Electrophysiological mechanisms of HIV-mediated neuropathogenesis

描述（由申请人提供）：尽管接受了抗逆转录病毒治疗，但美国约50%的HIV+患者被诊断患有HIV相关神经认知障碍（HAND）。HIV改变了认知和精神活动的两个关键调节器，内侧前额叶皮层（mPFC）和尾壳核（CPu; a.k.a.背侧纹状体）。过量的Ca 2+内流（部分通过NMDA受体，NMDAR）是神经元兴奋性毒性的关键，但HIV神经发病机制尚不完全清楚。我们发表的和试点数据指出了HIV介导的神经元超兴奋性的另一种机制，其涉及电压门控L型Ca 2+通道，独立于NMDAR。我们发现，HIV-1转录反式激活因子（达特）通过上调mPFC锥体神经元中的L-通道增加Ca 2+内流，使这些细胞更容易受到过度兴奋的影响。达特还加强NMDAR和L通道之间的相互作用，并且神经元显示出与衰老或使用HIV-1转基因（Tg）大鼠建模的类似的过度兴奋。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L-通道阻滞剂和NMDAR拮抗剂都不能单独治疗晚期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感染使得mPFC锥体神经元和CPu中棘神经元（MSN）更容易受到兴奋性刺激，通过过度激活L-通道; L-通道和NMDAR阻滞剂的联合治疗将改善mPFC/CPu神经病理生理学，在青少年中比在老年大脑中更有效。我们将通过三个目标使用综合电生理，光学/荧光成像和免疫/生化方法来测试这一假设。在目标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，HIV-1感染细胞释放的蛋白介导的L-通道过度激活，部分通过参与多巴胺D1受体（D1 R），并将揭示的达特的结构域介导的达特对L-通道的影响。在目标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Ca 2+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。在目标3中，我们将确定老年Tg大鼠中L通道过度激活和mPFC/CPu神经病理生理学的年龄（HAND的重要风险因素）相关恶化。我们假设，mPFC/CPu神经病理生理学将更严重的老化Tg大鼠比青春期Tg大鼠（和老化非Tg大鼠）。最近，我们预计L-通道/NMDAR阻断剂的联合治疗将减少/改善HIV介导的神经元过度兴奋性。总的来说，我们的研究将在HIV介导的神经病理学中建立一个新的范式，并为HIV介导的神经病理学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.