ASTROCYTE-DERIVED EXTRACELLULAR MITOCHONDRIA MAY SUPPORT NEURONAL FUNCTION UNDER CHRONIC OXIDATIVE STRESS

Spontaneous intercellular mitochondrial transfer from astrocytes to neurons has been reported in several pathological contexts, including ischaemic stroke and peripheral neuropathic pain, where donated mitochondria contribute to neuronal recovery. However, whether astrocyte-derived mitochondria can functionally support neurons under chronic neurodegenerative-like oxidative stress, and through which routes this occurs, remains unclear.
We established a chronic oxidative stress model in rat primary hippocampal neurons using sub-lethal H₂O₂ treatment, which consistently reduced mitochondrial membrane potential (TMRE) and metabolic activity (CCK8). Astrocyte-conditioned medium (ACM) showed a protective effect on stressed neurons, improving CCK8 readouts and sustaining neurite complexity in surviving MAP2⁺ neurons.
To assess the contribution of putative extracellular mitochondrial components, ACM was filtered through a 0.22 μm membrane to produce mitochondria-depleted ACM (mdACM). The protective effect was markedly attenuated after filtration, suggesting that >0.22 μm fractions contribute to the ACM-mediated support. In parallel, live-cell imaging in mixed hippocampal cultures with astrocyte mitochondria fluorescently labelled revealed astrocyte-to-neuron mitochondrial transfer events along tunnelling nanotube–like structures, supporting contact-dependent routes.
Ongoing work is characterising the astrocyte-derived large-particle fraction responsible for the ACM effect, including whether it contains functional mitochondria and/or is EV-associated. Add-back reconstitution experiments (mdACM supplemented with concentrated particulate fractions) are being performed to test causality and quantify contributions to neuronal mitochondrial and morphological outcomes under chronic oxidative stress.
Together, our findings suggest astrocytes partially protect stressed neurons via extracellular mitochondria delivered through particle- and potentially contact-dependent mechanisms, highlighting intercellular mitochondrial transfer as a dynamic response in neurodegenerative-like conditions.