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

细胞骨架失调是神经退行性疾病进展的显著标志。它在这方面的作用 然而，像阿尔茨海默病(AD)和亨廷顿病(HD)这样的疾病仍然知之甚少。 因此，需要新的工具来研究细胞骨架失调与脑出血之间的联系。 疾病状态。为了满足这一需求，我们创造了一种基因编码、光和压力门控开关 受到粘连蛋白-肌动蛋白棒(“辅因子”)的生化现象的启发，这种现象是对高水平的反应而形成的 与神经退行性疾病相关的氧化和能量应激，包括AD、HD和 帕金森氏病(PD)。辅因子是一种有用的工具，可以快速和可逆地读出氧化和 细胞中的能量应激水平和评估细胞应激依赖的肌动蛋白依赖的变化 流程。此外，辅因子可以通过空间和时间控制来激活，这比 目前可用的研究粘连蛋白-肌动蛋白棒的方法。我们通过偶联Cofilin蛋白产生了辅因子 和肌动蛋白与光遗传开关CryptoChrome 2(Cry2)-CIB，并在最近的 该传感器在永生化细胞系和分离的海马体中的活性。 神经元培养。在本提案中，我们将研究辅因子在剖析上行信令中的作用 通过确定其对突触功能和树突棘的影响而导致粘连蛋白-肌动蛋白棒形成的途径 用毒性淀粉样低聚物和淀粉样蛋白低聚体处理分离的神经元培养重建AD细胞模型 神经细胞培养来源于AD模型小鼠(3xTg-AD)。这项提议将使用余因来检验假设。 粘附素-肌动蛋白棒的形成通过破坏神经元功能，导致突触，从而参与AD的病理 树突棘丢失和结构可塑性受损。这项建议的具体目的是：(一)调查 氧化、能量和兴奋性毒性应激(包括过氧化氢、三磷酸腺苷耗竭、Aβ42纤维)的各种触发因素 和寡聚体和过度的谷氨酸受体刺激)影响辅因子介导的粘连蛋白-肌动蛋白簇 永生化的辅因子系统的抑制和激活小分子的形成和研究 细胞和AD的细胞模型中。(Ii)研究肌动蛋白ATP结合口袋的新突变 调节辅因子系统的应激反应，然后研究突变体诱导的能力 阿尔茨海默病(3xTg-AD)小鼠模型的应激非依赖性神经元功能障碍；以及(Iii)研究 辅因子介导的粘连蛋白-肌动蛋白簇形成对与树突棘相关的树突棘重塑的影响 器官型海马片培养中双光子谷氨酸去化诱导的结构可塑性。这 该提案将阐明粘连蛋白-肌动蛋白棒的形成，这是一种重要的生化和结构现象 神经退行性疾病(特别是阿尔茨海默病)进展的基础，这可能导致 用于延缓或阻止疾病进展的靶向治疗。

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.