THE ROLE OF SHANK3 IN CEREBELLAR GLUTAMATERGIC SIGNALING: IMPLICATIONS FOR AUTISM SPECTRUM DISORDER

Author

Rajaram Kshetri, Southern Illinois University CarbondaleFollow

Date of Award

8-1-2025

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Richardson, Ben

Abstract

Mutations in SHANK3 are a primary cause of Phelan-McDermid Syndrome (PMS), and the majority of individuals with PMS have autism spectrum disorder (ASD) like symptoms. Although Shank3 is highly expressed in cerebellar granule cells (CGCs), its function remains poorly understood. Our overarching hypothesis is that the loss of Shank3 alters mossy fiber-CGC (MF-CGC) glutamatergic synapses, which leads to CGC excitotoxicity, astrocyte reactivity, and microglial activation, leading to a subset of cerebellar-dependent ASD-like changes in behavior. This study aimed to determine: (1) the effect of Shank3 deletion in the whole brain or CGCs on naturalistic behaviors in both sexes over late development, (2) the role of Shank3 in the glutamate receptor function in CGCs of germline adult Shank3 knockout (KO) mice, and (3) the effects of Shank3 deletion on astrocyte reactivity, microglia activation, and CGC excitotoxicity in adult Shank3 KO mice.We investigated behavioral changes in germline Shank3Δex4–22 mice that lack all major Shank3 isoforms. Our behavioral experiments revealed age and sex dependent impairments in motor function, anxiety-like behavior, and repetitive behavior. These behavioral changes were more pronounced in adult mice (3-4 months old) compared to juveniles (5-7 weeks old), indicating age-related behavioral impairments. Additionally, we observed an increase in self-grooming in Shank3 KO males in both age groups and reduced social preference, particularly in adult male Shank3 KO mice. In parallel with our behavioral findings, whole-cell patch-clamp recordings demonstrated an increase in the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) only in adult Shank3Δex4–22 mice, suggesting changes in the postsynaptic receptor function of Shank3 at the adult stage. Significant increases in miniature excitatory postsynaptic currents (mEPSCs) at the MF-CGC synapse and in the AMPA/NMDA ratio of adult homozygous Shank3Δex4–22 KO mice support the upregulation of the MF-CGC synaptic AMPAR function in Shank3Δex4-22 mice. Next, our evoked excitatory postsynaptic current (eEPSCs) recordings showed faster AMPAR decay kinetics in the Shank3 KO group, suggesting a possible change in biophysical properties of AMPARs. This was further confirmed by inward rectification in the current-voltage (I-V) relationship of the eEPSC and the sensitivity of eEPSC amplitude to IEM-1460, a selective pharmacological blocker of calcium-permeable AMPA receptors (CP-AMPARs), indicating an elevated level of CP-AMPARs. In contrast, the probability of presynaptic neurotransmitter release was not affected in Shank3 KO mice, as the paired-pulse ratio and mEPSC frequency were similar between the two genotypes. Overall, electrophysiology results suggest that Shank3 plays an essential role in maintaining balance between CP- and calcium-impermeable (CI)-AMPARs at the MF-CGC synapse for synaptic maturation and cerebellar circuitry function. Dysregulation of this balance may underlie cerebellar-related behavioral deficits in Shank3 KO mice and contribute to ASD pathophysiology. We also explored the impact of germline Shank3 deletion on glial cell reactivity and possible neurotoxic effects in CGCs. Deletion of Shank3 did not result in astrocyte reactivity or changes in astrocyte numbers in the cerebellar cortex. However, a significant reduction in IBA1-stained microglial surface area was observed in Shank3 KO mice, suggesting a possible microglial activation. No significant difference was observed in NeuN-positive CGCs between the inner cerebellar cortex of wildtype (WT) and Shank3 KO mice, suggesting that the loss of Shank3 did not induce CGC cell loss or death. This observation suggests a potential role for cerebellar microglia in the neuropathology of Shank3-associated ASD, highlighting the need for further investigation into inflammatory signaling and neuro-glial interactions. Finally, we characterized and validated three Cre-driver lines (Atoh1-Cre, Nse-CreERT2, and Atoh1-CreERTM) for CGC-specific conditional knockout (cKO) models. Among these lines, Atoh1-CreERTM showed the highest specificity in targeting CGCs. However, Cre-mediated Shank3 deletion was dramatically less efficient in cKO mice (Atoh1-CreERTM;Shank3fl/fl). Furthermore, the behavioral performance of Shank3 cKO mice was similar to that of Cre-negative controls. This work highlights the role of Shank3 in modulating glutamatergic synaptic function and AMPAR composition in CGCs over late-stage development. The findings shed light on the age-dependent decline in behavior observed in Shank3Δex4–22 KO mice, along with specific microglial changes in the cerebellum. These findings enhance our understanding of the cerebellar mechanisms involved in SHANK3-related neurodevelopmental disorders, such as ASD and PMS, offering potential treatment targets.