Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, C09
TDP-43 causes defects in motor axon projections
1. King's College London, London, United Kingdom.
Progress in the MND field has been fuelled by the discoveries that mutations in RNA-processing proteins are causative in ALS. We and others have identified mutations in the genes encoding TDP43 in patients with ALS and FTLD-U. TDP43 is a RNA-processing protein, with DNA and RNA binding properties and multiple roles in neuronal survival and function. In this study, we have analysed the role of TDP43 in an embryonic in vivo system by testing the effects of over-expression of wild-type and mutant TDP43 on motor neuron viability, axon outgrowth, branching and cytoskeletal integrity. By understanding the relative impact of TDP43 mutant and wild-type forms on cytoskeletal integrity, we demonstrate a link between TDP43 proteinopathy and axon cytoskeleton. We have used the chick embryo as an acute model for TDP43 function. We electroporated GFP labelled TDP43 wild-type and mutant constructs in the chick embryo spinal cord (GFP-TDP-43WT, GFP-TDP-43Q331K and GFP-TDP-43M337V). The GFP fluorescence was widespread in the spinal cord demonstrating that almost 90-95% of NPCs were transfected. TUNEL staining showed a statistically significant increase in the number of apoptotic nuclei in embryos electroporated with TDP-43WT (p-value 0.0337), TDP-43Q331K (p-value 0.0026) and TDP-43M337V (p-value 0.0045) as compared to those expressing pEGFPC1. We next tested if there was any mis-localisation of TDP-43 proteins in our in vivo chick spinal cord model system. At E3.5 wild-type TDP-43, myc/HA and GFP tagged, protein was predominantly nuclear. However, the mutant forms of TDP 43 (particularly those tagged with GFP) showed more frequent cytoplasmic localisation aggregates while retaining the nuclear expression. We also demonstrate that at E6.5 all forms were detected cytoplasmically and in axons, suggesting an enhanced cytoplasmic localisation over time. We have demonstrated that at E6.5, TDP43 wild type and mutant forms lead to down-regulation of neurofilament associated protein. Careful confocal analysis demonstrated that the mis-localisation of TDP43 protein to the axon cytoplasm is accompanied by premature truncation of the axon projections and premature de-fasciculation of the axon bundles. In order to achieve statistical values for the effect of TDP43 on the length of axon projections, we carried out transfection of TDP43 in primary chick spinal motor neurons. Our in vitro data suggests a decrease in the total process length which is actually due the decrease in length of the longest process. The chick model offers a relatively quick and cost-effective means of validating genetic discoveries and will provide valuable insights into disease mechanisms.
Where applicable, experiments conform with Society ethical requirements