P2X4 Drives Sex-Specific neuroprotection in autoimmune neuroinflammation

• Non-internalized P2X4 receptors enhances purinergic signaling and dampens inflammatory pathways in microglia. • P2X4 receptor gain-of-function alleviates EAE symptoms in a sex-dependent manner. • Progesterone directly modulates P2X4 receptor gating, prolonging receptor activation. • Sex hormone-driven modulation of P2X4 receptor function underlies female-specific neuroprotection in the EAE model. Microglia critically influence multiple sclerosis (MS) pathophysiology through debris clearance, myelin repair, and modulation of neuroinflammation. These processes are partly regulated by ATP-gated ion channel P2X4, predominantly expressed in microglia. We previously reported that ivermectin (IVM), a positive allosteric modulator of P2X4, modulates microglia activation and function in myelin phagocytosis and promotes following lysolecithin-induced demyelination, and ameliorates neurological symptoms in experimental autoimmune encephalomyelitis (EAE). Here, we dissected the molecular and cellular basis of this protective effect using P2X4mCherryIN knock-in (P2X4KI) mice, in which P2X4 is replaced by a non-internalized variant (P2X4KI), leading to increased surface localization at the plasma membrane. Indeed, ATP-evoked currents were increased in P2X4KI microglia. Transcriptomic analyses revealed that P2X4KI microglia exhibit suppressed inflammatory and immune signaling pathways, suggesting that P2X4 orchestrates microglial responses to injury. Both constitutive and myeloid-specific P2X4KI mice showed a significant amelioration of EAE motor deficits, although exclusively in females. Notably, ovariectomy abolished P2X4-mediated protection in females whereas administration of progesterone gave protection to P2X4KI males, confirming the requirement of female hormones for P2X4-mediated protection. Indeed, progesterone potentiated P2X4 currents and prolonged channel deactivation, revealing direct hormonal modulation of P2X4 gating. These findings identify P2X4 as a key regulator of neuroinflammatory outcomes and reveal a previously unrecognized interaction between female hormones and P2X4 that underlies sex-specific disease modulation. Targeting this pathway may enable the development of precision therapies for MS.