Schematic diagram summarizing the mechanism by which TDP-43 acts as a stress-response switch to regulate protein homeostasis.

<p>(A) In the resting state, FOXO is retained significantly in the cytoplasm. TDP-43, which is predominantly nuclear, inhibits nuclear FOXO transcriptional activity. (B) In response to stress, TDP-43 undergoes cytoplasmic translocation and helps maintain protein homeostasis by both recruiting untranslated mRNAs to stress granules and promoting protein quality control. The recruitment of TDP-43 and target mRNAs to cytoplasmic granules contributes to slower translation and reduced protein-folding burden. The competitive binding of TDP-43 to 14-3-3 proteins drives nuclear translocation and activation of FOXOs. Consequently, FOXO-mediated protein quality control is activated.</p

Loss of function of TDP-1 extends the lifespan and reduces protein aggregation in <i>C. elegans</i> in a DAF-16-dependent manner.

<p>(A) Survival curves of wild-type (black) and <i>tdp-1(ok803lf)</i> loss-of-function mutant (red) demonstrate a significant lifespan extension. n>100, <i>p</i><0.001 The lifespan-extending effect of <i>tdp-1(ok803lf)</i> is blocked by a loss of function of DAF-16 in <i>daf-16(mu86lf)</i>, as shown by the survival curves (green and blue). n>100, <i>p</i><0.001. (B) Survival curves of <i>daf-2(e1370lf)</i> loss-of-function mutant alone (black) and the <i>daf-2(e1370lf);tdp-1(ok803lf)</i> double mutant (blue) show a further lifespan extension with the loss of TDP-1. n>100, <i>p</i><0.001. (C) Schematic diagram of the genetic pathway for TDP-1 regulation of the lifespan in relation to the DAF-2–DAF-16 pathway. (D) Loss of <i>daf-16</i> abolished the reduction of protein aggregation conferred by loss of function of TDP-1. Transgenic TDP-C25-YFP expressed in <i>C. elegans</i> neurons was fractionated in to soluble supernatant and insoluble pellet before western blot analysis using a YFP antibody. The effects of single mutant <i>tdp-1(ok803lf)</i> and double mutant <i>tdp-1(ok803lf); daf-16(mu86lf)</i> were compared. (E) Quantitative measurement of mRNA levels of select DAF-16 targets genes in wild-type and <i>tdp-1(ok803lf) C. elegans</i>. n>6, *<i>p</i><0.001; error bars represent SEM.</p

RNA-Processing Protein TDP-43 Regulates FOXO-Dependent Protein Quality Control in Stress Response

<div><p>Protein homeostasis is critical for cell survival and functions during stress and is regulated at both RNA and protein levels. However, how the cell integrates RNA-processing programs with post-translational protein quality control systems is unknown. Transactive response DNA-binding protein (TARDBP/TDP-43) is an RNA-processing protein that is involved in the pathogenesis of major neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we report a conserved role for TDP-43, from <i>C. elegans</i> to mammals, in the regulation of protein clearance via activation of FOXO transcription factors. In response to proteotoxic insults, TDP-43 redistributes from the nucleus to the cytoplasm, promoting nuclear translocation of FOXOs and relieving an inhibition of FOXO activity in the nucleus. The interaction between TDP-43 and the FOXO pathway in mammalian cells is mediated by their competitive binding to 14-3-3 proteins. Consistent with FOXO-dependent protein quality control, TDP-43 regulates the levels of misfolded proteins. Therefore, TDP-43 mediates stress responses and couples the regulation of RNA metabolism and protein quality control in a FOXO-dependent manner. The results suggest that compromising the function of TDP-43 in regulating protein homeostasis may contribute to the pathogenesis of related neurodegenerative diseases.</p></div

TDP-43 regulates levels of misfolded proteins.

<p>(A) Ectopic expression of TDP-43 increases the aggregation formation of SOD1 G85R. HEK293T cells were co-transfected with plasmids expressing SOD1 G85R and WT TDP-43. The cells were detergent-extracted to separate soluble proteins (unaggregated and oligomers) and insoluble aggregates. (B) Knockdown of TDP-43 suppressed the aggregation of SOD1 G85R. HEK293T cells were co-transfected with plasmids expressing SOD1 G85R and shRNA against TDP-43 or a scrambled control. The cells were detergent-extracted as described and subjected to western blotting analysis.</p

<i>C. elegans</i> TDP-1 forms cytoplasmic granules in neurons under proteotoxic stress.

<p>Transgenic <i>C. elegans</i> expressing TDP-1-YFP under a neuronal promoter were treated with the indicated proteotoxic stressors prior to fixation, DAPI staining, and visualization by florescence microscopy. (A) In untreated <i>C. elegans</i>, TDP-1-YFP shows primarily nuclear localization. Ventral cord neurons are shown. (B) When heat-stressed at 28°C for 16 h, TDP-1-YFP shows cytoplasmic localization and forms granular structures. Nerve ring neurons are shown. (C) When treated with 0.4 M NaCl for 24 h, TDP-1-YFP also translocates to the cytoplasm and forms granules. Ventral cord neurons are shown. (D) When crossed to a transgenic strain stably expressing the ALS mutant SOD1-G85R in neurons, a subset of animals shows cytoplasmic translocation and granule formation of TDP-1-YFP. Arrowheads point to stress-induced TDP-1-YFP granules. Scale bar: 5 µM.</p

TDP-43 negatively regulates the transcriptional activity of FOXOs in mammalian cells.

<p>(A) The FOXO transcriptional activity is significantly increased by the knockdown of endogenous TDP-43 in HEK293T cells. Cells were transfected with an FHRE-Luc reporter, a <i>Renilla</i> luciferase control, a FOXO3a-expressing construct, and an shRNA construct against endogenous TDP-43 or a scrambled control shRNA. Cell lysates were subjected to dual luciferase assays, and the ratio of firefly <i>to Renilla</i> luciferase activity was used to measure the FOXO transcriptional activity. (B) The knockdown of TDP-43 in HEK293T cells was confirmed by western blotting. GAPDH was used as a loading control. (C) The FOXO transcriptional activity was inhibited by the expression of TDP-43 in HEK293T cells. Cells were transfected with the FHRE-Luc reporter, the <i>Renilla</i> luciferase control, and one of the FOXO family members (FOXO1, FOXO3a, or FOXO4) for measurement of their respective activities in the presence or absence of Myc-TDP-43. (D) The protein levels of FOXOs, as represented by FOXO3a, were not reduced with the expression of TDP-43, as shown by western blotting. (E) The FOXO transcriptional activity, as represented by FOXO3a, was inhibited by TDP-43 in a dose-dependent manner. Cells were transfected as described above but with increasing amounts TDP-43-expressing constructs (0, 50, 100, or 200 ng DNA per well on 24-well plates). (F) The protein levels of FOXOs, as represented by FOXO3a, were not reduced by increasing levels of TDP-43, as shown by western blotting. n>3, *<i>p</i><0.05; error bars represent SEM.</p