Abstract Body: Background: The selective autophagic removal of mitochondria, lysosomes, Golgi are termed mitophagy, lysophagy, Golgiphagy, respectively. This study aims to investigate the role of organelle-specific autophagy in rodent brain ischemia models.

Methods: Rats and mice were subjected to either sham surgery, 20 min of global ischemia, or 40/120 min of focal brain ischemia, followed by 0.5-, 4-, 24- and 72-h reperfusion, respectively. Spatial and temporal colocalization of organelle-specific markers, autophagosome (AP) markers, late endolysosomal markers, and AP-to-lysosomal fusion markers were studied under confocal microscopy. Autophagic flux was examined by the buildup of p62 or ubiquitinated organelle protein. Endolysosomal structures were examined by electron microscopy. Cell-specific autophagy was evaluated by co-labeling autophagy markers (p62 and ubiquitinated proteins) with neuron-, oligodendrocyte-, astrocyte-; microglial cell- and macrophage-, and micro vessel-specific markers under confocal microcopy. Cell death was examined by histopathology.

Results: Autophagic flux was impaired in hippocampal CA1 neurons destined to die after brain ischemia, as indicated by the buildup of p62 and ubiquitinated proteins, depletion of NSF, and buildup of endolysosomal structures. In the same CA1 neurons where autophagic flux was impaired, Golgi and mitochondrial networks were irreversibly fragmented. However, only a fraction of Golgi or mitochondrial fragments were colocalized with autophagy marker p62, indicating the post-ischemic impairment of AP formation. Furthermore, a significant buildup of late endolysosomal markers was observed in post-ischemic neurons destined to die, likely due to NSF depletion-induced disruption of lysosomal degradation. In contrast to post-ischemic neurons, NSF was upregulated in Iba1-immunopositive inflammatory cells after brain ischemia.

Conclusion: Organelle-specific autophagy is persistently impaired in post-ischemic neurons destined to die, but only transiently impaired in neurons that survive the same ischemic periods. The irreversible impairment of autophagy is likely due to inactivation of NSF ATPase. In contrast, NSF is upregulated in increased inflammatory cells. These results gain valuable insights into the differential autophagic changes, which is useful for the development of therapeutics against ischemic brain injury.