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Aims: Congenital myasthenic syndromes (CMS) are characterized by muscle weakness, ptosis and episodic apnoea. Mutations affect integral protein components of the neuromuscular junction (NMJ). Here we searched for the genetic basis of CMS in female monozygotic twins. Methods: We employed whole-exome sequencing for mutation detection and Sanger sequencing for segregation analysis. Immunohistology was done with antibodies against CHD8, rapsyn, ��-catenin (��CAT) and golgin on fi-bro-blasts, human and mouse muscle. We recorded superresolution images of the NMJ using 3D-structured illumination microscopy. Results: We discovered a spontaneous missense mutation in CHD8 [chr14:g.21,884,051G>A, GRCh37.p11 | c.1732C>T, NM_00117062 | p.(R578C)], the gene encoding chromodomain helicase DNA-binding protein 8. This is the first missense mutation affecting Duplin, the short 110 kDa isoform of CHD8. It is known that CHD8/Duplin negatively regulates ��CAT signalling in the WNT pathway and plays a role in chromatin remodelling. Inactivating CHD8 mutations are associated with autism spectrum disorder and intellectual disability in combination with facial dysmorphism, overgrowth and macrocephalus. No muscle-specific phenotype has been reported to date. Co-immunostaining with rapsyn on human and mouse muscle revealed a strong presence of CHD8 at the NMJ being located towards the sarcoplasmic side of the rapsyn cluster, where it co-localizes with ��CAT. Conclusion: We hypothesize CHD8 to have a role in the maintenance of the structural integrity and function of the NMJ. Both patients benefited from treatment with 3,4-diaminopyridine, a reversible blocker of voltage-gated potassium channels at the nerve terminal that prolongs the action potential and increases acetylcholine release.

RNA viruses in the Picornaviridae family express a large 250 kDa viral polyprotein that is processed by virus-encoded proteinases into mature functional proteins with specific functions for virus replication. One of these proteins is the highly conserved enteroviral transmembrane protein 3A that assists in reorganizing cellular membranes associated with the Golgi apparatus. Here, we studied the molecular properties of the Coxsackievirus B3 (CVB3) protein 3A with regard to its dimerization and its functional stability. By applying mutational analysis and biochemical characterization, we demonstrate that protein 3A forms DTT-sensitive disulfide-linked dimers via a conserved cytosolic cysteine residue at position 38 (Cys38). Homodimerization of CVB3 protein 3A via Cys38 leads to profound stabilization of the protein, whereas a C38A mutation promotes a rapid proteasome-dependent elimination of its monomeric form. The lysosomotropic agent chloroquine (CQ) exerted only minor stabilizing effects on the 3A monomer but resulted in enrichment of the homodimer. Our experimental data demonstrate that disulfide linkages via a highly conserved Cys-residue in enteroviral protein 3A have an important role in the dimerization of this viral protein, thereby preserving its stability and functional integrity.

Recent advances in imaging technology have highlighted that scaffold proteins and receptors are arranged in subsynaptic nanodomains. The synaptic membrane-associated guanylate kinase (MAGUK) scaffold protein membrane protein palmitoylated 2 (MPP2) is a component of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor–associated protein complexes and also binds to the synaptic cell adhesion molecule SynCAM 1. Using superresolution imaging, we show that—like SynCAM 1—MPP2 is situated at the periphery of the postsynaptic density (PSD). In order to explore MPP2-associated protein complexes, we used a quantitative comparative proteomics approach and identified multiple γ-aminobutyric acid (GABA)A receptor subunits among novel synaptic MPP2 interactors. In line with a scaffold function for MPP2 in the assembly and/or modulation of intact GABAA receptors, manipulating MPP2 expression had effects on inhibitory synaptic transmission. We further show that GABAA receptors are found together with MPP2 in a subset of dendritic spines and thus highlight MPP2 as a scaffold that serves as an adaptor molecule, linking peripheral synaptic elements critical for inhibitory regulation to central structures at the PSD of glutamatergic synapses.

Summary In developing neurons, phosphoinositide 3-kinases (PI3Ks) control axon growth and branching by positively regulating PI3K/PI(3,4,5)P3, but how neurons are able to generate sufficient PI(3,4,5)P3 in the presence of high levels of the antagonizing phosphatase PTEN is difficult to reconcile. We find that normal axon morphogenesis involves homeostasis of elongation and branch growth controlled by accumulation of PI(3,4,5)P3 through PTEN inhibition. We identify a plasma membrane-localized protein-protein interaction of PTEN with plasticity-related gene 2 (PRG2). PRG2 stabilizes membrane PI(3,4,5)P3 by inhibiting PTEN and localizes in nanoclusters along axon membranes when neurons initiate their complex branching behavior. We demonstrate that PRG2 is both sufficient and necessary to account for the ability of neurons to generate axon filopodia and branches in dependence on PI3K/PI(3,4,5)P3 and PTEN. Our data indicate that PRG2 is part of a neuronal growth program that induces collateral branch growth in axons by conferring local inhibition of PTEN., Graphical Abstract, Highlights • Neuronal axon growth and branching is globally regulated by PI3K/PTEN signaling • PRG2 inhibits PTEN and stabilizes PIP3 and F-actin • PRG2 localizes to nanoclusters on the axonal membrane and coincides with branching • PRG2 promotes axonal filopodia and branching dependent on PI3K/PTEN, PTEN globally suppresses growth in multiple cell types and inhibits neuronal axon growth and branching. Brosig et al. describe a mechanism for developing neurons to inhibit PTEN function by upregulating PRG2 in the axon. Although the overall growth capacity of neurons remains constant, PRG2 redirects growth to axon branches.

CLC chloride/proton exchangers may support acidification of endolysosomes and raise their luminal Cl− concentration. Disruption of endosomal ClC‐3 causes severe neurodegeneration. To assess the importance of ClC‐3 Cl−/H+ exchange, we now generate Clcn3 unc/unc mice in which ClC‐3 is converted into a Cl− channel. Unlike Clcn3 −/− mice, Clcn3 unc/unc mice appear normal owing to compensation by ClC‐4 with which ClC‐3 forms heteromers. ClC‐4 protein levels are strongly reduced in Clcn3 −/−, but not in Clcn3 unc/unc mice because ClC‐3unc binds and stabilizes ClC‐4 like wild‐type ClC‐3. Although mice lacking ClC‐4 appear healthy, its absence in Clcn3 unc/unc/Clcn4 −/− mice entails even stronger neurodegeneration than observed in Clcn3 −/− mice. A fraction of ClC‐3 is found on synaptic vesicles, but miniature postsynaptic currents and synaptic vesicle acidification are not affected in Clcn3 unc/unc or Clcn3 −/− mice before neurodegeneration sets in. Both, Cl−/H+‐exchange activity and the stabilizing effect on ClC‐4, are central to the biological function of ClC‐3., ClC‐3 antiporter activity and heterodimerization with ClC‐4 are critical for endolysosomal acidification and central nervous system integrity.

Objective Hormone secretion from metabolically active tissues, such as pancreatic islets, is governed by specific and highly regulated signaling pathways. Defects in insulin secretion are among the major causes of diabetes. The molecular mechanisms underlying regulated insulin secretion are, however, not yet completely understood. In this work, we studied the role of the GTPase ARFRP1 on insulin secretion from pancreatic β-cells. Methods A β-cell-specific Arfrp1 knockout mouse was phenotypically characterized. Pulldown experiments and mass spectrometry analysis were employed to screen for new ARFRP1-interacting proteins. Co-immunoprecipitation assays as well as super-resolution microscopy were applied for validation. Results The GTPase ARFRP1 interacts with the Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). Both proteins are co-localized at the trans-Golgi network and regulate the first and second phase of insulin secretion by controlling the plasma membrane localization of the SNARE protein SNAP25. Downregulation of both GOPC and ARFRP1 in Min6 cells interferes with the plasma membrane localization of SNAP25 and enhances its degradation, thereby impairing glucose-stimulated insulin release from β-cells. In turn, overexpression of SNAP25 as well as GOPC restores insulin secretion in islets from β-cell-specific Arfrp1 knockout mice. Conclusion Our results identify a hitherto unrecognized pathway required for insulin secretion at the level of trans-Golgi sorting., Highlights • β-cell specific deletion of the trans-Golgi residing small GTPase ARFRP1 leads to elevated blood glucose levels in mice. • GOPC is a newly identified ARFRP1 dependent scaffolding protein. • ARFRP1 and GOPC are required for glucose-stimulated insulin secretion from pancreatic β-cells.

Pathological aggregation of amyloid‐β (Aβ) is a main hallmark of Alzheimer's disease (AD). Recent genetic association studies have linked innate immune system actions to AD development, and current evidence suggests profound gender differences in AD pathogenesis. Here, we characterise gender‐specific pathologies in the APP23 AD‐like mouse model and find that female mice show stronger amyloidosis and astrogliosis compared with male mice. We tested the gender‐specific effect of lack of IL12p40, the shared subunit of interleukin (IL)‐12 and IL‐23, that we previously reported to ameliorate pathology in APPPS1 mice. IL12p40 deficiency gender specifically reduces Aβ plaque burden in male APP23 mice, while in female mice, a significant reduction in soluble Aβ1–40 without changes in Aβ plaque burden is seen. Similarly, plasma and brain cytokine levels are altered differently in female versus male APP23 mice lacking IL12p40, while glial properties are unchanged. These data corroborate the therapeutic potential of targeting IL‐12/IL‐23 signalling in AD, but also highlight the importance of gender considerations when studying the role of the immune system and AD., Abrogating IL‐12/IL‐23 signaling ameliorates amyloidogenesis gender‐specifically in APP23 mice, indicating therapeutic potential of targeting IL‐12/IL‐23 in Alzheimer's disease and highlighting the importance of gender considerations in AD.

Compartmentalization of membrane transport and signaling processes is of pivotal importance to eukaryotic cell function. While plasma membrane compartmentalization and dynamics are well known to depend on the scaffolding function of septin GTPases, the roles of septins at intracellular membranes have remained largely elusive. Here, we show that the structural and functional integrity of the Golgi depends on its association with a septin 1 (SEPT1)-based scaffold, which promotes local microtubule nucleation and positioning of the Golgi. SEPT1 function depends on the Golgi matrix protein GM130 (also known as GOLGA2) and on centrosomal proteins, including CEP170 and components of γ-tubulin ring complex (γ-Turc), to facilitate the perinuclear concentration of Golgi membranes. Accordingly, SEPT1 depletion triggers a massive fragmentation of the Golgi ribbon, thereby compromising anterograde membrane traffic at the level of the Golgi., Highlighted Article: SEPT1 associates with the Golgi complex through GM130, and is required for maintaining Golgi architecture and function by connecting cis-Golgi membranes to the microtubule-nucleating machinery.

All synapses require fusion-competent vesicles and coordinated Ca2+-secretion coupling for neurotransmission, yet functional and anatomical properties are diverse across different synapse types. We show that the presynaptic protein RIM-BP2 has diversified functions in neurotransmitter release at different central murine synapses and thus contributes to synaptic diversity. At hippocampal pyramidal CA3-CA1 synapses, RIM-BP2 loss has a mild effect on neurotransmitter release, by only regulating Ca2+-secretion coupling. However, at hippocampal mossy fiber synapses, RIM-BP2 has a substantial impact on neurotransmitter release by promoting vesicle docking/priming and vesicular release probability via stabilization of Munc13-1 at the active zone. We suggest that differences in the active zone organization may dictate the role a protein plays in synaptic transmission and that differences in active zone architecture is a major determinant factor in the functional diversity of synapses.

Background Antagonists of the V1a vasopressin receptor (V1aR) are emerging as a strategy for slowing progression of CKD. Physiologically, V1aR signaling has been linked with acid-base homeostasis, but more detailed information is needed about renal V1aR distribution and function. Methods We used a new anti-V1aR antibody and high-resolution microscopy to investigate Va1R distribution in rodent and human kidneys. To investigate whether V1aR activation promotes urinary H + secretion, we used a V1aR agonist or antagonist to evaluate V1aR function in vasopressin-deficient Brattleboro rats, bladder-catheterized mice, isolated collecting ducts, and cultured inner medullary collecting duct (IMCD) cells. Results Localization of V1aR in rodent and human kidneys produced a basolateral signal in type A intercalated cells (A-ICs) and a perinuclear to subapical signal in type B intercalated cells of connecting tubules and collecting ducts. Treating vasopressin-deficient Brattleboro rats with a V1aR agonist decreased urinary pH and tripled net acid excretion; we observed a similar response in C57BL/6J mice. In contrast, V1aR antagonist did not affect urinary pH in normal or acid-loaded mice. In ex vivo settings, basolateral treatment of isolated perfused medullary collecting ducts with the V1aR agonist or vasopressin increased intracellular calcium levels in ICs and decreased luminal pH, suggesting V1aR-dependent calcium release and stimulation of proton-secreting proteins. Basolateral treatment of IMCD cells with the V1aR agonist increased apical abundance of vacuolar H + -ATPase in A-ICs. Conclusions Our results show that activation of V1aR contributes to urinary acidification via H + secretion by A-ICs, which may have clinical implications for pharmacologic targeting of V1aR.

SummaryNeurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis. Here we demonstrate that loss of the Syb2-specific endocytic adaptor AP180 causes a moderate activity-dependent reduction of vesicular Syb2 levels, defects in SV reformation, and a corresponding impairment of neurotransmission that lead to excitatory/inhibitory imbalance, epileptic seizures, and premature death. Further reduction of Syb2 levels in AP180−/−/Syb2+/− mice results in perinatal lethality, whereas Syb2+/− mice partially phenocopy loss of AP180, indicating that reduced vesicular Syb2 levels underlie the observed defects in neurotransmission. Thus, a large vesicular Syb2 pool maintained by AP180 is crucial to sustain efficient neurotransmission and SV reformation.

Neurotransmission relies on the calcium-triggered exocytic fusion of non-peptide neurotransmitter-containing small synaptic vesicles (SVs) with the presynaptic membrane at active zones (AZs) followed by compensatory endocytic retrieval of SV membranes. Here, we study the diffusional fate of newly exocytosed SV proteins in hippocampal neurons by high-resolution time-lapse imaging. Newly exocytosed SV proteins rapidly disperse within the first seconds post fusion until confined within the presynaptic bouton. Rapid diffusional spread and confinement is followed by slow reclustering of SV proteins at the periactive endocytic zone. Confinement within the presynaptic bouton is mediated in part by SV protein association with the clathrin-based endocytic machinery to limit diffusional spread of newly exocytosed SV proteins. These data suggest that diffusion, and axonal escape of newly exocytosed vesicle proteins, are counteracted by the clathrin-based endocytic machinery together with a presynaptic diffusion barrier., Neurotransmission is mediated by synaptic vesicles (SVs) fusion with the plasma membrane near active zones. Here, Gimber et al. observe that rapid diffusional spread and confinement is followed by slow reclustering of SV proteins at the periactive endocytic zone through SV protein association with the clathrin-based machinery.

Chloride Balancing Act The ionic composition of the cytosol and intracellular organelles must be regulated in the face of ongoing membrane traffic into and out of the cell. Now, two papers address the consequences of a change in the transport phenotype of an intracellular Cl − transport protein from a coupled exchanger to a passive Cl − conductor (see the Perspective by Smith and Schwappach ). Novarino et al. (p. 1398 , published online 29 April) investigated the consequence of a knock-in of the uncoupled ClC-5 transporter into mouse. The knock-out mouse of this endosomal kidney transporter has a severe endocytic phenotype believed to be due to a defect in vesicular acidification. The current study shows a similarly impaired endocytic phenotype for the uncoupled mutant, but the acidification of endosomes was unaffected. Weinert et al. (p. 1401 , published online 29 April) used a similar strategy to investigate the consequence of the equivalent mutation in the lysosomal transporter ClC-7, which is highly expressed in the resorption lacuna of osteoclasts and whose knock-out in mice produces lysosomal storage disease and severe osteopetrosis. A similar (though less severe) phenotype was observed in the knock-in mice containing the uncoupled ClC-7, indicating that coupled transport plays a critical role in lysosomes.

Excessive osteoclastic resorption is a defining feature of rheumatoid arthritis (RA), resulting in devastating tissue damage and pain. Omega-3 fatty acids (Ω3-FA) protect against RA and reduce bone pain, however the mechanism through which this occurs remains unknown. Cyclopentenone isoprostanes (IsoPs) form in situasoxidationproducts ofΩ3-FA.Wehypothesize that a beneficial effect of Ω3-FA in RA occurs following the formation of IsoPs, to suppress osteoclast formation. To test this, we used GC-MS to analyze the IsoP profile in serum from age-, sex-matchedRApatientsor controls (n=7).RApatient sampleswere confirmed asCEP1andCCP positive. In additionwe employedosteoclast formationassays using RAW264.7 cells treatedwith 15-A3-IsoP in addition to RANKL andM-CSF. Themolecularmechanism throughwhich IsoPmay affect osteoclast formation was assessed using an NFκB-reporter macrophage cell line, to recapitulate and quantify NFκB signaling in osteoclast precursor cells. These studies were supported by immunofluorescent staining for nuclear/cytoplasmic p65 andwestern blot analysis for phosphorylated IκBα. To confirm the role of NFκB in IsoPmediated osteoclast inhibition, sitedirected mutagenesis was employed to alter a putative IsoP binding region of IKK (C179). Mutated RAW 264.7 cells were assessed for osteoclast formation capacity and NFκB activation status, compared to unmodified cells. Our studies show that the IsoP levels within RA serum are significantly altered, compared to normal control patients. In addition, osteoclast precursor cells treated with 15-A3-IsoP (15 uM) formed fewer TRAP+ multinucleated cells (26.3±5.3) compared to vehicle treated controls (46.3±4.3, p