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• W3010165610 abstract "Article2 March 2020Open Access Source DataTransparent process Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration Stefanie Weinert Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Niclas Gimber orcid.org/0000-0001-9456-3063 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Dorothea Deuschel Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Till Stuhlmann Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Dmytro Puchkov Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Search for more papers by this author Zohreh Farsi Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Carmen F Ludwig Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Gaia Novarino Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Karen I López-Cayuqueo Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Rosa Planells-Cases Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Thomas J Jentsch Corresponding Author [email protected] orcid.org/0000-0002-3509-2553 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany NeuroCure Cluster of Excellence, Charité Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Stefanie Weinert Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Niclas Gimber orcid.org/0000-0001-9456-3063 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Dorothea Deuschel Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Till Stuhlmann Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Dmytro Puchkov Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Search for more papers by this author Zohreh Farsi Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Carmen F Ludwig Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Gaia Novarino Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Karen I López-Cayuqueo Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Rosa Planells-Cases Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Thomas J Jentsch Corresponding Author [email protected] orcid.org/0000-0002-3509-2553 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany NeuroCure Cluster of Excellence, Charité Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Author Information Stefanie Weinert1,2, Niclas Gimber1,2,†,‡, Dorothea Deuschel1,2,‡, Till Stuhlmann1,2,‡, Dmytro Puchkov1,‡, Zohreh Farsi2,†, Carmen F Ludwig1,2, Gaia Novarino1,2,†,‡, Karen I López-Cayuqueo1,2, Rosa Planells-Cases1,2 and Thomas J Jentsch *,1,2,3 1Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany 2Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany 3NeuroCure Cluster of Excellence, Charité Universitätsmedizin Berlin, Berlin, Germany †Present address: Charité Universitätsmedizin Berlin, Berlin, Germany †Present address: Broad Institute, Cambridge, MA, USA †Present address: Institute of Science and Technology Austria (IST), Klosterneuburg, Austria ‡These authors contributed equally to this work *Corresponding author. Tel: +49 30 9406 2961; E-mail: [email protected] EMBO J (2020)39:e103358https://doi.org/10.15252/embj.2019103358 See also: B Schwappach (May 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Introduction Ion homeostasis of intracellular organelles like endosomes, lysosomes, and synaptic vesicles (SVs) is important for luminal enzyme activity, ligand–receptor interactions, transmembrane voltage, transport of neurotransmitters, and other substrates across their limiting membranes, as well as vesicle budding, fusion, and trafficking. To date, most studies concentrated on luminal pH that drops along the endosomal/lysosomal pathway. This acidification is driven by the proton pump activity of V-type ATPases that need a neutralizing counter current. This current is believed to be carried mainly by chloride ions, although acidification of lysosomes prominently depends on cation channels (Steinberg et al, 2010; Weinert et al, 2010). The Cl−-dependent shunt was previously thought to be mediated by CLC Cl− channels (Günther et al, 1998; Piwon et al, 2000; Kornak et al, 2001), but the vesicular ClC-3 to ClC-7 proteins, in contrast to the plasma membrane Cl− channels (ClC-1, ClC-2, and ClC-K), are rather 2Cl−/H+-exchangers (Picollo & Pusch, 2005; Scheel et al, 2005; Neagoe et al, 2010; Weinert et al, 2010; Leisle et al, 2011; Rohrbough et al, 2018). Both, CLC channels and exchangers function as dimers with two independent ion permeation pathways that are entirely contained within each CLC monomer (Ludewig et al, 1996; Middleton et al, 1996; Weinreich & Jentsch, 2001; Dutzler et al, 2002; Zdebik et al, 2008; Jentsch & Pusch, 2018). The physiological importance of vesicular CLCs is highlighted by mouse models and patients carrying CLCN mutations. Their pathologies range from impaired renal endocytosis and kidney stones (ClC-5) (Lloyd et al, 1996; Piwon et al, 2000; Wang et al, 2000) to severe neurodegeneration (ClC-3) (Stobrawa et al, 2001), intellectual disability and epilepsy (ClC-4) (Veeramah et al, 2013; Hu et al, 2016; Palmer et al, 2018), to lysosomal storage disease (ClC-6) (Poët et al, 2006) or osteopetrosis associated with lysosomal storage and neurodegeneration (ClC-7) (Kornak et al, 2001; Kasper et al, 2005). A gain-of-function mutation in CLCN7 causes lysosomal storage disease and hypopigmentation without osteopetrosis (Nicoli et al, 2019). Not only Cl− channels, but also electrogenic 2Cl−/H+-exchangers are able to shunt proton pump currents. Indeed, the 2Cl−/H+-exchanger ClC-5 may support the acidification of renal endosomes (Günther et al, 2003; Hara-Chikuma et al, 2005a; Novarino et al, 2010) and ClC-7 is required to acidify the resorption lacuna of osteoclast (Kornak et al, 2001). However, more recent experiments demonstrate that the exchange activity, rather than the provision of an electrical shunt, is crucial for most biological roles of ClC-5 and ClC-7 (Novarino et al, 2010; Weinert et al, 2010). Clcn5unc and Clcn7unc/unc knock-in mice which carry uncoupling point mutations in the “gating glutamate”, a pore residue critically involved in 2Cl−/H+-exchange (Accardi & Miller, 2004), revealed that the conversion of 2Cl−/H+-exchange into a pure Cl− conductance causes similar pathologies as the disruption of Clcn5 and Clcn7, respectively (Novarino et al, 2010; Weinert et al, 2010). Comparable uncoupling mutations in CLCN5 were subsequently identified in patients with Dent's disease (Sekine et al, 2014; Bignon et al, 2018). These observations suggest an important role for proton-driven endosomal–lysosomal Cl− accumulation. Indeed, lysosomal Cl− concentration, but not luminal pH, was reduced in both Clcn7−/− and Clcn7unc/unc mice (Weinert et al, 2010). The transport properties of ClC-3 had been highly controversial (for review, see (Jentsch, 2008; Jentsch & Pusch, 2018)), but it is now established that ClC-3 is a vesicular 2Cl−/H+-exchanger (Matsuda et al, 2008; Guzman et al, 2013; Jentsch & Pusch, 2018; Rohrbough et al, 2018) like the close homologs ClC-4 and ClC-5 (Picollo & Pusch, 2005; Scheel et al, 2005) and the other vesicular CLCs. In addition to its presence on endosomes, ClC-3 may also be found on SVs (Stobrawa et al, 2001; Salazar et al, 2004; Seong et al, 2005; Grønborg et al, 2010) and synaptic-like microvesicles (SLMVs) of neuroendocrine cells (Salazar et al, 2004; Maritzen et al, 2008). However, more recent work questioned a significant presence of ClC-3 on SVs (Schenck et al, 2009). Furthermore, these authors suggested that the reduced acidification of SVs from Clcn3−/− mice (Stobrawa et al, 2001) rather results from a secondary decrease in the vesicular glutamate transporter VGLUT1 which may also conduct chloride (Schenck et al, 2009). We now asked whether the biological role of ClC-3 depends on its Cl−/H+ exchange activity or on its electrical conductance and whether ClC-3 is important for SV function. In stark contrast to the severe neurodegeneration of Clcn3−/− mice (Stobrawa et al, 2001; Dickerson et al, 2002; Yoshikawa et al, 2002), newly generated Clcn3unc/unc mice carrying an uncoupling mutation in ClC-3 lacked detectable phenotypes. This could be explained by a compensation by ClC-4 with which ClC-3 forms heteromers in vivo. Disruption of ClC-3 leads to increased ER retention and degradation of ClC-4, suggesting that a reduction of ClC-4 levels contributes to the severe neurodegeneration of Clcn3−/− mice. We also demonstrated that ClC-3 is expressed on a fraction of SVs and that miniature postsynaptic currents and SV acidification were not affected in young Clcn3−/− mice before the onset of neurodegeneration. Proton-driven Cl− transport by ClC-3, if not compensated by ClC-4, is crucial for the integrity of the CNS. ClC-3 overwhelmingly localizes to endosomes and apparently has no significant role in SVs. Results Clcn3unc/unc mice do not display neurodegeneration To elucidate whether the electrical conductance or the Cl−/H+-exchange activity of ClC-3 is crucial for its biological role, we generated Clcn3unc/unc knock-in mice carrying the E224A mutation in the “gating glutamate” (Appendix Fig S1A–D). When studied in a ClC-3 construct that partially localizes to the plasma membrane (Zhao et al, 2007) (Fig EV1A), this mutation linearized the normally strongly outwardly rectifying ClC-3 currents (Fig EV1B and C). As previously observed with the bacterial ecClC exchanger (Accardi & Miller, 2004) and mammalian ClC-4 through ClC-7 (Picollo & Pusch, 2005; Scheel et al, 2005; Neagoe et al, 2010; Leisle et al, 2011), this mutation also uncouples Cl− currents from H+ countertransport (Rohrbough et al, 2018). “Uncoupled” CLC exchangers mediate channel-like Cl− conductances without appreciable transport of H+. Clcn3unc/unc mice were viable and expressed the mutant ClC-3unc protein at wild-type (WT) levels (Appendix Fig S1E). These mice were born at Mendelian ratios, were fertile, and had no obvious phenotype. Even at 20 months of age, they neither displayed the severe degeneration of the CNS nor of the retina (Fig 1A and B) that is observed in Clcn3−/− mice (Stobrawa et al, 2001; Dickerson et al, 2002; Yoshikawa et al, 2002). This contrasts with findings for Clcn5unc and Clcn7unc/unc mice (Novarino et al, 2010; Weinert et al, 2010) which phenotypically largely resemble the respective null mice (Piwon et al, 2000; Kornak et al, 2001). These observations raised the possibility that another vesicular CLC protein might compensate for the loss of ClC-3 2Cl−/H+-exchange in Clcn3unc/unc, but not in Clcn3−/− mice. Click here to expand this figure. Figure EV1. Currents of partially plasma membrane directed “WT” ClC-3 and its E224A mutant When transfected into HeLa cells, wild-type ClC-3 (green) localized to enlarged Lamp-1-positive (red) late endosomes/lysosomes, GFP-ClC-34xLA (green) co-localizes with Lamp-1 (red) in normal sized vesicular structures, but is also partially redirected to the plasma membrane. DNA stained with DAPI [scale bar at bottom: 20 μm (for all panels); scale bar in inset: 5 μm]. Current–voltage relationships of HeLa cells, either non-transfected controls or transiently transfected with GFP-ClC-34xLA (“WT”) and GFP-ClC-34xLA E224A (“E224A”). Cells were examined 48–72 h post-transfection by whole-cell patch-clamp using 1-s voltage pulses and 20-mV increments from −80 to +140 mV. n.t., not transfected. Mean values ± SEM “WT”, n = 8; “E224A”, n = 4; n.t., n = 4, *P < 0.05, MW test. Currents of surface-directed GFP-ClC-34xLA were strongly outwardly rectifying and similar to ClC-3 currents reported by others (Li et al, 2002; Picollo & Pusch, 2005; Matsuda et al, 2008; Guzman et al, 2013) and as described for a ClC-5/ClC-3 chimera (Rohrbough et al, 2018). They resembled currents of ClC-4 and ClC-5 (Steinmeyer et al, 1995; Friedrich et al, 1999) but were only barely above background. Mutating the “gating glutamate” E224 to alanine abolished rectification, as reported for ClC-3 (Li et al, 2002; Matsuda et al, 2008; Rohrbough et al, 2018), ClC-4, and ClC-5 (Friedrich et al, 1999) and ClC-7 (Leisle et al, 2011). These low expression levels precluded measurements of H+-transport, but recent studies using ClC-5/3 chimeras convincingly demonstrate that ClC-3 is a Cl−/H+-antiporter which can be uncoupled by the E224A (“unc”) mutation (Rohrbough et al, 2018). Example traces of GFP-ClC-34xLA (“WT”) and GFP-ClC-34xLA E224A (“E224A”). Download figure Download PowerPoint Figure 1. Hippocampal morphology and expression of intracellular CLC proteins of Clcn3 mouse models Nissl-stained sagittal brain sections reveal no change in hippocampal morphology between Clcn3unc/unc and Clcn3+/+ mice at P45 or after 20 months. In contrast, the hippocampus was absent (indicated by asterisk) in 3-month-old Clcn3−/− mice (scale bar: 200 μm). Nissl-stained paraffin sections show intact retinal layers in 20-month-old Clcn3unc/unc mice. Neurodegeneration in Clcn3−/− mice, however, results in a loss of retinal structure already at 11 weeks of age (scale bar: 100 μm). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, photoreceptor outer segments; RPE, retinal pigment epithelium. Immunoblots for ClC-3, ClC-4, and CIC-5 of membrane fractions of WT (+/+), Clcn3unc/unc (unc/unc), Clcn3unc/+ (unc/+), and Clcn3−/− (−/−) mice. β-Actin, loading control. Representative Western blot for ClC-4 of membrane fractions from brain and kidney of WT (+/+), Clcn3+/− (+/−), and Clcn3−/− (−/−) mice. β-Actin, loading control. Quantification of ClC-4 immunoblots including those shown in (C, D) (normalized to actin). Mean values ± SEM. Average from ≥ 5 animals per genotype and at ≥ 2 immunoblots per animal. ***P < 0.0005, **P < 0.005, *P < 0.05 (two-tailed unpaired t-test). Source data are available online for this figure. Source Data for Figure 1 [embj2019103358-sup-0003-SDataFig1.zip] Download figure Download PowerPoint ClC-4 levels are reduced in Clcn3−/−, but not in Clcn3unc/unc mice We therefore examined the expression levels of ClC-4 and ClC-5, the closest homologs of ClC-3 (~ 76% identity), in tissues of Clcn3unc/unc and Clcn3−/− mice. Although ClC-4 mRNA levels are not changed in the brain of Clcn3−/− mice (Stobrawa et al, 2001), ClC-4 protein levels were strongly and more moderately decreased in brain and kidney of Clcn3−/− mice, respectively (Fig 1C–E). Depending on the tissue and experimental conditions, the ClC-4 band often appeared as doublet in Clcn3−/− mice (Fig EV2B). Western blot quantifications of the upper band, which likely reflects the mature glycosylated form of ClC-4 (see below), revealed that it was reduced down to 30 and 60% in brain and kidney of Clcn3−/− mice, respectively (Fig 1E). In heterozygous Clcn3+/− mice, ClC-4 protein amounts were reduced down to ~ 65% in brain and ~ 80% in kidney (Fig 1C and D). Expression of ClC-5, which is found in kidney but is almost absent from brain, appeared unchanged upon Clcn3 disruption (Fig 1C). ClC-4 protein levels were markedly reduced in all other Clcn3−/− tissues examined, including liver, pancreas, adrenal gland, spleen, lung, skeletal muscle, and heart (Fig EV2B). Treatment with PNGase F, which removes all N-linked glycans, showed that differences in size of ClC-4 were due to differential glycosylation (Fig EV2C). In contrast to the larger ClC-4 species, the lower band, which was much more prominent in most Clcn3−/− tissues, was sensitive to digestion with Endo H that cleaves oligosaccharides of core-glycosylated ER-resident membrane proteins (Fig EV2D). This indicated that in Clcn3−/− mice, a sizeable portion of ClC-4 does not leave the ER, where it is subject to degradation. Importantly, no reduction of ClC-4 protein levels was observed in brain or kidney of Clcn3unc/unc mice (Figs 1C and EV2A) which express an “uncoupled”, but otherwise intact ClC-3 protein. Importantly, whereas ClC-4 levels depended on ClC-3, ClC-3 levels were unchanged in both brain and kidney of Clcn4−/− mice (Fig EV2A). Click here to expand this figure. Figure EV2. Effect of ClC-3 on ClC-4 levels and glycosylation in various tissues Western blots for ClC-3, ClC-4, and ClC-5 of membrane fractions isolated from brain and kidney from mice of the indicated genotypes. Whereas ClC-4 protein levels depend on ClC-3, ClC-3 levels do not depend on ClC-4. β-Actin served as a loading control. Immunoblots for ClC-3 and ClC-4 of membrane fractions isolated from indicated organs of WT and Clcn3−/− mice (indicated by +/+ and −/−). β-Actin, loading control. The different sizes of ClC-4-positive bands most likely result from differences in glycosylation between tissues. Disruption of ClC-3 leads to the appearance, or increase in intensity, of the lowest band that represents the immature protein. The ratio between ClC-3 and ClC-4 proteins apparently differs between tissues. Effect of PNGase F on ClC-4 in brain and liver membrane fractions of WT (+/+) and Clcn3−/− (−/−) mice. Removal of N-linked glycans by PNGase F reveals that apparent size differences of ClC-4 between WT and Clcn3−/− tissues are owed to less glycosylation of ClC-4 in the absence of ClC-3. Treatment of brain and liver membranes with EndoH reveals that a large portion of ClC-4 in Clcn3−/− tissue has not left the ER. The arrows are pointing to the fractions of ClC-4 protein that are sensitive to endoglycosidase H treatment, i.e., has not been modified in the Golgi. Download figure Download PowerPoint ClC-3 and ClC-3unc heteromerize with ClC-4 CLC proteins function as dimers (Ludewig et al, 1996; Middleton et al, 1996; Dutzler et al, 2002). With the exception of ClC-6 and ClC-7, heterodimers have been observed upon heterologous co-expression of members of the same homology branch (Lorenz et al, 1996; Weinreich & Jentsch, 2001; Mohammad-Panah et al, 2003; Suzuki et al, 2006; Guzman et al, 2017). We therefore suspected that ClC-3 stabilizes ClC-4 by forming heterodimers in vivo. Indeed, ClC-3 antibodies co-immunoprecipitated ClC-4 from both WT and Clcn3unc/unc brain and vice versa (Fig 2A). These results were corroborated by Förster resonance energy transfer (FRET) measurements with fluorescently tagged ClC-3 and ClC-4 in transfected COS-7 cells (Fig 2B). Agreeing with previous results obtained with overexpressing HEK cells (Okkenhaug et al, 2006; Guzman et al, 2017), ClC-4 showed typical ER-like reticular staining when expressed in COS-7 cells (Fig 2C). In contrast, ClC-3 localized to vesicular structures, which were partially positive for the late endosomal/lysosomal marker Lamp-1 (Fig 2C). When both CLCs were co-transfected, ClC-4 co-localized with ClC-3 in vesicles (Fig 2D) (Guzman et al, 2017). A similar change in ClC-4 localization was observed upon co-expression with the ClC-3unc mutant (Fig 2D), as expected from our observation that the mutant ClC-3 protein retains its physical interaction with ClC-4 (Fig 2A). Hence, both WT ClC-3 and ClC-3unc associate with ClC-4 and thereby promote the transport of ClC-4 from the ER to endosomal–lysosomal compartments and protects it from degradation. Figure 2. Formation of ClC-3/ClC-4 heteromers Co-immunoprecipitation reveals a ClC-3–ClC-4 complex. Ten percent of solubilized brain membranes of WT and Clcn3unc/unc mice were directly loaded on the gel (input, or first immunoprecipitated (IP) with antibodies against ClC-3 or ClC-4). Western blots were probed for ClC-3 and ClC-4. Equivalent amounts of lysates and precipitates were loaded. * unspecific band/contamination. FRET experiments show homo- and heteromerization of ClC-3 and ClC-4 constructs [fused to yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)] overexpressed in COS-7 cells. Co-expressed constructs are indicated. Graph represents energy transfer efficiencies from acceptor photobleaching, depicted as bleach corrected values (subtraction of ClC-4CFP alone). Mean values ± SEM. N = 20 (ClC-3YFP/ClC-4CFP); 33 (ClC-3YFP/ClC-3CFP); 26 (ClC-4YFP/ClC-4CFP); 42 (ClC-3YFP/Lamp-1CFP) cells. ***P < 0.0005 (two-tailed unpaired t-test). Immunolabeling shows subcellular localization of hClC-4 (top, green in merge), hClC-3 (middle, green in merge), and hClC-3unc (bottom, green in merge) of transiently transfected COS-7 cells, in comparison with either PDI or Lamp-1 (both red) as marker for the ER and late endosomes/lysosomes, respectively. Co-localization of hClC-3 and hClC-4 in cytoplasmic vesicles of COS-7 cells transfected with hClC-4 cDNA [hemagglutinin (HA) tagged] together with either hClC-3 or hClC-3unc. Immunostaining used antibodies against ClC-3 and HA tag (green and red in merge, respectively). Data information: DNA stained with DAPI in (C) and (D), [scale bar in (C) and (D): 20 μm]. Source data are available online for this figure. Source Data for Figure 2 [embj2019103358-sup-0004-SDataFig2.zip] Download figure Download PowerPoint Severe neurodegeneration in Clcn3unc/unc/Clcn4−/− mice The above experiments suggested that ClC-4 may compensate for a loss of ClC-3 function in Clcn3unc/unc, but not in Clcn3−/− mice in which brain ClC-4 levels are decreased to ~ 30% of WT (Fig 1E). To completely eliminate this potential compensation, we crossed Clcn3unc/unc mice with Clcn4−/− mice. Although Clcn4−/− mice appear to be normal (Rickheit et al, 2010), we reassessed potential effects of Clcn4 disruption because mutations in CLCN4 were recently associated with X-linked intellectual disability and epileptic encephalopathy (Veeramah et al, 2013; Hu et al, 2016; Palmer et al, 2018). However, Clcn4−/− mice neither displayed discernible anatomical changes in the brain (Appendix Fig S2), nor obvious behavioral abnormalities. Clcn3unc/unc/Clcn4−/− mice were born at Mendelian ratio. Similar to Clcn3−/− mice (Stobrawa et al, 2001; Dickerson et al, 2002; Yoshikawa et al, 2002), they were growth-retarded. Most died within 5 weeks after birth and only ~ 20% became older than 10 weeks (Fig 3A). Brains from Clcn3unc/unc/Clcn4−/− mice displayed severe neurodegeneration (Fig 3B). Like in Clcn3−/− mice (Stobrawa et al, 2001), degeneration became first apparent in the hippocampus, but progressed much faster. Whereas the hippocampus began to show mild degeneration at P21 and almost totally disappeared at 10 weeks of age in Clcn3−/− mice (Stobrawa et al, 2001), Clcn3unc/unc/Clcn4−/− mice showed severe hippocampal degeneration at P21 and mice had lost their hippocampi already 4 weeks after birth (Fig 3B). Again similar to Clcn3−/− mice, neurodegeneration in Clcn3unc/unc/Clcn4−/− mice was accompanied by an activation of astrocytes as indicated by GFAP staining. Clcn3unc/unc/Clcn4−/− mice showed severe retinal degeneration (Fig 3C), similar to Clcn3−/− mice (Stobrawa et al, 2001). Figure 3. Life span and neurodegeneration of Clcn3/Clcn4 mouse models Clcn3unc/unc/Clcn4−/− and Clcn3−/− mice died within 3–4 weeks after birth. Approximately 20% of the animals survived in either line (Clcn3unc/unc/Clcn4−/−, n = 136, and Clcn3−/−, n = 187). All Clcn3−/−/Clcn4−/− mice (n = 4) died within 1–2 weeks after birth. Nissl-stained paraffin sections show progressive neuronal cell loss (arrows) that begins in hippocampal CA1 region of P14 Clcn3unc/unc/Clcn4−/− mice and results in a complete loss of the hippocampus at P28. Neurodegeneration progresses slower in Clcn3−/− mice (Stobrawa et al, 2001) (scale bar: 200 μm). Semi-thin sections of P28 retinae revealed degeneration of photoreceptor cells in the outer nuclear layer and outer and inner segment of Clcn3unc/unc/Clcn4−/−, but not of Clcn3unc/unc or Clcn4−/− mice (scale bar: 50 μm). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, photoreceptor outer segments; RPE, retinal pigment epithelium. Source data are available online for this figure. Source Data for Figure 3 [embj2019103358-sup-0005-SDataFig3.zip] Download figure Download PowerPoint The more severe phenotype of Clcn3unc/unc/Clcn4−/− compared to Clcn3−/− mice suggested that the severity of neurodegeneration depends on the expression levels of ClC-4. Clcn3unc/unc/Clcn4+/− mice, in which ClC-4 protein levels are reduced to 75% of normal, did not show neurodegeneration even at 12 weeks of age (Appendix Fig S3A). Likewise, on a Clcn4−/− background, heterozygous Clcn3unc alleles did not cause pathologies (Appendix Fig S3B). In contrast, only very few Clcn3−/−/Clcn4−/− mice were born and then died shortly after birth (Fig 3A). Hence, ClC-3 and ClC-4 have partially overlapping functions. The occurrence of neurodegeneration in Clcn3−/−, but not Clcn4−/− mice might be largely explained by the fact that ClC-4 levels are strongly decreased in Clcn3−/− mice, whereas ClC-3 abundance does not depend on ClC-4. As revealed only in the absence of ClC-4, the pure Cl− conductance of ClC-3unc cannot functionally replace the 2Cl−/H+-exchange of WT ClC-3. However, this conductance may substitute for some aspect of ClC-3 function as revealed by the milder phenotype of Clcn3unc/unc/Clcn4−/− compared to Clcn3−/−/Clcn4−/− mice. However, we cannot exclude that the more severe phenotype of the double knock-out is in part owed to" @default.
• W3010165610 created "2020-03-13" @default.
• W3010165610 creator A5016021031 @default.
• W3010165610 creator A5016899192 @default.
• W3010165610 creator A5022849603 @default.
• W3010165610 creator A5027504599 @default.
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• W3010165610 creator A5045974509 @default.
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• W3010165610 creator A5061950306 @default.
• W3010165610 creator A5075639449 @default.
• W3010165610 creator A5081710490 @default.
• W3010165610 date "2020-03-02" @default.
• W3010165610 modified "2023-10-16" @default.
• W3010165610 title "Uncoupling endosomal <scp>CLC</scp> chloride/proton exchange causes severe neurodegeneration" @default.
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