Depolarization causes the formation of a ternary complex between GlialCAM, MLC1 and ClC-2 in astrocytes: implications in megalencephalic leukoencephalopathy

Abstract

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM. GlialCAM is necessary for the correct targeting of MLC1, but also for the targeting of the Cl- channel ClC-2. Furthermore, GlialCAM modifies ClC-2 functional properties in vitro. However, in vivo studies in GlialCAM-/- mice have shown that the modification of ClC-2 activity only occurs in oligodendrocytes, despite GlialCAM and ClC-2 being expressed in astrocytes. Thus, the relationship between GlialCAM, MLC1 and ClC-2 in astrocytes is unknown. Here, we show that GlialCAM, ClC-2 and MLC1 can form a ternary complex in cultured astrocytes, but only under depolarizing conditions. We also provide biochemical evidences that this ternary complex exists in vivo. The formation of this complex changes ClC-2 localization in the membrane and its functional properties. ClC-2 association with GlialCAM/MLC1 depends on calcium flux through L-type calcium channels and activation of calcium-dependent calpain proteases. Based on these studies, we propose that the chloride influx mediated by GlialCAM/MLC1/ClC-2 in astrocytes may be needed to compensate an excess of potassium, as occurs in conditions of high neuronal activity. We suggest that a defect in this compensation may contribute to the pathogenesis of MLC disease.

Leukoencephalopathy‐causing CLCN2 mutations are associated with impaired Cl− channel function and trafficking

“…Furthermore, we provide new evidence that GlialCAM, ClC‐2 and MLC1 may form a ternary complex (Sirisi et al . ), as shown by MLC1 stabilising WT ClC‐2 in the presence of GlialCAM.…”

“…GlialCAM, MLC1 and ClC‐2 form a ternary complex in astrocytes (Sirisi et al . ), raising the possibility that MLC1 co‐expression might also contribute to the stability of the complex. Indeed, MLC1 co‐expression increased the PM density of WT, but not mutant ClC‐2 in HeLa cells (Fig.…”

“…Furthermore, recent results have indicated that ClC‐2 may form a ternary complex with GlialCAM and MLC1 in astrocytes (Sirisi et al . ). It is currently unknown whether GlialCAM and MLC1 have a role in stabilising ClC‐2.…”

Drosophila ClC‐a is required in glia of the stem cell niche for proper neurogenesis and wiring of neural circuits

“…Uptake of K + occurs concomitantly with uptake of Cl − and water, producing transient astrocyte swelling (Bellot-Saez, Kékesi, Morley, & Buskila, 2017). Based on its expression in astrocytic glia, the ClC-2 channel has been proposed as one of the channels that might participate in this Cl − uptake (Blanz et al, 2007;Hoegg-Beiler et al, 2014;Sirisi et al, 2017). Mutations in CLCN2, which codes for ClC-2, are responsible for leukoencephalopathy with ataxia (LKPAT) (Depienne et al, 2013) and ClC-2 has been related to megalencephalic leukoencephalopathy with subcortical cysts (MLC) (Hoegg-Beiler et al, 2014;Jeworutzki et al, 2012;Sirisi et al, 2017).…”

“…Based on its expression in astrocytic glia, the ClC-2 channel has been proposed as one of the channels that might participate in this Cl − uptake (Blanz et al, 2007;Hoegg-Beiler et al, 2014;Sirisi et al, 2017). Mutations in CLCN2, which codes for ClC-2, are responsible for leukoencephalopathy with ataxia (LKPAT) (Depienne et al, 2013) and ClC-2 has been related to megalencephalic leukoencephalopathy with subcortical cysts (MLC) (Hoegg-Beiler et al, 2014;Jeworutzki et al, 2012;Sirisi et al, 2017). Both conditions are characterized by vacuolization of white matter and edema, most probably as a consequence of impaired K + buffering, but patients can also present learning disabilities and mild to moderate intellectual impairment.…”