<p>Extracellular vesicles (EVs) are membrane-enclosed, nanoscale structures released by cells and play a key role in intercellular communication under both normal physiological and pathological conditions. They serve as conduits for transferring molecular cargo between neighboring cells, thereby modulating recipient cell function. While the HIV Transactivator of transcription (Tat) protein has been shown to induce ferroptosis in microglia, the role of Tat-activated microglia-derived EVs (Tat-MEVs) in transferring iron-handling and ferroptosis-associated cargo to neurons and promoting neuronal injury remains unexplored. In this study, we sought to evaluate the impact of cargo derived from Tat-MEVs on neuronal synaptodendritic degeneration. Rat primary cortical and hippocampal neurons were exposed to either control MEVs or Tat-MEVs and subsequently assessed for synaptodendritic degeneration, expression of key ferroptotic mediators, and mitochondrial dysfunction associated with neuronal injury. Neurons exposed to Tat-MEVs demonstrated increased expression of the key iron-handling and ferroptosis-associated proteins (transferrin, TF; transferrin receptor 1, TFR1; Six-Transmembrane Epithelial Antigen of the Prostate 3, STEAP3; divalent metal transporter 1, DMT1; and ferritin heavy chain 1, FTH1); inhibitory synaptic markers (GAD65, Gephyrin), Fe²⁺/total iron content, neuronal cytotoxicity and mitochondrial reactive oxygen species (ROS) compared to neurons exposed to control MEVs. These findings suggest a link between mitochondrial dysfunction and neuronal iron accumulation. The expression of these mediators was downregulated in neurons exposed to MEVs derived from iron chelator, deferoxamine (DFO)-pretreated BV2 cells. </p><p>Electrophysiological recordings further revealed reduced miniature excitatory postsynaptic currents in neurons exposed to Tat-MEVs, an effect that was attenuated in neurons exposed to DFO-derived MEVs. Additionally, dendritic spine analyses of neurons exposed to Tat MEVs revealed a reduction in mushroom and stubby spine subtypes, suggesting synaptodendritic injury.</p><p>Collectively, these findings demonstrate that Tat-MEVs transfer iron-handling and ferroptosis-associated cargo that promotes neuronal iron dysregulation, oxidative stress, mitochondrial dysfunction, and synaptodendritic degeneration. These changes are consistent with ferroptosis-associated neuronal stress and contribute to functional impairment in recipient neurons. This EV-based communication axis provides mechanistic insight into how HIV Tat-induced microglial dysfunction propagates iron-dependent neurotoxic signaling within the central nervous system and identifies EV-mediated iron dysregulation as a potential therapeutic target in NeuroHIV.</p>