Periodic Reporting for period 1 - UCUPA (Unraveling the Molecular Changes that Drive the Repression of the Unfolded Protein Response with Ageing)
Okres sprawozdawczy: 2020-09-01 do 2022-08-31
Ageing is the greatest risk factor for a wide range of diseases, such as dementia, COVID-19, and cardiovascular diseases. According to several governmental reports, it is expected that the European population with more than 65 years old will significantly increase over the next decades. Therefore, it is imperative that we increase our understanding of how humans and animals age, so we can generate novel therapeutic approaches to increase human healthspan and to prevent age-related disorders. The main question that we addressed in this project was to understand why animals lose their ability to sense and respond to stress when they age. With ageing, protein quality control pathways become less efficient, leading to the accumulation of misfolded proteins and toxic aggregates. A conserved hub in the proteostasis network is the UPRER (Unfolded Protein Response of the Endoplasmic Reticulum). Previously, it was identified that the UPRER pathway becomes repressed with ageing in animals, but the molecular mechanism of why its activity decreases with ageing remains elusive. This proposal tries to address this question by (i) Monitoring the activity of the different steps of the IRE-1/XBP-1 axis with ageing, and (ii) Identifying interventions that sustain UPRER activation in older animals and test their impact on lifespan.
Our new results show that UPRER activation is lost during the first day of adulthood as C. elegans enters the reproductive period. This was confirmed by measuring hsp-4::GFP levels and the levels of hsp-4 and xbp-1s mRNA at Day 1 and Day 2 of adulthood in animals exposed to tunicamycin (N-glycosilation inhibitor). Because previous data from our group showed that aged worms constitutively expressing a spliced version of xbp-1 are also capable of inducing hsp-4p::gfp we investigated the activity of other components of the pathway upstream of the transcriptional regulation of target genes by XBP-1. We observed that IRE-1 endoribonuclease activities (RIDD and xbp-1 mRNA splicing) are downregulated during the first day of adulthood as animals enter the reproductive period. To understand why IRE-1 activity was decreased in these animals, we measured its mRNA levels by qRT-PCR, and generated by CRISPR-Cas9, a strain expressing IRE-1 tagged with 3xFLAG. We found no decrease in IRE-1 mRNA or protein levels between day 1 and day 2 of adulthood that would explain the loss of IRE-1 activity between these ages. We also didn’t find differences in the RTCB-1 mRNA levels, enzyme responsible for ligating the xbp-1 mRNA exons during its splicing. We also had as main goal identifying interventions that prevented the age-related UPR decline, and potentially increase lifespan. We tested the mutant glp-1(e2141), because previous data from the literature show that it can prevent the decline of other stress response pathways, such as the Heat Shock Response. Surprisingly, glp-1 mutation did not prevent the decline in the UPR. This argues for the idea that alterations at the chromatin level are not the main responsible for the alterations observed in the UPR. We also tried if the pharmacological activation of IRE-1 by IXA4 could prevent the decline of the pathway; however, it was not successful. As IXA4 was initially identified based on assays using mammalian Ire1, which is significantly different from C. elegans IRE-1, it is possible that the drug is not activating IRE-1 as effectively as mammalian Ire1. However, we were able to show that the activation of the UPR by some odorant molecules, secreted by pathogenic bacteria, such as 1-undecene was capable extending C. elegans lifespan through an xbp-1 dependent manner. We are now investigating the mechanism by which these odorant molecules can modulate the UPR of these animals to promote longevity.
Our findings highlight how UPR decline differs from other cellular stress responses, and uncouples the regulation of somatic maintenance pathways from reproductive status, with implications for evolutionary theories of aging that rely on germline/soma tradeoff. Overall, this work expands our understanding of how the UPR is regulated in aging and pinpoints IRE-1's decreased endoribonuclease activity as a major contributing element to age-related proteostasis loss. These findings are important for our understanding of the nature of proteostasis failure in aging, and may have future implications for the development of drugs to treat age-associated diseases. We are confident that these results will be of interest to researchers in the field, and we hope that you find our contribution similarly intriguing.