doi: 10.1038/s43587-023-00383-4.
Epub 2023 Apr 3.
Cold temperature extends longevity and prevents disease-related protein aggregation through PA28γ-induced proteasomes
Affiliations
- PMID: 37118550
- PMCID: PMC10191861
- DOI: 10.1038/s43587-023-00383-4
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Cold temperature extends longevity and prevents disease-related protein aggregation through PA28γ-induced proteasomes
Nat Aging.
2023 May.
Abstract
Aging is a primary risk factor for neurodegenerative disorders that involve protein aggregation. Because lowering body temperature is one of the most effective mechanisms to extend longevity in both poikilotherms and homeotherms, a better understanding of cold-induced changes can lead to converging modifiers of pathological protein aggregation. Here, we find that cold temperature (15 °C) selectively induces the trypsin-like activity of the proteasome in Caenorhabditis elegans through PSME-3, the worm orthologue of human PA28γ/PSME3. This proteasome activator is required for cold-induced longevity and ameliorates age-related deficits in protein degradation. Moreover, cold-induced PA28γ/PSME-3 diminishes protein aggregation in C. elegans models of age-related diseases such as Huntington's and amyotrophic lateral sclerosis. Notably, exposure of human cells to moderate cold temperature (36 °C) also activates trypsin-like activity through PA28γ/PSME3, reducing disease-related protein aggregation and neurodegeneration. Together, our findings reveal a beneficial role of cold temperature that crosses evolutionary boundaries with potential implications for multi-disease prevention.
© 2023. The Author(s).
Conflict of interest statement
The authors declare no competing interests.
Figures
a–c, Trypsin-like (a), caspase-like (b) and chymotrypsin-like (c) proteasome activities in control sterile fer-15(b26);fem-1(hc17) C. elegans at day 6 of adulthood (mean ± standard error of the mean (s.e.m.) relative slope to 25 °C, n = 3 independent experiments). d–f, Trypsin-like (d), caspase-like (e) and chymotrypsin-like (f) proteasome activities in day 6 adult wild-type worms (mean ± s.e.m. relative slope to 20 °C, n = 5 independent experiments). g, Western blot of PA28γ/PSME-3, 19S RPN-6.1 and 20S α6/PAS-6 in day 6 adult control sterile worms. Graphs represent relative percentage values of proteasome subunits (corrected for α-tubulin loading control) to 25 °C (mean ± s.e.m., n = 3 independent experiments). h, mRNA levels in day 6 adult control sterile worms (mean ± s.e.m. relative expression to 20 °C, n = 4 independent experiments). i, Knockdown levels in day 6 adult control sterile worms on psme-3 RNAi initiated at day 1 of adulthood (mean ± s.e.m. relative expression to 20 °C vector RNAi, n = 4 independent experiments). j, Trypsin-like activity in day 6 adult control sterile worms on psme-3 knockdown (mean ± s.e.m. relative slope to 20 °C vector RNAi, n = 4 independent experiments). k, Somatic overexpression (OE) of psme-3 increases trypsin-like activity in adult worms at 15 °C (mean ± s.e.m. relative slope to control GFP(OE), n = 4 independent experiments). Two independent psme-3,GFP(OE) lines were tested. l, psme-3 overexpression does not increase trypsin-like activity at 20 °C (mean ± s.e.m. relative slope to control GFP(OE), n = 4 independent experiments). m, psme-3 overexpression increases trypsin-like activity at 25 °C (mean ± s.e.m. relative slope to control GFP(OE), n = 4 independent experiments). Control sterile worms were raised at 25 °C during development and then grown at the indicated temperatures until day 6 of adulthood. Wild-type and psme-3,GFP(OE) worms were raised at 20 °C during development and then grown at the indicated temperatures until day 6 of adulthood. Statistical comparisons were made by two-tailed Student’s t-test for paired samples. P value: *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant (P > 0.05). All the significant changes were also significant after correction for multiple testing by the false discovery rate (FDR) approach (FDR-adjusted P value (q value) < 0.05 was considered significant). Source Data contains exact P and q values. Source data
a, Trypsin-like proteasome activity in day 3-adult wild-type and trpa-1(ok999) mutant worms (mean ± s.e.m. relative slope to 15 °C vector RNAi, n = 4 independent experiments). b, Western blot of PA28γ/PSME-3 in day 6 adult wild-type and trpa-1(ok999) mutant worms. Graph represents the relative percentage values of PSME-3 (corrected for α-tubulin loading control) to 25 °C wild-type (mean ± s.e.m., n = 3 independent experiments). c, qPCR analysis of psme-3 mRNA levels in day 6 adult wild-type and trpa-1(ok999) mutant worms. Graph (relative expression to 25 °C wild-type) represents the mean ± s.e.m. of seven independent experiments. d, psme-3 mRNA levels in day 6 adult control sterile worms upon knockdown of distinct transcriptional regulators involved in cold-induced longevity. Graph (relative expression to 20 °C vector RNAi) represents the mean ± s.e.m. of 9 independent experiments. e, Knockdown of nhr-49 decreases cold-induced trypsin-like proteasome activity in control sterile worms (mean ± s.e.m. relative slope to 20 °C vector RNAi, n = 3 independent experiments). f, pbs-2 mRNA levels in day 6 adult control sterile worms. Graph (relative expression to 20 °C Vector RNAi) represents the mean ± s.e.m. of 9 independent experiments. g, psme-3 mRNA levels in day 6 adult wild-type and trpa-1(ok999) mutant worms. Graph (relative expression to 20 °C Vector RNAi) represents the mean ± s.e.m. of 8 independent experiments. In all the experiments, worms were raised at 20 °C until day 1 of adulthood and then grown at the indicated temperatures until day 3 (a) or 6 (b–g) of adulthood. Statistical comparisons were made by two-tailed Student’s t-test for paired samples. P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, P > 0.05. All the significant changes were also significant after correction for multiple testing by FDR approach (q value < 0.05). Source Data contains exact P and q values. Source data
a, Knockdown of psme-3 shortens cold-induced longevity (15 °C) in wild-type worms. Vector RNAi mean ± s.e.m.: 28.26 days ± 0.69, psme-3 RNAi: 24.19 ± 0.67. b, psme-3 RNAi does not reduce the lifespan of wild-type worms at 20 °C. Vector RNAi mean ± s.e.m.: 18.92 ± 0.34, psme-3 RNAi: 18.71 ± 0.42. c, psme-3 RNAi does not further shorten the lifespan of wild-type worms at 25 °C. Vector RNAi mean ± s.e.m.: 13.03 ± 0.30, psme-3 RNAi: 13.02 ± 0.30. d, rpn-6.1 RNAi shortens lifespan of wild-type worms at 15 °C. Vector RNAi mean ± s.e.m.: 21.13 ± 0.62, rpn-6.1 RNAi: 13.73 ± 0.36. e, rpn-6.1 RNAi shortens lifespan of wild-type worms at 20 °C. Vector RNAi mean ± s.e.m.: 16.60 ± 0.55, rpn-6.1 RNAi: 10.72 ± 0.23. f, rpn-6.1 RNAi shortens lifespan of wild-type worms at 25 °C. Vector RNAi mean ± s.e.m.: 12.75 ± 0.44, rpn-6.1 RNAi: 7.98 ± 0.15. In panels a–f, RNAi was initiated at day 1 of adulthood because rpn-6.1 is required for larval development. g, Lifespan at 15 °C upon psme-3 RNAi treatment in wild-type animals (vector RNAi mean ± s.e.m.: 28.83 ± 0.58, psme-3 RNAi: 23.03 ± 0.67) or RNAi-deficient animals in which RNAi efficiency has been rescued in specific tissues. Tissue-specific knockdown (KD) of psme-3 in the germline (vector RNAi mean ± s.e.m.: 28.84 ± 0.68, psme-3 RNAi: 23.04 ± 0.58), neurons (vector RNAi: 26.04 ± 0.67, psme-3 RNAi: 22.56 ± 0.59), intestine (vector RNAi: 26.37 ± 0.62, psme-3 RNAi: 22.85 ± 0.62) or muscle (vector RNAi: 26.35 ± 1.21, psme-3 RNAi: 23.37 ± 1.05) shortens cold-induced longevity at 15 °C. Knockdown was initiated from hatching. h, Somatic overexpression of psme-3 under the sur-5 promoter slightly shortens lifespan at 20 °C. GFP(OE) mean ± s.e.m.: 17.19 ± 0.41, psme-3,GFP(OE) #1: 15.48 ± 0.31, psme-3,GFP(OE) #2: 16.31 ± 0.31. i, Somatic overexpression of psme-3 is deleterious for adult lifespan at 25 °C. GFP(OE) mean ± s.e.m.: 13.83 ± 0.31, psme-3,GFP(OE) #1: 10.60 ± 0.19, psme-3,GFP(OE) #2: 10.67 ± 0.19. j, Overexpression of psme-3 in somatic tissues extends lifespan at 15 °C. GFP(OE) mean ± s.e.m.: 23.26 ± 0.43, psme-3,GFP(OE) #1: 26.65 ± 0.33, psme-3,GFP(OE) #2: 26.38 ± 0.31. Worms were raised at 20 °C and shifted to the indicated temperatures after development. In all the experiments, P values were calculated by two-sided log-rank test, n = 96 worms per condition. NS, P > 0.05). Supplementary Table 3 contains statistical analysis and replicate data of independent lifespan experiments.
a, Filter trap analysis of day 6 adult worms that express polyQ67::YFP or control polyQ19::CFP (detected by anti-GFP antibody) in neurons. Representative of three independent experiments. b, Western blot of day 6 adult worms to assess total polyQ67::YFP and polyQ19::CFP levels (detected by anti-GFP antibody). Graphs represent the relative percentage values of polyQ67 and polyQ19 protein levels (corrected for α-tubulin loading control) to 25 °C (mean ± s.e.m., n = 3 independent experiments). c, Filter trap of polyQ67::YFP aggregation upon psme-3 RNAi at the indicated temperatures. Worms were analyzed at day 6 of adulthood. Representative of three independent experiments. d, Western blot of total polyQ67 protein levels on psme-3 RNAi. Graph represents the relative percentage of polyQ67 protein levels (corrected for α-tubulin loading control) to 20 °C Vector RNAi (mean ± s.e.m., n = 4 independent experiments). Worms were analyzed at day 6 of adulthood. e, Thrashing movements over a 30-s period at day 3 of adulthood (n = 50 worms per condition from three independent experiments). The box plot represents the 25th–75th percentiles, the line depicts the median and the whiskers show the min–max values. f, Filter trap analysis of C. elegans that express polyQ40::YFP in the muscle alone (detected by anti-GFP antibody). Worms were analyzed at day 6 of adulthood. Representative of four independent experiments. g, Thrashing movements over a 30-s period at day 3 of adulthood (n = 50 worms per condition from three independent experiments). The box plot represents the 25th–75th percentiles, the line depicts the median and the whiskers show the min–max values. In all the experiments, worms were shifted at the indicated temperatures after development and RNAi was initiated at day 1 of adulthood. Statistical comparisons were made by two-tailed Student’s t-test for paired (b,d) or unpaired samples (e,g). P value: *P < 0.05, ***P < 0.001, ****P < 0.0001; NS, P > 0.05). All the significant changes were also significant after correction for multiple testing by FDR approach (q value < 0.05). Source Data contains for exact P and q values. Source data
a, Western blot with anti-FUS antibody of day 6 adult worms expressing ALS-related FUSP525L mutant variant in neurons. Graph represents the relative percentage of FUSP525L protein levels (corrected for α-tubulin loading control) to 25 °C (mean ± s.e.m., n = 3 independent experiments). b, Cold temperature decreases FUSP525L aggregation in day 6 adult worms (detected by filter trap with anti-FUS antibody). Representative of three independent experiments. c, Western blot of FUSP525L levels upon psme-3 RNAi at different temperatures. Graph represents the relative percentage of FUSP525L levels (corrected for α-tubulin) to 20 °C vector RNAi (mean ± s.e.m., n = 4 independent experiments). d, Filter trap of FUSP525L aggregation upon psme-3 RNAi in day 6 adult worms. Representative of five independent experiments. e, Thrashing movements over a 30-s period at day 3 of adulthood (n = 50 worms from three independent experiments). The box plot represents the 25th–75th percentiles, the line depicts the median and the whiskers show the min–max values. f, Western blot with anti-TDP-43 antibody of day 6 adult worms expressing ALS-related TDP-43M331V mutant variant in neurons. Graph represents the relative percentage of TDP-43M331V protein levels (corrected for α-tubulin) to 25 °C (mean ± s.e.m., n = 3 independent experiments). g, Cold temperature decreases TDP-43M331V aggregation in day 6 adult worms (detected by filter trap with anti-TDP-43 antibody). Representative of three independent experiments. h, Western blot of TDP-43M331V levels upon knockdown of PA28γ/psme-3 at different temperatures. Graph represents the relative percentage of TDP-43M331V levels (corrected for α-tubulin) to 20 °C Vector RNAi (mean ± s.e.m., n = 3 independent experiments). i, Filter trap analysis of TDP-43M331V aggregation upon psme-3 RNAi in day 6 adult worms. Representative of three independent experiments. In all the experiments, worms were shifted to the indicated temperatures after development and RNAi was initiated after development. Statistical comparisons were made by two-tailed Student’s t-test for paired (a,c,f,h) or unpaired samples (e). P value: *P < 0.05, **P < 0.01, ****P < 0.0001; NS, P > 0.05). Significant changes were also significant after correction for multiple testing by FDR (q value < 0.05). Source Data contains for exact P and q values. Source data
a, Trypsin-like activity in HEK293 cells at cold temperature (36 °C) for 24 h (mean ± s.e.m. relative slope to 37 °C, n = 12 independent experiments). b, Caspase-like activity in HEK293 cells (mean ± s.e.m. relative to 37 °C, n = 18 independent experiments). c, Chymotrypsin-like activity in HEK293 cells (mean ± s.e.m. relative to 37 °C, n = 9 independent experiments). d, Trypsin-like activity in HEK293 cells upon 25 µM HC-030031 for 24 h (mean ± s.e.m. relative to 37 °C + DMSO vehicle control, n = 7 independent experiments). e, mRNA levels of proteasome subunits in HEK293 cells (mean ± s.e.m. relative expression to 37 °C, n = 8 independent experiments). f, Western blot of PA28γ/PSME3, 19S PSMD11, and 20S α6/PSMA1 in HEK293 cells. Graph represents relative percentage values of proteasome subunits (corrected for β-actin loading control) to 37 °C (mean ± s.e.m., n = 5 independent experiments). g, Native gel electrophoresis of HEK293 cells followed by immunoblotting with anti-PSME3 antibody. Representative of 5 independent experiments. h, Knockdown levels in HEK293 cells expressing PSME3 shRNA (mean ± s.e.m. relative to non-targeting (NT) shRNA, n = 4 independent experiments). i, PSME3 protein levels in PSME3 shRNA-HEK293 cells. β-actin loading control. Representative of three independent experiments. j, Trypsin-like activity in PSME3-shRNA-HEK293 cells (mean ± s.e.m. relative to 37 °C NT shRNA, n = 5 independent experiments). k, PSME3 mRNA levels in HEK293 cells overexpressing (OE) PSME3 at 37 °C (mean ± s.e.m. relative to empty vector, n = 3 biological replicates). l, PSME3 protein levels in PSME3(OE)-HEK293 cells at 37 °C. α-tubulin loading control. Representative of four independent experiments. m, PSME3 overexpression increases trypsin-like activity at 37 °C and 36 °C (mean ± s.e.m. relative to 37 °C + empty vector, n = 4 independent experiments). In all the experiments, cells were cultured at 37 °C and then shifted to cold temperature (36 °C) or maintained at 37 °C for 24 h before the analysis. Statistical comparisons were made by two-tailed Student’s t-test for paired samples, except Fig. 6k (unpaired t-test). P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, P > 0.05). All the significant changes were also significant after correction for multiple testing by FDR (q value < 0.05). Source Data contains exact P and q values. Source data
a, Western blot with anti-HTT antibody in HEK293 cells expressing control Q23-HTT-GFP or aggregation-prone Q100-HTT-GFP. Graphs: mean ± s.e.m. relative percentage of Q23-HTT or Q100-HTT levels (corrected for β-actin loading control) to 37 °C, n = 5 independent experiments. b, Filter trap with anti-GFP antibody of HEK293 cells expressing Q23-HTT-GFP or Q100-HTT-GFP. Representative of five independent experiments. c, Western blot with anti-HTT antibody in Q100-HTT-GFP HEK293 cells upon TRPA1 shRNA. Graph: mean ± s.e.m. relative percentage of Q100-HTT levels (corrected for β-actin) to 37 °C + non-targeting (NT) shRNA, n = 3 independent experiments. d, Filter trap with anti-GFP of Q100-HTT-GFP HEK293 cells upon TRPA1 shRNA. Representative of three independent experiments. e, Western blot with anti-HTT in Q100-HTT-GFP HEK293 cells treated with 25 µM HC-030031 (24 h). Graph: mean ± s.e.m. relative percentage of Q100-HTT levels (corrected for β-actin) to 37 °C + DMSO vehicle control, n = 3 independent experiments. f, Filter trap with anti-GFP of Q100-HTT-GFP aggregates in HEK293 cells treated with 25 µM HC-030031 (24 h). Representative of three independent experiments. g, Western blot with anti-HTT antibody in HEK293 cells expressing control Q23-HTT-GFP or Q100-HTT-GFP upon PSME3 shRNA. Graphs: mean ± s.e.m. relative percentage of Q23-HTT (n = 4) or Q100-HTT (n = 5) levels (corrected for β-actin) to the corresponding 37 °C + NT shRNA. h, Filter trap with anti-GFP of HEK293 cells expressing Q23-HTT-GFP or Q100-HTT-GFP upon PSME3 shRNA. Representative of six independent experiments. i, Western blot with anti-HTT of PSME3(OE)-HEK293 cells expressing Q100-HTT-GFP. Graph: mean ± s.e.m. relative percentage of Q100-HTT levels (corrected for β-actin) to 37 °C + Empty vector, n = 4 independent experiments. j, Filter trap with anti-GFP of PSME3(OE)-HEK293 cells expressing Q100-HTT-GFP. Representative of four independent experiments. In all the experiments, cells were cultured at 37 °C and then shifted to cold temperature (36 °C) or maintained at 37 °C for 24 h before the analysis. Comparisons were made by two-tailed Student’s t-test for paired samples. P value: *P < 0.05, **P < 0.01, ***P < 0.001; NS, P > 0.05). All the significant changes were also significant after correction for multiple testing by FDR (q value < 0.05). Source Data contains exact P and q values. Source data
a, Western blot with anti-FUS antibody in HEK293 cells expressing wild-type FUS (WT) or ALS-related mutant FUSP525L. Graphs represent the relative percentage of FUS levels (corrected for β-actin loading control) to the corresponding 37 °C + non-targeting (NT) shRNA (mean ± s.e.m, FUS(WT): n = 3; FUS(P525L): n = 4). b, Filter trap with anti-FUS of HEK293 cells expressing wild-type or mutant FUS. Representative of five independent experiments. c, Western blot with anti-FUS of PSME3(OE)-HEK293 cells expressing mutant FUS. Graph represents the relative percentage of FUS levels (corrected for β-actin) to 37 °C + Empty vector (mean ± s.e.m. of 4 independent experiments). d, Filter trap with anti-FUS of PSME3(OE)-HEK293 cells expressing mutant FUS(P525L). Representative of four independent experiments. e, Immunocytochemistry of ALS (FUSP525L/P525L) and isogenic control (FUSWT/WT) iPSC-derived motor neurons. Cleaved caspase-3 (red), MAP2 (green) or Hoechst (blue) staining was used as a marker of apoptosis, neurons and nuclei, respectively. Scale bar: 20 µm. Graph represents the percentage of cleaved caspase-3-positive cells/total nuclei (mean ± s.e.m. of eight biological replicates, FUS(WT) 37 °C: 998 total nuclei; FUS(WT) 36 °C: 661; FUS(P525L) 37 °C: 331, FUS(P525L) 36 °C: 265). f, Western blot with anti-PSME3 of ALS-iPSCs expressing PSME3 shRNA. β-actin loading control. Representative of three independent experiments. g, Trypsin-like activity in ALS iPSC-derived motor neurons on PSME3 knockdown (mean ± s.e.m. relative slope to 37 °C NT shRNA, n = 3 independent experiments). h, Immunocytochemistry of ALS iPSCs-derived motor neurons with anti-cleaved caspase-3 (red), anti-MAP2 (green) and Hoechst (blue). Scale bar: 20 µm. Graph represents the percentage of cleaved caspase-3-positive cells/total nuclei (mean ± s.e.m. of nine biological replicates, 37 °C + NT shRNA: 822 total nuclei; 37 °C + psme-3 shRNA: 669; 36 °C + NT shRNA: 803; 36 °C + psme-3 shRNA: 567). In all the experiments, cells were cultured at 37 °C and then shifted to cold temperature (36 °C) or maintained at 37 °C for 24 h before the analysis. Two-tailed Student’s t-test for paired (a,c,g) or unpaired samples (e,h). P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, P > 0.05. All the significant changes were also significant after correction for multiple testing by FDR (q value< 0.05). Source Data contains exact P and q values. Source data
a, Log2-transformed fold changes in label-free quantification (LFQ) of proteasome subunits comparing day 6 adult control sterile fer-15(b26);fem-1(hc17) worms at 15 °C and 20 °C (n = 3). Data from ref. , P values calculated by two-tailed Student’s t-test. Worm orthologues of human proteasome subunits were identified with Ortholist2 according to InParanoid8 program,. b, Western blot with anti-PSME3 antibody in day 6 control sterile adult worms on psme-3 RNAi. Control sterile worms were raised at 25 °C during development and then grown at the indicated temperatures until day 6 of adulthood. RNAi initiated from day 1 adulthood. α-tubulin loading control. Representative of three independent experiments. c, Trypsin-like activity in day 3-adult control sterile worms (mean ± s.e.m. relative slope to 20 °C Vector RNAi, n = 4 independent experiments). Control sterile worms were raised at 25 °C until day 1 of adulthood and then grown at the indicated temperatures until day 3. d, Trypsin-like activity in day 3-adult wild-type worms (mean ± s.e.m. relative slope to 20 °C Vector RNAi, n = 4 independent experiments). Wild-type worms were raised at 20 °C during development and then grown at the indicated temperatures until day 3 of adulthood. e, Trypsin-like activity in day 1-adult wild-type worms (mean ± s.e.m. relative slope to 20 °C Vector RNAi, n = 4 independent experiments). Worms were raised on RNAi at the indicated temperatures during development. f, Trypsin-like activity in day 6 adult wild-type and trpa-1(ok999) mutant worms at 15 °C (mean ± s.e.m. relative slope to wild-type, n = 7 independent experiments). Worms were raised at 20 °C during development and then grown at 15 °C until day 6 of adulthood. g, Trypsin-like activity in day 6 adult worms at 20 °C (mean ± s.e.m. relative slope to wild-type, n = 7 independent experiments). h, Chymotrypsin-like activity in day 6 adult worms at 15 °C (mean ± s.e.m. relative slope to wild-type, n = 7 independent experiments). In c-h, statistical comparisons were made by two-tailed Student’s t-test for paired samples. P value: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS = not significant (P > 0.05). In c-e, all the significant changes were also significant after correction for multiple testing by FDR (q value< 0.05). Source Data contains exact P and q values. Source data
a, Images of day 5-adult worms expressing endogenous PSME-3 tagged with GFP at different temperatures. Scale bar: 100 µm. b, Higher magnification images of the head of day 5-adult worms expressing PSME-3::GFP at different temperatures. Scale bar: 20 µm. c, Higher magnification images of the body of day 5-adult worms expressing PSME-3::GFP at different temperatures. Scale bar: 20 µm. d, Intracellular localization of PSME-3 varies depending on the cell type. In germline and muscle cells, PSME-3 is mostly localized in the nucleus. In the intestine, PSME-3 is localized in the cytoplasm and nucleus. Likewise, PSME-3 is present in both the soma and nucleus of neurons. However, we could not detect PSME-3 in neuronal extensions. Images were obtained from day 5-adult worms expressing endogenous PSME-3::GFP at 15 °C. Scale bar: 10 µm. All the images are representative of three independent experiments. e, Knockdown (KD) of psme-3 in the germline decreases trypsin-like proteasome activity at 15 °C (mean ± s.e.m. of the relative slope to Vector RNAi, n = 4 independent experiments). f, Knockdown (KD) of psme-3 in muscle decreases trypsin-like proteasome activity at 15 °C (mean ± s.e.m. of the relative slope to Vector RNAi, n = 4 independent experiments). g, Knockdown (KD) of psme-3 in the intestine alone decreases trypsin-like proteasome activity at 15 °C (mean ± s.e.m. of the relative slope to Vector RNAi, n = 4 independent experiments). h, Knockdown (KD) of psme-3 in neurons decreases trypsin-like proteasome activity at 15 °C (mean ± s.e.m. of the relative slope to Vector RNAi, n = 4 independent experiments). All the statistical comparisons were made by two-tailed Student’s t-test for paired samples. P value: *P < 0.05, **P < 0.01. In all the experiments, worms were raised at 20 °C until day 1 of adulthood and then grown at the indicated temperatures until day 5 of adulthood. See Source Data for exact P and q values. Source data
a, Percentage survival of C. elegans following return to 20 °C after exposure to 4 °C cold shock (12 h) at day 1 of adulthood. Overexpression of PSME-3 does not increase resistance to extreme low temperature (4 °C). GFP(OE) mean ± s.e.m.: 5.65 days ± 0.31, PSME-3, GFP(OE) #1: 5.49 ± 0.26. b, psme-3 RNAi does not reduce the lifespan of wild-type or trpa-1(ok999) worms at 20 °C. Wild-type Vector RNAi mean ± s.e.m.: 18.35 ± 0.43, Wild-type psme-3 RNAi: 19.23 ± 0.41, trpa-1 Vector RNAi mean ± s.e.m.: 16.08 ± 0.30, trpa-1 psme-3 RNAi: 16.73 ± 0.39. c, psme-3 RNAi does not reduce the lifespan of wild-type or trpa-1(ok999) worms at 25 °C. Wild-type Vector RNAi mean ± s.e.m.: 13.33 ± 0.39, Wild-type psme-3 RNAi: 13.60 ± 0.33, trpa-1 Vector RNAi mean ± s.e.m.: 12.84 ± 0.33, trpa-1 psme-3 RNAi: 13.35 ± 0.30. d, psme-3 RNAi decreases the lifespan of wild-type worms at 15 °C. trpa-1(ok999) mutant worms live shorter than wild type animals at 15 °C, but psme-3 RNAi does not further decrease the short lifespan of trpa-1 mutants. Wild-type Vector RNAi mean ± s.e.m.: 27.92 ± 0.64, Wild-type psme-3 RNAi: 26.19 ± 0.61, trpa-1 Vector RNAi mean ± s.e.m.: 24.31 ± 0.80, trpa-1 psme-3 RNAi: 24.30 ± 0.85. e, Somatic overexpression of psme-3 under the sur-5 promoter shortens lifespan of trpa-1(ok999) mutant worms at 20 °C. trpa-1(ok999);GFP(OE) mean ± s.e.m.: 12.66 ± 0.32, trpa-1(ok999);psme-3,GFP(OE): 10.79 ± 0.41. f, Somatic overexpression of psme-3 shortens lifespan of trpa-1(ok999) mutant worms at 25 °C. trpa-1(ok999);GFP(OE) mean ± s.e.m.: 8.51 ± 0.15, trpa-1(ok999);psme-3,GFP(OE): 6.50 ± 0.22. g, psme-3 overexpression slightly increases the lifespan of trpa-1(ok999) mutant worms at 15 °C, but the lifespan extension is not significant. trpa-1(ok999);GFP(OE) mean ± s.e.m.: 17.16 ± 0.56, trpa-1(ok999);psme-3,GFP(OE): 18.62 ± 0.68. In all the experiments, P values were calculated by two-sided log-rank test, n = 90 worms per condition (a), n = 96 worms per condition (b-g). Supplementary Data 3 contains statistical analysis and replicate data of independent survival and lifespan experiments.
a, Western blot analysis of IFB-2 protein levels in wild-type adult animals cultured at the indicated temperatures after development. Graph represents the relative percentage values of IFB-2 protein levels (corrected for α-tubulin loading control) to 20 °C day 1-adult worms (mean ± s.e.m., n = 3 independent experiments). b, Filter trap analysis with antibody against IFB-2 of wild-type worms at different ages. Representative of four independent experiments. c, Western blot analysis of IFB-2 protein levels in day-10 adult wild-type worms upon psme-3 RNAi at cold temperature (15 °C). Graph represents the relative percentage values of IFB-2 protein levels (corrected for α-tubulin loading control) to Vector RNAi (mean ± s.e.m., n = 5 independent experiments). d, Filter trap with anti-IFB-2 antibody of day-10 adult wild-type worms upon psme-3 RNAi at the indicated temperatures. Representative of three independent experiments. In all the experiments, worms were raised at 20 °C and then shifted to the indicated temperatures after development. RNAi was initiated at day 1 of adulthood. All the statistical comparisons were made by two-tailed Student’s t-test for paired samples. P value: *P < 0.05, NS = not significant (P > 0.05). All the significant changes were also significant after correction for multiple testing by FDR approach (q value < 0.05). See Source Data for exact P and q values. Source data
a, Filter trap of neuronal polyQ67::YFP aggregation with anti-GFP in day 6 adults upon neuronal-specific psme-3 knockdown. Representative of three independent experiments. b-c, Western blot of polyQ67 protein levels upon psme-3 overexpression (OE) at 15 °C (b) or 25 °C (c). Graphs represent the relative percentage of polyQ67 protein levels upon PSME-3 overexpression (corrected for α-tubulin loading control) to control Q67 (mean ± s.e.m., n = 3 independent experiments). d, Filter trap of polyQ67 aggregation upon psme-3 overexpression at 15 °C and 25 °C. Representative of three independent experiments. e, Thrashing movements over a 30-s period (n = 100 worms from two independent experiments). The box plot represents the 25th–75th percentiles, the line depicts the median and the whiskers show the min–max values. f, Western blot of polyQ67 levels upon psme-3 overexpression at 20 °C. Graph represents the relative percentage of polyQ67 protein levels (corrected for α-tubulin) to control Q67 (mean ± s.e.m., n = 3 independent experiments). g, Filter trap of neuronal polyQ67 aggregation at 20 °C (representative of three independent experiments). h, Thrashing movements over a 30-s period (n = 50 worms per condition). The box plots and lines represent the 25th–75th percentiles and median, respectively. The whiskers show the min–max values. In b-h, we analyzed day 3-adult worms. i, Western blot of day 6 adult worms expressing polyQ40::YFP in muscle cells (detected by anti-GFP antibody). Graph represents the relative percentage of polyQ40 protein levels (corrected for α-tubulin) to 25 °C (mean ± s.e.m., n = 3 independent experiments). j, Western blot of polyQ40 protein levels in day 6 adult worms upon psme-3 RNAi at 15 °C. Graph represents the relative percentage of polyQ40 levels (corrected for α-tubulin) to 15 °C Vector RNAi (mean ± s.e.m., n = 3 independent experiments). RNAi was initiated at day 1 of adulthood. Statistical comparisons were made by two-tailed Student’s t-test for paired (b, c, f, i, j) or unpaired samples (e, h). P value: *P < 0.05, **P < 0.01, ****P < 0.0001, NS = not significant (P > 0.05). All the significant changes were also significant after correction for multiple testing by FDR (q value< 0.05). Source Data contains exact P and q values. Source data
a, Lowering temperature to 35 °C for 24 h decreases trypsin-like proteasome activity in HEK293 human cells (mean ± s.e.m. of the relative slope to 37 °C, n = 4 independent experiments). b, Lowering temperature to 35 °C (24 h) also decreases caspase-like proteasome activity in HEK293 cells (mean ± s.e.m. of the relative slope to 37 °C, n = 4 independent experiments). c, TRPA1 mRNA levels comparing human HEK293 cells and iPSC-derived motor neurons at standard temperature (37 °C). Graph represents the mean ± s.e.m. of the relative expression to motor neurons (n = 3 biological replicates). d, Western blot analysis with anti-TRPA1 antibody comparing human HEK293 cells and iPSC-derived motor neurons at standard temperature (37 °C). α-tubulin is the loading control. Representative of two independent experiments. e, qPCR analysis of knockdown levels in stable HEK293 cell lines expressing TRPA1 shRNA at the indicated temperatures (mean ± s.e.m. of the relative expression to control non-targeting (NT) shRNA, n = 6 independent experiments). f, Moderate cold temperature (36 °C, 24 h) induces trypsin-like proteasome activity in control HEK293 cells but not in TRPA1 shRNA cells (mean ± s.e.m. of the relative slope to 37 °C NT shRNA, n = 3 independent experiments). In all the experiments, cells were cultured at 37 °C and then shifted to colder temperatures or maintained at 37 °C for 24 h before the analysis. Statistical comparisons were made by two-tailed Student’s t-test for paired samples (a, b, e, f) or unpaired samples (c). P value: **P < 0.01, ***P < 0.001, ****P < 0.0001, NS = not significant (P > 0.05). All the significant changes were also significant after correction for multiple testing by FDR approach (q value < 0.05). See Source Data for exact P and q values. Source data
a, Western blot analysis with anti-FUS antibody of control non-targeting (NT) and TRPA1 shRNA-HEK293 cells expressing mutant FUS(P525L). Graph represents the relative percentage values of FUS protein levels (corrected for β-actin loading control) to 37 °C + NT shRNA (mean ± s.e.m., n = 3 independent experiments). Statistical comparisons were made by two-tailed Student’s t-test for paired samples. P value: *P < 0.05, NS = not significant (P > 0.05). All the significant changes were also significant after correction for multiple testing by FDR approach (q value < 0.05). See Source Data for exact P and q values. b, Filter trap analysis with anti-FUS antibody of NT and TRPA1 shRNA-HEK293 cells expressing mutant FUS(P525L). Knockdown of TRPA1 attenuates the ameliorative effects of cold temperature on FUS(P525L) aggregation levels. Representative of three independent experiments. c, Filter trap analysis with anti-FUS antibody of NT and PSME3 shRNA-HEK293 cells expressing mutant FUS(P525L) treated with 25 µM HC-030031 (TRPA1 antagonist) or DMSO vehicle control for 24 h. Representative of three independent experiments. Source data
a, Trypsin-like proteasome activity comparing human HEK293 cells and iPSC-derived motor neurons at standard temperature (37 °C). Bar graph represents the mean ± s.e.m. (relative slope to HEK293, n = 3 independent experiments). b, Caspase-like proteasome activity at standard temperature (37 °C). Graph represents the mean ± s.e.m. (relative slope to HEK293, n = 3 independent experiments). c, Chymotrypsin-like proteasome activity at standard temperature (37 °C). Graph represents the mean ± s.e.m. (relative slope to HEK293, n = 3 independent experiments). d, Cold temperature (36 °C) does not induce changes in the caspase-like proteasome activity of ALS iPSC-derived motor neurons (mean ± s.e.m. of the relative slope to 37 °C, n = 3 independent experiments). e, Cold temperature does not induce changes in the chymotrypsin-like proteasome activity of ALS iPSC-derived motor neurons (mean ± s.e.m. of the relative slope to 37 °C, n = 3 independent experiments). All the statistical comparisons were made by two-tailed Student’s t-test for paired samples. P value: **P < 0.01, ****P < 0.0001, NS = not significant (P > 0.05). See Source Data for exact P and q values. Source data
a, Immunocytochemistry with anti-PSME3 and anti-FUS antibodies of control (FUSWT/WT) and ALS (FUSP525L/P525L) iPSC-derived motor neurons. Hoechst staining was used as a nuclear marker. PSME3 is present in both the soma and nucleus of human motor neurons, but we did not detect PSME3 in neuronal extensions. Scale bar: 20 µm. Representative of two independent experiments. b, Immunocytochemistry with anti-PSME3 antibody of HEK293 cells at standard (37 °C) and cold temperature (36 °C). PSME3 is mostly accumulated in the nucleus of HEK293 cells. Higher exposure images indicate that a smaller amount of PSME3 is also located in the cytoplasm. Scale bar: 10 µm. Representative of four independent experiments. c, Immunocytochemistry with anti-HA tag antibody of HEK293 cells expressing either FUS(WT)-HA or FUS(P525L)-HA. Wild-type FUS is essentially located in the nucleus, whereas ALS-linked FUSP525L mutant variant is present in both the nucleus and cytoplasm. Cold temperature does not change the subcellular distribution of mutant FUSP525L. Hoechst staining was used as a nuclear marker. Scale bar: 10 µm. Representative of three independent experiments. In all the experiments, cells were cultured at 37 °C and then shifted to 36 °C or maintained at 37 °C for 24 h before the analysis.
a, Immunocytochemistry with anti-HA tag antibody of HEK293 cells expressing mutant FUS(P525L)-HA treated with either vehicle control (EtOH) or 20 nM leptomycin B for 6 h. Hoechst staining was used as a nuclear marker. The treatment with 20 nM leptomycin B (6 h), an inhibitor of nuclear export, increases the accumulation of mutant FUSP525L in the nucleus at both standard (37 °C) and cold temperature (36 °C). Scale bar: 10 µm. Representative of three independent experiments. b, Western blot analysis with anti-FUS antibody of HEK293 cells expressing mutant FUS(P525L)-HA treated with either vehicle control (EtOH) or 20 nM leptomycin B for 6 h. Bar graph represents the mean ± s.e.m. of relative percentage values of FUS protein levels (corrected for β-actin loading control) to 37 °C + EtOH vehicle control, n = 4 independent experiments. Statistical comparisons were made by two-tailed Student’s t-test for paired samples. P value: **P < 0.01, ***P < 0.001, NS = not significant (P > 0.05). All the significant changes were also significant after correction for multiple testing by FDR approach (q value < 0.05). See Source Data for exact P and q values. c, Images of HEK293 cells expressing Q100-HTT tagged with mCherry treated with either vehicle control (EtOH) or 20 nM leptomycin B for 6 h. Mutant HTT remained in the cytoplasm upon leptomycin B treatment. Hoechst staining was used as a nuclear marker. Scale bar: 10 µm. Representative of two independent experiments. d, Western blot analysis with anti-HTT antibody in HEK293 expressing mCherry-Q100-HTT. β-actin is the loading control. Representative of two independent experiments. Source data
Comment in
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Cold-induced longevity through proteasome boosting.Nat Rev Mol Cell Biol. 2023 Jun;24(6):381. doi: 10.1038/s41580-023-00610-1. Nat Rev Mol Cell Biol. 2023. PMID: 37185986 No abstract available.
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