Autophagy and Disease

Question 1

What happens when a cellular component/organelle becomes damaged?

Answer 1

Eliminate or repair it

Question 2

How does a cell eliminate an organelle?

Answer 2

Secretes it out of the cell

Proteasomal degradation

Lysosomal degradation

Question 3

Where does proteasomal degredation occur and what does it degrade?

Answer 3

Only proteins degraded in the CYTOSOL

Question 4

What are 3 lysosomal degradation pathways?

Answer 4

Autophagy = degrades intracellular components

Endocytosis = degrades components from cell surface

Phago/pinocytosis = degrades extracellular components

Question 5

How is a autolysosome formed?

Answer 5

Phagophore develops into autophagosome

Autophagosome and lysosome fuse to make an autolysosome

Question 6

Which stage of autolysosome formation has a DOUBLE-membrane?

Answer 6

Autophagosome

Question 7

What does ATG1 gene code for?

Answer 7

ULK1

Question 8

What is autophagy capable of degrading?

Answer 8

Protein
Lipid
Carbohydrate
Nucleic acid = whole organelles

Question 9

What forms of stress induce autophagy?

Answer 9

Nutrient derprivation

ER stress

Oxidative stress

Question 10

What are the 3 complexes needed to initiate autophagy?

Answer 10

Protein kinase ULK1

Lipid kinase VPS34

Ubiquitin-like system

Question 11

What is required for ULK activity and localization to phagophore?

Answer 11

ATG13
ATG101
FIP200

Question 12

What is required for VPS34 activity and localization to phagophore?

Answer 12

ATG14
VPS15
Beclin1

Question 13

What does ULK1 phosphoylate?

Answer 13

Beclin1, ATG14 and VPS34

Question 14

What are the two Ubiquin-like elements?

Answer 14

ATG12
ATG8

Question 15

For ubiquitin-like element ATG12 what are the E1, E2, and E3?

Answer 15

E1 = Atg7
E2 = ATG10
E3 = ?

Question 16

For ubiquitin-like element ATG8 what are the E1, E2, and E3?

Answer 16

E1 = Atg7
E2 = Atg3
E3 = Atg12/5

Question 17

What amino acid does Ub bind?

Answer 17

Glycine

Question 18

What amino acid does E2 bind?

Answer 18

Cysteine

Question 19

What proteins are most commonly mutated in Parkinson’s?

Answer 19

LRRK2

PINK1

Parkin

Question 20

What ATG subfamilies help the phagophore membrane grow?

Answer 20

Different ATG8s may have distinct roles for elongation and fusion

ATG8 subfamilies = LC3 and GATE-16/GABARAP

Question 21

What mutations in LRRK2 cause Parkinson’s?

Answer 21

LRRK2 is a kinase

Mutation of G2019S cause increased kinase activity

The increased kinase activity of LRRK2 interferes with the proper recruitment of autophagy-related proteins (such as PINK1, Parkin, and others) to damaged mitochondria.
This disrupts the signaling needed to initiate mitophagy.

The accumulation of defective mitochondria results in increased oxidative stress and cellular dysfunction, contributing to the neurodegeneration seen in Parkinson’s disease.

Question 22

What are potential Parkinson’s therapies to improve mitophagy?

Answer 22

LRRK2 inhibitor

PINK1 activator

UPS30 inhibitor = a mitochondrial deubiquitylating enzyme that negatively regulates mitophagy—the process by which cells remove damaged mitochondria. By inhibiting USP30, MTX325 aims to enhance mitophagy, thereby improving mitochondrial quality and function.

Question 23

What is the source of the autophagosomal membrane?***

Answer 23

Endoplasmic reticulum

Question 24

What is DFCP1?

Answer 24

PI3P binding protein that marks the site of autophagosome formation

Question 25

What does DFCP1 mark?

Answer 25

The omegasome

Question 26

What is the omegasome?

Answer 26

The omegasome is a membrane structure that serves as a scaffold for the formation of autophagosomes, crucial for autophagy. It has an omega-like shape and is enriched with phosphatidylinositol 3-phosphate (PI(3)P), a lipid produced by the Vps34 complex. DFCP1 marks the omegasome by binding to PI(3)P through its FYVE domains. This binding recruits other autophagy-related proteins (like WIPI) to the omegasome, enabling the membrane to curve and expand around damaged cellular material. The omegasome forms near the endoplasmic reticulum (ER), which provides lipids and scaffolding necessary for autophagosome formation. The omegasome thus acts as a scaffold, allowing the phagophore to emerge, which eventually matures into an autophagosome that engulfs and degrades unwanted cellular components.

Question 27

Can autophagy be selective and if so how?

Answer 27

Yes it can via Sequestosome-like Receptors (SLR)

Question 28

Name some sequestosome-like receptors

Answer 28

p62 OPTINEURIN Tax1BP1 NDP52 NBR1

Question 29

What happens with loss of Beclin1?

Answer 29

Promotes tumourigenesis

Question 30

Why is autophagy a double-edged sword?

Answer 30

Can also be applied to tumour cell survival Blocking autophagy in TUMOURS is a good thing but only in tumours

Question 31

What is mitophagy?

Answer 31

Autophagy of mitochondria

Question 32

What are PINk1 and Parkin?

Answer 32

Protein kinase = PINK1 E3 ligase = Parking

Question 33

What happens to PINK1 and Parkin when mutated in Parkinson's?

Answer 33

PINK1 is a mitochondrial kinase that normally accumulates on the outer mitochondrial membrane when mitochondria are damaged. It acts as a signal to recruit Parkin, an E3 ubiquitin ligase, to the damaged mitochondria. Mutations in PINK1 impair its ability to recognize and bind to damaged mitochondria, preventing the initiation of mitophagy. As a result, damaged mitochondria accumulate, leading to oxidative stress and cellular damage, contributing to Parkinson's symptoms. Parkin is normally recruited by PINK1 to damaged mitochondria, where it ubiquitinates mitochondrial proteins, marking them for degradation. Mutations in Parkin prevent it from effectively tagging damaged mitochondria for removal. This leads to defective mitophagy, allowing damaged mitochondria to persist, further increasing cellular stress and promoting neurodegeneration.

Question 34

What causes parkinsonism in humans and mitochondrial dysfunction in model organisms?

Answer 34

Loss-of-function mutations in PINK1 and Parkin cause parkinsonism and mitochondrial dysfunction.

Question 35

What is Parkin’s role in relation to damaged mitochondria?

Answer 35

Parkin (Ub E3 ligase) is recruited from the cytosol to damaged mitochondria to trigger their autophagy.

Question 36

What is currently unknown about Parkin's recruitment to damaged mitochondria?

Answer 36

How Parkin recognizes damaged mitochondria is unknown.

Question 37

How is the expression of PINK1 regulated on mitochondria?

Answer 37

The expression of PINK1 on individual mitochondria is regulated by voltage-dependent proteolysis, which maintains low levels on healthy mitochondria but facilitates rapid accumulation on damaged ones.

Question 38

What happens when PINK1 accumulates on mitochondria?

Answer 38

The accumulation of PINK1 on mitochondria is both necessary and sufficient for the recruitment of Parkin to mitochondria.

Question 39

What is mitophagy critical for maintaining?

Answer 39

Mitophagy is the selective removal of damaged mitochondria, which is critical for maintaining neuronal homeostasis.

Question 40

What disease is mitophagy mutation associated with?

Answer 40

Mutations in proteins involved in mitophagy cause neurodegenerative diseases, suggesting defective mitochondrial turnover contributes to neurodegeneration.

Question 41

What is the localization pattern of mitophagy events in neurons?

Answer 41

The localization of mitophagy events had a robust somal bias, meaning they were predominantly occurring near the cell body.

Question 42

In primary rat hippocampal neurons, we developed a mitophagy induction paradigm where mild oxidative stress induced low levels of mitochondrial damage, how did mitophagy react compared to basal conditions?

Answer 42

Mild oxidative stress is applied to the neurons, which causes low levels of mitochondrial damage. As a result, damaged mitochondria trigger the mitophagy process, but the key difference here is that the lysosomal fusion and acidification (the degradation step) happens more slowly. The autophagosomes efficiently engulf the damaged mitochondria, but they stay in an undigested state (non-acidified) for hours to days before proper degradation happens.

Question 43

What is optineurin?

Answer 43

A scaffold protein that helps in recruiting other proteins (like Parkin) to damaged mitochondria. It helps mediate the signaling between damaged mitochondria and the cell's cleanup machinery, playing a crucial role in Parkin activation for mitophagy. Optineuin-mediated mitophagy occurs in NEURONES (optineurine is also found in other tissues)

Question 44

When is PINK1/Parkin activated?

Answer 44

Does not normally regulate a majority of mitophagy under normal (basal) conditions It is a stress reponse pathway to activate mitophagy