Neuroimmune responses and therapeutics of alpha-synucleinopathies of the aging population

老年人群α-突触核蛋白病的神经免疫反应和治疗

基本信息

批准号：
10688878
负责人：
Eliezer Masliah
金额：
$ 8.36万
依托单位：
NATIONAL INSTITUTE ON AGING
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10688878
关键词：
2019-nCoV Academia Address Affect Aging Alzheimer&apos s Disease Alzheimer&apos s disease related dementia Amygdaloid structure Amyloid beta-Protein Animal Model Animals Antibodies Antibody titer measurement Apolipoprotein E Area Astrocytes Axon B-Lymphocytes Behavior Behavioral Binding Blood - brain barrier anatomy Brain COVID-19 COVID-19 pandemic effects COVID-19 patient CSF1 gene CSF1R gene CSF2 gene Cell Aging Cell model Cells ChIP-seq Chronic Disease Collaborations Combination immunotherapy DNA Damage DNA Vaccines Dementia with Lewy Bodies Disease Outcome Disease model Elderly Epitopes Gene Expression Profile Genes Genetic Genetically Engineered Mouse Goals Human IL6 gene Immune Immune response Immune system Immunologic Receptors Immunomodulators Immunotherapeutic agent Immunotherapy In Vitro Inflammation Inflammatory Inflammatory Response Injections Innate Immune Response Interneurons LRRK2 gene Laboratories MAP Kinase Gene MAPK12 gene Manuscripts Mediating Mediator of activation protein Methods Microglia Modeling Molecular Mouse Strains Mus NF-kappa B National Institute of Neurological Disorders and Stroke Natural Immunity Nerve Degeneration Neurodegenerative Disorders Neuroglia Neuroimmune Neuroimmune system Neurologic Neurons Nuclear Translocation Paper Parkinson Disease Parkinson&apos s Dementia Pathogenesis Pathogenicity Pathologic Pathology Pathway interactions Peptides Peripheral Pharmacology Phenotype Phosphotransferases Play Preparation Publications Publishing Research Role Science Signal Pathway Signal Transduction Synapses System T cell response T-Lymphocyte TLR2 gene TNF gene Technology Testing Therapeutic Therapeutic Effect Toll-like receptors Transgenic Mice Transgenic Model Tumor-infiltrating immune cells Vaccinated Vaccines Viral Vector Wild Type Mouse adaptive immunity age effect age related aged aging population alpha synuclein base brain tissue cell type combinatorial cytokine differential expression env Gene Products experimental study extracellular gene network genetic architecture immunomodulatory therapies immunoregulation immunosenescence in vitro Model in vivo in vivo Model inhibitor neurogenetics neuroinflammation neuropathology neurotoxic neurotoxicity novel novel therapeutics nuclear factors of activated T-cells p38 Mitogen Activated Protein Kinase protein TDP-43 protein aggregation proteostasis receptor response sex single-cell RNA sequencing small molecule synaptic function synucleinopathy targeted treatment tau Proteins therapeutic target therapy development trafficking transcription factor transcriptome sequencing transcriptomics transmission process γδ T cells

项目摘要

The main objective of the Molecular Neuropathology unit (MNU) at Laboratory of Neurogenetics (LNG) is to harness the immune system to better understand the mechanisms of neurodegeneration and to develop therapies for synucleinopathies of the aging population such as DLB, PD and AD. We propose 3 Aims, the first investigating the role of innate immune responses and combinatorial immunotherapy targeting LRRK2, Toll-like receptors, p38, NFAT and the protein aggregates (eg: a-synuclein, Abeta, tau) in PD/DLB; the second assessing downstream pro-inflammatory signaling pathways including MAPK-p38, NFAT and NFkB and the third evaluating the role of aging in T cell mediated adaptive immune cell responses in PD/DLB pathogenesis and for developing immunotherapies for synucleinopathies. During this period, we published 9 manuscripts mostly focusing understanding the pathogenesis of PD/DLB and developing novel pharmacological and immunotherapeutical approaches. Progress for Aim1. In previous studies we identified several immune receptors that mediate neuroinflammation in synucleinopathies, including Toll-like receptor 2 (TLR2) and have shown which species of a-synuclein bind TLR2 and mediates neuroinflammation. These results suggest that cell type-specific modulation of TLR2 may be a multifaceted and promising therapeutic strategy for synucleinopathies; inhibition of neuronal and astroglial TLR2 decreases pathogenic a-syn transmission, but activation of microglial TLR2 enhances microglial extracellular a-syn clearance. Along these lines we recently published a manuscript (Torres et al Neurobiol of Dis 2022) characterizing in vivo in our a-synuclein transgenic model the relationship between degeneration of interneurons and neuroinflammation in the amygdala. As an extension and given the impact of the COVID19 pandemic and potential role of SARS-CoV2 in AD/ADRD we investigated (in collaboration with the Nath laboratory) inflammation in the brains of COVID19 patients with evidence of neurological involvement, we found increased a-synuclein accumulation in axons and glial cells as well as astrocytic activation and T cell trafficking in affected CNS areas. Moreover, preliminary studies indicate that the SARS-CoV2 envelope protein might interact with TLR2 and play a role in inflammation and a-synuclein driven pathology, we published a manuscript addressing this possibility (Szabo et al Exp Mol Med 2022). Finally, this year in collaborative projects with academia we made considerable progress developing novel ADRD immunotherapies that either target 3RTau (Spencer et al Methods Mol Biol 2022) or simultaneously target multiple epitopes of a-synuclein. For Tau we developed a novel brain penetrating single chain antibody packed in a viral vector that can be applied peripherally. For a-synuclein, we used the MultiTEP based DNA vaccine technology which is an approach especially useful for the elderly with immunosenescence. All vaccines induced high titers of antibodies specific to a-synuclein that significantly reduced PD/DLB-like pathology in mice. The most significant effects were observed in mice vaccinated with PV-1949D and PV-1950D in a sex-dependent manner. We published two recent studies (Kim et al Vaccines 2022 and Zagorski et al Int J Mol Scie 2022). Progress for Aim 2. In collaboration with the Cookson group (Kim et al Science TM 2020) showed that LRRK2 plays a critical role in the activation of microglia by extracellular a-synuclein. We determined that LRRK2 promoted a neuroinflammatory cascade by selectively phosphorylating and inducing nuclear translocation of the immune transcription factor nuclear factor of activated T cells, cytoplasmic 2 (NFATc2). Our results suggest that modulation of LRRK2 and its downstream signaling mediator NFATc2 might be therapeutic targets for treating synucleinopathies. Next, we investigated the hypothesis that blocking NFATc2 translocation might ameliorate a-synuclein-mediated microglial neurotoxicity. Preliminary studies identified a novel peptide compound (11r VIVIT) that when administered peripherally into -syn tg mice reduced neuroinflammation, restores NFATc2 cytoplasmic localization and ameliorates behavioral deficits. We are currently expanding these studies to include detailed characterization of microglial transcriptomic, phenotypic profiling (M1 vs M2), neuropathological and functional examination. Our results suggest that modulation of LRRK2 and its downstream signaling mediator NFATc2 might be therapeutic targets for treating synucleinopathies. This paper is currently under preparation for submission. The next step is to investigate the immunomodulatory role of p38 inhibitors in models of synucleinopathy. We have previously shown that the synaptic MAPK p38-gamma plays a role synaptotoxicity in DLB/PD, while p38-alpha plays a role in neuroinflammation. We treated our animal models of DLB/PD with a p38 inhibitor that crosses the blood brain barrier and evaluated the effects on synaptic function and neuroinflammation. The results of this study were presented at the intramural NIA retreat and at SfN and a manuscript was recently submitted to Science TM. Progress for Aim 3. In collaboration with the Sen laboratory we showed that T cell infiltration with potential participation of NKT cells play an important role in DLB/PD. Next, we explored the role of aging in the pathogenesis of PD/DLB by analyzing, behavior, neuropathology and transcriptomics and immune responses in young and aged wildtype mice that received intrastriatal injections with a-synuclein preformed fibrils (pff). We found that aged mice showed more extensive accumulation of a-synuclein and behavioral deficits that was associated with greater infiltration of T cells and microgliosis. Distinct inflammatory patterns of gene expression in microglia showed that a-synuclein pff-induced genes networks in young mice (eg: CSF2, TNF, IL1b, IL6) that overlaps with genes differentially expressed in microglia in the aged mice. These results indicate that the aged and more inflamed brain micro-environment directly influences the disease outcome of pathological accumulation of a-syn in age-related chronic diseases such as DLB and PD. This manuscript was recently re-submitted to Molecular Neurodegeneration. Other experiments underway includes investigating the effects of aging and deleting microglia with the PLX3387 (a compound that targets CSF1R) on synucleinopathies in DLB/PD models.We are also investigating the effects of aging and inflammation in models where T cells and microglia are deleted or where CSF1 and CSF2 are modulated, manuscripts for all these studies are under preparation. Other collaborations at LNG are with Drs. Cookson, Singleton, Traynor and Scholz on the genetic architecture of FTD, DLB and MSA by providing expert neuropathological assessment, animal models and human postmorten brain tissues. In addition to the collaborations with LNG, we have been collaborating with the laboratory of Dr. Avi Nath at NINDS investigating the neuropathology of COVID19 that resulted on a recent publication on the microvascular alterations in NeuroCOVID (Nath et al NEJM 2021) and with Dr. Robert Tycho on modeling mechanisms of a-syn protein aggregation.

神经遗传学实验室 (LNG) 分子神经病理学部门 (MNU) 的主要目标是利用免疫系统更好地了解神经退行性变的机制，并开发针对老年人群突触核蛋白病（如 DLB、PD 和 AD）的疗法。我们提出了 3 个目标，第一个研究先天免疫反应和针对 LRRK2、Toll 样受体、p38、NFAT 和蛋白质聚集体（例如：a-synuclein、Abeta、tau）的组合免疫疗法在 PD/DLB 中的作用；第二个评估下游促炎信号通路，包括 MAPK-p38、NFAT 和 NFkB，第三个评估衰老在 T 细胞介导的适应性免疫细胞反应中在 PD/DLB 发病机制中的作用，并开发针对突触核蛋白病的免疫疗法。在此期间，我们发表了 9 篇手稿，主要关注了解 PD/DLB 的发病机制以及开发新的药理学和免疫治疗方法。目标 1 的进展。在之前的研究中，我们鉴定了几种在突触核蛋白病中介导神经炎症的免疫受体，包括 Toll 样受体 2 (TLR2)，并显示了哪些种类的 α-突触核蛋白与 TLR2 结合并介导神经炎症。这些结果表明，TLR2 的细胞类型特异性调节可能是一种多方面且有前途的突触核蛋白病治疗策略。抑制神经元和星形胶质细胞 TLR2 会减少致病性 a-syn 传递，但激活小胶质细胞 TLR2 会增强小胶质细胞细胞外 a-syn 清除。沿着这些思路，我们最近发表了一篇手稿（Torres et al Neurobiol of Dis 2022），在我们的α-突触核蛋白转基因模型中描述了体内神经元变性与杏仁核神经炎症之间的关系。作为延伸，考虑到 COVID19 大流行的影响以及 SARS-CoV2 在 AD/ADRD 中的潜在作用，我们（与 Nath 实验室合作）调查了 COVID19 患者大脑中的炎症，并有神经系统受累的证据，我们发现 a-轴突和神经胶质细胞中突触核蛋白的积累以及受影响的中枢神经系统区域的星形胶质细胞活化和 T 细胞运输。此外，初步研究表明 SARS-CoV2 包膜蛋白可能与 TLR2 相互作用，并在炎症和 α-突触核蛋白驱动的病理学中发挥作用，我们发表了一篇探讨这种可能性的手稿 (Szabo et al Exp Mol Med 2022)。最后，今年在与学术界的合作项目中，我们在开发新型 ADRD 免疫疗法方面取得了相当大的进展，这些疗法要么靶向 3RTau（Spencer 等人Methods Mol Biol 2022），要么同时靶向 a-突触核蛋白的多个表位。对于 Tau，我们开发了一种新型脑穿透单链抗体，包装在病毒载体中，可以外周应用。对于α-突触核蛋白，我们使用了基于MultiTEP的DNA疫苗技术，该技术对于患有免疫衰老的老年人特别有用。所有疫苗都能诱导高滴度的α-突触核蛋白特异性抗体，显着减少小鼠体内的 PD/DLB 样病理。在以性别依赖性方式接种 PV-1949D 和 PV-1950D 疫苗的小鼠中观察到最显着的效果。我们最近发表了两项研究（Kim et al Vaccines 2022 和 Zagorski et al Int J Mol Scie 2022）。目标 2 的进展。与 Cookson 小组合作 (Kim et al Science TM 2020) 表明 LRRK2 在细胞外 α-突触核蛋白激活小胶质细胞中发挥着关键作用。我们确定 LRRK2 通过选择性磷酸化和诱导活化 T 细胞的免疫转录因子核因子胞质 2 (NFATc2) 的核转位来促进神经炎症级联反应。我们的结果表明，LRRK2 及其下游信号传导介质 NFATc2 的调节可能是治疗突触核蛋白病的治疗靶点。接下来，我们研究了阻断 NFATc2 易位可能改善 α-突触核蛋白介导的小胶质细胞神经毒性的假设。初步研究发现了一种新型肽化合物 (11r VIVIT)，当将其外周注射到 -syn tg 小鼠体内时，可减少神经炎症、恢复 NFATc2 细胞质定位并改善行为缺陷。我们目前正在扩展这些研究，包括小胶质细胞转录组的详细表征、表型分析（M1 与 M2）、神经病理学和功能检查。我们的结果表明，LRRK2 及其下游信号传导介质 NFATc2 的调节可能是治疗突触核蛋白病的治疗靶点。目前该论文正在准备提交中。下一步是研究 p38 抑制剂在突触核蛋白病模型中的免疫调节作用。我们之前已经证明突触 MAPK p38-gamma 在 DLB/PD 中发挥突触毒性作用，而 p38-alpha 在神经炎症中发挥作用。我们使用能够穿过血脑屏障的 p38 抑制剂治疗 DLB/PD 动物模型，并评估其对突触功能和神经炎症的影响。这项研究的结果在 NIA 校内务虚会和 SfN 上发表，并且最近向 Science TM 提交了一份手稿。目标 3 的进展。与 Sen 实验室合作，我们发现 T 细胞浸润和 NKT 细胞的潜在参与在 DLB/PD 中发挥重要作用。接下来，我们通过对接受纹状体内注射α-突触核蛋白预制原纤维 (pff) 的年轻和老年野生型小鼠进行行为、神经病理学、转录组学和免疫反应分析，探讨了衰老在 PD/DLB 发病机制中的作用。我们发现，老年小鼠表现出更广泛的α-突触核蛋白积累和行为缺陷，这与 T 细胞浸润和小胶质细胞增多有关。小胶质细胞中基因表达的独特炎症模式表明，年轻小鼠中α-突触核蛋白pff诱导的基因网络（例如：CSF2、TNF、IL1b、IL6）与老年小鼠小胶质细胞中差异表达的基因重叠。这些结果表明，衰老和炎症加剧的大脑微环境直接影响DLB和PD等与年龄相关的慢性疾病中a-syn病理性积累的疾病结果。该手稿最近重新提交给分子神经变性。正在进行的其他实验包括研究衰老和使用 PLX3387（一种针对 CSF1R 的化合物）删除小胶质细胞对 DLB/PD 模型中突触核蛋白病的影响。我们还在研究衰老和炎症对删除 T 细胞和小胶质细胞或删除 T 细胞和小胶质细胞的模型的影响。其中 CSF1 和 CSF2 受到调节，所有这些研究的手稿正在准备中。 LNG 的其他合作是与 Drs. Cookson、Singleton、Traynor 和 Scholz 通过提供专家神经病理学评估、动物模型和人类死后脑组织来研究 FTD、DLB 和 MSA 的遗传结构。除了与 LNG 的合作外，我们还与 NINDS 的 Avi Nath 博士实验室合作研究 COVID19 的神经病理学，并于最近发表了一篇关于 NeuroCOVID 微血管改变的文章 (Nath et al NEJM 2021)。 Robert Tycho 关于 a-syn 蛋白聚集的建模机制。