Reactivating Chaperone-mediated Autophagy:

the advantages of preserving a selective autophagy

Ana Maria Cuervo MD PhD

Department of Anatomy and Structural Biology
Marion Bessin Liver Research Center

Albert Einstein College of Medicine, Bronx, NY

 Protein
damage/repair
Young Old
ATP
O. Free
Insult
O.
.
radicals
O
Protein
Protein

Chaperones

Repaired
Proteases Chaperones

Amino
acids
Proteases
Aggregates
Chaperone-mediated Autophagy and Aging

 Overview of CMA

 CMA and Aging

 Changes
 Consequences
 Causes
 Restorative efforts
Types of Autophagy in Mammals
ENDOCYTOSIS
lysosome
endosome

MICROAUTOPHAGY

CHAPERONE-
lysosome MEDIATED
AUTOPHAGY
Golgi

autophagic
vacuole

endoplasmic
MACROAUTOPHAGY reticulum
Chaperone-Mediated Autophagy (CMA)
substrate proteins cytosolic chaperone(s)
KFERQ-motif
hsc70/cochaperones
membrane receptor
lamp2a

lys-hsc70

proteases
lysosome
 Types of Autophagy

Microautophagy Macroautophagy Chaperone-mediated

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
substrate CYTOSOL
KFERQ-motif

Animal tissues (liver, kidney, spleen)

Regulation: Nutrient deprivation
(Stress) Toxic exposure Bag-1
Oxidative stress Hip
Hsp90

Substrates: Selective cytosolic proteins (30%) Hsp40

LysHsc70
Hop

Target signal: KFERQ-like

Lamp2a
LUMEN
Malfunctioning: Toxic-induced nephropathy LysHsc70

Galactosialidosis
Aging
Parkinson’s Disease
Chaperone-mediated Autophagy and Aging

 Overview of CMA
 CMA andAging
CMA and Aging
 Changes
 Consequences
 Causes
 Restorative efforts
 CMA in old Fibroblasts
DEGRADATION OF PROTEINS IN FIBROBLASTS
[ H]Leu
3

35
YOUNG FIBROBLASTS 35 OLD FIBROBASTS
30 30

DPM IN CELLS (%)

DPM IN CELLS (%) 25 25

Fibroblast 20 20

15 15

10 10
+ Serum - Serum Serum +
5 Serum + 5
Serum - Serum -

0 0
0 5 10 15 20 25 30
[3H]amino acids 0 5 10 15 20 25 30
T IM E (h ) T IM E (h )

Okada & Dice, 1984

Degradation of CMA Substrates by Lysosomes
Intact lysosomes Broken lysosomes

50 100
YOUNG (3m) YOUNG
40 OLD (22m) OLD
80
Proteolysis (%)

Proteolysis (%)
30 60
**
20 40

10 20

0 0
GAPDH GAPDH

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

Chaperone-mediated Autophagy and Aging

 Overview of CMA
 CMA and Aging
 Changes
 Consequences
Consequences
 Causes
 Restorative efforts
 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

Cytosol

Nuclei Proteasome
CMA

MICROAUTOPHAGY

Lysosome

MACROAUTOPHAGY
 Oxidized proteins in lysosomes
Roberta
Kiffin
In vivo Lysosomes
Liver Incubated
Washed
24h 24h Cytosol
Cytosol Membrane Matrix
Matrix Matrix
Membrane Membranes
paraquat
Mitochondria/
lysosomes
PQ
PQ -- + + - - ++ - - + + - + - +

Lysosomes
Saline serum
115
82

62
Hypotonic
shock 49

37
26

Membrane Matrix 19

NaCl 37 °C
1 2 3 4 5 6 7 8 9 10

Anti-DNPH
Kiffin et al. (2004) Mol. Biol. Cell
 CMA During Mild Oxidative Stress
Substrate
Lysosome saline
Lysosome
24h 24h

paraquat

40
Cyt Ctr 60 Lys Ctr
***
Proteolysis (%)

Lys PQ

Proteolysis (%)
Cyt H2O2 50
30
* 40
**
20 30

20
10
10
0 0
Intact Broken
lysosomes lysosomes GAPDH RNase A
CMA and Oxidative Stress Bag-1
Hip
Hsp90

LysHsc70
substrate
Hsp40 Hop

Lamp2a
LUMEN
chaperones
LysHsc70

L. Membr

Fed Strv PQ

lamp2a
lamp1
lamp2a
proteases
1 2 3
lysosome
Consequences of declined CMA with age

Impaired elimination of oxidized proteins

_
 Consequences of CMA blockage
substrate
chaperones
Ashish Massey

lamp2a CMA

lys-hsc70
MACROAUTOPHAGY

Lysosome
lysosome

CMA blockage (fibroblasts RNAi lamp2a)

Lamp2a(-) c1 Lamp2a(-) c2
Massey et al. (submitted)
 Effect of CMA Blockage
WILD TYPE CMA (-)
autophagic
vacuole

lysosome

CHAPERONE MEDIATED
MACROAUTOPHAGY
AUTOPHAGY
• Selective • Nonselective
• Low capacity • High capacity

Are cells OK with this switch?

 Consequences of CMA blockage
Deregulation of the response to stress

MTT- Viability assay

Apoptosis
140 50

AnnV(+) 7ADD(-) (% cells))

120 wt
wt 40
CMA (-)
Viable cells (%)

100
CMA (-)
80 30

60 20
40
10
20

0 0
H202 Paraquat Cadmium 42 °C none H2O2 pq UV 42 °C
UV
(50 mM) (1 mM) (10 mM)

24 hours post-insult
Massey et al. (submitted)
Consequences of declined CMA with age

Impaired elimination of oxidized proteins

_ Deregulation of the response to stress
Chaperone-mediated Autophagy and Aging

 Overview of CMA
 CMA and Aging
 Changes
 Consequences
 Causes
Causes
 Restorative efforts
 CMA and Aging: Step by Step
AGE (months)
3m 22 m 3m 22 m
chaperones

lamp2a
substrate

lamp2a

lys-hsc70

OK
OK
proteases

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

Regulation of CMA
Normal Nutrient
Conditions Deprivation

protease lamp2a Degradation

Substrate
OK

Redistribution

lysosome
 s ome
s
so Lys. membrane
Proteomics of the
Ly
Lysosomal Membrane
Guy Sovak NaCl Na2CO3 Detergent
lysosomal membranes

Lysosomal
Lysosomal
membrane
membrane
Rat liver

Lysosomal
matrix

3m 22 m 22m CR
Chaperone-mediated Autophagy and Aging
AGE (months)

lamp2a
3 m 22 m 3 m 22 m
 Overview of CMA
 CMA and Aging
lamp2a
 Changes
 Consequences lys-hsc70

OK
 Causes
proteases
 Restorative efforts

Can we repair the deffect?

Anna Kim
Caloric Restriction and CMA
14
GAPDH degradation
Age
9
3 12 12CR 22 22CR
8 Add Libitum

lamp2a
Caloric Restricted
7
Proteolysis(%)

lamp2s
5

lamp1
2

0 1 2 3 4 5
3m 12m 12m 22m 22m
CR CR
 The CMA-Regulated Animal Model
“Judy” Zhang

The “tet-off”
-Dox lamp2a mouse

+ Dox
+ Doxycycline - Doxycycline
100 Lamp2a Lamp2a (+) WT Lamp2a (+) WT
Levels lamp2a

Lamp2a
75
tTA

PALB tetR VP16

50

Lamp1
+ dox - dox

25

0 5 10
lamp2a 15 20
lamp2a
tet0 PhCMV*-1 PhCMV*-1
Monthstet0
 The CMA-Regulated Animal Model
Cong Zhang
IP 3 10
MW
-Dox 150
100
75
50
100 37
Levels lamp2a

25
75
20
WT 6months TL2a (tet-) 22monts
50
150
100
25 75

50

37
0 5 10 15 20
Months 25

20
WT 22months TL2a (tet+) 22months

Cytosol (Oxyblot)
Levels lamp2a

100
-Dox
Late restoration
75
Oxyblot-2D cytosol 26 months
50

25

0 5 10 15 20
Months

TL2a
1 month 3 months WT TL2A-2 1 month activation

Activation WT 1 2 3
Pool
Lysosomes

CMA+
Lysosomes

CMA-
Lysosomes TL2A-1 1 month activation TL2A-3 3months activation
There is hope for CMA……

…is that all?

 Autophagy and Aging

Microautophagy Macroautophagy Chaperone-mediated

AGING
?
Young Old

Glucagon
?
Lysosome

autophagosome autophagolysosome Lamp2a

E. Bergamini
 Autophagy Crosstalking
Susmita Kaushik
INTACT BROKEN
9
8
Proteolysis (%)

7
6 CMA
5
4
3
2
1
0
wt ATG5(-/-) wt ATG5(-/-)

WT ATG5(-/-)
Lysosome

MACROAUTOPHAGY
 Proteolytic cross-talking
TISSUE TIME
CMA

Proteasome

MICROAUTOPHAGY

Lysosome

MACROAUTOPHAGY
 The Faces of CMA
Ashish Judy
Massey Zhang Guy Sovak
Roberta
Kiffin
Oxidative Stress

Blockage
of CMA
Lysosome
Lamp2a Proteome and
Transgenic Aging
Mouse
Anna Kim
CR

Urmi
Marta Martinez Bandyopadhyay
CMA and
Neurodegeneration Identification Susmita
New components lysosome Kaushik Cross-talking
 Acknowledgements
COLLABORATORS
J. Fred Dice (Tufts University, MA) CMA

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

CMA and PD
Peter Lansbury (Harvard University, MA)
Harry Ischiropoulos (U. Penn, PA)
Ralph Nixon (New York University, NY) Autophagy and AD
Noburu Mizushima (Tokyo MI, Japan) Cross-talking

FUNDING
NIH/NIA, NIH/NIDK
Howard Hughes Biomedical Institute
Ellison Medical Foundation
Huntington’s Disease Society of America