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, 125 (2), 521-38

PINK1 Deficiency Impairs Mitochondrial Homeostasis and Promotes Lung Fibrosis

PINK1 Deficiency Impairs Mitochondrial Homeostasis and Promotes Lung Fibrosis

Marta Bueno et al. J Clin Invest.

Abstract

Although aging is a known risk factor for idiopathic pulmonary fibrosis (IPF), the pathogenic mechanisms that underlie the effects of advancing age remain largely unexplained. Some age-related neurodegenerative diseases have an etiology that is related to mitochondrial dysfunction. Here, we found that alveolar type II cells (AECIIs) in the lungs of IPF patients exhibit marked accumulation of dysmorphic and dysfunctional mitochondria. These mitochondrial abnormalities in AECIIs of IPF lungs were associated with upregulation of ER stress markers and were recapitulated in normal mice with advancing age in response to stimulation of ER stress. We found that impaired mitochondria in IPF and aging lungs were associated with low expression of PTEN-induced putative kinase 1 (PINK1). Knockdown of PINK1 expression in lung epithelial cells resulted in mitochondria depolarization and expression of profibrotic factors. Moreover, young PINK1-deficient mice developed similarly dysmorphic, dysfunctional mitochondria in the AECIIs and were vulnerable to apoptosis and development of lung fibrosis. Our data indicate that PINK1 deficiency results in swollen, dysfunctional mitochondria and defective mitophagy, and promotes fibrosis in the aging lung.

Figures

Figure 11
Figure 11. PINK1 deficiency increases susceptibility to lung fibrosis.
(A) Representative Masson trichrome staining in Pink1+/+, Pink1+/–, and Pink1–/– lung sections showed increased collagen deposition (blue) at day 15 after MHV68 infection. Scale bars: 10 μm (40×); 50 μm (10×). (B) Higher Col1a1 transcript levels in lungs of PINK1-deficient mice infected with MHV68 compared with control littermates. (C) Increased collagen deposition (assessed by hydroxyproline level) in lungs of PINK1-deficient mice after infection. (D) Weight loss after MHV68 infection was more severe in Pink1–/– mice. (E) Viral load (assessed by qPCR) of individual mouse lungs 15 days after MHV68 infection. Bars represent geometric mean. (F and G) Relative change in lung lysate Tgfb (F) and Fgf2 (G) transcript levels after infection. (H) Relative change in Tnfa, Il6, and Il10 mRNA levels after infection. Data represent mean ± SEM (BD and FH). *P < 0.05, **P < 0.01, 1-way ANOVA (B, C, and EH) or 1-way repeated-measures ANOVA (D) with post-hoc Bonferroni.
Figure 10
Figure 10. Mitochondrial dysfunction and increased cell apoptosis in PINK1-deficient mice.
(A) Complex I and complex IV activity, both basal and after MHV68 infection, was reduced in Pink1–/– versus Pink1+/+ lung mitochondria. CS, citrate synthase. (B) Mitochondrial mass (assessed by mtDNA/gDNA ratio) in lungs of infected Pink1+/+, Pink1+/–, and Pink1–/– mice. (C) Representative in situ TUNEL assay in lung sections at day 15 after MHV68 infection. Note the increase in positive signal (brown) in PINK1-deficient lungs. Scale bars: 50 μm. (D) Semiquantitative analyses showed significantly higher TUNEL-positive signal in PINK1-deficient versus control mice. (E and F) Immunoblot analyses in whole lung lysates from naive (E) and MHV68-infected (F) Pink1+/+, Pink1+/–, and Pink1–/– mice for BAX, OPA1, and the autophagic markers LC3I/LC3II and p62. Blots were stripped and reblotted with β-actin for loading normalization. Each lane represents an individual mouse. (G) Density analyses of LC3 and p62. Data represent mean ± SEM (A, B, D, and G). #P < 0.05 vs. Pink1+/+; *P < 0.05; 1- (B and D) or 2-way (A and G) ANOVA with post-hoc Bonferroni.
Figure 9
Figure 9. Altered mitochondrial quality control in AECIIs from PINK1-deficient mice.
(A) Representative TEM (n = 3 per group) of AECIIs from Pink1+/+, Pink1+/–, and Pink1–/– mice. Mitochondrial profiles showed enlarged swollen mitochondria in Pink1–/– AECIIs. Boxed regions are shown enlarged at right. Scale bars: 500 nm. (B) Frequency of mitochondria sizes, mitochondrial area, and percentage of abnormal mitochondria (swollen with evidence of severely disrupted cristae over all mitochondria) in Pink1+/+, Pink1+/–, and Pink1–/– AECIIs (from TEM images). (C) Mitochondrial mass (number of mitochondria per cell and percentage of cytoplasm area occupied by mitochondria; assessed by quantitative morphometry of TEM images) and relative mtDNA/gDNA ratio. (D) Representative images of Masson trichrome staining in lung slides showing increased collagen deposition (blue) around airways in Pink1+/– and Pink1–/– mice. Scale bars: 50 μm. (E) Significant increase in collagen deposition (assessed by hydroxyproline level) and Col1a1 expression in PINK1-deficient versus control mice. (F) Representative TEM of Pink1+/+ and Pink1–/– mice. Collagen fibers in alveolar septa (arrows) surrounded AECIIs in Pink1–/– mice. Scale bars: 500 nm. Data represent mean ± SEM (C and E). *P < 0.05, **P < 0.01, 1-way ANOVA with post-hoc Bonferroni (B, C, and E).
Figure 8
Figure 8. PINK1 modulates mitochondrial homeostasis.
(A) Mitochondrial mass and depolarization in A549 cells transfected with scramble control (Mock) or PINK1 shRNA (shPINK1), assessed by MitoTracker Green FM and JC-1 staining, respectively. Downmodulation of PINK1 was detrimental upon TM treatment 48 hours after transfection, inducing a higher accumulation of depolarized mitochondria. Bafilomycin A1 (10 nM) further increased mitochondrial numbers and depolarization. Data represent mean ± SEM of 16 replicates per condition. (B) Mitochondrial mass and depolarization induced by TM and/or bafilomycin A1 treatment was improved by PINK1 overexpression. Data represent mean ± SEM of 16 replicates per condition. (C and D) Increased levels of TGFB (C) and FGF2 (D) mRNA in A549 cells after PINK1 downregulation by shRNA or after treatment with mtDNA. (AD) Data represent mean ± SEM. §P < 0.05 vs. respective no-TM control, #P < 0.05 vs. no-TM Mock; *P < 0.05 as indicated, 1-way ANOVA with post-hoc Bonferroni.
Figure 7
Figure 7. Downregulation of PINK1 expression in AECIIs and lungs from aging and TM-treated mice.
(A) Microarray analyses of the LGRC cohort showed significantly decreased PINK1 expression in IPF patients (#P < 0.0001 vs. control). Data are presented as box-and-whisker plots, with horizontal bars representing medians, top whisker representing maximal expression, and bottom whisker representing the 5th percentile. (B) Significant reduction of PINK1 transcripts, assessed by quantitative RT-PCR, in whole IPF lungs. (C) Significant reduction of PINK1 transcripts in isolated IPF AECIIs. (D) Immunoblot of lysates of isolated AECIIs from donor control and IPF lungs showing lower levels of full-length (FL) PINK1 in IPF lungs. (E) PINK1 transcript levels, assessed by quantitative RT-PCR, in isolated lung fibroblasts from donor control and IPF patients. (F) Representative immunoblot of isolated lung fibroblasts from young (<50 years) and old (>50 years) donor controls and IPF patients, showing similar protein levels of full-length PINK1 and isoforms ΔN1 and ΔN2. (G) Quantitative RT-PCR showed diminished Pink1 expression in murine lungs with age and after TM treatment. (H) In vitro TM treatment diminished PINK1 expression in A549 cells. Data represent mean ± SEM (B, C, E, G, and H). *P < 0.05, **P < 0.01, unpaired 2-tailed Student’s t test (B, C, and E) or 1- (H) or 2-way (G) ANOVA with post-hoc Bonferroni.
Figure 6
Figure 6. ER stress stimulation recapitulates aging-associated susceptibility to lung fibrosis.
(A) Representative TEM (n = 4) of lungs from young and old mice at 0 (naive) and 15 days after MHV68 infection. Scale bars: 500 nm. (B) Representative TEM (n = 4) of AECIIs from young mice treated with TM only (2 μg/mouse) or TM (2 μg/mouse) followed by MHV68 infection 48 hours later. Scale bars: 500 nm. (C) Quantitative morphometry showed increased frequency of large mitochondria in young infected AECIIs when pretreated with TM. (D) Number of mitochondria per AECII (from TEM images) of MHV68-infected mice. Old mice and young TM-pretreated mice showed high numbers of mitochondria. (E) mtDNA/gDNA in lung samples showed increased mitochondrial mass after MHV68 infection in old mice and with TM treatment in young mice. (F) Representative Masson trichrome staining from lungs at day 15 after MHV68 infection, showing increased pneumonitis and collagen deposition (blue) in old mice and young TM-pretreated mice. Scale bars: 50 μm. (G) Relative change in collagen deposition (assessed by hydroxyproline level) upon MHV68 infection. (H) Relative change in Tgfb transcription upon MHV68 infection. Data represent mean ± SEM (D, E, G, and H). *P < 0.05, **P < 0.01, 2-tailed Student’s t test (C) or 1- (D, E, right, G, and H) or 2-way (E, left) ANOVA with post-hoc Bonferroni.
Figure 5
Figure 5. Stimulation of ER stress deteriorates mitochondrial function and impairs mitophagy in lung epithelial cells.
(A) A549 cells were treated with or without TM (1 μg/ml for 24 hours), and mitochondrial mass was determined by MitoTracker Green. Induction of autophagy by serum starvation reduced mitochondrial mass in TM-treated cells. The autophagy inhibitor bafilomycin A1 increased mitochondrial mass in untreated and TM-treated cells. (B) TM induced dose-dependent depolarization of mitochondria in A549 cells (assessed by JC-1 dye staining). Depolarization was increased in the presence of bafilomycin A1, but was not affected by starvation conditions. (C) Increased doses of TM induce apoptosis of A549 cells (assessed by annexin V staining). (D) Representative Western blot analyses showing increased levels of the mitochondrial marker TOM20 and autophagy markers p62 and LC3I/LC3II in lung lysates from aging and young mice after vehicle and TM treatment (2 μg/mouse). The β-actin blot was obtained from parallel samples run on a separate gel from the TOM20 and p62 blots. (E) Density analyses of Western blots in D. Data represent mean ± SEM (AC and E). *P < 0.05, **P < 0.01, 1- (AC) or 2-way (E) ANOVA with post-hoc Bonferroni.
Figure 4
Figure 4. Impaired mitochondrial function and fission/fusion dynamics in AECIIs with age.
(A) Mitochondrial respiration parameters in isolated primary AECIIs from young and old C57BL/6 mice with vehicle control or TM treatment (1 μg/ml for 1 hour). (B) Representative TEM (n = 3 per group) showing enlarged mitochondria but preserved structure in AECIIs from naive old mice. Boxed regions are shown enlarged at right. Scale bars: 500 nm. (C) Quantitative morphometry showed significantly increased frequency of large mitochondria in AECIIs from naive old mice (n ≥ 100 per condition). (D) Representative TEM (n = 3 per group) showing enlarged mitochondria in AECIIs from TM-treated young and old mice. Boxed regions are shown enlarged at right. Scale bars: 500 nm. (E) Morphometry showed increased frequency of large mitochondria in young and old AECIIs after TM treatment (n ≥ 100 per condition). (F) Area of AECII mitochondria and percentage of abnormal mitochondria (swollen with evidence of severely disrupted cristae over all mitochondria) from TEM images. (G) Representative Western blot membranes showing higher expression levels of mitochondrial fusion modulators (p-DRP1, MTF2, OPA1, and MTF1) in lungs from young and old mice treated with TM. (H) Density analyses of Western blots for fusion and fission mitochondrial modulators. Data represent mean ± SEM (A, F, and H). *P < 0.05, **P < 0.01 vs. young; #P < 0.05, ##P < 0.01 as indicated, 2-tailed Student’s t test (A, C, and E) or 1-way ANOVA with post-hoc Bonferroni (F and H).
Figure 3
Figure 3. Defective autophagy in AECIIs from IPF lungs.
(A) Representative immunostaining of lung sections from donor and IPF patients using anti-LC3 (red; autophagosomal marker) and anti–ATP synthase (green; mitochondrial marker) antibodies. Yellow puncta denote colocalization. Scale bars: 10 μm. (B) x-z coordinate image of z stack of merged LC3 and ATP synthase image of the IPF lung section in A. Partial colocalization was seen for the mitochondrial and autophagosomal markers (arrow). (C) Western blot analyses of p62 and LC3I/LC3II in isolated AECIIs from donor age-matched control and IPF lungs. Each lane represents an individual AECII preparation. Blots were stripped and reblotted using an anti–β-actin antibody as loading control. Results are also quantified below. Data represent mean ± SEM. *P < 0.05, unpaired, 2-tailed Student’s t test. (D) Representative immunostaining of donor and IPF patient lung sections using anti–SP-C (green) and anti-p62 (red). Yellow indicates colocalization of the markers. Scale bars: 10 μm.
Figure 2
Figure 2. Accumulation of dysmorphic and dysfunctional mitochondria in AECIIs from IPF lungs.
(A) Representative TEM (n = 4 per group) in donor and IPF AECIIs (identified by the presence of lamellar bodies). Boxed regions are shown enlarged at right. Scale bars: 500 nm. (B) Quantitative analyses of morphometric data from TEM images. (C) Frequency of mitochondria sizes in donor control and IPF lungs obtained from TEM images. (D) Isolated lung mitochondria showed reduced mitochondrial complex I and complex IV activity in IPF patients relative to age-matched donor controls. (E) Representative TEM (n = 3 per group) in AECIIs from young (<50 years) and older (>50 years) donor control lungs. Boxed regions are shown enlarged at right. Scale bars: 500 nm. (F) Frequency of mitochondria sizes and mitochondrial area from AECIIs in young and old donor controls (obtained from TEM images). (G) No significant difference in number of mitochondria per cell (obtained from TEM images) between young and old donor control AECIIs. (H) Percentage of abnormal mitochondria (swollen with evidence of severely disrupted cristae over all mitochondria) (obtained from TEM images). (I) Increased mitochondrial mass in IPF lungs, assessed by mtDNA/gDNA ratio. (J) Mitochondrial mass, assessed by mtDNA/gDNA ratio, in isolated AECIIs and lung fibroblasts from donor and IPF lungs. Data represent mean ± SEM (B, D, and FJ). *P < 0.05, **P < 0.01, unpaired, 2-tailed Student’s t test (BD, F, G,and J) or 1-way ANOVA with post-hoc Bonferroni (H and I).
Figure 1
Figure 1. Mitochondrial accumulation in AECIIs from dense fibrotic areas in IPF lungs.
(A) Representative immunofluorescence using anti–SP-C (AECII marker; green) and anti–ATP synthase (mitochondrial marker; red) antibodies, showing mitochondrial accumulation in hyperplasic AECIIs from honeycombs in IPF lung (n = 7 per group). Representative cells (asterisks) are shown in detail in the insets. Scale bars: 10 μm. (BE) Representative immunohistochemistry images (n = 6) in consecutive sections from IPF (BD) and donor control (E) lungs using anti–SP-C and anti-TOM20 (mitochondrial marker) antibodies. Epithelial cells from honeycomb areas (B and C) showed positive staining for both markers. Epithelial cells from areas with mild fibrosis (D) and donor control lung (E) showed less positive signal for the mitochondrial marker. Note the high positivity for TOM20 in macrophages located in alveolar spaces in donor control lung. Scale bars: 50 μm. (F) Semiquantitative scoring of SP-C/ATP synthase double-positive cells as a percentage of total SP-C–stained cells from 5 cases. MMF, mild moderated fibrosis; HC, honeycomb; DF, dense fibrosis. Data represent mean ± SEM. *P < 0.01 vs. normal, #P < 0.01 as indicated, 1-way ANOVA with post-hoc Bonferroni. (G) Representative images (n = 3) of immunohistochemistry analyses in consecutive sections from IPF lungs using BiP (ER stress marker), anti–SP-C, and anti-TOM20 antibodies. Arrows denote SP-C–positive cells on mild fibrosis cases. Scale bars: 50 μm.

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