Ana Maria Cuervo MD PhDDepartment of Anatomy and Structural Biology

Marion Bessin Liver Research Center

Albert Einstein College of Medicine, Bronx, NY

Reactivating Chaperone-mediated Autophagy: the advantages of preserving a selective autophagy

Protein damage/repair

Aggregates

Young

Protein

Insult

Chaperones

RepairedProteases

Amino acids

Protein

Old

Chaperones

Proteases

cv ATPFree radicalsO.

O.

O.

Overview of CMA

CMA and Aging Changes Consequences Causes Restorative efforts

Overview of CMA

Chaperone-mediated Autophagy and Aging

Types of Autophagy in Mammals

lysosome

Golgi

endoplasmicreticulumMACROAUTOPHAGY

ENDOCYTOSIS

autophagicvacuole

endosome

MICROAUTOPHAGY

lysosome

CHAPERONE-MEDIATED

AUTOPHAGY

Chaperone-Mediated Autophagy (CMA)

substrate proteinsKFERQ-motif

lysosome

membrane receptor lamp2a

proteases

lys-hsc70

cytosolic chaperone(s)hsc70/cochaperones

Types of Autophagy

Chaperone-mediatedMacroautophagyMicroautophagy

Constitutive Inducible Inducible

Vesicle-mediated Vesicle-mediated Direct transport

Proteins/organelles Proteins/organelles Proteins

Nonselective Nonselective? Selective

Characteristics of Chaperone-Mediated Autophagy

Described: Fibroblasts in culture, other cells type

Animal tissues (liver, kidney, spleen)

Regulation: Nutrient deprivation (Stress) Toxic exposure

Oxidative stress

Substrates: Selective cytosolic proteins (30%)

Target signal: KFERQ-like

Malfunctioning: Toxic-induced nephropathy

Galactosialidosis

Aging

Parkinson’s Disease

Hsp90

HopHsp40

LysHsc70

Bag-1

Hip

KFERQ-motif

substrate CYTOSOL

LUMENLamp2a

LysHsc70

Overview of CMA

CMA and Aging Changes

Consequences

Causes

Restorative efforts

CMA and Aging

Chaperone-mediated Autophagy and Aging

CMA in old Fibroblasts

[3H]Leu

Fibroblast

- Serum+ Serum

[3H]amino acids

DEGRADATION OF PROTEINS IN FIBROBLASTS

Okada & Dice, 1984

Serum -

0

5

10

20

25

30

35

0 5 10 15 20 25 30

T IM E (h )

DP

M I

N C

EL

LS

(%

)

Serum +

YOUNG FIBROBLASTS

15

OLD FIBROBASTS

0

5

10

20

25

30

35

0 5 10 15 20 25 30

T IM E (h )

DP

M I

N C

EL

LS

(%

)

Serum +

15

Serum -

Degradation of CMA Substrates by Lysosomes

Pro

teo

lysi

s (%

)

0

20

40

60

80

100YOUNG

OLD

GAPDH

OLD (22m)

**

0

10

20

30

40

50YOUNG (3m)

GAPDH

Pro

teo

lysi

s (%

)Intact lysosomes Broken lysosomes

Cuervo & Dice (2000) J. Biol. Chem.

Overview of CMA

CMA and Aging Changes

Consequences

Causes

Restorative efforts

Chaperone-mediated Autophagy and Aging

Consequences

Protein Degradation and Aging

Intracellular Protein Content Increases with Age

Accumulation of Damaged Proteins is a Common

Feature of Old Tissues

Does CMA participate in damaged protein removal?

Proteolytic systems inside the cell

Nuclei

Cytosol

Proteasome

Lysosome

MICROAUTOPHAGY

MACROAUTOPHAGY

CMA

Oxidized proteins in lysosomes

- + - + - +

Cytosol

115

82

62

49

37

Anti-DNPH

PQ - +

Cytosol

PQ

26

19

In vivo

paraquat

24h 24h

Saline serum

Lysosomes

Membrane MatrixMembrane Matrix- + - +

Liver

Mitochondria/lysosomes

Ly

so

so

me

sHypotonic

shock

Membrane Matrix

37 °C

- +

WashedMembranes

NaCl

1 2 3 4 5 6 7 8 9 10

IncubatedMatrix

- +

Roberta Kiffin

Kiffin et al. (2004) Mol. Biol. Cell

Lysosome

paraquat

24h 24h

salineLysosomeSubstrate

CMA During Mild Oxidative Stress

Broken lysosomes

0

10

20

30

40

Intact lysosomes

Pro

teo

lysi

s (%

)

Cyt Ctr

Cyt H2O2

*

Lys Ctr

GAPDH

0

10

20

30

40

50

60

Pro

teo

lysi

s (%

)

RNase A

Lys PQ***

**

CMA and Oxidative Stress

lysosome

proteases

lamp2a

substrate

chaperones

Hsp90

HopHsp40

LysHsc70

Bag-1

Hip

LUMENLamp2a

LysHsc70

L. Membr

lam

p2a

lam

p1

1 2 3

Fed Strv PQ

Consequences of declined CMA with age

Impaired elimination of oxidized proteins

_

Consequences of CMA blockage

lysosome

lys-hsc70

substratechaperones

lamp2a

Lysosome

MACROAUTOPHAGY

CMA

Ashish Massey

Mas

sey

et a

l. (s

ubm

itte

d)

Lamp2a(-) c1 Lamp2a(-) c2

CMA blockage (fibroblasts RNAi lamp2a)

Effect of CMA Blockage

CHAPERONE MEDIATED AUTOPHAGY

lysosome

WILD TYPE

• Selective• Low capacity

MACROAUTOPHAGY

autophagicvacuole

CMA (-)

• Nonselective• High capacity

Are cells OK with this switch?Are cells OK with this switch?

Consequences of CMA blockage

Massey et al. (submitted)

Deregulation of the response to stress

MTT- Viability assay

24 hours post-insult

0

20

40

60

80

100

120

140

H202

(50 M)Paraquat (1 mM)

Cadmium (10 M)

42 °C

Via

ble

ce

lls

(%) wt

CMA (-)

UV

CMA (-)

0

10

20

30

40

50Apoptosis

wt

none H2O2 UV 42 °CpqA

nn

V(+

) 7A

DD

(-)

(% c

ells

))

Consequences of declined CMA with age

Impaired elimination of oxidized proteins

Deregulation of the response to stress_

Overview of CMA

CMA and Aging Changes

Consequences

Causes

Restorative efforts

Chaperone-mediated Autophagy and Aging

Causes

CMA and Aging: Step by Step

lysosome

proteases

lys-hsc70

OK

OK

substratechaperones

lamp2a

lam

p2a

3 m 22 m 3 m 22 m

AGE (months)

Cuervo and Dice (2000) J. Biol. Chem.

lysosome

NormalConditions

protease

NutrientDeprivation

lamp2a

Substrate

Regulation of CMA

Redistribution

Degradation

OK

Lysosomalmembrane

Lysosomal matrix

Lysosomalmembrane

Proteomics of the Lysosomal Membrane

Guy Sovak NaCl Na2CO3 Detergent

Ra

t li

ve

r ly

so

so

ma

l m

em

bra

ne

sLysosomes

Lys. membrane

3 m 22 m 22m CR

Overview of CMA

CMA and Aging Changes

Consequences

Causes

Restorative efforts

Chaperone-mediated Autophagy and Aging

Can we repair the deffect?

proteases

lys-hsc70

OK

lamp2a

lam

p2a 3 m 22 m 3 m 22 m

AGE (months)

Caloric Restriction and CMA

3 12 12CR 22 22CR

Age

lam

p2a

lam

p1

1 2 3 4 5

lam

p2s

22mCR

12m CR

Caloric Restricted

14GAPDH degradation

0

1

2

3

4

5

6

7

8

9

3m 12m 22m

Pro

teo

lys

is(%

)

Add Libitum

Anna Kim

PALB tetR VP16

tTA

The “tet-off” lamp2a mouse

+ Dox

Lamp2a

The CMA-Regulated Animal Model

lamp2a

tet0 PhCMV*-1

- dox+ dox

lamp2a

tet0 PhCMV*-1

- DoxycyclineLamp2a (+) WT

La

mp

2a

La

mp

1

Lamp2a (+) WT

+ Doxycycline

20151050

Months

Lev

els

lam

p2a

100

75

25

50

-Dox

“Judy” Zhang

The CMA-Regulated Animal Model Cong Zhang

20151050

Months

Lev

els

lam

p2a

100

75

25

50

-Dox

TL2a (tet-) 22monts

TL2a (tet+) 22months

10

Cytosol (Oxyblot)

WT 6months

WT 22months

3

100

75

50

37

25

20

150

10075

50

37

25

20

150

MWIP

1 month

Pool Lysosomes

CMA+ Lysosomes

CMA- Lysosomes

3 months

TL2a

Activation WT 1 2 3

20151050Months

Lev

els

lam

p2

a

100

75

25

50

-DoxLate restoration

Oxyblot-2D cytosol 26 months

WT TL2A-2 1 month activation

TL2A-1 1 month activation TL2A-3 3months activation

There is hope for CMA……

…is that all?

AGING

Autophagy and Aging

Chaperone-mediatedMacroautophagyMicroautophagy

????

Lamp2a

Young Old

autophagosome autophagolysosome

Lysosome

?

E. Bergamini

Glucagon

Autophagy Crosstalking

Lysosome

MACROAUTOPHAGY

CMA

Susmita Kaushik

wt ATG5(-/-)

BROKEN

ATG5(-/-)

ATG5(-/-)

INTACT

0

1

2

3

4

5

6

7

8

9

Pro

teo

lysi

s (%

)

wt

WT

Proteolytic cross-talking

Lysosome

MICROAUTOPHAGY

MACROAUTOPHAGY

CMA

Proteasome

TISSUE TIME

The Faces of CMA

lysosomeCMA and

Neurodegeneration

Marta Martinez

Oxidative Stress

RobertaKiffin

Blockageof CMA

AshishMassey

Lamp2aTransgenic

Mouse

JudyZhang

IdentificationNew components

UrmiBandyopadhyay

Lysosome Proteome and

Aging

Guy Sovak

Susmita Kaushik

Cross-talking

Anna Kim

CR

Acknowledgements

J. Fred Dice (Tufts University, MA)

David Sulzer, Serge Przedborski (Columbia U., NY)

Peter Lansbury (Harvard University, MA)

Harry Ischiropoulos (U. Penn, PA)

Ralph Nixon (New York University, NY)

Noburu Mizushima (Tokyo MI, Japan)

COLLABORATORS

CMA

CMA and PD

FUNDING

NIH/NIA, NIH/NIDKHoward Hughes Biomedical InstituteEllison Medical FoundationHuntington’s Disease Society of America

Autophagy and AD

Cross-talking