Novel mechanisms and Drosophila model of APOL1-HIV-1 nephropathies in children

项目总结 携带两个名为G1或G2的APOL1风险等位基因(RA)的个体患上APOL1的几率是对照组的30倍 HIV肾病(HIVAN)与HIV+对照组的比较。我们目前对APOL1在 HIVAN主要来源于对培养的肾脏细胞的研究或临床遗传学研究，以及新的 需要实验模型来更深入地了解HIV-1之间的相互作用 和体内的APOL1-RA。我们的儿科肾病项目一直在研究艾滋病毒-肾脏疾病(HIV- 研发)25年，并处于填补这一空白的独特地位。最近，我们育成了果蝇APOL1-G0 和G1转基因(TG)系，发现APOL1-G1在肾细胞中的异位表达(相当于 指哺乳动物足细胞)增强了这些细胞的内吞活性和大小，削弱了酸化 细胞内的空泡，加速了它们的死亡。此外，我们发现HIV-1诱导的水平较低 HIV阳性儿童培养的足细胞通过动力蛋白依赖的内吞作用进行生产性感染 这是一种独立于CD4的机制，可增加这些细胞中APOL1-G1的表达。 此外，我们还发现，在转基因果蝇和培养的足细胞中过表达APOL1-G1会增加 参与细胞内吞作用、调节细胞骨架网络和细胞运输的GTP酶的活性。基座 根据这些发现，我们假设HIV-1在儿童中起到了“二次攻击”的作用，引发了HIV-RD。 通过内吞作用介导的机制感染足细胞，增加表达/ APOL1-RA活性超过其毒性阈值。反过来，这些更改会损害密钥 内吞、细胞运输、酸化和自噬途径破坏细胞的内稳态 并加速它们的死亡，导致蛋白尿和HIV-RD。为了验证这个假设，在目标1中 我们将确定表达APOL1-G0/G1/G2的果蝇TG系的表型和HIV基因 TAT、Nef和VPR，特别是在肾细胞中，并评估它们如何影响其结构和功能。在AIM 2，我们将定义APOL1-RA和HIV-基因在体内如何相互作用来调节肾细胞的功能 确定APOL1-RA是否通过以下途径加速表达HIV基因的肾细胞的死亡 影响它们的自噬通量，并进行RNA-Seq分析以确定新的转录途径， 规范这些相互作用。这些发现将在携带自噬报告的HIV-TG26小鼠身上得到验证 构建RFP-EGFP-LC3。在目标3中，我们将选择最好的果蝇模型并进行基于RNAi的 APOL1-HIV基因相互作用筛选以确定影响细胞功能和存活的新途径 肾细胞，确定APOL1-RA如何影响足细胞感染，并验证所有临床相关 来自HIV-RD儿童的样本或组织中的发现。这些研究将发现新的 APOL1-RA和HIV-1相互作用感染足细胞和沉淀HIV-RD的机制，以及 开发新的临床相关TG-Fly模型以a)研究儿童和青年HIV-RD的发病机制 成人；b)开发高通量筛查系统，以确定新的APOL1-艾滋病毒遗传调节因子 在活体内的相互作用；以及c)作为未来确定和筛选针对艾滋病毒-RD的新药靶点的平台。

PROJECT SUMMARY Individuals carrying two APOL1 risk alleles (RA) named G1 or G2 have ~ 30-fold higher odds of developing HIV-nephropathy (HIVAN) compared to HIV+ controls. Our current knowledge regarding the role of APOL1 in HIVAN is mostly derived from studies done in cultured renal cells or clinical genetic studies, and new experimental models are needed to gain a more in depth understanding of the interactions between HIV-1 and the APOL1-RA in vivo. Our pediatric nephrology program has been studying HIV-renal diseases (HIV- RD) for 25 years and is in a unique position to fill this gap. Recently, we developed Drosophila APOL1-G0 and G1 transgenic (Tg) lines, and found that ectopic expression of APOL1-G1 in nephrocytes (the equivalent of mammalian podocytes) enhanced the endocytic activity and size of these cells, impaired the acidification of intracellular vacuoles, and accelerated their death. In addition, we found that HIV-1 induces a low level productive infection of podocytes cultured from HIV+ children through a dynamin-dependent endocytosis mechanism that is independent of CD4, and increases the expression of APOL1-G1 in these cells. Furthermore, we found that overexpression of APOL1-G1 in Tg-flies and cultured podocytes increases the activity of GTPases involved in endocytosis, regulation of cytoskeletal networks, and cell trafficking. Based on these findings, we hypothesize that HIV-1 acts as a “second hit” to precipitate HIV-RD in children by infecting podocytes through an endocytosis mediated mechanism that increases the expression / activity of the APOL1-RA beyond their toxic threshold levels. In turn, these changes impair key endocytic, cell trafficking, acidification, and autophagy pathways that disrupt the homeostasis of podocytes and accelerate their dead, causing proteinuria and HIV-RD. To test this hypothesis, in aim 1 we will determine the phenotype of Drosophila Tg lines expressing APOL1-G0/G1/G2, and the HIV- genes Tat, Nef, and Vpr, specifically in nephrocytes, and assess how they affect their structure and function. In aim 2, we will define how APOL1-RA and HIV-genes interact in vivo to modulate the function of nephrocytes in dual Tg-fly lines, define whether APOL1-RA precipitate the death of nephrocytes expressing HIV-genes by affecting their autophagic flux, and perform RNA-Seq analysis to identify new transcriptional pathways that regulate these interactions. These findings will be validated in HIV-Tg26 mice carrying the autophagy reporter construct RFP-EGFP-LC3. In aim 3, we will select the best Drosophila model and perform an RNAi-based APOL1-HIV genetic interaction screening to identify new pathways that affect the function and survival of nephrocytes, determine how the APOL1-RA affect the infection of podocytes, and validate all clinical relevant findings in samples or tissues derived from children with HIV-RD. These studies will identify new mechanisms through which APOL1-RA and HIV-1 interact to infect podocytes and precipitate HIV-RD, and develop new clinically relevant Tg-fly models to a) study the pathogenesis of HIV-RD in children and young adults; b) develop a high throughput screening system to identify new genetic modulators of the APOL1-HIV interactions in vivo; and c) serve as future platforms to identify and screen new drug targets against HIV-RD.