Electrophysiological mechanisms of HIV-mediated neuropathogenesis

描述(申请人提供)：尽管接受抗逆转录病毒治疗，但美国约50%的HIV+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器，内侧前额叶皮质(MPFC)和尾壳核(CPU；又名)。背侧纹状体)。过度的钙内流(部分通过NMDAR)在神经元兴奋性毒性中起关键作用，但HIV神经发病机制尚不完全清楚。我们已发表的和初步的数据表明，HIV介导的神经元超兴奋性还有一个额外的机制，它涉及电压门控L型钙通道，而不依赖于NMDAR。我们发现，HIV-1转录反式激活因子(TAT)通过上调mPFC锥体神经元的L通道来增加钙内流，使这些细胞更容易受到过度兴奋的影响。TAT还增强了NMDAR和L通道之间的相互作用，神经元表现出与衰老或用HIV-1转基因(TG)大鼠建立的模型相似的过度兴奋。总之，这些发现指出了一种独特的机制，艾滋病毒通过这种机制诱导过度的神经兴奋。鉴于无论是L通道阻滞剂还是NMDAR拮抗剂都不能在晚期治疗HAND，了解艾滋病毒和L通道之间的复杂相互作用可能为HAND提供新的治疗策略。我们的中心假设是，艾滋病毒感染大脑使mPFC锥体神经元和中央处理器中的棘神经元(MSN)通过过度激活L通道而更容易受到兴奋性刺激的影响；L通道和NMDAR阻断剂联合治疗将改善mPFC/CPU的神经病理生理学，在青少年组比在老年人脑中更有效。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验证这一假说。在目标1中，我们将确定艾滋病病毒介导青春期TG大鼠mPFC/CPU区L通道过度激活从而导致神经元过度兴奋的细胞/分子机制(S)。我们预计，感染细胞释放的HIV-1蛋白部分通过与多巴胺D1受体(D1R)结合来介导L通道的过度激活，并将揭示TAT结构域介导TAT对L通道的影响。在目标2中，我们将评估NMDAR和L通道在调节神经元超兴奋性中的相互作用。我们假设N-甲基-D-天冬氨酸诱导的钙信号被过度放大，并被LVA-L通道传递到最终控制放电的其他离子通道。在目标3中，我们将确定年龄(手部的一个重要危险因素)在老年TG大鼠L通道过度激活和mPFC/CPU神经病理生理学中的相关性加剧。我们推测，老年TG大鼠的mPFC/CPU神经病理生理学将比青春期TG大鼠(和老年非TG大鼠)更严重。最近，我们预计L通道阻滞剂/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。总之，我们的研究将在HIV介导的神经发病机制中建立一个新的范例，并为未来HIV介导的神经病理学的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.