Selective neuronal autophagy in phosphorylated tau degradation and Alzheimer's disease

项目概要/摘要： 阿尔茨海默病（AD）病理学定义为细胞外淀粉样蛋白的异常蛋白聚集， β斑块和脑内细胞内过度磷酸化的tau缠结，导致进行性认知障碍， 下降然而，过多的β淀粉样蛋白靶向疗法产生的临床益处有限， AD中tau聚集的重要性的证据。具体来说，蛋白质降解网络， 包括自噬-溶酶体系统（自噬）和泛素-蛋白酶体系统（UPS）， 被认为是AD的缺陷，因此可能影响神经变性和细胞内tau蛋白的清除。 大脑探讨神经元蛋白质稳态和AD I中选择性蛋白质降解的机制， 使用诱导多能干细胞（iPSC）衍生的神经元系统（iNs）。具体来说，我们的实验室已经生成了 来自宗教秩序研究和拉什记忆和衰老项目（ROSMAP）队列的50多个人类iN系 其代表了晚发型AD的全部临床和神经病理学谱。在这些基因上 不同的iNs，我们以前测定了基础自噬通量和UPS能力。此外，蛋白质组学分析 这些相同iN的蛋白质印迹显示磷酸化tau蛋白种类和 与蛋白质聚集体的选择性自噬（aggregophagy）相关的蛋白质。特别感兴趣的是 泛素依赖的聚集性衔接蛋白，视神经磷酸酶（OPTN），显示最显着的 与我们的AD神经元中相关的聚集的磷酸化tau形式负相关。BCL-2相关 产气基因3（BAG 3）是另一种相关的聚集吞噬伴侣蛋白， 与兴奋性神经元内的tau稳态，并显示出对UPS的强代偿关系 在我们人类神经系统中的抑制作用。因此，我假设AD衍生的神经元具有减少的 暴露于AD相关神经毒性蛋白质和遗传毒性蛋白质后蛋白质降解活化的能力 自噬的调节，这导致磷酸化tau的积累和神经元活性的降低。 为了解决这个假设，我有两个目的：1）确定OPTN调节对神经元tau蛋白的影响 蛋白质稳态和神经元功能。为此，我将AD和非AD iN暴露于含有 神经毒性蛋白质和测量几种形式的tau和神经元放电。2)为了研究BAG 3- 分子伴侣介导的选择性自噬在调节人类蛋白质降解补偿中的作用 神经元在这里，我将调节BAG 3在AD和非AD iNs中的表达，并将这些iNs暴露于AD脑中。 含有神经毒性蛋白质的提取物。然后我将测量tau蛋白的积累，神经元放电和蛋白质周转。 总的来说，这项提议的发现不仅将进一步了解tau蛋白降解， 但将定义在细胞中控制OPTN和BAG 3调节和功能的分子机制。 神经元，并可能导致新的治疗方式。

Project Summary/Abstract: Alzheimer’s disease (AD) pathology is defined by aberrant protein aggregation of extracellular amyloid- beta plaques and intracellular hyperphosphorylated tau tangles within the brain, resulting in progressive cognitive decline. However, a plethora of amyloid-beta targeting therapies have yielded limited clinical benefit, lending credence to the significance of tau aggregation in AD. Specifically, the protein degradation network, which includes the autophagy-lysosomal system (autophagy) and the ubiquitin-proteasome system (UPS), is frequently cited as deficient in AD, and may therefore affect neurodegeneration and intracellular tau protein clearance within the brain. To investigate the mechanisms of selective protein degradation in neuronal proteostasis and AD I will use an induced pluripotent stem cell (iPSC)-derived neuronal system (iNs). Specifically, our lab has generated over 50 human iN lines from the Religious Order Study and Rush Memory and Aging Project (ROSMAP) cohorts which represent the full clinical and neuropathological spectrum of late onset AD. Across these genetically diverse iNs, we previously assayed basal autophagic flux and UPS capacity. Additionally, proteomic analysis and western blotting of these same iNs revealed strong associations between phosphorylated tau species and proteins relevant in selective autophagy of protein aggregates (aggrephagy). Of particular interest is the ubiquitin-dependent aggrephagy adaptor protein, optineurin (OPTN), which displays the most significant negative correlation with aggregated phospho-tau forms relevant in our AD neurons. BCL-2-associated athanogene 3 (BAG3) is another relevant aggrephagy chaperone protein that has previously been associated with tau homeostasis within excitatory neurons and shows a strong compensatory relationship upon UPS inhibition in our human neuronal system. Therefore, I hypothesize that AD derived neurons have a diminished capacity for protein degradation activation following exposure to AD relevant neurotoxic proteins and genetic modulation of autophagy, which results in an accumulation of phospho-tau and a reduction in neuronal activity. To address this hypothesis, I have two aims: 1) To determine the influence of OPTN regulation on neuronal tau proteostasis and neuronal function. To this end I will expose both AD and non-AD iNs to brain extract containing neurotoxic proteins and measure several forms of tau and neuronal firing. 2) To investigate the role of BAG3- mediated chaperone-assisted selective autophagy in modulating protein degradation compensation in human neurons. Here, I will modulate BAG3 expression in both AD and non-AD iNs and expose these iNs to AD brain extract containing neurotoxic proteins. I then will measure tau accumulation, neuronal firing, and protein turnover. Taken together, the findings of this proposal will not only further the understanding of tau protein degradation via aggrephagy but will define the molecular mechanisms governing OPTN and BAG3 regulation and function in neurons and may lead to novel therapeutic modalities.