Mutual regulation between RE1 Silencing Transcription Factor (REST) and Mitochondria in Neurodegenerative Disease

阿尔茨海默病（AD）是老年人中最常见的痴呆形式，全世界有超过4000万人受到影响。AD被认为是由β-淀粉样蛋白（Ab）和tau的神经毒性蛋白聚集体引起的，这些蛋白聚集体在衰老的大脑中积累。然而，这一观点受到临床观察和针对这些神经毒性蛋白的药物测试的AD临床试验的反复失败的挑战。最近的证据表明，线粒体功能障碍先于Ab斑块和神经元tau缠结的形成，并且Ab和tau不是AD的主要原因，而是通过加剧氧化损伤促进神经变性。AD发展的主要危险因素是衰老，衰老大脑的共同特征是神经元代谢活性降低、氧化应激增加和氧化损伤累积。 据推测，这种扰动的神经元代谢触发了进一步的线粒体功能障碍和氧化应激的粘性循环，最终导致神经变性和AD。最近的一项发现表明，RE 1沉默转录因子（REST）在健康老化的大脑中被重新激活，并控制着一个广泛的基因网络，这些基因网络可以防止细胞死亡，炎症和AD病理。 然而，神经元REST水平在衰老过程中如何调节的确切机制仍然难以捉摸。这一提议是基于初步证据，表明REST通过限制线粒体功能来控制细胞能量水平。有趣的是，线粒体应激增加导致神经元REST的完全缺失。这些发现表明，REST是一种新的反馈机制的一部分，该机制通过删除REST蛋白将初始线粒体生物能量扰动与转录崩溃耦合。这种调节机制可能将健康的大脑衰老与神经退行性变和认知能力下降区分开来，应该进一步阐明。因此，目的是首先使用CRISPR/Cas9系统建立REST敲除诱导的多能干细胞（iPSC）系。随后，它们将分化为神经元，以评估REST敲除条件下的线粒体功能。其次，我将评估自噬对REST表达和降解的参与。最后，内源性REST将通过CRISPR/Cas9在源自AD患者和年龄匹配对照的iPSC系中用荧光蛋白标记。这些细胞系将首先用于表征AD体外模型中的REST表达。最后，它们将用于阐明线粒体应激和自噬对REST蛋白水平的影响。

Alzheimers disease (AD) is the most common form of dementia among the elderly with over 40 million individuals affected worldwide. AD is considered to be caused by neurotoxic protein aggregates of beta-amyloid (Ab) and tau that accumulate in the aging brain. However, this view is challenged by clinical observations and by the repetitive failure of AD clinical trials testing drugs that target these neurotoxic proteins. Recent evidence suggests that mitochondrial dysfunction precedes the formation of Ab plaques and neurofibrillary tau tangles, and that Ab and tau are not the primary cause of AD but rather promote neurodegeneration by exacerbating oxidative damage. The main risk factor for the development of AD is aging, and a common feature of the aging brain is reduced neuronal metabolic activity, increased oxidative stress and accumulating oxidative damage. It is hypothesized that such perturbed neuronal metabolism triggers a viscous cycle of further mitochondrial dysfunction and oxidative stress, which eventually results in neurodegeneration and AD. A recent discovery demonstrated that the RE1 silencing transcription factor (REST) is reactivated in the healthy ageing brain and controls a broad network of genes that protect from cell death, inflammation and AD pathology. However, the exact mechanism how neuronal REST levels are regulated during ageing remained elusive. This proposal is based on preliminary evidence indicating that REST controls cellular energy levels by restricting mitochondrial function. Interestingly, increased mitochondrial stress leads to the complete deletion of neuronal REST. These findings suggest that REST is part of a novel feedback mechanism that couples initial mitochondrial bioenergetic perturbations with a transcriptional collapse by deleting REST protein. This regulatory mechanism might distinguish healthy brain ageing from neurodegeneration and cognitive decline and should be further elucidated. Thus, the aim is to first establish REST knock out induced pluripotent stem cell (iPSC) lines using the CRISPR/Cas9-system. Subsequently they will be differentiated into neurons to assess mitochondrial function in REST knock out conditions. Secondly, I will assess the involvement of autophagy on REST expression and degradation. Lastly, endogenous REST will be tagged with a fluorescent protein in iPSC lines derived from AD patients and age matched controls, by CRISPR/Cas9. These lines will be used to firstly characterize REST expression in an in vitro model of AD. Finally, they will be used to clarify the implication of mitochondrial stress and autophagy on REST protein level.