Abstract
HIV-associated neurocognitive disorders (HAND) are exacerbated by comorbid substance abuse, particularly cocaine use. However, the mechanisms by which HIV-1 proteins and cocaine synergize to induce neuronal dysfunction remain poorly understood. Emerging evidence suggests that astrocytes regulate neuronal health through metabolic and mitochondrial communication. Primary rat astrocytes were exposed to HIV-1 Tat protein and cocaine to assess changes in metabolic phenotype and mitochondrial dynamics. Mitochondrial morphology was evaluated via confocal microscopy, while extracellular vesicles (EVs) were isolated and characterized. Cell-type-specific mitochondrial labeling and live-cell imaging were used to track mitochondrial transfer from astrocytes to neurons. Neuronal viability, mitochondrial function, and bioenergetic profiles were assessed following exposure to astrocyte-derived EVs. Astrocytic mitochondrial fragmentation was inhibited using a GTPase-inactive Drp1 mutant or dominant-negative Orai1 to test rescue effects. Tat and cocaine co-exposure induced a hypermetabolic shift and mitochondrial fragmentation in astrocytes. Fragmented mitochondria were incorporated into astrocyte-derived extracellular vesicles (AEVs) and transferred to neurons. Neuronal uptake of these AEVs led to impaired mitochondrial function and reduced viability. Inhibition of astrocytic mitochondrial fission significantly decreased mitochondrial transfer and preserved neuronal bioenergetics and health. These findings identify a novel mechanism of transcellular mitochondrial transfer from astrocytes to neurons under pathological conditions. Mitochondria contained within AEVs act as neurotoxic agents in the context of HIV-1 Tat and cocaine exposure. Targeting astrocytic mitochondrial dynamics may provide a promising therapeutic approach for preventing or mitigating neurodegeneration in HAND and substance use disorders. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-025-02589-y.