Chloride Accumulation in Endosomes and Lysosomes: Facts Checked in Mice

News & Views

Chloride accumulation in endosomes and lysosomes: facts checked in mice

Blanche Schwappach

Loss of the ClC-3 chloride/proton exchanger counts (in contrast to the proton electromo- 2003) or the osteoclast resorption lacuna, found in intracellular compartments leads tive force that is harnessed by mitochondria sometimes considered to be an “extracellular to marked neurodegeneration. New to make ATP), then send any negatively lysosome” (Kornak et al, 2001). So, why genetic work by Weinert et al (2020) now charged ion in parallel to the protons and look any further and wonder about a shows that selective impairment of ClC-3’s you can accumulate them to a higher putative role of chloride beyond being nega- ion exchange activity is sufficient to elicit concentration, i.e., acidify the vesicle more tive? In science, as in real life, the most obvi- this severe phenotype in vivo. ClC-3 cooper- efficiently. This mechanism is called “chlo- ous explanation is not always the correct ates with the closely related ClC-4 in ride-dependent shunt”. For the longest time, one or at least not the full truth. To echo protecting endolysosomal chloride balance Ockham’s razor seemed to suggest very George Gershwin, “It ain’t necessarily so”. and neuronal integrity. plausibly that endosomal or lysosomal chlo- The normal lysosomal pH of mice lacking ride was just this, any negatively charged lysosomal ClC-7 or its b-subunit Ostm1 The EMBO Journal (2020) 39:e104812 ion compensating for the protons’ positive (Kasper et al, 2005; Lange et al, 2006), See also: S Weinert et al (May 2020) charge. which display severe lysosomal storage It seemed unnecessary to challenge this disease, suggested that CLC exchangers not ndosomes and lysosomes are compart- concept and has, indeed, proven experimen- only serve as chloride-dependent shunts. ments with critical functions in nutri- tally difficult to do so. The impulse to revisit Addressing the question if coupled transport E ent uptake, hormone signaling, a seemingly solved problem came from a activity is per se physiologically relevant, detoxification, and the recycling of metabo- seminal discovery by Accardi and Miller the Jentsch Lab has exploited the discovery lites or cellular building blocks. The ion (2004), their amazing demonstration that of uncoupling CLC mutations (Accardi & composition of the aqueous phase inside prokaryotic CLC homologues act as Miller, 2004) that convert the exchangers these compartments is critical to the broad secondary active transporters and are able to into a passively conducting, chloride- spectrum of biochemical reactions that occur use the energy stored in one ion’s gradient permeable pore: They embarked on the in both of them—from uncoupling receptors to move a second one against its electro- construction of a series of transgenic mouse and their ligands to the activity of many dif- chemical potential (secondary active trans- models expressing uncoupled intracellular ferent enzymes. For decades, the concentra- port). Prior to this discovery, CLC proteins CLC proteins and started a careful and tion of protons, or pH, inside endosomes were thought to be chloride channels, systematic comparison to the corresponding and lysosomes has received intense atten- passively allowing chloride ions to move in knockout mouse models. This strategy is tion. While any biochemist would agree that the energetically favored direction. elegant not only because it segregates the pH is one of the key parameters for optimal The unexpected discovery raised many physiological relevance of two biophysically enzyme function, it is well known that the questions regarding the biophysical trans- distinct transport modes—passive chloride correct concentration of other ions can be an port mechanism and the physiological rele- leak as opposed to proton gradient-driven equally important factor. In the past decade, vance of animal or plant intracellular chloride accumulation. It also segregates the it has emerged that the chloride ion is membrane proteins of the CLC family, such effects of losing the best-characterized and indeed critical for endosomal and lysosomal as mammalian ClC-3, ClC-4, ClC-5, ClC-6, presumably central function of a protein function (Novarino et al, 2010; Weinert and ClC-7. Indeed, these proteins were from the effect of losing the protein per se, À et al, 2010). found to function as 2Cl /H+ exchangers, with all its protein–protein interactions, The accumulation of protons is an too. This made the hypothesis that chloride regulation, or unknown functions. electrogenic (potential-building) process. “just” provides negative charge mechanisti- The work of Weinert et al (2020) now Hence, it is more effective when ions of the cally more complicated but still tenable reveals just how hard one had to look to opposite charge are allowed to accompany since it was observed that losing such trans- tackle the relevance of the exchange mode the protons inside the vesicular compart- porters did in some cases impair proper to the neuroprotective capacity of ClC-3. ment. Simply speaking—if the pH is all that acidification of endosomes (Gu¨nther et al, They replaced ClC-3 with the uncoupled

Department of Molecular Biology, Universitätsmedizin Göttingen, Göttingen, Germany. E-mail: [email protected] DOI 10.15252/embj.2020104812 | The EMBO Journal (2020) 39:e104812 | Published online 8 April 2020

Soma of CA3 neuron

WILD TYPE MUTANT MUTANT Clcn3 +/+ Clcn3 unc/unc Clcn3 unc/unc Clcn4+/+ Clcn4+/+ Clcn4 KO H+ H+ H+ Endosome ClC-4 ClC-3 ClC-4 ClC-3unc ClC-3unc ClC-3unc

Cl– Cl– Cl– Cl– Cl– Cl– Late endosome Late endosome Late endosome H+ H+ H+

CA3 neurons

ATP ADP ATP ADP ATP ADP

NORMAL DEVELOPMENT NORMAL DEVELOPMENT NEURONAL LOSS

CA1 CA1 Hippocampus

DG CA3 DG CA3

Figure 1. Assembly-dependent trafficking and chloride accumulation by the CLC-3 and CLC-4 exchanger proteins support neuronal health.

À À variant of the protein ClC-3unc/unc. Clcn3 / but so far not shown to be essential for any masked by co-assembly with ClC-4 and only knockout mice lose their hippocampus and CLC transporter’s cellular function. Biochemi- revealed in a mouse model combining the turn blind due to retinal degeneration cal investigation of the steady-state levels and knock-in of uncoupled ClC-3 with a knockout (Stobrawa et al, 2001). Surprisingly, and in glycosylation status of ClC-4, a close homo- of the Clcn4 gene (which does not give rise to contrast to the work on ClC-5 and ClC-7 logue of ClC-3 also robustly expressed in a severe phenotype illustrating that ClC-3 can where the replacement by the uncoupled neurons, enabled Weinert et al (2020) to fulfill its basic function without ClC-4). transporter phenocopies the knockout reveal that ClC-4 has to co-assemble with In conclusion, this work unequivocally (Novarino et al, 2010; Weinert et al, 2010), ClC-3 to leave the early secretory pathway shows the central role of CLC-mediated unc unc this was not the case in Clcn3 / and to provide its ion transport activity to the chloride/proton exchange and hence vesic- mice, which turned out to be unexpectedly right cellular compartment in the endolysoso- ular chloride accumulation in the neuro- normal (Fig 1). mal system. Indeed, mice expressing protective function of ClC-3. It provides a unc unc CLC membrane proteins are obligatory Clcn3 / but lacking Clcn4 show severe clear example of assembly-dependent traf- dimers with an ion-conducting pathway in neurodegeneration (Fig 1). As with the intra- ficking of a multimeric membrane protein each monomer. The overlapping expression cellular ClC-5 and ClC-7, the exchanger trans- and thereby illustrates that forward protein profiles of several CLC proteins raise the port mode, and hence the capacity to transport along the secretory pathway is possibility of heterodimerization, which has accumulate chloride inside vesicles, is critical anything but default. Like all intriguing been demonstrated for some combinations to ClC-3’s physiological function. This is scientific work, the article by Weinert et al

2 of 3 The EMBO Journal 39:e104812 | 2020 ª 2020 The Author Blanche Schwappach The EMBO Journal

(2020) opens new questions: Why is the Günther W, Piwon N, Jentsch TJ (2003) The ClC-5 rather than chloride conductance is crucial ClC-4 protein so strictly attended by ClC-3? chloride channel knock-out mouse – an animal for renal endocytosis. Science 328: Is there a specific functional or regulatory model for Dent’s disease. Pflugers Arch 445: 1398 – 1401 capacity endowed by ClC-4 to ClC-3/ClC-4 456 – 462 Stobrawa SM, Breiderhoff T, Takamori S, Engel D, heterodimers? Genetic links to human Kasper D, Planells-Cases R, Fuhrmann JC, Scheel O, Schweizer M, Zdebik AA, Bösl MR, Ruether K, brain disorders suggest that this may be Zeitz O, Ruether K, Schmitt A, Poët M, Steinfeld Jahn H, Draguhn A et al (2001) Disruption of the case. One can hope that this and R, Schweizer M et al (2005) Loss of the chloride ClC-3, a chloride channel expressed on synaptic related questions around the biochemical channel ClC-7 leads to lysosomal storage vesicles, leads to a loss of the hippocampus. function of chloride ions inside endosomal disease and neurodegeneration. EMBO J 24: Neuron 29: 185 – 196 or lysosomal compartments will be 1079 – 1091 Weinert S, Jabs S, Supanchart C, Schweizer M, pursued by a similarly rich portfolio of Kornak U, Kasper D, Bösl MR, Kaiser E, Schweizer M, Gimber N, Richter M, Rademann J, Stauber T, cutting-edge approaches in gene targeting, Schulz A, Friedrich W, Delling G, Jentsch TJ (2001) Kornak U, Jentsch TJ (2010) Lysosomal biochemistry, histology, cell biology, and Loss of the ClC-7 chloride channel leads to pathology and osteopetrosis upon loss of H+- physiology to elucidate even unexpected osteopetrosis in mice and man. Cell 104: driven lysosomal ClÀ accumulation. Science 328: facts. 205 – 215 1401 – 1403 Lange PF, Wartosch L, Jentsch TJ, Fuhrmann JC Weinert S, Gimber N, Deuschel D, Stuhlmann T, References (2006) ClC-7 requires Ostm1 as a beta-subunit Puchkov D, Farsi Z, Ludwig CF, Novarino G, Accardi A, Miller C (2004) Secondary active to support bone resorption and lysosomal López-Cayuqueo KI, Planells-Cases R et al transport mediated by a prokaryotic function. Nature 440: 220 – 223 (2020) Uncoupling endosomal CLC chloride/ homologue of ClC Cl- channels. Nature Novarino G, Weinert S, Rickheit G, Jentsch TJ proton exchange causes severe 427: 803 – 807 (2010) Endosomal chloride-proton exchange neurodegeneration. EMBO J 39:e103358

ª 2020 The Author The EMBO Journal 39:e104812 | 2020 3 of 3