4. Results
4.3. Mitophagy in murine model of AD-induced pulmonary fibrosis
4.3.3. AD induces differential expression of mitophagy proteins in murine AECII
56 (Figure 4.6I). Altogether, the data point towards increased mitochondrial biogenesis in AECII following AD treatment.
4.3.3. AD induces differential expression of mitophagy proteins in murine
Results
57 In addition, the expression of conventional macroautophagy proteins, ATG12-ATG5 and ATG7 was also analyzed, as deficiency of these proteins has been proven to induce mitochondrial defects and hinder the clearance of damaged mitochondria (Wu et al., 2009, Liu et al., 2012). Immunoblot analysis of the AD treated mice lung homogenates, murine AECII and MLE12 treated with AD in vitro displayed proficient increase in the expression of ATG12-ATG5 (~ 1.5-fold) in comparison to the Veh treated controls (Figures 4.7A and G, 4.8A and G, 4.9A and G). Similar to AD-induced ATG12-ATG5 expression, immunoblot analysis of AD (day 7, 21 and 28) treated mice lung homogenates exhibited significant upregulation (~ 2-fold) of ATG7 expression versus the Veh (Figures 4.7A and H).
Immunohistochemical analysis for ATG7 and proSP-C (AECII marker) on the serial lung sections revealed an increased staining of ATG7 in AECII in lung sections of AD treated (day 7, 14, 21 and 28) mice compared to the Veh treated (day 28) control mice (Figure 4.7J).
In line with this, in vitro AD treated murine AECII (Figure 4.8A and H) and AD treated MLE12 cells (Figures 4.9A and H) also showed a significant increase (~ 2 and ~ 1.5-fold respectively) in the ATG7 expression after 24 hours.
Thus, these results collectively imply unaltered expression of the mitophagy receptor proteins Bnip3L & Bnip3 and the cargo receptor protein NBR1, but an increased synthesis of autophagy adaptor protein p62 and other mitophagy proteins such as ATG12-ATG5 and ATG7 in murine AECII following AD treatment in vivo and in vitro.
Bnip3 25
35
70 Bnip3L (Homodimer)
55 ATG12- ATG5
70
ATG7
AD D7 AD D14 Veh D28 AD D21 AD D28 Veh D28
MW (kDa)
β-actin 40
Bnip3L (Monomer) 40
100
NBR1
70 p62
A.
58
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
50 100 150 200
IDV % of ß-actin nsIDV % of ß-actin
ns ns ns
Bnip3
AD D7 AD D14 AD D21 AD D28 Veh D28
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
50 100 150 200
IDV % of ß-actin
ns
IDV % of ß-actin ns
ns ns
Bnip3L - Monomer
AD D7 AD D14 AD D21 AD D28 Veh D28 AD D7
AD D14 AD D21
AD D 28
Veh D28 0
20 40 60 80
IDV % of ß-actin
ns
IDV % of ß-actin ns ns ns
Bnip3L - Homodimer
AD D7 AD D14 AD D21 AD D28 Veh D28
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
50 100 150
IDV % of ß-actin
*
IDV % of ß-actin ns *
**
p62
AD D7 AD D14 AD D21 AD D28 Veh D28
B. C.
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
50 100 150 200
IDV % of ß-actin ns
IDV % of ß-actin
ns
ns ns
Bnip3
AD D7 AD D14 AD D21 AD D28 Veh D28
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
50 100 150 200
IDV % of ß-actin
ns
IDV % of ß-actin ns
ns ns
Bnip3L - Monomer
AD D7 AD D14 AD D21 AD D28 Veh D28 AD D7
AD D14 AD D21
AD D 28
Veh D28 0
20 40 60 80
IDV % of ß-actin
ns
IDV % of ß-actin ns ns ns
Bnip3L - Homodimer
AD D7 AD D14 AD D21 AD D28 Veh D28
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
50 100 150
IDV % of ß-actin
*
IDV % of ß-actin ns *
**
p62
AD D7 AD D14 AD D21 AD D28 Veh D28
D. E.
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
50 100 150 200
IDV % of ß-actin **IDV % of ß-actin
ns **
*
ATG7
AD D7 AD D14 AD D21 AD D28 Veh D28 AD D7
AD D14 AD D21
AD D 28 Veh D28 0
50 100 150 200
IDV % of ß-actin
*
IDV % of ß-actin
ATG12-ATG5
AD D7 AD D14 AD D21 AD D28 Veh D28
*
*
*
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
20 40 60 80 100
IDV % of ß-actin
*
IDV % of ß-actin ns
*
**
NBR1
AD D7 AD D14 AD D21 AD D28 Veh D28
F. G.
H.
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
50 100 150 200
IDV % of ß-actin **IDV % of ß-actin
ns **
*
ATG7
AD D7 AD D14 AD D21 AD D28 Veh D28 AD D7
AD D14 AD D21
AD D 28 Veh D28 0
50 100 150 200
IDV % of ß-actin
*
IDV % of ß-actin
ATG12-ATG5
AD D7 AD D14 AD D21 AD D28 Veh D28
*
*
*
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
20 40 60 80 100
IDV % of ß-actin *IDV % of ß-actin ns
*
**
NBR1
AD D7 AD D14 AD D21 AD D28 Veh D28
F. G.
H.
Results
59
Figure 4.7. Differential expression of mitophagy proteins in AD treated mice lung tissues.
(A) Western blot analysis of lung homogenates of (left) AD treated mice (day 7, 14) and Veh treated mice (day 28) and (right) AD treated mice (day 21, 28) and Veh treated mice (day 28) for Bnip3L (homodimer and monomer), Bnip3, p62, NBR1, ATG12-ATG5, ATG7 and -actin (loading control). n = 5 mice per group. (B, C, D, E, F, G, H) Densitometry analysis of the target proteins’ blots namely Bnip3L (homodimer and monomer), Bnip3, p62, NBR1, ATG12-ATG5, ATG7 and -actin (loading control) and target protein*100/-actin ratio was calculated and represented as bar graphs. **p < 0.01, *p < 0.05, ns - no significance. (I) Immunohistochemical analysis of serial lung sections of AD (day 7, 14, 21 and 28) and Veh (day 28) treated mice for p62 and proSP-C. Arrows indicate AECII showing increased p62 expression from day 7 of AD treatment. n = 5 mice per group. Scale bar = 50 µm. Original magnification: x400 (J) Immunohistochemical analysis of serial lung sections of AD (day 7, 14, 21 and 28) and Veh (day 28) treated mice for ATG7 and proSP-C. Arrows indicate AECII showing increased ATG7 expression from day 7 of AD treatment. n = 5 mice per group. Scale bar = 50 µm. Original magnification: x400.
AD D14 AD D21 AD D28 Veh D28
p62proSP-C
AD D7
I.
AD D14 AD D21 AD D28 Veh D28
ATG 7
AD D7
proSP-C
J.
60
AD Veh
0 20 40 60 80 100
IDV % of ß-actinIDV % of GAPDH
AD Veh BNIP3L- Monomer ns
AD Veh
0 10 20 30
IDV % of ß-actin IDV % of GAPDH
AD Veh Bnip3L- Homodimer
A. ns
C.
B.
ATG12 - ATG5 55
AD Veh
AECII MW
(kDa)
70 ATG7
pro- SP C 25
BNIP3 35
25
p62 70
GAPDH 35
BNIP3L (Monomer) 40
BNIP3L (Homodimer) 70
NBR1 100
AD Veh
0 50 100 150
IDV % of ß-actin **IDV % of GAPDH
AD Veh ATG12-ATG5
AD Veh
0 5 10 15 20 25
IDV % of ß-actin
ns
IDV % of GAPDH
AD Veh NBR1
AD Veh
0 20 40 60 80 100
IDV % of ß-actin
*
IDV % of GAPDH
AD Veh
AD Veh p62
0 10 20 30 40 50
IDV % of ß-actin
ns
IDV % of GAPDH
AD Veh Bnip3
D. E.
F. G.
AD Veh
0 50 100 150
IDV % of ß-actin
**
IDV % of GAPDH
AD Veh ATG12-ATG5
AD Veh
0 5 10 15 20 25
IDV % of ß-actin
ns
IDV % of GAPDH
AD Veh NBR1
AD Veh
0 20 40 60 80 100
IDV % of ß-actin
*
IDV % of GAPDH
AD Veh
AD Veh p62
0 10 20 30 40 50
IDV % of ß-actin
ns
IDV % of GAPDH
AD Veh Bnip3
D. E.
F. G.
Results
61
Figure 4.8. Differential expression of mitophagy proteins in AD treated AECII.
(A) Representative western blot images for Bnip3L (homodimer & monomer), Bnip3, p62, NBR1, ATG12-ATG5, ATG7, proSP-C and GAPDH (loading control) from 24 hours of AD or Veh treated AECII isolated from C57Bl/6 mice. n = 3 independent experiments. (B, C, D, E, F, G and H) Densitometry analysis of the triplicate blots of the target proteins viz Bnip3L (homodimer & monomer), Bnip3, p62, NBR1, ATG12-ATG5, ATG7 and GAPDH (loading control) and target protein*100/GAPDH ratio was calculated and represented as bar graphs.
**p < 0.01,*p < 0.05, ns - no significance.
**
IDV % of GAPDH
AD Veh
AD ATG7 Veh
0 10 20 30 40 50
IDV % of ß-actin
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
50 100 150 200
IDV % of ß-actin
ns
IDV % of ß-actin ns
ns ns
Bnip3L - Monomer
AD D7 AD D14 AD D21 AD D28 Veh D28 AD D7
AD D14 AD D21
AD D 28 Veh D28 0
20 40 60 80
IDV % of ß-actin
ns
IDV % of ß-actin ns ns ns
Bnip3L - Homodimer
AD D7 AD D14 AD D21 AD D28 Veh D28
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
50 100 150
IDV % of ß-actin
*
IDV % of ß-actin ns *
**
p62
AD D7 AD D14 AD D21 AD D28 Veh D28
H. E.
AD 8h
AD 16h
AD 24h
Veh D24 0
50 100 150
IDV % of ß-actin nsIDV % of ß-actin
AD 8h AD 16h AD 24h Veh 24h Bnip3L - Monomer
ns
ns AD 8h
AD 16h
AD 24h
Veh D24 0
10 20 30 40
IDV % of ß-actin
ns IDV % of ß-actin
AD 8h AD 16h AD 24h Veh 24h Bnip3L - Homodimer
ns
ns
A.
C.
B.
55
ATG 12- ATG5 AD
8 16 24 24 MW (kDa)
Veh MLE12
Hours
70 ATG7
β-actin 40
35
25 Bnip3
NBR1 100
40 Bnip3L
(Monomer)
70 Bnip3L (Homodimer)
70
p62
62
Figure 4.9. Differential expression of mitophagy proteins in AD treated MLE12 cells.
(A) Representative western blot images for Bnip3L (homodimer & monomer), Bnip3, p62, NBR1, ATG12-ATG5, ATG7, proSP-C and -actin (loading control) from AD (8, 16 and 24 hours) and Veh (24 hours) treated MLE12 cells. n = 3 independent experiments. (B, C, D, E, F, G and H) Densitometry analysis of the triplicate blots of the target proteins viz Bnip3L (homodimer & monomer), Bnip3, p62, NBR1, ATG12-ATG5, ATG7 and
-actin (loading control) and target protein*100/-actin ratio was calculated and represented as bar graphs. **p
< 0.01,*p < 0.05, ns - no significance.
AD 8h
AD 16h
AD 24h
Veh D24 0
20 40 60 80 100
IDV % of ß-actin
*
IDV % of ß-actin
ATG12 - ATG5
AD 8h AD 16h AD 24h Veh 24h
* *
AD 8h
AD 16h
AD 24h
Veh D24 0
20 40 60
IDV % of ß-actin
ns
IDV % of ß-actin
*
NBR1
AD 8h AD 16h AD 24h Veh 24h ns
ns
AD 8h
AD 16h
AD 24h
Veh D24 0
50 100 150
IDV % of ß-actin **IDV % of ß-actin
*
p62
AD 8h AD 16h AD 24h Veh 24h
**
AD 8h
AD 16h
AD 24h
Veh D24 0
20 40 60 80
IDV % of ß-actin
ns
IDV % of ß-actin
AD 8h AD 16h AD 24h Veh 24h Bniip3
ns ns
D. E.
F. G.
AD 8h
AD 16h
AD 24h
Veh D24 0
20 40 60 80 100
IDV % of ß-actin
*
IDV % of ß-actin
ATG12 - ATG5
AD 8h AD 16h AD 24h Veh 24h
* *
AD 8h
AD 16h
AD 24h
Veh D24 0
20 40 60
IDV % of ß-actin
ns
IDV % of ß-actin
*
NBR1
AD 8h AD 16h AD 24h Veh 24h ns
ns
AD 8h
AD 16h
AD 24h
Veh D24 0
50 100 150
IDV % of ß-actin **IDV % of ß-actin
*
p62
AD 8h AD 16h AD 24h Veh 24h
**
AD 8h
AD 16h
AD 24h
Veh D24 0
20 40 60 80
IDV % of ß-actin
ns
IDV % of ß-actin
AD 8h AD 16h AD 24h Veh 24h Bniip3
ns ns
D. E.
F. G.
AD 8h
AD 16h
AD 24h
Veh D24 0
50 100 150 200
IDV % of ß-actin
ns
IDV % of ß-actin
*
ATG7
AD 8h AD 16h AD 24h Veh 24h ns
AD D7
AD D14 AD D21
AD D 28
Veh D28 0
20 40 60 80
IDV % of ß-actin
ns
IDV % of ß-actin ns ns ns
Bnip3L - Homodimer
AD D7 AD D14 AD D21 AD D28 Veh D28
AD D7 AD D14
AD D21 AD D 28
Veh D28 0
50 100 150
IDV % of ß-actin
*
IDV % of ß-actin ns *
**
p62
AD D7 AD D14 AD D21 AD D28 Veh D28
H. E.
Results
63