Pathophysiological role of MLC1, a protein involved in megalencephalic leukoencephalopathy with subcortical cysts

Megalencephalic leukoencephalopathy with subcortical cysts (MLC), is a rare congenital and incurable leukodystrophy characterized by macrocephaly, subcortical fluid cysts and myelin vacuolation. The majority of MLC patients carry mutations in the MLC1 gene encoding a membrane protein named MLC1 that is highly expressed in brain astrocytes contacting blood vessels, ependyma and meninges. Although the neuropathological features of MLC disease, the molecular structure and the cellular localization of MLC1 suggest a possible involvement of this protein in astrocyte-mediated osmoregulatory processes, the function of MLC1 is still unknown. Understanding the function of MLC1 protein whose mutations are the main cause of MLC is an essential step toward identification of disease mechanisms and development of effective therapies. During the course of this thesis project we generated new data on MLC1 expression, distribution and functional associated pathways in astrocytes that are deregulated by pathological mutations, paving the way for the identification of the specific MLC1 function. We found that: i) endogenous MLC1 protein is expressed in cultured astrocytes, particularly in the plasma membrane where it interacts with caveolin-1 and proteins of the dystrophin/dystroglycan complex (DCG), and also in intracellular organelles and endoplasmic reticulum (Lanciotti et al., 2010); ii) MLC1 undergoes endolysosomal trafficking and, most of the missense mutations found in patients hamper MLC1 intracellular trafficking and localization at the plasma membrane (Lanciotti et al., 2010, 2012); iii) MLC1 directly binds the beta-1 subunit of the Na, K-ATPase enzyme and is part of a multiprotein complex that includes the inward rectifying potassium channel 4.1 (Kir4.1), caveolin-1 and syntrophin, and is involved in astrocyte response to hyposmotic stress (Brignone et al., 2011). Moreover, we generated a human pathological model based on astrocytoma cell lines overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. Using this new MLC disease model we found that WT, but not mutated MLC1, functionally interacts with the transient receptor potential cation channel-4 (TRPV4) to activate swelling-induced calcium influx in astrocytes during hyposmotic stress (Lanciotti et al, 2012). These findings, together with a recent study showing defects in a chloride current in patient-derived lymphoblast cell lines subjected to hyposmosis (Ridder et al, 2011), represent the first evidence that the MLC1 protein is involved in the molecular pathways regulating astrocyte response to osmotic changes