Electrophysiological mechanisms of HIV-mediated neuropathogenesis

描述（由申请人提供）：尽管抗逆转录病毒治疗，在美国大约50%的HIV阳性患者被诊断为HIV相关神经认知障碍（HAND）。HIV改变了认知和精神运动活动的两个关键调节因子，内侧前额叶皮层（mPFC）和尾状壳核（CPu；又称背纹状体）。过量的Ca2+内流（部分通过NMDA受体，NMDAR）是神经元兴奋性毒性的关键，但潜在的HIV神经发病机制尚不完全清楚。我们发表的和试验数据指出了hiv介导的神经元高兴奋性的另一种机制，该机制涉及电压门控的l型Ca2+通道，独立于NMDAR。我们发现HIV-1转录反激活因子（Tat）通过上调mPFC锥体神经元中的l通道来增加Ca2+内流，使这些细胞更容易受到超兴奋性的影响。这也增强了NMDAR和l通道之间的相互作用，并且神经元表现出与衰老或使用HIV-1转基因（Tg）大鼠相似的过度兴奋。总之，这些发现指出了HIV诱导过度神经兴奋的独特机制。鉴于无论是l通道阻滞剂还是单独的NMDAR拮抗剂都无法治疗晚期HAND，了解HIV和l通道之间复杂的相互作用可能为HAND提供新的治疗策略。我们的中心假设是，大脑的HIV感染使mPFC锥体神经元和CPu中棘神经元（msn）更容易受到l通道过度激活的兴奋性刺激的影响；l通道和NMDAR阻滞剂的联合治疗将改善mPFC/CPu神经病理生理，在青少年中比在老年人中更有效。我们将通过综合电生理、光学/荧光成像和免疫/生化方法的三个目标来检验这一假设。在目的1中，我们将定义HIV介导青春期Tg大鼠mPFC/CPu中l通道过度激活和神经元过度兴奋的细胞/分子机制。我们预计受感染细胞释放的HIV-1蛋白部分通过参与多巴胺D1受体（D1R）介导l通道过度激活，并将揭示介导Tat对l通道作用的Tat结构域。在目的2中，我们将评估NMDAR和l通道在介导神经元超兴奋性中的相互作用。我们假设nmda诱发的Ca2+信号被过度放大，并通过LVA-L通道传递到最终控制放电的其他离子通道。在目的3中，我们将确定年龄（HAND的一个重要危险因素）与老年Tg大鼠l通道过度激活和mPFC/CPu神经病理生理相关的恶化。我们假设衰老Tg大鼠的mPFC/CPu神经病理生理比青春期Tg大鼠（和衰老的非Tg大鼠）更严重。最近，我们期望l通道/NMDAR阻滞剂的联合治疗将减少/改善hiv介导的神经元超兴奋性。总的来说，我们的研究将为hiv介导的神经发病机制建立一个新的范式，并为hiv介导的神经病理的未来治疗干预确定新的靶点。

DESCRIPTION (provided by applicant): Despite antiretroviral therapy, approximately 50% of HIV+ patients in the USA are diagnosed with HIV-associated neurocognitive disorders (HAND). HIV alters two key regulators of cognition and psychomotor activity, the medial prefrontal cortex (mPFC) and caudate-putamen (CPu; a.k.a. dorsal striatum). Excessive Ca2+ influx (partly via NMDA receptor, NMDAR) is critical in neuronal excitotoxicity, but the mechanisms underlying HIV neuropathogenesis are not entirely clear. Our published and pilot data point to an additional mechanism in HIV-mediated neuronal hyper-excitability which involves the voltage-gated L-type Ca2+ channel, independent of NMDAR. We showed that HIV-1 transactivator of transcription (Tat) increases Ca2+ influx by upregulating L-channels in mPFC pyramidal neurons, rendering these cells more susceptible and vulnerable to hyper-excitability. Tat also potentiates the interaction between NMDAR and L-channel, and neurons showed similar over-excitation as aged or modeled using HIV-1 transgenic (Tg) rats. Together, these findings point to a unique mechanism by which HIV induces excessive neuroexcitation. Given that neither an L-channel blocker nor a NMDAR antagonist alone was able to treat HAND at late stage, understanding the intricate interplay between HIV and the L-channels may provide novel therapeutic strategies for HAND. Our central hypothesis is that HIV infection of the brain renders mPFC pyramidal neurons and CPu medium spiny neurons (MSNs) more susceptible and vulnerable to excitatory stimuli via over-activating the L-channels; and that combined treatments of an L-channel and NMDAR blocker will ameliorate the mPFC/CPu neuropathophysiology, more effectively in adolescent than in older brain. We will test this hypothesis via three aims using integrated electro- physiological, optic/fluorescence imaging and immuno/biochemical approaches. In Aim 1 we will define the cellular/molecular mechanism(s) by which HIV mediates L-channel over-activation and consequently neuronal hyper-excitation in the mPFC/CPu of adolescent Tg rats. We expect that HIV-1 proteins released by infected cells mediate L-channel over-activation in part via engaging dopamine D1 receptor (D1R), and will reveal the domain of Tat that mediates the Tat effects on L-channels. In Aim 2 we will evaluate the interplay between NMDAR and L-channel in mediating neuronal hyper-excitability. We hypothesize that NMDA-evoked Ca2+ signal is over-amplified and relayed by LVA-L channels to other ion channels that ultimately control firing. In Aim 3 we will ascertain age (a significant risk factor for HAND)-associated exacerbation in L-channel over-activation and mPFC/CPu neuropathophysiology in older Tg rats. We hypothesize that mPFC/CPu neuropathophysiology will be more severe in aging Tg rats than in adolescent Tg rats (and aging non-Tg rats). Lately, we expect that combined treatments of L-channel/NMDAR blockers will diminish/ameliorate HIV-mediated neuronal hyper-excitability. Collectively, our studies will establish a novel paradigm in HIV-mediated neuropathogenesis and identify novel targets for future therapeutic intervention for HIV-mediated neuropathology.