Function and Mechanisms of Autophagy-Lysosomal Pathway in Traumatic Brain Injury

 描述(申请人提供)：开发治疗创伤性脑损伤(TBI)的新疗法的一个主要障碍是对损伤和恢复机制的不完全理解。我们研究的总体目的是确定自噬-溶酶体途径缺陷在脑外伤后继发性损伤中的分子机制和作用，以便在其操作的基础上进一步发展合理的治疗方法。自噬-溶酶体途径是细胞内降解和质量控制的重要组成部分，在包括神经退化在内的各种条件下发挥保护作用。然而，当溶酶体功能受损时，自噬也会导致细胞死亡。已经注意到脑外伤后自噬小体的积累，但其功能和机制尚不清楚。此外，脑外伤后溶酶体功能和溶酶体依赖自噬降解(通量)的效率尚未得到评估。因此，本研究的目的是验证以下假设：脑创伤后早期自噬-溶酶体通路的功能障碍导致神经细胞死亡，其恢复可以促进长期的恢复。我们的项目将使用自噬报告基因(GFP-LC3)和自噬缺陷(Beclin1/-)转基因小鼠模型来确定脑损伤后自噬-溶酶体途径的功能，并采用体内和体外模型系统来确定该途径的中断影响脑损伤后预后的机制。目的1确定颅脑损伤后溶酶体和自噬功能障碍的机制。体内和体外的互补方法，包括药理和遗传调节，将与体外自噬通量分析等新技术相结合，以检验自噬通量在脑创伤后早期受损的假设，反映细胞质磷脂酶A2(CPLA2)介导的溶酶体膜通透性(LMP)。目的2将确定脑外伤后增强溶酶体功能和自噬通量的功能后果。自噬通量和溶酶体生物发生的化学调节剂雷帕霉素和Torin1将用于野生型和自噬缺陷小鼠，以检验以下假设：脑损伤后自噬-溶酶体途径的恢复功能将导致功能结果的改善。目的3确定自噬-溶酶体途径对脑创伤后神经细胞应激和存活的影响。体内和体外对自噬、溶酶体功能和内质网应激的药理学和遗传学操作将被用来验证溶酶体活性和自噬清除受损加剧内质网应激导致脑外伤后神经元凋亡的假说。我们的研究将首次确定脑创伤后自噬-溶酶体途径的功能和机制。此外，我们将证明，增加溶酶体功能和自噬通量可以改善脑外伤后的组织学和行为结果，从而开辟潜在的新的治疗途径。

 DESCRIPTION (provided by applicant): A major barrier to development of novel treatments against traumatic brain injury (TBI) 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 defects in the autophagy-lysosomal pathway to secondary injury after TBI, in order to allow future development of rational therapeutic approaches based on its manipulation. The autophagy-lysosomal pathway is an essential component of intracellular degradation and quality control, and plays a protective function against a variety of conditions including neurodegeneration. However, when lysosomal function is compromised autophagy can also contribute to cell death. Accumulation of autophagosomes has been noted after TBI, but its function and mechanisms remain unknown. Additionally, lysosomal function and the efficiency of lysosome-dependent autophagic degradation (flux), has not been assessed after TBI. Therefore, the purpose of this study is to test the hypothesis that early after TBI dysfunction of the autophagy-lysosomal pathway contributes to neuronal cell death and its restoration can promote long-term recovery. Our project will use autophagy-reporter (GFP-LC3) and autophagy-deficient (Beclin1+/-) transgenic mouse models to determine the function of autophagy-lysosomal pathway after TBI and employ both in vivo and in vitro model systems to determine the mechanisms by which disruption of this pathway affects outcomes after TBI. AIM 1 will determine the mechanisms of lysosomal and autophagy dysfunction after TBI. Complimentary in vivo and in vitro approaches, including pharmacological and genetic modulations, will be combined with novel techniques such as ex vivo autophagy flux analysis to test the hypothesis that autophagy flux is impaired early after TBI, reflecting cytoplasmic phospholipase A2 (cPLA2) mediated lysosomal membrane permeabilization (LMP). AIM 2 will determine the functional consequences of enhancing lysosomal function and autophagy flux after TBI. Chemical modulators of autophagy flux and lysosomal biogenesis, Rapamycin and Torin1, will be used in wild type and autophagy deficient mice to test the hypothesis that restoring function of the autophagy-lysosomal pathway after TBI will result in improved functional outcomes. AIM 3 will determine the influence of autophagy-lysosomal pathway on neuronal cell stress and survival after TBI. Pharmacological and genetic manipulation of autophagy, lysosomal function and ER stress in vivo and in vitro will be used to test the hypothesis that impaired lysosomal activity and autophagic clearance exacerbate ER stress resulting in neuronal apoptosis after TBI. Our study will for the first time determine the functio and the mechanisms of autophagy-lysosomal pathway after TBI. Additionally, we will demonstrate that increasing lysosomal function and autophagy flux can improve histological and behavioral outcomes after TBI, thus opening potential novel treatment avenues.