Mechanisms of autophagy in iPS cell-derived cerebral cortical neurons

Theme: World-Class Underpinning Bioscience

Autophagy 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.