Astrocytic PDE5 impairs mitochondrial transfer to neurons by disrupting biogenesis through the PGC-1α/Nrf1/TFAM axis in ischemic stroke

Abstract Background Although mitochondria transfer between astrocytes and neurons has been widely reported, it remains uncertain whether this intercellular transfer is bidirectional, along with the ultimate biological outcome and the underlying molecular mechanisms after ischemic stroke (IS). Phosphodiesterase 5 (PDE5) is a cyclic guanosine monophosphate‑hydrolyzing enzyme that is upregulated in the ischemic brain, but whether it regulates this process and thereby contributes to ischemic brain injury remains unknown. Methods To investigate the role of PDE5 in ischemic injury, we generated cell-type-nonspecific PDE5 knockout (KO) mice and monoclonal PDE5-KO C8–D1A astrocytic lines, combined with permanent middle cerebral artery occlusion (pMCAO) in vivo and oxygen-glucose deprivation (OGD) in vitro. The specificity of PDE5-mediated effects was further validated by reconstitution assays. To investigate mitochondrial transfer between astrocytes and neurons and the contribution of PDE5 in this process, we established Mito-mCherry-labeled donor cell lines and collected conditioned medium from donor cells to treat recipient cells, with transfer verified by fluorescence microscopy and flow cytometry. Furthermore, small interfering RNA‑mediated knockdown of peroxisome proliferator‑activated receptor γ coactivator 1α (PGC‑1α) was employed to define the involvement of the PGC‑1α pathway in PDE5‑mediated effects. Results This study demonstrates that PDE5 is highly expressed in astrocytes of the mouse cortex, being equivalent to neurons. PDE5 KO is a benefit for IS dysfunction induced by pMCAO in mice, which was possibly mediated by astrocyte-derived extracellular mitochondria rather than primarily through a direct neuronal effect. Furthermore, ischemia induces mitochondrial dysfunction in astrocytes, followed by its transfer to neurons and consequent neuronal injury. Reciprocally, the impaired mitochondria transfer from OGD-challenged neurons subsequently suppressing astrocytic mitochondrial biogenesis. Notably, by activating the PGC-1α/nuclear respiratory factor 1 (Nrf1)/mitochondrial transcription factor A (TFAM) pathway, PDE5 KO in astrocytes not only promotes the transfer of functional mitochondria to neurons but also reverses the suppression of mitochondrial biogenesis in response to neuron-derived damaged mitochondria, ultimately improving ischemic neuronal impairment. Conclusions These findings reveal that there exists bidirectional transfer of damaged mitochondria between astrocytes and neurons after IS, in which astrocytic PDE5 plays a critical role, providing a theoretical basis for astrocytic PDE5-targeted neuroprotective strategies that regulate mitochondrial transfer to ameliorate ischemic injury.