Impact of N-acetyl cysteine ethyl ester on a-synuclein pathology in the olfactory system

 描述（由申请人提供）：自 20 世纪 70 年代以来，N-乙酰基-L-半胱氨酸 (NAC) 一直被用于治疗对乙酰氨基酚过量，并且从数十年的经验中我们已经知道它具有出色的安全性。 NAC 还被用于治疗患有神经系统疾病的患者。例如，NAC被发现可以促进阿尔茨海默氏病的认知功能。此外，NAC 被发现有助于解决士兵爆炸伤的症状。然而，关于 NAC 的生物利用度低以及需要高剂量才能渗透到大脑的问题已经出现。我们将开发细胞渗透性衍生物 NAC 乙酯 (NACET) 在基于 α-突触核蛋白原纤维的新型帕金森病模型中的应用。我们还将建立分子水平的保护作用机制。我们关于 NAC 的试验数据已经表明，NAC 可以以热休克蛋白依赖性方式保护神经元和星形胶质细胞。也就是说，NAC 升高 Hsp70A1/2，而 Hsp70 的泛抑制剂则消除其保护作用。 NAC 的这种新作用机制尚未在文献中被其他人描述过，但可能有助于解释为什么 NAC 在多项双盲、安慰剂对照临床试验中有效。在这里，我们将检验 NACET 可以以 Hsp70 依赖性方式保护嗅球神经元免受 α-突触核蛋白病的假设。我们选择在这些研究中检查嗅球，因为嗅球萎缩、细胞丢失和嗅觉功能障碍在帕金森病的早期发展。目标 1a) 用 α-突触核蛋白原纤维和不同浓度的 NACET 治疗初级嗅球神经元。确定 NACET 是否可以防止细胞损失、增加 Hsp70 家族成员并减轻泛素化蛋白和路易样内含物的增加。目标 1b) 使用 3 种独立的 Hsp70 抑制剂来测试 NACET 介导的保护是否会随着 Hsp70 活性的丧失而消失。目标 1c) 测试 NACET 是否通过激活转录因子 Nrf2 和 HSF1 来提高 Hsp70 水平。如果 Nrf2 或 HSF1 在 NACET 处理后易位到细胞核，则用 RNA 干扰将其敲低，看看 NACET 介导的保护是否被消除。目标 2a) 将 α-突触核蛋白原纤维注入小鼠嗅球并在饮用水中施用 NACET。 6 个月后通过体视学计数 NeuN 神经元和 Lewy 样内含物。目标 2b) 重复原纤维注射和 NACET 治疗并收集小鼠组织进行蛋白质印迹。测量 Hsp70 家族成员和泛素化蛋白的水平。测试以下假设：NACET 将减少体内神经元损失和路易样内含物，并且它将提高 Hsp70A1/2 并降低嗅球组织中的泛素化蛋白。 NACET 可能是第一个安全增强 Hsp70 防御并减少人类蛋白质错误折叠压力的治疗方法，有可能彻底改变帕金森病的治疗方法。这些研究体现了“从床边到工作台”的研究，即在实验模型中对已知对人体有效的化合物进行事后检查，以更深入地了解疾病病因学和药物作用。这种方法在精神病学领域已经成功多年，现在是时候利用它来对抗帕金森病了。

 DESCRIPTION (provided by applicant): N-acetyl-L-cysteine (NAC) has been used to treat acetaminophen overdose since the 1970s and we already know that it has an excellent safety profile from these many decades of experience. NAC has also been used to treat patients with neurological conditions. For example, NAC was found to facilitate aspects of cognition in Alzheimer's disease. Furthermore, NAC was found to help resolve symptoms of blast injury in soldiers. However, questions have arisen about the low bioavailability of NAC and the need for high doses to penetrate the brain. We will develop use of the cell-permeable derivative, NAC ethyl ester (NACET), in a novel α-synuclein fibril-based model of Parkinson's disease. We will also establish the mechanism of protective action at the molecular level. Our pilot data on NAC already suggest that NAC can protect neurons and astrocytes in a heat shock protein-dependent manner. That is, NAC raises Hsp70A1/2 and pan-inhibitors of Hsp70 abolish its protective effects. This novel mechanism of NAC action has not been described by others in the literature, but may help explain why NAC has been effective in multiple double blind, placebo-controlled clinical trials. Here we will test the hypothesis that NACET can protect olfactory bulb neurons against α-synucleinopathy in an Hsp70-dependent manner. We have chosen to examine the olfactory bulb in these studies, as bulb atrophy and cell loss and olfactory dysfunction develop early in the course of Parkinson's disease. Aim 1a) Treat primary olfactory bulb neurons with a-synuclein fibrils and various concentrations of NACET. Determine if NACET protects against cell loss, raises Hsp70 family members, and mitigates an increase in ubiquitinated proteins and in Lewy-like inclusions. Aim 1b) Use 3 independent Hsp70 inhibitors to test if NACET-mediated protection is abolished with loss of Hsp70 activity. Aim 1c) Test if NACET raises Hsp70 levels by activating the transcription factors Nrf2 and HSF1. If Nrf2 or HSF1 is translocated to the nucleus after NACET treatment, knock it down with RNA interference to see if NACET-mediated protection is abolished. Aim 2a) Infuse α-synuclein fibrils into the mouse olfactory bulb and administer NACET in drinking water. Count NeuN+ neurons and Lewy-like inclusions by stereology after 6 months. Aim 2b) Repeat the fibril injections and NACET treatments and collect mouse tissue for Western blotting. Measure levels of Hsp70 family members and ubiquitinated proteins. Test the hypothesis that NACET will reduce neuron loss and Lewy-like inclusions in vivo and that it will raise Hsp70A1/2 and lower ubiquitinated proteins in olfactory bulb tissue. NACET may be the first treatment to safely boost Hsp70 defenses and reduce protein-misfolding stress in humans, with the potential to revolutionize Parkinson's treatment. These studies exemplify "bedside-to-bench" research, where compounds that are known to work in humans are examined post hoc in experimental models to yield deeper insights into disease etiology and drug action. This approach has been successful in the field of psychiatry for many years and it is time to leverage it in the battle against Parkinson's disease.