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

Types of autophagy

?Endosomal/MVB entry?

Schematic diagram by SJ Cherra & CT Chu

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

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Ctrl siRNA

Atg7 siRNA

Propidium iodide cell death assay

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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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CtrlMPP+

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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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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)