Abstract
<jats:p>Multiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by demyelination, neurodegeneration, and progressive neurological disability. Galanin, a neuropeptide with immunomodulatory properties, signals through G protein-coupled receptors, among which galanin receptor 2 (GALR2) has been implicated with neuroprotective and anti-inflammatory functions. A rare homozygous single nucleotide variant in GALR2 (rs61745847; p.W249L) has been identified in a patient diagnosed with relapsing-remitting MS, however, the biological relevance of this variant in neuroinflammation remains unknown. Here, we investigated the impact of the orthologous GALR2 W248L mutation using a knock-in mouse model and experimental autoimmune encephalomyelitis (EAE). GALR2 W248L knock-in (KI) mice exhibited a more severe clinical course of EAE, accompanied by enhanced inflammatory infiltration, exacerbated demyelination, and increased microglial activation in the spinal cord compared with wild-type (WT) mice. Despite comparable lymphoid and myeloid cell frequencies in the central nervous system, alterations in microglial density and morphology suggested an important contribution of the innate immune system to disease exacerbation in the KI mice. Ex vivo analyses revealed that bone marrow-derived macrophages from KI animals exhibited a pronounced shift toward a pro-inflammatory phenotype, characterized by enhanced M1 polarization, impaired M2-associated responses, and increased NLRP3 inflammasome activation. In parallel, live-cell imaging of primary hippocampal neurons demonstrated reduced galanin binding in mutant cells, consistent with impaired GALR2 functional availability at the plasma membrane. Together, these findings identify GALR2 as a modulator of the neuroinflammatory response and indicate that disruption of galanin-GALR2 signaling promotes sustained innate immune activation, highlighting the relevance of this pathway for MS pathogenesis and its potential as a therapeutic target in neuroinflammatory disorders.</jats:p>