PROJECT SUMMARY A major barrier to development of novel treatments against spinal cord injury (SCI) is incomplete understanding of the mechanisms of injury and recovery. The overall aim of our research is to determine the molecular mechanisms and contribution of autophagy to neuronal cell damage and death after SCI, in order to allow future development of rational therapies. Autophagy is a lysosome-dependent degradation pathway essential for normal cellular homeostasis and protection from neurodegeneration. However, when lysosomal function is compromised autophagy can also contribute to cell death. Accumulation of autophagosomes has been noted after SCI, but its mechanisms and function remain unknown. Additionally, lysosomal function and the efficiency of autophagic degradation (flux), has not been assessed after SCI. Based on our preliminary data, we propose and will test the hypothesis that early after SCI dysfunction of the autophagy-lysosomal pathway contributes to neuronal cell damage and death and its restoration can promote long-term recovery. We will use autophagy-reporter and autophagy-deficient transgenic mice and in vivo and in vitro pharmacological and genetic manipulations to determine the mechanisms of autophagy after SCI and demonstrate its influence on neuronal cell death and functional outcomes after SCI. AIM 1 will determine the mechanisms of lysosomal and autophagy dysfunction after SCI. Complimentary in vivo and in vitro approaches will be combined with novel techniques such as ex vivo spinal cord slice cultures to test the hypothesis that autophagy flux is impaired early after SCI, reflecting cytoplasmic phospholipase A2 (cPLA2) mediated lysosomal membrane permeabilization (LMP). AIM 2 will determine functional consequences of restoring autophagy-lysosomal pathway after SCI. Pharmacological inducers of lysosomal biogenesis and autophagy flux, Trehalose and Torin1, will be used in wild type and autophagy deficient Becn1+/- mice to test the hypothesis that stimulating lysosomal biogenesis will restore autophagy-lysosomal pathway and result in improved functional outcomes. AIM 3 will determine the influence of autophagy-lysosomal pathway on axonal damage and neuronal cell survival after SCI. The contribution of impaired autophagy to axonal damage and neuronal cell death after SCI will be examined in vivo; we will also determine whether improving autophagic flux can attenuate neuronal cell damage and death after SCI. We hypothesize that impaired autophagy flux contributes to ER stress induced axonal damage and neuronal apoptosis after SCI. Our study will for the first time determine the function and the mechanisms of autophagy in neuronal cell damage and death after SCI. Additionally we will determine the optimal approaches for manipulation of autophagy-lysosomal pathway to improve functional outcomes after SCI, thus opening potential novel treatment avenues.