Extracellular Vesicles-Dependent Secretion Regulates Intracellular CYFIP2 Protein Homeostasis in Cortical Neurons

<b>Background</b>: Fragile X Syndrome (FXS) is the most common monogenic cause of autism spectrum disorders, and is characterized by the excessive immature excitatory synapses in cortical neurons, leading to excitatory/inhibitory imbalance and core autistic behaviors. This synaptic pathology has been attributed to dysregulated levels of synaptic proteins, including CYFIP2: a key regulator of synaptic structure and plasticity. However, the mechanism underlying the increased CYFIP2 protein level in FXS neurons remains unclear. Neurons abundantly secrete extracellular vesicles (EVs) enriched with bioactive cargos (proteins and miRNAs). <b>Objectives</b>: the goal of this research is to identify whether EV-dependent secretion plays important roles in regulating the intracellular CYFIP2 protein level in WT and FXS neurons. <b>Methods and Results</b>: our proteomic analysis reveals that CYFIP2 protein is packaged in EVs released by mouse cortical neurons. Pharmacological and genetic blockades of neuronal EV release significantly elevated intracellular CYFIP2 levels by 78 ± 14% and 168 ± 39%, respectively. Glutamate-evoked EV release significantly reduced the CYFIP2 level by 24 ± 2%. Neurons from <i>Fmr1</i> KO mice, an FXS model, secreted significantly less EVs (46 ± 5%) than the wild type, and showed significantly elevated CYFIP2 (by 155 ± 31%). Evoking EV release in FXS neurons significantly lowered the intracellular CYFIP2 (by 53 ± 6%). <b>Conclusions</b>: these findings identify an EV-secretion-dependent mechanism that controls neuronal CYFIP2 level, implicating EV-mediated export in the regulation of synaptic protein homeostasis, synaptic remodeling, and FXS-associated synaptic deficits.