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Liu-Yesucevitz L, Bilgutay A, Zhang YJ, Vanderweyde T, Vanderwyde T, Citro A, Mehta T, Zaarur N, McKee A, Bowser R, Sherman M, Petrucelli L, Wolozin B.
Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue. PLoS One.
2010;5(10):e13250.
PubMed Abstract
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Comments on Paper and Primary News |
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Comment by: Brian Freibaum
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Submitted 26 October 2010
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Posted 26 October 2010
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This paper by Liqun Liu-Yesucevitz and colleagues is an important contribution in understanding the role that TDP-43 plays in human diseases such as ALS and FTLD. This is a solid publication that confirms previous studies that demonstrate TDP-43 is present in stress granules and that its localization to these granules is enhanced during cell stress (1,2). Additionally, they demonstrate that mutants of TDP-43 associated with ALS have increased stress granule formation both in the presence and absence of stressors. Strikingly, they show the accumulation of RIPA insoluble TDP-43 following arsenite treatment. This is also exacerbated in the disease-causing mutants. This is the first report of TDP-43 colocalization with stress granule proteins found in cytoplasmic inclusions from the brain and spinal cord of patients with FTLD and ALS.
Future studies will be needed to assess the importance and impact of stress granule proteins in the pathogenesis of these diseases and the mechanism by which TDP-43 is recruited to these granules. Additionally, this study raises the questions of...
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This paper by Liqun Liu-Yesucevitz and colleagues is an important contribution in understanding the role that TDP-43 plays in human diseases such as ALS and FTLD. This is a solid publication that confirms previous studies that demonstrate TDP-43 is present in stress granules and that its localization to these granules is enhanced during cell stress (1,2). Additionally, they demonstrate that mutants of TDP-43 associated with ALS have increased stress granule formation both in the presence and absence of stressors. Strikingly, they show the accumulation of RIPA insoluble TDP-43 following arsenite treatment. This is also exacerbated in the disease-causing mutants. This is the first report of TDP-43 colocalization with stress granule proteins found in cytoplasmic inclusions from the brain and spinal cord of patients with FTLD and ALS.
Future studies will be needed to assess the importance and impact of stress granule proteins in the pathogenesis of these diseases and the mechanism by which TDP-43 is recruited to these granules. Additionally, this study raises the questions of what specific role TDP-43 plays in response to cellular stresses and whether TDP-43 is a bona fide stress response protein.
References:
1. Elvira, G. et al. Characterization of an RNA granule from developing brain. Mol Cell Proteomics 5, 635-51 (2006). Abstract
2. Moisse, K. et al. Divergent patterns of cytosolic TDP-43 and neuronal progranulin expression following axotomy: implications for TDP-43 in the physiological response to neuronal injury. Brain Res 1249, 202-11 (2009). Abstract
 View all comments by Brian Freibaum |
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Comment by: George Perry (Disclosure)
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Submitted 28 October 2010
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Posted 2 November 2010
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 I recommend this paper
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REAGENTS/MATERIAL:
To determine whether the inclusions co-localized with SGs, we co-labeled the cells with antibodies to SG markers, including TIA-1, TIAR, eIF3 and poly-A binding protein (PABP). Double labeling experiments indicated that inclusions composed of WT TDP-43 co-localized with SG markers under arsenite-induced conditions; TDP-43 inclusions also co-localized with SG markers under basal conditions, but the fraction of cells (<10%) exhibiting TDP-43 inclusions under basal conditions. Co-localization with PABP is considered a strong indication of coincident localization of mRNA. Co-localization of TDP-43 with SG markers is a robust observation that can be observed with multiple independent markers. We observed co-localization using antibodies to eIF3, TIA and PABP
Immunocytochemistry: Primary antibodies used were: rabbit anti-TDP-43 (Proteintech Group, Inc, Chicago, IL),
rabbit anti-TDP-43, C-terminus (aa 405-415, Cosmo Bio. Co., Carlsbad, California),
goat anti-TIA-1 (Santa Cruz Biotechnology, Santa Cruz, CA sc-1751)
or goat anti-eIF3η (Santa Cruz, sc-16377).
Immunohistochemistry of human tissue: Incubation was performed with primary antibodies (described above) overnight in 5% donkey serum/PBS.
LDH assay: 293-FT cells were transfected, and after 24 hrs the cells were trypsinized, counted and plated on a 96 well plate at 10,000 cells/well. The following day LDH release was measured using the
Cyto Tox 96 NonRadioactive Cytotoxicity Assay Kit (Promega) as per manufacturers directions. Released LDH was normalized to total LDH, and the results were further normalized to transfection efficiency for each plasmid.
Immunoblot: Immunoblots were performed using gradient PAGE on a 15-well, 4–20% Tris-Glycine gel (Invitrogen) as described previously. Antibody incubation was at 4°C overnight with rabbit polyclonal TDP-43 antibody (ProteinTech Group) or
mouse monoclonal anti-actin (C4) (Millipore Corporation, Billerica, MA ) in TBS-T plus 5% nonfat dry milk
.
Immunoprecipitation: Lysates were cleared by protein G Dynabeads (Invitrogen) for 1 hour at 4°C; 1 µl of TDP-43 antibody (anti TDP-43 C-terminus 405–414, Cosmo Bio Co.) was added to each cell lysate
. . . the Dynabeads were pulled down magnetically and washed three times in co-IP buffer. Eluting buffer (Invitrogen) was added to the protein-bead complexes to elute the binding proteins from the beads. The eluted samples were boiled at 95°C for 5 min in SDS-sample buffer. Proteins were then analyzed by western blot.
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