Why did we fund this project?
This award supports the transfer of the use of a non-invasive Drosophila imaging approach to replace mice and zebrafish in live imaging studies of organelle function and interaction in neuronal ageing.
Dr Alessio Vagnoni previously developed procedures for imaging axonal mitochondrial transport in the sensory neurons of the marginal nerve in the Drosophila wing. Since the wing is translucent and the fly has a short lifespan, axonal transport can be studied in the intact nervous system throughout ageing. During his David Sainsbury Fellowship, Alessio expanded the utility of the fly wing model as a potential replacement for the use of mice by developing new assays to assess interactions between the mitochondria and endoplasmic reticulum, which are often perturbed in neurodegenerative diseases, and to measure intracellular calcium levels following neuronal stimulation. The relatively short lifespan of Drosophila makes longitudinal studies of neuronal ageing more feasible and functional genetic studies can be performed by creating transgenic lines of flies. To validate the replacement potential of the fly model, comparative studies were conducted in the exposed sciatic nerve of ageing MitoMice – transgenic mice in which mitochondrially-targeted fluorescent proteins are selectively expressed in neurons.
Dr Tito Cali at the University of Padova works on mitochondria-ER interactions. With NC3Rs funding, Alessio will work with Tito to transfer the Drosophila model and imaging techniques replacing the use of some experiments using mice and zebrafish in his laboratory.
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, with NC3Rs funding, 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.
Mattedi F and Vagnoni A (2019). Temporal Control of Axonal Transport: The Extreme Case of Organismal Ageing. Frontiers in cellular neuroscience 13:393. doi: 10.3389/fncel.2019.00393
Mórotz GM et al. (2019). Kinesin light chain-1 serine-460 phosphorylation is altered in Alzheimer’s disease and regulates axonal transport and processing of the amyloid precursor protein. Acta Neuropathologica Communications 7(200). doi: 10.1186/s40478-019-0857-5