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2013-06-25
1
Autophagy: Friend or foe in asthma and COPD?
Augustine M.K. Choi, MD
Parker B. Francis Professor of Medicine
Harvard Medical School
Pulmonary and Critical Care Medicine
Brigham and Women’s Hospital
Airway Vista 2013
Asan Medical Center
Seoul, Korea
March 30, 2013
Disclosures
• Proterris
2013-06-25
2
Outline
• Introduction
• Autophagy: Regulation and function in experimental
and human lung disease (e.g. COPD)
• Selective Autophagy: Mitophagy and “ciliophagy” in
experimental and human lung disease (e.g. COPD)
• Translational/Clinical Implications
J. Cell Biol, 1962 J. Cell Biol, 1967
2013-06-25
3
NEJM, 2013
Autophagy
NEJM, 2013
Autophagy
2013-06-25
4
NEJM, 2013
Selective Autophagy
Nature 2008
Autophagy and Human Diseases
Cancer
Infection and immunity
Heart disease
Liver disease
Aging
Myopathies
Neurodegeneration
2013-06-25
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Mizushima N et al., Cell, 2011
Physiologic or Pathologic Roles of Autophagy
NEJM, 2013
2013-06-25
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Ryter and Choi, Annual Review of Physiology, 2012
Functional Role of Autophagy in Lung Disease
2013-06-25
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Autophagy in Smoke Induced COPD
Chen et al., PLoS ONE 2008
Chen, et al. PNAS 2010
Kim et al., Autophagy 2008
Ryter et al., Autophagy 2009
Kim et al., Methods Enzymol 2009
LC3B
Histone acetylation
Egr-1 E2F4
LC3B-II
Atg4
Autophagy
HDAC Activity
COPD
Lung cell apoptosis
2013-06-25
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Chen* & Lam* et al. PNAS, 2010 (Track II)
Emphysema Apoptosis
Autophagy
Autophagy
Less lung injury Less apoptosis
LC3 LC3
LC3
Atg5
Atg12
LC3 Cav-1
Cav-1
Cav-1
Chen* & Lam* et al. PNAS, 2010 (Track II)
Autophagy in Smoke Induced Emphysema
2013-06-25
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Bax
Caspase-8
LC3B Cav-1 Cav-1 LC3B FAS
FADD
Autophagosome
Cigarette Smoke COPD Autophagy
Mitochondrial ROS
t-Bid
Apoptosis
Caspase-3
Caspase-9
Bid
Cyto-c
LC3B
Cardiolipin Ox-cardiolipin
ROS
LC3B
LC3B
LC3B secretion
CS CS
Autophagy
Autophagic Flux
Blood biomarker
Chen* & Lam* et al. PNAS, 2010 (Track II)
Nat Med 2010
2013-06-25
10
a. Control CSE
0 10 2 10 3 10 4 10 5 0
20
40
60
80
100
0
0.5
1
1.5
2
2.5
Control CSE2h CSE4h
c.
Even
ts (
% o
f m
ax)
MitoSOX
Unstained
Control
CSE 2h
CSE 4h R
elat
ive
MF
I
*
*
Unstained Control
CSE 2h
CSE 4h
MitoTracker Deep Red MitoTracker Green
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Control CSE 2h CSE 4h Control CSE 2h CSE4h
Mitotracker Deep Red Mitotracker Green
Rel
ativ
e M
FI
*
*
0 10 2 10 3 10 4 10 5 0
20
40
60
80
100
0 10 2 10 3 10 4 10 5 0
20
40
60
80
100
Even
ts (
% o
f m
ax) d.
Cigarette smoke causes mitochondrial dysfunction
Never
Smokers
COPD
e.
PINK1
β-Actin
d. Never smokers Patients with COPD (GOLD2)
LC3B
0 0.5 1 2 4 8 12 24 (h)
CSE
PINK1
a.
β-Actin
full length
processed
0 0.5 1 2 4 8 12 24 (h)
CSE
β-Actin
PINK1
b.
PINK1, a mitochondrial membrane protein is regulated in CSE treated cells and in
COPD lung tissues
2013-06-25
11
a.
mt-mKeima
ex. 458 nm
mt-mKeima
ex. 543 nm
mt-mKeima
ratio
(543/458)
CSE Control
2.0
0
Control 2h 4h
CSE
Blue: Hoechest33258 (nucleus) Green: Tom20 (mitochondria)
b.
Cigarette Smoke Extract induces mitophagy
Cont CSE Cont CSE
Mitochondrial
fraction
Cytosolic
fraction
PINK1
Ubiqutin
LC3B
Tom20
β-Actin
a.
Protein staining
(membrane)
Cont CSE Cont CSE
Mitochondrial
fraction
Cytosolic
fraction
WT PINK1 KO
Air
CS
(Rel
ativ
e JC
-1 u
pta
ke
to r
oom
air
con
trol
of
WT
)
0%
20%
40%
60%
80%
100%
120%
ψm
(n=8) (n=6) (n=6) (n=6)
* # c.
b.
0 102
103
104
105
PE-A
0
20
40
60
80
100
% o
f M
ax
Isolated alveolar
epithelial cells
from WT mice
Isolated alveolar
epithelial cells
from PINK1 KO
mice
MitoSOX
Even
ts (
% o
f m
ax)
0
0.2
0.4
0.6
0.8
1
1.2
WT PINK1 KO
Rel
ativ
e M
FI
CS regulates PINK1 and mitochondrial dysfunction in vivo
2013-06-25
12
b.
WT PINK1 KO
Ch
ord
Len
gth
(μm)
15
20
25
30
35 Air
CS
(n=6) (n=4) (n=5) (n=5)
* #
0
5
10
15
20
25
30
Air
CS
WT PINK1 KO
% 3
h C
lear
ance
* #
(n=5) (n=5) (n=3) (n=4)
d.
PINK1 deficient mice exhibit decreased CS-induced emphysema and MCC dysfunction.
The Model: Mouse tracheal-bronchial epithelial
cells (MTECs) grown at air-liquid interface (ALI)
Seed
(0d)
ALI
(10d)
Experiment
(24d)
CS
Non-ciliated Cell Basal Cell Ciliated Cell
a
bb m
n
m
s
mv
m
ALI
D0
0
1000
2000
3000
4000
5 10 15 20 25
Time (d)
Res
ista
nc
e (
Ω/c
m2)
ALI
(10d)
1h
Low Dose CS
High Dose CS
24h 8h 4h
2013-06-25
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CS disrupts intercellular contacts and induces cilia loss in
MTEC cultures
CTL
4h
24h
0
5
10
15
20
25
30
CTL LD CS HD CS
% C
ilia
ted
Cell
s
*
0
500
1000
1500
2000
2500
3000
5 10 15 20 25
TE
R (
Ω/c
m2)
Time (h)
*
*
* * *
CTL LD CS HD CS
CTL LD CS HD CS
CS
m
bb a
RA
bb
m
a
n
bb
m
n
e
f
CTL 50
bb
a
m
100
bb
m
bb
a
m
β-actin
CQ Time (h)
LC3B
p62
p62
0 1 1 3 6 3 6 0
CTL CS
I
194
111
59
59
II
HMW p62
0
10
20
30
% C
ilia
ted
Cell
s *
1 2 3 Time (d)
CT
L C
S
c a
i
CT
L
1
2
3
CS
(d
)
ac α-tub F-actin nuclei
b
Mucin 5AC
Centrin 1
CTL CS
β-actin
d
0.0 CTL CS
Pu
ncta
/ 1
00 μ
m2
0.2
0.4
0.6
0.8
1.0 **
g
CTL CS
GFP-LC3B nuclei
h
0.03
0
0.01
0.02
0.04
R
A
1w 2m
ΔL
C3B
II (n
g)
*
0 h
0
0.01
0.02
0.03
0.04
0.05
0.06
RA 1w 2m
*
ΔL
C3B
II (n
g)
24 h
CS induces cilia loss and autophagy in MTEC cultures
2013-06-25
14
Centrin 1
LC3B
CTL
W C L
LAMP2
50
W C L
100
W C L
a
bb
a
bb
CT
L
CS
ac α-tub LC3B nuclei
IP:LC3B CTL IP IP IN IN IgG IgG
CSE
199 IB:Pericentrin
IB:IFT88
IB:LC3B
Colocalization of cilia and autophagic markers following CS exposure
0
0.5
1.0
1.5 Becn1+/+
Becn1+/-
**
CTL CS
Cil
iate
d C
ell
s
(no
rmalized
to
CT
L)
e
Becn
1+
/-
CTL CS
Becn
1+
/+
Autophagic degradation of cilia components (ciliophagy) promotes CS-induced cilia loss
f
Becn1+/- 0
0.005
0.010
0.015
Becn1+/+
Au
top
hag
oso
mes/1
00 μ
m2
CS RA
c
***
**
Becn1+/+ Becn1+/-
ΔL
C3B
(n
g)
0
0.02
0.04
0.06
0.08
*
RA CS
d a
Becn
1+
/+
Becn
1+
/-
s
m
bb
a
n
bb m
a
m bb
a
m
g
CTL CS
a
0
2
4
6
8
10
Au
top
hag
oso
mses/1
00μ
m2
CTL CS
Becn1+/+ Becn1+/-
*
b
2013-06-25
15
194 111
Ubiquitin
β-actin
7
17
59
30
25
Hdac6+/Y Hdac6-/Y
CTL CS CTL CS MW
(kDa)
b
Hd
ac6
+/Y
H
dac6
-/Y
CTL CS
PA nuclei
e
f
c
0
0.5
1.0
1.5
Cil
iate
d C
ell
s
(no
rmalized
to
CT
L)
CTL CS
**
*
Hdac6-/Y
Hdac6+/Y
g h C
TL
C
S
Hdac6+/Y Hdac6-/Y
a d
RA
C
S
Hdac6+/Y Hdac6-/Y
n m m
m m
n HDAC6
W IgG IP: LC3B CTL
IP W IgG IP
CSE
IB:LC3B
Hdac6-/Y
0.02
0.04
0.06
0.08
ΔL
C3B
(n
g)
Hdac6+/Y
CS RA
***
*
0 Hdac6+/
Y
0
0.005
0.010
0.015
**
**
Au
top
hag
osm
es/1
00 μ
m2
Hdac6-/Y
CS RA
CS induced ciliophagy and aggrephagy is mediated by HDAC6
% A
cti
vit
y R
em
ain
ing
0 1 2 3 85
90
95
100
105
Time (h)
RA
CS
*
** L
S
L S
L
Sagittal
Coronal
.
0
5
10
15 RA CS
*
* *
WT HDAC6 -/Y Tubastatin Vehicle
*
% 3
h C
lea
ran
ce
Rescue of mucociliary clearance dysfunction by HDAC6 inhibition
2013-06-25
16
0
10
20
30
RA
CS
WT PBA Becn+/- Maplc3b-/-
*
*
*
*
% 3
h C
lea
ran
ce
Rescue of mucociliary clearance dysfunction in beclin or LC3 deficient mice
Pathogenesis of COPD
Oxidant
-Antioxidant
Protease
-Antiprotease
Vascular Autoimmunity Microbial pathogen
Inflammation Cell death Autophagy
2013-06-25
17
TEAM
Zhihua Chen, PhD
Hilaire C. Lam, PhD candidate
Hong-Pyo Kim, PhD
Stefan W. Ryter, PhD
Jeffrey A. Haspel, MD, PhD
Tamas Dolinay, MD, PhD
Seon-Jin Lee, PhD
Akaya Smith, MD
Young-Sam Kim, MD, PhD
Kiichi Nakahira, MD, PhD
Joshua Englert, MD
Emeka Ifeidgbo, MS
Roberto Landazury, Bsc
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