Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: Relevance to MLC disease pathogenesis

Abstract

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1+, Rab5+) and recycling (Rab11+) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis.

Rescuing the negative impact of human endogenous retrovirus envelope protein on oligodendroglial differentiation and myelination

“…Specific V‐ATPase inhibition was shown to prevent TLR signaling dependent Salmonella typhimurium virulence (Arpaia et al, ) and a reduced TLR4 surface expression was accompanied by inhibited nitric oxide production in murine cells (Eswarappa et al, ), which is in accordance with our previous data showing ENV dependent nitrosative stress induction in oligodendroglial cells (Kremer et al, , ). On the other hand, V‐ATPase was also identified as target for the treatment of cancer, osteoporosis, and sepsis (Cotter et al, ; Forgac, ; Kartner & Manolson, ) and a contribution of V‐ATPase in different white matter pathologies such as the megalencephalic leukoencephalopathy with subcortical cysts (MLC) is suggested based on modulated intracellular pH‐homeostasis and protein trafficking in this disease (Brignone et al, ). Of note, an involvement of V‐ATPase in white matter homeostasis and brain function is further supported by observations on ATP6ap2.…”

MLC1 protein: a likely link between leukodystrophies and brain channelopathies

“…Our studies demonstrated that in astrocytes exposed to hyposmotic stress MLC1 is translocated to the plasma membrane and then internalized by caveolar endocytosis to be sorted to recycling or degradation pathways ( Figure 3 ). By cell fractionation, immunofluorescence and electron microscopy analysis of rat primary astrocytes and human astrocytoma cells, MLC1 protein was detected in early endosomes (EEA1 + and Rab5 + ; Figures 3 and 4A,B ; Lanciotti et al, 2010 ; Brignone et al, 2014 ) and in Lamp-1 + organelles and multivesicular bodies ( Figure 4C ; Lanciotti et al, 2010 ; Brignone et al, 2014 ), but not, or at very low levels in Golgi apparatus and clathrin vesicles ( Lanciotti et al, 2010 ). More recently, we found that in unstimulated astrocytes MLC1 is abundantly expressed in the Rab11 + perinuclear storage/recycling compartment from which it is recruited back to the plasma membrane when cells are exposed to hyposmotic stress ( Figure 3 ; Brignone et al, 2014 ).…”

“…By cell fractionation, immunofluorescence and electron microscopy analysis of rat primary astrocytes and human astrocytoma cells, MLC1 protein was detected in early endosomes (EEA1 + and Rab5 + ; Figures 3 and 4A,B ; Lanciotti et al, 2010 ; Brignone et al, 2014 ) and in Lamp-1 + organelles and multivesicular bodies ( Figure 4C ; Lanciotti et al, 2010 ; Brignone et al, 2014 ), but not, or at very low levels in Golgi apparatus and clathrin vesicles ( Lanciotti et al, 2010 ). More recently, we found that in unstimulated astrocytes MLC1 is abundantly expressed in the Rab11 + perinuclear storage/recycling compartment from which it is recruited back to the plasma membrane when cells are exposed to hyposmotic stress ( Figure 3 ; Brignone et al, 2014 ). Cytoskeletal elements are involved in MLC1 intracellular trafficking since nocodazole treatment, which perturbs microtubule organization, hampers MLC1 accumulation in the Rab11 + endosomal recycling compartment in rat primary astrocytes ( Lanciotti et al, 2010 ).…”

“… Modeling of MLC1 intracellular trafficking . Our previous results indicated that MLC1 is internalized via caveolae-mediated endocytosis and traffics through Rab5 + and EEA1 + early endosomes where it is sorted to the recycling (Rab11 + ) or degradative pathway (Lamp-1 + ; Lanciotti et al, 2010 ; Brignone et al, 2014 ) (modified from Brignone et al, 2014 ). …”

“…MLC1 protein structural features, molecular interactors (see below) and brain alterations observed in MLC patients (edema, fluid cysts, astrocyte, and myelin vacuolation) suggest that MLC1 can play a role in the regulation of ion and water fluxes and cell volume. However, although several recent studies support this hypothesis ( Ridder et al, 2011 ; Lanciotti et al, 2012 ; Brignone et al, 2014 ; Hoegg-Beiler et al, 2014 ; Dubey et al, 2015 ), the exact function of MLC1 is still unknown.…”