Novel EPO peptide therapy for a new mouse model of dementia

针对新痴呆症小鼠模型的新型 EPO 肽疗法

• 批准号: 9040015
• 负责人: PETER C DOWLING
• 金额: --
• 依托单位: VA NEW JERSEY HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-10-01 至 2018-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9040015
• 关键词: Adverse effects; Age-Months; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Animal Model; Animals; Antibodies; Biological Assay; Bioluminescence; Blood - brain barrier anatomy; Brain; Characteristics; Chronic; Clinical; Data; Dementia; Development; Disease; Down-Regulation; Drops; Erythropoietin; Exhibits; Foundations; Glial Fibrillary Acidic Protein; Health Care Costs; Healthcare; Hippocampus (Brain); Human; ITGAM gene; ITGAX gene; Immune; Immune response; Immune system; Immunohistochemistry; Inflammation; Inflammatory; Inflammatory Response; Lectin; Limb structure; Long-Term Effects; Luciferases; Mediating; Microdialysis; Modeling; Monitor; Monoclonal Antibodies; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Onset of illness; Outcome; Paresis; Pathologic; Pathology; Peptide Fragments; Peptides; Permeability; Pharmaceutical Preparations; Plant Roots; Population; Process; Radiolabeled; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Staining method; Stains; Synaptophysin; Testing; Therapeutic Effect; Time; Transcript; Transgenes; Transgenic Mice; Transgenic Organisms; United States; Ventricular; Veterans; Western Blotting; Work; Writing; aged; early childhood; effective therapy; hippocampal atrophy; immunoreactivity; improved; macrophage; mouse model; neurobehavioral; novel; novel therapeutics; prevent; public health relevance; radiotracer; response; small molecule; tau Proteins; tau-1; therapy duration

DESCRIPTION (provided by applicant): Project Summary Objectives: This proposal is written in direct response to a specific request for new therapeutic directions for neurodegenerative disease (Alzheimer's) treatment as well as the development of new animal models of neurodegenerative disease useful for monitoring the effects of new therapies on the dementing process. We plan to test a well-characterized small molecule peptide fragment, JM4 synthesized from erythropoietin for long-term therapy in three transgenic mouse models of neurodegenerative disease. We believe that part of the progression in Alzheimer's disease is related to protracted inflammatory processes and that our newly discovered potent Immunomodulatory compound, JM4 that appears to work through downregulation of the innate immune system may be beneficial. Our compound may or may not attack a root cause of Alzheimer's disease but if secondary inflammation is critical to onset or progression in this disorder, then our compounds may be of long-term benefit. Specific aims S.A.1: Determine the therapeutic effect of our small EPO peptide on three animal models of neurodegenerative disease and/or AD with different clinical and pathologic characteristics. The drug will be tested against the triple transgenic AD mouse model, a more aggressive 5xFAD beta-amyloid model and early on in a third model (P301S) where tau-mediated pathology, microglial activation and hind limb paresis are prominent. The functional effects of long-term therapy will be assessed using PBS sham-treated animals as controls. (Neurobehavioral assessment, time to disease onset, Kaplan- Meier survival curve, drug effect on preventing both ventricular enlargement and hippocampal atrophy. We will also employ immunohistochemistry and flow cytometric assays for determining the effects of therapy on microglial/macrophage immunoreactivity using MHCII, Lectin, Iba1, CD11b, CD11c, F4/80, on neurons (NeuN) using monoclonal antibodies for phosphorylated tau (AT8), conformational specific tau antibody (MC1), synaptophysin, 6E10 for ¿-amyloid products and APP, and by ThioS staining). S.A. 2: Develop a refined animal model of dementia using both 5xFAD amyloid and P301S tau mice further encoded with an additional transgene specific for GFAP-luciferase. These enhanced neurodegenerative models can be followed long-term in single animals non-invasively from early childhood (asymptomatic) through disease onset to full-blown disease. Our pilot data indicates that the GFAP-luc/P301S tau animals will manifest markedly increased bioluminescence as they become symptomatic. Importantly, positive effects of therapy induced by our small JM4 EPO peptide, JM4 should be reflected by suppressed or greatly reduced bioluminescence when compared to bioluminescent levels in symptomatic sham treated animals. Controls for the bioluminescence studies will include immunohistochemistry for GFAP, Western Immunoblots for GFAP and RT-PCR of GFAP transcripts to control for message abundance and extent of bioluminescent signal. S.A. 3: In aged symptomatic 5xFAD or P301S tau animals, determine if interventional JM4 therapy (3 month) initiated at 6 or 9 months of age is as effective as chronic very long duration therapy in delaying onset or progression of the neurodegenerative process using the criteria enumerated in S.A. 1 and 2 (neurobehavioral, drug effect on degree of GFAP bioluminescence and immunohistochemical assessment). S.A. 4: Using IV administered radiolabeled JM4, determine JM4 blood-brain barrier permeability in normal mice (whole brain counts from post-perfused animals and as an alternate by microdialysis in hippocampus). Potential Impact on Veterans Health Care: There is a pressing need for a therapy for Alzheimer's disease where there is currently no effective treatment. The Alzheimer foundation states that if the dementing process could be slowed by 5 years, US health care costs for this condition would drop by 50%. lf this is true, the remarkable delays we have been observing in our novel compound (JM4) treated AD mice, while well short of a complete cure, could likely slow progression by 5 years or more. lf positive findings are consistently confirmed in the proposed animal studies, then it would be a clear cut indication that the immune response and its modulation in Alzheimer's are likely critical components of the puzzle, Our compounds appear to work like no other immunomodulating compounds described to date and we believe since it is a naturally occurring motif hidden within the human and animal EPO molecule, its efficacy will be favorable and side effect profile minimal.

描述（由申请人提供）： 项目摘要目标：本提案是针对神经退行性疾病（阿尔茨海默氏症）治疗新治疗方向的具体要求而编写的，以及开发新的神经退行性疾病动物模型，可用于监测新疗法对痴呆过程的影响我们计划测试一种由促红细胞生成素合成的、已充分表征的小分子肽片段 JM4，用于三种神经退行性疾病转基因小鼠模型的长期治疗。阿尔茨海默病与长期炎症过程有关，我们新发现的强效免疫调节化合物 JM4 似乎通过下调先天免疫系统发挥作用，可能是有益的，我们的化合物可能会也可能不会攻击阿尔茨海默病的根本原因，但如果是继发性炎症。对于这种疾病的发病或进展至关重要，那么我们的化合物可能具有长期益处。具体目标 S.A.1：确定我们的小 EPO 肽对三种神经退行性疾病动物模型的治疗效果。该药物将针对三重转基因 AD 小鼠模型（一种更具侵袭性的 5xFAD β-淀粉样蛋白模型）以及早期的第三种模型（P301S）进行测试，其中 tau 介导的病理学、小胶质细胞激活。长期治疗的功能影响将使用 PBS 假治疗的动物作为对照进行评估（神经行为评估、发病时间、Kaplan-Meier 存活率）。曲线，药物对预防心室扩大和海马萎缩的作用我们还将采用免疫组织化学和流式细胞术测定使用 MHCII、凝集素、Iba1、CD11b、CD11c、F4/80 治疗对神经元的小胶质细胞/巨噬细胞免疫反应性的影响。 (NeuN) 使用磷酸化 tau (AT8) 的单克隆抗体，构象特异性 tau 抗体(MC1)，突触素，6E10 为 ¿ -淀粉样蛋白产物和 APP，并通过 ThioS 染色）。2：使用 5xFAD 淀粉样蛋白和 P301S tau 小鼠开发一种精致的痴呆动物模型，并进一步编码 GFAP 荧光素酶特异性的额外转基因。这些增强的神经退行性模型可以长期跟踪。 -从幼儿期（无症状）到疾病发作到疾病全面发展，对单个动物进行非侵入性治疗。 GFAP-luc/P301S tau 动物在出现症状时将明显增加生物发光。重要的是，与有症状的假治疗中的生物发光水平相比，我们的小 JM4 EPO 肽 JM4 诱导的治疗的积极效果应该通过生物发光的抑制或大大减少来反映。生物发光研究的对照包括 GFAP 的免疫组织化学、GFAP 的免疫印迹和对 GFAP 转录物进行 RT-PCR，以控制生物发光信号的信息丰度和范围 3：在老年有症状的 5xFAD 或 P301S tau 动物中，确定在 6 或 9 个月龄开始的介入 JM4 疗法（3 个月）是否与使用 S.A. 1 和 2 中列举的标准（神经行为、药物效应）进行长期持续治疗，以延迟神经退行性过程的发生或进展S.A. 4：使用静脉注射放射性标记的 JM4，确定正常小鼠的 JM4 血脑屏障通透性（来自灌注后动物的全脑计数，并作为海马微透析的替代方案）。关于退伍军人医疗保健：阿尔茨海默氏病目前尚无有效的治疗方法，因此迫切需要一种治疗方法。减缓 5 年，美国治疗这种疾病的医疗费用将下降 50%，如果这是真的，我们在新型化合物 (JM4) 治疗的 AD 小鼠中观察到的显着延迟，尽管远未完全治愈，但可能会下降。如果在拟议的动物研究中证实了一致的积极结果，那么这将明确表明阿尔茨海默病的免疫反应及其调节可能是这个难题的关键组成部分。工作与众不同迄今为止描述的免疫调节化合物，我们相信，由于它是隐藏在人类和动物 EPO 分子中的天然存在基序，因此其功效将是有利的，并且副作用最小。