31 research outputs found
Collybistin and gephyrin are novel components of the eukaryotic translation initiation factor 3 complex
<p>Abstract</p> <p>Background</p> <p>Collybistin (CB), a neuron-specific guanine nucleotide exchange factor, has been implicated in targeting gephyrin-GABA<sub>A </sub>receptors clusters to inhibitory postsynaptic sites. However, little is known about additional CB partners and functions.</p> <p>Findings</p> <p>Here, we identified the p40 subunit of the eukaryotic translation initiation factor 3 (eIF3H) as a novel binding partner of CB, documenting the interaction in yeast, non-neuronal cell lines, and the brain. In addition, we demonstrated that gephyrin also interacts with eIF3H in non-neuronal cells and forms a complex with eIF3 in the brain.</p> <p>Conclusions</p> <p>Together, our results suggest, for the first time, that CB and gephyrin associate with the translation initiation machinery, and lend further support to the previous evidence that gephyrin may act as a regulator of synaptic protein synthesis.</p
Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability
The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR a2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant collybistin CB3SH3-R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3-R356Q and CB3SH3-R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of collybistin PH domain phosphoinositide binding
Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar purkinje cells
Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABA(A) receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABA(A) receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABA(A) receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABA(A) receptors and is abolished by preventing CaMKII phosphorylation of GABA(A) receptors. Our results reveal a novel GABA(A) receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum
Oligodeoxynucleotides containing unmethylated cytosine-guanine motifs are effective immunostimulants against pneumococcal meningitis in the immunocompetent and neutropenic host
Background!#!Bacterial meningitis is a fatal disease with a mortality up to 30% and neurological sequelae in one fourth of survivors. Available vaccines do not fully protect against this lethal disease. Here, we report the protective effect of synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG ODN) against the most frequent form of bacterial meningitis caused by Streptococcus pneumoniae.!##!Methods!#!Three days prior to the induction of meningitis by intracerebral injection of S. pneumoniae D39, wild-type and Toll-like receptor (TLR9)!##!Results!#!Pre-treatment with 100 μg CpG ODN prolonged survival of immunocompetent and neutropenic wild-type mice but not of TLR9!##!Conclusions!#!Pre-conditioning with CpG ODN strengthened the resistance of neutropenic and immunocompetent mice against S. pneumoniae meningitis in the presence of TLR9. Administration of CpG ODN decreased bacterial burden in the cerebellum and reduced the degree of bacteremia. Systemic administration of CpG ODN may help to prevent or slow the progression to sepsis of bacterial CNS infections in healthy and immunocompromised individuals even after direct inoculation of bacteria into the intracranial compartments, which can occur after sinusitis, mastoiditis, open head trauma, and surgery, including placement of an external ventricular drain
Metabarcoding of eukaryotic parasite communities describes diverse parasite assemblages spanning the primate phylogeny
Despite their ubiquity, in most cases little is known about the impact of eukaryotic parasites on their mammalian hosts. Comparative approaches provide a powerful method to investigate the impact of parasites on host ecology and evolution, though two issues are critical for such efforts: controlling for variation in methods of identifying parasites and incorporating heterogeneity in sampling effort across host species. To address these issues, there is a need for standardized methods to catalogue eukaryotic parasite diversity across broad phylogenetic host ranges. We demonstrate the feasibility of a metabarcoding approach for describing parasite communities by analysing faecal samples from 11 nonhuman primate species representing divergent lineages of the primate phylogeny and the full range of sampling effort (i.e. from no parasites reported in the literature to the best‐studied primates). We detected a number of parasite families and regardless of prior sampling effort, metabarcoding of only ten faecal samples identified parasite families previously undescribed in each host (x̅ = 8.5 new families per species). We found more overlap between parasite families detected with metabarcoding and published literature when more research effort—measured as the number of publications—had been conducted on the host species' parasites. More closely related primates and those from the same continent had more similar parasite communities, highlighting the biological relevance of sampling even a small number of hosts. Collectively, results demonstrate that metabarcoding methods are sensitive and powerful enough to standardize studies of eukaryotic parasite communities across host species, providing essential new tools for macroecological studies of parasitism.Peer Reviewe
Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin
The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA(A) receptor (GABA(A)R) subtypes. We report a direct interaction between the GABA(A)R α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA(A)R α3 M3–M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABA(A)R α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA(A)Rs in recombinant systems. Interestingly, motifs responsible for interactions with GABA(A)R α2, GABA(A)R α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA(A)R α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABA(A)R α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABA(A)R α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses
Immune hyperreactivity of Aβ plaque-associated microglia in Alzheimer's disease
Alzheimer's disease (AD) is strongly associated with microglia-induced neuroinflammation. Particularly, Aβ plaque-associated microglia take on an "activated" morphology. However, the function and phenotype of these Aβ plaque-associated microglia are not well understood. We show hyperreactivity of Aβ plaque-associated microglia upon systemic inflammation in transgenic AD mouse models (i.e., 5XFAD and APP23). Gene expression profiling of Aβ plaque-associated microglia (major histocompatibility complex II+ microglia) isolated from 5XFAD mice revealed a proinflammatory phenotype. The upregulated genes involved in the biological processes (gene ontology terms) included: "immune response to external stimulus" such as Axl, Cd63, Egr2, and Lgals3, "cell motility", such as Ccl3, Ccl4, Cxcr4, and Sdc3, "cell differentiation", and "system development", such as St14, Trpm1, and Spp1. In human AD tissue with similar Braak stages, expression of phagocytic markers and AD-associated genes, including HLA-DRA, APOE, AXL, TREM2, and TYROBP, was higher in laser-captured early-onset AD (EOAD) plaques than in late-onset AD plaques. Interestingly, the nonplaque parenchyma of both EOAD and late-onset AD brains, the expression of above-mentioned markers were similarly low. Here, we provide evidence that Aβ plaque-associated microglia are hyperreactive in their immune response and phagocytosis in the transgenic AD mice as well as in EOAD brain tissue. We suggest that Aβ plaque-associated microglia are the primary source of neuroinflammation related to AD pathology
Long-lasting pro-inflammatory suppression of microglia by LPS-preconditioning is mediated by RelB-dependent epigenetic silencing
Microglia, the innate immune cells of the central nervous system (CNS), react to endotoxins like bacterial lipopolysaccharides (LPS) with a pronounced inflammatory response. To avoid excess damage to the CNS, the microglia inflammatory response needs to be tightly regulated. Here we report that a single LPS challenge results in a prolonged blunted pro-inflammatory response to a subsequent LPS stimulation, both in primary microglia cultures (100 ng/ml) and in vivo after intraperitoneal (0.25 and 1 mg/kg) or intracere-broventricular (5 mu g) LPS administration. Chromatin immunoprecipitation (ChIP) experiments with primary microglia and microglia acutely isolated from mice showed that LPS preconditioning was accompanied by a reduction in active histone modifications AcH3 and H3K4me3 in the promoters of the IL-10 and TNF-alpha genes. Furthermore, LPS preconditioning resulted in an increase in the amount of repressive histone modification H3K9me2 in the IL-1 beta promoter. ChIP and knock-down experiments showed that NF-kappa B subunit RelB was bound to the IL-1 beta promoter in preconditioned microglia and that RelB is required for the attenuated LPS response. In addition to a suppressed pro-inflammatory response, preconditioned primary microglia displayed enhanced phagocytic activity, increased outward potassium currents and nitric oxide production in response to a second LPS challenge. In vivo, a single i.p. LPS injection resulted in reduced performance in a spatial learning task 4 weeks later, indicating that a single inflammatory episode affected memory formation in these mice. Summarizing, we show that LPS-preconditioned microglia acquire an epigenetically regulated, immune-suppressed phenotype, possibly to prevent excessive damage to the central nervous system in case of recurrent (peripheral) inflammation. (C) 2015 Elsevier Inc. All rights reserved
Identification and expression of a unique neonatal variant of the GABAA receptor α3 subunit
The GABAA receptor provides the majority of inhibitory neurotransmission in the adult central nervous system but in immature brain is responsible for much of the excitatory drive, a requirement for normal brain development. It is well established that GABAA receptor subunit expression changes across the course of brain development. In the present study, we have identified a splice variant of the GABAA receptor α3 subunit which appears unique to the developing brain, referred to here as the GABAA receptor α3 subunit neonatal variant (GABAA receptor α3N). RT-PCR and sequence analysis revealed splicing of exon 8 of the α3 subunit. Western blot analysis showed expression of GABAA receptor α3N in the cortex of several neonatal species and significantly reduced expression of this splice variant in the corresponding adult brains. Expression was evident in multiple brain regions and decreased across development in the pig. Fractionation revealed differential cellular localisation in the parietal cortex, hippocampus and thalamus of the full-length GABAA receptor α3 and GABAA receptor α3N. Immunoprecipitation showed direct interaction with the GABAA receptor subunits α1 and γ2 but not with gephyrin