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Autophagy
Charleen T. Chu, MD, PhD
Associate Professor of Pathology
University of Pittsburgh School of Medicine
Summer Academy 2010Molecular Mechanisms of Human Disease
Lecture Goals
Regulation of macroautophagy as a physiologic
response to stress
Dysregulation of autophagy in disease pathogenesis
A tale of two mitophagies: the importance of brakes
Lysosomal Garbage Disposal
Housekeeping functions
Expansion due to undigestible remnants
Lipofuscin - lipid peroxidation
Lysosomal storage diseases
Drug induced deficits - chloroquine
Oxidative stress, protein & organelle damage
Protein aggregates (+/- ubiquitin)
Pathogenic organisms
Destroy organism
Used by organism for life cycle/replication
Autophagy “Self-eating” by lysosomal degradation
Macroautophagy
Chaperone-mediated autophagy
Microautophagy
Basal turnover - long-lived proteins & organelles
Physiologic stress response Starvation - generate carbon sources and reduce
unneeded structures
Clearance of damaged constituents includingprotein aggregates
Defense against intracellular pathogens
A brief history of Macroautophagy
Christian de Duve
1955 - discovers lysosome (liver)
1963 - endocytosis and autophagy named
1973-1976 –autophagy in developmental and
chemotherapy-induced cell death
Ohsumi lab 1997 –clones first yeast autophagy-related gene 1 (Atg1)
1998 – first mammalian Atg homolog (Atg12)
2000 – ubiquitin-like conjugation of Atg12 and Atg8 (LC3)
2004 Rubinsztein -autophagy inhibits polyglutamine
aggregates in Huntington disease models
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Autophagy Papers in Scopus
Bcl2
Atg8/LC3
conjugation
Autophagy for the new millenium
Apoptosis took off
in the 1990s
Macroautophagy: Processes
Autolysosome
Fusion of
Amphisome/
Lysosome
Autophagosome
Nucleation
“W-some”Isolation membrane
Extension and closure
EM image from Florez-McClue, M et al J of Neurosci 2004, 24:4498-4509 with
permission from Society for Neuroscience © All Rights Reserved.
Ubiquitin fold proteins
Ubiquitin
http://en.wikipedia.org/wiki/File:Ubiquitin_cartoon-2-.png
Microtubule associated protein
1 light chain 3 (LC3)
C-terminus
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*
The core conjugation machinery
E1 Ubi activating enzyme
–Create high energy thiol ester bond
E2 Ubi conjugating enzymes (30-40)
–Transfers Ubi to substrate
E3 Ubi protein ligases (hundreds)
–Bring together substrate and E2
–May function as intermediate for transfer
Atg7
Atg3
E1-like:
E2-like: Atg10
E3-like:
Atg12-Atg5LC3Atg16L
LC3-Phosphatidylethanolamine
Canonical Pathway of Induction/Nucleation
mTOR
ULK (Atg1)-
Atg13-
FIP200 (Atg17)
Beclin1-
PIK3C (Vps34)
PI(3)P-rich
NucleationAtg16L
Atg12 -Atg5
Ubiquitin-like conjugations
LC3-PE
mTOR – mammalian target of rapamycin; ULK - UNC-51-like kinase; FIP2-- - focal
adhesion kinase family interacting protein of 200 kD; PIK3C – class III
phosphoinositide 3-kinase
Signaling regulation of autophagy
Beclin1-
PIK3C
Beclin1-Bcl2
Atg4ROS
Insulin
PIK1C
Akt
mTOR
Rapamycin Amino acids
AMP Kinase
P-JNK
P-ERKP-MEKDagda R et al Autophagy
2008; 4(6): 770-782
Selective autophagyPexophagy, Mitophagy, Reticulophagy, Lipophagy, Aggregophagy, etc.
Bulk phase entrapment during starvation
Peroxisomes in yeast; mitochondria in red
blood cell and lens maturation
Yeast specific genes for pexophagy, mitophagy
Pathologic situations
Mitochondrial clearance elicited by apoptotic
stimuli (Tolkovsky)
Degree of depolarization as signal for mitophagy
vs apoptosis (Lemasters)
Clearance of impaired mitochondria protective in
PINK1 deficiency (Chu)
Mechanisms for selectivity
Trafficking hypothesis (Kopito)
Damaged organelles/protein aggregates are targeted
to perinuclear microtubule assembly areas
(aggresomes) – apparent selectivity by enrichment
Receptors for mammalian mitophagy
Nix for erythrocytes, BNIP in hypoxia (Zhang, Dikic)
Specific adapter proteins discovered that
bridge LC3 and ubiquitinated cargo
p62, NBR1 (Johansen, Komatsu)
Recruitment of the parkin ubiquitin ligase to
depolarized mitochondria (Youle)
Role(s) of autophagy
Autophagic cell death?Tissue regression/remodeling
Gatekeeper role upstream of apoptosis
Excessive degradation in neurodegeneration?
•Prolongs cell survival during starvation
•Tumor cell resistance to chemotherapy
•Sequester mitochondrial death factors
•Clear protein aggregates
•Clear damaged mitochondria
EM of dying cells
Cell with
organelle swellingApoptotic cells –
committed &
beyond rescue
Autophagic
morphology – failed
adaptation vs.
suicide?Injured, but
rescuable?
Beyond rescue
Image courtesy of Donna Beer Stolz
Type 1 Type 2 Type 3
Potential role(s) of autophagy in cell death
Pro-survival - death from failed compensation.
Atrophic response to limiting resources.
Sequester damaged mitochondria
Pro-death
Gatekeeper role upstream of apoptosis
Mechanism of controlling death-related debris?
Alternative executionary system?
Context dependent, “autophagic stress”
Excessive or imbalanced induction/clearance
Most stress responses lead to repair or cell suicide
Correlation ≠ Causation
Dysregulation of Autophagy
Insufficient autophagy
“Excessive” autophagy Anabolic-Catabolic Imbalance
Induction Completion
Autophagic stress
Chu, C. Autophagic Stress in Neuronal Injury and Disease.
J. Neuropathol Exp Neurol. Vol. 65(5):423-432
Lippincott Williams & Wilkins © 2006
Insufficient autophagy
Rapamycin reduces aggregates and
improves Huntington’s models (Rubinsztein)
Caveat: rapamycin regulates protein synthesis and Akt signaling
Conditional mouse autophagy
deficiency: Atg7 KO (Komatsu); Atg5 KO (Mizushima)
Protein aggregates in brain and liver
Diminished beclin 1 expression in
Alzheimer’s, Huntington’s and aging (Wyss-Coray, Yuan)
Autophagic Stress
Chloroquine or bafilomycin treatment
toxic to cells experimentally
Lysosomal storage diseases
Glycogen storage diseases
Mucolipidosis
Autophagy induction by starvation OK
Clearance after refeeding impaired (J Biol Chem, 281, 39041-50, supplement)
Acute MPP+ complex I inhibition
Increased mitochondrial turnover overall,
but induction exceeds clearance
“Excessive” Autophagy
Bcl2 binding of beclin 1 inhibits its
autophagy-promoting role
Rheostat to prevent overactivation of
autophagy (Levine)
But what determines “excessive”
Cell-type specific threshold
Intrinsic capacity for lysosomal fxn?
Context of other injury/aging effects
Redox impairment of nuclear import and
transcription (J Neuropathol Exp Neurol, 66, 873-83)
Cherra, S et al Future Neurology 2008 3:309-323
with permission from Future Medicine.
© All Rights Reserved.
Parkinson’s Disease
~ 1 million people in North America
Neurodegenerative movement disorder
Symptomatic therapy eventually stops working
Dopaminergic
SNc neurons
A Tale of Two Mitophagies
PTEN-induced kinase 1 (PINK1) 2004: Mutated in PARK6 (1p36) (Valente...Wood)
Early onset, L-Dopa responsive (Bonifati et al 2005)
N-terminal mitochondrial targeting sequence
1-Methyl-4-phenylpyridinium (MPP+) 1982: MPTP contaminant in synthetic heroin
Inhibitor of mitochondria complex I
Autophagy imbalance in disease
Not just a question of on or off...
Stable PINK1 lines
A series shRNA
D series shRNA
V series nontargetingA D
Modeling PINK1 loss of function
RNAi resistant
PINK1 plasmid
PINK1 deficient cells show
mitochondrial dysfunction
Ruben Dagda
Aaron Gusdon
Seahorse XF24 Extracellular Flux Analyzer,
A Gusdon, RK Dagda & CT Chu,
unpublished data
Dagda, RK et al J Biol Chem Vol. 284
No. 3:13843-13855 Copyright © 2009, by
the American Society for Biochemistry and
Molecular Biology
Autophagosome Markers
Isolation membrane Autophagosome Autolysosome
Lysosome
LC3-I (Atg 8), diffuse cytoplasmic pool
LC3-II puncta, membrane conjugated
LC3-ILC3-II
+ - Food
CT Chu, modified from N Mizushima
Bafilomycin
PINK1 LOF induces autophagy and
mitochondrial degradation
Ruben Dagda
Dagda, RK et al J Biol Chem Vol. 284 No. 3:13843-13855 Copyright © 2009,
by the American Society for Biochemistry and Molecular Biology
CT Chu, unpublished data
Inhibiting Autophagy in PINK1 shRNA lines
LC3
Atg3
Ubiquitin-like conjugation
Atg7
Beclin1-
PIK3C (Vps34)
Atg10
Atg12 -Atg5
Dagda, RK et al J Biol Chem Vol. 284 No. 3:13843-
13855 Copyright © 2009, by the American Society
for Biochemistry and Molecular Biology
SJ Cherra and CT Chu,
unpublished data
Autophagy reduces cell death in PINK1
deficient cells
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V17 PINK1 shRNA
D14Cell D
eath
(%
tra
nsfe
cte
d c
ells)
Ctrl siRNA
Atg7 siRNA
Propidium iodide cell death assay
*
†
Dagda, RK et al J Biol Chem Vol. 284 No. 3:13843-13855 Copyright ©
2009, by the American Society for Biochemistry and Molecular Biology
PINK1 loss >>
Mitochondrial Dysfunction & ROS
Fission & Autophagy sequester &
clear damaged mitochondria,
reducing cell death
The MPP+ model
Targets mitochondrial complex I.
Causes an active form of AIF-mediated, caspase-
independent cell death.
TH (red) MAP2 (green)
CTL 5 mM MPP+
Jianhui Zhu
J Zhu and CT Chu, unpublished images
Chu, C et al J. Neurochem 2005, 94: 1685-169
Zhu J et al Am J Pathol 2007, 170:75-86
MPP+ N+ CH3
MPP+ induced autophagy and
mitochondrial degradationAcute MPP+
(acute model)
LC3- Autophagosomes
PDH
Calnexin
Cytp450R
Ctrl 250 500 250 5001 wk 2 wk
Chronic MPP+, RA differentiated
60 kD complex IV protein
Jianhui Zhu
Zhu J et al Am J Pathol
2007, 170:75-86 with
permission from the
American Society for
Investigative Pathology
J Zhu & CT Chu, unpublished data
Autophagy contributes to
MPP+ toxicity
SH-SY5Y cells
RA-differentiated SH-SY5Y cellsSam Cherra
Jianhui Zhu
Zhu J et al Am J Pathol 2007, 170:75-86
with permission from the American Society
for Investigative Pathology
SJ Cherra & CT Chu, unpublished data
Pathologic Mitophagy?
Autophagy of damaged mitochondria viewed
as pro-survival mechanism
Neuronal cells may be hyper-sensitive to
excess mitochondrial degradation (Tolkovsky 2002
Biochimie 84: 233)
SNc - low mitochondrial mass (Liang 2007 Exp Neurol 203: 370)
Differences in regulation of “physiologic” vs.
“pathologic” autophagy/mitophagy?
Inhibitors of beclin 1/PI3K signaling
But, inhibitors of MAPK/ERK Kinase did...
1° TH neurons
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Vehicle PD 3-MA WT
TH
neu
ron
s w
ith
gra
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lar
LC
3
(fo
ld in
du
cti
on
)
CtrlMPP+
**
SH-SY5Y cells
did not inhibit MPP+ induced AVsJianhui Zhu
Zhu J et al Am J Pathol 2007, 170:75-86 with permission from the American Society for Investigative Pathology
Inhibition of MAPK/ERK kinase (MEK)
prevents MPP+ -induced autophagic death
SH-SY5Y cells
1° TH neurons
Mitochondrial injury
Where do MAPK/ERK
kinase inhibitors act?
MPP+
Mitophagy
Cell death?
or failed adaptation?
Zhu J et al Am J Pathol 2007, 170:75-86 with permission from the American Society for Investigative Pathology
MEK inhibitors reduce mitophagy, downstream
of mitochondrial injury in acute MPP+ toxicity...
Control cells MPP+, 48 h
MPP+ UO, 48 hMEKi UO126 and PD98059
Zhu J et al Am J Pathol 2007, 170:75-86 with permission from the American Society for Investigative Pathology
Is ERK sufficient to drive mitophagy?
Fold Elk-1 Reporter Activity
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PCMV5.0 ERK2-WT ERK2-CA MEK-CA
Ruben Dagda
Dagda R et al Autophagy 2008;
4(6): 770-782 Copyright ©
2008 Landes Bioscience
GFP-LC3 MTR
Effects of ERK2 overexpression on Mitophagy
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PCMV5.0 ERK2-WT ERK2-CA
% o
f G
FP
-LC
3 p
un
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co
locali
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wit
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ito
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dri
a'
Pathologic Autophagy: A Role for Brakes?
Beclin1-
PIK3C (Vps34)
PI(3)P-rich
LC3-PE
Beclin1-
Bcl2
1. Competition for by Bcl2 -
preventing “overactivation”Pattingre...Levine 2005 Runaway car??
2. Jumpy
dephosphorylates PI(3)P -
acting as “brakes”Vergne... Deretic 2009
PINK1 deficiency MPP+
Conclusions
As with any essential process, either too
little or too much autophagy is harmful.
Research Frontiers include:
Harnessing selective autophagy mechanisms
Coordination of autophagy and regenerative
biosynthesis
Jianhui (Jeffrey) Zhu, MD, PhD
Edward Plowey, MD, PhD
Ruben Dagda, PhD
Jason Callio
Salvatore (Sam) Cherra III
Vivek Patel
Gaelle Guilloux-Douillard. PhD
Aaron Gusdon
Irene Pien
Scott Kulich, MD, PhD, VAMC
Craig Horbinski, MD, PhD, U Kentucky
Liz Chalovich, PhD, Wheeling U
Former Trainees
Chu Lab: Cell Biology of Parkinsonian Neurodegeneration
NIH (R01 AG026389 and -03W1;
P01 NS059806; R56 NS065789)
The Ellison Medical Foundation
(AFAR Julie Martin Mid-Career Award
in Aging Research)