# Behavioral regression in shank3Δex4−22 mice during early adulthood corresponds to cerebellar granule cell glutamatergic synaptic changes

## TL;DR

This study shows that Shank3 gene deletion in mice leads to worsening behavioral issues in adulthood, linked to changes in cerebellar synapses, suggesting a role for the cerebellum in autism.

## Contribution

The study reveals age-related behavioral regression and synaptic changes in cerebellar granule cells in Shank3-deficient mice, linking them to autism-like symptoms.

## Key findings

- Behavioral impairments in Shank3Δex4−22 mice worsen with age, including motor deficits, anxiety, and repetitive behaviors.
- SHANK3 is localized in cerebellar granule cells and its deletion enhances glutamatergic synaptic activity in these cells.
- Reduced social preference and elevated self-grooming in males were observed in adult Shank3Δex4−22 mice.

## Abstract

Shank3, a gene encoding a synaptic scaffolding protein, is implicated in autism spectrum disorder (ASD) and is disrupted in Phelan-McDermid syndrome (PMS). Despite evidence of regression or worsening of ASD-like symptoms in individuals with PMS, the underlying mechanisms remain unclear. Although shank3 is highly expressed in the cerebellar cortical granule cells, its role in cerebellar function and contribution to behavioral deficits in ASD models are unknown. This study investigates behavioral changes and cerebellar synaptic alterations in shank3Δex4−22 mice at two developmental stages.

Shank3Δex4−22 wildtype, heterozygous, and homozygous knockout mice lacking exons 4–22 (all functional isoforms) were subjected to a behavioral battery in both juvenile (5–7 weeks old) and adult (3–5 months old) mouse cohorts of both sexes. Immunostaining was used to show the expression of SHANK3 in the cerebellar cortex. Spontaneous excitatory postsynaptic currents (sEPSCs) from cerebellar granule cells (CGCs) were recorded by whole-cell patch-clamp electrophysiology.

Deletion of shank3ex4−22 caused deficits in motor function, heightened anxiety, and repetitive behaviors. These genotype-dependent behavioral alterations were more prominent in adult mice than in juveniles. Reduced social preference was only identified in adult shank3Δex4−22 knockout mice and self-grooming was uniquely elevated only in males across both age groups. Immunofluorescence staining indicates the presence of SHANK3 predominantly in the dendrite-containing rosette-like structures in CGCs, colocalizing with presynaptic markers of glutamatergic mossy fiber. Electrophysiological findings identify a parallel relationship between the age-related exacerbation of behavioral impairments and the enhancement of sEPSC amplitude in CGCs.

Other behavioral tests of muscle strength (grip strength test), memory (Barnes/water maze), and communication (ultrasonic vocalization), were not performed. Further study is necessary to elucidate how SHANK3 modulates synaptic function at the mossy fiber-granule cell synapse in the cerebellum.

Our findings reveal an age-related exacerbation of behavioral impairments in shank3Δex4−22 mutant mice. These results suggest that SHANK3 may play a role in maintaining glutamatergic receptors and synapses in CGCs, as well as the potential involvement of the cerebellum in ASD.