PAF: Presynaptic and Postsynaptic Mechanisms of Injury in HAND

描述（由申请人提供）：即使在抗逆转录病毒联合治疗（cART）的存在下，年轻人和老年人的hiv相关神经认知障碍（HAND）仍然是一个巨大的和日益增长的关注。尽管cART可以抑制病毒复制，但神经系统内仍会产生TAT和gp120等病毒蛋白，它们具有神经毒性并刺激炎症（1,2）。HIV诱导的神经炎症被认为是大约50%的HIV阳性个体表现出认知能力下降的一个主要因素（3,4），但是关于这种持续认知能力下降的分子和细胞机制的更多信息仍然需要解决。血小板活化因子（PAF）是一种炎症介质，也可以改变神经传递（5-8）。与没有认知障碍的HIV阳性个体相比，HAND患者的慢性高水平存在(9)。在HAND和其他神经炎性疾病的小鼠模型中，PAF受体（PAFR）拮抗已被证明具有神经保护作用（10-17）。因此，PAF可能是与神经炎症和HAND相关的神经元损伤的主要贡献者，因为它具有调节突触强度的能力。然而，关于突触中PAF信号传导的机制，我们还不清楚，包括它主要发生在突触前还是突触后室，还是两者都发生。我假设慢性PAF暴露增加神经传递，通过作用于突触前和突触后隔室导致神经元损伤。增强的神经递质释放和增强的信号接收使突触处于高活动状态，即使存在生理水平的刺激，它们也容易受到兴奋毒性损伤和能量衰竭。我将使用体外和体内技术来验证这一假设。在Aim 1中，我将首先确定PAFR在突触前和/或突触后区室中的精确定位。然后，我将研究PAF诱导的突触囊泡组织的变化以及调节突触前腔室内突触囊泡释放的蛋白质。在Aim 2中，我将测试突触后腔室中的PAF信号是否对神经元损伤是必要的或充分的。我将进一步研究PAF信号是否会改变NMDA和AMPA受体的转运或激活状态，从而改变突触后细胞接收的信号强度。在Aim 3中，我将使用尖端技术，包括使用皮质窗和光遗传学的活体多光子显微镜，在体内和HAND模型中确定PAF诱导的突触损伤的急性和长期影响。这些研究的结果验证了PAFR信号通路作为治疗HAND和其他神经炎性疾病的可行靶点，并增加了我们对导致HAND患者认知能力下降的分子机制的理解。

DESCRIPTION (provided by applicant): HIV-associated neurocognitive disorders (HAND) for both the young and aging population remain a huge and growing concern even in the presence of combination antiretroviral therapy (cART). Although viral replication is inhibited by cART, vira proteins such as TAT and gp120 are still produced within the nervous system which are neurotoxic and stimulate inflammation (1, 2). HIV induced neuroinflammation is thought to be a major contributing factor in the cognitive decline exhibited by roughly 50% of HIV-positive individuals (3, 4), but more information on the molecular and cellular mechanisms relevant to this persistent cognitive decline still needs to be addressed. Platelet activating factor (PAF) is n inflammatory mediator that can also alter neurotransmission (5-8). It is present at chronically high levels in patients with HAND as compared to HIV positive individuals without cognitive impairments (9). PAF receptor (PAFR) antagonism has been shown to be neuroprotective in mouse models of HAND and other neuroinflammatory diseases (10-17). Thus, PAF may be a major contributor to the neuronal injury associated with neuroinflammation and HAND because of its ability to modulate synaptic strength. Yet, there is much about the mechanisms of PAF signaling at the synapse that we do not understand including whether it occurs primarily in the presynaptic or postsynaptic compartment or both. I hypothesize that chronic PAF exposure increases neurotransmission leading to neuronal injury by acting on both the pre- and postsynaptic compartments. Enhanced neurotransmitter release and enhanced signal reception places synapses in a high activity state where they are vulnerable to excitotoxic injury and energy failure even in the presence of physiologic levels of stimulation. I will test this hypothess using both in vitro and in vivo techniques. In Aim 1, I will first determining the precise localizaion of the PAFR within the presynaptic and/or postsynaptic compartments. Then I will investigate PAF induced changes to synaptic vesicle organization and the proteins that regulate synaptic vesicle release within the presynaptic compartment. In Aim 2, I will test whether PAF signaling within the postsynaptic compartment is necessary or sufficient for neuronal injury. I will further investigate whether PAF signaling alters NMDA and AMPA receptor trafficking or activation state that would change the signal strength received by the postsynaptic cell. In Aim 3, I will determine the acute and long-term effects of PAF- induced synaptic injury in vivo and in a model of HAND using cutting-edge techniques including intravital multiphoton microscopy using cortical windows and optogenetics. Results from these studies should validate the PAFR signaling pathway as a viable target for the treatment of HAND and other neuroinflammatory diseases as well as increase our understanding of the molecular mechanisms leading to the cognitive decline in HAND patients.