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

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

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

<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

Suppression of neurodegeneration associated with diverse misfolded proteins in invertebrate models by <i>ufd-2</i> and <i>spr-5</i> loss-of-function mutations.

<p>(<b>A</b>) Top: schematic drawing depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the <i>C</i>. <i>elegans</i> body plan. Left: micrographs show the SOD1^G85R-YFP proteins expressed in WT or the <i>spr-5(by134);ufd-2(tm1380)</i> mutant background. The double-mutant worms show a marked decrease in protein aggregation in neurons. Enlarged sections of head neurons (red framed) and ventral cord neurons (white framed) are shown. Middle: quantification of locomotion in the <i>spr-5(by134);ufd-2(tm1380)</i> and the WT <i>C</i>. <i>elegans</i> with neuronal expression of SOD1^G85R-YFP (<i>n</i> = 30). Right: a decrease in the protein levels of SOD1^G85R-YFP in the presence of <i>spr-5(by134);ufd-2(tm1380)</i> is shown by western blots of the supernatant (S) and the pellet (P) fractions. (<b>B and C</b>) Analyses of the <i>spr-5(by134);ufd-2(tm1380)</i> and the WT <i>C</i>. <i>elegans</i> with neuronal expression of TDP-43-c25-YFP (<i>n</i> = 30) or PolyQ-YFP (<i>n</i> = 12) as in (A). (<b>D</b>) Neurodegenerative rough-eye phenotype in adults is alleviated by the knockdown of the <i>Drosophila</i> homologs of <i>ufd-2</i>/UBE4B and <i>spr-5</i>/LSD1—CG9934 and Su(Var)3-3, respectively, compared to the control (CTRL). Eye-specific expression of TDP-43^M337V, FUS^R521C, and RNA interference (RNAi) was driven by GMR-Gal4. (<b>E</b>) <i>Drosophila</i> eye pigment quantitation. Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p

Regulation of Protein Quality Control by UBE4B and LSD1 through p53-Mediated Transcription

<div><p>Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, and its failure is associated with many neurodegenerative disorders. Here, we identify two genes, <i>ufd-2</i> and <i>spr-5</i>, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in <i>Caenorhabditis elegans</i>. Loss of human orthologs ubiquitination factor E4 B (UBE4B) and lysine-specific demethylase 1 (LSD1), respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries. An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity. These studies identify a new protein quality control pathway via regulation of transcription factors and point to the augmentation of protein quality control as a wide-spectrum antiproteotoxicity strategy.</p></div

Identification and characterization of a robust suppressor that ameliorates the locomotion defects in the <i>C</i>. <i>elegans</i> model of SOD1-associated ALS.

<p>(<b>A</b>) Workflow of the suppressor screen identifying mutant <i>C</i>. <i>elegans</i> (red) with saliently improved movement. (<b>B</b>) Locomotor behavior, measured by thrashing rates in liquid medium, in the <i>C</i>. <i>elegans</i> strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1^G85R, in the presence (M1/M1) or absence (+/+) of the suppressor mutation (<i>n</i> = 16). (<b>C</b>) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1^G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1^G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure. (<b>D</b>) Sequence analysis of the M1 strain, revealing that independent mutations in two genes, a lysine-specific demethylase, <i>spr-5 (R646Q)</i>, and a ubiquitin ligase, <i>ufd-2 (W824X)</i>, are required for the full suppressor phenotype. (<b>E</b>) <i>C</i>. <i>elegans</i> SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant <i>C</i>. <i>elegans</i> are indicated. (<b>F</b>) Locomotor behavior, measured by thrashing rates, of the <i>C</i>. <i>elegans</i> carrying the SOD1^G85R transgene on the normal background (WT), with the null mutation of either <i>ufd-2(tm1380)</i> or <i>spr-5(by134)</i>, the double mutation <i>ufd-2(tm1380);spr-5(by134)</i>, or the M1 suppressor <i>ufd-2(W824X);spr-5(R646Q</i>) (<i>n</i> = 16). (<b>G</b>) Western blotting of the supernatant (S) and pellet (P) protein fractions from the <i>C</i>. <i>elegans</i> carrying the SOD1^G85R transgene with the double mutation <i>ufd-2(tm1380);spr-5(by134)</i> compared with controls. While SOD1^G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1^G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis. (<b>H</b>) The overexpression (OE) of WT UFD-2 or SPR-5 in the <i>C</i>. <i>elegans</i> nervous system blocks the protection conferred by the double mutation <i>ufd-2(tm1380);spr-5(by134)</i> (<i>n</i> = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p

p53 promotes the clearance of misfolded SOD1 mutant proteins.

<p>(<b>A</b>) p53 small molecule activators Tenovin-1 and CP-31398 reduce the levels of misfolded SOD1 proteins, as determined by the SOD1^G85R solubility assay in HEK293 cells. Increasing concentrations of the p53 activators significantly decrease the levels of SOD1^G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions. (<b>B</b>) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1^G85R but not WT SOD1 proteins in the SOD1^G85R aggregation assay, as shown by western blots of both supernatant (S) (<i>n</i> = 2) and pellet (P) (<i>n</i> = 3) fractions. (<b>C</b>) A complete absence of p53 increases the accumulation of SOD1^G85R mutant proteins in p53–/– HCT116 cells when compared with controls. Representative western blots (left panels) and quantification of SOD1^G85R levels in the supernatant lysates are shown. The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1. The right graph panel shows the same data as shown in the middle panel, but normalized to the average SOD1^G85R level for each amount of the transfected plasmid. Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p