The Function and Mechanisms of Autophagy in Spinal Cord Injury

项目概要 开发针对脊髓损伤（SCI）的新疗法的主要障碍是不完整 了解损伤和恢复的机制。我们研究的总体目标是确定 自噬对 SCI 后神经细胞损伤和死亡的分子机制及其贡献 允许未来合理疗法的发展。自噬是一种溶酶体依赖性降解途径 对于正常细胞稳态和防止神经变性至关重要。然而，当溶酶体 功能受损时，自噬也会导致细胞死亡。自噬体的积累 SCI后被注意到，但其机制和功能仍不清楚。此外，溶酶体功能和 SCI 后尚未评估自噬降解（通量）的效率。根据我们的初步 根据数据，我们提出并将检验以下假设：SCI 后早期自噬-溶酶体功能障碍 途径导致神经元细胞损伤和死亡，其恢复可以促进长期恢复。 我们将使用自噬报告基因和自噬缺陷转基因小鼠进行体内和体外实验 药理学和基因操作以确定 SCI 后自噬的机制 证明其对 SCI 后神经细胞死亡和功能结果的影响。 AIM 1 将确定 SCI 后溶酶体和自噬功能障碍的机制。体内和体外免费 方法将与离体脊髓切片培养等新技术相结合，以测试 假设 SCI 后早期自噬流受损，反映细胞质磷脂酶 A2 (cPLA2) 介导的溶酶体膜透化（LMP）。 AIM 2 将确定以下功能的后果 SCI后恢复自噬-溶酶体途径。溶酶体生物发生的药理学诱导剂和 自噬流、海藻糖和 Torin1 将用于野生型和自噬缺陷 Becn1 /- 小鼠进行测试 刺激溶酶体生物合成将恢复自噬-溶酶体途径并导致 改善功能结果。 AIM 3将确定自噬-溶酶体途径对 SCI 后轴突损伤和神经元细胞存活。自噬受损对轴突的贡献 SCI后的损伤和神经元细胞死亡将在体内进行检查；我们还将确定是否改进 自噬流可以减轻 SCI 后神经元细胞的损伤和死亡。我们假设受损 自噬通量导致 SCI 后 ER 应激诱导的轴突损伤和神经元凋亡。 我们的研究将首次确定神经细胞自噬的功能和机制 SCI 后损伤和死亡。此外，我们将确定操纵的最佳方法 自噬-溶酶体途径可改善 SCI 后的功能结果，从而开辟潜在的新途径 治疗途径。

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.