Characterization of Alternative Isoforms and Inclusion Body of the TAR DNA-binding Protein-43

Department of Neurology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2009; 285(1):608-19. DOI: 10.1074/jbc.M109.022012
Source: PubMed


TAR DNA-binding protein-43 (TDP-43) has been recently identified as a major component of the ubiquitinated inclusions found in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis, diseases that are collectively termed TDP-43 proteinopathies. Several amyotrophic lateral sclerosis-linked mutations of the TDP-43 gene have also been identified; however, the precise molecular mechanisms underlying the neurodegeneration remain unclear. To investigate the biochemical characteristics of TDP-43, we examined truncation, isoforms, and cytoplasmic inclusion (foci) formation using TDP-43-expressing cells. Under apoptosis, caspase-3 generates two 35-kDa (p35f) and 25-kDa (p25f) fragments. However, in caspase-3(-/-) cells, novel caspase-3-independent isoforms of these two variants (p35iso and p25iso) were also detected under normal conditions. With a deletion mutant series, the critical domains for generating both isoforms were determined and applied to in vitro transcription/translation, revealing alternate in-frame translation start sites downstream of the natural initiation codon. Subcellular localization analysis indicated that p35 (p35f and p35iso) expression leads to the formation of stress granules, cellular structures that package mRNA and RNA-binding proteins during cell stress. After applying proteasome inhibitors, aggresomes, which are aggregates of misfolded proteins, were formed in the cytoplasm of cells expressing p35. Collectively, this study demonstrates that the 35-kDa isoforms of TDP-43 assemble in stress granules, suggesting that TDP-43 plays an important role in translation, stability, and metabolism of mRNA. Our findings provide new biological and pathological insight into the development of TDP-43 proteinopathies.

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    • "1245 (EF_434182.1) and 783 (EF_434183.1) nucleotides encoding 43, 34 and 25 kDa isoforms, respectively, of TDP-43 (Nishimoto et al. 2010). "
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    ABSTRACT: Loss of function of TAR DNA-binding protein (TDP-43) has been implicated in neurodegenerative disorders in both humans and animal models. TDP-43 has also been shown to be cis-acting transcriptional repressor of the acrosome vesicle (Acrv) gene in mice. In the present study, we investigated the expression of the TDP-43 transcript (TARDBP) and protein in germ cells from 11 fertile and 98 subfertile men to verify its potential association with poor seminograms. The expression profile of TDP-43 was characterised in immature germ cells and spermatozoa from semen from fertile and subfertile men using reverse transcription-polymerase chain reaction, western blotting and immunofluorescence. Although germ cells from subfertile men tested negative for TARDBP, the full-length message of the same was detected in fertile men. TDP-43 was detected in spermatozoa from fertile men using western blot analysis and immunofluorescence. The expression of this protein was negligible in spermatozoa from men with primary spermatogenic dysfunction. We conclude that a deficiency in the TDP-43 expression is associated with defective spermatogenesis and male infertility. We propose that TDP-43 could be used as a marker of male factor infertility.
    Reproduction Fertility and Development 10/2014; DOI:10.1071/RD14090 · 2.40 Impact Factor
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    • "To ask whether transcriptional down-regulation by STS is the only mechanism for reduced levels of TDP- 43, we further looked at involvement of caspase-3 in TDP-43 proteolysis as it has been shown that TDP-43 is cleaved by caspase-3 (Dormann et al., 2009; Zhang et al., 2009; Nishimoto et al., 2010). As expected, 5uM STS induced activation of caspase-3 in U87 cells at 8 hours after treatment (Figure 3A), in addition, increased level of caspase-3 was activated when concentration of STS increased after 8 hours treatment (Figure 3B). "
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    ABSTRACT: TDP-43 is a ubiquitously expressed DNA/RNA binding protein that has recently attracted attention for its involvement in neurodegenerative diseases. While TDP-43 has been found to participate in various important cellular activities including stress and apoptosis, little is known about its role in cancer cells. Here we report that staurosporine (STS) induced apoptosis in U87 glioma cells is associated with rapid downregulation of TDP-43 at both mRNA and protein levels. The latter is dependent on activation of caspase 3. More importantly, we have shown that knockdown of TDP-43 by specific siRNA dramatically enhanced cytotoxicity of STS. These results suggest that normal level of TDP-43 may be protective for cancer cells under apoptotic insult.
    Asian Pacific journal of cancer prevention: APJCP 04/2014; 15(8):3575-9. DOI:10.7314/APJCP.2014.15.8.3575 · 2.51 Impact Factor
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    • "TDP-43 is a DNA/RNA binding protein of 43 kDa mainly localized in the nucleus, and has been implicated in transcriptional repression, pre-mRNA splicing and translational regulation [3], [4], [5]. In cell culture, overexpressed full length TDP-43 is localized mainly in the nucleus, whereas C-terminal fragments containing RNA recognition motif 2 (RRM2) and the glycine-rich domain are localized both in the nucleus and cytoplasm with formation of ubiquitinated inclusions in the latter compartment [6], [7], [8], [9], . Recent studies have shown that the TDP-43 C-terminal fragments are prone to aggregation and may serve as a seed to facilitate aggregation of full-length TDP-43 [10]. "
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    ABSTRACT: TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43M337V mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43wt or GFP-TDP-43M337V or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43M337V allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.
    PLoS ONE 03/2014; 9(3):e91269. DOI:10.1371/journal.pone.0091269 · 3.23 Impact Factor
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