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

Aging significantly increases the risk of neurodegenerative diseases characterized by protein aggregation. Lowering body temperature has consistently shown to extend lifespan in various organisms, including poikilotherms (*C. elegans*, *Drosophila*, fish) and homeotherms (rodents). In *C. elegans*, shifting from 20°C to 15°C leads to substantial lifespan extension. This longevity effect is not solely attributed to reduced metabolic rate but involves regulated processes. For example, the cold-sensitive TRPA-1 channel plays a role in activating lifespan extension. Cold temperatures also upregulate molecular chaperones, supporting proteostasis. This study hypothesized that a better understanding of cold-induced effects could reveal mechanisms to modify pathological protein aggregation, potentially leading to therapeutic interventions for preventing various age-related diseases. The proteasome, responsible for degrading misfolded proteins, is a key player in maintaining proteostasis and preventing age-related pathologies. The proteasome's core (20S) has three catalytic subunits with different specificities (caspase-like, trypsin-like, chymotrypsin-like). The 20S can be activated by various regulatory particles, including PA28 (11S). PA28γ/PSME3 forms homo-heptameric rings and promotes ubiquitin-independent protein degradation. While less studied than 26S proteasomes, its conserved nature suggests a critical role.

Extensive research demonstrates the correlation between reduced body temperature and increased longevity across diverse species. Studies on *C. elegans*, *Drosophila melanogaster*, and various fish species have shown that lower temperatures extend lifespan. Rodent studies have also corroborated this finding, suggesting a conserved mechanism. The impact of temperature on lifespan is not simply a consequence of slower metabolic rates; instead, it is governed by regulated biological processes. The role of cold-sensitive channels like TRPA-1 and the upregulation of molecular chaperones under cold conditions have been established. Previous research has also highlighted the proteasome's importance in maintaining proteostasis and preventing age-related diseases, with its different activation mechanisms being a focal point of study. However, the specific mechanisms by which cold temperature impacts the proteasome and contributes to longevity remained unclear.

The study employed both *C. elegans* and human cell models. *C. elegans* experiments used wild-type, mutant (*fer-15(b26); fem-1(hc17)* for sterility), and RNAi-treated worms. Lifespan assays were performed by shifting worms to various temperatures (15°C, 20°C, 25°C) after reaching adulthood. Proteasome activity (trypsin-like, caspase-like, chymotrypsin-like) was measured using fluorogenic substrates. Western blotting and qPCR quantified protein and mRNA levels of proteasome subunits, including PSME-3. Filter trap assays assessed protein aggregation. Tissue-specific RNAi was utilized to determine the role of PSME-3 in different tissues (*germline, neurons, intestine, muscle*). Human HEK293 cells were cultured at 37°C and shifted to 36°C for 24 hours to mimic moderate cold conditions. Similar assays were conducted, including proteasome activity measurements, protein and mRNA quantification, and filter trap analysis using constructs expressing disease-related proteins (polyQ-expanded huntingtin, mutant FUS). Stable shRNA lines for PSME3 and TRPA1 were generated for knockdown studies. Motor neurons were differentiated from human iPSCs (both control and ALS-linked FUS mutation) to assess cold temperature's effects on neurodegeneration. Immunocytochemistry quantified apoptosis levels. Subcellular localization of PSME3 was analyzed through immunofluorescence microscopy. Statistical analysis included paired and unpaired Student's t-tests, with FDR correction for multiple comparisons.

In *C. elegans*, cold temperatures (15°C) selectively increased trypsin-like proteasome activity. This increase depended on PSME-3, the worm ortholog of human PA28γ/PSME3. Knockdown of *psme-3* reduced cold-induced longevity and impaired protein degradation. Cold-induced PSME-3 prevented aggregation of disease-related proteins (polyQ peptides in Huntington's disease and ALS models). In human cells, moderate cold temperatures (36°C) also increased trypsin-like proteasome activity via PA28γ/PSME3. This reduction in temperature also decreased the aggregation of disease-related proteins, including huntingtin (Huntington's disease) and FUS (ALS). Knockdown of PSME3 or TRPA1 blocked the cold-induced degradation of these proteins. In ALS-iPSC-derived motor neurons, cold temperature reduced apoptosis, which was also dependent on PSME3. PSME3 is present in both nucleus and cytoplasm in different cell types, suggesting it can act in different cellular compartments. Leptomycin B, blocking nuclear export, showed that PSME3 preferentially promotes cold-induced degradation of FUS in the nucleus.

The study's findings demonstrate a conserved mechanism by which cold temperature enhances proteostasis and longevity. TRPA-1, a cold-sensitive channel, is involved in activating PSME-3 expression and subsequent increase in trypsin-like proteasome activity in both *C. elegans* and human cells. The ability of PA28γ-activated proteasomes to degrade ubiquitin-independent proteins provides a mechanism to counteract the age-related decline in ubiquitination and proteasomal degradation. The contrasting effects of PSME-3 overexpression at different temperatures in *C. elegans* suggest a regulatory mechanism that balances proteasome activities based on environmental conditions. The successful translation of the findings from *C. elegans* to human cells highlights the potential for targeting PSME3 as a therapeutic strategy to combat protein aggregation in age-related diseases.

This study reveals a conserved mechanism where cold temperature, through the TRPA-1-PSME-3 pathway, enhances proteasomal activity and reduces protein aggregation. The implications for therapeutic intervention in age-related diseases are significant, particularly given the potential to target PSME3 at normal temperatures. Future studies could focus on identifying specific activators and inhibitors of PSME-3, understanding the optimal temperature range for its therapeutic effects, and clarifying the precise mechanisms of its action in different cellular compartments.

The study primarily used *in vitro* models and cell lines. Extrapolating the findings directly to the *in vivo* situation in humans requires further research. The specific mechanisms by which TRPA-1 regulates PSME-3 expression and the role of other potential regulatory factors need further investigation. The long-term consequences of PSME-3 modulation on other cellular processes beyond protein aggregation are also not completely addressed.