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

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：中枢神经系统内的HIV-1 TAT蛋白在神经毒性和认知障碍证据中起关键作用，在与HIV相关的神经认知障碍（HAND）中起着关键作用。由成像，神经心理学表现和验尸检查支持的临床观察线的融合线已经实现了多巴胺（DA）信号的失调是确定手的风险。诸如可卡因之类的滥用药物已被证明通过增强病毒复制来加剧手的严重程度。 TAT和可卡因通过直接抑制DA转运蛋白（DAT）活性来协同提高合成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系统功能障碍的干预有可能改善手工早期患者的神经认知功能。目前，目前尚不清楚TAT如何影响艾滋病毒感染患者手的大脑中的DA系统，从而产生神经认知障碍。我们的研究将确定TAT与人DAT（HDAT）之间的分子间相互作用，并探讨TAT潜在可卡因诱导的DA转运的抑制，从而导致DA失调用于机械和介入目的。最近，我们确定了HDAT与TAT相互作用的密钥保留，这对于TAT诱导的DAT摄取抑制作用至关重要。例如，我们发现被苯丙氨酸（Y88F）取代的DAT酪氨酸88显示出正常的表面表达和DA摄取，但减弱了TAT诱导的DA转运的抑制作用。此外，我们发现Y88F部分衰减了新型DAT变构调节器SRI-20041对DAT功能的变构调节作用。这些研究使我们提出了这样一种新的假设，即TAT通过独特的酪氨酸88位点起作用，以扰动通常保持集中DA转运的DAT调节网络，从而导致DA连接的神经精神病功能障碍在手中突出出现。我们将生成带有强力霉素诱导的TAT转基因的DAT Y88F敲入小鼠线，并确定DAT Y88对TAT诱导的DA传输抑制的影响，以及相关的认知/行为定义TAT转基因小鼠（AIM 1）。此外，我们将确定将SRI-20041微灌注到前额叶皮层中是否减轻了认知和行为定义TAT转基因小鼠（AIM 2）。这项探索性研究将发现对TAT诱导的认知/行为定义的DAT的关键功能定义并提供了新的机械基础，以识别DAT的靶标，以开发出HDAT中专门阻止TAT结合位点的化合物，从而稳定物理DA的传播。