Abstract

We have successfully developed the chick embryo model of ALS. We carried out overexpression of HA and Myc tagged TDP-43WT, TDP-43Q331K and TDP-43M337V constructs (Sreedharan et al, 2008). We also carried out over-expression of pEGFPC1 as a control. TUNEL staining demonstrated that GFP transfection resulted in low levels of apoptotic TUNEL +ve cells. However, embryos electroporated with the WT and MT forms of TDP-43 showed a marked increase. A two-tailed t test of GFP vs HA-TDP-43WT-Myc, GFP vs HA-TDP-43Q331K-Myc and GFP vs HA-TDP-43M337V-Myc gave p-values of 0.1252, 0.0179 and 0.0079 respectively. We also carried out electroporation of N-terminal GFP tagged constructs expressing the same wild-type and mutant genes (GFP-TDP-43WT, GFP-TDP-43Q331K and GFP-TDP-43M337V). TUNEL staining showed a statistically significant increase in the number of apoptotic nuclei in embryos electroporated with TDP-43WT, TDP-43Q331K and TDP-43M337V as compared to those expressing pEGFPC1. A two-tailed t test of GFP vs GFP-TDP-43WT gave a p-value of 0.0337; GFP vs GFP-TDP-43Q331K gave a p-value of 0.0026 and GFP vs GFP-TDP-43M337V gave a p-value of 0.0045. The mutant forms of TDP-43 showed predominantly cytoplasmic localisation as compared to TDP-43WT. Moreover, we observed nuclear aggregates in the sections of embryos electroporated with mutant TDP-43. A two-tailed t-test of GFP-TDP-43WT vs GFP-TDP-43Q331K gave a p-value of 0.012 and that of GFP-TDP-43WT vs GFP-TDP-43M337V gave a p-value of 0.009.