Autophagy (Autofagia)
Mohamed Elgendy MD, PhD
Autophagy (Autofagia)
Mohamed Elgendy MD, PhD
[email protected]
“Auto+phagy” Greek for “Self Eating”
Autophagy = Recycling
Types of Autophagy
• 1-Macro-autophagy
• 2-Micro-autophagy
• 3-Chaperon-mediated autophagy
Macro-autophagy
1-Induction
Lysosome
Autophagosome
Fusion
LC
3
LC3 II PE
LC
3
LC
3
Phagophore
2-Nucleation
Autolysosome
LC3 I
3-Maturation
Other types of Autophagy
• Macro-autophagy
Delivery of cytoplasmic cargo to the lysosome through the intermediary of a
double membranebound vesicle, referred to as an autophagosome, that
fuses with the lysosome to form an autolysosome.
• Micro-autophagy
Cytosolic components are directly taken up by the lysosome itself through
invagination of the lysosomal membrane
• Chaperon-mediated autophagy
Targeted proteins are translocated across the lysosomal membrane in a
complex with chaperone proteins (such as Hsc-70) that are recognized by
the lysosomal membrane receptor LAMP-2A, resulting in their unfolding and
degradation
Roles of autophagy
• Housekeeping roles
Removal of misfolded or aggregated proteins, clearing damaged organelles,
such as mitochondria and ER.
• Host-defense mechanism
Degradation of intracellular pathogens
• Role during embryonic development
Balancing sources of energy at critical times
• As a component of cellular integrated stress
responses.
Regulation of Autophagy
1-Induction
Lysosome
Autophagosome
LC
3
LC
3
Phagophore
Fusion
Autolysosome
2-Nucleation
3-Maturation
Regulation of Autophagy
Starvation
1-Induction
mTOR
Rapamycin
Promotes autophagy
Inhibits autophagy
AMPK
Rheb
TSC 2
Low Energy
Akt
PTEN
Regulation of Autophagy
Starvation
1-Induction
mTOR
Rapamycin
Promotes autophagy
Inhibits autophagy
AMPK
Rheb
TSC 2
Low Energy
Akt
PTEN
Regulation of Autophagy
1-Induction
Lysosome
Autophagosome
LC
3
LC
3
Phagophore
Fusion
Autolysosome
2-Nucleation
3-Maturation
Regulation of Autophagy
2-Nucleation
Autophagosome
LC
3
Phagophore
LC3 II
Atg 7
Atg 12
Atg 7
Atg 5
Atg 4
Atg 16
Atg 3
Atg 10
Conjugation Systems
LC3 I
Detection of Autophagy
1-Induction
Lysosome
Autophagosome
Fusion
LC
3
LC3 II PE
LC
3
LC
3
Phagophore
2-Nucleation
Autolysosome
LC3 I
3-Maturation
Regulation of Autophagy
-Relatively understudied
-Requires the small G protein Rab7 in its
GTP-bound state
Inactivation of LAMP-2 is the causative genetic
lesion associated with Danon disease in humans, an
X-linked condition that causes cardiomyocyte hypertrophy
and accumulation of autophagosomes in heart
muscle. Similar cardiac defects are observed in Lamp2-null mice, as well as skeletal abnormalities and
periodontitis associated with inflammation arising from a
failure to eliminate intracellular pathogens in the oral
mucosa
-Within the lysosome, cathepsin proteases B
and D are required for turnover of
autophagosomes
LC
3
Fusion
LC
3
-Requires Lamp-1 and Lamp-2 at the
lysosome
Lysosome
Autolysosome
3-Maturation
Detection of Autophagy
Electron Microscopy
Detection of Autophagy
Fluorescence microscopy
(increase in punctate LC3)
Detection of Autophagy
Fluorescence microscopy
(Acidotropic dyes)
Acridine Orange
Lysotracker Red
Detection of Autophagy
Immunoblotting
Detection of Autophagy
Other methods
-mTOR kinase activity
(Kinase assays western blot or immunoprecipitation)
-Transcriptional regulation of autophagy genes
(qRT-PCR or Northern blot)
Detection of Autophagy
Flux measurement
Klionsky et al., Autophagy 2008
Crosstalk between autophagy and
other cellular processes
Autophagy and Apoptosis
- Autophagy as an alternative mode of programmed
cell death :
Apoptosis PCD type I
Autophagic cell death PCD type II
Autophagy : a matter of life or death?
Life
Death
-Starvation
-Role in development
(e.g Atg5 KO, Atg7 KO)
(Regression of salivary glands in
Drosophila)
-Role in tumor suppression
Negative regulators :PI3K, Akt, Bcl-2
Positive regulators: Beclin-1, UVRAD,
Bif1, PTEN.
-Autophagy inducers as
anticancer agents :
e.g. Arsenic Trioxide, Temozolomide,
SAHA, Ceramide, Obatoclax,
Rapamycin
• Brief and mild vs prolonged and excessive
• Mechanistically and functionally different autophagy
programs
-Selective (specific) vs non-selective (non-specific)
- Atg5-,Atg7-indepdent , Beclin-1-depedenet autophagy (nature 2009)
-The magnitude of Beclin-1 induction:
mild  survival
strong death
• Intactness of apoptotic machinery
Bcl-2 family links apoptosis to
autophagy
Stress/Damage DNA damage Oncogenic
Growth Factor
stress
Decision
Proapoptotic
Noxa
Puma
Bmf
Depreivation
Bim
Bad
Bcl-2 family
Mcl-1
Antiapoptotic
Death/Survival
A1
Bcl-2
Bcl-xL
Bcl-B
Regulation of Autophagy
Starvation
1-Induction
mTOR
Rapamycin
Promotes autophagy
Inhibits autophagy
AMPK
Rheb
TSC 2
Low Energy
Akt
PTEN
Bcl-2 family as regulators of autophagy
Mcl-1
Bcl-X
Bcl-2
pLC3-GFP
BH3
Beclin-1
Autophagy
Beclin-1 Links apoptosis to autophagy
• Interaction with the Bcl-2 family proteins
• Cleavage by caspases
Kroemer et al., Mol. Cell 2010
Autophagy and senescence
-Autophagy mediates oncogene-induced senescence
Young et al., Genes and Dev 2009
-Senescent cells may also die by autophagy
Autophagy and ER stress
Segredi et al., EMBO reports 2006
Autophagy and ER stress
• The role of ATF4 in transcriptional regulation of
autophagy genes LC3 and Beclin-1.
• Crosstalk between autophagy and unfolded
protein response (UPR).
• Autophagy counterbalances ER expansion.
• ER as a source of phagophore membrane
biogenesis
Autophgay and DNA damage response
-Autophagy induced by HDACi counteracts DNA
damage response
-Autophagy may promote genomic stability by
clearing damaged mitochondria
Autophagy in health and disease
•
Cardiovascular and ischemic diseases
Starvation during ischemia
•
Neurodegenrative diseases
Intracellular aggregate accumulation plays a particularly significant role in the
aetiology of neurodegenerative diseases, including dementia, Alzheimer’s,
Huntington’s and Parkinson’s
For example, polyglutamine-expansion repeats, as seen in mutant huntingtin
(Huntington’s disease), mutant forms of α-synuclein (familial Parkinson’s disease)
and different forms of tau (Alzheimer’s disease) are dependent on autophagy for their
clearance from neurons.
Neuronal-specific inactivation of the key autophagy genes Atg5 or Atg7 results in
intracellular aggregate accumulation and neurodegeneration in mice
•
Cancer
Autophagy and Cancer
A friend or Enemy?
• Autophagy regulators as tumor suppressors
or oncogenes
Morselli et al. / Biochimica et Biophysica Acta 2009
Morselli et al. / Biochimica et Biophysica Acta 2009
Targeting autophagy for therapeutic intervention
Roy et al., Semin Immunopathol 2010
Autophagy and Cancer
A friend or Enemy?
• Brief specific versus prolonged nonspecific autophagy?
• Different roles during different stages of
tumorigenesis?
Autophagy and immunity
• Infection induces autophagy in many cases.
• Autophagy induction during infection is regulated by
cytokines (e.g IFN gamma) and pathogen recognition
receptors (PRRs) that recognize conserved components
of pathogens or products of their replication(PAMPs) and
DAMPs (e.g. products of necrotic cells, abnormal
reactive oxygen species, misfolded proteins)
• The transcription factor NF-kB and some of its upstream
regulators function to integrate diverse stress signals
including immune signals with the autophagy pathway
Autophagy and aging
• Autophagy promotes longevity
Rubinsztein et al., Cell 2011
Thank you (Grazie)
Questions?
[email protected]