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Description
Environmental stress can severely challenge the ability of an organism to survive and reproduce. One such, potentially dangerous, environmental stressor is acute cold, whose cellular impact, in contrast to excessive heat, remains poorly understood. To mitigate stress-induced damage, an affected organism launches diverse cellular responses. Crucially, in order to preserve protein homeostasis (proteostasis), cells must coordinate protein synthesis with protein degradation. How this happens in response to cold in the main focus of this proposal. Many animals respond to cold by entering a state of inactivity and metabolic depression, known as hibernation. One conserved feature of hibernation is a global repression of translation. Using the powerful genetic model of Caenorhabditis elegans, which can display a hibernationlike behavior, our first goal is to determine which pathways repress general translation in the cold and uncover how specific transcripts evade the general block of translation. Compared with protein synthesis, even less is known about protein stability/turnover during hibernation and recovery. Recently, another conserved mechanism has been identified in C. elegans, which extracts unnecessary proteins and organelles from cells in large vesicles called exophers. Using the C. elegans model, we observed the potential roles for both UPS and exopher-based pathways in the adaptation of animals to low temperatures. Thus, our second goal is to dissect the mechanisms underlying hibernation-related remodeling of the C. elegans proteome. To achieve both goals, we will employ a range of approaches, including genomics, proteomics, and imaging. Summing up, we propose a highly complementary research plan that aims to break new grounds in the understanding of cellular adaptation to hypothermia, which has potential biomedical implications.
Summary of project results
The project aimed to address several critical issues related to the cellular adaptation to cold temperatures, focusing on gene expression, the ubiquitin-proteasome system (UPS) and related proteostasis mechanisms in Caenorhabditis elegans. The primary challenges were:
Gene expression: Examining the significance of post-transcriptional regulation of cold-specific gene expression and identifying mechanisms responsible for cold-specific gene activation functioning despite the general repression of protein synthesis.
Protein homeostasis under cold stress: Understanding how cells maintain protein balance during cold conditions, particularly through the UPS and autophagy-lysosome pathways. This included identifying how these systems are regulated and how they contribute to the overall stress resilience and survival of organisms in cold environments.
Cold adaptation mechanisms: Investigating how the UPS and other proteostasis mechanisms, such as exophers (large vesicles expelling unnecessary proteins and organelles), help organisms adapt to cold temperatures.
Pharmacological enhancement of cold tolerance: Exploring how pharmacological agents, like floxuridine (FUdR), can enhance proteasome function and cold tolerance, even when proteasome subunits are compromised.
Inter-tissue dynamics and environmental cues: Elucidating the regulation of extracellular vesicles (exophers) in response to environmental cues, such as temperature changes and pheromone signals, and understanding the complex dynamics between different tissues that govern these processes.
Genetic contributions to cold adaptation: Identifying genetic components, such as proteasome subunits, that play a role in cold adaptation and understanding their regulation and function.
The project undertook several key activities and produced notable outputs, including:
Experimental studies on C. elegans: Utilized C. elegans as a model organism to study the roles of gene regulation, UPS, autophagy-lysosome pathway, and exophers in cold adaptation. This involved genetic screens, genomics, RNA interference (RNAi), various cell biology techniques, and temperature treatments to observe physiological and molecular changes.
Identification of key proteasome subunits and activators: Discovered proteasome subunits and activators linked to heat resistance, survival at low temperatures, and lifespan extension at moderate temperatures. Specifically, highlighted the role of FUdR in enhancing proteasome function under cold stress.
Pharmacological interventions: Demonstrated that FUdR treatment improves UPS activity and cold tolerance, even in the absence of certain proteasome subunits, by activating a distinct proteostasis-modulating pathway and a detoxification pathway.
Regulation of exophers: Established that exopher production in C. elegans is influenced by environmental cues, such as temperature and pheromones. Identified the molecular pathways and neurons involved in this regulation, providing insights into the adaptive response system of extracellular vesicles.
Genetic analysis and human linkages: Through collaboration, linked findings in C. elegans to human health by studying a de novo FEM1C mutation associated with neurodevelopmental disorders. Used genetic and phenotypic analyses to understand the mutation’s impact on protein function and locomotion in worms, providing evidence for its pathogenicity in humans.
Phosphatase role in cold adaptation: Investigated the roles of phosphatases PAA-1 and VHP-1 in cold adaptation, using RNAi screening and phosphoproteomic studies to map their activity at different temperatures and stages of cold stress. This included detailed LC-MS/MS analyses to identify potential substrates and regulatory mechanisms.
The project achieved significant results with broad impacts for multiple beneficiaries:
Enhanced understanding of cold adaptation: Provided deep insights into how gene regulation, UPS, and exophers contribute to cellular adaptation to cold temperatures. This knowledge advances the field of molecular and cellular biology, particularly in understanding stress resilience and proteostasis. We also identified a conserved pathway (involving a ferritin variant) promoting resistance to cold, which opened up the door to studies involving human cells.
Therapeutic potential of FUdR: Identified FUdR as a potential therapeutic agent for conditions characterized by proteasome impairment. Demonstrated its ability to enhance proteasome function and cold tolerance, opening new avenues for medical research and treatment strategies.
Insights into exopher regulation: Clarified how environmental cues regulate exopher production, contributing to the understanding of inter-tissue communication and adaptive responses. This knowledge has implications for research into cellular waste management and disease mechanisms.
Link to human neurodevelopmental disorders: Established a connection between C. elegans findings and human health by linking FEM1C mutations to neurodevelopmental disorders. This research benefits medical genetics by providing a model for studying the functional impacts of specific mutations.
Foundational knowledge for further research: Laid the groundwork for future studies on phosphatases in cold adaptation, with detailed phosphoproteomic maps and identified regulatory pathways. This foundational knowledge is valuable for researchers exploring stress biology and potential therapeutic targets.
Collaborative and cross-disciplinary impact: The project fostered international collaboration, enhancing scientific cooperation and knowledge exchange. Beneficiaries include researchers, medical professionals, and potentially patients with conditions related to proteasome dysfunction and cold adaptation mechanisms.
Overall, the project has significantly advanced the understanding of cellular responses to cold stress, with implications for aging, stress resilience, and potential medical applications. The findings offer a robust platform for future research and therapeutic developments, benefiting both the scientific community and broader society.
Summary of bilateral results
Collaboration between the teams was active: joint research was published with the participation of both teams, joint workshops and symposia were organized and publicity activities were undertaken in relation to their joint research. The two teams have plans for further scientific collaboration with each other, which is expected to focus on translational research. Moreover, as a result of our collaboration with Professor Rafał Ciosk from the University of Oslo (UiO), we have secured funding for a new project under the Preludium Bis grant from the National Science Centre in Poland. The details of the project are as follows:Project Title: Cold Stress Adaptation in C. elegans: The Roles of PAA-1 and VHP-1 Phosphatases.