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.

The endoplasmic reticulum (ER) stress-mediated pathway is involved in a wide range of human neurodegenerative disorders. Hence, molecules that regulate the ER stress response represent potential candidates as drug targets to tackle these diseases. In previous studies we demonstrated that upon acetylation the reticulon-1C (RTN-1C) variant of the reticulon family leads to inhibition of histone deacetylase (HDAC) enzymatic activity and endoplasmic reticulum stress-dependent apoptosis. Here, by microarray analysis of the whole human genome we found that RTN-1C is able to specifically regulate gene expression, modulating transcript clusters which have been implicated in the onset of neurodegenerative disorders. Interestingly, we show that some of the identified genes were also modulated in vivo in a brain-specific mouse model overexpressing RTN-1C. These data provide a basis for further investigation of RTN-1C as a potential molecular target for use in therapy and as a specific marker for neurological diseases.