Endocytic Control of Autophagosome Formation in Lupus T cells

 描述(申请人提供)：系统性红斑狼疮(SLE)是一种病因不明的自身免疫性疾病。其发病机制至少部分归因于T细胞功能障碍。因此，拟议的研究将集中在一个关键的知识缺口上--调节正常T细胞发育的自噬途径如何导致SLE中的促炎性人群倾斜和功能障碍。该项目的中心假设是基于最近的证据，即内噬体对自噬小体的形成起作用，以及Rab4A在狼疮T细胞自噬增加中的作用。Rab4A是一种调节内体循环的小GTP酶。它在系统性红斑狼疮患者中明显过度表达，甚至在狼疮易发小鼠中也是如此。虽然自噬基因，特别是ATG5基因与系统性红斑狼疮有遗传关联，但它们在发病机制中的作用尚不清楚。值得注意的是，ATG5是由CD71+内吞体内转运的，后者由Rab4A回收。携带结构性活性Rab4AQ72L的新发育小鼠表现出CD71表达增加和ATG5缺失。这些小鼠的脾细胞和胸腺细胞表现为mTORC1的激活和FoxP3的耗竭。虽然mTORC1通常被认为是自噬的抑制因子，但它是在溶酶体表面被激活的。因此，基于令人信服的初步数据，特定的目标将检验这一工作假说，即Rab4A的过度表达是加速自噬的原因，并通过促进mTORC1到溶酶体的交通，导致SLE中T细胞的异常发育。鉴于GTP酶抑制剂3-PEHPC在体外可使Rab4A失活并在体内阻止狼疮发病，拟议的实验将确定1)Rab4A是否促进SLE患者依赖CD71/ATG5的自噬小体的形成；2)mTORC1激活和FoxP3耗尽通过内吞运输到溶酶体；以及3)Rab4A失活是否阻止狼疮易感小鼠的发病。根据目标1，在系统性红斑狼疮患者的纵向研究中，将评估Rab4A的过度表达是通过保留CD71而增强自噬小体生物发生的原因。在目标2中，将评估Rab4A启动的mTOR从抑制性Rab5+内体到Rheb+溶酶体的运输是系统性红斑狼疮患者mTORc1激活和由此产生的Treg→Th17反式分化的机制。在目标3中，将通过组成活性的Rab4AQ72L对自身免疫的发展的影响、发病机制对Rab4A、ATG5和ATG7的依赖以及mTORC1的下游激活和mTORC2的抑制来模拟Rab4A驱动的自噬在狼疮中的作用。这项研究具有重要意义，因为它将促进我们对自噬小体生物发生及其对T细胞发育和可塑性的影响的基本理解，并对狼疮的发病和治疗具有广泛的翻译相关性。这种方法是创新的，因为它将使用基因定义的内体交通检查点来影响狼疮的发病机制。这一结果将为我们理解内吞体运输、它如何控制自噬小体的形成及其在疾病发病机制中的作用带来新的视角，并将为SLE的治疗找到新的机制靶点。

 DESCRIPTION (provided by applicant): Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology. The pathogenesis is at least partly attributed to T-cell dysfunction. Therefore, the proposed studies will focus on a critical gap in knowledge - how autophagy pathways that regulate normal T-cell development cause pro-inflammatory population skewing and dysfunction in SLE. The central hypothesis of this project has been based on recent evidence for the contribution of endosomes to the formation of autophagosomes and the role of Rab4A in increased autophagy in lupus T cells. Rab4A is a small GTPase that regulates endosome recycling. It is markedly overexpressed in SLE patients and, even prior to disease onset, in lupus-prone mice. Although autophagy genes, in particular ATG5, have been genetically linked to SLE, their role in pathogenesis is undefined. Notably, ATG5 is transported by CD71+ endosomes which are recycled by Rab4A. Newly developed mice carrying constitutively active Rab4AQ72L show increased expression of CD71 and depletion of ATG5. Splenocytes and thymocytes of these mice show activation of mTORC1 and depletion of FoxP3. While mTORC1 is generally regarded as an inhibitor of autophagy, it is activated on the surface of lysosomes. Thus, based on compelling preliminary data, the Specific Aims will test the working hypothesis that Rab4A overexpression is responsible for accelerated autophagy and, through promoting the traffic of mTORC1 to lysosomes, for abnormal T-cell development in SLE. Given that the GTPase inhibitor 3-PEHPC inactivates Rab4A in vitro and blocks lupus pathogenesis in vivo, the proposed experiments will determine whether 1) Rab4A promotes CD71/ATG5-dependent autophagosome formation in SLE patients; 2) mTORC1 activation and FoxP3 depletion occur through endocytic traffic to lysosomes; and 3) inactivation of Rab4A blocks pathogenesis in lupus-prone mice. Under Aim 1, Rab4A overexpression will be assessed in longitudinal studies of SLE patients as the cause of enhanced autophagosome biogenesis via the retention of CD71. Under Aim 2, Rab4A-initiated traffic of mTOR from inhibitory Rab5+ endosomes to Rheb+ lysosomes will be evaluated as the mechanism underlying mTORC1 activation and the resultant Treg→Th17 trans-differentiation in SLE patients. Under Aim 3, the role of Rab4A-driven autophagy in lupus will be modeled by the impact of constitutively active Rab4AQ72L on development of autoimmunity and the dependence of pathogenesis on Rab4A, ATG5 and ATG7 and downstream activation of mTORC1 and inhibition of mTORC2. The proposed research is significant because it will advance our basic understanding of autophagosome biogenesis and its impact on T-cell development and plasticity with broad translational relevance for the pathogenesis and treatment of lupus. The approach is innovative as it will employ genetically defined checkpoints of endosomal traffic to influence lupus pathogenesis. The results will bring new perspectives to our understanding of endosome traffic, how it controls autophagosome formation and its role in disease pathogenesis, and will identify new mechanistic targets for treatment of SLE.