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

 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)明显的神经毒性和认知障碍中发挥关键作用。在影像、神经心理学表现和尸检的支持下，临床观察的趋同路线表明，多巴胺(DA)信号调节失调是手部疾病的一个风险决定因素。滥用药物，如可卡因，已被证明通过促进病毒复制而加剧手部疾病的严重程度。TAT和可卡因通过直接抑制DA转运体(DAT)活性而协同增加突触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系统功能障碍的干预有可能改善早期手部患者的神经认知功能。目前尚不清楚TAT如何影响 HIV感染者手部大脑中的DA系统，从而产生神经认知功能障碍。我们的研究将确定TAT和人DAT(HDAT)之间的分子间相互作用，并探索TAT如何加强可卡因诱导的DA转运抑制，从而导致机制和干预目的的DA失调。最近，我们确定了HDAT与TAT相互作用的关键残基，这些残基对于TAT诱导的DAT摄取抑制至关重要。例如，我们发现苯丙氨酸(Y88F)取代的DAT酪氨酸88显示出正常的表面DAT表达和DA摄取，但减弱了TAT对DA转运的抑制。此外，我们发现Y88F部分减弱了一种新型的DAT变构调节剂SRI-20041对DAT功能的变构调制作用。这些研究使我们提出了一个新的假设，即TAT通过独特的酪氨酸88位点作用于扰乱DAT调节网络，该网络通常维持集中的DA运输，导致与DA相关的神经精神障碍，突出表现在手部。我们将建立携带强力霉素诱导的TAT转基因的DAT Y88F敲入鼠系，并确定DAT Y88在TAT诱导的TAT转基因小鼠的DA运输抑制和相关的认知/行为缺陷中的影响(目标1)。此外，我们将确定将SRI-20041微量注入前额叶皮质是否可以缓解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在TAT诱导的认知/行为障碍中的重要作用，并为在DAT上识别靶点以开发特异性阻断HDAT中TAT结合位点(S)的化合物，从而稳定生理性DA传递提供新的机制基础。

 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.