HIV-1 disrupts oligodendrocyte functions: Ca2+ mediated mechanisms

描述（由申请人提供）：自1996年联合抗逆转录病毒疗法（cART）问世以来，HIV感染患者的死亡率和神经发病机制的初始进展已显著降低。然而，药物穿透血脑屏障的能力较差，使大脑成为艾滋病毒的储存库和慢性炎症的场所。艾滋病仍然是美国非老年人痴呆症的最常见原因。髓鞘或少突胶质细胞（OLs）的损伤会加重神经功能障碍。HIV患者的白色损害可发生在髓鞘苍白之前的早期阶段，表明OL/髓鞘可能是病毒或病毒蛋白的直接靶点。HIV-1蛋白达特在感染的早期和晚期都由CNS中的感染性脑膜炎病毒主动分泌。据报道，达特通过神经元、星形胶质细胞和小胶质细胞中的钙（Ca 2+）不稳定诱导毒性和功能变化。CaMKII近年来发现，作为调节钙离子信号转导的核心的OLs的细胞组分，在OL分化和髓鞘形成中起重要作用。我的中心假设是，Tat诱导的OL/髓鞘损伤是通过过度的Ca 2+内流介导的，随后是CaMKII过度激活，这可以通过阻断达特和多巴胺能受体之间的相互作用或通过抑制CaMKII激活来减弱。为了支持这一假设，我们的初步数据表明：1）在转基因小鼠中短时间的达特诱导导致OL的形态和超微结构异常; 2）暴露于100 nM Tat 1 -86的培养的小鼠OL具有减少的髓鞘样膜延伸; 3）1 nM - 100 nM Tat 1 -86可以诱导OL中[Ca 2 +]i的持续的、剂量依赖性的增加; 4）OLs在我们的培养条件下表达功能性离子型谷氨酸受体（iGluRs）。这些结果表明，HIV-1达特可能通过与iGluRs相互作用，通过[Ca 2 +]i失调直接损伤OL/髓鞘。拟议的研究调查，如果 CaMKII作为在OL中表达的主要CaMKII同种型，是Tat诱导的介导OL/髓鞘损伤的[Ca 2 +]i增加的下游信号分子。目的1通过评估CaMKII磷酸化的变化来研究HIV-1 Tat 1 -86对CaMKII激活的测试效果。目的2研究iGluR拮抗剂是否能减弱CaMKII？激活和Tat诱导的OL [Ca 2 +]i增加。目的3研究测试抑制iGluRs或CaMKII？活化是否可以减轻Tat诱导的OL/髓鞘损伤。药理学方法和siRNA干扰将用于抑制iGluRs和CaMKII？。将使用包含所有CNS细胞的脑细胞聚集体模型结合z-堆叠共聚焦成像和3-D重建来比较不同实验条件下的髓鞘结构。还将评估典型髓鞘蛋白水平以及OL过程复杂性。由于HIV是一种人类疾病，因此所有实验都将在鼠和人OL上进行。研究结果将指导未来的研究方向，有关艾滋病毒患者的OL发病机制的基础，并有利于开发新的治疗方法，以减轻HIV-1感染的中枢神经系统的后果。

DESCRIPTION (provided by applicant): Since the advent of combined anti-retroviral therapy (cART) in 1996, the incidence of mortality and initial progress of neurological pathogenesis has been dramatically decreased in HIV-infected patients. However, the inferior penetration of drugs across the blood-brain barrier makes brain a reservoir of HIV and a site of chronic inflammation. AIDS remains the most common cause of dementia in non-aged individuals in the US. Injury to myelin or oligodendrocytes (OLs) would enhance neurologic dysfunction. White matter damage in HIV patients can occur at early stages, prior to myelin pallor, suggesting that OLs/myelin may be direct targets of virus or viral proteins. HIV-1 protein Tat is actively secreted by infected cels in the CNS at both early and late stages of infection. Tat has been reported to induce toxicity and functional changes via calcium (Ca2+) destabilization in neurons, astrocytes and microglia. CaMKII¿, the cellular component of OLs that is central to the coordination of Ca2+ signal transduction, has recently been found to play an important role in OL differentiation and myelination. My central hypothesis is that Tat-induced OL/myelin damage is mediated via excessive Ca2+ influx followed by CaMKII¿ over-activation, which can be attenuated by blocking the interaction between Tat and glutamatergic receptors, or by inhibition of CaMKII¿ activation. To support this hypothesis, our preliminary data show that: 1) short periods of Tat induction in a transgenic mouse caused abnormal morphology and ultrastructure in OLs; 2) cultured murine OLs exposed to 100 nM Tat1-86 have reduced myelin-like membrane extensions; 3) 1 nM - 100 nM Tat1-86 can induce a sustained, dose-dependent increase of [Ca2+]i in OLs; and, 4) OLs under our culture conditions express functional ionotropic glutamatergic receptors (iGluRs). These findings suggest that HIV-1 Tat may injure OLs/myelin directly via [Ca2+]i dysregulation through interaction with iGluRs. Proposed studies investigate if CaMKII¿, the major CaMKII isoform expressed in OLs, is the downstream signaling molecule of Tat-induced [Ca2+]i increase that mediates OL/myelin injury. Aim 1 studies test effects of HIV-1 Tat1-86 on CaMKII¿ activation by assessing changes in CaMKII¿ phosphorylation. Aim 2 studies test whether iGluR antagonists can attenuate CaMKII¿ activation and Tat-induced OL [Ca2+]i increases. Aim 3 studies test whether inhibiting iGluRs or CaMKII¿ activation can alleviate Tat-induced OL/myelin injury. Pharmacological approaches and siRNA interference will be used to inhibit iGluRs and CaMKII¿. A brain cell aggregate model containing all CNS cells in combination with z-stack confocal imaging and 3-D reconstruction will be used to compare myelin structures under different experimental conditions. Levels of typical myelin protein as well as OL process complexity will also be assessed. Since HIV is a human disease, all experiments will be performed on both murine and human OLs. Findings will guide the direction of future studies concerning the basis of OL pathogenesis in HIV patients and benefit development of new therapies to alleviate central nervous system consequences of HIV-1 infection.