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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. [ABSTRACT FROM AUTHOR]

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 Clcn3unc/unc mice in which ClC‐3 is converted into a Cl− channel. Unlike Clcn3−/− mice, Clcn3unc/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 Clcn3unc/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 Clcn3unc/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 Clcn3unc/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. Synopsis: The importance of intracellular ClC‐3 channel conductance versus chloride/proton exchange activity for endosome and synaptic vesicle function remain unclear. Here, study of a new ClC‐3 uncoupling mouse mutant (Clcn3unc/unc) reveals that proton transport by ClC‐3/ClC‐4 heterodimers is crucial for central nervous system integrity. Clcn3unc/unc mice devoid of channel function do not exhibit neurodegeneration.ClC‐3 forms heterodimers with ClC‐4 and stabilizes it.Depletion of ClC‐4 in Clcn3unc/unc mice causes severe neurodegeneration.ClC‐3 has no functional role in synaptic vesicles. [ABSTRACT FROM AUTHOR]

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. Synopsis: 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. Female APP23 mice show stronger amyloidosis compared with male mice.Lack of IL‐12/IL‐23 signaling reduces Aβ plaque load in male APP23 mice and affects plasma IFNγ levels.In female APP23 mice, deficiency in IL‐12/IL‐23 signaling reduces soluble Aβ1‐40 and modulates various brain and plasma cytokines. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is characterized not only by extracellular amyloid plaques and neurofibrillary tangles, but also by microglia‐mediated neuroinflammation. Recently, autophagy has been linked to the regulation of the inflammatory response. Thus, we investigated how an impairment of autophagy mediated by BECN1/Beclin1 reduction, as described in Alzheimer's disease patients, would influence cytokine production of microglia. Acutely stimulated microglia from Becn1+/− mice exhibited increased expression of IL‐1beta and IL‐18 compared to wild‐type microglia. Becn1+/−APPPS1 mice also contained enhanced IL‐1beta levels. The investigation of the IL‐1beta/IL‐18 processing pathway showed an elevated number of cells with inflammasomes and increased levels of NLRP3 and cleaved CASP1/Caspase1 in Becn1+/− microglia. Super‐resolation microscopy revealed a very close association of NLRP3 aggregates and LC3‐positive vesicles. Interestingly, CALCOCO2 colocalized with NLRP3 and its downregulation increased IL‐1beta release. These data support the notion that selective autophagy can impact microglia activation by modulating IL‐1beta and IL‐18 production via NLRP3 degradation and thus present a mechanism how impaired autophagy could contribute to neuroinflammation in Alzheimer's disease. Synopsis: Decreased autophagy in microglia, the resident immune cells of the CNS, modulates production of the proinflammatory cytokines IL‐1 beta and IL‐18 by selective degradation of NLRP3. Primary microglia from Becn1+/− mice release a higher amount of IL‐1 beta and IL‐18 compared to wild‐type microglia upon a proinflammatory stimulus.Microglia from Becn1+/− mice display an increase in crucial components of the NLRP3 inflammasome.Super‐resolution microscopy (SIM) reveals the presence of NLRP3 aggregates in autophagosomes indicating their degradation.The putative autophagic linker protein murine CALCOCO2 but not the established autophagic adaptor p62/SQSTM1 colocalizes with NLRP3 and its downregulation increases IL‐1 beta release from stimulated microglia. Inflammasome degradation to curb production of IL‐1beta and IL‐18 cytokines indicates a brain immune cell role for autophagy, whose loss in Alzheimer's disease may contribute to neuropathology. [ABSTRACT FROM AUTHOR]