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TDP-43 loss of cellular function through aggregation requires additional structural determinants beyond its C-terminal Q/N prion-like domain.

Budini M, Romano V, Quadri Z, Buratti E, Baralle FE - Hum. Mol. Genet. (2014)

Bottom Line: To our knowledge, this is the only system that achieves full functional TDP 43 depletion with effects similar to RNAi depletion or gene deletion.As a result, this model will prove useful to investigate the loss-of-function effects mediated by TDP-43 aggregation within cells without affecting the expression of the endogenous gene.These data show for the first time that cellular TDP-43 aggregation can lead to total loss of function and to defective splicing of TDP-43-dependent splicing events in endogenous genes.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.

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Related in: MedlinePlus

(A) A schematic representation of the Flag-TDP-12xQ/N construct, describing all its different structural determinants. Residues 403–414 were eliminated due to cloning strategy. (B) An immunofluorescence of the Flag-TDP-43 WT and Flag-TDP-12xQ/N-induced proteins using anti-Flag (red) and anti-TDP-43 (green) antibodies. The cell nuclei were stained with the reagent TOPRO-3. A merge between anti-Flag/anti-TDP-43 antibodies or anti-Flag/anti-TDP-43/TOPRO-3 is also reported. (C) A co-immunofluorescence of differentiated NSC-34 motor neuron cell line co-transfected with constructs expressing Flag-TDP-12xQ/N and Myc-TDP-43 WT. The assay was performed using anti-Flag (red) and anti-Myc (green) antibodies. The cell nuclei were stained with the reagent TOPRO-3. A merge between anti-Flag/anti-Myc antibodies or anti-Flag/anti-Myc/TOPRO-3 is reported.
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DDU415F1: (A) A schematic representation of the Flag-TDP-12xQ/N construct, describing all its different structural determinants. Residues 403–414 were eliminated due to cloning strategy. (B) An immunofluorescence of the Flag-TDP-43 WT and Flag-TDP-12xQ/N-induced proteins using anti-Flag (red) and anti-TDP-43 (green) antibodies. The cell nuclei were stained with the reagent TOPRO-3. A merge between anti-Flag/anti-TDP-43 antibodies or anti-Flag/anti-TDP-43/TOPRO-3 is also reported. (C) A co-immunofluorescence of differentiated NSC-34 motor neuron cell line co-transfected with constructs expressing Flag-TDP-12xQ/N and Myc-TDP-43 WT. The assay was performed using anti-Flag (red) and anti-Myc (green) antibodies. The cell nuclei were stained with the reagent TOPRO-3. A merge between anti-Flag/anti-Myc antibodies or anti-Flag/anti-Myc/TOPRO-3 is reported.

Mentions: We have previously developed a cell-based aggregation model by cloning in-tandem 12 copies of the Q/N region from TDP-43 in the C-terminal of EGFP, resulting in the expression of a so-called EGFP-12xQ/N protein (16). In order to generally improve the aggregation process and to further study whether other regions in TDP-43 could contribute to it we considered to include the 12xQ/N repetitions into the C-terminus of the Flag-TDP-43 protein itself (Fig. 1A).Figure 1.


TDP-43 loss of cellular function through aggregation requires additional structural determinants beyond its C-terminal Q/N prion-like domain.

Budini M, Romano V, Quadri Z, Buratti E, Baralle FE - Hum. Mol. Genet. (2014)

(A) A schematic representation of the Flag-TDP-12xQ/N construct, describing all its different structural determinants. Residues 403–414 were eliminated due to cloning strategy. (B) An immunofluorescence of the Flag-TDP-43 WT and Flag-TDP-12xQ/N-induced proteins using anti-Flag (red) and anti-TDP-43 (green) antibodies. The cell nuclei were stained with the reagent TOPRO-3. A merge between anti-Flag/anti-TDP-43 antibodies or anti-Flag/anti-TDP-43/TOPRO-3 is also reported. (C) A co-immunofluorescence of differentiated NSC-34 motor neuron cell line co-transfected with constructs expressing Flag-TDP-12xQ/N and Myc-TDP-43 WT. The assay was performed using anti-Flag (red) and anti-Myc (green) antibodies. The cell nuclei were stained with the reagent TOPRO-3. A merge between anti-Flag/anti-Myc antibodies or anti-Flag/anti-Myc/TOPRO-3 is reported.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262490&req=5

DDU415F1: (A) A schematic representation of the Flag-TDP-12xQ/N construct, describing all its different structural determinants. Residues 403–414 were eliminated due to cloning strategy. (B) An immunofluorescence of the Flag-TDP-43 WT and Flag-TDP-12xQ/N-induced proteins using anti-Flag (red) and anti-TDP-43 (green) antibodies. The cell nuclei were stained with the reagent TOPRO-3. A merge between anti-Flag/anti-TDP-43 antibodies or anti-Flag/anti-TDP-43/TOPRO-3 is also reported. (C) A co-immunofluorescence of differentiated NSC-34 motor neuron cell line co-transfected with constructs expressing Flag-TDP-12xQ/N and Myc-TDP-43 WT. The assay was performed using anti-Flag (red) and anti-Myc (green) antibodies. The cell nuclei were stained with the reagent TOPRO-3. A merge between anti-Flag/anti-Myc antibodies or anti-Flag/anti-Myc/TOPRO-3 is reported.
Mentions: We have previously developed a cell-based aggregation model by cloning in-tandem 12 copies of the Q/N region from TDP-43 in the C-terminal of EGFP, resulting in the expression of a so-called EGFP-12xQ/N protein (16). In order to generally improve the aggregation process and to further study whether other regions in TDP-43 could contribute to it we considered to include the 12xQ/N repetitions into the C-terminus of the Flag-TDP-43 protein itself (Fig. 1A).Figure 1.

Bottom Line: To our knowledge, this is the only system that achieves full functional TDP 43 depletion with effects similar to RNAi depletion or gene deletion.As a result, this model will prove useful to investigate the loss-of-function effects mediated by TDP-43 aggregation within cells without affecting the expression of the endogenous gene.These data show for the first time that cellular TDP-43 aggregation can lead to total loss of function and to defective splicing of TDP-43-dependent splicing events in endogenous genes.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.

Show MeSH
Related in: MedlinePlus