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

• 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
• 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' 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

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.

在许多受感染的个体中，人类免疫缺陷病毒1型（HIV-1）进入大脑并可引起 广泛的HIV-1相关神经认知障碍（HAND），从轻度损伤到重度损伤 艾滋病相关性痴呆（HAD）。虽然抗逆转录病毒联合治疗（cART）的广泛使用， 有效地延长了艾滋病毒1/艾滋病（获得性免疫缺陷综合症）感染者的寿命， 在cART时代，轻度HAND的流行也增加了。事实上，最近的流行病 研究表明在美国超过50%的HIV-1感染者发展为HAND。因为HIV-1不 被感染神经元、被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）被认为有助于 通过直接机制（病毒蛋白的产生）或间接机制（病毒蛋白的产生）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）。病理学研究也表明， HIV在大脑中复制的状态增加了细胞内和可能的细胞外β-淀粉样蛋白（Aβ），这是一个经典的标志， 阿尔茨海默病（AD）和痴呆症。然而，HIV-1如何以及为什么会增加Aβ的产生， 感染，以及这是否有助于神经变性，仍然不清楚。我们的初步数据表明 淀粉样前体蛋白作为人类HIV-1感染早期和晚期的先天性限制因子 包括小胶质细胞。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。在查明 为进一步研究其潜在机制，我们发现APP是一种新的HIV-1基质（MA）相关蛋白， 阻止早期感染。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。为了逃避这一限制，HIV-1感染或 单独的Gag促进APP的γ-分泌酶依赖性加工，这导致Aβ40分泌增加， 通过蛋白质印迹和ELISA测定Aβ42，并可被γ-分泌酶抑制剂阻断。分馏 来自表达GAG的细胞的上清液在培养的原代皮层神经元中引起毒性，并且被 用γ-分泌酶抑制剂处理表达GAG的细胞。最后，回归分析表明， 在该系统中，在各种条件下，上清液中的A β与Aβ水平精确相关。本提案中 我们的目标是确定APP限制HIV-1感染的潜在机制，以及病毒如何逃逸， 这种限制通过APP降解导致Aβ代谢改变和神经元损伤。这些 aims将利用广泛的创新方法，包括高分辨率活细胞成像和联合 在微流控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。 流程.我们的研究结果将为HIV感染如何以及为什么诱导神经元凋亡提供新的线索。 损害，对我们对神经认知障碍和神经艾滋病的一般理解有更广泛的影响。