The Function and Mechanisms of Autophagy in Spinal Cord Injury

项目摘要 脊髓损伤（SCI）新疗法开发的一个主要障碍是不完整 了解损伤和恢复的机制。我们研究的总体目标是确定 自噬在脊髓损伤后神经细胞损伤和死亡中的分子机制和作用， 允许未来合理治疗的发展。自噬是一种溶酶体依赖的降解途径 对于正常细胞内稳态和保护神经变性至关重要。然而，当溶酶体 功能受损自噬也可导致细胞死亡。自噬体的积累 脊髓损伤后，它被注意到，但其机制和功能仍然未知。此外，溶酶体功能和 自噬降解（通量）的效率在SCI后还没有被评估。根据我们初步的 数据，我们提出并将测试这一假设，即SCI后早期自噬-溶酶体功能障碍， 该通路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， 药理学和遗传操作，以确定SCI后自噬的机制， 证实其对SCI后神经元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。免费体内和体外试验 这些方法将与新的技术相结合，例如离体脊髓切片培养，以测试 假设SCI后早期自噬通量受损，反映了细胞质磷脂酶A2（cPLA 2） 介导的溶酶体膜透化（LMP）。AIM 2将确定 恢复SCI后的自噬-溶酶体途径。溶酶体生物发生的药理学诱导剂和 自噬通量海藻糖和Torin 1将用于野生型和自噬缺陷Becn 1 +/-小鼠中进行测试 刺激溶酶体生物发生将恢复自噬-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 脊髓损伤后轴突损伤和神经细胞存活。受损的自噬对轴突的贡献 我们将在体内检测SCI后的损伤和神经元细胞死亡;我们还将确定是否改善 自噬流可以减轻SCI后神经细胞的损伤和死亡。我们假设 自噬流参与了脊髓损伤后内质网应激引起的轴突损伤和神经元凋亡。 本研究将首次阐明自噬在神经细胞中的作用及其机制 SCI后的损伤和死亡。此外，我们将确定最佳的方法来操纵 自噬-溶酶体途径改善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.