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

Abstract

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

Niemann–Pick type C disease: cellular pathology and pharmacotherapy

“…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.…”

“…Accordingly, the sub‐population of GlcCer on the cytosolic face of membranes ('cytosolic GlcCer') emerges as an important factor in NPC disease. We have recently demonstrated that cytosolic GlcCer may be responsible for regulating vATPase and hence that GBA2 inhibition results in correction of defects in both lysosomal acidification and endocytic trafficking in NPCD (Wheeler et al ). Potentially this ties in well with the ideas that NPC1 is a sphingosine exporter (Lloyd‐Evans et al ; Höglinger et al ; Höglinger et al ; Wheeler et al ) and that Sph inhibits GBA2 (Schonauer et al ).…”

“…Potentially this ties in well with the ideas that NPC1 is a sphingosine exporter (Lloyd‐Evans et al ; Höglinger et al ; Höglinger et al ; Wheeler et al ) and that Sph inhibits GBA2 (Schonauer et al ). Thus reduced Sph export removes an endogenous negative regulator of GBA2 leading to a reduction in cytosolic GlcCer and consequently reduced vATPase activation and increased LEL pH (Chakraborty et al ; Tharkeshwar et al ; Wheeler et al ).…”

Role of Endolysosomes in Severe Acute Respiratory Syndrome Coronavirus-2 Infection and Coronavirus Disease 2019 Pathogenesis: Implications for Potential Treatments

“…Coronaviruses enter host cells by pH-dependent endocytosis ( Yang et al, 2004 ; Burkard et al, 2014 ; Hoffmann et al, 2020b ) and the acidic environment of endolysosomes is regulated not only by v-ATPase ( Mindell, 2012 ), but also by Na + /K + -ATPase ( Cain et al, 1989 ), mucolipin (TRPML1) channels ( Li M. et al, 2017 ), big potassium channels (BK and MaxiK) ( Khan et al, 2019b ), Niemann-Pick type C (NPC1) ( Wheeler et al, 2019a ; Wheeler et al, 2019b ; Höglinger et al, 2019 ; Lim et al, 2019 ), and two-pore channels (TPCs) ( Marchant and Patel, 2015 ; Grimm et al, 2017 ; Khan et al, 2020 ). To date, TPCs and NPC1 have both been implicated in coronavirus infectivity.…”

“…Niemann-Pick disease type C1 (NPC1) is an endolysosome-resident protein ( Higgins et al, 1999 ) that regulates trafficking of late endosomes and lysosomes ( Ko et al, 2001 ; Zhang et al, 2001 ; Ganley and Pfeffer, 2006 ; Sztolsztener et al, 2012 ), membrane trafficking of essential cellular factors such as cholesterol and sphingolipids ( Chen et al, 2005 ; Infante et al, 2008 ; Kwon et al, 2009 ; Lange et al, 2012 ; Höglinger et al, 2019 ), and regulation of endolysosome pH and calcium ( Elrick et al, 2012 ; Liu and Lieberman, 2019 ; Wheeler et al, 2019a ). Impaired NPC1 is an underlying cause of Niemann-Pick disease; a lysosome storage disease ( Lloyd-Evans et al, 2008 ; Schuchman and Desnick, 2017 ).…”