Role of APOL1 in HIVAN pathogenesis

描述（由申请人提供）：HIV-1相关肾病（HIVAN）是一种几乎完全局限于非洲人后裔的疾病。HIVAN的发病机制尚不完全清楚，但形成肾小球血液滤过屏障的足细胞功能障碍和损伤对该病的发展至关重要。HIV-1介导足细胞损伤的机制尚不清楚，主要是由于这些细胞上缺乏经典的HIV-1进入受体的表达。然而，在实验模型中发现HIV-1 Nef重现了一种hiv表型，这表明足细胞可被其他有效感染细胞释放的Nef损伤。此外，局灶节段性肾小球硬化（FSGS）和HIVAN发病率的显著种族差异已经导致APOL1基因的遗传变异的发现，该基因易使非裔美国人纯合这些风险等位基因发展为这些肾病。然而，野生型（WT） APOL1蛋白的细胞功能以及APOL1风险突变体在HIV-1感染时对足细胞损伤的贡献尚不清楚。该应用着眼于APOL1通路，该通路可用于治疗HIVAN中nef介导的足细胞损伤。我们发现足细胞中的APOL1与溶酶体和自噬体强烈共定位，表明APOL1可能在自噬中发挥作用。我们还证明了APOL1 WT刺激分化足细胞的自噬。由于APOL1基因风险变异的遗传是完全隐性的，分析APOL1对自噬的影响将需要APOL1风险等位基因的足细胞模型纯合子。我们已经开发了一种从尿液中分离和培养人足细胞的方法，该方法将用于探索APOL1在足细胞中的作用。在这个应用中，我们提出野生型APOL1通过激活自噬通量和抵消Nef对自噬的抑制作用来保护HIVAN的发展。相比之下，缺乏这些功能的APOL1风险变异允许Nef的有害影响。我们建议：1)通过比较APOL1和高风险双突变体（APOL1 DM）分子之间的相互作用和自噬机制，利用免疫沉淀（IP）、western blotting和共聚焦显微镜分析APOL1和高风险双突变体（APOL1 DM）促进自噬启动的机制。2)为了研究APOL1抵消HIV-1 Nef抑制自噬体成熟的机制，我们将测试APOL1 WT是否与Nef竞争从而刺激自噬体成熟。将确定Nef和APOL1在Beclin1/UVRAG/C-Vps复合物中的结合伙伴。自噬体与溶酶体的融合将使用新型串联荧光mRFP-EGFP-LC3结构进行分析。肾脏致病性SF2 Nef和突变体将用于识别SF2 Nef抑制自噬的机制以及APOL1 WT如何克服Nef的抑制。我们相信，我们的研究结果将导致一种新的治疗策略，旨在通过刺激自噬通量和在APOL1风险等位基因存在的情况下靶向Nef来预防HIVAN的足细胞损伤。

DESCRIPTION (provided by applicant): HIV-1 associated nephropathy (HIVAN) is a disease almost entirely limited to individuals of African descent. The pathogenesis of HIVAN is not fully understood, however dysfunction and injury to podocytes that form the glomerular blood filtration barrier are essential to the development of this disease. The mechanism of HIV-1- mediated podocyte injury is unclear mostly due to the lack of expression of classical HIV-1 entry receptors on these cells. However, findings that HIV-1 Nef recapitulates a HIVAN phenotype in experimental models, suggest that podocytes can be injured by Nef that is released by other productively infected cells. Further, striking racial disparities in the frequency of focal segmentl glomerulosclerosis (FSGS) and HIVAN have led to the discovery of genetic variants of the APOL1 gene that predispose African Americans homozygous for these risk alleles to the development of these nephropathies. However, the cellular function of the wild type (WT) APOL1 protein and the contribution of APOL1 risk mutants to podocyte damage in the face of HIV-1 infection remain unknown. This application looks at the APOL1 path that could be therapeutically exploited to protect against Nef-mediated podocyte injury in HIVAN. We show that APOL1 in podocytes strongly colocalizes with lysosomes and autophagosomes, suggesting that APOL1 may play a role in autophagy. We also demonstrate that APOL1 WT stimulates autophagy in differentiated podocytes. Since inheritance of the APOL1 gene risk variants is fully recessive, analysis of the effect of the APOL1 on autophagy will require a podocyte model homozygous for the APOL1 risk alleles. We have developed a method for isolation and culture of human podocytes from urine that will be used to explore the role of APOL1 in podocytes. In this application, we propose that wild type APOL1 is protective against HIVAN development by activating autophagic flux and counteracting the inhibitory effect of Nef on autophagy. In contrast, APOL1 risk variants lacking these functions permit the deleterious effects of Nef. We propose: 1) To dissect the mechanism(s) by which APOL1 and a high risk double mutant (APOL1 DM) contribute to autophagy initiation by comparing the interactions between these two molecules and the autophagy machinery, using immunoprecipitation (IP), western blotting and confocal microscopy. 2) To investigate the mechanism(s) by which APOL1 counteracts the suppression of autophagosome maturation by HIV-1 Nef, we will test whether APOL1 WT competes with Nef and thus stimulates autophagosome maturation. Binding partners of Nef and APOL1 in Beclin1/UVRAG/C-Vps complexes will be identified. Autophagosome fusion with lysosomes will be analyzed using a novel tandem fluorescent mRFP-EGFP-LC3 construct. Renal pathogenic SF2 Nef and mutants will be used to discern mechanism(s) by which SF2 Nef inhibits autophagy and how APOL1 WT overcomes Nef's inhibition. We believe that results of our proposal will lead to a novel therapeutic strategy aimed at preventing podocyte injury in HIVAN by stimulation of autophagic flux and by targeting Nef in the presence of APOL1 risk alleles.