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）已用于治疗对乙酰氨基酚过量，我们已经从几十年的经验中了解到它具有极好的安全性。NAC也被用于治疗神经系统疾病患者。例如，发现NAC促进阿尔茨海默病的认知方面。此外，NAC被发现有助于解决士兵爆炸伤的症状。然而，关于NAC的低生物利用度和需要高剂量渗透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-突触核蛋白纤维的新型帕金森病模型中的应用。我们还将在分子水平上建立保护作用的机制。我们关于NAC的初步数据已经表明，NAC可以以热休克蛋白依赖的方式保护神经元和星形胶质细胞。也就是说，NAC提高了Hsp 70 A1/2，而Hsp 70的泛抑制剂消除了其保护作用。这种新的NAC作用机制尚未在文献中被其他人描述，但可能有助于解释为什么NAC在多个双盲，安慰剂对照临床试验中有效。在这里，我们将测试的假设，NACET可以保护嗅球神经元对α-突触核蛋白病的热休克蛋白70依赖的方式。在这些研究中，我们选择检查嗅球，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。目的1a）用α-突触核蛋白纤维和不同浓度的NACET处理原代嗅球神经元。确定NACET是否能防止细胞丢失，提高Hsp 70家族成员，并减轻泛素化蛋白和Lewy样包涵体的增加。目的1b）使用3种独立的Hsp 70抑制剂来测试NACET介导的保护是否随着Hsp 70活性的丧失而被消除。目的1c）测试NACET是否通过激活转录因子Nrf 2和HSF 1来提高Hsp 70水平。如果Nrf 2或HSF 1在NACET处理后易位到细胞核，用RNA干扰将其敲除，以观察NACET介导的保护是否被废除。目的2a）将α-突触核蛋白纤维注入小鼠嗅球并在饮用水中给予NACET。6个月后用体视学方法计数NeuN+神经元和Lewy样包涵体。目的2b）重复原纤维注射和NACET处理，并收集小鼠组织用于Western印迹。测量Hsp 70家族成员和泛素化蛋白的水平。测试NACET将减少体内神经元损失和Lewy样包涵体，以及它将提高嗅球组织中的Hsp 70 A1/2和降低泛素化蛋白的假设。NACET可能是第一种安全地增强Hsp 70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氏症的治疗方法。这些研究被称为“床边到长凳”研究，其中已知对人类有效的化合物在实验模型中进行事后检查，以更深入地了解疾病病因和药物作用。这种方法在精神病学领域已经成功多年，现在是时候在与帕金森病的斗争中利用它了。

 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.