AXOTRANSMAP · Assembling a functional map of axonal signalling endosomes

Assembling a functional map of axonal signalling endosomes

Due to their specialised morphology made of somatodendritic and axonal compartments, neurons are able to propagate signals over long distances. Despite the important role played by electrical signals, long-range communication between the soma and distal synapses also occurs thanks to a specific process: the fast axonal transport. The so called retrograde fast axonal transport is carried out by specialized vesicles called signalling endosomes, which convey neuronal growth and differentiation factors or neurotrophins, along the axon towards the cell body. At this level, they would modulate genetic responses to regulate neuronal physiology and survival. Despite their crucial role, little is known about the identity and the dynamics of these carriers. Given the growing evidence linking axonal transport deficits to neurodegeneration, gaining a better mechanistic understanding of the make-up of these vesicles and their regulation is crucial. The aim of this project was to characterise the molecular content of the signalling endosomes and to build a physical and functional map of their transport in motor neurons, which are the target of important human diseases. For this purpose, I exploited a new developed strategy using a fragment of the tetanus neurotoxin (HCT) conjugated to ferromagnetic beads. Since the HcT-beads complex is transported in signalling endosomes with the neurotrophins, it constitutes the tool of choice to pull-down and magnetically isolate these vesicles. The main objective of my project was to purify those retrograde carriers from motor neurons generated from embryonic stem cells and analyse their molecular composition during their transport. I improved the purification protocol previously established in the laboratory, and demonstrated that the technique was highly specific as proteins already known to be involved in fast axonal transport were present. To further study the dynamics of these carriers and assess in more details how the proteins composing the signalling endosomes were changing during transport, I used different experimental time points and a highly quantitative strategy to study their molecular content. Our results showed the fast and progressive maturation of signalling endosomes with time, via the recruitment of specific proteins, and also highlighted potential new components that might play a role during axonal transport. Strikingly, retrograde carriers are specifically enriched in proteins known to be involved in neurodegeneration and infection of the central nervous system. The proteins whose recruitment is temporal-dependent had been experimentally validated and we showed that they are mostly involved in intracellular transport and maturation processes and also neuronal development and differentiation. This group of proteins include our novel candidates of interest, whose precise temporal recruitment might be essential for the transport of signalling endosomes and their regulation. Further investigation is under process to precise the function of those novel factors and their role in retrograde transport. This project allowed me to provide for the first time a quantitative analysis of the proteins contained in motor neuron signalling endosomes, and to assemble a map of these carriers mediating axonal transport. Since our data revealed the presence of many proteins known to be involved in neurodegenerative diseases, it constitutes a valuable resource to further our understanding of retrograde carrier molecular composition, dynamics and regulation in neurons, with direct links to a wide range of neurological diseases. Providing a complete characterisation of these vesicles contribute to a better understanding of axonal transport and to the identification of novel reliable biomarkers for neurodegenerative disorders, given that defects in axonal transport are known to contribute to a wide range of neurodegenerative diseases such as Huntington’s and Alzheimer’s diseases and motor neuron disease. Importantly, this project was made truly European thanks to many collaborations with leading researchers from the European Research Area (ERA), who contributed to achieve my research goals and thereby precise the causal relationship between deficits in axonal transport and neurodegeneration. This is of crucial importance since the general ageing of the European Union population is paralleled by an increase in the number of patients suffering from these devastating and extremely costly diseases. This imposes an ever-increasing burden on the health services of the Member States and on their social stability, as existing treatments are still limited and only alleviate the symptoms, rather than addressing the cause. Our findings and their exploitation will aid the understanding of the early phases of the pathogenesis of these neurodegenerative disorders and provide important clues for the development of novel therapeutic approaches, therefore contributing to ERA research excellence in the field. Since this project also revealed possible links with neurotropic infections, new collaborations within the ERA are also in process to further investigate how viral infection and spreading could occur in the central nervous system, thereby contributing to another field of research excellence. Our results are included and described in a peer-review publication currently in press, in Molecular and Cellular Proteomics (MCP). MCP Publication link (also attached as pdf): http://www.mcponline.org/content/early/2015/12/18/mcp.M115.051649.full.pdf+html Webpage for host laboratory projects (including this one): https://www.ucl.ac.uk/ion/departments/sobell/Research/GSchiavo/GScurrentProjects Relevant contact details: s.debaisieux@ucl.ac.uk giampietro.schiavo@ucl.ac.uk

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз