Loss of Mitochondrial Adenine Nucleotide Translocase (ANT1) Regulates Cellular Senescence and Protects against Chronic Obstructive Pulmonary Disease

Abstract

Chronic obstructive pulmonary disease (COPD) is a destructive lung disease resulting in alveolar tissue loss, airway remodeling and inflammation with cigarette smoke (CS) being a major causative factor. Mitochondrial dysfunction can lead to changes in cell fate implicated in COPD. Cellular senescence, a state of cell cycle arrest, results in abnormal responses in lung epithelial cells. An important protein in mitochondrial metabolism and cell fate is adenine nucleotide translocase (ANT), a mitochondrial ATP transporter. Our studies suggest that levels of ANT are altered in COPD. We hypothesize that loss of ANTs results in increased cellular senescence thus altering COPD pathogenesis and airway dysfunction. Methods Utilizing siRNA knockdown of SLC25A4 (ANT1) and SLC25A5 (ANT2) in human bronchial epithelial cells, Beas-2bs, we assessed cellular senescence (p53, p21, p16) via western blot, qt-PCR and B-galactocidase analysis. Cells were also exposed to CS extract. Wildtype (WT) and ant1-null mice were exposed to CS for 2 and 6mo or air control with lung histology and morphometry analysis. We performed single cell RNA sequencing on lungs from 2mo air and CS-exposed mice. Single-cell library preparation was performed using 10X Genomics 5prime v1 reagents to analyze 5000 cells/mouse (n=3 mice/group). Alignment was performed using Cell Ranger and standard workflow analysis using R package Seurat v3.2. Cell clusters were generated using unsupervised UMAP. Pathway analysis on differential gene expression lists by cell type was performed using Ingenuity Pathway Analysis. Results Knockdown of ANT1 or ANT2 in Beas-2b cells results in an increase in cell senescence proteins p16, p21, p53 and beta-galactosidase staining. Surprisingly, ant1-null mice exposed to 6months of CS exposure are protected from alveolar destruction (fig.1), airway remodeling and chronic inflammation with fewer macrophages. Single cell RNA sequencing of WT lung cells demonstrates that CS (2 mo) causes a significant increase in genes associated with senescence in alveolar type 1 epithelial cells (AT1, p-value 1.0x10-6) with dysregulation of mitochondrial-related gene pathways when compared to air controls: mitochondrial dysfunction, upregulation of Eif2 signaling and NRF2-mediated oxidative stress, and downregulation in sirtuin signaling. Alveolar type 2 cells (AT2) from WT-smoked mice show downregulation of Eif2 signaling, unfolded protein response, NRF2-mediated oxidative stress, and sirtuin signaling. Expression of slc25a4 (ant1) is upregulated in AT2 cells in WT-smoked mice (p-value 1.7x10-17). Mitochondrial and cell fate pathways were significantly altered in AT1 and AT2 cells from WT-smoked mice but not from ant1-null mice, suggesting a key role in lung epithelial responses in COPD. Conclusions ANTs alter cell senescence in alveolar epithelial cells. An In vivo mouse model shows that ant1 is upregulated in AT2 cells after CS and loss of ant1 is protective against CS lung injury. Ant1 regulates epithelial cell fate, mitochondrial function and CS-related cell injury, providing a potential therapeutic avenue for COPD.