Targeted Sequencing of 10,198 Samples Confirms Abnormalities in Neuronal Activity and Implicates Voltage-Gated Sodium Channels in Schizophrenia Pathogenesis.
ABSTRACT: BACKGROUND:Sequencing studies have pointed to the involvement in schizophrenia of rare coding variants in neuronally expressed genes, including activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-D-aspartate receptor (NMDAR) complexes; however, larger samples are required to reveal novel genes and specific biological mechanisms. METHODS:We sequenced 187 genes, selected for prior evidence of association with schizophrenia, in a new dataset of 5207 cases and 4991 controls. Included among these genes were members of ARC and NMDAR postsynaptic protein complexes, as well as voltage-gated sodium and calcium channels. We performed a rare variant meta-analysis with published sequencing data for a total of 11,319 cases, 15,854 controls, and 1136 trios. RESULTS:While no individual gene was significantly associated with schizophrenia after genome-wide correction for multiple testing, we strengthen the evidence that rare exonic variants in the ARC (p = 4.0 × 10-4) and NMDAR (p = 1.7 × 10-5) synaptic complexes are risk factors for schizophrenia. In addition, we found that loss-of-function variants and missense variants at paralog-conserved sites were enriched in voltage-gated sodium channels, particularly the alpha subunits (p = 8.6 × 10-4). CONCLUSIONS:In one of the largest sequencing studies of schizophrenia to date, we provide novel evidence that multiple voltage-gated sodium channels are involved in schizophrenia pathogenesis and confirm the involvement of ARC and NMDAR postsynaptic complexes.
Project description:A small number of rare, recurrent genomic copy number variants (CNVs) are known to substantially increase susceptibility to schizophrenia. As a consequence of the low fecundity in people with schizophrenia and other neurodevelopmental phenotypes to which these CNVs contribute, CNVs with large effects on risk are likely to be rapidly removed from the population by natural selection. Accordingly, such CNVs must frequently occur as recurrent de novo mutations. In a sample of 662 schizophrenia proband-parent trios, we found that rare de novo CNV mutations were significantly more frequent in cases (5.1% all cases, 5.5% family history negative) compared with 2.2% among 2623 controls, confirming the involvement of de novo CNVs in the pathogenesis of schizophrenia. Eight de novo CNVs occurred at four known schizophrenia loci (3q29, 15q11.2, 15q13.3 and 16p11.2). De novo CNVs of known pathogenic significance in other genomic disorders were also observed, including deletion at the TAR (thrombocytopenia absent radius) region on 1q21.1 and duplication at the WBS (Williams-Beuren syndrome) region at 7q11.23. Multiple de novos spanned genes encoding members of the DLG (discs large) family of membrane-associated guanylate kinases (MAGUKs) that are components of the postsynaptic density (PSD). Two de novos also affected EHMT1, a histone methyl transferase known to directly regulate DLG family members. Using a systems biology approach and merging novel CNV and proteomics data sets, systematic analysis of synaptic protein complexes showed that, compared with control CNVs, case de novos were significantly enriched for the PSD proteome (P=1.72 × 10??. This was largely explained by enrichment for members of the N-methyl-D-aspartate receptor (NMDAR) (P=4.24 × 10??) and neuronal activity-regulated cytoskeleton-associated protein (ARC) (P=3.78 × 10??) postsynaptic signalling complexes. In an analysis of 18?492 subjects (7907 cases and 10?585 controls), case CNVs were enriched for members of the NMDAR complex (P=0.0015) but not ARC (P=0.14). Our data indicate that defects in NMDAR postsynaptic signalling and, possibly, ARC complexes, which are known to be important in synaptic plasticity and cognition, play a significant role in the pathogenesis of schizophrenia.
Project description:Correct function of neuronal networks is enabled by a delicate interplay among neurons communicating with each other. One of the keys is the communication at chemical synapses where neurotransmitters like glutamate, GABA, and glycine enable signal transfer over the synaptic cleft. Thereby, the neurotransmitters are released from the presynapse and bind as ligands to specific receptors at the postsynaptic side to allow for modulation of the postsynaptic membrane potentials. The postsynaptic electrical signal, which is highly modulated by voltage-gated ion channels, spreads over the dendritic tree and is thus integrated to allow for generation of action potentials at the axon hillock. This concert of receptors and voltage-gated ion channels depends on correct function of all its components. Misfunction of receptors and/or voltage-gated potassium channels (VGKC) leads to diverse adverse effects in patients. Such malfunctions can be the result of inherited genetic alterations or pharmacological side effects by drugs. Recently, autoantibodies targeting receptor or channel complexes like NMDAR, AMPAR, GABA-receptors, glycine receptors, LGI1 or CASPR2 (previously termed as VGKC-complex antibodies) have been discovered. The presence of specific autoantibodies against these targets associates with severe forms of antibody-mediated encephalitis. Understanding the molecular details of autoantibody actions on receptor and VGKC complexes is highly desirable and may open the path to develop specific therapies to treat humoral autoimmune encephalitis. Here, we summarize the current knowledge and discuss technical approaches to fill the gap of knowledge. These techniques include electrophysiology, biochemical approaches for epitope mapping, and in silico modeling to simulate molecular interactions between autoantibody and its molecular target.
| S-EPMC4491625 | BioStudies
Project description:Targeted Sequencing of Salamander Voltage-Gated Sodium Channels
Project description:Arc is an activity regulated neuronal protein yet little is known about its protein interactions, assembly into multiprotein complexes, role in human disease and cognition. We applied an integrated proteomic and genetic strategy using targeted tagging of a Tandem Affinity Purification (TAP) tag and Venus fluorescent protein into the endogenous Arc gene in mice, biochemical and proteomic characterization of native complexes in wild type and knockout mice, and human genetic analyses of disease and intelligence. TAP tagging enabled efficient purification of complexes and identification of many novel Arcinteracting proteins, of which PSD95 was the most abundant. PSD95 was essential for Arc assembly into 1.5 MDa complexes and activity-dependent recruitment to excitatory synapses. Integrating human genetic data with proteomic data showed postsynaptic Arc- PSD95 complexes are enriched in schizophrenia, intellectual disability, autism and epilepsy mutations and normal variants in intelligence. Arc-PSD95 postsynaptic complexes are a molecular substrate for the convergence of normal and pathological genetic variants impacting on human cognitive function.
Project description:In the rodent olfactory bulb the smooth dendrites of the principal glutamatergic mitral cells (MCs) form reciprocal dendrodendritic synapses with large spines on GABAergic granule cells (GC), where unitary release of glutamate can trigger postsynaptic local activation of voltage-gated Na<sup>+</sup>-channels (Na<sub>v</sub>s), that is a spine spike. Can such single MC input evoke reciprocal release? We find that unitary-like activation via two-photon uncaging of glutamate causes GC spines to release GABA both synchronously and asynchronously onto MC dendrites. This release indeed requires activation of Na<sub>v</sub>s and high-voltage-activated Ca<sup>2+</sup>-channels (HVACCs), but also of NMDA receptors (NMDAR). Simulations show temporally overlapping HVACC- and NMDAR-mediated Ca<sup>2+</sup>-currents during the spine spike, and ultrastructural data prove NMDAR presence within the GABAergic presynapse. This cooperative action of presynaptic NMDARs allows to implement synapse-specific, activity-dependent lateral inhibition, and thus could provide an efficient solution to combinatorial percept synthesis in a sensory system with many receptor channels.
Project description:Inherited alleles account for most of the genetic risk for schizophrenia. However, new (de novo) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case-control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders.
Project description:Voltage-gated ion channels are important mediators of physiological functions in the central nervous system. The cyclic activation of these channels influences neurotransmitter release, neuron excitability, gene transcription, and plasticity, providing distinct brain areas with unique physiological and pharmacological response. A growing body of data has implicated ion channels in the susceptibility or pathogenesis of psychiatric diseases. Indeed, population studies support the association of polymorphisms in calcium and potassium channels with the genetic risk for bipolar disorders (BPDs) or schizophrenia. Moreover, point mutations in calcium, sodium, and potassium channel genes have been identified in some childhood developmental disorders. Finally, antibodies against potassium channel complexes occur in a series of autoimmune psychiatric diseases. Here we report recent studies assessing the role of calcium, sodium, and potassium channels in BPD, schizophrenia, and autism spectrum disorders, and briefly summarize promising pharmacological strategies targeted on ion channels for the therapy of mental illness and related genetic tests.
Project description:The large-conductance calcium- and voltage-activated K<sup>+</sup> (BK) channel has a requirement of high intracellular free Ca<sup>2+</sup> concentrations for its activation in neurons under physiological conditions. The Ca<sup>2+</sup> sources for BK channel activation are not well understood. In this study, we showed by coimmunopurification and colocalization analyses that BK channels form complexes with NMDA receptors (NMDARs) in both rodent brains and a heterologous expression system. The BK-NMDAR complexes are broadly present in different brain regions. The complex formation occurs between the obligatory BK? and GluN1 subunits likely via a direct physical interaction of the former's intracellular S0-S1 loop with the latter's cytosolic regions. By patch-clamp recording on mouse brain slices, we observed BK channel activation by NMDAR-mediated Ca<sup>2+</sup> influx in dentate gyrus granule cells. BK channels modulate excitatory synaptic transmission via functional coupling with NMDARs at postsynaptic sites of medial perforant path-dentate gyrus granule cell synapses. A synthesized peptide of the BK? S0-S1 loop region, when loaded intracellularly via recording pipette, abolished the NMDAR-mediated BK channel activation and effect on synaptic transmission. These findings reveal the broad expression of the BK-NMDAR complexes in brain, the potential mechanism underlying the complex formation, and the NMDAR-mediated activation and function of postsynaptic BK channels in neurons.
Project description:Dopaminergic hyperfunction and N-methyl-D-aspartate receptor (NMDAR) hypofunction have both been implicated in psychosis. Dopamine-releasing drugs and NMDAR antagonists replicate symptoms associated with psychosis in healthy humans and exacerbate symptoms in patients with schizophrenia. Though hippocampal dysfunction contributes to psychosis, the impact of NMDAR hypofunction on hippocampal plasticity remains poorly understood. Here, we used an NMDAR antagonist rodent model of psychosis to investigate hippocampal long-term potentiation (LTP). We found that single systemic NMDAR antagonism results in a region-specific, presynaptic LTP at hippocampal CA1-subiculum synapses that is induced by activation of D1/D5 dopamine receptors and modulated by L-type voltage-gated Ca(2+) channels. Thereby, our findings may provide a cellular mechanism how NMDAR antagonism can lead to an enhanced hippocampal output causing activation of the hippocampus-ventral tegmental area-loop and overdrive of the dopamine system.
Project description:Arc is an activity-regulated neuronal protein, but little is known about its interactions, assembly into multiprotein complexes, and role in human disease and cognition. We applied an integrated proteomic and genetic strategy by targeting a tandem affinity purification (TAP) tag and Venus fluorescent protein into the endogenous Arc gene in mice. This allowed biochemical and proteomic characterization of native complexes in wild-type and knockout mice. We identified many Arc-interacting proteins, of which PSD95 was the most abundant. PSD95 was essential for Arc assembly into 1.5-MDa complexes and activity-dependent recruitment to excitatory synapses. Integrating human genetic data with proteomic data showed that Arc-PSD95 complexes are enriched in schizophrenia, intellectual disability, autism, and epilepsy mutations and normal variants in intelligence. We propose that Arc-PSD95 postsynaptic complexes potentially affect human cognitive function.