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

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.

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.

The work that we propose in this application will have a significant impact on each of the 3Rs.

- Replacement.
This proposal is based on a new in vivo system that I independently established in my current laboratory in order to study neuronal ageing in Drosophila. This represents a unique alternative to existing vertebrate (mostly murine) models, which will therefore be completely replaced by the Drosophila system in the initial phase of the project (Phase1 of the Case for Support). A PubMed search revealed that in the two years from May 2013 to May 2015, at least 45 research groups worldwide resorted to vertebrate models to study mitochondrial transport. Therefore, I believe that the Drosophila system has the potential to replace vertebrate models in many future studies.

During the second phase of our project, I aim to partially replace mouse models by exploiting the availability in the Schiavo lab of motor neurons differentiated from mouse embryonic stem cells (Terenzio et al, 2014). I will use these neurons to validate key findings from Drosophila and to establish the appropriate dosage of agonists of the cAMP/PKA pathway for imaging of sciatic nerves in live mice.

-Refinement.
It is important to validate the physiological relevance of our study to humans. We therefore cannot bypass the use of an in vivo mammalian system for imaging and pharmacological studies. However, to refine the experiments in a way that would be the least harmful for the animals, I will use the in vivo imaging procedure established in the Schiavo lab (Bilsland et al, 2010). Because imaging is performed on the exposed sciatic nerve of anaesthetised mice, it will be possible to perform pharmacological studies initially by local application of drugs on the exposed nerve just prior to imaging. By avoiding oral or intravenous administration of the drugs, possible side effects and unnecessary animal suffering will be minimised.

-Reduction.
In the first part of the project, our replacement strategy will lead to a 100% reduction of mice used. We estimate that we will image neurons from at least 400 flies and that many more will be used for biochemical studies and genetic crossing. Thus, a large number of animals would be necessary if this study were to be conducted in mice.

The use of motor neurons during the second part of the project will result in a significant reduction of the number of mice used. Based on previous studies in which axonal transport was imaged from tissue explants of mice (see, for instance, (Milde et al, 2015)), the use of cultured cells is thereby predicted to reduce by >85% the number of animals that would otherwise be required to achieve the aims of this part of the project (Phase2 of the Case for Support).

Crucially, during our imaging experiments in mice, three treatments - vehicle and drug at two different concentrations - will be applied sequentially in the same mouse (Aim 2.2 of the Case for Support). The sciatic nerve from the same animal will also act as a control (i.e. pre-treatment) before being repeatedly challenged. Therefore, with our protocol one mouse will be used for work that could require four mice (i.e. a net 75% reduction in animal usage for these experiments). Based on my power calculations (Supporting Information to the Animal Species section), this will save 162 mice for this set of experiments.

The costs attached to studies of neuronal ageing in mice are prohibitive for many labs. This, along with the time required for those studies, is one of the reasons why the field of neuronal ageing is not very advanced compared to other areas of research. Our approach will therefore be of a broader appeal as it significantly cuts the costs involved for this type of research, allows sophisticated genetic experiments to be performed and provides a reasonable timeframe for planning and completing a project. We believe our approach will significantly expand the interest in neuronal ageing research.