Lecture layout

• Part 1 : Autophagy definition, functions, regulation and methods of detection

• Part 2: The crosstalk between autophagy and other cellular processes

• Part 3: Autophagy in health and disease

Part 1 Autophagy definition (what?),

functions (why?) , regulation (how?)

and methods of detection (how we see it?)

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

Oxidative, replicative, oncogenic stress, ..etc

Damaged organelles (e.g mitochondria) and misfolded proteins

Autophagy

Degradation and recycling

Recycled materials

Amino acids and energy

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?)

LC3 I

LC3 II PE

Fusion

Lysosome

Autolysosome

Phagophore Autophagosome

1-Induction

2-Nucleation

3-Maturation

Macro-autophagy

Fusion

Lysosome

Autolysosome

Phagophore Autophagosome

1-Induction

2-Nucleation 3-Maturation

Regulation of Autophagy

1-Induction (selective)

Regulation of Autophagy

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

?

1-Induction (non selective)

Regulation of Autophagy

mTOR

Promotes autophagy Inhibits autophagy

Rheb TSC 2 Akt PTEN

AMPK Starvation Low Energy

Rapamycin

LKB1 Nutrients

ERK Ras MEK

ULK1 Beclin 1 (Atg 6)

ATG13

ATG101

FIP200

ATG14L

Vps34

Ambra1

Bif 1

UVRAG

Bcl-2/xL

Rubicon

Fusion

Lysosome

Autolysosome

Phagophore Autophagosome

1-Induction

2-Nucleation 3-Maturation

Regulation of Autophagy

Regulation of Autophagy

Phagophore Autophagosome

2-Nucleation

Atg 12

Atg 5

Atg 16

Atg 7

Atg 4

Atg 3 LC3 I

LC3 II

Atg 10

Atg 7

Conjugation Systems

Fusion

Lysosome

Autolysosome

Phagophore Autophagosome

1-Induction

2-Nucleation

3-Maturation

Detection of Autophagy

Fusion

Lysosome

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 Lamp- 2-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

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?)

LC3 I

LC3 II PE

Fusion

Lysosome

Autolysosome

Phagophore Autophagosome

1-Induction

2-Nucleation

3-Maturation

Macro-autophagy

Fluorescence microscopy (increase in punctate LC3

Detection of Autophagy

Immunoblotting Detection of Autophagy

LC3 I

LC3 II PE

Fusion

Lysosome

Autolysosome

Phagophore Autophagosome

1-Induction

2-Nucleation

3-Maturation

Macro-autophagy

Fluorescence microscopy (Acidotropic dyes)

Detection of Autophagy

Acridine Orange

Lysotracker Red

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?

-Starvation (e.g Atg5 KO, Atg7 KO)

-Role in development (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

Life Death

• 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 Æ survival strongÆ death

• Intactness of apoptotic machinery

Cells dying by apoptosis

Cells dying by autophagy

Bcl-2 family links apoptosis to autophagy

Beclin-1 Links apoptosis to autophagy

• Interaction with the Bcl-2 family proteins

• Cleavage by caspases

Mcl-1 Bcl-X Bcl-2

Beclin-1

Autophagy

BH3

Bcl-2 family as regulators of autophagy

pLC3-GFP

Kroemer et al., Mol. Cell 2010

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

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

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