Cold temperature extends longevity and prevents disease-related protein aggregation through PA28γ-induced proteasomes

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.

Extreme low temperatures are detrimental, but a moderate decrease in body temperature can have beneficial effects for the organism 1 . In fact, lowering body temperature extends longevity in both poikilotherms (for example Caenorhabditis elegans [2][3][4] , Drosophila melanogaster 5 , and distinct fish species 6,7 ) and homeotherms such as rodents 8 . For instance, C. elegans lives for a shorter period of time when shifted from the standard temperature (20 °C) to warmer temperatures, whereas exposure to low temperature (15 °C) induces a remarkable lifespan extension [9][10][11][12] . Exposure of rodents to hot ambient temperature can result in 0.5 °C higher body temperature that shortens lifespan 13,14 . In contrast, a mild decrease of 0.5 °C in body temperature prolongs lifespan in mice, supporting a conserved role of temperature reduction in longevity 8 . Correlations between body temperature and lifespan are also reported for humans [15][16][17] . The normal human body temperature ranges between 36.5 and 37 °C 18 . Whereas an acute drop in body temperature below 35 °C leads to hypothermia, the human body temperature slightly varies during the day and even reaches moderate cold temperatures (36 °C) during sleep 19 . Interestingly, the human body temperature has decreased monotonically by 0.03 °C per decade since the Industrial Revolution, providing a potential link with the progressive increase in human longevity over the last 160 years 15 .
Although the longevity effects of low temperature were reported more than a century ago 20 , little is known about how cold temperature influences lifespan and health. The conventional view was that coldinduced longevity ensues from a reduction in the rate of chemical reactions and metabolism, leading to slower energy expenditure and Given that exposure of C. elegans to low temperature (15 °C) after development is sufficient to induce lifespan extension 9,12 , we asked whether cold temperature influences proteasome activity during adulthood. To prevent the development of progeny, we used fer-15(b26);fem-1(hc17) mutant worms 9,33 , which are sterile when raised at the restrictive temperature (25 °C) during development. Similar to wild-type animals, cold temperature after development also extends lifespan in control sterile worms 9 . Thus, we raised control sterile worms at 25 °C until day 1 of adulthood and then shifted them to different temperatures. At day 6 of adulthood, worms exhibited a dramatic increase in trypsin-like proteasome activity at cold temperature (15 °C) when compared with 20 °C or 25 °C (Fig. 1a). However, adult control sterile worms had similar caspase-like and chymotrypsin-like proteasome activities across temperatures (Fig. 1b,c). The cold-induced effects on trypsin-like activity were not linked with sterility, as wild-type worms also had a similar increase when shifted from 20 °C to 15 °C during adulthood (Fig. 1d). In contrast, caspase-like and chymotrypsin-like activities remained similar or decreased in wild-type animals at cold temperature, respectively (Fig. 1e,f).
To assess whether cold temperature changes the levels of proteasome subunits, we used available quantitative proteomics data 9 . We did not find substantial differences in 20S subunits comparing adult worms at cold (15 °C) and standard (20 °C) temperatures, including β2/ PBS-2 that has trypsin-like activity (Extended Data Fig. 1a). Moreover, proteomic analysis did not detect significant changes in 19S subunits. Among PA28 subunits, C. elegans only expresses psme-3, the orthologue of human PA28γ/PSME3 (refs. 34,35). Notably, quantitative proteomics revealed increased levels of PSME-3 at cold temperature (Extended Data Fig. 1a). By western blot, we confirmed that worms exhibit higher protein levels of PSME-3 at 15 °C when compared with 20 °C or 25 °C (Fig. 1g). The increase in PSME-3 protein levels correlated with an upregulation of the mRNA amounts at cold temperature (Fig. 1h). Because PA28γ/PSME3 selectively activates trypsin-like activity in vitro 31,36 , we asked whether PSME-3 is required for the elevated trypsin-like activity at 15 °C. Indeed, knockdown of psme-3 after development decreased trypsin-like activity at cold temperature, but not at standard temperature (Extended Data Fig. 1b and Fig. 1i,j).
Because low temperature extends lifespan, we could not discard that the changes observed in day 6 adults ensue from differences in pace of living 21 . However, cumulative evidence in C. elegans demonstrates that cold-induced longevity is a regulated process that cannot be only explained by passive changes in chemical reactions 9,11,22 . For instance, the cold-sensitive channel TRPA-1 detects low temperature in the nervous system and non-excitable tissues such as the intestine, actively leading to lifespan extension in C. elegans 11,12 . Moreover, low temperature induces molecular chaperones in C. elegans that maintain protein homeostasis (proteostasis) and cell function, including the chaperonin TRiC/CCT and the co-chaperone DAF-41/p23 (refs. 9,23).
Aging is a major risk factor for distinct neurodegenerative disorders linked with protein aggregation, including Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (ALS) 24,25 . Here, we hypothesize that a better understanding of cold-induced effects can lead to converging modifiers of pathological protein aggregation, with therapeutic implications for multidisease prevention. To this end, we examine whether cold temperature influences proteasome activity, a determinant of cell function and viability 25 . The proteasome can prevent aging and pathological conditions through degrading unwanted, damaged and misfolded proteins which are prone to aggregation, including disease-related mutant proteins 24,26 . The structure of the proteasome is highly conserved among eukaryotes 27,28 . The proteasome core (20S) contains three catalytic subunits with different cleavage specificities (β1, β2 and β5 that respectively exhibit caspase-like (hydrolysis after acidic amino acids), trypsin-like (hydrolysis after basic amino acids) and chymotrypsin-like activities (cleavage after hydrophobic amino acids) 29 . Activation of proteolytic sites occurs through the assembly of the 20S with regulatory particles 25,28 . The major regulatory complex is the 19S, which is composed of multiple distinct subunits. The assembly of the 20S with the 19S regulatory complex forms active 26S proteasomes, which recognize and degrade proteins tagged with ubiquitin. In addition to the 19S complex, the 20S can also be activated by other regulatory particles such as PA200/PSME4 or PA28 (also known as 11S) 30 . PA28 can be formed by either hetero-heptameric rings of 28-kDa proteins (PA28α/PSME1, PA28β/PSME2), which are characteristic of the immune system, or homo-heptameric rings of PA28γ/PSME3 subunits, which are expressed throughout the entire organism [30][31][32] . In contrast to the 19S, PA28γ promotes protein degradation in a ubiquitin-independent manner 31 . Although the function and substrates of the PA28γ-activated proteasome are less understood than those of 26S proteasome, its activity and evolutionary conservation indicate an important biological role [30][31][32] .
Here, we find that the worm orthologue of PA28γ/PSME3 is required for cold-induced longevity and attenuates age-related deficits in protein degradation by the 26S proteasome. Moreover, cold-induced PA28γ prevents aggregation of disease-related proteins in C. elegans models of Huntington's disease and ALS. Notably, a moderate reduction  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. Article https://doi.org/10.1038/s43587-023-00383-4 aging rates rather than a direct effect of PSME-3. To disentangle these possibilities, we examined worms at a younger age (that is, day 3 of adulthood). Importantly, cold temperature after development was sufficient to induce trypsin-like activity in both control sterile and wild-type animals at day 3 of adulthood (Extended Data Fig. 1c   cold-induced trypsin-like activity in day 3 adults but had no effects at 20 °C (Extended Data Fig. 1c,d). To assess younger ages, we started the cold temperature and RNAi treatments during development and assessed proteasome activity at day 1 of adulthood. Indeed, day 1 adults exhibited higher levels of trypsin-like activity at 15 °C when compared with age-matched worms at 20 °C (Extended Data Fig. 1e). Although knockdown of psme-3 during development slightly decreased basal trypsin-like activity in day 1 adults at 20 °C, its inhibitory effects were much stronger at cold temperature (Extended Data Fig. 1e). Thus, these data support a direct effect of cold-induced PSME-3 in the upregulation of trypsin-like proteasome activity. Along these lines, overexpression of psme-3 was sufficient to further increase trypsin-like activity at 15 °C (Fig. 1k). In contrast, psme-3 overexpression did not induce trypsin-like activity at 20 °C (Fig. 1l), suggesting that PSME-3 function requires its activation by other factors that occurs upon cold temperature. Additionally, worms may also have mechanisms to inactivate PSME-3 at 20 °C. However, worms did not inhibit overexpressed PSME-3 when subjected to mild heat stress, resulting in a pronounced increase of trypsin-like activity at 25 °C (Fig. 1m). Altogether, we found that elevated PA28γ/PSME-3 function underlies the high levels of trypsin-like activity induced by cold temperature in C. elegans, while overexpression of PSME-3 can also promote trypsin-like proteasome activity at warm temperature, but not standard temperature.

TRPA-1 is required for cold-induced trypsin-like activity
In C. elegans, low temperature activates the cold-sensitive channel TRPA-1, which promotes lifespan extension 11,12 . We found that mutant worms lacking trpa-1 have lower trypsin-like activity when compared with wild-type animals at cold temperature, either at day 3 or 6 of adulthood ( Fig. 2a and Extended Data Fig. 1f). In contrast, loss of trpa-1 did not reduce basal trypsin-like activity at standard temperature (Extended Data Fig. 1g). Moreover, lack of trpa-1 did not affect a different proteasome activity at 15 °C, supporting that activation of TRPA-1 channels contributes to the selective induction of trypsin-like activity at low temperature (Extended Data Fig. 1h). Because loss of trpa-1 did not further decrease the low trypsin-like activity of psme-3 RNAi-treated worms at 15 °C, these results indicate that TRPA-1 channels modulate proteasome activity through PSME-3 (Fig. 2a).
Intrigued by these findings, we asked in which tissues PSME-3 acts to regulate organismal lifespan. Although TRPA-1 is expressed in multiple tissues, its activation in the intestine and neurons is particularly important to promote longevity 11 . However, activation of TRPA-1 in neurons also signals distal tissues such as the germline to delay reproductive aging 9 . In turn, the germline releases factors to induce pro-longevity genes in somatic tissues, including the intestine and muscle 9 . According to its key role in cell non-autonomous regulation of cold-induced longevity 9 , knockdown of psme-3 in the germline alone resulted in the strongest decrease of lifespan at 15 °C (Fig. 3g). In addition, tissue-specific knockdown of psme-3 in neurons, intestine or muscle also significantly decreased lifespan at 15 °C (Fig. 3g). Thus, PSME-3 has a pro-longevity role in all the tissues which are known to influence cold-induced lifespan extension (that is germline, neurons, intestine and muscle). To assess whether PSME-3 is expressed in these tissues, we tagged endogenous PSME-3 protein with GFP. We found that worms exhibit a robust basal expression of PSME-3 across tissues, even at standard temperature (Extended Data Fig. 2a). Because cold temperature only induces a moderate increase in PSME-3 levels ( Fig. 1g), we could not use the reporter strain to assess whether PSME-3 is upregulated in a particular tissue at 15 °C (Extended Data Fig. 2a-c). Nevertheless, we confirmed that PSME-3 is highly expressed not only in the germline and intestine but also in muscle and neurons, although to a lesser extent (Extended Data Fig. 2a-d). Importantly, specific knockdown of psme-3 in the aforementioned tissues decreased trypsin-like activity (Extended Data Fig. 2e-h), further supporting a functional role of PSME-3 in the germline, intestine, muscle and neurons. in C. elegans at cold temperature. 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. We then assessed whether PSME-3 overexpression in somatic tissues is sufficient to promote longevity. Interestingly, PA28γ/psme-3 overexpression slightly decreased lifespan at standard temperature (20 °C) and was strongly detrimental at warmer temperature (Fig. 3h,i). On the contrary, PA28γ/psme-3 overexpression further extends longevity at 15 °C (Fig. 3j) 39 . To examine whether PA28γ/PSME-3 can confer resistance to acute cold shock, we exposed worms to extreme low temperature (4 °C) for 12 h and then shifted them back to 20 °C. We found that overexpression of PA28γ/psme-3 did not increase survival after exposure to acute cold shock (Extended Data Fig. 3a). Thus, our data indicate that PA28γ/ PSME-3 does not protect from extreme low temperatures but contributes to the longevity effects induced by moderate cold temperature. However, PSME-3 overexpression can have negative effects on lifespan at higher temperatures. Given that activation of TRPA-1 promotes cold-induced longevity, we performed epistasis experiments to assess its functional links with PSME-3 in lifespan regulation. Similar to wild-type animals, knockdown of psme-3 did not shorten lifespan of trpa-1-lacking worms at standard (20 °C) and warmer (25 °C) temperatures (Extended Data Fig. 3b,c). Whereas loss of psme-3 shortened the cold-induced long lifespan of wild-type animals, it did not further decrease the short lifespan of trpa-1 mutant worms at cold temperature (15 °C) (Extended Data Fig. 3d). Moreover, overexpression of psme-3 also shortened the lifespan of trpa-1 mutant animals at both standard and warm temperatures, but not at cold temperature (Extended Data Fig. 3e-g). Although psme-3 overexpression slightly increased the mean lifespan of trpa-1 mutant worms at 15 °C, this extension was not significant (Extended Data Fig. 3g). Together, our results suggest that TRPA-1 acts upstream of PSME-3 and is required for its longevity effects in C. elegans at moderate low temperature.

Low temperature prevents disease-related protein aggregation
In contrast to 26S proteasomes, activation of the 20S proteasome through PA28γ induces protein degradation in a ubiquitin-independent manner 31 . Aging triggers a global loss of ubiquitination through the proteome in C. elegans, reducing the degradation of multiple proteins by the 26S proteasome 26 . Subsequently, these dysregulated proteasome targets accumulate during aging and impair cellular function 26 . For instance, the intermediate filament IFB-2 escapes the clean-up by the 26S proteasome with age, leading to its aggregation within intestinal cells. Conversely, knockdown of IFB-2 during adulthood delays the decline in intestinal integrity characteristic of aging animals and extends lifespan at standard temperature 26 . Notably, we found that cold temperature prevents the accumulation of IFB-2 and its subsequent aggregation in adult wild-type animals with age (Extended Data Fig. 4a,b). However, knockdown of PA28γ/psme-3 reduces the degradation of IFB-2, suppressing the ameliorative effects of low temperature on IFB-2 aggregation (Extended Data Fig. 4c,d). Thus, these results suggest that cold-induced PA28γ/PSME-3 can ameliorate age-related deficits in protein degradation.
In vitro, PA28γ preferentially promotes the degradation of unstructured proteins over their native/folded counterparts, including unfolded variants of β-casein and insulin-like growth factor 1 (IGF-1) 40 . Moreover, in vitro experiments demonstrated that PA28γ promotes the degradation of peptides containing expanded polyglutamine repeats (polyQ) 41 , which are linked with different human diseases. Thus, we asked whether cold-induced PA28γ/PSME-3 modulates the levels of disease-related mutant proteins which are prone to misfolding and aggregation. To this end, we used C. elegans that specifically express expanded polyQ peptides in neurons. These worms replicate key features of Huntington's disease, as protein aggregation and neurotoxicity correlate with increased length of the polyQ peptide, with a pathogenic threshold of 40 repeats [42][43][44][45] . By filter trap assay, we did not observe aggregation of control polyQ19 peptides at any of the tested temperatures. In contrast, polyQ67-expressing worms had a strong aggregation phenotype at standard temperature (20 °C) which was further increased by mild heat-stress (25 °C) (Fig. 4a). Notably, cold temperature (15 °C) attenuated polyQ67 aggregation when compared with standard temperature, correlating with a downregulation in the total amounts of polyQ67 protein ( Fig. 4a,b). On the contrary, low temperature did not decrease the protein levels of control polyQ19 when compared with standard temperature (Fig. 4b).
Knockdown of PA28γ/psme-3 diminished the cold-induced degradation of polyQ67, increasing its aggregation at 15 °C (Fig. 4c,d). The aggregation of polyQ67 in neurons causes neurotoxicity and subsequently impairs motility, a disease-like phenotype 42,43 . We found that loss of PA28γ/psme-3 reduces the motility of neuronal polyQ67-expressing worms at cold temperature, but does not affect control polyQ19 worms (Fig. 4e). We then asked whether PSME-3 influences neuronal aggregation of polyQ67 through its intracellular activity in neurons or via cell non-autonomous effects triggered by its function in other tissues. Notably, neuronal-specific knockdown of psme-3 was sufficient to induce aggregation of polyQ67 protein in neurons (Extended Data Fig. 5a). Thus, these results indicate that intracellular levels of PSME-3 can directly regulate proteostasis of polyQ-expanded proteins in neurons. Because overexpression of psme-3 induces trypsin-like activity at both 15 °C and 25 °C (Fig. 1k,m), we assessed the effects of psme-3 overexpression in polyQ67-expressing worms. We found that psme-3 overexpression reduces the total levels of neuronal polyQ67 at both cold and warm temperatures (Extended Data Fig. 5b,c). Concomitantly, psme-3 overexpression diminished the accumulation of aggregated polyQ67 peptides as well as motility defects at 25 °C (Extended Data Fig. 5d,e). Given the low levels of aggregated polyQ67 at cold temperature, we could not detect a further decrease upon psme-3 overexpression at 15 °C (Extended Data Fig. 5d). Nevertheless, psme-3 overexpression was sufficient to further ameliorate motility defects at 15 °C (Extended Data Fig. 5e). In contrast, psme-3 overexpression   did not influence the levels, aggregation and neurotoxicity of polyQ67 at 20 °C (Extended Data Fig. 5f-h), according to its lack of effects on proteasome activity at standard temperature (Fig. 1l).
In addition to neurons, low temperature also decreased polyQ levels and aggregation in C. elegans that specifically express expanded polyQ peptides in the muscle ( Fig. 4f Article https://doi.org/10.1038/s43587-023-00383-4 Moreover, knockdown of PA28γ/psme-3 reduced the degradation of muscle polyQ peptides (Extended Data Fig. 5j). Conversely, loss of psme-3 suppressed the inhibitory effects of cold temperature over polyQ aggregation in the muscle (Fig. 4f). The aggregation of expanded polyQ within muscle cells has intracellular detrimental effects in C. elegans, reducing muscle function and organismal motility 46 . In correlation with polyQ aggregates levels in the muscle, cold temperature ameliorated the deficits in coordinated movement whereas loss of psme-3 suppressed these beneficial effects (Fig. 4g). Therefore, coldinduced PA28γ/PSME-3 can prevent the aggregation of expanded polyQ proteins in distinct tissues, alleviating their pathological effects. Besides expanded polyQ proteins, we asked whether coldinduced PA28γ/PSME-3 can also diminish aggregation of other disease-related proteins.  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.
Article https://doi.org/10.1038/s43587-023-00383-4 express an ALS-related mutant variant of FUS protein (FUS P525L ) in neurons 47 . These worms replicate pathological phenotypes of ALS such as protein aggregation and neurodegeneration, as reflected by loss of motility 43,47 . We found that cold temperature decreases the total levels of mutant FUS protein, resulting in lower disease-related aggregation compared to either 20 °C or 25 °C (Fig. 5a,b). However, knockdown of PA28γ/psme-3 diminished the cold-induced degradation of mutant FUS, triggering the accumulation of FUS aggregates at 15 °C (Fig. 5c,d). Subsequently, knockdown of psme-3 suppressed the beneficial effects of cold temperature on ALS-related motility deficits (Fig. 5e). In addition to mutant FUS, cold temperature also decreased the protein levels and aggregation of ALS-related TDP-43 M331V variant ( Fig. 5f,g), another mutant protein which is prone to aggregation 48 . Conversely, knockdown of PA28γ/psme-3 reduced the cold-induced degradation of mutant TDP-43, promoting its aggregation at 15 °C (Fig. 5h,i). Collectively, our data indicate that cold-induced PA28γ/ PSME-3 can attenuate the pathological aggregation of distinct diseaserelated proteins in C. elegans models.

Cold temperature induces PA28γ-proteasomes in human cells
The human body can physiologically reach moderate cold temperatures (36 °C) during sleep, but a pathological acute drop in temperature (≤ 35 °C) leads to hypothermia 19 . To assess whether a moderate cooling also influences proteasome activity in human cells, we shifted HEK293 human cells from the standard temperature (37 °C) to moderate cold temperature (36 °C) for 24 h. Similar to C. elegans, moderate cold temperature increased trypsin-like activity in human cells, whereas the other two activities of the proteasome remained similar (Fig. 6a-c). However, a further decrease in temperature to 35 °C not only downregulated trypsin-like activity but also caspase-like activity when compared with 37 °C (Extended Data Fig. 6a,b). Thus, these results indicate that moderate cold temperature (36 °C) can selectively induce trypsin-like activity in human cells, but lower temperatures can have a general negative effect on proteasome activities. Because HEK293 cells expressed endogenous levels of TRPA1 (Extended Data Fig. 6c,d), we asked whether this cold-sensitive channel also regulates the induction of trypsin-like activity in human cells at moderate cold temperature. Indeed, either knockdown of TRPA1 or treatment with HC-030031, a selective antagonist of TRPA1 49,50 , blocked the trypsinlike activity induced by cold temperature in HEK293 cells (Extended Data Fig. 6e,f and Fig. 6d). Moderate cold temperature (36 °C) resulted in increased transcript and protein levels of PSME3, the human orthologue of PA28γ (Fig. 6e,f). Moreover, we found increased assembly of PSME3 subunits into proteasome-activator 11S/PA28γ 7 complexes at 36 °C (Fig. 6g). To further determine whether PSME3 underlies the upregulation of trypsin-like activity triggered by moderate cold temperature, we generated stable PSME3-shRNA expressing HEK293 lines.
These cells exhibited robust knockdown of PSME3 at either standard or cold temperatures (Fig. 6h,i). Remarkably, loss of PSME3 prevented the upregulation of trypsin-like activity induced by moderate cold temperature, but did not have strong effects at standard temperature (Fig. 6j). Moreover, PSME3 overexpression further increased trypsinlike activity at moderate cold temperature (Fig. 6k-m). In contrast to C. elegans, PSME3 overexpression was also sufficient to induce trypsinlike activity at 37 °C (Fig. 6k-m), raising the possibility that PSME3 can have valuable effects in human cells even at normal temperature.

PA28γ-proteasomes degrade human disease-related proteins
Given the beneficial impact of cold temperature in C. elegans, we asked whether low temperature also prevents disease-related protein aggregation in human cells. To this end, we generated HEK293 cell models that express either control (Q23) or polyQ-expanded (Q100) huntingtin (HTT), the mutant protein underlying Huntington's disease 51,52 . In these cells, expression of mutant Q100-HTT resulted in the accumulation of polyQ aggregates, whereas control Q23-HTT did not form aggregates (Fig. 7a,b). Moderate cold temperature (36 °C) substantially reduced the amounts of Q100-HTT protein and its aggregation, but it did not influence control Q23-HTT levels (Fig. 7a,b). We found that either the knockdown or inhibition of TRPA1 channels is sufficient to block the cold-induced degradation of mutant HTT, leading to its aggregation at 36 °C (Fig. 7c-f). Likewise, stable PSME3-knockdown HEK293 cells lost their cold-induced ability to promote degradation of mutant HTT, resulting in similar levels of polyQ-expanded aggregates at cold and standard temperatures (Fig. 7g,h). In contrast, loss of PSME3 did not further increase the protein levels and aggregation of mutant HTT at standard temperature (37 °C) (Fig. 7g,h). Moreover, knockdown of PSME3 did not affect control Q23-HTT levels at either standard or low temperature, supporting that cold-induced PA28γ/PSME3 preferentially promotes the degradation of mutant HTT (Fig. 7g,h). Given that PSME3 overexpression induces trypsin-like activity even at normal temperature, we assessed whether increasing PSME3 levels prevents mutant HTT aggregation at 37 °C. Indeed, PSME3 overexpression was sufficient to promote the degradation of mutant HTT, reducing its aggregation at normal temperature (Fig. 7i,j). Because the aggregated amounts of mutant HTT were very low at 36 °C, it was difficult to interpret whether PSME3 overexpression further decreases its aggregation at cold temperature (Fig. 7j).
Besides polyQ-expanded HTT, cold temperature also diminished the levels and aggregation of the ALS-related mutant FUS P525L variant in HEK293 human cells (Fig. 8a,b). Knockdown of PSME3 suppressed the cold-induced degradation of aggregation-prone FUS P525L , resulting in the accumulation of FUS aggregates in HEK293 cells at cold temperature (Fig. 8a,b). In contrast, cold-induced PA28γ/ PSME3 did not change wild-type FUS levels (Fig. 8a,b). Importantly, either knockdown or pharmacological inhibition of TRPA1 also 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.
Prompted by these findings, we asked whether cold temperature can ameliorate disease-related neurodegeneration. ALS is characterized by the selective degeneration of motor neurons 53 . Accordingly, motor neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) exhibit increased cell death phenotypes [54][55][56][57] . Indeed, iPSC-derived motor neurons harboring the severe ALSlinked FUS P525L mutation 58 had increased apoptotic rates compared with isogenic controls (FUS WT/WT ) (Fig. 8e). Notably, cold temperature (36 °C) attenuated the elevated apoptotic rates of ALS motor neurons (Fig. 8e). To assess whether these effects are mediated by PA28γ/PSME3, we generated stable PSME3-shRNA ALS-iPSCs and differentiated them into motor neurons (Fig. 8f). The nervous system express the highest levels of PA28γ/PSME3 compared with other tissues 32 . Consistently, we found that iPSC-derived motor neurons have higher (over threefold) basal trypsin-like activity than HEK293 cells at standard temperature, whereas the other proteasome activities were decreased (Extended Data Fig. 8a-c). Although iPSC-derived motor neurons already displayed elevated trypsin-like activity at standard conditions, cold temperature could further increase this specific activity without affecting chymotrypsin-like and caspase-like activities ( Fig. 8g and Extended Data Fig. 8d,e). Knockdown of PA28γ/PSME3 decreased trypsin-like activity in motor neurons at both standard and cold temperature, but the reduction was significantly stronger at cold temperature (Fig. 8g). In correlation with this decline in trypsin-like activity, knockdown of PSME3 reduced the ameliorative effects of cold temperature in the neurodegeneration phenotype of ALS motor neurons (Fig. 8h). Together, our results indicate that cold temperature prevents pathological phenotypes such as protein aggregation and neurodegeneration, a process mediated by the induction of trypsin-like activity in a PA28γdependent manner.

PA28γ promotes protein degradation in nucleus and cytoplasm
Because proteasomes are active in both the cytoplasm and nucleus 59 , we asked in which subcellular compartment PA28γ/PSME3 promotes degradation of disease-related proteins. Although PA28γ/PSME3 is mostly located in the nucleus of distinct human cell lines 60,61 , cumulative evidence demonstrates that PSME3 also becomes more prominent in the cytoplasm depending on the conditions and cell type 62,63 . Indeed, we found that the subcellular distribution varies depending on the cellular type. In the C. elegans germline and muscle, PSME-3 mostly accumulated in the nucleus (Extended Data Fig. 2d). In intestinal cells, PSME-3 was mostly present in the cytoplasm but also detected in the nucleus. Likewise, PSME-3 was present in both the soma and nucleus of C. elegans neurons, but we did not detect PSME-3 in neuronal extensions (Extended Data Fig. 2d). We observed a similar distribution of PSME3 in the soma and nucleus of human iPSC-derived motor neurons (Extended Data Fig. 9a). In contrast, PSME3 was mostly accumulated in the nucleus of HEK293 cells, although we also observed cytoplasmic PSME3 to a lesser extent (Extended Data Fig. 9b). Importantly, cold temperature did not change the subcellular distribution of PSME3 in C. elegans or human cells (Extended Data Fig. 2a-c and Extended Data Fig. 9b).
Article https://doi.org/10.1038/s43587-023-00383-4 we observed increased cytoplasmic localization of mutant FUS P525L in both human motor neurons and HEK293 cells, but a fraction of mutant FUS remained in the nucleus (Extended Data Fig. 9a,c).
We found that cold temperature does not change the subcellular distribution of mutant FUS P525L in human HEK293 cells (Extended Data Fig. 9c)   distribution of PSME3, which remained mostly located in the nucleus (Extended Data Fig. 9b). Thus, we assessed whether cold-induced PSME3 prevents aggregation of mutant FUS by inducing its degradation in the cytoplasm or nucleus. To this end, we treated HEK293 cells with leptomycin B, an inhibitor of nuclear export 69 . Notably, leptomycin B treatment for 6 hours was sufficient to induce the accumulation of mutant FUS P525L in the nucleus (Extended Data Fig. 10a). Subsequently, leptomycin B treatment further increased the cold-induced degradation of mutant FUS P525L protein (Extended Data Fig. 10b). Although leptomycin B also accumulated FUS P525L in the nucleus at normal temperature (37 °C), it did not promote FUS degradation under this condition (Extended Data Fig. 10a,b). Thus, PSME3 preferentially promotes cold-induced degradation of FUS P525L in the nucleus, a process that could eventually reduce the loading of mutant FUS into the cytoplasm and subsequent aggregation. In contrast to FUS, mutant HTT is mainly cytoplasmic 70 and remained in the cytoplasm upon leptomycin B treatment (Extended Data Fig. 10c). Concomitantly, leptomycin B did not further increase the cold-induced degradation of mutant HTT (Extended Data Fig. 10d). Because cold temperature was sufficient to promote HTT degradation without leptomycin B treatment (Extended Data Fig. 10d), these data suggest that even the relative low amounts of PSME3 in the cytoplasm of HEK293 cells can scavenge HTT. Thus, cold-induced PSME3 may promote degradation of aggregation-prone proteins in either the nucleus or cytoplasm, depending on the main subcellular localization of the disease-related proteins.

Discussion
Previous studies in C. elegans demonstrated that cold-induced longevity is not a passive thermodynamic process but depends on the ability of the animal to sense low temperature through the cold-sensitive TRPA-1 channel 11,12,71 . Here, we discovered that TRPA-1 induces the expression of the proteasome-activator PA28γ/PSME-3 in C. elegans specifically at cold temperature (15 °C), contributing to extended longevity. In worms, TRPA-1 channels open when temperature drops to ~20 °C 72 . Accordingly, mutant worms lacking TRPA-1 have a shorter lifespan at both 15 °C and 20 °C, but not at warmer temperature, than do wild-type worms 11 . However, we found that PSME-3 expression is only induced at 15 °C. Although TRPA-1 is functional at both 15 °C and 20 °C, worms live much longer at 15 °C. Thus, TRPA-1 could trigger further pro-longevity mechanisms when the temperature drops to 15 °C. For instance, DAF-16 is required for the role of TRPA-1 in lifespan regulation at both 15 °C and 20 °C 11 , whereas other transcription factors such as NHR-49 are only required for lifespan extension at 15 °C 22 . Along these lines, PSME-3 levels are upregulated through NHR-49/TRPA-1 activation, whereas DAF-16 is dispensable for this phenotype. Although further work is required to define whether NHR-49 directly regulates psme-3 transcription or acts indirectly by regulating other factors that in turn control psme-3 expression, these data supports that TRPA-1 activates additional mechanisms at 15 °C. Moreover, neuronal activation of TRPA-1 delays germline aging at cold temperature but not at 20 °C 9 . In turn, the germline releases signals to promote somatic fitness at 15 °C 9 . Besides the intracellular effects of PSME-3 within somatic tissues, we observed that knockdown of psme-3 in the germline alone induces the strongest decrease in cold-induced longevity. Because PA28γ/PSME-3 is only required for lifespan extension at 15 °C but not at 20 °C, these results could explain why TRPA-1 activation induces a longer lifespan at 15 °C.
The biological roles of PA28γ-activated proteasomes are understood less well than those of 19S-activated proteasomes (26S). Whereas the 19S induces the three proteolytic activities of the proteasome, PA28γ preferentially promotes trypsin-like activity. Another important difference is that 19S-activated proteasomes selectively recognize and degrade ubiquitinated proteins, whereas PA28γ promotes degradation in a ubiquitin-independent manner 31 . With age, alterations in distinct ubiquitin ligases and deubiquitinating enzymes lead to the accumulation of aggregation-prone proteins that cannot be degraded by 26S proteasomes 25,26 . Thus, we speculate that the capacity of PA28γ-activated proteasomes to terminate targets regardless of their ubiquitinated state can be advantageous to prevent accumulation of these proteins with age. In support of this hypothesis, cold-induced PA28γ attenuates age-related deficits in the degradation of IFB-2, a protein that becomes less ubiquitinated and degraded by 26S proteasomes during aging 26 . Beyond the effects on C. elegans longevity, cold-induced PA28γ/PSME-3 also prevents the aggregation of distinct mutant proteins involved in age-related diseases such as Huntington's and ALS.
Although PA28γ/PSME3 levels are upregulated and required for cold-induced beneficial effects, it is important to note that overexpression of PSME-3 does not increase trypsin-like activity at standard temperature (20 °C) in C. elegans. Therefore, these data suggest that upregulation of PSME-3 is not sufficient to promote trypsin-like activity in C. elegans and additional cold-induced factors are required to activate PSME-3. In addition, worms could also induce inhibitory mechanisms to prevent PSME-3 activity at standard temperature. It will be fascinating to define which are these activators and/or inhibitors of PA28γ/PSME-3 induced by cold and normal temperature, respectively. An intriguing question is why worms inactivate PSME-3 at normal temperature. Although PA28γ/PSME-3 induces C. elegans longevity under cold temperature, overexpression of psme-3 has detrimental effects on viability under standard temperature (20 °C) and particularly mild heat stress (25 °C). Given that PA28γ promotes protein degradation in a ubiquitin-independent manner, a potential explanation is that changes in the balance between PA28γ and 19S-induced proteasomes could increase the degradation of distinct regulatory proteins regardless their ubiquitination state. Although this characteristic might be beneficial to diminish aggregation-prone proteins that accumulate 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. Article https://doi.org/10.1038/s43587-023-00383-4 during aging (for example IFB-2), maintaining high levels of other proteasome targets may be required for survival of young animals at 20 °C and 25 °C. In contrast, an advantageous environment such as cold temperature could circumvent the requirement for high levels of these regulatory proteins. We speculate that worms inhibit PSME-3 function at 20 °C to reduce its deleterious effects on viability under standard temperature, while sacrificing its capacity to terminate aggregation-prone proteins that will eventually accumulate and contribute  to organismal aging at older ages. Indeed, psme-3 overexpression does not decrease aggregation of polyQ-expanded proteins at standard temperature. In contrast, worms do not inactivate psme-3 overexpression under mild heat stress (25 °C), leading to an increase in trypsin-like activity similar to cold temperature. Although PSME-3 overexpression has detrimental effects for survival of wild-type worms at 25 °C, it prevents aggregation and neurotoxicity of polyQ-expanded proteins at this temperature. These results suggest a trade-off between the effects of PSME-3 in lifespan and protein aggregation when temperature rises above 15 °C, which is neutralized by cold temperature.
In addition to C. elegans, moderate cold temperature (36 °C) also induces PA28γ/PSME3 in human cells. Notably, inhibition of TRPA1 channel blocks the effects of cold temperature on proteasome activity in human cells, suggesting that TRPA1 influences proteostasis across species. Whereas cold-induced activation of TRPA1 is also required for PSME3 function in human cells at low temperature, PSME3 overexpression can circumvent this limitation and upregulate trypsin-like activity, even at normal temperature (37 °C). The reasons underlying this mismatch between C. elegans and human cells are unknown, but could ensue from differences between species. Moreover, the normal temperatures are also very different between C. elegans and humans (20 °C and 37 °C, respectively), and this factor could impinge on PSME3 regulation. Finally, additional regulatory mechanisms could be engaged at the organismal level in C. elegans. Such interorgan processes might be conserved in vertebrates, but they are lost in cultured cells.
Importantly, the cold-induced role of PSME3 to diminish protein aggregation is also conserved in human cells, indicating that cold temperature could be a converging mechanism to prevent distinct human disorders with age. Because PSME3 overexpression is sufficient to increase trypsin-like proteasome activity and prevent diseaserelated aggregation in human cells even at 37 °C, these findings open the possibility that PSME3 can be therapeutically targeted at normal temperatures. However, further studies are required to define activating mechanisms of basal PSME3 levels at normal temperatures and understand its therapeutic potential. Together, our findings demonstrate a beneficial role of cold temperature that crosses evolutionary boundaries to maintain proteostasis.

C. elegans psme-3 overexpression plasmid
To construct the C. elegans psme-3 overexpression plasmid, pPD95.77 from the Fire Lab kit was digested with SphI and XmaI to insert 3.6 kb of the sur-5 promoter. The resultant vector was then digested with KpnI and EcoRI to excise GFP and insert a multi-cloning site containing KpnI, NheI, NotI, XbaI and EcoRI. psme-3 was PCR amplified from cDNA to include 5′ NheI and 3′ NotI restriction sites and then cloned into the vector (primers: 5′-TTGGCTAGCATGGTCAAGAA-GCAAAGTATGCCGG-3′ and 5′-CAAGCGGCCGCTTAATAAAGATG TTCGGTGTTGG-3′).

Lifespan studies
Synchronized larvae by egg laying protocol were raised and fed OP50 E. coli at 20 °C until day 1 of adulthood. Once hermaphrodite worms reached adulthood, they were shifted to a given temperature (that is, 15 °C, 20 °C or 25 °C) on plates with HT115 E. coli carrying empty vector or RNAi constructs. Adult worms (n = 96 per condition) were scored every day or every other day 80 . From the initial population, the worms that were lost or burrowed into the medium and those with 'protruding vulva' or that underwent bagging were censored.

Cold-shock survival assay
Synchronized larvae were raised and fed OP50 E. coli at 20 °C until day 1 of adulthood. Then, worms were transferred to fresh plates and exposed to 4 °C for 12 h. Worms were returned to 20 °C and checked every day for viability. Article https://doi.org/10.1038/s43587-023-00383-4

Motility assays
At day 3 of adulthood, worms were transferred to a drop of M9 buffer. After 30 s of adaptation, we counted the number of body bends for 30 s. A body bend is defined as a change in direction of the bend at the mid-body 42 .

Proteasome activity
Worms were lysed in proteasome activity assay buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 5 mM MgCl 2 , 2 mM ATP and 1 mM dithiothreitol) using a Precellys 24 homogenizer (Bertin technologies). Human cells were collected in proteasome activity assay buffer and lysed by passing 10 times through a 27 G needle attached to a 1 ml syringe. C. elegans and human cell lysates were centrifuged at 10,000 × g for 10 min at 4 °C. For each sample, 25 µg of total protein were transferred to a 96-well microtiter plate (BD Falcon) and incubated with fluorogenic proteasome substrates. To measure trypsin-like proteasome activity, we used Ac-Arg-Leu-Arg-AMC (Enzo, BWL-AW9785-0005). For chymotrypsin-like and caspaselike proteasome activities, we used Z-Gly-Gly-Leu-AMC (Enzo, BML-ZW8505-0005) and Z-Leu-Leu-Glu-AMC (Enzo, BWL-ZW9345-0005), respectively. Fluorescence accumulation upon proteasomal degradation of the fluorogenic substrate (380 nm excitation, 460 nm emission) was measured on a microplate fluorometer (EnSpire, Perkin Elmer) every 5 min for 1 h. Then, the slope of fluorescence accumulation over time was calculated. To average independent replicate experiments and perform statistical analysis, we normalized the slope from the test conditions to the respective control condition of the same replicate experiment.
Blue native gel immunoblotting of 11S/PA28γ complex HEK293 cells were collected in lysis buffer (50 mM Tris-HCl at pH 7.5, 1 mM dithiothreitol, 10% glycerol, protease inhibitor cocktail) and then lysed by passing 10 times through a 27-gauge needle attached to a 1 ml syringe. After centrifugation of the lysates at 16,000 g for 10 min at 4 °C, the supernatant was collected and protein concentration was determined. 50 µg of total protein was run on a 3-13% gel in deep blue cathode buffer (50 mM Tricine, 7.5 mM Imidazole and 0.02% Coomassie G250) at 4 °C for 3 h at 100 V. Then, we exchanged deep blue cathode buffer to slightly blue cathode buffer (50 mM Tricine, 7.5 mM Imidazole, and 0.002% Coomassie G250) and run at 100 V overnight. We transferred proteins to a polyvinylidene difluoride membrane at 400 mV for 3 h by semi-dry blotting. Immunoblotting analysis was performed with anti-PSME3 antibody (Abcam, ab97576 1:1,000).

Filter trap and western blot of aggregation-prone proteins
Adult C. elegans were collected with M9 buffer, and then worm pellets were frozen with liquid N2. Frozen worm pellets were thawed on ice, and extracts were generated by glass-bead disruption on ice in non-denaturing lysis buffer (50 mM Hepes at pH 7.4, 150 mM NaCl, 1 mM EDTA and 1% Triton X-100) supplemented with protease inhibitor cocktail. Worm debris was removed with 8,000 g spin for 5 min at 4 °C. Protein concentrations were measured with BCA protein assay. 100 µg protein extract was supplemented with SDS at a final concentration of 0.5% and loaded onto a cellulose acetate membrane assembled in a slot-blot apparatus (Bio-Rad). Then, the membrane was washed with 0.2% SDS and SDS-resistant protein aggregates were assessed by immunoblotting using antibodies against IFB-2 (Developmental Studies Hybridoma Bank, MH33, 1:1,000), GFP (AMSBIO, TP401, 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000), and TDP43 (Abcam, ab225710, 1:1,000). Extracts were also analyzed by SDS-PAGE/Western blot with anti-GFP, anti-FUS, anti-TDP43, and anti-α-tubulin (Sigma-Aldrich, T6199, 1:5,000) to quantify total levels of the corresponding proteins.
Likewise, HEK293T cells were collected and lysed in non-denaturing lysis buffer supplemented with protease inhibitor cocktail. Cell lysates were homogenized by passing 10 times through a 27-gauge needle. Lysates from HEK293T cells expressing pARIS-mCherry-httQ23-GFP or pARIS-mCherry-httQ100-GFP were centrifuged at 8,000 g for 5 min at 4 °C. Lysates from HEK293T cells expressing FUS-HA-WT or FUS-HA-P525L were centrifuged at 1,000 g for 5 min at 4 °C. Then, we collected the supernatants and measured protein concentrations with BCA protein assay. 100 µg of HEK293T protein extracts were supplemented with SDS at a final concentration of 0.5% and loaded onto a cellulose acetate membrane assembled in a slot-blot equipment. The membrane was washed with 0.2% SDS and aggregates were assessed by immunoblotting with anti-GFP (AMSBIO, TP401, 1:5000) and anti-FUS (Abcam, ab154141, 1:1,000) antibodies. To quantify total levels of proteins, cell extracts were also analyzed by SDS-PAGE/western blot with anti-HTT (Cell Signaling, 5656, clone D7F7, 1:1,000), anti-FUS (Abcam, ab154141, 1:1,000), and anti-β-actin (Abcam, ab8226, 1:5,000). Uncropped images of all western blots are presented in Source Data.

Real-time quantitative PCR
For C. elegans experiments, total RNA was isolated from 200 synchronized day 6 adult worms using RNAbee (Tel-Test Inc., CS501-B). For human cell samples, total RNA was also extracted using RNAbee. Then, we generated cDNA from isolated RNA using a qScript Flex cDNA synthesis kit (Quantabio). SYBR green real-time quantitative PCR (qPCR) assays were performed with a 1:20 dilution of cDNA using a CFC384 Real-Time System (Bio-Rad). Data from C. elegans and human cells were normalized to the geometric mean of cdc-42 and Y45F10D.4 (ref. 87) or β-actin and GADPH 83 as housekeeping genes, respectively. Data was analyzed using the comparative 2ΔΔC t method, which provides relative changes in gene expression to the control condition after correction for housekeeping genes 88 . The 2ΔΔC t method allows to compare relative expression data between different conditions in qPCR assays. Supplementary Table 2 contains details about the primers used for qPCR.

Statistics and reproducibility
For proteasome activities, protein levels and mRNA levels, we presented the data as relative changes to the corresponding control condition. To average independent experiments, we normalized test conditions to the corresponding control sample measured at the same time in each replicate experiment. Accordingly, we performed statistical analysis of changes in proteasome activities, protein levels or mRNA levels by two-tailed Student's t-test for paired samples. Regarding motility data, we used two-tailed Student's t-test for unpaired samples as the thrashing movements of multiple control worms were compared to another condition across different experiments without normalization for each replicate experiment. In graphs containing more than one statistical comparison, we performed correction for multiple testing by controlling the FDR using the two-stage step-up method of Benjamini, Krieger and Yekutieli 89 . The figures present whether the changes are significant according to the P values obtained from Student's t-test, but all the indicated significant changes were also significant after correction for multiple testing by the FDR method (FDR-adjusted P value (q value) < 0.05 was considered significant). Data distribution was assumed to be normal but this was not formally tested. Source Data contains individual data points as well as exact P values and q values for each statistical comparison presented in the figures. All the aforementioned statistical analyses were performed using GraphPad Prism (version 9.4.1).
For lifespan and cold-shock survival data analysis, we used GraphPad Prism (version 6.0) to determine median lifespan and Article https://doi.org/10.1038/s43587-023-00383-4 generate lifespan graphs. OASIS software (version 1) 90 was used to determine mean lifespan. P values were calculated with GraphPad Prism (6.0). The P values refer to experimental and control animals in a single lifespan experiment. Each graph shows a representative experiment. Supplementary Table 3 contains number of total/censored worms and statistical analysis for each replicate lifespan experiment.
No statistical methods were used to predetermine sample size, but our sample sizes are similar to those reported in previous publications using the same procedures 9,26,44,80,82,91,92 . No animals or data points were excluded from the analyses. Worms and cells were distributed to the various groups of all experiments from single pulls. Data collection was not randomized. Data collection and analysis were not performed blind to the conditions of the experiments.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
Article https://doi.org/10.1038/s43587-023-00383-4 Extended Data Fig. 7 | Knockdown or pharmacological inhibition of TRPA1 triggers mutant FUS aggregation at cold temperature. 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.