Investigating the role of the cell cycle protein E2F1 in HIV-induced neurotoxicit

研究细胞周期蛋白 E2F1 在 HIV 诱导的神经毒性中的作用

• 批准号: 8140804
• 负责人: Jacob Zyskind
• 金额: $ 4.18万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8140804
• 关键词: AIDS Dementia Complex; Alzheimer' s Disease; Amino Acid Sequence; Apoptosis; Attenuated; Behavioral; Binding; Brain; Calcium; Calpain; Cell Cycle; Cell Cycle Progression; Cell Cycle Proteins; Cell Death; Cell division; Cells; Cessation of life; Cleaved cell; Co-Immunoprecipitations; Cognitive; Collection; Complex; Cysteine Protease; Cytosol; DNA Binding Domain; Data; Disease; E2F Transcription Factor 1; E2F1 gene; Environment; Enzyme-Linked Immunosorbent Assay; Exhibits; Gene Targeting; Genetic Transcription; Goals; HIV; Hand; Huntington Disease; Immunoprecipitation; Impaired cognition; In Vitro; Infiltration; Lead; Length; Mass Spectrum Analysis; Mediating; Messenger RNA; Microglia; Mitotic; Modeling; Molecular Weight; Motor; Nerve Degeneration; Neuraxis; Neurocognitive; Neurodegenerative Disorders; Neurologic; Neurons; Parkinson Disease; Pathologic; Patients; Peptide Sequence Determination; Physiologic pulse; Play; Proliferating; Protein Isoforms; Proteins; Role; S Phase; Signal Transduction; Site; Small Interfering RNA; Syndrome; Testing; Therapeutic; Work; base; extracellular; in vitro Model; inhibitor/antagonist; macrophage; monocyte; neuron loss; neurotoxic; neurotoxicity; novel therapeutics; overexpression; prevent; protein p73; research study; response; therapeutic target; transcription factor

DESCRIPTION (provided by applicant): HIV-associated neurocognitive disorder (HAND) is a neurological syndrome consisting of cognitive, motor and behavioral deficits. Neuronal loss in HAND follows the infiltration of the central nervous system by HIV-infected/activated monocytes-derived macrophages. These macrophages and subsequently activated resident microglia secrete a collection of soluble factors that alter the extracellular environment and determine neuronal viability. The neuronal response to this complex signaling environment involves activation of the cell cycle regulatory machinery, notably increased expression of the transcription factor E2F1, which is known to activate transcription of genes necessary for G1 to S phase progression of the cell cycle. This project proposal aims to determine the role of E2F1 in HIV-mediated neuronal loss with the long-term goal of producing a therapeutic strategy for HAND that involves blocking E2F1 pathological function. Preliminary data for this proposal shows that E2F1 is cleaved by calpain in primary neuronal cultures following an HIV insult. Furthermore, both inhibition of calpain activation and deletion of E2F1 attenuate HIV-induced neuronal death in primary neuronal cultures. Finally, treatment of primary neurons with HIV insult leads to reduced levels of pro-survival protein MDMx and accumulation of pro-death protein p73. These results suggest the hypothesis that cleaved neuronal E2F1 plays a contributing role in HIV-induced neuronal death, possibly by destabilizing the pro-survival protein MDMx and/or stabilizing the pro-death protein p73. To determine whether cleaved E2F1 contributes to HIV-induced neurotoxicity, the calpain cleavage site in E2F1 will be identified using an immunoprecipitation approach combined with protein sequencing and mass spectrometry analyses. Stability of cleaved E2F1 protein will be compared to stability of full length E2F1 by pulse-chase. A lentiviral approach will then be used to infect primary cortical cultures with a construct encoding the cleaved E2F1 sequence, allowing for subsequent analysis of the effects of cleaved E2F1 expression on neuronal viability by a MAP2 cell-based ELISA and hand counts of infected cells. To determine whether observe alterations in MDMs and p73 stability are due to cleaved E2F1, stability of each protein will be compared between wildtype and E2F1-/- cortical cultures treated with HIV insult using a pulse-chase approach. Sensitivity of MDMx-E2F1 binding to calpain activation will be assessed through a co-immunoprecipitation approach utilizing pharmacological inhibitors to calpain. Finally, the contribution of MDMx and p73 protein level alterations to HIV- induced neurotoxicity will be determined by assessing the effects of pharmacological inhibition of p73 and siRNA-mediated knockdown of MDMx on HIV-induced neuronal death. Together, the results from these experiments will define the role of calpain-cleaved E2F1 in HIV-induced neurotoxicity and indicate its potential as a therapeutic target in HAND. PUBLIC HEALTH RELEVANCE: Patients with HIV-associated dementia exhibit increased expression of cell cycle-related protein E2F1. However, its pathologic role in neurodegeneration is unclear. We believe that defining its contribution to neuronal loss will lead to novel therapeutic strategy of targeting E2F1 to prevent the neuronal damage and subsequent cognitive decline characterized by HIV-associated dementia.

描述（由申请人提供）：HIV相关神经认知障碍（HAND）是一种神经系统综合征，包括认知、运动和行为缺陷。HIV感染/活化的单核细胞衍生的巨噬细胞浸润中枢神经系统后，HAND中的神经元丢失。这些巨噬细胞和随后激活的常驻小胶质细胞分泌一系列可溶性因子，这些因子改变细胞外环境并决定神经元活力。神经元对这种复杂信号环境的反应涉及细胞周期调节机制的激活，特别是转录因子E2 F1的表达增加，已知转录因子E2 F1激活细胞周期的G1至S期进展所必需的基因的转录。该项目提案旨在确定E2 F1在HIV介导的神经元损失中的作用，其长期目标是为HAND产生一种治疗策略，包括阻断E2 F1病理功能。该提议的初步数据显示，E2 F1在HIV损伤后的原代神经元培养物中被钙蛋白酶切割。此外，抑制钙蛋白酶激活和E2 F1的缺失都能减弱原代神经元培养物中HIV诱导的神经元死亡。最后，用HIV损伤处理原代神经元导致促存活蛋白MDMx的水平降低和促死亡蛋白p73的积累。这些结果表明，裂解的神经元E2 F1在HIV诱导的神经元死亡中起着重要作用，可能是通过使促生存蛋白MDMx不稳定和/或使促死亡蛋白p73稳定。为了确定切割的E2 F1是否有助于HIV诱导的神经毒性，将使用免疫沉淀方法结合蛋白质测序和质谱分析来鉴定E2 F1中的钙蛋白酶切割位点。通过脉冲追踪将切割的E2 F1蛋白的稳定性与全长E2 F1的稳定性进行比较。然后使用慢病毒方法用编码切割的E2 F1序列的构建体感染原代皮质培养物，允许随后通过基于MAP 2细胞的ELISA和感染细胞的手动计数分析切割的E2 F1表达对神经元活力的影响。为了确定观察到的MDM和p73稳定性的改变是否是由于切割的E2 F1引起的，将使用脉冲追踪方法在野生型和用HIV损伤处理的E2 F1-/-皮质培养物之间比较每种蛋白质的稳定性。MDMx-E2 F1结合钙蛋白酶激活的敏感性将通过免疫共沉淀法利用钙蛋白酶的药理学抑制剂进行评估。最后，将通过评估p73的药理学抑制和siRNA介导的MDMx敲低对HIV诱导的神经元死亡的影响来确定MDMx和p73蛋白水平变化对HIV诱导的神经毒性的贡献。总之，这些实验的结果将确定钙蛋白酶裂解的E2 F1在HIV诱导的神经毒性中的作用，并表明其作为HAND治疗靶点的潜力。 公共卫生相关性：HIV相关性痴呆患者表现出细胞周期相关蛋白E2 F1表达增加。然而，其在神经变性中的病理作用尚不清楚。我们相信，确定其对神经元损失的贡献将导致靶向E2 F1的新的治疗策略，以防止神经元损伤和随后的认知功能减退，其特征在于HIV相关性痴呆。