1,25-Dihydroxyvitamin D modulates L-type voltage-gated calcium channels in a subset of neurons in the developing mouse prefrontal cortex.

Schizophrenia has been associated with a range of genetic and environmental risk factors. Here we explored a link between two risk factors that converge on a shared neurobiological pathway. Recent genome-wide association studies (GWAS) have identified risk variants in genes that code for L-type voltage-gated calcium channels (L-VGCCs), while epidemiological studies have found an increased risk of schizophrenia in those with neonatal vitamin D deficiency. The active form of vitamin D (1,25(OH)2D) is a secosteroid that rapidly modulates L-VGCCs via non-genomic mechanisms in a range of peripheral tissues, though its non-genomic effects within the brain remain largely unexplored. Here we used calcium imaging, electrophysiology and molecular biology to determine whether 1,25(OH)2D non-genomically modulated L-VGCCs in the developing prefrontal cortex, a region widely implicated in schizophrenia pathophysiology. Wide-field Ca2+ imaging revealed that physiological concentrations of 1,25(OH)2D rapidly enhanced activity-dependent somatic Ca2+ levels in a small subset of neurons in the developing PFC, termed vitamin D-responsive neurons (VDRNs). Somatic nucleated patch recordings revealed a rapid, 1,25(OH)2D-evoked increase in high-voltage-activated (HVA) Ca2+ currents. Enhanced activity-dependent Ca2+ levels were mediated by L-VGCC but not associated with any changes to Cacna1c (L-VGCC pore-forming subunit) mRNA expression. Since L-VGCC activity is critical to healthy neurodevelopment, these data suggest that suboptimal concentrations of 1,25(OH)2D could alter brain maturation through modulation of L-VGCC signalling and as such may provide a parsimonious link between epidemiologic and genetic risk factors for schizophrenia.

The association between neonatal vitamin D status and risk of schizophrenia.

Clues from the epidemiology of schizophrenia, such as the increased risk in those born in winter/spring, have led to the hypothesis that prenatal vitamin D deficiency may increase the risk of later schizophrenia. We wish to explore this hypothesis in a large Danish case-control study (n = 2602). The concentration of 25 hydroxyvitamin D (25OHD) was assessed from neonatal dried blood samples. Incidence rate ratios (IRR) were calculated when examined for quintiles of 25OHD concentration. In addition, we examined statistical models that combined 25OHD concentration and the schizophrenia polygenic risk score (PRS) in a sample that combined the new sample with a previous study (total n = 3464; samples assayed and genotyped between 2008-2013). Compared to the reference (fourth) quintile, those in the lowest quintile (<20.4 nmol/L) had a significantly increased risk of schizophrenia (IRR = 1.44, 95%CI: 1.12-1.85). None of the other quintile comparisons were significantly different. There was no significant interaction between 25OHD and the PRS. Neonatal vitamin D deficiency was associated with an increased risk for schizophrenia in later life. These findings could have important public health implications related to the primary prevention of schizophrenia.

USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors.

USP9X, is highly expressed in neural progenitors and, essential for neural development in mice. In humans, mutations in USP9X are associated with neurodevelopmental disorders. To understand USP9X's role in neural progenitors, we studied the effects of altering its expression in both the human neural progenitor cell line, ReNcell VM, as well as neural stem and progenitor cells derived from Nestin-cre conditionally deleted Usp9x mice. Decreasing USP9X resulted in ReNcell VM cells arresting in G0 cell cycle phase, with a concomitant decrease in mTORC1 signalling, a major regulator of G0/G1 cell cycle progression. Decreased mTORC1 signalling was also observed in Usp9x-null neurospheres and embryonic mouse brains. Further analyses revealed, (i) the canonical mTORC1 protein, RAPTOR, physically associates with Usp9x in embryonic brains, (ii) RAPTOR protein level is directly proportional to USP9X, in both loss- and gain-of-function experiments in cultured cells and, (iii) USP9X deubiquitlyating activity opposes the proteasomal degradation of RAPTOR. EdU incorporation assays confirmed Usp9x maintains the proliferation of neural progenitors similar to Raptor-null and rapamycin-treated neurospheres. Interestingly, loss of Usp9x increased the number of sphere-forming cells consistent with enhanced neural stem cell self-renewal. To our knowledge, USP9X is the first deubiquitylating enzyme shown to stabilize RAPTOR.

Experience-Dependent Accumulation of N6-Methyladenosine in the Prefrontal Cortex Is Associated with Memory Processes in Mice.

UNLABELLED: The RNA modification N(6)-methyladenosine (m(6)A) influences mRNA stability and cell-type-specific developmental programming, and is highly abundant in the adult brain. However, it has not been determined whether m(6)A is dynamically regulated by experience. Based on transcriptome-wide profiling of m(6)A, we report that the level of m(6)A increases in the medial prefrontal cortex (mPFC) of mice in response to behavioral experience. The modulation was enriched near the stop codon of mRNAs, including genes related to neuronal plasticity. In primary cortical neurons, in vitro, modulation of m(6)A by the RNA demethylase FTO influenced the degradation profiles of a subset of transcripts with modulated sites. In vivo, the expression of Fto and the m(6)A methyltransferase, Mettl3 correlated with the observed increase in m(6)A levels post-training. Furthermore, targeted knockdown of FTO in the mPFC led to enhanced consolidation of cued fear memory. Thus, together with its role in early development, the dynamic regulation of m(6)A in the adult brain serves as an important epitranscriptomic mechanism associated with behavioral adaptation. SIGNIFICANCE STATEMENT: N(6)-methyladenosine (m(6)A) is the most prevalent internal modification on RNA, however, its cellular dynamics in vivo remains elusive. Here we provide the first demonstration of m(6)A upregulation in the mouse medial prefrontal cortex (mPFC) following behavioral training. Knocking down the m(6)A demethylase FTO in the mPFC, which increases total m(6)A level, results in enhanced consolidation of fear memory. Our findings suggest that m(6)A is regulated in an activity-dependent manner in the adult brain, and may function to fine-tune mRNA turnover during memory-related processes.

Seizures are regulated by ubiquitin-specific peptidase 9 X-linked (USP9X), a de-ubiquitinase.

Epilepsy is a common disabling disease with complex, multifactorial genetic and environmental etiology. The small fraction of epilepsies subject to Mendelian inheritance offers key insight into epilepsy disease mechanisms; and pathologies brought on by mutations in a single gene can point the way to generalizable therapeutic strategies. Mutations in the PRICKLE genes can cause seizures in humans, zebrafish, mice, and flies, suggesting the seizure-suppression pathway is evolutionarily conserved. This pathway has never been targeted for novel anti-seizure treatments. Here, the mammalian PRICKLE-interactome was defined, identifying prickle-interacting proteins that localize to synapses and a novel interacting partner, USP9X, a substrate-specific de-ubiquitinase. PRICKLE and USP9X interact through their carboxy-termini; and USP9X de-ubiquitinates PRICKLE, protecting it from proteasomal degradation. In forebrain neurons of mice, USP9X deficiency reduced levels of Prickle2 protein. Genetic analysis suggests the same pathway regulates Prickle-mediated seizures. The seizure phenotype was suppressed in prickle mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of fat facets a USP9X orthologue. USP9X mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a de novo missense mutation and another a novel coding mutation. Both USP9X variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress prickle-mediated seizure activity, and that USP9X variants may predispose to seizures. These studies point to a new target for anti-seizure therapy and illustrate the translational power of studying diseases in species across the evolutionary spectrum.