High lumenal chloride in the lysosome is critical for lysosome function

Krishnan, Yamuna; Chakraborty, Kasturi; Leung, Ka‐Ho

doi:10.1101/141788

Cited by 23 publications

(42 citation statements)

References 75 publications

(84 reference statements)

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“…It is not known why different pools of GlcCer have opposite effects on endolysosomal pH. GlcCer storage within the lysosome increases endolysosomal pH [10][11][12][13]20] whereas increasing cytosolic GlcCer decreases pH (current study). Possibly different pools of GlcCer bind different vATPase domains with opposite effects.…”

Section: Accepted Manuscriptmentioning

confidence: 75%

Cytosolic glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease

Wheeler

Haberkant

Bhardwaj

et al. 2019

Neurobiology of Disease

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Niemann-Pick type C disease (NPCD) is a neurodegenerative disease associated with increases in cellular cholesterol and glycolipids and most commonly caused by defective NPC1, a late endosomal protein. Using ratiometric probes we find that NPCD cells show increased endolysosomal pH. In addition U18666A, an inhibitor of NPC1, was found to increase endolysosomal pH, and the number, size and heterogeneity of endolysosomal vesicles. NPCD fibroblasts and cells treated with U18666A also show disrupted targeting of fluorescent lipid BODIPY-LacCer to high pH vesicles. Inhibiting non-lysosomal glucocerebrosidase (GBA2) reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking in NPCD fibroblasts. GBA2 KO cells also show decreased endolysosomal pH. NPCD fibroblasts also show increased expression of a key subunit of the lysosomal proton pump vATPase on GBA2 inhibition. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic-facing GlcCer, which are reduced in NPC disease. Niemann-Pick type C disease (NPCD) is a devastating neurodegenerative condition most commonly due to mutations in NPC1 [1,2] a protein of late endosomes and lysosomes [3]. (Due to difficulties in precisely distinguishing these two sets of organelles the term endolysosome will be used here to include both.) Mutations in NPC1 are associated with impaired endocytic transport via decreased endolysosomal calcium release [4,5]. In turn, endocytosis and luminal calcium are dependent on correct endolysosomal acidification [4] and have been found to be controlled by glycolipids in neurons [6], melanocytes [7], plant vacuoles [8] and C. elegans [9]. It is increasingly apparent that aberrant lysosomal GlcCer in Gaucher disease is associated with elevated endolysosomal pH [10-13]. Glycolipids, vital for mammals [14], are also implicated in membrane trafficking [10,15]. Similarly in yeast the NPC1 homologue ncr1 regulates both vacuolar pH [16] and glycolipid transport [17]. An overview of sphingolipid metabolism highlighting the connection with endocytosis is offered in Fig. S1. Does NPC1 affect endolysosomal pH? Conflicting evidence has been found both in disease fibroblasts [5,16,18-20] and in cells treated with U18666A, a putative inhibitor of NPC1 [5,21-23]. If endocytic traffic is delayed then probes which permeate all acidic organelles will show increased pH values even though fully mature lysosomes still acidify correctly. We used such a general probe to

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Section: Accepted Manuscriptmentioning

confidence: 75%

Cytosolic glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease

Wheeler

Haberkant

Bhardwaj

et al. 2019

Neurobiology of Disease

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“…Another key protein is the vATPase proton pump through which these organelles acquire the requisite acidic pH. The pH of lysosomes in NPCD cells has been reported as elevated by ourselves (Wheeler et al ) as well as other workers (Tharkeshwar et al ; Chakraborty et al ) although other studies find it normal (Bach et al ; Lloyd‐Evans et al ; Elrick et al ). (We propose that these differences are because of heterogeneity of lysosomal vesicles.…”

Section: Npc1mentioning

confidence: 86%

Niemann–Pick type C disease: cellular pathology and pharmacotherapy

Wheeler

Sillence

2019

Journal of Neurochemistry

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Niemann–Pick type C disease (NPCD) was first described in 1914 and affects approximately 1 in 150 000 live births. It is characterized clinically by diverse symptoms affecting liver, spleen, motor control, and brain; premature death invariably results. Its molecular origins were traced, as late as 1997, to a protein of late endosomes and lysosomes which was named NPC1. Mutation or absence of this protein leads to accumulation of cholesterol in these organelles. In this review, we focus on the intracellular events that drive the pathology of this disease. We first introduce endocytosis, a much‐studied area of dysfunction in NPCD cells, and survey the various ways in which this process malfunctions. We briefly consider autophagy before attempting to map the more complex pathways by which lysosomal cholesterol storage leads to protein misregulation, mitochondrial dysfunction, and cell death. We then briefly introduce the metabolic pathways of sphingolipids (as these emerge as key species for treatment) and critically examine the various treatment approaches that have been attempted to date.

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“…Given that Cl − /H + ‐exchange accumulates Cl − in acidic vesicles in a secondary active process, we expect that luminal Cl − concentrations in Clcn3 −/− and Clcn3 unc/unc / Clcn4 −/− endosomes are decreased. Lower Cl − concentrations have been measured in lysosomes of cells derived from Clcn7 −/− and Clcn7 unc/unc mice (Weinert et al , , ) and in Caenorhabditis elegans lysosomes lacking the ClC‐7 ortholog (Chakraborty et al , ). Whereas lysosomes can be easily loaded with dextran‐coupled indicators, we are currently unable to specifically target Cl − sensors to endosomes.…”

Section: Discussionmentioning

confidence: 99%

Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration

et al. 2020

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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.

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High lumenal chloride in the lysosome is critical for lysosome function

Cited by 23 publications

References 75 publications

Cytosolic glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease

Cytosolic glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease

Niemann–Pick type C disease: cellular pathology and pharmacotherapy

Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration

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