Impaired Lysosomal Function Caused by Subarachnoid Hemorrhage Attenuates the Beneficial Effects of Autophagy

Introduction: Autophagy is generally considered to be a process that facilitates cell survival by maintaining cellular homeostasis through the involvement of lysosomes in the recycling of cellular components and organelle renewal. Autophagy is often thought to play a protective role in early brain injury (EBI) in subarachnoid hemorrhage (SAH). Normally, p62, a receptor for autophagosomes, decreases due to the increase of autophagosomes, but in SAH both autophagosome markers LC3Ⅱ/Ⅰ and autophagic substrate p62 are increased. This seemingly paradoxical phenomenon aroused our interest, and we studied the complete autophagic lysosomal pathway, from the formation of autophagosomes, the fusion of autophagosomes with lysosomes, and the degradation of autophagic lysosomes to investigate the causes of this phenomenon.

Methods: We used an in vivo model of intravascular perforation and an in vitro model of hemoglobin (HB) exposure to illustrate the potential causes of altered autophagy after SAH.

Results: Western blot analysis showed that HB significantly promotes LC3Ⅱ/Ⅰ elevation and autophagic substrate p62 accumulation. 3-Methyladenine (3-MA), which blocks the upstream steps of autophagy, remarkably reduced HB-induced LC3Ⅱ/Ⅰ elevation and further increased p62 accumulation. Chloroquine (CQ), which blocks the downstream steps of autophagy, has no significant effect on HB-induced LC3Ⅱ/Ⅰ elevation, but further promotes p62 accumulation. This suggests that HB causes increased autophagosome formation and impaired autophagosome clearance. Immunofluorescence co-localization analysis shows that HB impairs autophagosome- lysosome fusion. We then found that lysosome-associated membrane protein (LAMP1) and mature-cathepsin D (CTSD)/pro-CTSD, which both maintain lysosomal stability, decreased both in vivo and in vitro after SAH.

Conclusion : The intact autophagic lysosomal pathway is the basis for the protective effect of autophagy. The impairment of lysosomal function caused by SAH leads to the failure of autophagosomes to degrade smoothly and complete the recycling of cellular components, which weakens the protective effect of autophagy in EBI.