CofActor: Investigation of the Role of Cytoskeletal Dysregulation in Neurodegenerative Disease

CofActor：细胞骨架失调在神经退行性疾病中作用的研究

• 批准号: 10359367
• 负责人: Robert M Hughes
• 金额: $ 45.3万
• 依托单位: EAST CAROLINA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10359367
• 关键词: 3xTg-AD mouse; Actins; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease pathology; Amino Acids; Amyloid; Amyloid beta-42; Amyloid beta-Protein; Binding; Binding Sites; Biochemical; Biochemical Phenomena; Biochemical Process; Biosensor; Cell Line; Cell model; Cells; Cellular Stress; Couples; Coupling; Cytoskeleton; Dendritic Spines; Development; Disease; Disease Progression; Engineering; Environment; Event; Functional disorder; Glutamate Receptor; Glutamates; Hippocampus (Brain); Huntington Disease; Hydrogen Peroxide; Impairment; Investigation; Lead; Light; Longitudinal Studies; Mediating; Mediator of activation protein; Methods; Monitor; Mutagenesis; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Dysfunction; Neurons; Optics; Organism; Oxidation-Reduction; Parkinson Disease; Pathway interactions; Process; Proteins; Publications; Reagent; Reporter; Reporting; Research; Rod; Role; Signal Pathway; Site-Directed Mutagenesis; Slice; Source; Stress; Synapses; System; Testing; Therapeutic; base; biological adaptation to stress; cell immortalization; cell type; cofactor; cofilin; cryptochrome 2; excitotoxicity; experimental study; inhibitor; innovation; insight; light gated; mouse model; mutant; novel; novel therapeutics; optogenetics; protein aggregation; protein protein interaction; response; sensor; small molecule; small molecule inhibitor; synaptic function; targeted treatment; temporal measurement; therapeutic target; tool; two-photon

Cytoskeletal dysregulation is a prominent hallmark of neurodegenerative disease progression. Its role in conditions like Alzheimer's Disease (AD) and Huntington's Disease (HD), however, remains poorly understood. New tools are therefore required to investigate the connections between cytoskeletal dysregulation and the disease state. In response to this need, we have created a genetically encoded, light and stress-gated switch inspired by the biochemical phenomenon of cofilin-actin rods (“CofActor”), which form in response to high levels of oxidative and energetic stress associated with neurodegenerative disorders, including AD, HD and Parkinson's Disease (PD). CofActor can be a useful tool for rapid and reversible readout of oxidative and energetic stress levels in cells and to assess the cellular stress-dependent changes in actin-dependent processes. Furthermore, CofActor can be activated with both spatial and temporal control, a major advance over currently available methods of studying cofilin-actin rods. We created CofActor by coupling the proteins cofilin and actin with the well-characterized optogenetic switch Cryptochrome 2 (Cry2) – CIB, and in a recent publication, characterized the activity of this sensor in immortalized cell lines and in dissociated hippocampal neuron cultures. In this proposal, we will investigate the utility of CofActor for dissecting the upstream signaling pathways leading to cofilin-actin rod formation by determining its impacts on synaptic function and dendritic spine remodeling in cellular models of AD using dissociated neuron cultures treated with toxic amyloid oligomers and neuron cultures derived from AD model mice (3xTg-AD). This proposal will use CofActor to test the hypothesis that cofilin-actin rod formation contributes to AD pathology by disrupting neuronal function, resulting in synapse loss and impaired structural plasticity of dendritic spines. The specific aims of this proposal are: (i) Investigate how various triggers of oxidative, energetic and excitotoxic stress (including H2O2, ATP depletion, Aβ42 fibrils and oligomers and excessive glutamate receptor stimulation) impact CofActor-mediated cofilin-actin cluster formation and investigate small molecules as inhibitors and activators of the CofActor system in immortalized cells and in cellular models of AD. (ii) Investigate novel mutations of the ATP-binding pocket of actin for modulation of the stress response of the CofActor system, followed by investigating the mutants' ability to induce stress-independent neuronal dysfunction in a mouse model of AD (3xTg-AD); and (iii) Investigate the effect of CofActor-mediated cofilin-actin cluster formation on dendritic spine remodeling associated with dendritic spine structural plasticity induced by 2-photon glutamate uncaging in organotypic hippocampal slice cultures. This proposal will shed light on cofilin-actin rod formation, an important biochemical and structural phenomenon underlying the progression of neurodegenerative diseases (specifically AD), which could lead to the development of targeted therapeutics to slow or stop disease progression.

细胞骨架失调是神经退行性疾病进展的重要标志。它的作用 然而，像阿尔茨海默氏病（AD）和亨廷顿氏病（HD）这样的疾病仍然对此知之甚少。 因此，需要新工具来研究细胞骨架失调与 疾病状态。为了应对这种需求，我们创建了一个遗传编码的，光明和应力门的开关 灵感来自Cofilin-Actin棒的生化现象（“辅助因子”），该现象是响应高水平的 与神经退行性疾病有关的氧化和能量应力，包括AD，HD和 帕金森氏病（PD）。辅助因子可能是快速且可逆的氧化和可逆读数的有用工具 细胞中的能量应力水平，并评估肌动蛋白依赖性的细胞应激依赖性变化 过程。此外，可以通过空间和临时控制来激活辅因子，这是一个重大进展 当前研究Cofilin-actin杆的可用方法。我们通过耦合蛋白质Cofilin创建辅因子 和肌动蛋白具有良好的光遗传学开关加密色素2（CRY2） - CIB，以及最近的 出版物，表征了该传感器在永生的细胞系中的活性和分离的海马 神经元文化。在此提案中，我们将研究辅助因子剖析上游信号的实用性 通过确定其对突触功能和树突状脊柱的影响，导致Cofilin-actin杆形成的途径 使用有毒淀粉样蛋白低聚物治疗的解离神经元培养物在AD的细胞模型中进行重塑 源自AD模型小鼠（3XTG-AD）的神经元培养物。该建议将使用辅助因子检验假设 Cofilin-肌动蛋白杆的形成通过破坏神经元功能有助于AD病理，导致突触 树突状棘的损失和结构可塑性受损。该提案的具体目的是：（i）调查 氧化，能量和兴奋性应激的各种触发因素如何（包括H2O2，ATP耗竭，Aβ42原纤维 以及低聚物和多余的谷氨酸受体刺激）影响辅助因子介导的cofilin-actin簇 形成并研究小分子作为永生化的辅因子系统的抑制剂和活化剂 细胞和AD的细胞模型。 （ii）研究肌动蛋白ATP结合口袋的新型突变 调节辅因子系统的应力反应，然后研究突变体诱导的能力 AD的小鼠模型（3XTG-AD）中的不依赖应力的神经元功能障碍； （iii）调查 与树突状脊柱相关的树突状脊柱重塑上的辅因子介导的Cofilin-Actin簇形成 有机海马切片培养物中的2光谷氨酸脉冲诱导的结构可塑性。这 提案将阐明Cofilin-Actin杆形成，这是一种重要的生化和结构现象 神经退行性疾病的进展（特别是AD）的基础，这可能导致发展 靶向疗法的减慢或停止疾病进展。