Modelling the neuronal cell biology of human ageing in vitro

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Aim 1 - Side-by-side comparison of protocol efficiencies to differentiate human dermal fibroblasts into neurons (YEAR 1). Two main routes are currently available to obtain fibroblast-induced neurons that retain the ageing signature of the donor. Route 1 involves direct reprogramming of the fibroblasts into neurons (PMID: 26456686); with Route 2, fibroblasts are first converted into induced neuronal progenitor cells (iNPCs) before differentiation into a neuronal lineage (PMID: 24379375). Both methods have been shown to yield neurons that retain a degree of the molecular age of the donor cells. However, a side-to-side comparison of the two methods have not yet been undertaken. This is a very important step towards modelling the cell biology of human ageing in vitro and to establish if the neurons obtained with the two methods are functionally equivalent. Therefore, as part of the assay setup, we aim to perform an initial comparison of the two methodologies. After differentiation, we will confirm neuronal identity by immunostaining for specific neuronal markers. The aged phenotype will be assessed via RNAseq to reveal the ageing signature of the neurons. Aim 2 - To measure mitochondrial transport and function in young and old neurons (YEAR 2). We will initially measure mitochondrial transport in induced neurons derived from human donors of different ages ('young' cells < 20 years, 'old' cells > 60 years). Mitochondria will be stained with the fluorescent vital dye MitoTracker and the axonal transport of these organelles recorded by time-lapse spinning disk microscopy for 3-5 minutes. We and others have showed that reduced mitochondrial transport is a hallmark of neuronal ageing in invertebrates and mice. This set of experiments is important to establish whether defective axonal transport is in fact an evolutionary conserved hallmark of neuronal ageing from invertebrates to humans. It has been suggested that mitochondria may display functional defects during ageing of model organisms, although it is not clear whether this is also true in ageing human neurons. To answer this question, the student will use a combination of vital dyes and reporters to measure the mitochondrial membrane potential and Ca2+ homeostasis in these cells. Aim 3 - To identify agonists of the cAMP/PKA pathway with a neuroprotective role in old neurons (YEAR 3). We found that chronic activation of the cAMP/PKA pathway in Drosophila neurons in vivo strongly stimulates mitochondrial transport during later life (PMID: 29606421). Remarkably, sustained feeding with the pathway agonist 8-Br-cAMP, while stimulating mitochondrial transport, also reduces the appearance of protein aggregation, indicative of improved protein homeostasis. We will undertake a pilot screen for chemical agonists of the cAMP/PKA pathway that would increase mitochondrial motility in older neurons, which is predicted to be neuroprotective. Pathway antagonists will be assayed in a secondary screen, depending on time availability towards the end of the project. As the cAMP/PKA pathway is involved in gene activation, we will take the most promising hits from our screen and perform RNAseq experiments in drug-treated old cells. Our aim is to match the functional data to transcriptomic analyses in order to discover potential mediators of pathway-induced neuroprotection during ageing.