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