Shank3 establishes AMPA receptor subunit composition at cerebellar mossy fiber-granule cell synapses and shapes regional microglia activation.

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 is critical for excitatory glutamatergic synapse function by interacting with AMPARs, NMDARs, and mGluRs. While Shank3 deficiency has been extensively studied in forebrain regions, its role in the cerebellum, a brain area increasingly implicated in ASD pathobiology, remains comparatively underexplored. Cerebellar granule cells (CGCs) exhibit high Shank3 expression. However, its role in cerebellar glutamatergic synapses is poorly understood. This study aims to investigate how Shank3 loss affects mossy fiber-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{Delta}ex4-22 KO were performed to record miniature, evoked, and glutamate uncaged responses. Similarly, the current-voltage (I-V) relationship was analyzed with intracellular spermine and pharmacological validation of calcium-permeable AMPARs (CP-AMPARs) was done by IEM-1460. Immunofluorescence staining was performed for microglia using IBA1 labeling. We found a significant increase in mEPSC amplitude and AMPAR-mediated response to glutamate uncaging, which indicates that the loss of Shank3 enhances postsynaptic AMPAR function. Furthermore, the KO group showed 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. Furthermore, KO mice showed less ramified microglia suggesting the possible presence of activated microglia in the cerebellar cortex. Together, these findings highlight a critical 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 possible presence of activated microglia in the cerebellum, may underscore cerebellar-related behavioral deficits in Shank3 KO mice and may suggest a potential mechanism contributing to ASD pathophysiology.