Context-dependent roles of mitochondrial LONP1 in orchestrating the balance between airway progenitor versus progeny cells

Abstract

While all eukaryotic cells are dependent on mitochondria for function, in a complex tissue, which cell type and which cell behavior are more sensitive to mitochondrial deficiency remain unpredictable. Here, we show that in the mouse airway, compromising mitochondrial function by inactivating mitochondrial protease gene Lonp1 led to reduced progenitor proliferation and differentiation during development, apoptosis of terminally differentiated ciliated cells and their replacement by basal progenitors and goblet cells during homeostasis, and failed airway progenitor migration into damaged alveoli following influenza infection. ATF4 and the integrated stress response (ISR) pathway are elevated and responsible for the airway phenotypes. Such context-dependent sensitivities are predicted by the selective expression of Bok, which is required for ISR activation. Reduced LONP1 expression is found in chronic obstructive pulmonary disease (COPD) airways with squamous metaplasia. These findings illustrate a cellular energy landscape whereby compromised mitochondrial function could favor the emergence of pathological cell types.

Keywords: ATF4; BOK; COPD; airway homeostasis; differentiated progeny cells; influenza infection; integrated stress response; lung epithelial cells; mitochondria; progenitor basal cells.

Figure 1.. Lonp1 is required for airway epithelial cell differentiation and homeostasis

(A-B) Western blot and quantification of ETC components in E13.5 lungs. n=3 for each. (C) Quantification of mitochondrial DNA copy number in E13.5 lungs. n=10 for each. (D-G) Immunostaining and quantification of club, ciliated and basal cells (quantification of luminal KRT5) in E18.5 trachea. n=3 for each. Scale bars, 50 μm. (H) Experimental scheme for adult inactivation of Lonp1. (I-P) Immunostaining and quantification of club, ciliated, basal (quantification of luminal KRT5) and goblet cells at indicated days after the last dose of tamoxifen. Boxed regions are magnified on the right. n=3 for each. Scale bars in (I), 50 μm for trachea, 200 μm for lung and 20 μm for magnified panels on the right. Scale bars in (L), 30 μm for trachea and 500 μm for lung. Scale bars in (M) and (O), 50 μm. Student’s t test was used for (B, C, E, G, J, K, N and P). See also Figure S1.

Figure 2.. Lonp1 is required cell-autonomously for ciliated cell survival

(A) Experimental scheme for analyzing apoptosis in Sox2creER;Lonp1. (B-C) Immunostaining and quantification of cleaved Caspase3 for apoptotic cells (denoted by arrowheads) at D14 post gene inactivation with quantification at multiple time points in (C). Regions marked by yellow asterisks were magnified in the upper inserts. Scale bars, 50 μm. (D-E) Immunostaining and quantification of cleaved Caspase3 with ciliated, club and basal cells at D14 post gene inactivation. n=3 for each. Scale bars, 30 μm. (F) Experimental scheme for inactivating Lonp1 in ciliated cells. (G-H) Immunostaining and quantification of cleaved Caspase3 in ciliated cells at multiple time points. n=3 for each. Scale bars, 30 μm. (I) Experimental scheme for continuous tamoxifen administration in Foxj1creER;Lonp1. (J-L) Immunostaining and quantification of club and ciliated cells at denoted time points. n=3 for each. Scale bars, 30 μm. (M-O) Immunostaining and quantification of basal cells at D60 on tamoxifen. Boxed regions are magnified in (N). Asterisks denote non-specific staining underneath the airway. n=3 for each. Scale bars, 25 μm for trachea, 1 mm for lung and 50 μm for magnified images in (N). Student’s t test was used for (C, H, K, L and O). One-way ANOVA test was used for (E). See also Figure S2.

Figure 3.. Transcriptomic analysis reveals the activation of ISR in airway epithelium of Lonp1 mutants

(A) Volcano plot showing differentially expressed genes from bulk RNA-seq analysis. ISR pathway genes were highlighted. (B) Heatmap showing differential expression of cell stress- and cell cycle-related genes. (C) Top biological processes from GO analysis of differentially expressed genes. (D) RT-qPCR quantification of stress-related genes at E13.5. n=4 for each. (E-F) ATF4 immunostaining and quantification with epithelium outlined by E-Cadherin at E13.5. n=4 for each. Scale bars, 50 μm. (G) Integrated UMAP of control and Sox2creER;Lonp1 adult lungs with individual conditions shown on the right. Feature plot of lineage reporter tdTomato shown on the lower right. (H) UMAP with annotations of different cell types. (I) Dot plot showing expression levels of identified marker genes. (J) Ratio of control versus mutant cell numbers in each cell type. (K) Heatmap showing z-scores of differentially enriched pathways. (L) ISR pathway enrichment score projected onto UMAP. Red dashed lines outline mutant-specific clusters. (M) Feature plots of core ISR genes. (N) Violin plots showing enrichment scores for branches of the ER stress pathway. (O) Experimental scheme for analyzing ISR activation in Sox2creER;Lonp1. (P-Q) Immunostaining and quantification of ATF4 with FOXJ1 at denoted time points. Arrowheads denote ATF4+ ciliated cells. Scale bars, 50 μm. (R) Quantifications of ATF4 co-staining with ciliated, club and basal cell markers at D10 post gene inactivation. n=3 for each. Student’s t test was used for (D, F and Q). Wilcox test was used for (N). One-way ANOVA test was used for (R). See also Figure S3 and Table S2.

Figure 4.. Lonp1 maintains airway homeostasis through repressing ISR

(A, I) Experimental schemes for analyzing airway phenotypes in Sox2creER;Lonp1;Atf4 (A) and Sox2creER;Lonp1;Ddit3 (I). (B-P) Immunostaining and quantification of proteins as labeled. Quantification of luminal KRT5+TRP63− cells were shown in (H) and (P). Arrowheads denote apoptotic ciliated cells. n=3 for each. Scale bars, 30 μm. One-way ANOVA test was used for (C, E, F, H, K, M, N and P). See also Figure S4.

Figure 5.. Bok expression in ciliated cells is required for ISR activation and apoptosis in Lonp1 mutant

(A) Density plot showing Bok expression in the integrated UMAP. (B) Violin plot showing Bok expression across cell types. (C) Experimental scheme for analyzing Bok expression in Foxj1creER;tdTomato. (D-E) RNAscope staining and quantification of Bok RNA with lineage reporter tdTomato. Arrowheads denote Bok-expressing ciliated cells. n=3. Scale bars, 20 μm. (F) Experimental scheme for analyzing airway phenotypes in Sox2creER;Lonp1;Bok. (G-O) Immunostaining and quantification of proteins as labeled. Arrowheads denote co-staining. n=3 for each. Scale bars, 50 μm. One-way ANOVA test was used for (H, J, K, M and O). See also Figure S5.

Figure 6.. Lonp1 promotes influenza infection-induced pathologic basal progenitor cell migration through repressing ISR

(A) Experimental scheme for influenza infection. (B) H&E analysis (left) and lineage tracing of SOX2+ airway progenitor cells (right) in virus injured lungs. Boxed regions are magnified on the right to show that, compared to control, KRT5+ basal cells are stalled in the mutant airway. Scale bars, 1 mm for whole lung H&E and immunostaining. 50 μm for magnified images. AW: airway. (C) Quantification of KRT5+ basal cells in injured airways. n>=5 for each. (D) Quantification of damaged areas covered by KRT5+ basal cells. n>=5 for each. (E-I) Immunostaining and quantification of proteins as labeled. n=3 for each. Scale bars, 20 μm. AW: airway. (J) Experimental scheme for influenza infection. (K-M) Immunostaining of KRT5 and PDPN in virus injured lungs. Boxed regions are magnified in (L). Percentage of damaged areas covered by KRT5+ basal cells are quantified in (M). n=4 for each. Scale bars, 1 mm in (K) and 100 μm in (L). Student’s t test was used for (C, D, F, I and M). See also Figure S6.

Figure 7.

Bok is required for ISR activation and stalling of viral infection-induced airway basal cells in Lonp1 mutants (A) Experimental scheme for scRNA-seq analysis of virus injured lungs. (B) Integrated UMAP of cells from PBS control, D11 and D20 post viral infection. Clusters of ectopic basal cells were magnified on the left. (C) Feature plots showing marker genes in proliferative (denoted by red arrowheads) and migratory (denoted by blue arrowheads) infection-induced basal cells. (D) Volcano plot showing differentially expressed genes between proliferative and migratory ectopic basal cells. Arrowhead denotes Bok. (E) Feature plot of Bok showing its preferential expression in proliferative ectopic basal cells (denoted by arrowhead). (F-G) RNAscope staining and quantification of Bok expression at D14 post viral infection. Arrowheads denote Bok-expressing basal cells. n=3 for each. Scale bars, 50 μm. (H) Experimental scheme for influenza infection. (I-K) Immunostaining and quantification of KRT5 at D14 post viral infection. Boxed regions are magnified in (J). n=4 for each. Scale bars, 1 mm in (I) and 100 μm in (J). (L-M) Immunostaining of LONP1 protein (L) and KRT5+ basal cells (M) in the adjacent sections of human control and COPD airways. Dashed line denotes low-grade to high-grade transition of squamous epithelium. Scale bars, 30 μm. (N) Quantification of LONP1 protein levels in control and COPD airways. n=3 for each. (O) Cellular energy landscape of the airway epithelium. Student’s t test was used for (G and K). One-way ANOVA test was used for (N). See also Figure S7 and Table S1.