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. 2024 Jul 23;29(7):264.
doi: 10.31083/j.fbl2907264.

Neuronal CBP-1 is Required for Enhanced Body Muscle Proteostasis in Response to Reduced Translation Downstream of mTOR

Santina Snow  1   2 , Dilawar Ahmad Mir  2 , Zhengxin Ma  2 , Jordan Horrocks  2 , Matthew Cox  2 , Marissa Ruzga  2 , Hussein Sayed  2 , Aric N Rogers  2
Affiliations

Neuronal CBP-1 is Required for Enhanced Body Muscle Proteostasis in Response to Reduced Translation Downstream of mTOR

Santina Snow et al. Front Biosci (Landmark Ed). .

Abstract

Background: The ability to maintain muscle function decreases with age and loss of proteostatic function. Diet, drugs, and genetic interventions that restrict nutrients or nutrient signaling help preserve long-term muscle function and slow age-related decline. Previously, it was shown that attenuating protein synthesis downstream of the mechanistic target of rapamycin (mTOR) gradually increases expression of heat shock response (HSR) genes in a manner that correlates with increased resilience to protein unfolding stress. Here, we investigate the role of specific tissues in mediating the cytoprotective effects of low translation.

Methods: This study uses genetic tools (transgenic Caenorhabditis elegans (C. elegans), RNA interference and gene expression analysis) as well as physiological assays (survival and paralysis assays) in order to better understand how specific tissues contribute to adaptive changes involving cellular cross-talk that enhance proteostasis under low translation conditions.

Results: We use the C. elegans system to show that lowering translation in neurons or the germline increases heat shock gene expression and survival under conditions of heat stress. In addition, we find that low translation in these tissues protects motility in a body muscle-specific model of proteotoxicity that results in paralysis. Low translation in neurons or germline also results in increased expression of certain muscle regulatory and structural genes, reversing reduced expression normally observed with aging in C. elegans. Enhanced resilience to protein unfolding stress requires neuronal expression of cbp-1.

Conclusions: Low translation in either neurons or the germline orchestrate protective adaptation in other tissues, including body muscle.

Keywords: C. elegans; eIF4G; healthspan; ifg-1; lifespan; proteostasis; translation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.. Reduced translation in germline or neurons primes the heat shock response (HSR) and increases thermotolerance.
(A) Time course of heat shock gene expression in N2 animals under ifg-1 RNA interference (RNAi) normalized to control RNAi at each time point shown, beginning at the onset of adulthood. Unpaired t-tests using Welch’s correction were conducted on ΔCts for each gene at each time point (Supplementary Table 3). (B) Heat shock gene expression of tissue-specific strains on ifg-1 RNAi normalized to control RNAi at day 7 of adulthood. From left to right, panels show N2 wild type animals, MAH23 (germline-specific RNAi strain), TU3335 (neuron-specific RNAi strain), WM118 (body muscle-specific RNAi strain), VP303 (intestine-specific RNAi strain), and NR222 (hypodermis-specific RNAi strain). Two-way ANOVAs for each strain were used comparing ΔCts of all genes (Supplementary Table 4). (C) Thermotolerance of tissue-specific RNAi strains after 1 week on RNAi. Unpaired t-tests using Welch’s correction were run at each time point for each strain (Supplementary Table 5). (D) Similar to (B) except that heat shock gene expression was measured 1 hour after exposure to 35 °C for 4 hours. Two-way ANOVAs were conducted for each strain comparing heat shock gene expression (Supplementary Table 4). Error bars represent means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig. 2.
Fig. 2.. Reducing translation in the germline or neurons improves motility and restores youthful transcription of muscle maintenance genes.
(A) Paralysis assay of proteinopathy model expressing alpha-synuclein in body muscle (strain NL5901) on control or ifg-1 RNAi from the onset of adulthood. Kaplan-Meier survival curves were plotted for paralysis assays and compared using the Mantel-Cox log rank test, **** p < 0.0001. Additional replicates show similar results (Supplementary Table 6). (B) Representative microscope images of YFP-tagged alpha-synuclein in NL5901 on control and ifg-1 RNAi for 7 days. Scale bar = 200 μM. Images were collected from at least 9 animals per replicate across 4 replicates. (C) Quantification and comparison of fluorescence in NL5901 animals from (B) using an unpaired t-test with Welch’s Correction (Supplementary Table 7). (D) Western blot probing for alpha-synuclein and beta-tubulin proteins in NL5901 animals comparing conditions from (B) using an unpaired t-test with Welch’s Correction (Supplementary Table 8). (E) Expression of muscle structural and regulatory genes in NL5901 animals on control vs. ifg-1 RNAi at day 7 of adulthood compared using two-way ANOVA (Supplementary Table 9). (F) Aging-related changes in muscle-related gene expression of N2 animals on control RNAi using two-way ANOVA, **** p < 0.0001. (G) Expression of body muscle genes in N2 animals on control or ifg-1 RNAi from day 1 to day 7 of adulthood compared using two-way ANOVA, p < 0.0001. (H) From left to right, panels show body muscle gene expression on control or ifg-1 RNAi for the first 7 days of adulthood in MAH23 (germline-specific RNAi strain), TU3335 (neuron-specific RNAi strain), WM118 (body muscle-specific RNAi strain), VP303 (intestine-specific RNAi strain), and NR222 (hypodermis-specific RNAi strain). Two-way ANOVA was used (Supplementary Table 10). Error bars represent means ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001.
Fig. 3.
Fig. 3.. Lowering translation in the germline or neurons upregulates muscle-related gene expression and improves motility in a model of body muscle proteotoxicity.
(A) Expression of muscle-related genes after 7 days of ifg-1 RNAi in the proteinopathy model strain expressing alpha-synuclein in body muscle crossed with tissue-specific RNAi strains (strains ANR149, ANR168, ANR153). Comparisons between animals treated with control or ifg-1 RNAi were carried out using two-way ANOVAs. (B) Paralysis assays carried out for strains and conditions in (A). Kaplan-Meier survival curves were plotted for paralysis assays and compared using the Mantel-Cox log rank test. Replicates for each strain were conducted with similar results (see Supplementary Table 6). Error bars represent means ± SEM. ** p < 0.01. **** p < 0.0001.
Fig. 4.
Fig. 4.. cbp-1 RNAi in neurons both dampens muscle-related gene expression changes as well as enhanced survival to proteotoxicity under low translation conditions.
(A) Expression of genes enriched in muscle in the neuron-specific RNAi strain TU3335 crossed with NL5901 (ANR168). Day 1 adults were placed on control or ifg-1 RNAi for 2 days before transfer to control or cbp-1 RNAi for 5 days. Two-way ANOVAs were conducted comparing ΔCts of the genes shown (Supplementary Table 11). (B) Western blot of alpha-synuclein (top) and total protein measured from Ultraviolet (UV) shadowing (bottom) for conditions in (A). Quantification and comparison of alpha-synuclein was run using unpaired t-tests with Welch’s Correction (right) (Supplementary Table 12). (C) Paralysis assay of the strain and conditions from (A). Kaplan-Meier survival curves were plotted for the paralysis assay and compared using the Mantel-Cox log rank test. p < 0.0001 (blue asterisk, ctl vs. ifg1:ctl) and p < 0.0001 (yellow asterisk, ctl:cbp-1 vs. ifg-1:cbp-1) (Supplementary Table 13). (D) Thermotolerance assays (5 hours at 37 °C) using RNAi conditions from (A) in the neuron-specific RNAi strain TU3335. Comparisons at different time points were carried out with unpaired t-tests using Welch’s correction compared to control RNAi (Supplementary Tables 14,15). Error bars represent means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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