A new Drosophila-based strategy to study mitochondrial transport and neuronal ageing in vivo.

Lead Research Organisation: MRC Centre Cambridge
Department Name: LMB Cell Biology

Abstract

The world population is ageing rapidly. By 2047, the number of people aged 60 and over is expected to exceed the number of children and adolescents aged under 16 (UNDESA, World Population Ageing 2013). Ageing is the main risk factor for dementia and many other neuronal disorders affect individuals only in later life. Contrarily to other age-related diseases, a cure or treatment for dementia is not available. Finding measures to improve the health of ageing neurons is therefore crucial to ease the increasing societal and financial burdens associated with age-related diseases. How neurons age is also a fascinating, and poorly understood, intellectual problem.

A growing body of work suggests that correct distribution of cellular constituents is crucial for ensuring proper function of the nervous system in later life. For example, many studies have implicated defective axonal transport of organelles in the pathogenesis of various neurological disorders. A current hypothesis in the field is that interventions that increase transport of organelles would delay the onset of neuronal dysfunction during ageing. However, the mechanisms that regulate axonal transport in ageing neurons are poorly understood, partly because of lack of suitable models to perform longitudinal studies. Although appropriate mouse models exist, longitudinal studies in mice are very challenging because of time and costs involved. In addition, surgery is required to allow imaging of axonal transport in live mice.

I recently developed a new method to study in detail the intracellular transport of organelles, which uses the fruit fly Drosophila melanogaster. As this assay exploits the accessible position of neurons in the translucent wing, the procedure is non-invasive. Combining the powerful genetics of Drosophila with time-lapse live imaging, I am able to follow the transport of organelles in live animals of different ages. Importantly, the relatively short lifespan of Drosophila makes longitudinal studies feasible. Many research groups worldwide currently use vertebrate whole animal models, ex vivo explants and primary cultures to study axonal transport. I believe that the unique advantages of the Drosophila system mean that in can replace vertebrate models in many future studies of axonal transport and neuronal ageing.

I have discovered a remarkable age-related decline in the axonal transport of mitochondria in wing neurons. I increased transport of mitochondria in this system by manipulating the transport machinery an observed a substantial suppression of age-dependent neuronal dysfunction. During the course of my studies, I also found evidence of an evolutionarily conserved signalling pathway that upregulates mitochondrial transport in axons of ageing neurons. The main aim of my research will be to understand the molecular mechanisms linking this specific signalling cascade to mitochondrial transport and neuronal ageing. This would for the first time define a signaling cascade that could upregulate mitochondrial transport in ageing neurons and hence better inform future therapeutic efforts to combat age-related diseases..

To address this question, I will take a multidisciplinary approach by integrating the innovative assay in Drosophila with work in mammalian neurons. Initially, CRISPR genome-editing tools (optimised in the host lab) will be used in combination with biochemistry and quantitative time-lapse imaging of axonal transport in living Drosophila. Key findings will then be validated in motor neurons derived from mouse embryonic stem cells and in sciatic nerves of mice in vivo. By performing much of the work in Drosophila, only a small number of mice will be needed. These animals will be used to test the broader relevance of my findings, with the results potentially may have a significant translational impact on human ageing.

Technical Summary

This proposal is based on a new in vivo system that I have established to study axonal transport of organelles in wing sensory neurons of Drosophila melanogaster. This system allows, for the first time, organelle transport to be studied in intact adult neurons of living Drosophila over time. Longitudinal studies in this system have revealed a remarkable age-dependent decline in mitochondrial transport. My previous data suggest that experimental upregulation of mitochondrial motility delays age-associated protein aggregation and increases neuronal healthspan. I also found compelling evidence that an evolutionarily conserved signalling pathway can regulate mitochondrial transport in axons of ageing neurons. I propose to exploit this innovative imaging assay to understand the molecular mechanisms linking this specific signalling cascade to mitochondria transport and neuronal ageing. Initially, I will undertake a biochemical characterisation of this signalling pathway in Drosophila, including the identification of downstream targets that regulate transport. By using CRISPR genome engineering and tissue specific RNAi,I will attempt to identify the key regulatory nodes of the pathway. This will be followed by phenotypic analysis of neuronal function. After the Drosophila work, I will test the relevance of our findings in mammalian neurons. These experiments will be performed in cultured mouse motor neurons derived from embryonic stem cells in which mitochondria will be fluorescently labeled with a commercial dye. Finally, I will explore whether chemical activation of the pathway is sufficient to increase mitochondrial trafficking in single neurons of mouse sciatic nerve in vivo. To achieve this, I will use an available transgenic mouse strain, known as MitoMouse, which expresses a fluorescent marker of mitochondria in neurons. In these experiments,mitochondrial transport in young and old mice will be compared before and after challenging the neurons with pathway agonists.

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