Mitochondria-containing large extracellular vesicles target mouse motor neurons upon intramuscular injection

Pinky, P. P.; Wang, Z.-M.; Khare, P.; Jhaveri, J.; Basudkar, V.; Rao, K.; Lawrence, A.; Pingali, A.; Dave, K.; Stolz, D. B.; Sun, M.; Zheng, S.-y.; Shiva, S.; Milligan, C.; Delbono, O.; Soundara Manickam, D.

doi:10.1101/2025.09.15.675842

bioRxiv · 10.1101/2025.09.15.675842

Mitochondria-containing large extracellular vesicles target mouse motor neurons upon intramuscular injection

Pinky, P. P.·Wang, Z.-M.·Khare, P.·Jhaveri, J.·Basudkar, V.·Rao, K.·Lawrence, A.·Pingali, A.·Dave, K.·Stolz, D. B.·Sun, M.·Zheng, S.-y.·Shiva, S.·Milligan, C.·Delbono, O.·Soundara Manickam, D.

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Abstract

Amyotrophic Lateral Sclerosis (ALS) is a neurological disorder that causes progressive degeneration of motor neurons. Mitochondrial dysfunction accelerates neurodegeneration aggravating the severity of ALS. We hypothesized that increasing the mitochondrial function of motor neurons may promote neuronal survival. Therefore, we investigated the potential of neuron-derived mitochondria containing extracellular vehicles (EVs) as a novel therapeutic approach for ALS using differentiated NSC-34 cells as a surrogate for neurons. Neuron derived-large EVs (lEVs) but not small EVs (sEVs) contained mitochondria. However, we observed increased cell viability and oxygen consumption rates in heat-stressed neurons treated with both sEVs and lEVs suggesting improved mitochondrial function in recipient neurons. The increased oxygen consumption rates in sEV-treated heat-stressed neurons was accompanied by a greater proton leak compared to lEV treatment. The greater proton leak observed with sEVs likely suggests a lower efficiency of oxidative phosphorylation compared to that achieved by cells treated with mitochondria-containing lEVs. These findings suggest that mitochondrial components present in sEVs, such as proteins and mitochondrial DNA, may too contribute to improving cellular respiration. Furthermore, we have demonstrated that lEV mitochondria are transported into the lumbar spinal cord motor neurons following intramuscular injection in C57BL/6 mice in a EV dose-dependent manner. Collectively, for the first time, we have demonstrated the therapeutic effects of neuronal EVs in recipient heat-stressed neurons and the delivery of lEV mitochondria to spinal cord motor neurons in vivo without any EV surface modifications for neuronal targeting. Further studies will determine the therapeutic efficacy of mitochondria-containing EVs in the SOD1G93A transgenic mouse model of ALS. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/675842v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@af590aorg.highwire.dtl.DTLVardef@1fed11dorg.highwire.dtl.DTLVardef@52d339org.highwire.dtl.DTLVardef@1a97182_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Pinky, P. P., Wang, Z.-M., Khare, P., Jhaveri, J., Basudkar, V., Rao, K., Lawrence, A., Pingali, A., Dave, K., Stolz, D. B., Sun, M., Zheng, S.-y., Shiva, S., Milligan, C., Delbono, O., Soundara Manickam, D.. 2025-09-17. Mitochondria-containing large extracellular vesicles target mouse motor neurons upon intramuscular injection. https://doi.org/10.1101/2025.09.15.675842

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