Megalencephalic leukoencephalopathy with subcortical cysts protein-1: A new calcium-sensitive protein functionally activated by endoplasmic reticulum calcium release and calmodulin binding in astrocytes

MLC1 is a membrane protein highly expressed in brain perivascular astrocytes and whose mutations account for the rare leukodystrophy (LD) megalencephalic leukoencephalopathy with subcortical cysts disease (MLC). MLC is characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling which cause cognitive and motor dysfunctions and epilepsy. In cultured astrocytes, lack of functional MLC1 disturbs cell volume regulation by affecting anion channel (VRAC) currents and the consequent regulatory volume decrease (RVD) occurring in response to osmotic changes. Moreover, MLC1 represses intracellular signaling molecules (EGFR, ERK1/2, NF-kB) inducing astrocyte activation and swelling following brain insults. Nevertheless, to date, MLC1 proper function and MLC molecular pathogenesis are still elusive. We recently reported that in astrocytes MLC1 phosphorylation by the Ca /Calmodulin-dependent kinase II (CaMKII) in response to intracellular Ca release potentiates MLC1 activation of VRAC. These results highlighted the importance of Ca signaling in the regulation of MLC1 functions, prompting us to further investigate the relationships between intracellular Ca and MLC1 properties. We used U251 astrocytoma cells stably expressing wild-type (WT) or mutated MLC1, primary mouse astrocytes and mouse brain tissue, and applied biochemistry, molecular biology, video imaging and electrophysiology techniques. We revealed that WT but not mutant MLC1 oligomerization and trafficking to the astrocyte plasma membrane is favored by Ca release from endoplasmic reticulum (ER) but not by capacitive Ca entry in response to ER depletion. We also clarified the molecular events underlining MLC1 response to cytoplasmic Ca increase, demonstrating that, following Ca release, MLC1 binds the Ca effector protein calmodulin (CaM) at the carboxyl terminal where a CaM binding sequence was identified. Using a CaM inhibitor and generating U251 cells expressing MLC1 with CaM binding site mutations, we found that CaM regulates MLC1 assembly, trafficking and function, being RVD and MLC-linked signaling molecules abnormally regulated in these latter cells. Overall, we qualified MLC1 as a Ca sensitive protein involved in the control of volume changes in response to ER Ca release and astrocyte activation. These findings provide new insights for the comprehension of the molecular mechanisms responsible for the myelin degeneration occurring in MLC and other LD where astrocytes hav