Context-Dependent Roles of Mitochondrial LonP1 in Orchestrating the Balance Between Airway Progenitor Versus Progeny Cells
Background and Problem
In recent years, there has been significant progress in research on the multifaceted roles of mitochondria within cells. Aside from being the energy source of the cell, mitochondria also play crucial regulatory roles in processes such as cell proliferation, differentiation, and self-renewal, attracting widespread attention in stem cell biology and regenerative medicine. However, how different cell types’ sensitivity to mitochondrial defects manifests in complex tissue environments, and how different cell behaviors are affected by mitochondrial defects, remain unresolved mysteries. This study, led by scientists Le Xu, Chunting Tan, and others, is affiliated with multiple research institutions including the University of California, San Diego, Columbia University, and Boston Children’s Hospital. It was published in October 2024 in the journal “Cell Stem Cell.” The research focuses on the impact of LONP1 (AAA+ Lon protease 1) deactivation-induced mitochondrial dysfunction in the mouse airway and its effects on airway stem cells and differentiated cells in different environments. This discovery not only deepens the understanding of the pathology of respiratory diseases such as COPD (Chronic Obstructive Pulmonary Disease) but also uncovers unique regulatory mechanisms of specific cell types in response to mitochondrial damage.
Research Origin and Methodology
The authors employed various Cre-LoxP systems to knock out LONP1 in different cell types, allowing systematic observation of the composition and behavior of airway epithelial progenitor cells and mature progeny cells. They designed two mutants, shhCre and Sox2CreER, to inhibit LONP1 during developmental stages and in adult mice, respectively. The experiments included several assessments of mitochondrial functions such as levels of ETC (Electron Transport Chain) proteins, ROS (Reactive Oxygen Species) generation, oxygen consumption rate (OCR), and gene transcriptome analysis, aiming to comprehensively explore the effects of LONP1 loss on airway cells. Additionally, through single-cell RNA sequencing (scRNA-seq), researchers systematically revealed transcriptional characteristics and signaling pathway changes in different cell types in LONP1-mutant airways.
Main Research Procedures and Findings
Key Role of LONP1 in Balancing Airway Epithelial Progenitor and Progeny Cells
Analysis shows that LONP1 is widely expressed in all cell types in the lung during both development and adulthood, including epithelial, mesenchymal, endothelial, and immune cells. Researchers found that the loss of LONP1 impedes the proliferation and differentiation of airway progenitor cells, manifested as apoptosis of terminally differentiated ciliated cells during developmental stages, which are replaced by basal and goblet cells under homeostasis. Moreover, airway progenitor cells were unable to migrate into the injured alveoli after influenza virus infection when LONP1 is absent, leading to impaired airway repair. Single-cell transcriptome analysis revealed significant upregulation of the ATF4 and ISR (integrated stress response) pathways, with ISR pathway activation predicted by specific BOK protein expression. The results indicate massive ciliated cell apoptosis and progenitor/progeny cell imbalance in LONP1-deficient airways, resembling the airway phenotype of COPD patients.Mechanism for Maintaining Mitochondrial Protein Homeostasis and Health
Through Western blot analysis of the lung tissue of mutant mice, researchers found a significant reduction of ETC complex proteins in the mutant lung, leading to increased ROS levels. Further analysis showed decreased mitochondrial DNA copy number and reduced OCR in the mutant lung, highlighting LONP1 as essential for maintaining mitochondrial protein homeostasis and function. Additionally, Seahorse mitochondrial stress test results indicated significantly reduced mitochondrial respiration levels in the adult mutant airway, further supporting the link between LONP1 loss and mitochondrial dysfunction.Airway Cell Type Transformation Due to LONP1 Loss
Through immunohistochemistry staining, researchers observed that in the LONP1-deficient developmental airway, markers of normal ciliated cells were almost entirely absent, replaced by a large number of cells exhibiting basal cell characteristics. A similar prevalence of basal-like cells and a significant increase of goblet cells were observed in the adult mutant airway. The ciliated cells appeared particularly vulnerable in the absence of LONP1, with apoptosis closely linked to cytochrome C release from the mitochondrial membrane. Further stepwise analysis using the Cre-LoxP system demonstrated that inactivation of LONP1 in ciliated cells significantly affected cell survival but had a lesser impact on basal and goblet cells.Activation of the ISR Pathway and Its Effect on Airway Phenotype
To understand molecular changes induced by LONP1 deficiency, the authors conducted RNA sequencing and immunohistochemistry analyses, which showed significant upregulation of ISR pathway-related proteins such as ATF4 and DDIT3. This stress response selectively affects ciliated cells and can be partially reversed by the small molecule inhibitor ISRIB. Additionally, gene knockout experiments confirmed the central role of ATF4 and DDIT3 in LONP1-deficiency-induced ciliated cell apoptosis. LONP1 loss not only activates the ISR pathway but also leads to aberrant proliferation of basal-like cells and goblet cells, observed in COPD airways.Key Regulatory Role of BOK in ISR-Induced Apoptosis
To further unravel the molecular basis of ciliated cell-specific apoptosis, researchers found high BOK expression in both normal and LONP1-deficient ciliated cells and demonstrated through gene knockout experiments that BOK is a necessary factor for ISR-induced apoptosis. Moreover, depletion of BOK in airway stem cells inhibited ATF4 upregulation, indicating that BOK acts as a bridge between the ISR pathway and apoptosis.Role of LONP1 in Stem Cell Migration Post-Influenza Infection
In experiments involving influenza virus infection, it was found that airway stem cells lacking LONP1 were unable to migrate to the damaged alveolar regions. Further studies using a mouse infection model showed that ATF4 activation in basal cells of the injury zone inhibited cell migration, and double mutant experiments (knocking out ATF4 and BOK) confirmed the inhibitory role of ISR in the migration process.Potential Role of LONP1 in COPD Pathology
Researchers observed decreased levels of LONP1 protein in airway tissues of COPD patients, particularly in areas of squamous metaplasia, a finding highly consistent with the phenotypes in the mouse model. This suggests that LONP1 may play an important role in the pathological mechanism of COPD.
Conclusion and Significance
This study systematically reveals the key functions of LONP1 in airway progenitor and progeny cells and discovers that its loss induces stress responses in specific airway cell types through the ISR pathway. LONP1 plays an irreplaceable role in controlling the balance of airway cell types and in responding to injury repair, and its loss may lead to COPD-like airway phenotypes, providing new perspectives for further research into COPD pathogenesis. The study also uncovers the critical role of BOK in regulating cellular stress and apoptosis, providing an important foundation for developing treatments targeting LONP1 or BOK. Overall, this study elucidates the multiple roles of the mitochondrial protease LONP1 in maintaining airway health and its significant importance in regulating cell behavior in complex environments.