Unbiased High Content Screening for Novel Synaptotoxic Substances in Human Alzheimer's Disease Brain Lysates

对人类阿尔茨海默氏病脑裂解物中新型突触毒性物质进行公正的高内涵筛选

• 批准号: 9378165
• 负责人: David L Brody
• 金额: $ 22.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9378165
• 关键词: AD pathology; Age; Alzheimer' s Disease; Amyloid beta-Protein; Blinded; Brain; Brain Diseases; Brain region; Cells; Chromatography; Clinical; Disease; Effectiveness; Elderly; Event; Frontotemporal Dementia; Goals; Hippocampus (Brain); Human; Hydrophobic Interactions; Impaired cognition; Inflammation; Ion-Exchange Chromatography Procedure; Knowledge; Lewy Body Dementia; Mass Spectrum Analysis; Measures; Methods; Molecular; Molecular Sieve Chromatography; Mus; Neurons; Pathology; Patients; Peptides; Phase; Poison; Preclinical Drug Evaluation; Presynaptic Terminals; Prevention; Proteins; Proteomics; Research Personnel; Specificity; Symptoms; Synapses; Testing; Therapeutic; Toxic effect; Toxin; Work; aqueous; base; experimental study; frontal lobe; improved; insight; neuron loss; new therapeutic target; non-demented; novel; novel strategies; protein purification; screening; tau Proteins; therapeutic candidate

PROJECT SUMMARY Clinical dementia of the Alzheimer's type (DAT) is the most common dementing disorder in the elderly. DAT is most tightly correlated with synaptic loss in vulnerable brain regions, which has led to the hypothesis that loss of synaptic terminals is a key event in early cognitive decline. To date, the mechanisms of synaptic loss in DAT remain unknown. Previous researchers have assumed that specific molecular entities such as tau or amyloid-beta (Aβ) are responsible for synaptic degeneration in DAT, but have not performed unbiased screens. Our primary hypothesis is there are as-yet- uncharacterized synaptotoxic substances in brain lysates from patients with DAT that are not present in brain lysates from high pathology, age matched, non-demented controls. We further hypothesize these substances cause synapses to deteriorate in the early phase of DAT which eventually leads to neuronal cell death and severe cognitive decline. To test our hypothesis, we have recently developed a novel, unbiased, high content screening approach using live primary hippocampal neurons from mice genetically manipulated to express a red fluorescent pre-synaptic tag and a green fluorescent post-synaptic tag. This new approach allows us to measure the synapse gain/loss of cultured live neurons serially over several days in a highly sensitive and fully quantitative fashion. We therefore propose to use our novel, unbiased screening approach to identify as-yet-uncharacterized synaptotoxic substances in brain lysates from patients with DAT and to further characterize the synaptotoxic activity of the newly-identified substances. Specific aims of the proposed studies are: 1) To determine whether there are synaptotoxic substances in aqueous brain lysates from patients with DAT using our novel live cell screening approach for synaptic toxicity, as well as how consistent the toxicity is from patient to patient; 2) To determine the specificity of the toxic entities from patients with DAT by assessing brain lysates from non-demented control subjects with Aβ and tau pathology, and normal control subjects; 3) To use our live neuron screening of fractionated aqueous brain lysates from patients with DAT for synaptic toxicity to guide purification of the most potent and specific toxic entities, and then identify the previously uncharacterized synaptotoxic substances in patients with DAT. If successful, we expect to identify a list of previously-uncharacterized synaptotoxic proteins, peptides or other substances specific to patients with DAT. Once the synaptotoxic activities of these substances have been characterized, we will target these novel synaptotoxic agents as new therapeutic targets and perform a drug screening for new candidate therapeutic molecules. The results of our proposed experiments may fundamentally advance knowledge of DAT as well as improve the effectiveness of DAT treatment by identifying targets for new candidate therapeutics.

项目摘要 阿尔茨海默型临床痴呆（DAT）是老年人中最常见的痴呆症。DAT是最 与脆弱大脑区域的突触丢失密切相关，这导致了突触丢失的假设， 终端是早期认知衰退的关键事件。迄今为止，DAT中突触丢失的机制仍不清楚。 以前的研究人员认为，特定的分子实体，如tau蛋白或淀粉样蛋白β（Aβ）是负责 DAT中的突触变性，但尚未进行无偏筛选。我们的主要假设是 DAT患者脑裂解物中不存在的未表征突触毒性物质 年龄匹配的非痴呆对照组中我们进一步假设这些物质导致突触 在DAT的早期阶段恶化，最终导致神经元细胞死亡和严重的认知能力下降。 为了验证我们的假设，我们最近开发了一种新颖的，无偏见的，高内容的筛选方法， 来自小鼠的活的原代海马神经元经遗传操作以表达红色荧光突触前标签和 绿色荧光突触后标记。这种新方法使我们能够测量培养的活细胞的突触获得/损失。 神经元连续数天以高度敏感和完全定量的方式。因此，我们建议使用我们的 一种新的，无偏见的筛选方法，以确定尚未表征的突触毒性物质的脑裂解物， DAT患者，并进一步表征新鉴定的物质的突触毒性活性。 本研究的具体目的是：1）确定是否存在突触毒性物质， 使用我们的新的活细胞筛选突触毒性的方法，从DAT患者的含水脑裂解液，以及 患者之间的毒性一致性如何; 2）确定患者毒性实体的特异性 通过评估来自具有Aβ和tau病理学的非痴呆对照受试者和正常对照的脑裂解物， 3）使用我们的活神经元筛选从DAT患者中分离的含水脑裂解物用于突触 毒性，以指导纯化最有效和具体的有毒实体，然后确定以前的 DAT患者中未表征的突触毒性物质。 如果成功的话，我们希望能够鉴定出一系列以前没有特征的突触毒性蛋白质，肽或其他 DAT患者特有的物质。一旦这些物质的突触毒性活性被表征， 我们将把这些新的突触毒性剂作为新的治疗靶点， 候选治疗分子。我们提出的实验结果可能会从根本上推进知识， DAT以及通过确定新的候选治疗药物的靶点来提高DAT治疗的有效性。