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

描述（由申请方提供）：长期目标是表征一种称为PINCH的宿主衍生蛋白促进神经元适应性以响应CNS HIV感染的机制，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。突触树突损伤与HIV患者认知障碍的存在和严重程度密切相关。由于突触树突状细胞损伤在HIV感染的大脑是部分可逆的，可以在没有显着的神经元损失的情况下发生，从这些研究中获得的信息将提高我们的能力，以改善治疗HIV感染的大脑和减轻神经系统并发症在许多HIV患者中观察到的治疗。在HIV感染者的大脑中，PINCH仅由显示突触树突损伤迹象的神经元表达。PINCH是一种衔接蛋白，通过与整合素连接激酶（ILK）和Nck 2的相互作用介导细胞外基质和细胞内网络途径之间的双向信号转导。因此，我们的总体假设是，在响应于由CNS的HIV感染引起的神经元信号传导的破坏时，PINCH蛋白通过类小泛素化来稳定，以促进与ILK和Nck 2的适当相互作用。我们提出了两个目标，调查在体外I）PINCH表达的调节机制，和II）暴露于病毒蛋白质和宿主炎症因子的CNS HIV感染过程中产生的神经元上的PINCH表达的生物学后果。将通过qRT-PCR和北方和西方分析以及通过siRNA敲低来评估用TNF-α或达特处理以模拟脑的HIV感染的某些方面的原代神经元的PINCH表达。在暴露于TNF α和/或达特后，通过相互IP和Western分析评估PINCH的类小泛素化。我们进一步假设PINCH与ILK和Nck 2的正常通讯中断可能会减少神经元的恢复。将评估暴露于TNF α和/或达特的神经元的ILK和Nck 2表达以及与PINCH的功能相互作用。为了解决CNS HIV感染背景下PINCH的生物学后果，将使用定点诱变、PINCH蛋白蓄积和MALDI-TOF分析。将通过神经元的激光捕获显微切割和MALDI-TOF分析，对患有MCMD、HIVE、痴呆和重要的HAND（不存在HIVE）的HIV患者的大脑以及死亡但无CNS并发症的HIV患者进行扩展研究。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要长得多。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，许多艾滋病患者患有神经损伤。在这种情况下，PINCH蛋白可能有助于HIV感染大脑期间的神经元恢复。了解PINCH功能的机制将有助于改善治疗方法，以保护大脑免受HIV的损害，并可能改善HIV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.