Autophagy (Autofagia)

Autophagy (Autofagia)
Mohamed Elgendy MD, PhD
[email protected]
Lecture layout
• Part 1 : Autophagy definition, functions,
regulation and methods of detection
• Short Break 
• Part 2: The crosstalk between autophagy and
other cellular processes
• Short Break 
• Part 3: Autophagy in health and disease
Research on Autophagy
Part 1
Autophagy definition (what?),
functions (why?)
, regulation (how?)
and methods of detection (how we see it?)
“Auto+phagy” Greek for “Self Eating”
Why it could be a good idea for a cell to
eat itself?
Autophagy Definition
• Autophagy is a recycling process by which
cytoplasmic components are sequestered in
double membrane vesicles and degraded
upon fusion with lysosomal compartments
Autophagy = Recycling
Cellar damage/stress
Autophagy
Recycled materials
Oxidative, replicative,
oncogenic stress, ..etc
Degradation and recycling
Amino acids and energy
Damaged organelles (e.g
mitochondria) and
misfolded proteins
Types of Autophagy
• 1-Macro-autophagy
• 2-Micro-autophagy
• 3-Chaperon-mediated autophagy
Part 1
Autophagy definition (what?),
functions (why?),
regulation (how?),
and methods of detection (how we see it?)
Cellular functions 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.
Part 1
Autophagy definition (what?),
functions (why?)
, regulation (how?)
and methods of detection (how we see it?)
Macro-autophagy
1-Induction
Lysosome
Phagophore
Autophagosome
Fusion
LC3 II PE
2-Nucleation
Autolysosome
LC3 I
3-Maturation
Regulation of Autophagy
1-Induction
Lysosome
Phagophore
Autophagosome
Fusion
Autolysosome
2-Nucleation
3-Maturation
Regulation of Autophagy
1-Induction
(selective)
?
Phagophore
Autophagosome
Acetylation targets mutant huntingtin to autophagosomes for degradation
Cell, 137 (2009), pp. 60–72
Mature ribosomes are selectively degraded upon starvation by an
autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease
Nat Cell Biol, 10 (2008), pp. 602–610
Regulation of Autophagy
1-Induction
(non selective)
mTOR
Nutrients
Rheb
Rapamycin
ATG13
ULK1
FIP200
Bif 1
Ambra1
ATG101
Promotes autophagy
Inhibits autophagy
AMPK
LKB1
TSC 2
Akt
PTEN
ERK
MEK
Ras
ATG14L
Beclin 1
(Atg 6)
Vps34
UVRAG
Starvation
Low Energy
Bcl-2/xL
Rubicon
Regulation of Autophagy
1-Induction
Lysosome
Phagophore
Autophagosome
Fusion
Autolysosome
2-Nucleation
3-Maturation
Regulation of Autophagy
2-Nucleation
Autophagosome
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
Phagophore
Autophagosome
Fusion
2-Nucleation
Autolysosome
3-Maturation
Regulation of Autophagy
-Relatively understudied
-Requires the small G protein Rab7 in its
GTP-bound state
-Requires Lamp-1 and Lamp-2 at the
lysosome
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
Lysosome
Fusion
Autolysosome
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
Part 1
Autophagy definition (what?),
functions (why?)
, regulation (how?)
and methods of detection (how we see it?)
Detection of Autophagy
1-Induction
Lysosome
Phagophore
Autophagosome
Fusion
2-Nucleation
Autolysosome
3-Maturation
Detection of Autophagy
Electron Microscopy
Macro-autophagy
1-Induction
Lysosome
Phagophore
Autophagosome
Fusion
LC3 II PE
2-Nucleation
Autolysosome
LC3 I
3-Maturation
Detection of Autophagy
Fluorescence microscopy
(increase in punctate LC3
Detection of Autophagy
Fluorescence microscopy
(increase in punctate LC3)
Detection of Autophagy
Immunoblotting
Macro-autophagy
1-Induction
Lysosome
Phagophore
Autophagosome
Fusion
LC3 II PE
2-Nucleation
Autolysosome
LC3 I
3-Maturation
Detection of Autophagy
Fluorescence microscopy
(Acidotropic dyes)
Acridine Orange
Lysotracker Red
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
Part 2
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
Cells dying by apoptosis
Cells dying by autophagy
Annexin V/ Propodium Iodide
Bcl-2 family links apoptosis to autophagy
Mcl-1
Bcl-2 Family
Bax
Bcl-2
Bak
Bcl-xL
Noxa
Bcl-B
Puma
Bcl-W
Bid
A1
Bad
LIFE
DEATH
Bax
Mcl-1
Bak
Bcl-2
Noxa
Bcl-xL
Puma
Bcl-B
Bid
Bcl-W
Bad
A1
LIFE
DEATH
Bax
Mcl-1
Bak
Bcl-2
Noxa
Bcl-xL
Puma
Bcl-B
Bid
Bcl-W
Bad
A1
LIFE
DEATH
Bax
Mcl-1
Bak
Bcl-2
Noxa
Bcl-xL
Puma
Bcl-B
Bid
Bcl-W
Bad
A1
LIFE
DEATH
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
Mcl-1 …a unique member of the Bcl-2 family
PEST
BH4
BH3
BH1
BH2
TM
Mcl-1
Bcl-2
Bcl-xL
Bcl-W
A1
Bcl-B
Mcl-1 …a unique member of the Bcl-2 family
Upregulated in several tumors
Contributes to chemo-resistance and relapse
 Hanahan & Weinberg. Cell 2000
Mcl-1
A unique member of the Bcl-2 family
Contribution to Tumorigenesis
Upregulated in several tumors
Contributes to chemo-resistance and relapse
 Hanahan & Weinberg. Cell 2000
 Hanahan & Weinberg. Cell 2011
Mcl-1
Beclin 1
Mcl-1
Beclin 1
Mcl-1 & Beclin 1 compete for binding USP9X
USP9X
N1
N2
C1
Mcl-1
C2
Beclin 1
Mcl-1/Beclin 1 axis correlates with melanoma
progression in patients
Elgendy et al., Nature Comm. 2014
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
Part 3
Autophagy in health and disease
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?
• Genome integrity (removal of DNA damaging
materials).
• Brief specific versus prolonged non-specific
autophagy?
• Different roles during different stages of
tumorigenesis?
• Mediating response/resistance to several
chemotherapeutics
Regulation of Autophagy
Starvation
1-Induction
mTOR
Rapamycin
Promotes autophagy
Inhibits autophagy
AMPK
Rheb
TSC 2
Low Energy
Akt
PTEN
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 for attention
Questions?
[email protected]