Functional roles for E2F1 in neurons: mechanisms of synaptic damage in HIV-associ

hiv相关的神经认知障碍在临床上被定义为由进行性神经元损伤引起的运动、认知和行为缺陷的组合。尽管一系列因素导致HAND中发生的神经退行性变，但导致神经元损伤的确切机制仍不清楚。有趣的是，细胞周期机制，特别是E2F1，与包括HAND在内的许多神经退行性疾病有关。然而，这种细胞周期相关蛋白的生理和病理作用仍然知之甚少。最终，这一新知识将导致一种新的治疗策略，即靶向保存E2F1生理功能，同时阻断其治疗HAND和其他神经退行性疾病的病理活性。本文提供的初步数据表明，E2F1结合RNA并在突触特化中发挥作用，从而提出了E2F1通过其RNA结合活性在有丝分裂后神经元中调节突触特化的具体假说。为了确定E2F1在突触特化中的作用，我们将使用RNA干扰技术或基因敲除动物来去除E2F1，并检测树突树突、脊柱形态发生、PSD95聚类和突触数量的变化。为了确定E2F1 RNA结合活性在神经元中的作用，RNA结合活性将被缺乏RNA结合结构域或含有RNA竞争配体的显性阴性构建体所抑制，并测定神经元活力、靶配体稳定性和突触特化的变化。这些实验的结果将确定E2F1在有丝分裂后神经元中的生理作用及其在HAND中的潜在病理贡献。

HIV-associated neurocognitive disorder is clinically defined by a combination of motor, cognitive, and behavioral deficits that result from progressive neuronal damage. Although a constellation of factors contributes to the neurodegeneration that occurs in HAND, the precise mechanism leading to neuronal damage remains unelucidated. Interestingly, the cell cycle machinery, specifically E2F1, has been implicated in many neurodegenerative diseases including HAND. However, both the physiologic and the pathologic roles of this cell cycle-related protein remain poorly understood. Ultimately, this new knowledge will lead to a novel therapeutic strategy of targeting the preservation of E2F1 physiologic function while simultaneously blocking its pathologic activity for treating HAND and other neurodegenerative diseases. The preliminary data provided in this proposal suggests that E2F1 binds RNA and plays a role in synaptic specialization, thus leading to the specific hypothesis of which E2F1 regulates synaptic specialization through its RNA binding activity in postmitotic neurons. To determine the role of E2F1 in synaptic specialization, E2F1 will be depleted using RNA interference technology or genetic knockout animals and assay for changes in dendritic arborization, spine morphogenesis, PSD95 clustering, and synapse number. To determine the role of E2F1 RNA binding activity in neurons, the RNA binding activity will be inhibited with dominant negative constructs lacking RNA binding domain or containing RNA competitive ligand and assay for changes in neuronal viability, stability of target ligands, and synaptic specializations. The results from these experiments will identify the physiologic role of E2F1 in postmitotic neurons and its potential pathologic contributions in HAND.