ENDOGENOUS NEURONAL REPAIR MECHANISMS IN SIV-INFECTED MACAQUES

SIV 感染猕猴的内源性神经修复机制

• 批准号: 8358008
• 负责人: Susan V. Westmoreland
• 金额: $ 6.84万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8358008
• 关键词: Adult; Alzheimer' s Disease; Animal Model; Animals; Anti-Retroviral Agents; Apoptotic; Astrocytes; Binding; Brain; CD8B1 gene; Cessation of life; Chronic; Communication; Creatine; Dendritic Spines; Development; Disease; Elements; Encephalitis; EphA4 Receptor; Ephrin-B3; Ephrins; Experimental Autoimmune Encephalomyelitis; Funding; Genes; Grant; Growth Associated Protein 43; Growth Cones; HIV encephalitis; Hippocampus (Brain); Inflammatory; Injury; Ligands; Macaca; Macaca mulatta; Measures; Mediating; Membrane; Messenger RNA; Metabolic; Metabolism; Microglia; Modeling; Monkeys; Multiple Sclerosis; N-acetylaspartate; National Center for Research Resources; Nerve Growth Factors; Neuronal Plasticity; Neurons; Neuroprotective Agents; Neurotrophic Tyrosine Kinase Receptor Type 1; New England; Pathogenesis; Peripheral; Peripheral Nervous System Diseases; Personal Communication; Phosphotransferases; Presynaptic Terminals; Primates; Principal Investigator; Process; Proteins; Rattus; Recovery; Regulation; Research; Research Infrastructure; Resources; Role; SIV; Severities; Signal Pathway; Signal Transduction; Source; Spinal Ganglia; Stroke; Synapses; Synaptic Membranes; Synaptic plasticity; Synaptophysin; T-Lymphocyte; Testing; Therapeutic; Time; United States National Institutes of Health; Up-Regulation; Virus; Virus Diseases; antiretroviral therapy; axon growth; axon regeneration; axonal guidance; axonal pathfinding; axonal sprouting; base; cell type; cohort; cost; extracellular; frontal lobe; inhibitor/antagonist; mRNA Stability; macrophage; monocyte; neurotoxic; neurotransmitter release; presynaptic; protein expression; receptor; repaired

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。对子项目和子项目主要研究者的主要支持可能是由其他来源提供的，包括其他 NIH 来源。 子项目列出的总成本可能代表子项目使用的中心基础设施的估计金额，而不是 NCRR 拨款向子项目或子项目工作人员提供的直接资金。 GAP-43（神经调节蛋白）是突触前膜的一种成分，是轴突生长锥的主要蛋白质。其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释放。 GAP-43 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，并且已通过 EAE 动物模型用于测量多种慢性炎症性疾病（例如多发性硬化症、阿尔茨海默病和中风。 GAP-43 表达也已用于测试抗逆转录病毒引起的周围神经病变的发病机制以及神经保护药物对大鼠背根神经节的可能作用。 本研究的目的是使用标记物评估突触前末梢（GAP-43 和 SYN）和突触后膜（MAP-2）来评估与猿猴免疫缺陷病毒（SIV）脑炎相关的神经元和突触损伤。 SIV 已被确立为 HIV 脑炎 (HIVE) 的模型。还有文献记载，SIVE 严重程度的增加与突触素和 MAP-2 表达的减少以及神经元代谢标志物 N-乙酰天冬氨酸/肌酸比值 (NAA/Cr) 的减少呈负相关。对一组 CD8 耗尽的 SIVmac251 感染恒河猴（接受或不接受联合抗逆转录病毒治疗 (CART)）的 FC 和 HI 中的额叶皮层 (FC) 和海马 (HI) 中的 GAP-43 蛋白表达进行免疫组织化学检查。 我们之前已经证明，经过 28 天的抗逆转录病毒治疗后，CD8 耗尽的 SIV 感染的猕猴中 NAA/Cr 的下降得到逆转，这与大脑中 CD68+ SIV 感染的血管周围巨噬细胞几乎完全清除有关。 我们推测，经过治疗的猕猴 NAA 正常化的部分原因是突触前膜损失的逆转，这表现为突触素的增加。我们还假设 GAP-43 将作为 CART 的修复机制上调。 我们在此证明，虽然在 CART 的作用下，突触素损失并未逆转，但 GAP-43 上调，但这种上调发生在初始 SIV 感染时，并且 CART 不会增强这种上调。 GAP-43 的增加代表了初始损伤时发生的内源性修复机制，可能作为 SIV 模型中潜在的修复标志物。 我们还证明，SIV 感染期间激活的小胶质细胞和浸润巨噬细胞表达轴突引导分子肝配蛋白 B3。 Ephrin B3 是 EphA4 和 EphB3 的配体，参与突触和树突棘的形成。 它介导神经元中已证实的抗凋亡活性，但也可作为轴突生长的抑制剂，这是维持轴突稳定性的机制。尽管肝配蛋白 B3 可以通过排斥作用调节轴突萌芽，但其作用与 GAP-43 的诱导有关。 在大脑巨噬细胞和小胶质细胞中发现肝配蛋白 B3 并非出乎意料。 Ephrin B3 及其受体在外周 T 细胞和单核细胞/巨噬细胞中表达。这可能提供了一种机制，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，因为两种细胞类型都表达肝配蛋白 B3 受体 EphA4 和 EphB3。 尽管经治疗的动物中 NAA/Cr 正常化证明短期 CART 可以显着改善神经元代谢，但感染 SIV 的未治疗猴子和接受 CART 的猴子之间 GAP-43 表达没有显着差异。 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荷显着减少，但持续的损伤（以及修复尝试）继续诱导 GAP-43 表达。 我们观察到，在接受治疗的动物中，CART 显着降低了脑病毒负荷，但小胶质细胞激活和 TNFa（一种神经毒性因子）水平持续存在（L. Annamalai 个人通讯）。 改善参数、神经元代谢和 GAP-43/肝配蛋白 B3 的不匹配也可能反映了代谢改善的时间过程与促进神经适应过程的因子的诱导和抑制的时间过程的差异。 GAP-43 mRNA 由神经生长因子 (NGF) 诱导，神经生长因子依赖于 Mst3b，这是一种对轴突生长调节至关重要的神经元特异性激酶。 肝配蛋白作为促进或阻碍神经元修复机制的因素的作用存在争议，但很明显肝配蛋白参与神经元与星形胶质细胞和小胶质细胞的复杂通讯。 我们提出了一种模型，其中病毒感染激活的小胶质细胞（和肝配蛋白 B3 表达）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反应，从而诱导 GAP-43 并增加 mRNA 稳定性。 除肝配蛋白 B3 及其受体外，其他肝配蛋白及其受体也可能参与这种多细胞激活和信号传导途径。 这些细胞外轴突生长分子、其受体或下游信号元件的治疗性调节可能会增强成人中枢神经系统的轴突萌芽和再生。 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肝配蛋白，以及进一步表征内源性神经再生潜力。