Role of autophagy in survival and regeneration of adult sensory neurons

了解成熟神经系统神经元对应激和损伤反应的潜在机制是这一提议的重点。我们一直在研究成年啮齿动物背根神经节(DRG)感觉神经元中与神经元存活和轴突再生相关的机制，特别是热休克蛋白和细胞外环境在这些过程中的作用。DRG神经元及其轴突是我们外部环境的主要传感器，因此可以暴露在机械、热、化学和渗透刺激下，这些刺激可能是有害的。成年感觉神经元不同于胚胎或新生儿的感觉神经元，也不同于中枢神经系统的神经元，它们对各种环境应激源(热、高糖、营养饥饿)具有相对的抵抗力，因此确定这些细胞在这方面是否具有不同的应对机制是有意义的。然而，在某些情况下，这些刺激可导致轴突终末损伤，进展为轴突死亡，并可能导致神经元丢失。自噬是一种受调控的细胞过程，可降解和循环利用各种细胞成分。虽然神经系统中的自噬通常被认为是在神经退化和应激反应的背景下进行的，但越来越明显的是，健康的神经元也表现出基本的自噬，尽管调节这种自噬的机制并不完全清楚。也有人认为，与其他类型的细胞相比，自噬在神经元中可能受到独特的调节，并可能适应轴突的局部生理。与中枢神经系统自噬的研究相比，研究自噬在外周感觉神经元中的作用的研究相对较少。自噬与神经元存活有关，但这种影响在不同的神经元类型和实验环境中是不同的。虽然在受损的轴突中观察到自噬小体的数量增加，但自噬在损伤后再生中的作用尚不清楚。了解这些过程如何影响细胞对损伤和随后的轴突再生的反应是神经系统中的一个基本问题。我们的目标是调查自噬在基础条件下和随后的实验干预-营养剥夺和氧化应激-对DRG神经元反应的贡献。初步数据支持了所提出的实验的可行性。我们将通过使用自噬的化学调节剂(如雷帕霉素、3-MA、氯喹)、通过过度表达或RNAi改变已知的调节这一过程的蛋白质(如Beclin、mTOR、Ulk1、HMGB1)的表达来确定自噬对神经元存活和轴突再生是积极的还是消极的影响。我们将研究神经元存活和轴突再生。显然，如果神经元经历自噬或凋亡细胞死亡，就不会有轴突再生。然而，轴突对局部损伤的反应是成熟神经系统中的一个问题，了解如何预防轴突营养不良，促进轴突恢复和延长是促进功能恢复的关键。

Understanding the mechanisms underlying the response of neurons of the mature nervous system to stress and injury is the focus of this proposal. We have been studying mechanisms associated with neuronal survival and axonal regeneration in the dorsal root ganglion (DRG) sensory neurons of adult rodents, with particular interest in the role of heat shock proteins as well as the extracellular environment in these processes.DRG neurons and their axons are the primary sensors of our external environment and as such can be exposed to mechanical, thermal, chemical and osmotic stimuli that can potentially be deleterious. Adult sensory neurons, unlike their embryonic or neonatal counterparts or unlike neurons of the CNS, are relatively resistant to a variety of environmental stressors (heat, high glucose, nutrient starvation), and it is of interest to determine if these cells possess different coping mechanisms in this regard. However, in some situations these stimuli can cause axon terminal damage progressing to axonal dieback and potentially neuronal loss. Autophagy is a regulated cellular process for the degradation and recycling of various cellular components. While autophagy in the nervous system has often been considered in the context of neurodegeneration and stress responses, it is becoming clear that healthy neurons also exhibit basal autophagy although the mechanisms regulating this are not fully understood. It has also been suggested that autophagy may be uniquely regulated in neurons compared to other cell types and may be adapted to the local physiology in the axons. In contrast to studies of autophagy in the CNS there are relatively few studies investigating the role of autophagy in peripheral sensory neurons. Autophagy has been linked to neuronal survival, but the influence differs in different neuronal types and experimental contexts. Although increased numbers of autophagosomes have been observed in damaged axons, the role of autophagy in regeneration following injury remains unclear. Understanding how these processes can influence cellular responses to injury and subsequent neurite regrowth is a fundamental question in the nervous system.Our objectives are to investigate the contribution of autophagy to the response of DRG neurons under basal conditions and following experimental interventions–nutrient deprivation and oxidative stress. Preliminary data support the feasibility of the proposed experiments. We will determine whether autophagy has a positive or negative influence on neuronal survival and axonal regeneration by using chemical modulators of autophagy (eg., rapamycin, 3-MA, chloroquine), by overexpression or RNAi to alter expression of proteins known to regulate the process (eg., beclin, mTOR, Ulk1, HMGB1). We will investigate both neuronal survival and axonal regeneration. Obviously if the neurons undergo autophagic or apoptotic cell death, there will be no axonal regeneration. However, axonal dieback in response to local damage is a problem in the mature nervous system, and understanding how to prevent axonal dystrophy and promote axonal recovery and elongation is key to promoting recovery of function.