Kshetri Rajaram, Richardson Ben D
bioRxiv. 2025 Aug 1:2025.08.01.668222. doi: 10.1101/2025.08.01.668222.
Mutations in 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 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 expression. However, its role in cerebellar glutamatergic synapses is poorly understood. This study aims to investigate how loss affects mossy fiber-CGC glutamatergic synaptic function. Whole-cell patch clamp electrophysiological recordings from CGCs in cerebellar brain slices from adult (4-6 months old) wild type (WT) and homozygous 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 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 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 KO mice and may suggest a potential mechanism contributing to ASD pathophysiology.
SHANK3基因的突变是费伦-麦克德米德综合征（PMS）的主要遗传病因，PMS是一种常与自闭症谱系障碍（ASD）共病的神经发育障碍。作为突触后位点的关键支架蛋白，SHANK3通过与AMPA受体、NMDA受体和代谢型谷氨酸受体相互作用，对兴奋性谷氨酸能突触功能至关重要。虽然SHANK3缺乏在前脑区域已得到广泛研究，但其在小脑（一个越来越多地与ASD病理生物学相关的脑区）中的作用仍相对未被充分探索。小脑颗粒细胞（CGCs）表现出高SHANK3表达。然而，其在小脑谷氨酸能突触中的作用尚不清楚。本研究旨在探讨SHANK3缺失如何影响苔藓纤维-CGC谷氨酸能突触功能。对成年（4 - 6个月大）野生型（WT）和纯合SHANK3基因敲除（KO）小鼠的小脑脑片CGCs进行全细胞膜片钳电生理记录，以记录微小、诱发和谷氨酸光解笼反应。同样，用细胞内精胺分析电流-电压（I-V）关系，并用IEM - 1460对钙通透AMPA受体（CP-AMPARs）进行药理学验证。使用IBA1标记对小胶质细胞进行免疫荧光染色。我们发现微小兴奋性突触后电流（mEPSC）幅度和AMPA受体介导的对谷氨酸光解笼反应显著增加，这表明SHANK3缺失增强了突触后AMPA受体功能。此外，KO组显示出更快的AMPA受体衰减动力学、内向整流以及对IEM - 1460的敏感性增加，这表明在苔藓纤维-CGC突触处存在高比例具有独特生物物理特性的CP-AMPARs。此外，KO小鼠的小胶质细胞分支较少，表明小脑皮质中可能存在活化的小胶质细胞。总之，这些发现突出了SHANK3在维持苔藓纤维-CGC突触处CP-AMPARs和CI-AMPARs之间平衡中的关键作用，这对于突触成熟和正常的小脑回路功能至关重要。这种平衡失调，以及小脑中可能存在的活化小胶质细胞，可能是KO小鼠小脑相关行为缺陷的原因，并可能提示一种导致ASD病理生理的潜在机制。
