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

 说明(申请人提供)：N-乙酰-L-半胱氨酸(NAC)自20世纪70年代以来一直用于治疗对乙酰氨基酚过量，从这几十年的经验中我们已经知道它具有极好的安全性。NAC也被用于治疗患有神经疾病的患者。例如，NAC被发现有助于阿尔茨海默病患者认知的各个方面。此外，NAC还被发现有助于缓解士兵的爆炸伤症状。然而，NAC的生物利用度较低，以及需要高剂量才能渗透到大脑中的问题已经出现。我们将开发一种新的基于α-突触核蛋白纤维的帕金森氏病模型。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可以通过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上调了Hsp70A1/2，而Hsp70的PAN抑制剂取消了其保护作用。NAC的这种新的作用机制在文献中没有被其他人描述，但可能有助于解释为什么NAC在多个双盲、安慰剂对照的临床试验中有效。在这里，我们将测试假设，即NACET可以保护嗅球神经元免受α-突触核病症的影响，这是一种依赖于HSP70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是否可以防止细胞丢失，增加Hsp70家族成员，并减轻泛素化蛋白和路易样包涵体的增加。目的1b)使用3种独立的Hsp70抑制剂来检测NACET介导的保护作用是否随着Hsp70活性的丧失而消失。目的1c)检测NACET是否通过激活转录因子Nrf2和HSF1来提高Hsp70水平。如果NACET治疗后Nrf2或HSF1移位到细胞核，用RNA干扰将其击倒，看看NACET介导的保护是否被取消。目的2a)向小鼠嗅球内注入α-突触核蛋白纤维，并在饮用水中给予NACET。6个月后体视学计数Neun神经元和Lewis样包涵体。目的2b)重复原纤维注射和NACET处理，收集小鼠组织进行Western blotting。测量Hsp70家族成员和泛素化蛋白的水平。验证NACET将减少体内神经元丢失和路易样包涵体的假设，以及它将提高嗅球组织中Hsp70A1/2和降低泛素化蛋白的假设。NACET可能是第一种安全地增强人类Hsp70防御系统和减少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。这些研究例证了“床边到工作台”的研究，即已知对人类有效的化合物在实验模型中进行事后检查，以获得对疾病病因和药物作用的更深层次的见解。这种方法在精神病学领域已经成功了很多年，现在是时候利用它来对抗帕金森氏症了。

 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.