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

 描述（由申请方提供）：中枢神经系统内的HIV-1达特蛋白在HIV相关神经认知障碍（HAND）中明显的神经毒性和认知损害中起关键作用。由影像学、神经心理学表现和死后检查支持的临床观察结果表明，多巴胺（DA）信号转导失调是HAND的风险决定因素。滥用药物，如可卡因，已被证明通过增强病毒复制来加剧HAND的严重程度。达特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系统功能障碍的干预有可能改善早期HAND患者的神经认知功能。目前，尚不清楚达特如何影响 艾滋病病毒感染者大脑中的DA系统发生HAND，从而产生神经认知功能障碍.我们的研究将确定达特和人DAT（hDAT）之间的分子间相互作用，并探讨达特如何加强可卡因诱导的DA转运抑制，导致DA调节障碍的机制和干预的目的。最近，我们确定了hDAT与达特相互作用的关键残基，这是Tat诱导的DAT摄取抑制的关键。例如，我们发现DAT酪氨酸88被苯丙氨酸（Y88 F）取代显示正常的表面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Y88 F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。这些研究使我们提出了一个新的假设，即达特通过独特的酪氨酸88位点干扰DAT调节网络，该网络通常维持集中的DA转运，导致DA相关的神经精神功能障碍在HAND中突出表现。我们将产生携带多西环素诱导的达特转基因的DAT Y88 F敲入小鼠系，并确定DAT Y88对达特转基因小鼠中的DA转运和相关认知/行为缺陷的Tat诱导的抑制的影响（目的1）。此外，我们将确定将SRI-20041微输注到前额皮质中是否减轻达特转基因小鼠的认知和行为缺陷（目的2）。 这项探索性研究将揭示DAT对Tat诱导的认知/行为缺陷至关重要的关键功能，并为鉴定DAT上的靶点以开发特异性阻断hDAT中达特结合位点的化合物提供新的机制基础，从而稳定生理DA传递。

 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.