Development of Brain Organoids to Study the Impact of HIV-1, Drugs of Abuse and Aging on Cognitive Impairment

项目摘要 HIV-1相关的神经炎症和神经毒性导致认知障碍（HIV-1相关的神经毒性）。 神经认知障碍或HAND），甚至在那些处于抑制性抗逆转录病毒疗法（ART）下的患者中。随着人们 与HIV-1年龄一起生活，正在发生复合效应，与年龄相关的痴呆症增加到HAND， 导致复杂的神经认知缺陷这将对医疗保健产生巨大的影响 不仅在美国，而且在发展中国家。HAND如何发展，以及它如何成为 修饰仍然神秘，主要是因为在世界范围内研究HIV-1感染和HAND非常困难 人脑在过去的几年里，干细胞技术的新发展使人们能够在干细胞领域中发挥重要作用。 诱导多能干细胞（iPSC）分化为“脑类器官”，并且这些培养物可以是 在促进神经外胚层三维扩张的条件下体外生长， 或“微型大脑”形式。大脑类器官是异质的，形成各种大脑区域，包括 腹侧前脑、大脑皮层、海马和中脑和后脑边界。它们的神经元 是功能性的，能够电兴奋，并发展小胶质细胞。这些脑类器官也类似于 在基因表达水平上研究人类皮层发育，并允许深入分析神经网络、细胞 行为，药物筛选，疾病建模，以及大脑发育的变化。而脑区 不同iPSC系的类器官组成不同，区域基因表达模式在很大程度上仍然存在 可在个体间复制。这为研究艾滋病病毒感染创造了前所未有的新机会。 个脑袋我们建议开发我们的iPSC衍生的类器官模型，该模型包含小胶质细胞， 更好地代表了能够支持强烈HIV-1感染的成年成熟大脑。有了这个模型，我们可以测试 不同的病毒是否导致不同的神经毒性，以及小胶质细胞以外的细胞是否可以潜伏 感染了病毒我们将测试病毒中的单个蛋白质，包括HIV-1达特如何引起神经系统疾病。 损伤，以及ART或药物如双脱氢皮质抑素A（dCA），交叉中和达特活性， 影响了进程。有趣的是，接触达特也能增强可卡因介导的奖赏机制， 这进一步促进了HAND，揭示了HIV-1感染和药物之间复杂的相互作用网络， 滥用药物我们将确定达特和可卡因在神经损伤中是如何合作的， 由于大脑类器官提供了HIV-1在大脑中潜伏的模型，我们可以测试是否 给予dCA，可以“阻断和锁定”任何残留的病毒，最后是否HIV-1特异性细胞毒性T 细胞（CTL）可以清除类器官中的病毒。总之，这些研究有望提供新的见解， HAND的发病机制，达特介导的神经毒性，可卡因的作用，以及这些之间的潜在联系 流程.最后，该模型有望增加关于HIV-1在大脑中潜伏期的令人兴奋的发现，如果可以的话， 沉默或消灭。

PROJECT ABSTRACT HIV-1 associated neuroinflammation and neurotoxicity lead to cognitive impairments (HIV-1-associated neurocognitive disorders or HAND) even in those under suppressive antiretroviral therapy (ART). As people living with HIV-1 age, a compounding effect is occurring, with age associated dementia added to HAND, leading to a complex web of neurocognitive deficit. This will have tremendous implications for health care systems not only in the USA but also in the developing world. How HAND develops, and how it could be modified remain mysterious, largely because It has been very difficult to study HIV-1 infection and HAND in the human brain. Over the past few years new developments in stem cell technologies have permitted the differentiation of “cerebral organoids” from induced pluripotent stem cells (iPSCs), and these cultures can be grown in vitro in conditions that promote three-dimensional expansion of neuroectoderm, in cerebral organoid or “miniature brain” forms. Cerebral organoids are heterogeneous and form a variety of brain regions, including ventral forebrain, cerebral cortex, hippocampus, and mid- and hindbrain boundary. They exhibit neurons that are functional and capable of electrical excitation, and develop microglia. These brain organoids also resemble human cortical development at the gene expression level, and allow in depth analysis of neural networks, cell behavior, drug screening, disease modeling, and variations in brain development. While brain-region composition varies in organoids from different iPSC lines, regional gene-expression patterns remain largely reproducible across individuals. These create unparalleled new opportunities to study HIV-1 infection of the brain. We propose to develop our iPSC derived organoid model which incorporates microglia, into one that better represents an adult mature brain that can support robust HIV-1 infection. With this model, we can test whether different viruses lead to differential neurotoxicity and if cells other than microglia can be latently infected with virus. We will test how individual proteins from the virus, including HIV-1 Tat causes neurological damage, and how ART or drugs such as Didehydro-Cortistatin A (dCA), which cross-neutralizes Tat activity, affects the process. Interestingly, exposure to Tat also potentiates cocaine-mediated reward mechanisms, which further promotes HAND, revealing a complex web of interactions between HIV-1 infection and drugs of substance abuse. We will determine how Tat and cocaine collaborate in neurological damage and determine if dCA can reverse it. As cerebral organoids provide a model for HIV-1 latency in the brain we can test whether administration of dCA, can “block-and-lock” any residual virus, and finally whether HIV-1 specific cytotoxic T cells (CTL) can eliminate virus in the organoid. Together, these studies promise to provide novel insights into the pathogenesis of HAND, Tat mediated neurotoxicity, effects of cocaine, and the potential link between these processes. Finally, the model promises to add exciting findings on HIV-1 latency in the brain, and if it can be silenced or eliminated.