CaMKIIα inhibition facilitates functional recovery after preterm hypoxic-ischemic brain injury by disrupting microglial GPNMB signaling.

Background: Preterm brain injury involves white matter damage and aberrant synaptic development, yet the mechanisms linking these pathologies remain unclear. Our previous work demonstrated that hypoxia-ischemia (HI) upregulates microglial glycoprotein non-metastatic melanoma protein B (GPNMB), which is released and correlates with abnormal synaptogenesis and neurobehavioral deficits. However, the downstream signaling pathways by which GPNMB impairs synaptic function are unknown. Given the central role of hippocampal Ca2⁺/calmodulin-dependent protein kinase II (CaMKII) and its substrate, the glutamate receptor A1 (GluA1) subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, in synaptic signaling and spine development, we investigated whether they mediate GPNMB-induced impairments in immature brain post-HI. Methods: Preterm HI brain injury was induced in P3 C57BL/6 mice using a modified Rice-Vannucci model. Microglia-specific GPNMB overexpression mice were achieved via crosses between Cx3cr1-CreERT2 and R26-CAG-LSL-GPNMB mice. The α-isoform of calmodulin-dependent protein kinase II (CaMKIIα) inhibitor KN93 (10 mg/kg, i.p.) was administered. Synaptic markers [synaptophysin (SYN), postsynaptic density protein 95 (PSD95), GluA1], signaling molecules [p-CaMKIIα, cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF)], and ultrastructure were assessed by western blot, immunofluorescence, and electron microscopy. Protein interaction was predicted by AlphaFold 3. Long-term neurobehavioral function was evaluated using open field, rotarod, and Morris water maze tests. Results: HI increased the expression levels of synaptic proteins (SYN, PSD95), damaged the synaptic ultrastructure, and induced neurobehavioral deficits. All these deficits were exacerbated by microglial GPNMB overexpression, which was accompanied by increased expression levels of p-CaMKIIα, CaMKIIα and GluA1. Immunofluorescence analysis revealed that GPNMB overexpression significantly enhanced the co-localization and fluorescence intensity of GluA1 and PSD95 in the hippocampal CA1, CA3 and DG subregions, which was reversed by KN93 administration. KN93 reversed GPNMB-induced upregulation of p-CaMKIIα, CaMKIIα and GluA1, and enhanced CREB1 and BDNF expression. It also restored synaptic structure and ameliorated behavioral deficits. In the HI injury model, KN93 administration significantly reduced HI-induced upregulation of p-CaMKIIα, t-CaMKIIα, PSD95, and SYN, and improved behavioral outcomes in both wild-type and GPNMB-overexpressing HI mice, with a more pronounced effect in the latter group. Conclusions: Microglial GPNMB aggravates post-HI synaptic and behavioral deficits by promoting CaMKIIα phosphorylation and GluA1 dysregulation. Inhibition of CaMKIIα using KN93 reinstates synaptic integrity and neurobehavioral performance, demonstrating that the GPNMB-CaMKIIα-GluA1 axis is a pathogenic pathway and a potential target for intervention in preterm brain injury.