Knock-in mouse model of dopamine transporter-Tat interaction underlying NeuroAIDS

NeuroAIDS 背后的多巴胺转运蛋白与 Tat 相互作用的敲入小鼠模型

• 批准号: 9137163
• 负责人: Jun Zhu
• 金额: $ 22.89万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9137163
• 关键词: Address; Affect; Allosteric Site; Amino Acid Sequence; Anti-Retroviral Agents; Attention; Attenuated; Automobile Driving; Autopsy; Base Sequence; Behavioral; Binding; Binding Proteins; Binding Sites; Biological Preservation; Brain; Breeding; CRISPR/Cas technology; Clinical; Cocaine; Cognitive; Collaborations; Computer Simulation; Development; Dopamine; Doxycycline; Early Intervention; Effectiveness; Exhibits; Functional disorder; Genetic; Genetic Transcription; HIV; HIV Infections; HIV-1; HIV-associated neurocognitive disorder; Health; Homeostasis; Human; Image; Impaired cognition; Individual; Infection; Intervention; Knock-in; Knock-in Mouse; Learning; Life; Link; Locomotion; Mediating; Memory; Microinjections; Molecular Target; Mus; Neuraxis; Neurocognitive; Neurocognitive Deficit; Patients; Performance; Peripheral; Phenylalanine; Physiological; Play; Prefrontal Cortex; Prevalence; Proteins; Publishing; Research; Risk; Role; Sequence Alignment; Severities; Signal Transduction; Site; Staging; Surface; Synapses; Technology; Testing; Therapeutic Intervention; Trans-Activators; Transgenes; Transgenic Mice; Transgenic Organisms; Transmembrane Domain; Validation; Viral Proteins; Virus Replication; Wild Type Mouse; Work; antiretroviral therapy; base; dopamine system; dopamine transporter; dopaminergic neuron; drug of abuse; genetic manipulation; genetic regulatory protein; improved; in vivo; intermolecular interaction; mouse model; neuroAIDS; neurocognitive disorder; neuropathology; neuropsychiatry; neuropsychological; neurotoxicity; novel; preference; public health relevance; research study; reuptake; therapeutic target; transmission process; uptake

 DESCRIPTION (provided by applicant): HIV-1 Tat protein within the central nervous system plays a pivotal role in the neurotoxicity and cognitive impairment evident in HIV-associated neurocognitive disorders (HAND). Converging lines of clinical observations, supported by imaging, neuropsychological performance, and postmortem examinations, have implicated that dysregulation of dopamine (DA) signaling is a risk determinant of HAND. Drugs of abuse, such as cocaine have been shown to exacerbate the severity of HAND by enhancing viral replication. Tat and cocaine synergistically increase synaptic DA levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Therefore, an intervention for HIV infection-induced dysfunction of DA system has the potential to improve neurocognitive function in patients with the early- stage of HAND. Currently, it is unclear how Tat influences the DA system in the brains of HIV- infected patients with HAND, thereby producing neurocognitive impairment. Our studies will identify the intermolecular interactions between Tat and human DAT (hDAT) and explore how Tat potentiates cocaine-induced inhibition of DA transport, leading to DA dysregulation for both mechanistic and interventional purposes. Recently, we identified key residues for hDAT interacting with Tat, which are critical for Tat-induced inhibitio of DAT uptake. For example, we found that DAT Tyrosine88 replaced by Phenylalanine (Y88F) displays normal surface DAT expression and DA uptake but attenuates Tat-induced inhibition of DA transport. Moreover, we found that Y88F partially attenuates the allosteric modulatory effects of SRI-20041, a novel DAT allosteric modulator, on DAT function. These studies led us to the novel hypothesis that Tat acts via the unique tyrosine88 site to perturb the DAT regulatory network that normally sustains concentrative DA transport, resulting in DA-linked neuropsychiatric dysfunction prominently featured in HAND. We will generate DAT Y88F knock-in mouse line harboring a doxycycline- inducible Tat transgene, and determine the impact of DAT Y88 on Tat-induced inhibition of DA transport and associated cognitive/behavioral deficits in Tat transgenic mice (Aim 1). Moreover, we will determine whether microinfusion of SRI-20041 into prefrontal cortex alleviates cognitive and behavioral deficits in Tat transgenic mice (Aim 2). This exploratory research will uncover critical functions of DAT important for Tat-induced cognitive/behavioral deficits and provide a novel mechanistic basis to identify targets on the DAT for developing compounds that specifically block Tat binding site(s) in hDAT, thereby stabilizing physiological DA transmission.

 描述（由申请人提供）：中枢神经系统内的 HIV-1 Tat 蛋白在 HIV 相关神经认知障碍 (HAND) 中明显的神经毒性和认知障碍中发挥着关键作用。影像学、神经心理学表现和尸检支持的临床观察结果表明，多巴胺 (DA) 信号传导失调是 HAND 的风险决定因素。滥用药物（如可卡因）已被证明会通过增强病毒复制而加剧 HAND 的严重程度。 Tat 和可卡因通过直接抑制 DA 转运蛋白 (DAT) 活性，协同增加突触 DA 水平，最终导致多巴胺能神经元损伤。因此，对HIV感染引起的DA系统功能障碍进行干预有可能改善早期HAND患者的神经认知功能。目前尚不清楚 Tat 如何影响 HIV感染者大脑中的DA系统存在HAND，从而产生神经认知障碍。我们的研究将确定 Tat 和人类 DAT (hDAT) 之间的分子间相互作用，并探讨 Tat 如何增强可卡因诱导的 DA 转运抑制，从而导致 DA 失调，从而达到机械和干预目的。最近，我们鉴定了 hDAT 与 Tat 相互作用的关键残基，这对于 Tat 诱导的 DAT 摄取抑制至关重要。例如，我们发现 DAT 酪氨酸 88 被苯丙氨酸 (Y88F) 取代，表现出正常的表面 DAT 表达和 DA 摄取，但减弱了 Tat 诱导的 DA 转运抑制。此外，我们发现 Y88F 部分减弱了 SRI-20041（一种新型 DAT 变构调节剂）对 DAT 功能的变构调节作用。这些研究使我们得出了一个新的假设，即 Tat 通过独特的酪氨酸88 位点起作用，扰乱通常维持集中 DA 转运的 DAT 调节网络，导致 HAND 中突出的 DA 相关神经精神功能障碍。我们将产生含有多西环素诱导的 Tat 转基因的 DAT Y88F 敲入小鼠系，并确定 DAT Y88 对 Tat 诱导的 DA 运输抑制和 Tat 转基因小鼠相关认知/行为缺陷的影响（目标 1）。此外，我们将确定将 SRI-20041 微量输注到前额皮质是否可以减轻 Tat 转基因小鼠的认知和行为缺陷（目标 2）。 这项探索性研究将揭示 DAT 对 Tat 诱导的认知/行为缺陷的关键功能，并为识别 DAT 上的靶点提供新的机制基础，以开发特异性阻断 hDAT 中 Tat 结合位点的化合物，从而稳定生理 DA 传输。