Similar dose-dependence of motor neuron cell death caused by wild type human TDP-43 and mutants with ALS-associated amino acid substitutions

Journal of Biomedical Science, May 2013

Background TDP-43, a multi-functional DNA/ RNA-binding protein encoded by the TARDBP gene, has emerged as a major patho-signature factor of the ubiquitinated intracellular inclusions (UBIs) in the diseased cells of a range of neurodegenerative diseases. Mutations in at least 9 different genes including TARDBP have been identified in ALS with TDP-43 (+)-UBIs. Thus far, the pathogenic role(s) of the more than 30 ALS-associated mutations in the TARDBP gene has not been well defined. Results By transient DNA transfection studies, we show that exogenously expressed human TDP-43 (hTDP-43), either wild type (WT) or 2 different ALS mutant (MT) forms, could cause significantly higher apoptotic death rate of a mouse spinal motor neuron-like cell line (NSC34) than other types of cells, e.g. mouse neuronal Neuro2a and human fibroblast HEK293T cells. Furthermore, at the same plasmid DNA dose(s) used for transfection, the percentages of NSC34 cell death caused by the 2 exogenously expressed hTDP-43 mutants are all higher than that caused by the WT hTDP-43. Significantly, the above observations are correlated with higher steady-state levels of the mutant hTDP-43 proteins as well as their stabilities than the WT. Conclusions Based on these data and previous transgenic TDP-43 studies in animals or cell cultures, we suggest that one major common consequence of the different ALS-associated TDP-43 mutations is the stabilization of the hTDP-43 polypeptide. The resulting elevation of the steady state level of hTDP-43 in combination with the relatively low tolerance of the spinal motor neurons to the increased amount of hTDP-43 lead to the neurodegeneration and pathogenesis of ALS, and of diseases with TDP-43 proteinopathies in general.

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Similar dose-dependence of motor neuron cell death caused by wild type human TDP-43 and mutants with ALS-associated amino acid substitutions

Journal of Biomedical Science Similar dose-dependence of motor neuron cell death caused by wild type human TDP-43 and mutants with ALS-associated amino acid substitutions Lien-Szu Wu 0 1 Wei-Cheng Cheng 0 Che-Kun James Shen 0 1 0 Institute of Molecular Biology, Academia Sinica , Nankang, Taipei 115 , Taiwan 1 Institute of Molecular Medicine, National Taiwan University , Taipei , Taiwan Background: TDP-43, a multi-functional DNA/ RNA-binding protein encoded by the TARDBP gene, has emerged as a major patho-signature factor of the ubiquitinated intracellular inclusions (UBIs) in the diseased cells of a range of neurodegenerative diseases. Mutations in at least 9 different genes including TARDBP have been identified in ALS with TDP-43 (+)-UBIs. Thus far, the pathogenic role(s) of the more than 30 ALS-associated mutations in the TARDBP gene has not been well defined. Results: By transient DNA transfection studies, we show that exogenously expressed human TDP-43 (hTDP-43), either wild type (WT) or 2 different ALS mutant (MT) forms, could cause significantly higher apoptotic death rate of a mouse spinal motor neuron-like cell line (NSC34) than other types of cells, e.g. mouse neuronal Neuro2a and human fibroblast HEK293T cells. Furthermore, at the same plasmid DNA dose(s) used for transfection, the percentages of NSC34 cell death caused by the 2 exogenously expressed hTDP-43 mutants are all higher than that caused by the WT hTDP-43. Significantly, the above observations are correlated with higher steady-state levels of the mutant hTDP-43 proteins as well as their stabilities than the WT. Conclusions: Based on these data and previous transgenic TDP-43 studies in animals or cell cultures, we suggest that one major common consequence of the different ALS-associated TDP-43 mutations is the stabilization of the hTDP-43 polypeptide. The resulting elevation of the steady state level of hTDP-43 in combination with the relatively low tolerance of the spinal motor neurons to the increased amount of hTDP-43 lead to the neurodegeneration and pathogenesis of ALS, and of diseases with TDP-43 proteinopathies in general. TDP-43; ALS Mutations; Protein stability; Spinal motor neuron cells; Apoptosis Background The TAR-DNA-binding protein 43 (TDP-43)-encoding gene, TARDBP, is well conserved among the multicellular organisms from C. elegans to human [ 1,2 ]. Of the multiple isoforms encoded by the TARDBP gene, the 43 kDa TDP-43 protein is the most abundant one expressed in all tissues [ 3,4 ], mainly in the nucleus but some also residing in the cytoplasm [ 4,5 ]. TDP-43 appears to be a general transcription repressor [ 3,5,6 ], a splicing factor [ 7,8 ], and a neuronal activity-responsive factor [ 4 ]. Not surprisingly, intact TARDBP gene is indispensible for normal early development of the mouse embryos [ 9-12 ]. Lately, TDP-43 has emerged as the major patho-signature protein of the ubiquitinated intracellular inclusions (UBIs) in the diseased brain/ neuron cells of a range of neurodegenerative diseases, two major ones being the frontotemporal lobar degeneration with ubiquitin-positive, tau- and α-synuclein negative inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) [ 13-15 ]. Biochemical analyses have revealed that human TDP-43 (hTDP-43) is promiscuously modified/ processed in the affected regions of the brains and spinal cords of the FTLD-U and ALS patients, respectively [ 13-15 ]. Loss-of-function of TDP-43 as well as gain-of-cytotoxicity, as the result of the promiscuous modifications of TDP-43, have been suggested to lead to the pathogenesis of FTLD-U and as ALS with the TDP-43 (+) UBIs [6,15-18 and references therein]. The molecular and cellular basis for the pathogenesis of either ALS or FTLD-U is poorly understood yet. Mutations in 11 different genes, including the long studied superoxide dismutase 1 (SOD 1) and TARDBP, have been identified to be associated with 10% of ALS [ 19 ], which is a disease with age-dependent degeneration of the spinal cord motor neurons [ 20 ]. Furthermore, the majority of the ALS cases, including those the disease genes of which have not been identified yet, are signalized with the TDP-43(+)-UBIs [ 21 ]. Interestingly, more than 30 different ALS-associated TARDBP mis-sense substitutions have been identified, almost all of which are mapped in the glycine-rich domain of TDP-43 [ 15,16,22 ]. A number of DNA transfection/ microinjection experiments in cell cultures or cell lines have been carried out to analyze the cyto-toxicities of different ALS-associated hTDP-43 mutants in comparison to the wild type [ 23-27 ]. For instance, Q331K and M337V accelerate spontaneous hTDP-43 aggregation in yeast cells [ 23 ]. On the other hand, while both the wild type hTDP-43 and 3 mutant forms of hTDP-43 (A315T, G348C, and A382T) induce death of primary motor neurons but not cells from Neuro2a and COS cell lines, the mutant forms are more potent than the wild type hTDP- (...truncated)


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Lien-Szu Wu, Wei-Cheng Cheng, Che-Kun Shen. Similar dose-dependence of motor neuron cell death caused by wild type human TDP-43 and mutants with ALS-associated amino acid substitutions, Journal of Biomedical Science, 2013, pp. 33, 20, DOI: 10.1186/1423-0127-20-33