Iba1 deficiency impairs microglial synaptic remodeling and neuronal survival after axonal injury

Microglia remodel neuronal circuits in pathological conditions; however, the molecular requirements for these responses and their consequences for motoneuron survival remain unclear. Aif1 (Iba1) knockout mice were generated using CRISPR/Cas9-mediated deletion, and baseline phenotypes and responses to unilateral facial nerve axotomy were assessed using immunohistochemistry, transmission electron microscopy, and single-nucleus RNA sequencing of the facial motor nucleus. Motoneuron survival and nuclear γH2AX foci were evaluated 28 days post-axotomy. Under baseline conditions, Iba1−/− mice had reduced body weights and mild behavioral abnormalities compared to wild-type mice. After axotomy, microglial ensheathment of ChAT-positive facial motoneurons was reduced, with fewer neurons showing extensive perisomatic microglial coverage than in Iba1+/+ mice. Ultrastructurally, somatic synapse loss observed after injury in wild-type mice was not detected in Iba1−/− mice, and fewer injured motoneurons were in contact with microglial processes. Single-nucleus transcriptomics showed an exaggerated expansion of an interferon-responsive microglial state in Iba1−/− mice after axotomy, whereas injured motoneurons displayed altered transcriptional programs related to synapse organization and neurotransmission. At 28 days, Iba1−/− mice showed reduced motoneuron survival, lower ChAT expression, and increased nuclear γH2AX foci. Iba1 supports microglia-neuron cross-talk that enables effective perisomatic remodefling after axonal injury; disruption of this response is accompanied by inflammatory-state shifts and compromised motoneuron survival.