title: 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.
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Electronic address: mariastefania.brignone@gmail.com.
authors.forename: Maria S
authors.initials: MS
authors.lastname: Brignone
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Electronic address: angela.lanciotti@iss.it.
authors.forename: Angela
authors.initials: A
authors.lastname: Lanciotti
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Electronic address: sergio.visentin@iss.it.
authors.forename: Sergio
authors.initials: S
authors.lastname: Visentin
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Electronic address: chiara.denuccio@iss.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: De Nuccio
authors.affiliation: Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Electronic address: paola.molinari@iss.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Molinari
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Electronic address: serena.camerini@iss.it.
authors.forename: Serena
authors.initials: S
authors.lastname: Camerini
authors.affiliation: Department of Technology and Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Electronic address: marco.diociaiuti@iss.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Diociaiuti
authors.affiliation: Unit of Neurodegenerative Disorders, Laboratory of Molecular Medicine, Bambino Gesù Pediatric Research Hospital, Piazza S. Onofrio 4, 00165 Rome, Italy. Electronic address: stefania.petrini@opbg.net.
authors.forename: Stefania
authors.initials: S
authors.lastname: Petrini
authors.affiliation: Unit of Neurodegenerative Disorders, Laboratory of Molecular Medicine, Bambino Gesù Pediatric Research Hospital, Piazza S. Onofrio 4, 00165 Rome, Italy. Electronic address: gaetana.minnone@yahoo.it.
authors.forename: Gaetana
authors.initials: G
authors.lastname: Minnone
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Electronic address: marco.crescenzi@iss.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Crescenzi
authors.affiliation: Unit of Neurodegenerative Disorders, Laboratory of Molecular Medicine, Bambino Gesù Pediatric Research Hospital, Piazza S. Onofrio 4, 00165 Rome, Italy; Institute of Translational Pharmacology, CNR, Via del Fosso Cavaliere 100, 00133 Rome, Italy. Electronic address: luisa.braccilaudiero@inmm.cnr.it.
authors.forename: Luisa Bracci
authors.initials: LB
authors.lastname: Laudiero
authors.affiliation: Unit of Neurodegenerative Disorders, Laboratory of Molecular Medicine, Bambino Gesù Pediatric Research Hospital, Piazza S. Onofrio 4, 00165 Rome, Italy. Electronic address: ebertini@gmail.com.
authors.forename: Enrico
authors.initials: E
authors.lastname: Bertini
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Electronic address: tcpetrucci.petrucci61@gmail.com.
authors.forename: Tamara C
authors.initials: TC
authors.lastname: Petrucci
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Electronic address: elena.ambrosini@iss.it.
authors.forename: Elena
authors.initials: E
authors.lastname: Ambrosini
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D050916:TRPV Cation Channels / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
chemical_list: D002118:Calcium; D008565:Membrane Proteins; D050916:TRPV Cation Channels; D014168:Transferrin; D025262:Vacuolar Proton-Translocating ATPases; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C425069:MLC1 protein, human; C416986:TRPV4 protein, human; C093846:early endosome antigen 1
keywords: Calcium; Early/recycling endosomes; Hyposmosis; K-ATPase; Leukodystrophy; Na; Rab11; TRPV4; V-ATPase