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Published on: September 3, 2014
Shank3 establishes AMPA receptor subunit composition at cerebellar mossy fiber-granule cell synapses and is
Rajaram Kshetri1, Raheem F H Ai Aameri1, Ben D Richardson1
1Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL 62702, United States of America.
Abstract:
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.
Insights
Shank3 protein loss in mice alters cerebellar synapses, increasing specific AMPA receptors and changing microglia. This may explain Phelan-McDermid Syndrome and autism spectrum disorder-related behaviors.
Area of Science:
- Neuroscience
- Synaptic Plasticity
- Neurodevelopmental Disorders
Background:
- Mutations in Shank3 are a primary cause of Phelan-McDermid Syndrome (PMS), a neurodevelopmental disorder often linked with autism spectrum disorder (ASD).
- SHANK3 protein is crucial for excitatory synaptic function, but its role in the cerebellum, implicated in ASD, is understudied.
- Cerebellar granule cells (CGCs) have high Shank3 expression, making them a key focus for studying Shank3's cerebellar function.
Purpose of the Study:
- To investigate the impact of Shank3 loss on glutamatergic synaptic function in cerebellar granule cells (CGCs).
- To explore how Shank3 deficiency affects AMPA receptor composition and microglia morphology in the cerebellum.
Main Methods:
- Whole-cell patch clamp electrophysiology was used to record synaptic responses in CGCs from Shank3 knockout (KO) and wild-type (WT) mice.
- Immunohistochemistry was employed to assess AMPA receptor subunit expression (GluA2, GluA4) and microglia morphology (IBA1).
Main Results:
- Shank3 KO mice showed increased quantal synaptic response amplitudes and altered AMPA receptor kinetics in CGCs.
- Evidence suggests a higher proportion of calcium-permeable AMPA receptors (CP-AMPARs) at the mossy fiber-CGC synapse in Shank3 KO mice.
- Shank3 deficiency was associated with reduced GluA2 density and less ramified microglia in the cerebellar cortex.
Conclusions:
- Shank3 is vital for balancing CP- and CI-AMPARs at the mossy fiber-CGC synapse, impacting synapse maturation and cerebellar function.
- Altered synaptic function and microglial morphology in the cerebellum due to Shank3 loss may contribute to behavioral deficits seen in PMS and ASD.
