Shank3 establishes AMPA receptor subunit composition at cerebellar mossy fiber-granule cell synapses and is associated with altered regional microglial morphology.

Mutations in Shank3 are the primary genetic cause of Phelan-McDermid Syndrome (PMS), a neurodevelopmental disorder frequently comorbid with autism spectrum disorder (ASD). As a key scaffolding protein in the postsynaptic site, SHANK3 shapes excitatory glutamatergic synaptic function by interacting with AMPARs, NMDARs, and mGluRs. While Shank3 deficiency has been extensively studied in forebrain regions, relatively little is known about its role in the cerebellum, a brain area increasingly implicated in ASD pathobiology and involved in motor and non-motor processing. Since cerebellar granule cells (CGCs) exhibit high Shank3 expression, this study aims to investigate how Shank3 loss affects mossy fiber (MF)-CGC glutamatergic synaptic function. Whole-cell patch clamp electrophysiological recordings from CGCs in ex vivo cerebellar brain slices from adult (4-6 months old) wild type (WT) and homozygous Shank3∆ex4-22 KO mice were performed to record miniature, evoked, and photoactivated glutamatergic responses. Immunohistochemistry was used to determine AMPAR subunit (GluA2, GluA4) expression and microglia morphology (IBA1). Quantal mEPSC amplitudes and AMPAR-mediated response to photo-uncaged glutamate were increased in CGCs in the absence of Shank3. Evoked EPSCs recorded in CGCs from Shank3 KO mice had faster AMPAR decay kinetics, inward rectification, and increased sensitivity to IEM-1460, suggesting that a high proportion of CP-AMPARs with distinct biophysical properties are present at the MF-CGC synapse. A reduced density of GluA2 near active zones within cerebellar glomeruli regions containing MF-CGC synapses were consistent with electrophysiological findings. Shank3 KO mice also had less ramified microglia relative to control mice, suggesting a shift in microglial morphology in the cerebellar cortex in early adulthood. Together, these findings highlight a novel role of Shank3 in maintaining the balance between CP- and CI-AMPARs at the MF-CGC synapse, which is essential for synapse maturation and proper cerebellar circuitry function. Dysregulation of this balance, with an altered microglial morphological state in the cerebellum, may be a possible mechanism contributing to cerebellar-related behavioral deficits in Shank3 KO mice and may represent a component of ASD pathophysiology.