Engineering therapeutic TRIM11 disaggregases for Alzheimer's Disease-Related Dementias (ADRDs)

工程治疗 TRIM11 解聚酶治疗阿尔茨海默病相关痴呆症 (ADRD)

• 批准号: 10539674
• 负责人: James Shorter
• 金额: $ 44.69万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-20 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10539674
• 关键词: Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease related dementia; Amino Acid Motifs; Amyloid; Amyloid Fibrils; Amyotrophic Lateral Sclerosis; Autopsy; Biological Assay; Biotechnology; Cytoplasmic Inclusion; Directed Molecular Evolution; Disease; Engineering; Family; Frontotemporal Dementia; Human; Human Engineering; Huntington Disease; In Vitro; Lewy Bodies; Lewy Body Dementia; Libraries; Mediating; Methods; Modeling; Movement Disorders; Multiple System Atrophy; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathologic; Patients; Persons; Pharmacologic Substance; Proteins; Research; Structure; TRIM Motif; TRIM11 gene; Therapeutic; Therapeutic Agents; Toxic effect; Validation; Variant; Yeasts; alpha synuclein; conformer; effective therapy; in vivo; member; mutant; neuroprotection; neurotoxic; novel; presynaptic; prevent; protein misfolding; proteostasis; synucleinopathy; therapeutically effective; yeast genetics

Project Summary Our research objective is to engineer variants of a human tripartite motif (TRIM) protein, TRIM11, with enhanced ability to mitigate alpha-synuclein (alpha-syn) misfolding and toxicity that underlies synucleinopathies. Synucleinopathies are common, debilitating neurodegenerative disorders that afflict millions of people worldwide and include Parkinson's Disease, Multiple System Atrophy and Lewy Body Dementia, an Alzheimer's Disease-Related Dementia [ADRD]). In all synucleinopathies, the proteostasis network fails to counter the misfolding of the small presynaptic protein alpha-syn. alpha-Syn populates a range of misfolded conformers ranging from soluble toxic oligomers to self-templating amyloid fibrils capable of initiating and propagating disease de novo. alpha-Syn fibrils cluster into large cytoplasmic inclusions termed Lewy Bodies, a pathological hallmark of synucleinopathies. Unfortunately, there are no effective therapeutics for synucleinopathies. Remarkably, we have discovered that human TRIM11 can prevent and reverse the formation of alpha-syn fibrils and reduce their toxicity in vitro and in vivo. However, we suspect that TRIM11 becomes overwhelmed and fails to counter alpha-syn misfolding in synucleinopathies. Indeed, elevating expression of TRIM11 partially mitigates alpha-syn-mediated neurodegeneration in vivo, but neuroprotection is incomplete and significant neurodegeneration still occurs. Thus, methods to enhance TRIM11 disaggregase activity in the degenerating neurons of synucleinopathy patients could reverse deleterious accumulation of alpha-syn and offer a solution for synucleinopathies. We hypothesize that engineering TRIM11 with enhanced disaggregase activity will enable more effective disassembly of toxic oligomeric and amyloid forms of alpha-syn, which could confer therapeutic benefits in synucleinopathies. Thus, we will engineer TRIM11 with enhanced disaggregase activity against neurotoxic alpha-syn conformers. We will then assess the ability of these enhanced TRIM11 variants to antagonize alpha-syn aggregation and toxicity elicited by alpha-syn oligomers and fibrils in mammalian primary neurons. To do so, we will pursue three aims: (1) Engineer enhanced TRIM11 variants to mitigate alpha-syn aggregation and toxicity in yeast; (2) Define optimal TRIM11 variants that disassemble alpha-syn fibrils and oligomers in vitro; and (3) Define enhanced TRIM11 variants that mitigate alpha-syn aggregation and toxicity in primary neurons. The proposed project establishes a pipeline that begins by leveraging the power of yeast genetics to pinpoint optimal TRIM11 disaggregases from mutant libraries and culminates with their validation at the pure protein level and in mammalian neurons. By the end of these studies, there will be a clear “go/no go” decision for moving enhanced TRIM11 variants into mouse synucleinopathy models and ultimately synucleinopathy patients. Moreover, by revealing how TRIM11 disaggregase activity can be enhanced by specific mutations we will begin to clarify the mechanism of TRIM11 action.

项目概要 我们的研究目标是设计人类三联基序 (TRIM) 蛋白 TRIM11 的变体， 增强减轻 α-突触核蛋白 (α-syn) 错误折叠和突触核蛋白病潜在毒性的能力。 突触核蛋白病是一种常见的、使人衰弱的神经退行性疾病，影响数百万人 世界范围内，包括帕金森病、多系统萎缩症和路易体痴呆症（一种 阿尔茨海默氏病相关痴呆 [ADRD]) 在所有突触核蛋白病中，蛋白质稳态网络均无法发挥作用。 对抗小突触前蛋白 α-Syn 的错误折叠，形成一系列错误折叠。 构象异构体范围从可溶性有毒低聚物到能够启动和形成自模板淀粉样原纤维 将疾病从头传播到称为路易体的大细胞质内含物中。 不幸的是，没有有效的治疗方法。 值得注意的是，我们发现人类 TRIM11 可以预防和逆转突触核蛋白病。 α-syn 原纤维的形成并降低其体外和体内毒性然而，我们怀疑 TRIM11。 变得不堪重负，无法对抗突触核蛋白病中的α-syn错误折叠。 TRIM11 的表达部分减轻体内 α-syn 介导的神经变性，但神经保护作用 因此，增强 TRIM11 解聚酶的方法仍然会发生。 突触核蛋白病患者退化神经元的活性可以逆转 α-syn 的有害积累，并为突触核蛋白病提供了一种解决方案。 解聚酶活性将能够更有效地分解有毒的寡聚体和淀粉样形式的α-syn， 这可以为突触核蛋白病带来治疗益处，因此，我们将设计增强的 TRIM11。 然后我们将评估这些增强的α-syn构象异构体的解聚集活性。 TRIM11 变体拮抗 α-syn 聚集和 α-syn 寡聚物和原纤维引起的毒性 为此，我们将追求三个目标：(1) 设计增强的 TRIM11 变体 (2) 定义反汇编 alpha-syn 的最佳 TRIM11 变体 体外原纤维和寡聚物；以及 (3) 定义增强的 TRIM11 变体，以减轻 α-syn 聚集和 拟议的项目建立了一个管道，首先利用 酵母遗传学从突变文库中查明最佳 TRIM11 解聚酶，并最终得到其结果 在纯蛋白质水平和哺乳动物神经元中进行验证，到这些研究结束时，将会有一个明确的结果。 将增强的 TRIM11 变体转移到小鼠突触核蛋白病模型中的“进行/不进行”决定，并最终 此外，通过揭示如何通过增强 TRIM11 解聚酶活性。 具体突变我们将开始阐明TRIM11的作用机制。