Abstract DP266: Lysosome-Mitochondria Temporal and Spatial Regulation of Zinc Aggregation: A Novel Mechanistic Pathway in Ischemic Stroke

Introduction: Ischemia stroke is the second leading cause of death and disability globally. Mitochondria play a pivotal role in determining ischemia stroke outcomes. We have confirmed that zinc increased immediately after reperfusion in ischemic penumbra of the brain. However, the specific zinc transporters involved and the their mechanisms remain unclear. This study aims to elucidate how zinc accumulation induces post-stroke neuronal mitochondrial dysfunction. Methods: Sprague-Dawley adult rats were subjected to 90 min MCAO. Infarct volume and neurological deficits were determined by TTC staining and scoring systems, and the ischemic penumbra were isolated at 6/24 hours after reperfusion. Zinc levels and mitochondria function (mitochondria complex activity I/II/III/IV and ATP produnction) were measured via ELISA. Zinc transporters, including ZIP family, ZnT family and other zinc transportor (TRPML1) were analyzed by real time PCR, Western blot and immunofluorescence. Results: Zinc levels in ischemic penumbra of the brain were increased 6h after reperfusion (p<0.001). The only zinc ion transporter that showed significantly increase at this time point was TRPML (p<0.01), a receptor located on the lysosomal membrane, inducing an increase in zinc ion elevation. At 24h after reperfusion, zinc levels continued to rise (p<0.001), while zinc transporter 9 (ZnT9), located on the outer mitochondrial membrane, exhibited the most significant decrease among all members of the ZIP and ZnT families (p<0.001). Moreover, AAV-mediated ZnT9 overexpressing reduced zinc levels in the ischemic penumbra (p<0.01), rescued damage to mitochondrial complex activity (p<0.001), restored ATP production (p<0.001) and alleviated ischemic brain injuries (p<0.001). These findings suggest that zinc ions accumulation is dynamically regulated early by increased TRPML and later by decreased ZnT9, which maybe responsible for zinc accumulation and contribute to mitochondrial damage following stroke. Conclusions: This study reveals a novel mechanism underlying the sustained accumulation of zinc ions following stroke, including early activation of lysosomal TRPML and the later regulation of mitochondrial ZnT9 in the ischemic penumbra. Our study not only elucidates the intricate lysosome-mitochondria regulation of zinc aggregation and mitochondria dysfunction following stroke but also provides valuable insights into potential therapeutic targets for mitigating stroke-induced damage.