Autophagy—meaning "self-eating" in Greek—is a fundamental cellular mechanism that preserves cell health by recycling and degrading worn-out or dysfunctional components. Serving as an essential housekeeping process, autophagy also plays a key role in strengthening immunity, mobilizing when cells encounter stressors such as starvation or infection to eliminate bacteria, viruses, and other threats.
But autophagy is not monolithic. Scientists distinguish between "non-selective autophagy," which indiscriminately clears away chunks of cellular material, and "selective autophagy," which carefully targets damaged organelles, misfolded proteins, or invading pathogens for destruction. Diverse physiological and environmental stresses—ranging from nutrient deprivation to chemical toxins and infection—can activate this pathway.
While autophagy is crucial for cellular health, its dysregulation has serious consequences, contributing to diseases including cancer and neurodegenerative disorders.
Now, in a new study published in the Journal of Cell Biology, University of Ottawa Faculty of Medicine researchers unveil an optimized workflow that exposes novel signaling mechanisms regulating autophagy in response to numerous disease-related stress conditions.
By discovering stress-specific autophagy regulators, the team has laid the groundwork for new strategies to decipher autophagy's molecular regulation—potentially accelerating the translation of research from bench to bedside.
Additionally, the researchers have assembled a rich dataset designed to empower multidisciplinary investigations exploring a broad spectrum of biomedical questions.
Exploring a cellular mystery
Recent studies have revealed that defects in autophagy relevant to disease often stem from improper management of specific cellular cargo. For instance, protein aggregates accumulate in neurodegenerative disorders, while damaged mitochondria persist in cancer. These examples illustrate how the precise regulation of autophagy and its ability to target specific types of cellular cargo is essential for preventing disease and maintaining cellular function. Understanding these mechanisms is crucial for developing future therapeutic strategies aimed at correcting autophagy defects.
Recognizing this, Dr. Maxime Rousseaux , Assistant Professor in the Department of Cellular and Molecular Medicine (CMM ), and Dr. Ryan Russell , Associate Professor in the same department and the study's senior author, questioned whether modern gene-editing technologies could support the first comparative analysis of the pathways directing the fate of these distinct cargos.
Dr. Rousseaux, who holds the Canada Research Chair in Personalized Genomics of Neurodegeneration , co-authored this study. His expertise in genetic screening was instrumental in adapting the screening methodology for this research. His interest in the subject is deeply connected to his work on neurodegenerative diseases, with a particular focus on the autophagy defects characteristic of these conditions.
Together, their objective was to establish a comprehensive framework and resource to advance fundamental scientific understanding and open new avenues for therapeutic interventions.
Ambitious use of cutting-edge technology
To address this challenge, the team conducted kinome-wide CRISPR screens to pinpoint distinct signaling pathways that govern various forms of autophagy. A kinome-wide CRISPR screen employs pooled screening technology to systematically investigate the function of protein kinases—a key family of proteins that regulate numerous cellular processes.
Dr. Russell notes that kinases are particularly attractive for drug development due to their high level of "druggability," making them frequent targets for therapeutic interventions.
Truc Losier, a joint PhD student in the Rousseaux and Russell labs, developed a new workflow tailored to enable the comparative pathway mapping envisioned by the researchers. While traditional CRISPR screening can survey the whole genome and uncover novel genes and pathways, employing multiple parallel screens streamlines the process of validating targets.
According to Dr. Russell, the team's novel methodology incorporated four or five genetic screens simultaneously.
By integrating results from these multiple datasets, the researchers were able to perform direct, organelle-specific comparisons. This approach—meticulously implemented by Losier—involved generating large pools of genetically altered cells and subjecting them to brief, intense stress conditions lasting only about three to six hours, as opposed to the conventional seven-day protocol, capturing the early phases of autophagy.
Dr. Russell describes the scale and precision of Losier's work as exceptionally ambitious, with "a huge breadth of analysis in a very acute window." The study revealed a scope of unique regulatory mechanisms not previously observed in autophagy research.
Beyond advancing understanding of autophagy itself, the customized screening strategy developed by the team holds promise for investigators across disciplines who rely on integrated datasets and rapid response experiments. Dr. Russell emphasizes the broader impact of this innovative approach for researchers exploring complex cellular processes, underscoring his lab's commitment to uncovering the dynamics of autophagy regulation in healthy and pathological tissues.
The GEM core – rising to the research challenge
The team's hard-won screening methodology was accomplished through an innovative core at the Faculty of Medicine: The Genome editing and molecular biology (GEM) Facility . This uOttawa core facility provides access to cutting-edge genetic editing techniques and cDNA cloning.
"This was the first screen that came out of this core facility. We have a new genome editing facility where we're pushing the boundaries of what can be done technically," says Dr. Russell, who is co-director of the GEM core alongside Dr. Rousseaux.
Armed with new knowledge from this paper, a next step for the uOttawa team is to explore whether they can use the kinases and signaling pathways they discovered to perhaps regulate innate immunity.
"We'd like to see if we can pharmacologically regulate pathogen infection using some of those kinases," Dr. Russell says.
