autophagy

Question 1

Learning Objectives for Autophagy

Answer 1

To introduce the process of autophagy and why it is important.

Learning Objectives
● Understand the basic concept of autophagy
● Understand the need for regulation
● Understand the physiological roles of autophagy
● Understand how yeast genetics can be used as a model system to study
autophagy

To provide an overview of autophagy in disease

Learning Objectives
● Understand the involvement of autophagy in cancer.
● Understand the involvement of autophagy in neurodegenerative diseases such as Huntington’s and Parkinson’s disease.
● Understand the potential and limitations of autophagy-based therapies.

Question 2

For simple understanding, what is autophagy?

Answer 2

A process to digest intracellular material.

• vesicles are made in the cytosol that capture components of the cytosol such as organelles or proteins. Ultimately these are isolated from the rest of the cytosol, fusion with lysosomes occurs and the components are degraded

Question 3

What are the 6 steps of the process of autophagy?

Answer 3

1: Initiation - Autophagy is initiated in response to damaged organelles or cellular stressors such as nutrient deprivation or oxidative stress

2: Nucleation: Phagophore (isolation membrane) begins to form which involves recruitment of Beclin1 protein. The phagophore expands and engulfs portions of the cytoplasm, such as damaged organelles or protein aggregates

3: Elongation: Phagophore elongates and closes forming a a double-membrane vesicle known as autophagosome

4: Maturation - Autophagosome undergoes maturation which enables it to fuse with lysosomes. this forms an autolysosome

5: Degradation: within the autolysosome the sequestered material is exposed to lysosomal enzymes leading to the breakdown of the sequestered material. The resulting breakdown products are recycled back into the cell

6. Termination - the process ends with the recycling of the autolysosome and release of recycled material back into the cytoplasm

Question 4

What physiological processes is autophagy needed for?

Answer 4

• removing damaged components e.g. from daily wear and tear
• Signalling
• Recycling nutrients
• Differentiation of cells/cellular remodelling
  -Killing intracellular pathogens

Question 4

List the key characteristics of the 3 protein degradation pathways - proteasome pathway, autophagy pathway, chaperone mediated autopghagy

Answer 4

Macroautophagy
- Lysosomal
- bulk digestion
- can remove whole organelles
- molecules released support metabolism

proteosome pathway:
- non lysosomal
- degrades individual proteins
- Major turnover route for short lived proteins

chaperone mediated autophagy/microautophagy
- lysosomal
- degrades individual proteins
- turns over specific long lived proteins

Question 5

What is the ONLY mechanism of degrading organelles?

Answer 5

Autophagy

Question 6

What cell type is autophagy needed to form?

Answer 6

Erythrocytes

Question 7

Yeast experiment for autophagy

Answer 7

• If you took yeast strains with deficient proteases that could not digest things in the vacuole efficiently and you starve them, you start to see accumulation of material
• It was speculated these were autophagosomes catching material in the cytosol, fusing with the vacuole but they can’t be digested hence the accumulation

Question 8

What do cells lacking autophagy accumulate?

Answer 8

Protein aggregates

Question 9

What causes the common phenotype of neurodegenerative diseases?

Answer 9

Accumulation of protein aggregates

Question 10

What is Huntington’s disease caused by?
What does effect does this have on the protein?

Answer 10

PolyQ in Huntingtin protein

PolyQ causes Huntingtin protein to misfold - the longer the polyQ region the more likely the protein will misfold

Question 11

Explain the mechanism of Huntington’s disease
4 points

Answer 11

• With polyQ Huntingtin, misfolding and aggregation can occur (this only becomes a problem when rate of misfolding Huntingtin proteins is higher than the capacity to degrade them)

-With age autophagy capacity decreases hence why Huntington’s is more prevalent with age

• Usually when aggregation of Huntingtin proteins occurs, they are ubiquitinated and some undergo proteasomal degradation and are digested
• If there are too many aggregates for proteosomal degradation, the aggregates are clumped together into one big aggresome which would normally targeted by autophagy for degradation

Question 12

List and explain mechanisms of toxicity in relation to Huntington’s disease
5 points

Answer 12

• One way could be that due to the misfolding, the protein loses its original function
• Could be the toxic oligomers - perhaps they damage or override the protein
• Could be the aggresomes
• Proteins that normally binds huntingtin could also be included in the aggregates and then degraded by autophagy

-this ultimately leads to loss of neuronal activity

Question 13

Simply, what causes parkinsons?
What type of neurones does parkinson normally affect?
Compare cases of Parkinson’s and Huntington’s in terms of what % of cases are familial

Answer 13

• Aggregated proteins in cytosol of cells - mainly alpha-syn aggregates
• Mitochondria accumulation
• Specifically affects dopaminergic neurones

In Huntington, most cases are familial. In Parkinson’s, only 5-10% of cases are familial

Question 14

What happens in chaperone mediated autophagy, generally speaking?

Answer 14

Individual proteins go through LAMP2 receptor on lysosomes and enter the lysosome for degradation

Question 15

What protein is the main cause of Parkinson’s? How common is it that a mutation in this causes Parkinson’s?

Answer 15

Aggregates of alpha-syn

rare - 5-10% of cases

Question 16

How does mutated alpha-syn contribute to the development of the Parkinson’s disease?

Answer 16

• Alpha syn is normally degraded by chaperone mediated autophagy. in mutated form, alpha syn blocks the pathway

-Alpha-syn mutation blocks the chaperone mediated pathway which in turn also blocks other proteins being taken up via the pathway which causes dysregulation of cells

Question 17

What is the effect of alpha-syn on what pathway? How can this cause a general dysregulation of cells

Answer 17

Alpha-syn blocks the chaperone mediated pathway
-alpha-syn engages the receptor but it cannot be transferred across the membrane
- through alpha-syn blockage of the chaperone mediated pathway, other proteins are also blocked from being taken up via this pathway which can cause dysregulation of cells

Question 18

The accumulation of what 2 things leads to Parkinson’s?

Answer 18

-Accumulation of alpha-syn protein
- accumulation of damaged mitochondira

Question 19

Why is accumulation of damaged mitochondria a bad thing?

Answer 19

• Mitochondria are the main source of ROS in the body
• Damaged mitochondria release higher levels of ROS
• ROS damages cellular compartments

Question 20

What are the 2 most prominent genes in familial Parkinson’s?
What are they?
How often are they found in what cases?

Answer 20

PINK1
- Is a mitochondrial kinase
- mutation only found in 5-10% of sporadic early onset parkinsons

PARKIN
- is a ubiquitin ligase - responsible for tagging damaged mitochondria with ubiquitin which marks them for degradation through autophagy
- mutation found in 10-15% of sporadic early onset parkinson

Question 21

Explain how PINK1 and PARKIN play a role in Parkinson’s
3 points

Answer 21

• Normally when a mitochondria becomes damaged - PINK1 and PARKIN work together which leads to ubiquitination of that mitochondria and its ability to be sequestered within an autophagosome

-If you lack PINK1 or PARKIN, the mtch cannot be targeted. This means mtch accumulate over time which causes release of more ROS, more oxidative damage which results in more proteins being misfolded and more organelles being damaged

-This is a feedback system hence why symptoms worsen with time

Question 22

in leymans terms, what causes cancer?
What is the cause of most cancers

Answer 22

• cancer is caused by accumulation of DNA damage
• Most cancers begin with a mutation in a gene required for normal function e.g. growth, cell death, DNA repair

Question 23

Is autophagy tumour repressive?

Answer 23

Yes

Question 24

What mutation do patients with cancers often have?
What is the normal function of the gene when it isn’t mutated?
Mice example

Answer 24

• Beclin1 mutation often seen in cancer
  -Beclin1 is a gene required for autophagosome formation

Less Beclin1 = can accumulate more DNA damage

• Can knockout Beclin1 in mice = mouse die from tumours within 18 months

Question 25

What process do cells use inside growing tumours to survive?
why do they do this?

Answer 25

cells inside growing tumours use autophagy to survive - because the environment inside growing tumours is low in nutrients and oxygen

Question 26

Which common cell regulation process does autophagy inhibit, and vice versa?

Answer 26

Autophagy inhibits apoptosis

Question 27

What is the leyman purpose of autophagy vs apoptosis in cells?

Answer 27

Apoptosis keeps cells alive
apoptosis destroys cells

Question 28

What is Beclin1? What is its general function?

Answer 28

• Mutation in this Beclin1 gene is often seen in cancer patients
• Beclin1 is part of a complex with a lipid kinase that makes lipids which leads to autophagosome formation

Question 29

Is Beclin1 lost in tumours?

Answer 29

Yes

Question 30

Where are Bcl proteins normally found?
Describe the role of Bcl2 proteins in apoptosis
3 points

Answer 30

Bcl2 is usually found on the outer membrane of mitochondria

Bcl2 controls how much cytochrome is released from mitochondria, this is important for driving apoptosis
- Bcl2 suppresses release of cytochrome from mitochondria

Bcl2 is an inhibitor of apoptosis

Question 31

What happens when Beclin1 and Bcl2 interact?

Answer 31

• Bcl2 is taken away from the mitochondria, which means the mitochondria are free to cause apoptosis
• Beclin1 is taken away from its complex with lipid kinase, so autophagosome formation no longer occurs

Question 32

Why is ‘losing’ Beclin1 a bad thing when it comes to tumours?

Answer 32

if you lose Beclin1, bcl2 is free to bind to mitochondria which inhibits apoptosis - not what you want to happen inside tumour cells

Question 33

Beclin1 and Bcl2 are known to interact with each other. What happens when you activate autophagy?

Answer 33

When you activate autophagy, Beclin1 is phosphorylated and returns to its lipid kinase complex, therefore autophagosome formation needed for autophagy can occur. At the same time, Bcl2 returns to the mitochondria, inhibiting apoptosis

-When you activate autophagy you automatically inhibit apoptosis and the cell is driven towards a survival state

Question 34

Autophagy is anti-oncogenic and pro-oncogenic.
What does oncogenic mean?
List how is it anti-oncogenic and pro-oncogenic

Answer 34

Oncogenic = a mutated gene that has potential to cause cancer

anti-oncogenic:
- Cell homeostasis
- Damage removal
- Reduced ROS
- Reduced inflammation

pro-oncogenic
- Cells can survive in low oxygen/nutrient environments
- Prevention of apoptosis
- Survival during chemotherapy

Question 35

Explain 3 examples of autophagy therapeutics in cancer

Answer 35

• If you have tumours, inhibit autophagy to prevent cells from surviving metabolic stress inside the tumour as the tumour grows
• Inhibit autophagy to increase apoptosis e.g. during chemotherapy

-Elevate autophagy in healthy individuals to remove damaged cells and reduce likelihood of cancer occurring in the first place