Transcript Aging and selective degradation of cytosolic proteins in

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.
O.
Free radicals
Insult
.
O
Protein
Protein
Chaperones
Repaired
Proteases
Chaperones
Amino
acids
Aggregates
Proteases
Chaperone-mediated Autophagy and Aging
 Overview of CMA
 CMA and Aging




Changes
Consequences
Causes
Restorative efforts
Types of Autophagy in Mammals
ENDOCYTOSIS
endosome
lysosome
MICROAUTOPHAGY
lysosome
CHAPERONEMEDIATED
AUTOPHAGY
Golgi
autophagic
vacuole
MACROAUTOPHAGY
endoplasmic
reticulum
Chaperone-Mediated Autophagy (CMA)
substrate proteins
KFERQ-motif
cytosolic chaperone(s)
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 ChaperoneMediated Autophagy
 Described: Fibroblasts in culture, other cells type
substrate
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
CYTOSOL
KFERQ-motif
Bag-1
Hip
LysHsc70
Hsp40
Hsp90
Hop
Lamp2a
LUMEN
LysHsc70
Chaperone-mediated Autophagy and Aging
 Overview of CMA
 CMA
CMA and
andAging
Aging
 Changes
 Consequences
 Causes
 Restorative efforts
CMA in old Fibroblasts
DEGRADATION OF PROTEINS IN FIBROBLASTS
[3H]Leu
YOUNG FIBROBLASTS
35
30
+ Serum
- Serum
DPM IN CELLS (%)
DPM IN CELLS (%)
30
Fibroblast
25
20
15
10
Serum +
Serum -
5
25
20
15
10
Serum +
Serum -
5
0
0
[3H]amino acids
OLD FIBROBASTS
35
0
5
10
15
20
T IM E (h )
25
30
0
5
10
15
20
25
30
T IM E (h )
Okada & Dice, 1984
Degradation of CMA Substrates by Lysosomes
Intact lysosomes
Broken lysosomes
50
100
40
OLD (22m)
30
**
20
Proteolysis (%)
Proteolysis (%)
YOUNG (3m)
80
YOUNG
OLD
60
40
20
10
0
0
GAPDH
Cuervo & Dice (2000) J. Biol. Chem.
GAPDH
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
Proteasome
Nuclei
CMA
MICROAUTOPHAGY
Lysosome
MACROAUTOPHAGY
Oxidized proteins in lysosomes
Roberta
Kiffin
In vivo
Lysosomes
Liver
24h
Cytosol
Cytosol
24h
Mitochondria/
lysosomes
Lysosomes
paraquat
Saline serum
Hypotonic
shock
PQ
PQ
--
+ +
1
2
Membrane Matrix
Matrix
Membrane
- - ++ - - + +
Washed
Membranes
+
Incubated
Matrix
-
+
115
82
62
49
37
26
Membrane
NaCl
Matrix
19
37 °C
3
4
5
6
7
8
9
10
Anti-DNPH
Kiffin et al. (2004) Mol. Biol. Cell
CMA During Mild Oxidative Stress
Substrate
saline
Lysosome
24h
Lysosome
24h
paraquat
30
Cyt Ctr
60
Proteolysis (%)
Proteolysis (%)
40
Cyt H2O2
*
20
10
***
50
40
Lys Ctr
Lys PQ
**
30
20
10
0
Intact
lysosomes
0
Broken
lysosomes
GAPDH
RNase A
CMA and Oxidative Stress
Bag-1
Hip
LysHsc70
substrate
Hsp40
Hsp90
Hop
Lamp2a
LUMEN
chaperones
LysHsc70
L. Membr
Strv
PQ
2
3
lamp1
lamp2a
Fed
lamp2a
proteases
1
lysosome
Consequences of declined CMA with age
Impaired elimination of oxidized proteins
_
Consequences of CMA blockage
Ashish Massey
chaperones
CMA
lamp2a
lys-hsc70
MACROAUTOPHAGY
Lysosome
lysosome
CMA blockage (fibroblasts RNAi lamp2a)
Lamp2a(-) c1
Massey et al. (submitted)
substrate
Lamp2a(-) c2
Effect of CMA Blockage
WILD TYPE
CMA (-)
autophagic
vacuole
lysosome
CHAPERONE MEDIATED
AUTOPHAGY
• Selective
• Low capacity
MACROAUTOPHAGY
• Nonselective
• High capacity
Are cells OK with this switch?
Consequences of CMA blockage
Deregulation of the response to stress
MTT- Viability assay
Viable cells (%)
120
wt
100
Apoptosis
50
AnnV(+) 7ADD(-) (% cells))
140
CMA (-)
80
60
40
20
wt
40
CMA (-)
30
20
10
0
0
H202
(50 mM)
Paraquat
(1 mM)
Cadmium
(10 mM)
24 hours post-insult
Massey et al. (submitted)
UV
42 °C
none
H2O2
pq
UV
42 °C
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)
chaperones
substrate
22 m
lamp2a
3m
lamp2a
lys-hsc70
OK
OK
proteases
Cuervo and Dice (2000) J. Biol. Chem.
lysosome
3m
22 m
Regulation of CMA
Normal
Conditions
protease
Nutrient
Deprivation
Degradation
lamp2a
Substrate
OK
Redistribution
lysosome
Proteomics of the
Lysosomal Membrane
Lys. membrane
Guy Sovak
Rat liver
lysosomal membranes
NaCl
Na2CO3
Detergent
Lysosomal
Lysosomal
membrane
membrane
Lysosomal
matrix
3m
22 m
22m CR
 Overview of CMA
lamp2a
Chaperone-mediated Autophagy and Aging
AGE (months)
3 m 22 m 3 m 22 m
 CMA and Aging
 Changes
 Consequences
 Causes
lamp2a
lys-hsc70
OK
proteases
 Restorative efforts
Can we repair the deffect?
Caloric Restriction and CMA
Anna Kim
14GAPDH
degradation
Age
9
Add Libitum
Caloric Restricted
7
12
12CR
1
2
3
22
22CR
lamp2s
6
5
4
3
lamp1
Proteolysis(%)
3
lamp2a
8
2
1
0
3m
12m
12m
CR
22m 22m
CR
4
5
The CMA-Regulated Animal Model
“Judy” Zhang
The “tet-off”
-Dox lamp2a mouse
+ Dox
+ Doxycycline
Lamp2a
Lamp2a
75
Lamp2a (+)
tTA
PALB
50
tetR
VP16
+ dox
- dox
25
0
5
tet0 PhCMV*-1
10
lamp2a
Monthstet0
15
PhCMV*-1
20
lamp2a
Lamp1
Levels lamp2a
100
WT
- Doxycycline
Lamp2a (+)
WT
The CMA-Regulated Animal Model
Cong Zhang
IP 3
MW
-Dox
10
150
100
75
50
Levels lamp2a
100
37
25
75
20
WT 6months
TL2a (tet-) 22monts
WT 22months
TL2a (tet+) 22months
50
150
100
75
25
50
0
5
10
Months
15
20
37
25
20
Cytosol (Oxyblot)
100
Levels lamp2a
Late restoration
-Dox
75
Oxyblot-2D cytosol 26 months
50
25
0
5
10
15
20
Months
TL2a
1 month
Activation
WT
1
2
3 months
WT
TL2A-2
1 month activation
TL2A-3
3months activation
3
Pool
Lysosomes
CMA+
Lysosomes
CMALysosomes
TL2A-1
1 month activation
There is hope for CMA……
…is that all?
Autophagy and Aging
Microautophagy
AGING
?
Macroautophagy
Chaperone-mediated
Young
Glucagon
?
Lysosome
autophagosome
E. Bergamini
autophagolysosome
Lamp2a
Old
Autophagy Crosstalking
Susmita Kaushik
INTACT
9
BROKEN
Proteolysis (%)
8
7
6
CMA
5
4
3
2
1
0
wt
WT
ATG5(-/-)
wt
ATG5(-/-)
ATG5(-/-)
Lysosome
MACROAUTOPHAGY
Proteolytic cross-talking
TISSUE
TIME
CMA
Proteasome
MICROAUTOPHAGY
Lysosome
MACROAUTOPHAGY
The Faces of CMA
Ashish
Massey
Roberta
Kiffin
Judy
Zhang
Guy Sovak
Oxidative Stress
Blockage
of CMA
Lysosome
Proteome and
Aging
Lamp2a
Transgenic
Mouse
Anna Kim
CR
Marta Martinez
CMA and
Neurodegeneration
Urmi
Bandyopadhyay
Identification
New components
lysosome
Susmita
Kaushik
Cross-talking
Acknowledgements
COLLABORATORS
J. Fred Dice (Tufts University, MA)
CMA
David Sulzer, Serge Przedborski (Columbia U., NY)
Peter Lansbury (Harvard University, MA)
CMA and PD
Harry Ischiropoulos (U. Penn, PA)
Ralph Nixon (New York University, NY)
Noburu Mizushima (Tokyo MI, Japan)
FUNDING
NIH/NIA, NIH/NIDK
Howard Hughes Biomedical Institute
Ellison Medical Foundation
Huntington’s Disease Society of America
Autophagy and AD
Cross-talking