Schizophrenia is a highly heritable disorder with a polygenic pattern of inheritance and a population prevalence of ~1%. Previous studies have implicated synaptic dysfunction in schizophrenia. We tested the accumulated association of genetic variants in expert-curated synaptic gene groups with schizophrenia in 4673 cases and 4965 healthy controls, using functional gene group analysis. Identifying groups of genes with similar cellular function rather than genes in isolation may have clinical implications for finding additional drug targets. We found that a group of 1026 synaptic genes was significantly associated with the risk of schizophrenia (P=7.6 × 10(-11)) and more strongly associated than 100 randomly drawn, matched control groups of genetic variants (P<0.01). Subsequent analysis of synaptic subgroups suggested that the strongest association signals are derived from three synaptic gene groups: intracellular signal transduction (P=2.0 × 10(-4)), excitability (P=9.0 × 10(-4)) and cell adhesion and trans-synaptic signaling (P=2.4 × 10(-3)). These results are consistent with a role of synaptic dysfunction in schizophrenia and imply that impaired intracellular signal transduction in synapses, synaptic excitability and cell adhesion and trans-synaptic signaling play a role in the pathology of schizophrenia.

Major Depression is a prevalent mental disorder that is characterized by negative mood and reduced motivation, and frequently results in social withdrawal and memory-related deficits. Repeated stressors, such as adverse life events, increase the risk for development of the disorder. Consequently, individual variability in stress response greatly weighs on depression-vulnerability and -resilience. Here, we employed the social defeat-induced persistent stress (SDPS) paradigm to identify depression-prone individuals and to examine the temporal development of depression in the months following exposure to brief defeat stress. Male Wistar rats were socially defeated (5 defeat episodes) and single-housed for a prolonged period of time (~24 weeks). We assessed the emergence of a sustained depressive-like state by repeatedly evaluating social motivation (social approach avoidance) and spatial memory (object place recognition) in SDPS rats during the isolation period. Individual variability in the effects of SDPS yielded two extreme subpopulations: an SDPS-prone group that showed gradual affective and cognitive deterioration in terms of social approach and memory retention, and a SDPS-resilient group that did not develop this phenotype. Notably, in SDPS-prone individuals, the affective deficits preceded later cognitive impairments, providing a novel temporal profile of the development of pathology in this preclinical model of sustained depression.

The freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model.

Neurotransmission and synaptic strength depend on expression of post-synaptic receptors on the cell surface. Post-translational modification of receptors, trafficking to the synapse through the secretory pathway, and subsequent insertion into the synapse involves interaction of the receptor with A-kinase anchor proteins (AKAPs) and scaffolding proteins. Neurobeachin (Nbea), a brain specific AKAP, is required for synaptic surface expression of both glutamate and GABA receptors. Here, we investigated the role of Nbea-dependent targeting of postsynaptic receptors by studying Nbea interaction with synapse-associated protein 102 (SAP102/Dlg3) and protein kinase A subunit II (PKA II). A Nbea mutant lacking the PKA binding domain showed a similar distribution as wild-type Nbea in Nbea null neurons and partially restored GABA receptor surface expression. To understand the relevance of Nbea interaction with SAP102, we analysed SAP102 null mutant mice. Nbea levels were reduced by ~80% in SAP102 null mice, but glutamatergic receptor expression was normal. A single-point mutation in the pleckstrin homology domain of Nbea (E2218R) resulted in loss of binding with SAP102. When expressed in Nbea null neurons, this mutant fully restored GABA receptor surface expression, but not glutamate receptor expression. Our results suggest that the PKA-binding domain is not essential for Nbea's role in receptor targeting and that Nbea targets glutamate and GABA receptors to the synapse via distinct molecular pathways by interacting with specific effector proteins.

Synaptic transmission is the predominant form of communication in the brain. It requires functionally specialized molecular machineries constituted by thousands of interacting synaptic proteins. Here, we made use of recent advances in cross-linking mass spectrometry (XL-MS) in combination with biochemical and computational approaches to reveal the architecture and assembly of synaptic protein complexes from mouse brain hippocampus and cerebellum. We obtained 11,999 unique lysine-lysine cross-links, comprising connections within and between 2362 proteins. This extensive collection was the basis to identify novel protein partners, to model protein conformational dynamics, and to delineate within and between protein interactions of main synaptic constituents, such as Camk2, the AMPA-type glutamate receptor, and associated proteins. Using XL-MS, we generated a protein interaction resource that we made easily accessible via a web-based platform (http://xlink.cncr.nl) to provide new entries into exploration of all protein interactions identified.

Neurotrophins and their Trk receptors play a crucial role in the development and maintenance of the vertebrate nervous system, but to date no component of this signalling system has been found in invertebrates. We describe a molluscan Trk receptor, designated Ltrk, from the snail Lymnaea stagnalis. The full-length sequence of Ltrk reveals most of the characteristics typical of Trk receptors, including highly conserved transmembrane and intracellular tyrosine kinase domains, and a typical extracellular domain of leucine-rich motifs flanked by cysteine clusters. In addition, Ltrk has a unique N-terminal extension and lacks immunoglobulin-like domains. Ltrk is expressed during development in a stage-specific manner, and also in the adult, where its expression is confined to the central nervous system and its associated endocrine tissues. Ltrk has the highest sequence identity with the TrkC mammalian receptor and, when exogenously expressed in fibroblasts or COS cells, binds human NT-3, but not NGF or BDNF, with an affinity of 2.5 nM. These findings support an early evolutionary origin of the Trk family as neuronal receptor tyrosine kinases and suggest that Trk signalling mechanisms may be highly conserved between vertebrates and invertebrates.

Genome-wide association studies have suggested a role for a genetic variation in the presynaptic gene PCLO in major depressive disorder (MDD). As with many complex traits, the PCLO variant has a small contribution to the overall heritability and the association does not always replicate. One variant (rs2522833, p.Ser4814Ala) is of particular interest given that it is a common, nonsynonymous exon variant near a calcium-sensing part of PCLO. It has been suggested that the molecular effects of such variations penetrate to a variable extent in the population due to phenotypic and genotypic heterogeneity at the population level. More robust effects may be exposed by studying such variations in isolation, in a more homogeneous context. We tested this idea by modeling PCLO variation in a mouse knock-in model expressing the Pclo(SA)(/)(SA) variant. In the highly homogeneous background of inbred mice, two functional effects of the SA-variation were observed at the cellular level: increased synaptic Piccolo levels, and 30% increased excitatory synaptic transmission in cultured neurons. Other aspects of Piccolo function were unaltered: calcium-dependent phospholipid binding, synapse formation in vitro, and synaptic accumulation of synaptic vesicles. Moreover, anxiety, cognition and depressive-like behavior were normal in Pclo(SA)(/)(SA) mice. We conclude that the PCLO p.Ser4814Ala missense variant produces mild cellular phenotypes, which do not translate into behavioral phenotypes. We propose a model explaining how (subtle) cellular phenotypes do not penetrate to the mouse behavioral level but, due to genetic and phenotypic heterogeneity and non-linearity, can produce association signals in human population studies.

Absence of presynaptic protein MUNC18-1 (gene: Stxbp1) leads to neuronal cell death at an immature stage before synapse formation. Here, we performed transcriptomic and proteomic profiling of immature Stxbp1 knockout (KO) cells to discover which cellular processes depend on MUNC18-1. Hippocampi of Stxbp1 KO mice showed cell-type specific dysregulation of 2123 transcripts primarily related to synaptic transmission and immune response. To further investigate direct, neuron-specific effects of MUNC18-1 depletion, a proteomic screen was performed on murine neuronal cultures at two developmental timepoints prior to onset of neuron degeneration. 399 proteins were differentially expressed, which were primarily involved in synaptic function (especially synaptic vesicle exocytosis) and neuron development. We further show that many of the downregulated proteins upon loss of MUNC18-1 are normally upregulated during this developmental stage. Thus, absence of MUNC18-1 extensively dysregulates the transcriptome and proteome, primarily affecting synaptic and developmental profiles. Lack of synaptic activity is unlikely to underlie these effects, as the changes were observed in immature neurons without functional synapses, and minimal overlap was found to activity-dependent proteins. We hypothesize that presence of MUNC18-1 is essential to advance neuron development, serving as a 'checkpoint' for neurons to initiate cell death in its absence.Significance StatementPresynaptic protein MUNC18-1 is essential for neuronal functioning. Pathogenic variants in its gene, STXBP1, are among the most common found in patients with developmental delay and epilepsy. To discern the pathogenesis in these patients, a thorough understanding of MUNC18-1's function in neurons is required. Here, we show that loss of MUNC18-1 results in extensive dysregulation of synaptic and developmental proteins in immature neurons before synapse formation. Many of the downregulated proteins are normally upregulated during this developmental stage. This indicates that MUNC18-1 is a critical regulator of neuronal development, which could play an important role in the pathogenesis of STXBP1 variant carriers.

Major depressive disorder (MDD) is a common complex trait with enormous public health significance. As part of the Genetic Association Information Network initiative of the US Foundation for the National Institutes of Health, we conducted a genome-wide association study of 435 291 single nucleotide polymorphisms (SNPs) genotyped in 1738 MDD cases and 1802 controls selected to be at low liability for MDD. Of the top 200, 11 signals localized to a 167 kb region overlapping the gene piccolo (PCLO, whose protein product localizes to the cytomatrix of the presynaptic active zone and is important in monoaminergic neurotransmission in the brain) with P-values of 7.7 x 10(-7) for rs2715148 and 1.2 x 10(-6) for rs2522833. We undertook replication of SNPs in this region in five independent samples (6079 MDD independent cases and 5893 controls) but no SNP exceeded the replication significance threshold when all replication samples were analyzed together. However, there was heterogeneity in the replication samples, and secondary analysis of the original sample with the sample of greatest similarity yielded P=6.4 x 10(-8) for the nonsynonymous SNP rs2522833 that gives rise to a serine to alanine substitution near a C2 calcium-binding domain of the PCLO protein. With the integrated replication effort, we present a specific hypothesis for further studies.

Synapses are key neuronal elements of the brain. They are responsible for transmission, integration, and storage of information between nerve cells. A synapse is considered as the most complex cellular organelle consisting of approximately 1500 of proteins that are interacting in an activity dependent manner. We have initiated a series of immuno-precipitation experiments in conjunction with LC-MS/MS analysis in order to gain better insight into the organization of the synapse. In particular, we focused on proteins that have been implicated previously in the process of neuroplasticity, i.e., the glutamate receptor (GluR2), scaffolding proteins (PSD-95 and CASK), voltage gated potassium (KCNQ2 and Kv4.2) and calcium (CaV beta4) channel subunits, the signalling protein (GIT1) and synaptic vesicle protein (synaptophysin). This study confirms the previous reported protein-protein interactions and furthermore detects novel interactors. In conjunction with the literature reported protein-protein interaction a simple synaptic protein interactome was constructed. This model implicates the potential interaction of distinct protein complexes, and the engagement of single proteins, especially the scaffolding proteins, in multiple protein complexes.

Frontotemporal dementia (FTD) is a neurodegenerative disorder predominantly affecting the frontal and temporal lobes. Genome-wide association studies (GWAS) on FTD identified only a few risk loci. One of the possible explanations is that FTD is clinically, pathologically, and genetically heterogeneous. An important open question is to what extent epigenetic factors contribute to FTD and whether these factors vary between FTD clinical subgroup. We compared the DNA-methylation levels of FTD cases (n = 128), and of FTD cases with Amyotrophic Lateral Sclerosis (FTD-ALS; n = 7) to those of unaffected controls (n = 193), which resulted in 14 and 224 candidate genes, respectively. Cluster analysis revealed significant class separation of FTD-ALS from controls. We could further specify genes with increased susceptibility for abnormal gene-transcript behavior by jointly analyzing DNA-methylation levels with the presence of mutations in a GWAS FTD-cohort. For FTD-ALS, this resulted in 9 potential candidate genes, whereas for FTD we detected 1 candidate gene (ELP2). Independent validation-sets confirmed the genes DLG1, METTL7A, KIAA1147, IGHMBP2, PCNX, UBTD2, WDR35, and ELP2/SLC39A6 among others. We could furthermore demonstrate that genes harboring mutations and/or displaying differential DNA-methylation, are involved in common pathways, and may therefore be critical for neurodegeneration in both FTD and FTD-ALS.