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）的分子神经病理学科（MNS）的主要目标是利用免疫系统，以更好地了解神经变性的机理，并开发衰老人群的A-鼻核的疗法。我们提出了3个目标，这是第一个研究先天免疫反应和靶向LRRK2，TOLL样受体和蛋白质聚集体（例如：A-Synnuclei，Nabeta，nabeta，tau）的作用的作用；第二次评估包括MAPK-P38，NFAT和NFKB在内的下游促炎信号通路以及第三个评估T细胞介导的适应性免疫细胞反应在PD/DLB发病机理中的作用以及为A-突触核肾上腺病的免疫疗法的作用。在此期间，我们发表了超过24项手稿，主要集中于了解PD/DLB的发病机理，并开发了新型的药理和免疫治疗方法。 AIM1的进度。我们最近发表了我们的研究结果，表明在细胞外A-突触核蛋白聚集体与TLR2之间的相互作用与中和的免疫疗法靶向类似Toll样受体的免疫疗法2通过调节A-核蛋白传播和神经蛋白nirement andecent ander ander ander drode dodre dropn dodrede dropn ode dropn ode dropn odection 2靶向类似收费的受体。在19财年期间，我们还研究了其他单一和联合siRNA（Spencer等人，DIS的Neurobiol，2019年）药理（Hassan等人，Snow等人，Snow等，SCI Reports 2018）和免疫治疗方法（Mandler等人Alz和Dem 2019）。我们知道，正在开发针对帕金森氏病的阿尔茨海默氏病和 - 苏纳核蛋白（-syn）靶向淀粉样蛋白（A）蛋白（A）蛋白（A）蛋白（A）的免疫治疗方法。但是，尚不清楚靶向A和/或SYN的单一或组合免疫疗法是否有效。淀粉样蛋白前体蛋白/-SYN TG小鼠用特异性（AD02）或-Syn（PD-AFF1）的亲属（AFF）肽和组合对染色（AFF）肽进行免疫。 AD02更有效地减轻了A和PTAU负担；但是，该组合表现出一些添加效应。 AD02和PD -AFF1都有效地降低了-SHN，改善了锥体神经元的变性，并减少了神经炎症。 PD-AFF1更有效地改善胆碱能和多巴胺能纤维损失；合并的免疫表现出添加效应。 AD02更有效地改善了埋藏的颗粒测试行为，而PD-AFF1则更有效地改善了水平束检验；合并的免疫表现出添加效应。我们得出结论，靶向A和-SYN的主动免疫疗法联合可能对用Lewy身体治疗痴呆症具有潜在的兴趣。我们现在正在与DRS实验室合作进行调查。 Cookson和Singleton在细胞外A-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。这项工作在2018年11月在圣地亚哥举行的SFN会议上记录了。 AIM 2的进展。激酶途径的改变，包括有丝分裂原活化蛋白激酶（MAPK）p38在PD/DLB和AD中播放p38。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。尽管已在AD中研究了p38，但对于MAPK p38在DLB/PD和其他突触核管中的作用而言，p38已知。尽管在健康对照中，人类和非TG小鼠p38a与神经元和星形胶质细胞有关，但p38g定位于突触前末端以及在突触末端免疫染色的DLB和A-SYNNTG小鼠的大脑中，降低了P38G的p38G，并将其重新分布在神经元细胞的身体上。此外，双重免疫标记表明，p38g与DLB患者和A-Syn TG小鼠的A-SYN聚集体共定位。相反，在星形胶质细胞中，在DLB病例和A-Syn TG小鼠的大脑中，P38A增加了。这些结果表明，A-Syn可能会干扰p38途径，并且可能在DLB/PD中的炎症和突触功能障碍的机理中发挥作用。这项研究于2019年在壁内静修处介绍，手稿将提交给Acta Neuropath Comm。另外并与Drs合作。 Cookson和Singleton实验室我们正在研究小胶质细胞中LRRK2下游的促炎信号传导途径，具体是，我们发现LRRK2通过选择性地磷酸化和诱导核的核酸核的核酸转移因子，激活T-Cells的核转录因子的核转录因子，cytopoplatc，cytoplats 2（n cytoplats 2（n cytoplatc），通过选择性地磷酸化和诱导核的核酸核来促进这种神经炎的级联反应。同样，在包括帕金森氏病（PD）和痴呆症患者（DLB）以及突触核糖核模型的动物模型中，在包括帕金森氏病（PD）和痴呆症在内的大脑中发现了NFATC2级联反应的激活。因此，我们提出了LRRK2及其下游信号介质NFATC2的调节，作为突触核苷病的新型治疗策略。这项研究的结果在2018年11月在圣地亚哥的SFN和2019年的壁内撤退中介绍。修订后的手稿由科学TM考虑。 AIM 3的进展。作为了解周围免疫系统参与DLB/PD的第一步，我们评估了-SYN转基因（TG）小鼠中的T细胞群体（例如：THY1启动子第61行）。 FACS和神经病理学分析表明，大脑-Syn TG小鼠中CD3+ T细胞的增加，但没有增加CD20（B细胞）。此外，CD3+细胞共表达了干扰素，并且在大脑区域紧邻的星形胶质细胞过程中发现神经炎症细胞增加（例如，星形胶质细胞，小胶质细胞增多）和促炎细胞因子。这些结果表明，浸润的适应性免疫反应在突触核苷的神经炎和神经退行性中起重要作用，并且调节外周NKT和T细胞可能是DLB/PD的可行治疗策略。这些结果在2018年在圣地亚哥举行的SFN会议上提出。我们正在准备手稿以进行出版，并比较A-syn TG年轻（3-4 m/o）和老鼠（18-24 m/o）的大脑中的NKT。作为这些研究的后续和作为LAB项目间项目的一部分，我们正在研究各个年龄的PD/DLB类似A-Syn Prion样品的播种模型，并通过删除T细胞群体（与Jyoti Dr.Jyoti和Ranjan Sen合作）。我们假设-syn PFF模型中的神经退行性可能与细胞毒性T细胞反应的年龄依赖性增加有关。颅内注入-syn PFF进入纹状体中后，在比较两只小鼠同学后的3个月后，延伸到杏仁核，海马和新皮质的延伸到杏仁核，海马和新皮质的积累，但年龄较大的病理学伴随着幼年的病理学，与年轻的病理学相结合，与年轻的病理相结合，与年轻的病理相结合，该细胞伴随着幼年的病理学。小胶质细胞增多和星形胶质细胞增多。有趣的是，与年轻小鼠队列相比，在注射老年小鼠队列后1个月内，CD3抗体检测到的T细胞数量的增加更大。正在进行流式细胞仪和小胶质细胞RNA序列分析免疫细胞分布的其他研究，以更好地了解-SYN PFF颅内注射的免疫反应。这些结果将在2019年在芝加哥的SFN会议上提出。 LNG的其他合作是与Drs。 Andy Singleton，Bryan Traynor和Sonja Scholz关于FTD，DLB和MSA的遗传结构，通过提供专家的神经病理学评估，动物模型和人类后的乳腺脑组织。