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

• 批准号: 10003730
• 负责人: Eliezer Masliah
• 金额: $ 167.7万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 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/10003730
• 关键词: Active Immunotherapy; Adaptive Immune System; Age; Alzheimer' s Disease; Amygdaloid structure; Amyloid; Amyloid Proteins; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Animal Model; Animals; Antibodies; Area; Attenuated; B-Lymphocytes; Brain; CD3 Antigens; Cell Count; Cell Nucleus; Cell model; Cell physiology; Cells; ChIP-seq; Chicago; Collaborations; Combination immunotherapy; Corpus striatum structure; Cytotoxic T-Lymphocytes; Exhibits; FYN gene; Fiber; Flow Cytometry; Functional disorder; Genetically Engineered Mouse; Goals; Hippocampus (Brain); Human; Immune; Immune response; Immune system; Immunization; Immunize; Immunomodulators; Immunotherapeutic agent; Immunotherapy; Inflammation; Inflammatory; Injections; Innate Immune Response; Interferons; LRRK2 gene; Laboratories; Lewy Body Dementia; Link; MAPK14 gene; Manuscripts; Mediating; Mediator of activation protein; Microglia; Modeling; Molecular; Mouse Strains; Mus; NF-kappa B; Natural Immunity; Neocortex; Nerve Degeneration; Neurodegenerative Disorders; Neuroimmune; Neurons; Parkinson Disease; Parkinson' s Dementia; Pathogenesis; Pathology; Pathway interactions; Patients; Peptides; Peripheral; Pharmacology; Phosphotransferases; Play; Population; Presynaptic Terminals; Process; Proteins; Publications; Publishing; RNA Sequences; Reporting; Research; Role; Science; Signal Pathway; Signal Transduction; Small Interfering RNA; Susceptibility Gene; Synapses; System; T cell response; T-Lymphocyte; TLR2 gene; TREM2 gene; Testing; Therapeutic; Therapeutic Effect; Toll-like receptors; Transgenic Mice; Transgenic Organisms; Tumor-infiltrating immune cells; Work; adaptive immune response; adaptive immunity; age related; aged; aging population; alpha synuclein; astrogliosis; behavior test; brain tissue; cell type; cholinergic; cohort; combinatorial; cytokine; experimental study; extracellular; follow-up; genetic architecture; hippocampal pyramidal neuron; immunomodulatory therapies; immunoregulation; improved; in vitro Model; in vivo Model; interest; meetings; mitogen-activated protein kinase p38; neurogenetics; neuroinflammation; neuronal cell body; neuropathology; neurotoxic; novel; novel therapeutics; nuclear factors of activated T-cells; prion-like; promoter; protein aggregate; response; single-cell RNA sequencing; synuclein; synucleinopathy; targeted treatment; tau Proteins; tau phosphorylation; therapy development; transcription factor; transcriptome sequencing; transcriptomics; transmission process; γδ T cells

The main objective of the Molecular Neuropathology section (MNS) at Laboratory of Neurogenetics (LNG) is to harness the immune system to better understand the mechanisms of neurodegeneration and to develop therapies for a-synucleinopathies of the aging population. We propose 3 Aims, the first investigating the role of innate immune responses and combinatorial immunotherapy targeting LRRK2, Toll-like receptors and the protein aggregates (eg: a-synuclei,n 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 T cell mediated adaptive immune cell responses in PD/DLB pathogenesis and for developing immunotherapies for a-synucleinopathies. During this period we published over 24 manuscripts mostly focusing understanding the pathogenesis of PD/DLB and developing novel pharmacological and immunotherapeutical approaches. Progress for Aim1. We recently published the results of our studies demonstrating that blocking the interactions between extracellular a-synuclein aggregates and TLR2 with neutralizing Immunotherapy targeting Toll-like receptor 2 alleviates neurodegeneration in models of synucleinopathy by modulating a-synuclein transmission and neuroinflammation (Kim et al Molecular Neurodegeneration 2018). During FY19 we also investigated other single and combined siRNA (Spencer et al, Neurobiol of Dis 2019) pharmacological (Hassan et al, and Snow et al, Sci Reports 2018) and immunotherapeutical approaches (mandler et al Alz and Dem 2019). We know that Immunotherapeutic approaches targeting amyloid (A) protein and tau in Alzheimer's disease and -synuclein (-syn) in Parkinson's disease are being developed for treating dementia with Lewy bodies. However, it is unknown if single or combined immunotherapies targeting A and/or -syn may be effective. Amyloid precursor protein/-syn tg mice were immunized with AFFITOPEs (AFF) peptides specific to A (AD02) or -syn (PD-AFF1) and the combination. AD02 more effectively reduced A and pTau burden; however, the combination exhibited some additive effects. Both AD02 and PD-AFF1 effectively reduced -syn, ameliorated degeneration of pyramidal neurons, and reduced neuroinflammation. PD-AFF1 more effectively ameliorated cholinergic and dopaminergic fiber loss; the combined immunization displayed additive effects. AD02 more effectively improved buried pellet test behavior, whereas PD-AFF1 more effectively improved horizontal beam test; the combined immunization displayed additive effects. We conclude that combined active immunotherapy targeting A and -syn may be of potential interest for the treatment of dementia with Lewy bodies. We are now investigating in collaboration with the laboratory of Drs. Cookson and Singleton the interactions between extracellular a-synuclein, TLR2 and LRRK2 and their role in microglial activation and inflammation. This work was recemtly presented at the SFN meeting in San Diego Nov 2018. Progress for Aim 2. Alterations in kinase pathways including mitogen activated protein kinase (MAPK) p38 has been proposed to play a in PD/DLB and AD. While activation of p38a has been linked to neuro-inflammation, alterations in p38g has been associated with Tau phosphorylation in AD. Although p38 has been studied in AD less is known as to the role of MAPK p38 in DLB/PD and other synucleinopathies. While in healthy control human and Non-Tg mice p38a was associated with neurons and astroglial cells, p38g localized to pre-synaptic terminals and in the brains of DLB and a-syn Tg mice immunostaining in the synaptic terminals was reduced and p38g was redistributed to the neuronal cell bodies. Further, double immunolabeling showed that p38g co-localized with a-syn aggregates in DLB patient and a-syn Tg mice. In contrast, in the brains of DLB cases and a-syn Tg mice p38a was increased in astroglial cells. These results suggest that a-syn might interfere with the p38 pathway and might play a role in the mechanisms of inflammation and synaptic dysfunction in DLB/PD. This study was presented at the intra-mural retreat in 2019 and the manuscript will be submitted to Acta Neuropath Comm. In addition and in collaboration with Drs. Cookson and Singleton labs we are investigating the pro-inflammatory signaling pathways downstream of LRRK2 in microglia, specifically we found that LRRK2 promoted this neuroinflammatory cascade by selectively phosphorylating and inducing nucleus translocation of the immune transcription factor, nuclear factor of activated T-cells, cytoplasmic 2 (NFATc2). Likewise, activation of NFATc2 cascade was identified in the brains of synucleinopathies patients including Parkinsons disease (PD) and dementia with Lewy bodies (DLB) and in animal models of synucleinopathy. Therefore, we propose the modulation of LRRK2 and its downstream signaling mediator NFATc2 as a novel therapeutic strategy for synucleinopathies. The results of this study were presented at SFN in san Diego Nov 2018 and at the intramural retreat in 2019. The revised manuscript is under consideration by Science TM. Progress for Aim 3. As a first step to understand the involvement of the peripheral immune system in DLB/PD, we evaluated T cell populations in -syn transgenic (tg) mice (eg: Thy1 promoter line 61). FACS and neuropathologica analysis showed increase of CD3+ T cells but not CD20 (B cells) in the brains -syn tg mice. Furthermore, CD3+ cells co-expressed interferon and where found in close proximity of astroglial cells processes in areas of the brain displaying increased neuro-inflammatory cells (eg: astrogliosis, microgliosis) and pro-inflammatory cytokines. These results suggest that infiltrating adaptive immune responses play an important role in neuro-inflammation and neurodegeneration in synucleinopathies and that modulating peripheral NKT and T cells might be a viable therapeutic strategy for DLB/PD. These results were presented at the 2018 SFN meeting in San Diego. We are in the process of preparing the manuscript for publication as well as comparing NKT in the brains of a-syn tg younger (3-4 m/o) and older mice (18-24 m/o). As a follow up to these studies and as part of an inter-lab project we are investigating T cell responses in an a-syn prion-like seeding models of PD/DLB at various ages and by deleting T cell populations (in collaborations with Drs. Jyoti and Ranjan Sen). We hypothesize that neurodegeneration in the -syn pff model might be associated with an age dependent increase in cytotoxic T cell responses. Following intracranial injection of -syn pff into striatum there was extensive accumulation of -syn that extended to the amygdala, hippocampus and neocortex with greater pathology when comparing the 1 vs the 3 months post injection of both mice cohorts, but aged mice cohort showed significantly increased -syn pathology compare to young mice cohort that was accompanied by infiltration of T cells, microgliosis and astrogliosis. Interestingly we observed greater increase of T cell numbers detected by CD3 antibody in 1 month post injection of aged mice cohort compared to young mice cohort. Additional studies of analyzing immune cell distributions with flow cytometry and microglia RNA sequence are underway to better understand immune response of intracranial injection of -syn pff. These results will be presented at the SFN meeting in Chicago in 2019. Other collaborations at LNG are with Drs. Andy Singleton, Bryan Traynor and Sonja Scholz on the genetic architecture of FTD, DLB and MSA by providing expert neuropathological assessment, animal models and human postmorten brain tissues.

神经遗传学实验室 (LNG) 分子神经病理学部门 (MNS) 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，并开发针对老龄化人群的 α-突触核蛋白病的疗法。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 样受体和蛋白质聚集体（例如：a-synuclei、n 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 T 细胞介导的适应性免疫细胞反应在 PD/DLB 发病机制中的作用以及开发针对 a-突触核蛋白病的免疫疗法。在此期间，我们发表了超过 24 篇手稿，主要关注了解 PD/DLB 的发病机制并开发新的药理学和免疫治疗方法。 目标 1 的进展。我们最近发表的研究结果表明，通过针对 Toll 样受体 2 的中和免疫疗法阻断细胞外 α-突触核蛋白聚集体与 TLR2 之间的相互作用，可以通过调节 α-突触核蛋白传递和神经炎症来减轻突触核蛋白病模型中的神经变性（Kim 等人《分子神经变性》） 2018）。在 2019 财年期间，我们还研究了其他单一和组合 siRNA（Spencer 等人，Neurobiol of Dis 2019）药理学（Hassan 等人和 Snow 等人，Sci Reports 2018）和免疫治疗方法（mandler 等人 Alz 和 Dem 2019）。我们知道，针对阿尔茨海默病中的淀粉样 (A) 蛋白和 tau 蛋白以及帕金森病中的 -突触核蛋白 (-syn) 的免疫治疗方法正在开发中，用于治疗路易体痴呆症。然而，尚不清楚针对A和/或-syn的单一或联合免疫疗法是否有效。淀粉样前体蛋白/-syn tg 小鼠用 A (AD02) 或 -syn (PD-AFF1) 特异的 AFFITOPE (AFF) 肽及其组合进行免疫。 AD02更有效降低A和pTau负担；然而，这种组合表现出一些相加效应。 AD02 和 PD-AFF1 均有效减少 -syn，改善锥体神经元的变性，并减少神经炎症。 PD-AFF1更有效地改善胆碱能和多巴胺能纤维损失；联合免疫显示出累加效应。 AD02更有效地改善埋丸测试行为，而PD-AFF1更有效地改善水平梁测试；联合免疫显示出累加效应。我们的结论是，针对 A 和 -syn 的联合主动免疫疗法可能对治疗路易体痴呆具有潜在意义。我们现在正在与博士实验室合作进行调查。 Cookson 和 Singleton 研究了细胞外 α-突触核蛋白、TLR2 和 LRRK2 之间的相互作用及其在小胶质细胞激活和炎症中的作用。这项工作最近在 2018 年 11 月于圣地亚哥举行的 SFN 会议上进行了展示。 目标 2 的进展。包括丝裂原激活蛋白激酶 (MAPK) p38 在内的激酶途径的改变已被认为在 PD/DLB 和 AD 中发挥作用。虽然 p38a 的激活与神经炎症有关，但 p38g 的改变与 AD 中的 Tau 磷酸化有关。尽管 p38 在 AD 中的研究已被研究，但对于 MAPK p38 在 DLB/PD 和其他突触核蛋白病中的作用知之甚少。而在健康对照人和非 Tg 小鼠中，p38a 与神经元和星形胶质细胞相关，而 p38g 定位于突触前末梢，而在 DLB 和 a-syn Tg 小鼠的大脑中，突触末梢的免疫染色减少，p38g 重新分布到突触末端。神经元细胞体。此外，双重免疫标记显示 p38g 与 DLB 患者和 a-syn Tg 小鼠中的 a-syn 聚集体共定位。相反，在 DLB 病例和 a-syn Tg 小鼠的大脑中，星形胶质细胞中的 p38a 增加。这些结果表明 a-syn 可能干扰 p38 通路，并可能在 DLB/PD 炎症和突触功能障碍机制中发挥作用。这项研究在 2019 年的壁画静修会上提出，手稿将提交给 Acta Neuropath Comm。此外并与博士合作。 Cookson 和 Singleton 实验室正在研究小胶质细胞中 LRRK2 下游的促炎信号通路，具体来说，我们发现 LRRK2 通过选择性磷酸化和诱导免疫转录因子、活化 T 细胞核因子、细胞质2 (NFATc2)。同样，在帕金森病 (PD) 和路易体痴呆 (DLB) 等突触核蛋白病患者的大脑中以及突触核蛋白病动物模型中也发现了 NFATc2 级联的激活。因此，我们提出调节 LRRK2 及其下游信号传导介质 NFATc2 作为突触核蛋白病的新型治疗策略。这项研究的结果于 2018 年 11 月在圣地亚哥的 SFN 和 2019 年的校内务虚会上发表。Science TM 正在考虑修订后的手稿。 目标 3 的进展。作为了解 DLB/PD 中外周免疫系统参与的第一步，我们评估了 -syn 转基因 (tg) 小鼠（例如：Thy1 启动子系 61）中的 T 细胞群。 FACS 和神经病理学分析显示，syn tg 小鼠大脑中 CD3+ T 细胞增加，但 CD20（B 细胞）没有增加。此外，CD3+细胞共表达干扰素，并且在大脑区域的星形胶质细胞突起附近发现神经炎症细胞（例如：星形胶质细胞增生、小胶质细胞增生）和促炎细胞因子的增加。这些结果表明，浸润性适应性免疫反应在突触核蛋白病的神经炎症和神经变性中发挥重要作用，调节外周 NKT 和 T 细胞可能是 DLB/PD 的可行治疗策略。这些结果已在 2018 年圣地亚哥 SFN 会议上公布。我们正在准备出版手稿，并比较 a-syn tg 年轻小鼠 (3-4 m/o) 和老年小鼠 (18-24 m/o) 大脑中的 NKT。作为这些研究的后续工作和实验室间项目的一部分，我们正在研究不同年龄的 PD/DLB 的 a-syn 朊病毒样播种模型中的 T 细胞反应，并通过删除 T 细胞群（与 Drs 合作）乔蒂和兰詹·森）。我们假设 -syn pff 模型中的神经变性可能与年龄依赖性细胞毒性 T 细胞反应的增加有关。颅内注射 -syn pff 至纹状体后，-syn 大量积聚，并延伸至杏仁核、海马和新皮质，比较两组小鼠注射后 1 个月与 3 个月的病理情况，但老年小鼠组表现出显着增加-syn 病理学与伴有 T 细胞浸润、小胶质细胞增生和星形胶质细胞增生的年轻小鼠队列进行比较。有趣的是，与年轻小鼠组相比，我们观察到注射 CD3 抗体后 1 个月，老年小鼠组检测到的 T 细胞数量有更大的增加。使用流式细胞术和小胶质细胞 RNA 序列分析免疫细胞分布的其他研究正在进行中，以更好地了解颅内注射 -syn pff 的免疫反应。这些结果将于 2019 年在芝加哥举行的 SFN 会议上公布。 LNG 的其他合作是与 Drs. Andy Singleton、Bryan Traynor 和 Sonja 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。