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: 8436287
• 负责人: Jacob Zyskind
• 金额: $ 4.19万
• 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/8436287
• 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; public health relevance; 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.

描述（申请人提供）：hiv相关神经认知障碍（HAND）是一种由认知、运动和行为缺陷组成的神经系统综合征。HAND的神经元损失是由hiv感染/激活的单核细胞来源的巨噬细胞浸润中枢神经系统引起的。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的生存能力。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E2F1的表达增加，已知其激活细胞周期G1期到S期进展所需的基因转录。该项目提案旨在确定E2F1在hiv介导的神经元丢失中的作用，其长期目标是制定一种涉及阻断E2F1病理功能的HAND治疗策略。这一建议的初步数据表明，在HIV感染后的原代神经元培养中，E2F1被calpain切割。此外，在原代培养的神经元中，抑制calpain激活和删除E2F1都能减轻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水平降低和促死亡蛋白p73的积累。这些结果表明，劈裂的神经元E2F1可能通过破坏促存活蛋白MDMx和/或稳定促死亡蛋白p73，在hiv诱导的神经元死亡中发挥了重要作用。为了确定被切割的E2F1是否与hiv诱导的神经毒性有关，将使用免疫沉淀方法结合蛋白测序和质谱分析来鉴定E2F1中的calpain切割位点。用脉冲追踪法比较裂解后的E2F1蛋白与全长E2F1蛋白的稳定性。然后使用慢病毒方法用编码裂解E2F1序列的构建体感染原代皮质培养物，允许随后通过基于MAP2细胞的ELISA和感染细胞的手计数分析裂解E2F1表达对神经元活力的影响。为了确定MDMs和p73稳定性的观察变化是否由于E2F1的切割，将使用脉冲追踪方法比较野生型和E2F1-/-皮质培养物中每种蛋白的稳定性。MDMx-E2F1结合对calpain激活的敏感性将通过利用calpain药理抑制剂的共免疫沉淀方法进行评估。最后，MDMx和p73蛋白水平改变对HIV诱导的神经毒性的贡献将通过评估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。总之，这些实验的结果将确定calpain-cleaved E2F1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。