| Summary: | 博士 === 國立陽明大學 === 生化暨分子生物研究所 === 100 === TAR DNA-binding protein 43 (TDP-43) is a nuclear RNA/DNA-binding protein that is important for neuronal functions through regulating gene transcription, mRNA splicing, mRNA stability and microRNA biogenesis. Several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are characterized by inclusion bodies formed by TDP-43. Pathological TDP-43 undergoes several posttranslational modifications such as cytoplasmic translocation, proteolytic cleavage, hyper-phosphorylation at Ser379, 403, 404, 409 and 410 five residues. The effects of these posttranslational modifications on aggregation propensity of TDP-43 and their pathogenic roles in these diseases remained to be defined at the start of this thesis work. To address these issues, we established cell and transgenic Drosophila models with expression of full length or truncated forms of TDP-43. We demonstrated that the truncated forms of TDP-43 such as ND251 and ND207, with the N-terminal 251 and 207 amino acids deleted, respectively, had higher aggregation propensity than full length TDP-43 (fTDP), and formed aggregates recapitulating important features of TDP inclusions in ALS/FTLD-U. These results were consistent with the current literature that proteolytic C-terminal fragment facilitated TDP aggregation. Using various approaches including fluorescence resonance energy transfer technique, our data suggested that N-terminus of TDP-43 interacted with the C-terminus and prevented TDP-43 from self-aggregation. Moreover, overexpression of N-terminal fragment spanning the first 104 amino acids (N104) of TDP-43 significantly decreased the number of aggregates formed by ND251. These results showed that the N-terminus of TDP-43 might serve the basis for development of therapeutic peptide drug.
Hyper-phosphorylation was currently believed to promote TDP-43 aggregation because of its close link with pathogenic TDP species and previous studies which showed that phosphorylation of those five serine residues by casein kinase (CK) 1 and 2 promoted the propensity of TDP to form filaments in vitro. However, the effect of these phosphorylation sites on the aggregates formation in vivo remains to be determined. In this thesis, we demonstrated hyper-phosphorylation and hyper-ubiquitination indeed were tightly associated with aggregates formed by truncated TDP species. However, they occurred temporally later than aggregation in cell culture models. To our surprise, mutation of these serines to alanines (S5A) to eliminate the phosphorylation potential of these serine residues increased the number of aggregates of truncated TDPs; in contrast, mutations to aspartic acids (S5D) or glutamic acids (S5E) to simulate hyperphosphorylation status exerted the opposite effects. Western blot analyses showed a corresponding change in the aggregation propensity of the truncated TDPs. In addition, CK2α overexpression decreased the aggregation propensity of ND251 or ND207. Functionally, ND251 or ND207 aggregates decreased the number of neurites of Neuro2a cells induced by retinoic acid and slowed down the growth curve of Neuro2a cells by MTT assay, indicative of the cytotoxic effects of TDP aggregates. S5A mutation aggravated, but S5E mutation alleviated these cytotoxic effects. To verify these results in vivo, several lines of transgenic fly models including ND251, ND251S5A and ND251S5E were developed. Consistent with previous observations, ND251 and ND251S5A formed numerous aggregates in neurons, and salivary gland of transgenic Drosophila, but ND251S5E did not. Taken together, our data indicated that, contrary to the current thought, hyperphosphorylation likely represented a cellular defense mechanism to stop or prevent pathogenic TDP from aggregation. Therefore, enhancement of phosphorylation or delivery of N-terminal TDP peptide may serve as an effective therapeutic strategy against ALS/FTLD-U.
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