ROLE OF CELL CYCLE PROTEINS IN HIV ENCEPHALITIS

描述（由申请人提供）：HIV相关痴呆（HAD）患者的神经元损伤和丢失与CNS的巨噬细胞浸润和神经炎症最密切相关。这些因素中的大多数刺激细胞周期调节机制的变化，即使在没有细胞分裂的情况下，细胞周期调节机制也决定细胞的结果。这使我们提出了以下总体假设：HIV阳性HAD患者的神经元表现出改变的细胞周期蛋白活性，这种活性决定了神经元对HIV感染的巨噬细胞释放的因子的反应。细胞周期机制的诱导通常导致E2 F家族转录因子的转录活性增加，从而导致DNA合成和进展到S期所必需的基因产物的表达。E2 F家族的活性通过与视网膜母细胞瘤肿瘤抑制蛋白pRb及其家族成员的直接相互作用而受到抑制，当pRb过度磷酸化时，这种相互作用被破坏。为了支持我们的假设，我们已经观察到增加E2 F1和过度磷酸化pRb在中枢神经系统的艾滋病毒脑炎患者，在猿猴模型的疾病，并在我们的体外艾滋病毒神经变性模型的神经元。令人惊讶的是，E2 F1定位于细胞质，这与其已知的转录作用不一致。SIVE中E2 F DNA结合活性的改变进一步支持了E2 F功能的改变。E2 F1在神经元中的一致和主要的细胞质定位表明这种蛋白质的新作用。有趣的是，E2 F1已被报道与独特的RNA发夹结构结合，并介导至少一种mRNA种类Axin 2的稳定;然而，对E2 F1功能的这一方面知之甚少。因此，我们假设E2 F1，独立于pRb发挥作用，通过改变核酸结合活性，亚细胞分布和钙蛋白酶裂解在调节HIV感染中的神经变性中起着新的作用。为了验证这一假设，我们提出：1）确定E2 F1核酸结合结构域作为调节神经变性机制的潜在细胞质作用。2)确定钙蛋白酶切割的E2 F1调节E2 F1功能如核酸结合和神经元存活的能力。3)确定E2 F1定位在HIV诱导的神经毒性中的作用。鉴于其在有丝分裂后神经元中潜在的独特功能和调节，我们相信E2 F1可以作为预防HIV相关痴呆中神经元丢失的靶点。公共卫生相关性：HIV相关性痴呆患者表现出细胞周期调节蛋白E2 F1水平升高;然而，与其在分裂细胞中的已知作用相反，E2 F1表现出由神经元中的新机制调节的独特活性。鉴于其在神经元中潜在的独特功能和调节，我们相信E2 F1可以作为预防HIV相关性痴呆中神经元丢失和随后认知能力下降的靶点。

DESCRIPTION (provided by applicant): Neuronal damage and loss in patients with HIV-associated dementia (HAD) is most closely associated with macrophage infiltration and neuroinflammation of the CNS. Most of these factors stimulate changes in cell cycle regulatory machinery which determine cellular outcomes even in the absence of cell division. This has led us to propose the following overarching hypothesis: neurons in HIV-positive patients with HAD exhibit altered cell cycle protein activity and this activity determines neuronal survival in response to factors released by HIV infected macrophages. Induction of cell cycle machinery classically results in increased transcriptional activity of the E2F family of transcription factors leading to the expression of gene products necessary for DNA synthesis and progression to S-phase. Activity of the E2F family is repressed by direct interaction with the Retinoblastoma tumor suppressor protein, pRb, and its family members, an interaction that is disrupted when pRb is hyperphosphorylated. In support of our hypothesis, we have observed increased E2F1 and hyper-phosphorylated pRb in the CNS of patients with HIV encephalitis, in a simian model of disease, and in neurons in our in vitro HIV neurodegeneration model. Surprisingly, E2F1 is localized to the cytoplasm, a site inconsistent with its known transcriptional roles. Altered E2F function is further supported by altered E2F DNA binding activity in SIVE. The consistent and predominant cytoplasmic localization of E2F1 in neurons suggests a novel role for this protein. Interestingly, E2F1 has been reported to bind to a unique RNA hairpin structure and mediate stabilization of at least one mRNA species, Axin 2; however, little is known about this aspect of E2F1 function. We thus hypothesize that E2F1, acting independently of pRb, plays a novel role in modulating neurodegeneration in HIV infection via altered nucleic acid binding activity, subcellular distribution, and calpain cleavage. To test this hypothesis, we propose to: 1) determine the potential cytoplasmic role for the E2F1 nucleic acid binding domain as a mechanism modulating neurodegeneration. 2) determine the ability of calpain-cleaved E2F1 to modulate E2F1 functions such as nucleic acid binding and neuronal survival. 3) determine the role for E2F1 localization in HIV-induced neurotoxicity. Given its potentially unique function and regulation in post-mitotic neurons, we believe E2F1 may serve as a target to prevent neuronal loss in HIV associated dementia. PUBLIC HEALTH RELEVANCE: Patients with HIV-associated dementia exhibit increased levels of the cell cycle regulatory protein, E2F1; however, in contrast to its known roles in dividing cells, E2F1 exhibits distinct activities that are regulated by novel mechanisms in neurons. Given its potentially unique function and regulation in neurons, we believe E2F1 may serve as a target to prevent neuronal loss and subsequent cognitive decline in HIV associated dementia.