Evaluating autophagy modulation as a therapeutic strategy for Alzheimer's Disease using human neuronal culture models

摘要 到目前为止，还没有有效的治疗阿尔茨海默氏症的方法来减少认知能力的下降。尽管 现有药物在减少淀粉样β蛋白方面是有效的，多靶点药物方法更有可能在 影响认知功能。一种潜在的补充治疗方法是调节自噬 溶酶体途径(ALP)，负责蛋白质周转，并已被证明调节降解 中枢神经系统中错误折叠的蛋白质。自噬激活通常有助于清除蛋白质聚集体和受损的 细胞器通过隔离形成自噬小体，这些小泡最终与溶酶体融合，然后 将自噬货物降级。在长寿命的极化神经元中，这一途径更为复杂 自噬主要始于远端轴突，因此必须去除自噬溶酶体中间产物。 从轴突通过逆行运输到胞体，具有蛋白降解活性的溶酶体驻留在那里。中途中断 碱性磷酸酶中间产物的运输或成熟长期以来一直被认为与AD的发病有关，因为它们的堆积 在Aβ周围营养不良的神经突起中，Aβ聚集，可能与AD时A DNA的产生有关。我们的初步数据 IPSC来源的人i3Neurons的AD模型显示，ALP在轴突肿胀中积聚 在高通量筛选中确定为自噬的一种新化合物处理后被清除 Hela细胞中的上调因子。重要的是，我们的数据显示，这种化合物还可以减少细胞内和 在我们的AD模型I3神经元中，细胞外Aβ42和增加神经元自噬(增加LC3II/LC3I)。 来自Aldrich实验室的令人兴奋的新数据确定溶酶体膜蛋白1(LAMP1)是一个直接靶标 在院子里。鉴于这些初步研究，我们假设这种新化合物通过直接 与LAMP1的相互作用以及LAMP1与逆行机械的相互作用的潜在稳定性， 促进溶酶体逆行转运和自噬小体成熟，从而清除轴突碱性磷酸酶 囊泡并最终降低Aβ水平。我们将通过确定机制来检验这一假说 这种新型化合物改变了碱性磷酸酶的运输和碱性磷酸酶的组成和功能，并发现了新的潜力 神经元中的靶点(通过无偏见的蛋白质组学方法)。通过这些研究，我们还将确定是否 与新药相比，FDA批准的药物雷帕霉素可以改变轴突碱性磷酸酶积聚或Aβ42水平 化合物，从而使人们对这两种药物的翻译潜力有了新的认识。最后，我们将确定是否 在家族性AD模型中，新化合物可以降低Aβ42，以及它是如何改变这些神经元中的碱性磷酸酶的。给定 强有力的证据表明，该化合物可以调节轴突碱性磷酸酶运输，Aβ42和神经元自噬， 建议的实验结果可能与其他神经退行性疾病的治疗方法相关。 以碱性磷酸酶功能障碍为病理特征的疾病，如帕金森氏症， 肌萎缩侧索硬化症和额颞部痴呆。

ABSTRACT To date there is no effective treatment for Alzheimer’s disease that decreases cognitive decline. Although the available drugs are effective at reducing amyloid beta, a multi-target drug approach is more likely to succeed in impacting cognitive function. One potentially complementary treatment approach is modulation of the autophagic lysosomal pathway (ALP), which is responsible for protein turnover and has been shown to regulate degradation of misfolded proteins in the CNS. Autophagy activation normally helps clear protein aggregates and damaged organelles via sequestration into autophagosomes, these vesicles eventually fuse with lysosomes, which then degrade the autophagic cargo. This pathway is more complicated in long-lived, polarized neurons, where autophagy largely initiates in the distal axon and thus autophagic lysosomal intermediates must be removed from the axon via retrograde transport to the soma where proteolytically active lysosomes reside. Disruptions in the transport or maturation of ALP intermediates has long been implicated in AD pathogenesis, as they buildup in dystrophic neurites around Aβ aggregates and likely contribute to Aβ production in AD. Preliminary data in our AD model of iPSC-derived human i3Neurons shows that ALP intermediates that accumulate in axonal swellings are cleared upon treatment with a novel compound identified in a high throughput screen as an autophagy upregulator in Hela cells. Importantly, our data shows that this compound also reduces both intracellular and extracellular Aβ42 and increases neuronal autophagy (increased LC3II/LC3I) in our AD model i3Neurons. Exciting new data from the Aldrich lab has identified lysosomal membrane protein 1 (LAMP1) as a direct target of the compound. Given these preliminary studies, we hypothesize that the novel compound, through direct interaction with LAMP1 and potential stabilization of LAMP1 interactions with retrograde machinery, increases retrograde lysosomal transport and autophagosome maturation, and thus clears axonal ALP vesicles and ultimately lowers Aβ levels. We will test this hypothesis by determining the mechanism by which the novel compound alters ALP transport and ALP composition and function, as well as identify new potential targets in neurons (by an unbiased proteomics approach). Through these studies, we will also determine if the FDA-approved drug Rapamycin can alter axonal ALP buildup or Aβ42 levels in comparison to the novel compound, thus shedding new insight into the translational potential of both drugs. Lastly, we will determine if the novel compound can reduce Aβ42 in a familial AD model and how it alters ALP in these neurons. Given the strong evidence that this compound can modulate axonal ALP transport, Aβ42 and neuronal autophagy, the results from the proposed experiments could be relevant to therapeutic approaches in other neurodegenerative diseases that have ALP dysfunction as a contributing pathological feature, such as Parkinson’s disease, amyotrophic lateral sclerosis, and frontotemporal dementia.