Inhibition of Hsp70 ATPase activity reduces tau through the cellular degradation

描述(申请人提供)：分子伴侣是治疗蛋白质错误折叠引起的神经系统疾病的一个很有前途的治疗靶点。这是因为它们有能力与“客户”蛋白质相互作用，这些蛋白质容易错误折叠和聚集。这些客户之一，微管相关蛋白tau，在许多称为tauopathy的神经退行性疾病中异常聚集。一些伴侣蛋白在直立性变态模型中作为治疗靶点显示出良好的效果。然而，这项研究建议增加Hsp70介导的干预的证据，以发现降低tau水平的化合物。Hsp70的伴侣活性是由ATP的水解酶提供动力的。此外，许多调节Hsp70的ATP水解率的药理化合物已经被证明可以调节tau的水平：亚甲基蓝(MB)被证明是一种抑制Hsp70的ATPase活性的化合物。甲基溴已被证明可以降低不同模型的tau水平，并已被证明可以挽救认知。这些数据表明，Hsp70 ATPase的调节可能是开发抗tau疗法的一个可行的工具。然而，有大量文献描述了甲基溴的许多其他相互作用和影响。因此，目前的数据为MB作为一种有前途的候选治疗创造了更强的理由，而不是将HSP70的S ATPase作为治疗目标。Hsp70作为治疗靶点的有力证据将使药物开发商能够创造出可能治愈肥胖症的新化合物。因此，我们创造了新的基于遗传学的工具来研究这个问题，以便收集支持或反驳这一假说的数据。我们已经创建了Hsp70的单一氨基酸突变体，并测试了它们的ATPase和复性活性以及它们对tau水平的影响。Hsp70上的一个突变E175s降低了它的ATPase活性和复性活性。此外，它还可以降低tau病细胞模型中tau的水平。因此，我们复制了之前使用Hsp70抑制剂化合物的体外数据。本研究的目的是验证Hsp70 ATPase活性抑制通过细胞降解途径降解tau的假说。为了实现这一目标，我们的设计包括以下具体目标：1)我们将在Hsp70E175S过表达的情况下阻断和激活蛋白酶体、巨型自噬和伴侣辅助的自噬途径；2)我们将确定在体内抑制Hsp70的ATPase活性是否会减少tau，并评估这一策略的治疗潜力。 与公众健康相关：tau蛋白在脑细胞的正常活动中非常重要。由于未知的原因，tau失去了其正常的功能，并在许多神经系统疾病中产生毒性。患有这些疾病的人数很多，而且由于老龄化人口的增加，预计还会大幅增加。不幸的是，目前大多数这些疾病的治疗方法只能在很短的一段时间内减轻症状。因此，发现可以阻止、预防或显著减缓tau毒性反应的治疗方法是科学研究中的一个紧迫目标。 菲尔德。

DESCRIPTION (provided by applicant): Molecular chaperones are a promising therapeutic target in the treatment of neurological diseases of protein misfolding. This is because of their ability to interact with "client" proteins that become misfolded and aggregate-prone. One of these clients, the microtubule associated protein tau, abnormally aggregates in numerous neurodegenerative diseases termed tauopathies. Some chaperone proteins have shown auspicious results as therapeutic targets in models of tauopathy. However, this study proposes to add to the evidence for Hsp70-mediated interventions for the discovery of compounds that reduce tau levels. The chaperone activity of Hsp70 is powered by the hydrolysis of ATP. Moreover, many pharmacological compounds that modulate the rate of ATP hydrolysis of Hsp70 have been shown to modulate the levels of tau: One compound that was shown to inhibit the ATPase activity of Hsp70 is methylene blue (MB). MB has been shown to reduce tau levels in various models of tauopathy and has been shown to rescue cognition. These data suggest that Hsp70 ATPase modulation may be a viable tool for the development of anti-tau therapies. However, there is vast literature describing the many other interactions and effects of MB. As a result, the current data create a stronger case for MB as a promising therapeutic candidate than for Hsp70's ATPase as a therapeutic target. Strong evidence for Hsp70 as a therapeutic target will allow drug developers to create new compounds that may hold the cure for tauopathies. Thus, we have created new genetics-based tools with which to study this question in order to collect data that will either support or refute the hypothesis. We have created single amino acid mutants of Hsp70 and tested their ATPase and refolding activity as well as their effects on tau levels. One mutation, E175S, on Hsp70 reduces its ATPase activity and refolding activity. Furthermore, it reduces tau levels in cell models of tauopathy. Thus, we have replicated the previous in vitro data that used Hsp70 inhibitor compounds. The objective of this study is to test the hypothesis that the inhibition of Hsp70 ATPase activity degrades tau through the cellular degradation pathways. To achieve this, our design consists of the following specific aims: 1) We will block and activate the proteasome, macroautophagy and chaperone-assisted autophagy pathways in the presence of Hsp70E175S overexpression; 2) We will determine if inhibiting the ATPase activity of Hsp70 reduces tau in vivo and evaluate the therapeutic potential of this strategy. PUBLIC HEALTH RELEVANCE: The protein tau is very important in the normal activities of brain cells. Due to unknown reasons tau loses its normal function and becomes toxic in a large number of neurological diseases. The number of people suffering from these diseases is large and is projected to increase dramatically due to an increase in the aging population. Unfortunately, the current treatments available for most of these diseases only reduce symptoms for a brief period of time. As a result, the discovery of therapies that could halt, prevent or significantly slow the repercussions of tau toxicity is an urgent goal in the scientific field.