Novel Cytoskeletal Stabilizers as Potential Treatments for Limbic Lewy Body Disorders

摘要 路易体病是一组致命性神经退行性疾病，伴有α-共核病变型包涵体的扩散。 涉及嗅觉、情感、运动和认知的大脑区域。大约有130万名患者生活在美国 路易体痴呆(DLB)和另外约400万帕金森氏病(PD)患者。此外，一个 据估计，约50%的阿尔茨海默病患者表现出共存的路易氏病变(AD1b)。如果治疗可以是 开发出减缓α-突触核苷酸病的发病和扩散的药物，神经退行性变可能会得到缓解和恶化 数以百万计的患者会延迟功能的恢复。最近的研究表明，α-突触核蛋白是一种新型的微管。 动力酶，以其聚集的形式扰乱轴突在微管(MT)上的运输。因此，提示MT的动态性 趋向稳定已成为治疗神经退行性疾病的一种潜在的新疗法。因此，我们 观察到路易体疾病患者嗅球中去酪氨酸化(稳定)的α-微管蛋白的显著丢失。这个 嗅球/前嗅核复合体(OB/AON)与边缘系统联系密切，是 在这一系列疾病中，最早显示α-共核病理性包涵体的区域。我们最近还发现 两种新的MT稳定剂(AG161-41和47)减少了α-突触核蛋白聚集体的出现(高达48%) 在原代培养的海马区，暴露于预先形成的α-突触核蛋白纤维。据我们所知，这一观测是独一无二的， 由于其他MT稳定剂尚未被证明可以减轻路易氏病变。我们的人体组织和原代培养 试点数据为进一步的概念验证研究提供了令人信服的论据。因此，我们将合成改进的类比 并测试MT稳定化可缓解暴露于α-Synuclein纤维的病理后遗症的假设 边缘路易氏病理的细胞和动物模型。在目标1中，我们将合成另外的AG161-41和47和12 AG161-47的新类似物。在目标2A-C中，我们将确定是否通过以下措施使天平向MT稳定倾斜 14种化合物可降低原发疾病患者的包涵体密度、细胞丢失、蛋白质聚集和MT不稳定的标志物 用α-突触核蛋白纤维处理培养的边缘神经元。在Aim 2D中，我们将评估微管蛋白分析中的化合物 用长春新碱组装和置换。在目标3A中，我们将表征AG161的药代动力学特性。 47和4个最有希望的类似物。在目标3B中，我们将测试5种剂量的 AIMS 2-3A中唯一最有效、最安全的类似物，用于在OB/AON中注入α-Synuclein纤维的小鼠。认知能力， 焦虑、嗅觉和运动行为将在基线和每月间隔进行评估。α-突触核蛋白包涵体与细胞 计数将使用无偏见的体视学工具进行测量。AIM 3B将确定药理活性剂量(PAD) 这可以缓解包涵体、细胞丢失、蛋白质聚集和MT不稳定。在AIM 3C中，我们将在 一项更长期的干预性研究，旨在测试最有希望的候选者是否能够在行为障碍后挽救神经元 出现缺陷(例如，在3个月内出现嗅觉丧失)。AIM 2B的分析将重复进行。这些概念验证 研究的目的是1)为未来的SAR研究铺平道路，2)产生关于细胞骨架作用的新假设 体内对路易体疾病的稳定，以及3)有助于加速MT修饰剂向临床的转化。

Abstract Lewy body disorders are a family of fatal neurodegenerative conditions with α-synucleinopathic inclusions spreading across brain regions involved in smell, affect, movement, and cognition. There are ~1.3M patients living in the US with dementia with Lewy bodies (DLB) and another ~4M patients living with Parkinson's disease (PD). In addition, an estimated ~50% of Alzheimer's disease patients display coexisting Lewy pathology (ADLB). If therapies could be developed that slow the onset and spread of α-synucleinopathy, neurodegeneration might be mitigated and deterioration of function would be delayed in millions of patients. Recent studies reveal that α-synuclein is a novel microtubule (MT) dynamase, disrupting axonal transport across microtubules (MTs) in its aggregated form. Thus, tipping MT dynamicity towards stabilization has emerged as a potential new treatment for neurodegenerative disorders. Accordingly, we observed significant loss of detyrosinated (stable) α-tubulin in the olfactory bulb of men with Lewy body disorders. The olfactory bulb/anterior olfactory nucleus (OB/AON) complex is closely connected with the limbic system and is one of the earliest regions to display α-synucleinopathic inclusions in this family of conditions. We have also recently discovered that two novel MT stabilizers (AG161-41 & 47) reduce the emergence of α-synuclein aggregates (up to a remarkable 48%) in primary hippocampal cultures exposed to preformed α-synuclein fibrils. To our knowledge, this observation is unique, as other MT stabilizers have not been demonstrated to mitigate Lewy pathology. Our human tissue and primary culture pilot data make a compelling argument for further proof-of-concept studies. Thus, we will synthesize improved analogs and test the hypothesis that MT stabilization tempers the pathological sequelae of exposure to α-synuclein fibrils in cellular and animal models of limbic Lewy pathology. In Aim 1, we will synthesize additional AG161-41 & 47 and 12 novel analogs of AG161-47. In Aims 2A-C, we will determine if tipping the balance towards MT stabilization with these 14 compounds reduces inclusion density, cell loss, protein aggregation, and markers of MT destabilization in primary limbic neuron cultures treated with α-synuclein fibrils. In Aim 2D, we will evaluate the compounds in assays of tubulin assembly and displacement with vincristine. In Aim 3A, we will characterize the pharmacokinetic properties of AG161- 47 and 4 of the most promising analogs from Aim 2. In Aim 3B, we will test the neuroprotective potential of 5 doses of the single most effective, safe analog from Aims 2-3A, in mice infused with α-synuclein fibrils in the OB/AON. Cognitive, anxiety, olfactory, and motor behavior will be assessed at baseline and monthly intervals. α-synuclein inclusions and cell counts will be measured with unbiased stereological tools. Aim 3B will identify a pharmacologically active dose (PAD) that mitigates inclusions, cell loss, protein aggregation, and MT destabilization. In Aim 3C, we will use this PAD in a longer-term, interventional study designed to test if the most promising candidate can rescue neurons after behavioral deficits emerge (e.g., smell loss emerges within 3 months). The assays of Aim 2B will be repeated. These proof-of-concept studies are designed to 1) pave the way for future SAR studies, 2) generate new hypotheses about the role of cytoskeletal stabilization in Lewy body disorders in vivo, and 3) help accelerate the translation of MT-modifying agents to the clinic.