Mechanisms of autophagy in iPS cell-derived cerebral cortical neurons

主题：世界级支撑生物科学从信号到参与的主要蛋白质复合体和伴随自噬形成的膜重排，已经相当详细地描述了氨基酸退出或基础自噬(在充分营养的情况下形成自噬)引起的自噬。虽然大多数工作都是在标准的哺乳动物组织培养细胞(HeLa，HEK-293，永生化的小鼠胚胎成纤维细胞)中完成的，但可以认为，研究这一途径的最重要的细胞模型是神经元。多年来，人们一直认为神经元没有自噬活动，但最近的研究表明，这种活动非常快(Maday和Holzbaur 2014 Dev Cell 30：71)，成熟的自噬小体在被溶酶体清除之前没有机会积累到可见的水平。事实上，自噬受损的小鼠的一个主要表型是新生代变性疾病(Komatsu等人2006自然441：880；Hara等人2006自然441：885)，大概是因为通常被自噬清除的聚集体在这些动物的神经元中积聚。然而，应该注意的是，提出受损的自噬是所有神经退行性疾病的基础-相反，似乎情况是自噬参与了许多此类疾病的主要或辅助因素(Menzies等人2015年NAT Rev Neurosc 16：345)。这项研究的目的是利用iPS细胞产生神经干细胞的最新进展来研究人类大脑皮层神经元的自噬。剑桥一家名为Axol的公司的主要活动之一是生产前体细胞并分化为在大多数方面与皮质神经元相似的神经细胞。在与Axol的科学家的合作中，我们已经证明了自噬报告可以在分化前的前体细胞中表达，然后在活细胞中跟踪自噬动力学。这是一个非常令人兴奋的发展，因为它将使我们能够将我们在研究自噬动力学方面的相当专业知识从组织培养细胞(Karanasios等人2014方法S1046；Karanasios等人2014 Curr Prot Cytom 69：12.34.1)转移到神经元中。简而言之，我们将使用标准方法产生表达4个荧光标记的自噬报告的前体iPS细胞，这些报告代表自噬生物发生的不同阶段。产生这样稳定的品系是很重要的，因为大多数自噬记者在瞬时转染环境中不能很好地工作。然后，这些细胞将被分化为皮质神经元，并建立自噬小体形成的动力学。一旦实现了这一点，我们将对阿尔茨海默病或亨廷顿病患者的iPS细胞进行同样的实验，也可以通过Axol获得。已经证明，阿尔茨海默病患者的皮质神经元在培养20天内开始分泌AB42肽，这表明它们至少概括了疾病病理的某些方面。我们将使用这些细胞来提出一个基本的问题：自噬途径如何对疾病表型的发生做出反应？这个问题的答案将对自噬和神经退化产生非常重要的影响。

Theme: World-Class Underpinning BioscienceAutophagy induced upon amino acid withdrawal or basal autophagy (autophagosome formation in the presence of full nutrients) have been described in considerable detail, from the signals involved to the main protein complexes participating and the membrane re-arrangements that accompany formation of autophagosomes. Although the majority of the work has been done in standard mammalian tissue culture cells (HeLa, HEK-293, immortalised mouse embryonic fibroblasts) it can be argued that the most important cell models in which to study this pathway are neurons. For many years it was thought that neurons have no autophagic activity, but recent work has instead revealed that such activity is very fast (Maday and Holzbaur 2014 Dev Cell 30: 71) and mature autophagosomes do not have a chance to build up to visible levels before clearance by the lysosomes. In fact, a major phenotype of mice compromised for autophagy is neorodegeneration disease (Komatsu et al 2006 Nature 441: 880; Hara et al 2006 Nature 441:885), presumably because aggregates that are normally cleared away by autophagy build up in the neurons of these animals. It should be noted however that it would be an ovesimplification to suggest that compromised autophagy underlies all neurodegenerative disesases-instead what appears to be the case is that autophagy is involved in many of these diseases either as a primary or as an auxiliary factor (Menzies et al 2015 Nat Rev Neurosc 16: 345).The object of this studentship is to take advantage of recent advances in production or neural stem cells from iPS cells in order to investigate autophagy in human cerebral cortical neurons. Production of the precursors and differentiation into neuronal cells resembling in most respects cortical neurons is one of the main activities of a company in Cambridge termed Axol. In collaboration with scientists from Axol we have already shown that an autophagy reporter can be expressed in the precursor cells before differentiation and then autophagosome dynamics can be followed in live cells. This is a very exciting development because it will allow us to transfer our considerable expertise in studying autophagy dynamics from tissue culture cells (Karanasios et al 2014 Methods S1046; Karanasios et al 2014 Curr Prot Cytom 69: 12.34.1) into neurons. Briefly, we will generate using standard methods precursor iPS cells expressing 4 fluorescently tagged autophagy reporters representing distinct stages of autophagoosme biogenesis. It is important to generate such stable lines because most autophagy reporters do not work well in transient transfection settings. These cells will then be differentiated into cortical neurons and the dynamics of autophagosome formation will be established. Once this is achieved we will do the same experiment with iPS cells from Alzheimer's or Huntigton's disease patients, also availabe by Axol. It has already been shown that cortical neurons from the Alzheimer's patients start secreting Ab42 peptide within 20 days of culture indicating that they recapitulate at least some aspect of the disease pathology. We will use these cells to ask a fundamental question: how does the autophagic pathway respond to the onset of the disease phenotype? The answer to this will have very important implications for autophagy and for neurodegeneration.