Neurons connect to their remote targets via axons, which usually survive for the lifetime of an organism. Spatiotemporal regulation of the axonal proteome by local protein synthesis (LPS) plays a critical role in neuronal wiring and axon survival, raising the intriguing possibility that some neurological disorders involve LPS dysfunction. To visualise LPS in situ, I optimised multiple imaging techniques to investigate Netrin-1-induced translation in cultured retinal axons. Total axonal protein synthesis measured by metabolic and puromycin labelling indicates axons experience stage-dependent alterations in translation rate upon Netrin-1 stimulation. Remarkably, Netrin-1 triggers a burst of β-actin synthesis starting within 20 seconds of cue application at multiple non-repetitive sites visualised by single molecule translation imaging, an approach that allows direct visualisation of translation dynamics in response to external stimuli. Further studies have shown that local translation can occur on Rab7a-associated late endosomes, where mRNA recruitment and translation are coordinately regulated. Notably, mRNAs encoding mitochondria-related proteins are found translating on late endosomes docking in the vicinity of mitochondria, suggesting late endosomes act as ‘platforms’ for the localised synthesis of mitochondrial proteins necessary for maintaining mitochondrial integrity. Moreover, this process is affected in axons expressing the Charcot-Marie-Tooth disease type 2B (CMT2B)-related Rab7a mutants, leading to abnormal mitochondrial biogenesis and activity and compromised axon survival. Finally, attenuated de novo protein synthesis is observed in axons expressing amyotrophic lateral sclerosis (ALS)-associated fused in sarcoma (FUS) mutants and hypomethylated wild-type FUS. Live imaging reveals mislocalised mutant or hypomethylated FUS granules are transported along axons and accumulate at growth cones, possibly irreversibly trapping RNA molecules, resulting in reduced distance travelled by RNA granules in axons. Furthermore, mutant FUS expression results in defective retinal projections in vivo, highlighting the importance of RNA metabolism and local translation in axonal homeostatic mechanisms. In conclusion, aberrant translational activity in axons leads to prominent axonopathy, which recapitulates features of early stages of neurological diseases, providing the basis for novel therapeutic strategies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:745031 |
| Date | January 2018 |
| Creators | Lin, Qiaojin |
| Contributors | Holt, Christine |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/273662 |
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