psychiatric disorder pathogenesis. (A) Schematic of core biological processes affected by the loss of BCL11B summarizes phenotypes identified in current and previous studies, including depolarized mitochondria (green), abnormal calcium signaling, acute glutamatergic neurotransmission, disbalance in phosphatase levels, reduced cAMP-PKA signaling, and downstream dephosphorylation of DARPP32 and other targets [adapted from (11)]. (B) These core molecular changes are most prominent in MSNs compared with CTX neurons and show preferential enrichment for HD and SCZ genes, suggesting that they are likely to contribute to the pathogenesis in these disorders. In contrast, in CTX neurons, a fraction of these BCL11B-dependent phenotypes, although affected less severely, may also contribute to ASD pathogenesis. (C) In summary, we propose that MSNs play a distinct role in psychiatric disease development compared with CTX neurons, where BCL11B hypofunction acts in tandem with mHTT in HD and other risk variants in psychiatric disorders to contribute to disease pathogenesis. AMPAR, AMPA receptor; ASD, autism spectrum disorder; CTX, cortical glutamatergic; HD, Huntington's disease; KO, knockout; mDA, midbrain dopaminergic; mHTT, mutant huntingtin; MSNs, medium spiny neurons; NDD, neurodevelopmental disorder; NMDAR, NMDA receptor; PKA, protein kinase A; SCZ, schizophrenia.

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Biological Psychiatry: Global Open Science July 2023; 3:418-429 www.sobp.org/GOS and pathological responses to physiological stimuli. These BCL11B-associated glutamatergic and dopaminergic signaling phenotypes may also be present in MSNs in SCZ.
Furthermore, PKA signaling was previously implicated in modulating intracellular Ca 21 levels and Ca 21 oscillations (35,76). In agreement with these findings, we demonstrate that deficits in spontaneous activity-evoked Ca 21 oscillations in BCL11B-deficient MSNs can be rescued in a PKA-dependent manner. Thus, we propose that BCL11B deficiency-driven disruption of calcium homeostasis in MSNs, together with dysregulated levels of protein phosphatases/kinases (11), result from reduced PKA signaling and lead to a marked decrease in phosphorylation of its targets. In addition, increased intracellular Ca 21 levels would induce overactivation of PP3-dependent DARPP32-Thr34 dephosphorylation (36). Identified disruption of PKA-regulated intracellular Ca 21 signaling and phosphorylation of DARPP32 in MSNs has significant implications for many psychiatric and neurodegenerative diseases (11,77,78). These processes regulate transcriptional and behavioral responses of MSNs to pharmacological stimuli, including antidepressants, neuroleptics, and drugs of abuse (79). Reduced levels of full-length DARPP32 and increased levels of DARPP32 isoforms lacking the crucial residue Thr34 were reported in patients with SCZ (80,81), which suggests disruption of DARPP32-Thr34 phosphorylation in SCZ MSNs. Moreover, abnormal postsynaptic PKA activity due to accelerated maturation of corticostriatal circuits was demonstrated to cause behavioral abnormalities in a Shank3B 2/2 mouse model of ASD (82). Indeed, we demonstrate that BCL11B-dependent cAMP-PKA-calcium signaling and DA-DARPP32 signaling pathways in MSNs are enriched for psychiatric disorder risk variants, pointing to a potential role for BCL11B-associated phenotypes in neuropathology in these disorders.
In conclusion, we identify BCL11B-regulated molecular mechanisms in striatal and cortical neurons and further strengthen the hypothesis that MSN dysfunction may contribute separately from cortical neuron pathology to psychiatric disease development. We provide evidence that genetic susceptibility loci within BCL11B-regulated pathways may modulate a limited number of core disease-related biological processes in MSNs, including cAMP-PKA-calcium signaling, DA-DARPP32 signaling, and glutamate neurotransmission. We propose that BCL11B-associated phenotypes may contribute to neuropathology most significantly in HD and SCZ and identify modulation of PKA-dependent Ca 21 signals and protein phosphorylation as potential new therapeutic targets in the striatum.