Dr Alessio Vagnoni was awarded funding to build confidence in the use of Drosophila to study mitochondrial transport in neuronal ageing and neurodegeneration to replace some studies involving rodents.
Principal Investigator: Dr Alessio Vagnoni
Organisation: King's College London
Award type: David Sainsbury fellowship
Start date: 2016
Duration: 3 years
Deficiencies in the active intracellular transport of organelles, such as mitochondria, along axons are associated with neuronal ageing and are a hallmark of many neurodegenerative diseases. Understanding the cell biology of neuronal ageing could lead to the identification of new targets for therapeutic intervention. To date axonal transport has been extensively studied in cultured primary neurons and tissue explants, however, these models do not always consistently reflect what happens in vivo because of the lack of other cell types (e.g. muscle cells), myelination and excitatory/inhibitory input into cell bodies.
Recent advances in intravital techniques have allowed the process to be monitored in real time in vivo in a range of species. Studies in mice have focused on axonal transport in the sciatic nerve and axons of the spinal cord and dorsal roots. The procedures are invasive (involving surgery), technically demanding and require the generation of specific transgenic lines expressing fluorescent proteins to track the movement of ‘cargo’ along the axon. They are also limited by tissue transparency and imaging depth, and the relatively long lifespan of mice makes studying the cell biology of neuronal ageing difficult.
Working with Dr Simon Bullock at the MRC Laboratory of Molecular Biology, Alessio had previously developed non-invasive 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. This combined with the genetic tools available to manipulate the Drosophila genome (including the generation of neurological diseaserelated models) provides a powerful system for understanding neuronal ageing, with advantages over other in vivo approaches.
3Rs benefits (actual and potential)
During his Fellowship, Alessio expanded the utility of the fly wing model 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. To validate the model as a potential replacement for rodents, comparative mitochondrial trafficking studies were conducted in the exposed sciatic nerve of ageing MitoMice – transgenic mice in which mitochondrially-targeted fluorescent proteins are selectively expressed in neurons. The findings are being prepared for publication.
As an additional 3Rs advance, working with Professor Giampietro Schiavo at the Institute of Neurology, Alessio also maximised the use of the MitoMice. After the imaging studies, neurons from the dorsal root ganglion of young and old mice were rapidly extracted and cultured, providing an opportunity to compare the in vivo and in vitro properties of two subsets of sensory neurons derived from the same animal. This correlation between the animal and in vitro data could help to reduce the numbers of mice used as well as facilitating the use of functional assays that cannot be performed in vivo.
Scientific and technological benefits
Previous work by Alessio and colleagues had shown that the number of actively transported mitochondria declines significantly in the wing sensory neurons during ageing. In his Fellowship, Alessio demonstrated that the cAMP/protein kinase A (PKA) pathway promotes mitochondrial transport in adult Drosophila wing neurons and that feeding aged flies with a small molecule agonist of PKA is sufficient to suppress the decline in mitochondrial transport, with upregulation of the motor protein, kinesin-1, an important output for PKA activation.
Evidence suggests that axonal transport of Amyloid Precursor Protein (APP), which plays a major role in the development of Alzheimer’s disease, requires kinesin-1. Based on the strength of the fly wing model, Alessio has established a collaboration with Professor Chris Miller at King’s College London generating mutant Drosophila to measure in vivo axonal transport of vesicles containing APP in ageing animals. This has avoided the use of approximately 160 mice. Alessio also has active collaborations to replace the use of rodents and zebrafish with flies in laboratories in the UK and Italy.
Alessio has published two first author papers in Nature Protocols and Current Biology, highlighting key findings from the project, as well as being a co-author on a paper reviewing methodological advances in intravital imaging. He has given two public presentations on the importance of the 3Rs in research on neurodegeneration at ‘Pint of Science’ festivals held in Cambridge and London in 2017.
Alessio is now building an independent research career in neuronal ageing at King’s College London, securing £500k to start his own laboratory. The funding includes a van Geest Fellowship and a BBSRC Collaborative Training Partnership PhD award with Ely Lilly to study the neuronal cell biology of human ageing in vitro. Alessio has also received a UK-Israel Science Lectureship grant from the British Council and the UK Science & Innovation Network. The grant will support a series of lectures at Israeli universities and strengthen collaborations between laboratories, allowing Alessio to further disseminate his fly model.
This case study was published in our 2019 Research Review.