The role of amyloid precursor protein in HIV-1 replication and associated neurodegeneration

淀粉样前体蛋白在 HIV-1 复制和相关神经变性中的作用

基本信息

批准号：
9348763
负责人：
Mojgan Hosseini Naghavi
金额：
$ 41.85万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-02-01 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9348763
关键词：
AIDS Dementia Complex Abeta synthesis Acquired Immunodeficiency Syndrome Affect Alzheimer&apos s Disease Amyloid beta-Protein Amyloid beta-Protein Precursor Astrocytes Binding Binding Proteins Biological Assay Brain Bypass Bystander Effect Cell membrane Cells Characteristics Chronic Coculture Techniques Data Dementia Developed Countries Developing Countries Development Enzyme-Linked Immunosorbent Assay Functional disorder HIV HIV Infections HIV-1 HIV-associated neurocognitive disorder Human Impairment Incidence Individual Infection Integration Host Factors Lead Light Longevity Mediating Membrane Metabolism Microfluidics Microglia Modeling Mus Nerve Degeneration Neuraxis Neurocognitive Deficit Neuronal Dysfunction Neurons Organ Outcome Pathology Patients Plasmids Polyproteins Prevalence Process Production Proteins Public Health Regression Analysis Resolution Resources Role Senile Plaques Small Interfering RNA System Testing Toxic effect Transfection Viral Viral Proteins Virus Western Blotting Work abeta deposition amyloid precursor protein processing antiretroviral therapy base brain cell cell type chemokine cytokine design epidemiology study extracellular gamma secretase inhibitor/antagonist innovation live cell imaging macrophage neuroAIDS neurocognitive disorder neuron loss neurotoxic neurotoxicity novel novel strategies prevent protein expression

项目摘要

In many infected individuals, human immunodeficiency virus type 1 (HIV-1) enters the brain and can cause a broad spectrum of HIV-1-associated neurocognitive disorders (HAND) ranging from mild impairments to severe HIV-associated dementia (HAD). While widespread use of combination antiretroviral therapy (cART) has effectively increased the life span of people living with HIV-1/AIDS (acquired immunodeficiency syndrome), the prevalence of milder forms of HAND has also increased in the cART era. Indeed, recent epidemiological studies indicate that greater than 50% of HIV-1 infected people in the USA develop HAND. As HIV-1 does not infect neurons, HIV-1 infected macrophages and microglia (and possibly astrocytes) are thought to contribute to neuronal dysfunction and death via a direct mechanism (production of viral proteins) or an indirect “bystander” effect (production of cytokines and chemokines). Pathology studies have also shown that a chronic state of HIV replication in the brain increases intra and possibly extracellular β-amyloid (Aβ), a classic hallmark of Alzheimer's disease (AD) and dementia. However, how and why Aβ production is elevated by HIV-1 infection, and whether this contributes to neurodegeneration, remains unclear. Our preliminary data identifies amyloid precursor protein (APP) as an innate restriction factor for both early and late HIV-1 infection in human cells, including microglia. Moreover, we establish that HIV-1 overcomes this restriction by reducing APP levels, but in doing so generates Aβ40 and Aβ42 products that are toxic to primary cortical neurons. In identifying the underlying mechanism for further study, we find that APP is a novel HIV-1 matrix (MA) associated protein that blocks early infection. By transfection of an infectious HIV-1 clone or Gag-expressing plasmids, we find that increasing APP expression also binds HIV-1 Gag through its MA domain and potently suppresses late stage HIV-1 budding, trapping Gag in specific membrane compartments. To escape this restriction, HIV-1 infection or Gag alone promotes γ-secretase-dependent processing of APP. This results in elevated secretion of Aβ40 and Aβ42 as determined by western blotting and ELISA, and can be blocked by γ-secretase inhibitors. Fractionated supernatants from Gag-expressing cells cause toxicity in cultured primary cortical neurons, and is blocked by treating Gag-expressing cells with γ-secretase inhibitors. Finally, regression analysis shows that neurotoxicity in supernatants correlates precisely with Aβ levels under a variety of conditions in this system. In this proposal we aim to determine the underlying mechanisms by which APP restricts HIV-1 infection, and how viral evasion of this restriction through degradation of APP results in altered Aβ metabolism and neuronal damage. These aims will make use of a wide range of innovative approaches including high-resolution live cell imaging and co- culturing of microglia with neuronal cells in microfluidic chambers to determine the mechanistic basis of these processes. The outcome of our studies will shed new light on how and why HIV infection induces neuronal damage, with broader implications for our general understanding of neurocognitive disorders and neuroAIDS.

在许多感染者中，1 型人类免疫缺陷病毒 (HIV-1) 进入大脑并可导致广泛的 HIV-1 相关神经认知障碍 (HAND)，从轻度损伤到严重损伤 HIV 相关痴呆 (HAD) 已广泛使用联合抗逆转录病毒疗法 (cART)。有效延长了 HIV-1/AIDS（获得性免疫缺陷综合症）感染者的寿命，事实上，最近的流行病学表明，在 cART 时代，轻度手足口病的患病率也有所增加。研究表明，美国超过 50% 的 HIV-1 感染者会患上手足口病，而 HIV-1 则不会。感染的神经元、HIV-1感染的巨噬细胞和小胶质细胞（可能还有星形胶质细胞）被认为是造成这种情况的原因。通过直接机制（病毒蛋白的产生）或间接机制导致神经元功能障碍和死亡 “旁观者”效应（细胞因子和趋化因子的产生）也表明，慢性。 HIV 在大脑中的复制状态会增加细胞内和可能细胞外的 β-淀粉样蛋白 (Aβ)，这是一个典型的标志然而，HIV-1 如何以及为何增加 Aβ 的产生？感染以及这是否会导致神经退行性变，我们的初步数据仍不清楚。淀粉样前体蛋白 (APP) 作为人类早期和晚期 HIV-1 感染的先天限制因子此外，我们确定 HIV-1 通过降低 APP 水平克服了这一限制，但这样做会产生对初级皮质神经元有毒的 Aβ40 和 Aβ42 产物。为了进一步研究潜在机制，我们发现 APP 是一种新型的 HIV-1 基质（MA）相关蛋白，通过转染感染性 HIV-1 克隆或表达 Gag 的质粒，我们发现增加 APP 表达还可以通过其 MA 结构域结合 HIV-1 Gag，并有效抑制晚期阶段 HIV-1 出芽，将 Gag 捕获在特定的膜隔室中，以逃避这种限制，HIV-1 感染或。 Gag 单独促进 APP 的 γ 分泌酶依赖性加工，从而导致 Aβ40 和 Aβ40 的分泌增加。通过蛋白质印迹和 ELISA 测定，Aβ42 可以被 γ-分泌酶抑制剂阻断。来自表达 Gag 的细胞的上清液会对培养的原代皮质神经元产生毒性，并被用γ-分泌酶抑制剂处理Gag表达细胞最后，回归分析显示神经毒性。在该系统中，上清液中的 Aβ 水平与多种条件下的 Aβ 水平精确相关。我们的目标是确定 APP 限制 HIV-1 感染的潜在机制，以及病毒如何逃避通过 APP 的降解来打破这种限制会导致 Aβ 代谢改变和神经元损伤。目标将利用广泛的创新方法，包括高分辨率活细胞成像和联合在微流体室中培养小胶质细胞和神经元细胞，以确定这些的机制基础我们的研究结果将为艾滋病毒感染如何以及为何诱发神经元提供新的线索。损害，对我们对神经认知障碍和神经艾滋病的一般理解具有更广泛的影响。