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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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A schematic model of how aggregation does not necessarily correlate with loss of function. In particular, loss of TDP-43 function occurs only when the endogenous TDP-43 is able to interact with very high efficiency with Flag-TDP-12xQ/N ▵RRM1/2 aggregates and to be totally sequestered in the insoluble aggregate. The question mark reflects the fact that we still do not know whether the Q/N region and the N-terminus interact with each other or only among themselves. On the other hand, deletion of the 75 N-terminal residues of TDP-43 in the Flag-TDP-12xQ/N ▵RRM1/2, ▵N mutant abolished the ability of the aggregates to efficiently induce loss-of-function effects, being much less able to sequester endogenous TDP-43.
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DDU415F7: A schematic model of how aggregation does not necessarily correlate with loss of function. In particular, loss of TDP-43 function occurs only when the endogenous TDP-43 is able to interact with very high efficiency with Flag-TDP-12xQ/N ▵RRM1/2 aggregates and to be totally sequestered in the insoluble aggregate. The question mark reflects the fact that we still do not know whether the Q/N region and the N-terminus interact with each other or only among themselves. On the other hand, deletion of the 75 N-terminal residues of TDP-43 in the Flag-TDP-12xQ/N ▵RRM1/2, ▵N mutant abolished the ability of the aggregates to efficiently induce loss-of-function effects, being much less able to sequester endogenous TDP-43.

Mentions: Our new model, schematically represented in Figure 7, also allowed us to study the importance of other TDP-43 domains in the protein aggregation process. First of all, we confirmed previous data indicating that the RNA-binding activity is not necessary for TDP-43 aggregation (16). In addition, we also observed that deletion of the 75 N-terminal residues of Flag-TDP-12xQ/N abolished the ability of the aggregates to efficiently induce loss-of-function effects. This result is particularly interesting as is consistent with previous studies which demonstrated that the N-terminal domain (residues 1–105) is involved in TDP-43 protein oligomerization in vitro (51) and in TDP-43 intermolecular interactions in cell culture (29,52). Most recently, the involvement of the N-terminal domain in TDP-43 aggregation was also proposed in a cellular aggregation model where TDP-43 full-length aggregates were triggered by disrupting the NLS signals (GFP-TDP-43NLSm) as previously described (27). In this case, however, it should be noted that mutating the NLS is just a way to increase the amount of TDP-43 production through its autoregulation process (6,7) by increasing the amount of this protein that is made by the cell and exported to the cytoplasm with minimal return to the nucleus. Furthermore, Zhang et al. (53) found that the deletion of residues 1–10 (GFP-TDP-4310-4014-NLSm) eliminated the interaction with full-length TDP-43 (MYC-TDP-43). A difference between the Zhang et al. model and ours is that, in our case, the elimination of the residues 1–75 from Flag-TDP-12xQ/N did not disrupt aggregate formation, but reduced the efficiency of interaction with endogenous TDP-43. These results indicate that the N-terminal portion of TDP-43 fulfills an important role in an aggregation context by mediating the interaction between endogenous TDP-43 and the aggregates. It should nonetheless be noted that the Co-IP results presented in Figure 6 show that removal of this region does not completely abolish the ability of the mutant to interact with endogenous TDP-43. This further strengthens the concept that TDP-43 oligomerization is mediated by several regions of this protein that co-operate to mediate this self-interaction whose functional significance remains still to be explored.Figure 7.


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 schematic model of how aggregation does not necessarily correlate with loss of function. In particular, loss of TDP-43 function occurs only when the endogenous TDP-43 is able to interact with very high efficiency with Flag-TDP-12xQ/N ▵RRM1/2 aggregates and to be totally sequestered in the insoluble aggregate. The question mark reflects the fact that we still do not know whether the Q/N region and the N-terminus interact with each other or only among themselves. On the other hand, deletion of the 75 N-terminal residues of TDP-43 in the Flag-TDP-12xQ/N ▵RRM1/2, ▵N mutant abolished the ability of the aggregates to efficiently induce loss-of-function effects, being much less able to sequester endogenous TDP-43.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4262490&req=5

DDU415F7: A schematic model of how aggregation does not necessarily correlate with loss of function. In particular, loss of TDP-43 function occurs only when the endogenous TDP-43 is able to interact with very high efficiency with Flag-TDP-12xQ/N ▵RRM1/2 aggregates and to be totally sequestered in the insoluble aggregate. The question mark reflects the fact that we still do not know whether the Q/N region and the N-terminus interact with each other or only among themselves. On the other hand, deletion of the 75 N-terminal residues of TDP-43 in the Flag-TDP-12xQ/N ▵RRM1/2, ▵N mutant abolished the ability of the aggregates to efficiently induce loss-of-function effects, being much less able to sequester endogenous TDP-43.
Mentions: Our new model, schematically represented in Figure 7, also allowed us to study the importance of other TDP-43 domains in the protein aggregation process. First of all, we confirmed previous data indicating that the RNA-binding activity is not necessary for TDP-43 aggregation (16). In addition, we also observed that deletion of the 75 N-terminal residues of Flag-TDP-12xQ/N abolished the ability of the aggregates to efficiently induce loss-of-function effects. This result is particularly interesting as is consistent with previous studies which demonstrated that the N-terminal domain (residues 1–105) is involved in TDP-43 protein oligomerization in vitro (51) and in TDP-43 intermolecular interactions in cell culture (29,52). Most recently, the involvement of the N-terminal domain in TDP-43 aggregation was also proposed in a cellular aggregation model where TDP-43 full-length aggregates were triggered by disrupting the NLS signals (GFP-TDP-43NLSm) as previously described (27). In this case, however, it should be noted that mutating the NLS is just a way to increase the amount of TDP-43 production through its autoregulation process (6,7) by increasing the amount of this protein that is made by the cell and exported to the cytoplasm with minimal return to the nucleus. Furthermore, Zhang et al. (53) found that the deletion of residues 1–10 (GFP-TDP-4310-4014-NLSm) eliminated the interaction with full-length TDP-43 (MYC-TDP-43). A difference between the Zhang et al. model and ours is that, in our case, the elimination of the residues 1–75 from Flag-TDP-12xQ/N did not disrupt aggregate formation, but reduced the efficiency of interaction with endogenous TDP-43. These results indicate that the N-terminal portion of TDP-43 fulfills an important role in an aggregation context by mediating the interaction between endogenous TDP-43 and the aggregates. It should nonetheless be noted that the Co-IP results presented in Figure 6 show that removal of this region does not completely abolish the ability of the mutant to interact with endogenous TDP-43. This further strengthens the concept that TDP-43 oligomerization is mediated by several regions of this protein that co-operate to mediate this self-interaction whose functional significance remains still to be explored.Figure 7.

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