Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration

Abstract

CLC chloride/proton exchangers may support acidification of endolysosomes and raise their luminal Cl À concentration. Disruption of endosomal ClC-3 causes severe neurodegeneration. To assess the importance of ClC-3 Cl À /H + exchange, we now generate Clcn3 unc/unc mice in which ClC-3 is converted into a Cl À channel. Unlike Clcn3 À/À 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 À/À , but not in Clcn3 unc/unc mice because ClC-3 unc binds and stabilizes ClC-4 like wild-type ClC-3. Although mice lacking ClC-4 appear healthy, its absence in Clcn3 unc/unc /Clcn4 À/À mice entails even stronger neurodegeneration than observed in Clcn3 À/À 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 À/À mice before neurodegeneration sets in. Both, Cl À /H + -exchange activity and the stabilizing effect on ClC-4, are central to the biological function of ClC-3.

Neurodegeneration Upon Dysfunction of Endosomal/Lysosomal CLC Chloride Transporters

“…By contrast, the acidification of lysosomes does not necessarily depend on parallel Cl − influx as it can be supported by cation efflux (Steinberg et al, 2010 ; Weinert et al, 2010 ). In addition to the acidification of early endosomes, a pivotal role of the vesicular CLCs is the pH gradient-dependent secondary active accumulation of luminal Cl − , as highlighted in engineered mouse models in which individual CLC exchangers were mutated to pure chloride conductors (Novarino et al, 2010 ; Weinert et al, 2010 , 2020 ). The importance of lysosomal Cl − is still elusive, but it may affect further parameters of the vesicular ion homeostasis including Ca 2+ (Stauber and Jentsch, 2013 ; Chakraborty et al, 2017 ; Astaburuaga et al, 2019 ).…”

“…ClC-3 predominantly localizes on endosomes (Stobrawa et al, 2001 ; Hara-Chikuma et al, 2005 ; Shibata et al, 2006 ). Additionally, it is found on synaptic vesicles (SVs) (Stobrawa et al, 2001 ; Salazar et al, 2004 ; Weinert et al, 2020 ) and synaptic-like micro vesicles (Salazar et al, 2004 ; Maritzen et al, 2008 ). ClC-4 displays similarly broad tissue expression as ClC-3, but its relative abundance is lower in tissues other than brain and muscle (Van Slegtenhorst et al, 1994 ; Mohammad-Panah et al, 2002 , 2003 ; Weinert et al, 2020 ).…”

Chloride accumulation in endosomes and lysosomes: facts checked in mice

“…The work of Weinert et al () now reveals just how hard one had to look to tackle the relevance of the exchange mode to the neuroprotective capacity of ClC‐3. They replaced ClC‐3 with the uncoupled variant of the protein ClC‐3 unc/unc .…”

“…The overlapping expression profiles of several CLC proteins raise the possibility of heterodimerization, which has been demonstrated for some combinations but so far not shown to be essential for any CLC transporter's cellular function. Biochemical investigation of the steady‐state levels and glycosylation status of ClC‐4, a close homologue of ClC‐3 also robustly expressed in neurons, enabled Weinert et al () to reveal that ClC‐4 has to co‐assemble with ClC‐3 to leave the early secretory pathway and to provide its ion transport activity to the right cellular compartment in the endolysosomal system. Indeed, mice expressing Clcn3 unc / unc but lacking Clcn4 show severe neurodegeneration (Fig ).…”

“…It provides a clear example of assembly‐dependent trafficking of a multimeric membrane protein and thereby illustrates that forward protein transport along the secretory pathway is anything but default. Like all intriguing scientific work, the article by Weinert et al () opens new questions: Why is the ClC‐4 protein so strictly attended by ClC‐3? Is there a specific functional or regulatory capacity endowed by ClC‐4 to ClC‐3/ClC‐4 heterodimers?…”