Role of PINCH in neuronal response to HIV infection of the CNS

描述(申请人提供)：长期目标是描述一种名为Pinch的宿主衍生蛋白在应对HIV感染中枢神经系统时促进神经元健康的机制，总体目标是了解Pinch在暴露于病毒和炎症因子的神经元中的作用。突触树突触损伤与HIV患者认知损害的存在和严重程度密切相关。由于HIV感染对大脑的突触树突起损伤是部分可逆的，在没有明显神经元丢失的情况下可以发生，从这些研究中获得的信息将增强我们改进治疗HIV感染大脑的治疗方法的能力，并减少在许多HIV患者中观察到的神经并发症。在HIV感染者的大脑中，PINCH仅由出现突触树突损伤迹象的神经元表达。PINCH是一种适配器蛋白，通过与整合素连接激酶(ILK)和NKK2的相互作用，介导细胞外基质和细胞内网络通路之间的双向信号转导。因此，我们的总体假设是，为了应对HIV感染CNS导致的神经元信号转导中断，Pinch蛋白通过苏莫化作用稳定下来，以促进与ILK和Nck 2的适当相互作用。我们提出了两个目标，在体外研究1)调控PINCH表达的机制，以及II)在HIV感染中枢神经系统过程中，暴露于病毒蛋白和宿主炎症因子的神经元上PINCH表达的生物学后果。用肿瘤坏死因子-a或TAT处理的原代神经元模拟HIV感染大脑的某些方面，将通过qRT-PCR、Northern和Western分析以及siRNA敲除来评估Pinch的表达。在暴露于TNFa和/或TAT后，将通过相互的IP和Western分析来评估Pinch的SUMOA化。我们进一步假设，Pinch与ILK和NKK2正常通讯的中断可能会削弱神经元的恢复。暴露于TNFa和/或TAT的神经元将被评估ILK和Nock2的表达以及与Pinch的功能相互作用。在HIV感染中枢神经系统的背景下，为了解决PIPCH的生物学后果，将使用定点突变、PINCH蛋白积累和MALDI-TOF分析。将通过对神经元的激光捕获显微解剖和MALDI-TOF分析，对患有mcmd、蜂窝、痴呆(更重要的是在没有蜂巢的情况下患有手部)的艾滋病毒患者的大脑以及没有中枢神经系统并发症而死亡的艾滋病毒患者的大脑进行扩展研究。公共卫生相关性：由于有效的抗艾滋病毒药物，艾滋病毒患者的寿命大大延长。抗艾滋病毒药物在治疗大脑中的艾滋病毒方面效果较差。因此，许多艾滋病毒患者患有神经损伤。在此背景下，PINCH蛋白可能有助于HIV感染大脑期间神经元的恢复。了解Pinch功能将有助于改进治疗方法以保护大脑免受艾滋病毒损害的机制，并可能改善艾滋病毒患者的生活质量。

DESCRIPTION (provided by applicant): The long-term objective is to characterize mechanisms by which a host-derived protein called PINCH promotes neuronal fitness in response HIV infection of the CNS with the overall goal to understand PINCH's role in neurons exposed to viral and inflammatory factors. Synaptodendrtic injury correlates closely with the presence and severity of cognitive impairment in HIV patients. Since synaptodendritic damage in HIV infection of the brain is partially reversible and can occur in the absence of significant neuronal loss, information gained from these studies will enhance our ability to improve therapies to treat HIV infection of the brain and alleviate neurological complications observed in many HIV patients. In the brains of HIV infected individuals, PINCH is expressed exclusively by neurons showing signs of synaptodendritic damage. PINCH is an adapter protein that mediates bidirectional signal transduction between the extracellular matrix and intracellular networking pathways via interactions with integrin linked kinase (ILK) and Nck2. Thus, our overall hypothesis is that in response to disruptions in neuronal signaling caused by HIV infection of the CNS, PINCH protein is stabilized by sumoylation to promote proper interactions with ILK and Nck2. We propose two AIMS that investigate in vitro I) mechanisms by which PINCH expression is regulated, and II) the biological consequences of PINCH expression on neurons exposed to viral protein and host inflammatory factors produced during HIV infection of the CNS. Primary neurons treated with TNF-a or Tat to mimic some aspects of HIV infection of the brain will be assessed for PINCH expression via qRT-PCR, and Northern and Western analyses, and by siRNA knockdown. Sumoylation of PINCH will be assessed by reciprocal IP and Western analyses after exposure to TNFa and/or Tat. We further hypothesize that disruptions in PINCH's normal communication with ILK and Nck2 may diminish neuronal recovery. Neurons exposed to TNFa and/or Tat will be assessed for ILK and Nck2 expression and functional interactions with PINCH. To address biological consequences of PINCH in the context of HIV infection of the CNS, site-directed mutagenesis, PINCH protein accumulation and MALDI-TOF analyses will be used. Expanded studies of HIV patients' brains with MCMD, HIVE, dementia and importantly HAND in the absence of HIVE as well as HIV patients who died without CNS complications will be analyzed via laser capture microdissection of neurons and MALDI-TOF analyses will also be conducted. PUBLIC HEALTH RELEVANCE: RELEVANCE HIV patients are much living longer due to effective anti-HIV medications. Anti-HIV medications are less effective at treating HIV in the brain. So, many HIV patients suffer from neurological impairments. In this context, PINCH protein may contribute to neuron recovery during HIV infection of the brain. Understanding mechanisms by which PINCH functions will contribute to improved therapies to protect the brain from damage by HIV, and may improve the quality of life in HIV patients.