ENDOGENOUS NEURONAL REPAIR MECHANISMS IN SIV-INFECTED MACAQUES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 GAP-43(神经调制素)是突触前膜的一种成分，是轴突生长锥的主要蛋白质。它的主要功能包括轴突生长、轴突寻路、突触可塑性和调节神经递质的释放。GAP-43的表达已被证明在发育、神经元可塑性和神经元死亡过程中上调，并已通过EAE动物模型、阿尔茨海默病和中风被用于衡量各种慢性炎症性疾病如多发性硬化症的功能轴突恢复。GAP-43的表达也被用来测试抗逆转录病毒诱导的大鼠周围神经病的发病机制以及神经保护剂对大鼠背根神经节的可能影响。 本研究的目的是用标记评估突触前终末(GAP-43和SYN)和突触后膜(MAP-2)来评估与猴免疫缺陷病毒(SIV)脑炎相关的神经元和突触损伤。SIV已被公认为HIV脑炎(HIVE)的模型。研究还表明，SIVE的严重程度与突触素和MAP-2表达的降低以及神经元代谢的标志物N-乙酰天冬氨酸/肌酸比值(NAA/Cr)的降低呈负相关。采用免疫组织化学方法检测额叶皮质(FC)和海马区(HI)生长相关蛋白43(GAP-43)的表达。 我们之前已经证明，CD8缺失的SIV感染猕猴的NAA/Cr下降在28天的抗逆转录病毒治疗后被逆转，这与CD68+SIV感染的血管周围巨噬细胞从脑中几乎完全清除有关。我们推测，治疗猕猴NAA正常化的部分原因是突触前膜丢失的逆转，这将表现为突触素的增加。我们还假设GAP-43将被上调为CART的修复机制。我们在这里证明，虽然突触素的丢失在CART的这一时间框架内不能逆转，GAP-43被上调，但这种上调发生在最初的SIV感染时，并且不被CART增强。GAP-43的增加代表了一种内源性修复机制，这种机制发生在最初的损伤中，并可能作为SIV模型中潜在的修复标志。 我们还证明了在SIV感染过程中激活的小胶质细胞和浸润性巨噬细胞表达轴突引导分子ePhin B3。Ephin B3是EphA4和EphB3的配体，参与突触和树突的形成。它不仅能调节神经元的抗凋亡活性，而且还能抑制轴突生长，这是一种维持轴突稳定的机制。虽然ewitin B3可以通过排斥来调节轴突的萌发，但它的作用与GAP-43的诱导有关。在大脑中的巨噬细胞和小胶质细胞中发现了eparin B3并不是没有预料到的。肾上腺素B3及其受体在外周T细胞和单核/巨噬细胞中表达。这可能提供了一种机制，即巨噬细胞和小胶质细胞可以调节神经元和星形胶质细胞的功能，因为这两种细胞都表达EphA4和EphB3受体。 尽管短期CART治疗动物的NAA/Cr正常化证明了神经元代谢的显著改善，但感染SIV的未经治疗的猴子和接受CART治疗的猴子之间的GAP-43表达没有显著差异。我们推测，尽管在本研究中接受CART的动物中观察到脑病毒负担显著降低，但仍有持续的损伤(和修复尝试)继续诱导GAP-43的表达。我们观察到，在处理的动物(L.Annamalai个人交流)中，CART在显著减少脑病毒负担方面存在不匹配，但小胶质细胞激活和神经毒性因子TNFa的持续水平。与促进神经适应过程的因素的诱导和抑制相比，改善参数的不匹配，神经元代谢和GAP-43/EPhin B3，也可能反映了代谢改善的时间进程的不同。 GAP-43mRNA是由神经生长因子(NGF)诱导的，神经生长因子依赖于Mst3b，Mst3b是一种神经元特异性激酶，对轴突生长起中心调节作用。肾上腺素作为促进或阻碍神经元修复机制的因子的作用存在争议，但很明显，肾上腺素参与了神经元与星形胶质细胞和小胶质细胞之间复杂的通讯。我们提出了一个模型，在该模型中，病毒感染激活的小胶质细胞刺激星形胶质细胞分泌NGF，NGF与神经元上的TrkA受体结合，从而诱导神经保护信号级联，导致GAP-43的诱导和增加mRNA的稳定性。除了肾上腺素B3和它们的受体之外，其他的肾上腺素可能参与了这个多细胞激活和信号通路。这些细胞外轴突生长分子、它们的受体或下游信号元件的治疗调节可能会增强成年中枢神经系统中轴突的萌发和再生。成年SIV感染猕猴模型将有助于探索与突触可塑性相关的其他基因和肾上腺素，以及进一步表征内源性神经再生潜力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. GAP-43 (neuromodulin) is a component of the presynaptic membrane that is the major protein of axonal growth cones. Its primary functions include axonal growth, axonal pathfinding, synaptic plasticity, and regulating neurotransmitter release. GAP-43 expression has been shown to be up-regulated during development, neuronal plasticity and neuronal death, and it has been used to measure functional axonal recovery in various chronic inflammatory diseases such as multiple sclerosis via the EAE animal model, Alzheimer's disease, and stroke. GAP-43 expression has also been used to test the pathogenesis of antiretroviral-induced peripheral neuropathy and possible effects of neuroprotective drugs in the dorsal root ganglion in rats. The purpose of this study has been to assess neuronal and synaptic damage associated with simian immunodeficiency virus (SIV) encephalitis using markers to evaluate the pre-synaptic terminals (GAP-43 and SYN) and post-synaptic membranes (MAP-2). SIV has been well established as a model for HIV encephalitis (HIVE). It has also been documented that increased severity of SIVE inversely correlates with decreased synaptophysin and MAP-2 expression and with decreased N-acetylaspartate/creatine ratio (NAA/Cr), a marker of neuronal metabolism. Immunohistochemical examination of GAP-43 protein expression in frontal cortex (FC) and hippocampus (HI) was undertaken was evaluated in FC and HI from a cohort of CD8-depleted SIVmac251-infected rhesus with and without combined antiretroviral therapy (CART). We had previously demonstrated that NAA/Cr decline in CD8-depleted SIV-infected macaques is reversed after 28 days of antiretroviral therapy associated with near complete clearance of CD68+ SIV-infected perivascular macrophages from the brain. We hypothesized that part of the cause of the normalization of NAA in treated macaques was reversal of presynaptic membrane loss that would be represented by increased synaptophysin. We also hypothesized that GAP-43 would be upregulated as a repair mechanism with CART. We demonstrate here that although synaptophysin loss is not reversed in this time frame with CART, GAP-43 is upreglulated, but the upregulation occurs with initial SIV infection and is not augmented with CART. Increased GAP-43 represents an endogenous repair mechanism that occurs with initial injury and may serve as a potential marker of repair in the SIV model. We have also demonstrated that microglia and infiltrating macrophages activated during SIV-infection express the axonal guidance molecule ephrin B3. Ephrin B3 is a ligand for EphA4 and EphB3 and is involved in synapse and dendritic spine formation. It mediates demonstrated anti-apoptotic activity in neurons, but also functions as an inhibitor of axonal growth, a mechanism to maintain axonal stability. Although ephrin B3 can regulate axonal sprouting through repulsion, its action has been associated with induction of GAP-43. The discovery of ephrin B3 in macrophages and microglia in the brain was not unanticipated. Ephrin B3 and its receptors are expressed in peripheral T cells and monocytes/macrophages. This may provide a mechanism whereby macrophages and microglia could modulate neuron and astrocyte function, as both cell types express the ephrin B3 receptors, EphA4 and EphB3. Despite the documented significant improvement in neuronal metabolism with short-term CART evidenced by normalization of NAA/Cr in the treated animals, there was no significant difference in GAP-43 expression between SIV-infected untreated monkeys and those that received CART. We speculate that there is ongoing injury (and attempts at repair) that continues to induce GAP-43 expression despite the marked reduction in brain virus burden that is observed in the animals that received CART in this study. We observe a mismatch in marked reduction of brain virus burden with CART, but persistent levels of microglial activation and TNFa, a neurotoxic factor, in the treated animals (L. Annamalai personal communication). The mismatch in parameters of improvement, neuronal metabolism and GAP-43/ephrin B3, may also reflect differences in the time course of metabolic improvement, compared with the time course of induction and suppression of factors that promote neuroadaptive processes. GAP-43 mRNA is induced by nerve growth factor (NGF), which is dependent on Mst3b, a neuron-specific kinase central to regulation of axonal outgrowth. The role of ephrins as factors that can promote or hinder neuronal repair mechanisms is controversial, but it is clear that ephrins are involved in the intricate communication of neurons with astrocytes and microglia. We propose a model in which microglia activated by virus infection (and ephrin B3 expression) stimulate astrocytes to secrete NGF, which binds TrkA receptors on neurons, thus inducing neuroprotective signaling cascades that result in the induction and increased mRNA stability of GAP-43. Other ephrins in addition to ephrin B3 and their receptors are likely to be involved in this multicellular activation and signaling pathway. Therapeutic modulation of these extracellular axonal growth molecules, their receptors, or downstream signaling elements may augment axonal sprouting and regeneration in the adult CNS. The adult SIV-infected macaque model will be useful for exploring additional genes and ephrins that are associated with synaptic plasticity as well as for further characterization of endogenous neuroregenerative potential.