A novel Drosophila platform to replace the use of mice and zebrafish for the study of ER-mitochondria interactions.

A rapidly ageing population and the associated burden of age-related disease are two of the major societal and financial challenges facing developed and developing countries. Understanding the molecular processes that underpin ageing is a fundamental biological question and a critical step in designing interventions that could increase healthy life expectancy (healthspan). It is particularly important to understand the processes that contribute to ageing of neurons, as ageing is a major risk factor for many neurodegenerative diseases.

The proposed project will advance our basic understanding of the cell biology of neuronal ageing by shedding light on the molecular mechanisms by which the communication between the ER and the mitochondria affects neuronal functionality over time. Communication between the two organelles regulates a number of processes, for instance calcium and phospholipids exchange, essential for proper cellular functions. The project will take advantage of a new platform that we developed to monitor ER-mitochondria interactions in Drosophila melanogaster as part of a long-term project aimed at studying the cell biology of neuronal ageing.

Studying the cell biology of neurons in adult animals requires intravital or ex vivo imaging approaches, which in vertebrate model organisms are technically challenging and time-consuming. Moreover, such studies require a significant number of animals and often use surgical procedures. Our imaging system affords non-invasive, detailed imaging of intracellular dynamic processes in ageing neurons of ageing fruit flies. This system exploits the accessibility to microscopic observation of sensory neurons in the adult wing of Drosophila. The relatively short lifespan of fruit flies makes longitudinal studies feasible and the sophisticated genetic tools available in this organism greatly facilitate functional studies. Our methodology represents a significant advance for the field, allowing imaging of live neurons in an intact adult nervous system to be coupled to powerful genetic tools. With this system, we have been able to make significant progress towards understanding how specific neuronal functions decline during ageing.

In our past work, we have discovered a remarkable age-dependent decline in the axonal transport of mitochondria in adult neurons of Drosophila. Reduced transport contributes to the broader decline of neuronal homeostasis that occurs during ageing while upregulation of this process appears to be beneficial in older neurons. Although these findings provide a strong association between mitochondrial motility and neuronal function, it is still unknown how modulation of transport mechanistically affects neuronal ageing phenotypes. An exciting possibility is that the interactions between the mitochondria and the ER would directly regulate mitochondrial transport and functions thus significantly impacting on neuronal ageing.

By transferring our technology into the laboratory of Dr Tito Cali at the University of Padova (Italy), this work will reduce the number of mice and zebrafish used to study ER-mitochondria communications and further expand the utility of our Drosophila model to maximise its 3Rs potential.

Many functions essential for neuronal survival are regulated by specialised contact sites between two cellular compartments, the ER and the mitochondria. It has become increasingly clear that disrupting ER-mitochondria contacts sites is a mechanistic step in a number of neurodegenerative diseases. Restoring damaged ER-mitochondria associations is therefore an exciting therapeutic target to ameliorate neuronal functions in disease. However, it is not clear how to precisely modulate the association between the two compartments because the full repertoire of complexes that mediate ER-mitochondria interactions is yet to be discovered. Whether perturbing the physical connection between the two organelles can impact on the ageing process of neurons is still an open question.

The majority of studies to date have been performed in cultured cells and surprisingly little work has been done in model organisms or using in vivo approaches. This is a considerable limitation and it has prevented a full understanding of the ER-mitochondria axis and its implication in ageing and disease.

I have established a novel assay that allows imaging of live neurons in the intact nervous system of the adult Drosophila (PMID:26598558). Based on this assay, I recently developed a procedure suitable to image ER-mitochondria interaction and produced transgenic flies that harbour a new reporter of ER-mitochondria contact sites. By coupling this protocol to the powerful genetics of the fruit fly, we are able to specifically visualise and interfere with the ER-mitochondria interactions in vivo over the lifespan of the animal. Our new platform will also be crucial in future work aimed at discovering novel factors that affect ER-mitochondria interactions during ageing and in the diseases of the nervous system.

The work described in this application will replace a sizeable number of mice and zebrafish through transfer of competence from the 'developer' to the 'end-user' and have a significant impact on Replacement.

A large body of work from the Cali lab encompasses the use of primary neuronal cultures from mouse embryos and live imaging of zebrafish embryos. For ER-mitochondria studies only, approximately 30 pregnant mice and 50 adult zebrafish are used per year for dissection and breeding purposes, respectively. Our Drosophila model will initially replace the whole complement of rodents used to study ER-mitochondria biology and, by the end of the project, Dr Cali is keen to replace zebrafish for in vivo imaging due to its unsuitability for live imaging studies of neuronal ageing.
The immediate and deliverable 3Rs impact, achievable within the 18-month time frame set out in this project, is to replace 30 mice and 50 fish. Our Drosophila platform is ready to use, easily transferable into the Cali lab, and we do not foresee any barriers to adoption of the technique. We have published a detailed step-by-step protocol to encourage the uptake of the Drosophila system for the study of neuronal ageing and to promote reproducibility of the science. We believe this will further assist with the transfer process in the new lab.

Within the Padova Neuroscience Centre (PNC), Dr Cali collaborates with 5 groups using mouse neuronal cultures and zebrafish models to study neurodegeneration. These groups, who only use protected species as their model systems, have shown interest in our Drosophila platform as a valid alternative. This led to the idea of organising a training workshop at the PNC for future potential end-users, as well as a short course at the Neuroscience Graduate School, to encourage wider uptake of Drosophila in ageing and neurodegeneration studies.
At least 10 different research groups worldwide have resorted to primary neuronal cultures of rodents to study ER-mitochondria biology in the last year (PubMed search: November 2017-November 2018). In the same period, 9 different groups published studies on the cell biology of neuronal ageing in zebrafish. We therefore believe that, in a broader context, our Drosophila model has the potential to replace many more rodents that are used to obtain primary neuronal cultures as well as zebrafish used for breeding purposes. The uptake of our platform by the Cali lab is an essential step to achieve wider dissemination and to contribute bridging the '3Rs valley of death' (https://www.nc3rs.org.uk/news/bridging-3rs-valley-death-nc3rs-strategy-2017-2019).

We recognise that translating basic scientific findings to a clinical setting for the benefit of patients is a long process. With a view on improving the general translation and commercialisation of our science, and therefore increase the translatability of our 3R-minded strategy, we have established a collaboration with Eli Lilly through a concurrent BBSRC-funded project in the lab. They showed great interest in our Drosophila platform and in the topic discussed in this application (see attached letter of support). We agreed in principle to collaborate towards a pilot screen to find modifiers of ER-mitochondria interactions and, if successful in our application, we intend to include them as an additional 'end-user' in future studies. We believe that endorsement from our industry partner is crucial to build wider confidence in our model. The costs attached to studies of neuronal ageing in mice, but also zebrafish, are prohibitive for many labs. This, along with the time required for mice studies, is one of the reasons why the field of neuronal ageing is not very advanced compared to other areas of research. Our methodology will be of broad 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 completing a project.