Shank3 establishes AMPA receptor subunit composition at cerebellar mossy fiber-granule cell synapses and shapes
Rajaram Kshetri1, Ben D Richardson1
1Department of Pharmacology, Southern Illinois University - School of Medicine, Springfield, IL 62702.
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 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 Δex4-22 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.
Insights
Loss of Shank3 protein in mice enhances cerebellar synapse function and alters AMPA receptor properties, potentially contributing to neurodevelopmental disorders like autism spectrum disorder.
Area of Science:
- Neuroscience
- Synaptic Plasticity
- Neurodevelopmental Disorders
Background:
- Mutations in Shank3 are a primary cause of Phelan-McDermid Syndrome (PMS), often co-occurring with autism spectrum disorder (ASD).
- SHANK3 is a crucial postsynaptic scaffolding protein regulating excitatory synapse function, but its role in the cerebellum, implicated in ASD, is understudied.
- Cerebellar granule cells (CGCs) express high levels of Shank3, yet its function in their glutamatergic synapses remains unclear.
Purpose of the Study:
- To investigate the impact of Shank3 deficiency on glutamatergic synaptic function in cerebellar granule cells (CGCs).
- To explore alterations in AMPA receptor (AMPAR) properties and microglial morphology in Shank3 knockout mice.
Main Methods:
- Electrophysiological recordings (whole-cell patch clamp) were used to analyze miniature, evoked, and glutamate-uncaged responses in cerebellar slices from wild-type and Shank3 knockout mice.
- Current-voltage relationships, spermine effects, and sensitivity to IEM-1460 (a CP-AMPAR blocker) were assessed.
- Immunofluorescence staining for microglia (IBA1) was performed to examine microglial morphology.
Main Results:
- Shank3 deficiency led to increased miniature excitatory postsynaptic current (mEPSC) amplitude and enhanced AMPAR-mediated responses to glutamate uncaging.
- Shank3 knockout mice exhibited faster AMPAR decay kinetics, inward rectification, and increased sensitivity to IEM-1460, indicating a higher proportion of calcium-permeable AMPARs (CP-AMPARs).
- Less ramified microglia were observed in Shank3 knockout mice, suggesting potential microglial activation.
Conclusions:
- Shank3 is critical for maintaining the balance of CP-AMPARs and calcium-impermeable AMPARs (CI-AMPARs) at the mossy fiber-CGC synapse.
- This balance is essential for proper synapse maturation and cerebellar circuit function.
- Dysregulation of this synaptic balance, possibly involving activated microglia, may contribute to cerebellar deficits and ASD pathophysiology in Shank3-deficient individuals.
