Targeting astrocytic CLC2(CLCN2) restores myelin regeneration through inhibition of SPP1/CD44 signaling pathway in leukoencephalopathy

Accumulating myelin damage and impaired remyelination are central pathological features of leukoencephalopathies, including Multiple Sclerosis and Alexander Disease, where astrocytes play essential roles in maintaining central nervous system homeostasis and mediating astrocyte-oligodendrocyte interactions. Chloride Ion Channel 2 (ClC-2), encoded by CLCN2, is functionally expressed in astrocytes and is likely critical to white matter integrity; however, its precise roles and interactions remain unclear. Clarifying the mechanisms by which astrocytic ClC-2 influences white matter is essential for developing treatments for CLCN2-related leukoencephalopathy (CC2L) and potentially other white matter disorders. In this study, we demonstrate that dysfunctional ClC-2 in astrocytes—derived from both mouse models and human-induced pluripotent stem cells (hiPSCs)—impairs oligodendrocyte lineage cell development in vitro and delays remyelination in vivo. Transcriptomic analyses identified SPP1 as a key inhibitory factor on remyelination, secreted from ClC-2-deficient astrocytes. This inhibition was validated through astrocyte-specific modulation of SPP1 expression, where overexpression exacerbated, and downregulation alleviated, demyelination effects. Furthermore, we discovered that SPP1 upregulation in astrocytes with abnormal ClC-2 negatively impacts remyelination by interacting with CD44 on oligodendrocyte progenitor cells. Finally, we confirmed that increased SPP1 expression and the resulting suppression of oligodendrocyte lineage cell development were also present in hiPSC-derived astrocytes harboring a CLCN2 mutation from a CC2L patient. Collectively, these findings reveal that astrocytic ClC-2 is intricately linked to white matter integrity through SPP1 regulation, positioning it as a potential therapeutic target for CC2L and other leukoencephalopathies.