Dysregulation of synaptic and developmental transcriptomic/proteomic profiles upon depletion of MUNC18-1.

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.

Stitching the synapse: Cross-linking mass spectrometry into resolving synaptic protein interactions.

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.

From prenatal anxiety to parenting stress: a longitudinal study.

The objective of this study was to explore how maternal mood during pregnancy, i.e., general anxiety, pregnancy-specific anxiety, and depression predicted parenting stress 3 months after giving birth, thereby shaping the child's early postnatal environmental circumstances. To this end, data were used from 1073 women participating in the Dutch longitudinal cohort Generations2, which studies first-time pregnant mothers during pregnancy and across the transition to parenthood. Women filled out the State Trait Anxiety Inventory (STAI), Pregnancy-Related Anxiety Questionnaire-revised (PRAQ-R), and Beck Depression Index (BDI) three times during pregnancy: at 12, 22, and 32 weeks gestational age. Three months postpartum, a parenting stress questionnaire was filled out yielding seven different parenting constructs. Latent scores were computed for each of the repeatedly measured maternal mood variables with Mplus and parenting stress constructs were simultaneously regressed on these latent scores. Results showed that trait anxiety and pregnancy-specific anxiety were uniquely related to almost all parenting stress constructs, taking depression into account. Early prevention and intervention to reduce maternal anxiety in pregnancy could hold the key for a more advantageous trajectory of early postnatal parenting.

Cerebellar tDCS does not improve performance in probabilistic classification learning.

In this study, the role of the cerebellum in a cognitive learning task using transcranial direct current stimulation (tDCS) was investigated. Using a weather prediction task, subjects had to learn the probabilistic associations between a stimulus (a combination of cards) and an outcome (sun or rain). This task is a variant of a probabilistic classification learning task, for which it has been reported that prefrontal tDCS enhances performance. Using a between-subject design, all 30 subjects learned to improve their performance with increasing accuracies and shortened response times over a series of 500 trials. Subjects also became more confident in their prediction during the experiment. However, no differences in performance and learning were observed between subjects receiving sham stimulation (n = 10) or anodal stimulation (2 mA for 20 min) over either the right cerebellum (n = 10) or the left prefrontal cortex (n = 10). This suggests that stimulating the brain with cerebellar tDCS does not readily influence probabilistic classification performances, probably due to the rather complex nature of this cognitive task.

Cognitive flexibility deficits in a mouse model for the absence of full-length dystrophin.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full-length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full-length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full-length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients.

Cerebellar tDCS does not affect performance in the N-back task.

The N-back task is widely used in cognitive research. Furthermore, the cerebellum's role in cognitive processes is becoming more widely recognized. Studies using transcranial direct current stimulation (tDCS) have demonstrated effects of cerebellar stimulation on several cognitive tasks. Therefore, the aim of this study was to investigate the effects of cerebellar tDCS on cognitive performance by using the N-back task. The cerebellum of 12 participants was stimulated during the task. Moreover, the cognitive load was manipulated in N = 2, N = 3, and N = 4. Every participant received three tDCS conditions (anodal, cathodal, and sham) divided over three separated days. It was expected that anodal stimulation would improve performance on the task. Each participant performed 6 repetitions of every load in which correct responses, false alarms, and reaction times were recorded. We found significant differences between the three levels of load in the rate of correct responses and false alarms, indicating that subjects followed the expected pattern of performance for the N-back task. However, no significant differences between the three tDCS conditions were found. Therefore, it was concluded that in this study cognitive performance on the N-back task was not readily influenced by cerebellar tDCS, and any true effects are likely to be small. We discuss several limitations in task design and suggest future experiments to address such issues.

Neurobeachin Regulates Glutamate- and GABA-Receptor Targeting to Synapses via Distinct Pathways.

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.

Functional characterization of the PCLO p.Ser4814Ala variant associated with major depressive disorder reveals cellular but not behavioral differences.

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.

Cerebellar transcranial direct current stimulation effects on saccade adaptation.

Saccade adaptation is a cerebellar-mediated type of motor learning in which the oculomotor system is exposed to repetitive errors. Different types of saccade adaptations are thought to involve distinct underlying cerebellar mechanisms. Transcranial direct current stimulation (tDCS) induces changes in neuronal excitability in a polarity-specific manner and offers a modulatory, noninvasive, functional insight into the learning aspects of different brain regions. We aimed to modulate the cerebellar influence on saccade gains during adaptation using tDCS. Subjects performed an inward (n = 10) or outward (n = 10) saccade adaptation experiment (25% intrasaccadic target step) while receiving 1.5 mA of anodal cerebellar tDCS delivered by a small contact electrode. Compared to sham stimulation, tDCS increased learning of saccadic inward adaptation but did not affect learning of outward adaptation. This may imply that plasticity mechanisms in the cerebellum are different between inward and outward adaptation. TDCS could have influenced specific cerebellar areas that contribute to inward but not outward adaptation. We conclude that tDCS can be used as a neuromodulatory technique to alter cerebellar oculomotor output, arguably by engaging wider cerebellar areas and increasing the available resources for learning.

Hours in non-parental child care are related to language development in a longitudinal cohort study.

BACKGROUND: The effects of child care services on several domains of child development have been extensively investigated, but evidence regarding the effects of child care on language development remains inconclusive. METHODS: Within a large-scale population-based study, we examined the longitudinal associations between non-parental child care and language development from 1 to 6 years (n = 5375). RESULTS: Results showed that more hours in non-parental child care were associated with better language abilities. However, more hours in care in the first year of life were associated with less language proficiency at ages 1 to 1.5. At later ages, this effect disappeared and language proficiency increased. Furthermore, children who spent more hours in centre-based care had better language scores than children in home-based care. Ethnicity, socio-economic status, gender or parity did not change these results. CONCLUSIONS: This large, multi-ethnic study demonstrates beneficial effects of non-parental child care, particularly centre-based care, on language proficiency later in childhood.

The interrelationship between pregnancy-specific anxiety and general anxiety across pregnancy: a longitudinal study.

BACKGROUND: High levels of prenatal maternal anxiety - either pregnancy-specific anxiety or general anxiety - may have detrimental effects on both the mother and her child. It is currently unknown how these two different expressions of anxiety influence each other over time during pregnancy. AIMS: This study aimed to describe the relationship between state, trait and pregnancy-specific anxiety levels across pregnancy. METHODS: Longitudinal data from three data-waves of a large-scaled sample of nulliparous normal risk pregnant women were used to display associations over time by means of autoregressive and cross-lagged panel models. RESULTS: Cross-lagged, cross-time pathways from pregnancy-specific anxiety to state as well as trait anxiety were positively significant, while vice versa the most consistent links were found from trait anxiety to pregnancy-specific anxiety. CONCLUSIONS: We conclude that pregnancy-specific anxiety and general anxiety appear to influence each other over time, resulting in heightened anxiety for some soon-to-be mothers.

High-throughput phenotyping of avoidance learning in mice discriminates different genotypes and identifies a novel gene.

Recognizing and avoiding aversive situations are central aspects of mammalian cognition. These abilities are essential for health and survival and are expected to have a prominent genetic basis. We modeled these abilities in eight common mouse inbred strains covering ∼75% of the species' natural variation and in gene-trap mice (>2000 mice), using an unsupervised, automated assay with an instrumented home cage (PhenoTyper) containing a shelter with two entrances. Mice visited this shelter for 20-1200 times/24 h and 71% of all mice developed a significant and often strong preference for one entrance. Subsequently, a mild aversive stimulus (shelter illumination) was automatically delivered when mice used their preferred entrance. Different genotypes developed different coping strategies. Firstly, the number of entries via the preferred entrance decreased in DBA/2J, C57BL/6J and 129S1/SvImJ, indicating that these genotypes associated one specific entrance with the aversive stimulus. Secondly, mice started sleeping outside (C57BL/6J, DBA/2J), indicating they associated the shelter, in general, with the aversive stimulus. Some mice showed no evidence for an association between the entrance and the aversive light, but did show markedly shorter shelter residence times in response to illumination, indicating they did perceive illumination as aversive. Finally, using this assay, we screened 43 different mutants, which yielded a novel gene, specc1/cytospinB. This mutant showed profound and specific delay in avoidance learning. Together, these data suggest that different genotypes express distinct learning and/or memory of associations between shelter entrance and aversive stimuli, and that specc1/cytospinB is involved in this aspect of cognition.

Supporting the generalist genes hypothesis for intellectual ability/disability: the case of SNAP25.

Intellectual disability (ID) is an unresolved health care problem with a worldwide prevalence rate of 2-3%. For many years, research into the genetic causes of ID and related disorders has mainly focused on chromosomal abnormalities or X-linked genetic deficits. Only a handful of autosomal genes are known to cause ID. At the same time it has been suggested that at least some cases of ID represent an extreme form of normal intellectual ability and therefore that genes important for intellectual ability in the normal range may also play a role in ID. In this study, we tested whether the autosomal SNAP25 gene, which was previously associated with variation in intellectual ability in the normal range, is also associated with ID. The gene product of SNAP25 is an important presynaptic plasma membrane protein, is known to be involved in regulating neurotransmitter release, and has been linked to memory and learning by its effect on long term potentiation in the hippocampus. Allele frequencies of two genetic variants in SNAP25 previously associated with intellectual ability were compared between a group of 636 ID cases (IQ < 70) and a control group of 361 persons of higher than average intellectual ability. We observed a higher frequency of the putative risk allele of rs363050 (P = 0.02; OR = 1.24) in cases as compared to controls. These results are consistent with a role of SNAP25 in ID, and also support the notion that ID reflects the lower extreme of the quantitative distribution of intellectual ability.

Functional gene group analysis identifies synaptic gene groups as risk factor for schizophrenia.

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.

Genome-wide association for major depressive disorder: a possible role for the presynaptic protein piccolo.

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.

Reduced expression of neuropeptide genes in a genome-wide screen of a secretion-deficient mouse.

Activity-dependent changes in synapses rely on functional changes in resident proteins and on gene expression. We addressed the relationship between synapse activity and the expression of synaptic genes by comparing RNA levels in the neocortex of normal mice versus secretion-deficient and therefore synaptically silent munc18-1 (mammalian homologue of Caenorhabditis elegans uncoordinated locomotion-18) null mutants, using microarray expression analysis, real-time quantitative PCR and northern blotting. We hypothesized that genes under the control of synaptic activity would be differentially expressed between mutants and controls. We found that few synaptic genes were differentially expressed. However, most neuropeptide genes with detectable expression on the microarray were differentially expressed, being expressed 3-20-fold higher in control cortex. Several other secreted proteins were also differentially expressed, but genes encoding their receptors and many other synaptic components were not. Differential expression was confirmed by real-time quantitative PCR analysis. In situ hybridization indicated that the difference in neuropeptide expression was uniform and not due to the loss of specific cells in the mutant. In primary sensory neurons, which do not depend on synaptic activity for their input, the differential expression of neuropeptides was not observed. These data argue against a general relationship between the activity of synapses and the expression of their resident proteins, but suggest a link between secretion and the expression of genes encoding the secreted products.

Munc18-1 phosphorylation by protein kinase C potentiates vesicle pool replenishment in bovine chromaffin cells.

Activation of protein kinase C (PKC) after robust stimulation is necessary for vesicle pool replenishment in secretory cells. Here we studied the contribution of a prominent downstream PKC target, Munc18-1, to this process in bovine chromaffin cells. In these cells, both activation of endogenous PKC and overexpressing of Munc18-1 promote vesicle pool replenishment after an extensive stimulation. In order to study the physiological relevance of PKC-dependent Munc18-1 phosphorylation, we generated two Munc18-1 phospho-mutants; one that mimics a constitutively PKC-phosphorylated Munc18-1 (i.e. a phosphomimetic mutant; Munc18-1(S313D)) and a second that cannot be PKC-phosphorylated (Munc18-1(3A)). Overexpression of Munc18-1(3A) caused a significant decrease in vesicle pool replenishment following a depleting stimulation, while Munc18-1(S313D) caused a significant increase in vesicle pool replenishment. These findings suggested that the phosphorylation of Munc18-1 by PKC potentiates vesicle pool replenishment. This hypothesis was further strengthened by the finding that overexpression of wild type Munc18-1 in the presence of a PKC inhibitor caused a significant reduction in vesicle pool replenishment, similar to that observed with Munc18-1(3A). Moreover, overexpression of Munc18-1(S313D) in the presence of the PKC inhibitor partly alleviated this attenuation, elucidating Munc18-1's unique contribution to vesicle pool replenishment. Finally, we demonstrate that Munc18-1 promotes vesicle docking in a phosphorylation-independent manner. This is deduced from the findings that both the wild type and the two Munc18-1 phospho-mutants enhanced docking to the same extent in bovine chromaffin cells. We conclude that Munc18-1 facilitates docking in a PKC phosphorylation-independent manner, and that its phosphorylation by PKC potentiates vesicle pool replenishment following a depleting stimulation, at a post-docking stage.