A novel RNAi screen for neurotrophin receptor internalisation and trafficking in motor neurons.
Doctoral thesis, UCL (University College London).
A primary focus of the Molecular Neuropathobiology laboratory is the investigation of the long range trafficking of neurotrophins and neurotrophin receptors in motor neurons (MNs). The goal of my project was to deepen our understanding of the nature of the cellular machinery controlling long-range neurotrophin trafficking in MNs, by discovering new players involved in this process. In order to achieve this goal I performed an siRNA screen in MNs derived from mouse ES cells to monitor the cell surface binding and internalization of two fluorescently tagged reporters: the binding fragment of the Tetanus neurotoxin (HC), which enters an axonal transport compartment shared with neurotrophins and their receptors, and an antibody directed against the extracellular domain of the neurotrophin receptor p75NTR. A high-throughput, lipidbased siRNA transfection method was optimised for ES cell-derived MNs and used to screen a library of siRNAs directed against a pool of genes involved in endocytosis and membrane trafficking. The primary candidate genes were subsequently validated, and one gene in particular, Bicaudal D homolog 1 (BICD1), was selected for further analyses. BICD1 is a member of the Bicaudal D family, whose members function as molecular motor adaptors with pleiotropic roles in intracellular trafficking. Bicd1 expression at E12.5 and 13.5 was restricted to the nervous system, suggesting an important role for BICD1 in neurons. I used gene-trapped ES cells to derive MNs depleted of the BICD1 protein (Bicd1gt/gt MNs), which, when challenged with either HC or the p75NTR antibody, displayed an increased intracellular accumulation of both probes. Furthermore, I found that the level of neurotrophin receptors exposed at the plasma membrane was increased in these cells compared to their wild type counterparts, suggesting that BICD1 might be involved in the regulation of neurotrophin receptor dynamics in mammalian neurons. I also found that TrkB signalling upon stimulation with the brain-derived neurotrophic factor (BDNF) in Bicd1gt/gt MNs, was impaired. This suggests that the depletion of BICD1 not only affects the trafficking of neurotrophin receptors, but also their signalling capabilities. In conclusion, this thesis work has demonstrated that the concept of high throughput screening can be applied to cells notoriously difficult to handle and transfect, such as MNs. This approach was successful in unravelling a new role for BICD1 in neurons, where it appears to regulate the intracellular trafficking and signalling of neurotrophin receptors. Taken together with the in vivo expression data, these data suggest that BICD1 plays an important role in the development and function of the nervous system.
|Title:||A novel RNAi screen for neurotrophin receptor internalisation and trafficking in motor neurons|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences|
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