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

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Mizushima N et al., Cell, 2011

Physiologic or Pathologic Roles of Autophagy

NEJM, 2013

2013-06-25

6

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

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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

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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

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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

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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

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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

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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

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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

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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

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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