Mitochondrial dysfunction contributes to neurodegenerative diseases and developmental disorders such as Fragile X syndrome (FXS). The cross-talk between mitochondria and extracellular vesicles (EVs) suggests that EVs may transfer mitochondrial components as intermediators for intracellular communication under physiological and pathological conditions. In the present study, the ability of EVs to transfer mitochondrial components and their role in mitochondrial dysfunction in astrocytes were examined in the brains of Fmr1 knockout (KO) mice, a model of FXS. The amounts of mitochondrial transcription factor NRF-1, ATP synthases ATP5A and ATPB, and the mitochondrial membrane protein VDAC1 in EVs were reduced in cerebral cortex samples and astrocytes from Fmr1 KO mice. These reductions correspond to decreased mitochondrial biogenesis and transcriptional activities in Fmr1 KO brain, along with decreased mitochondrial membrane potential (MMP) with abnormal localization of vimentin intermediate filament (VIF) in Fmr1 KO astrocytes. Our results suggest that mitochondrial dysfunction in astrocytes is associated with the pathogenesis of FXS and can be monitored by depletion of components in EVs. These findings may improve the ability to diagnose developmental diseases associated with mitochondrial dysfunction, such as FXS and autism spectrum disorders (ASD).

The ADGRG1 gene encodes GPR56, an adhesion G-protein-coupled receptor essential for neuronal migration during cortical development. GPR56 interacts with extracellular matrix components such as collagen III, integrin α3β1, and tetraspanins CD81/CD9. While prior studies demonstrated cortical dysplasia in collagen III single knockout and GPR56/Itga3 double knockout mice, the role of CD81 in brain development remains unclear. This study investigates the functional interaction between CD81 and GPR56 in the embryonic brain. We found CD81 highly expressed in apical and basal regions and the meninges of the developing neocortex. CD81 was detected with GPR56 in neurons and astrocytes and expressed in collagen III⁺ meningeal fibroblasts (MFs) and CD34⁺ endothelial cells. CD81 directly interacted with GPR56 in neurons and with collagen III in MFs. In neurons, CD81 was required for collagen III-induced activation of the GPR56-RhoA signaling pathway. In MFs, CD81 depletion reduced vimentin and collagen III levels, indicating impaired meningeal integrity. Notably, Gpr56/Cd81 double knockout mice exhibited severe frontal brain malformations, including neuronal over-migration and cortical dyslamination. These findings show a cooperative role of CD81 with GPR56 and collagen III in both neural and meningeal compartments, contributing to proper cortical formation during embryonic development.