641. Microglial osteopontin is required for myelin structural integrity during brain development

Abstract

Abstract Background Microglia play essential roles in brain development, including regulation of synaptic pruning, phagocytosis, and myelination. A distinct population of white matter–associated microglia at early postnatal development stage in mouse brain expresses high levels of osteopontin (OPN, Spp1), a multifunctional protein implicated in immune modulation and tissue remodeling. Abnormal microglial function and alterations in white matter connectivity have been implicated in neurodevelopmental and neuropsychiatric disorders, such as schizophrenia, autism spectrum and mood disorders. While OPN expressions have been linked to myelination and white matter injury, the exact mechanism and specific role of microglial-derived OPN in developmental myelin formation and integrity remains poorly understood. Aims & Objectives This study aimed to investigate the role of microglial-derived OPN in regulating oligodendrocyte development and maintaining myelin structural integrity during early postnatal brain development in mice, a critical developmental window relevant to the establishment of long-range white matter connectivity. Method Microglia-specific OPN knockout mice were generated using Spp1 flox/flox crossed with Cx3cr1-Cre mice. O4+ oligodendrocytes were isolated from postnatal day (P) 7 mouse brains for quantitative RT-PCR analysis of genes related to oligodendrocyte differentiation and maturation. Myelin ultrastructure was examined at P35 using scanning electron microscopy. In addition, Western blot analysis was performed to assess the expression of key myelin-associated proteins at P35. Results Gene expression analysis of O4+ oligodendrocytes isolated at P7 revealed no significant differences in the expression of key markers associated with oligodendrocyte differentiation and maturation between control and microglial OPN knockout mice, including Pdgfra, Cspg4 (NG2), Nkx2.2, Sox17, Olig1, Olig2, Gpr56, Gpr17, Apc, Cnp, Mbp, and Plp1, indicating preserved oligodendrocyte lineage progression. Consistently, the expression of major myelin-associated proteins was confirmed at the protein level by Western blot analysis. In contrast, ultrastructural examination at P35 revealed pronounced abnormalities in myelin architecture in knockout mice, including myelin swelling, focal bulging, and loosening of myelin lamellae. These structural changes indicate impaired myelin integrity despite preserved oligodendrocyte differentiation and myelin protein expression. Discussion & Conclusions Our findings show that microglial-derived OPN is dispensable for oligodendrocyte differentiation and initial myelin formation but is essential for maintaining myelin structural integrity during postnatal brain development in mice. Despite preserved oligodendrocyte lineage progression and myelin protein expression, loss of microglial OPN results in ultrastructural myelin abnormalities, indicating a selective defect in myelin stability rather than developmental myelination. Such subtle yet persistent myelin instability may increase long-term vulnerability of white matter. Given the well-established links between myelin abnormalities, disrupted brain connectivity, neurodevelopmental and mood disorders, such as autism and schizophrenia, microglial OPN signaling may represent a developmentally relevant pathway contributing to neuropsychiatric disease risk.