Lecture 22 Autophagy 2

Question 1

Autophagy is what kind of process?

Answer 1

A housekeeping process

Question 2

What occurs with no autophagy?

Answer 2

Protein aggregates

Question 3

Which cell is particularly sensitive to autophagy?

Answer 3

Neurons

Question 4

Neuronal-specific autophagy in mice causes?

Answer 4

Accumulation of ubiquinated aggregates

Increased apoptosis

Question 5

What occurs to the autophagic capacity of cells as we age and this causes?

Answer 5

Decreases so ability to get rid of damage decreases until cells apoptose

Question 6

Name 3 proteinopathies in neurodegeneration?

What protein aggregates form in these diseases?

Answer 6

• α-synuclein in Parkinsons
• Huntingin aggregates in Huntington’s
• Amyloid β plaques in Alzheimer’s

Question 7

What is Huntingtons caused by?

Answer 7

• Mutation in a single gene – monogenic disease

* Caused by polyglutamine (polyQ) expansion in Huntingtin protein

Question 8

Number of Q in healthy Huntington protein vs mutant

Answer 8

• Q<18 = healthy, Q>35=disease-causing

Question 9

What occurs as a result of an expanded polyQ Huntington protein?

Answer 9

1. Huntington protein is misfolded
2. N terminus of the protein gets cleaved so proteins oligomerise and aggregate
3. Leads to ubiquitination
4. Causes proteasomal degradation
   OR aggresome formation and then autophagic degradation

Question 10

Where in this Huntington pathway is toxic?

Answer 10

It is unclear

Question 11

Name ways toxicity is caused in Huntington pathway:

Answer 11

• Loss of normal function of Huntingtin
• Toxic oligomers
• Proteasomal damage
• Aggresomes
• Aggresomes sequester adaptor proteins
• Protein endogenously binding to Huntingin

Question 12

How many people does Parkinson’s affect?

Answer 12

1-2 per 100 in UK

Question 13

Parkinsons is due to loss of what?

Answer 13

Dopaminergic neruons

Question 14

Main neuropathology of Parkinsons is?

Answer 14

• Main neuropathology is aggregates of α-synuclein (Lewy Bodies)

Question 15

Describe the genetics of Parkinsons?

Answer 15

• Complex genetics

• Only 5-10% of cases familial (i.e. clear genetic pathway)
• α-synuclein itself rarely mutated

Question 16

How often is α-synuclein itself mutated

Answer 16

Rarely

Question 17

What normally degrades α-synuclein?

Answer 17

Chaperone-mediated autophagy

Question 18

How does chaperone mediated autophagy work in Parkinsons?

Answer 18

• LAMP2 receptor on surface of lysosome directly recognises α-synuclein and is transported into the lysosome to be degraded.

Question 19

Example of mutated α-synuclein?

Answer 19

A53T

Question 20

What do mutated versions of α-synuclein do?

Answer 20

Block the chaperone-mediated pathway, causing toxicity. They are recongised but clog LAMP2 stopping other proteins being degraded too

Question 21

What else accumulates in Parkinsons?

Answer 21

Damaged mitochondria

Question 22

What are mitochondria the main source of?

Answer 22

Reactive Oxygen Species (ROS)

Question 23

What does ROS do?

Answer 23

Damages cellular components

Question 24

What is the hypothesis for mitochondrial disfunction in parkinsons?

Answer 24

Parkinsons may be caused by mitochondrial-derived oxidative damage

Question 25

What 2 genes regulate mitophagy

Answer 25

PINK1 | PARKIN

Question 26

What is PINK1?

Answer 26

A mitochondrial kinase that phosphorylates damaged mitochondria in the PINK1/PARKIN mediated mitophagy

Question 27

What is PARKIN?

Answer 27

Cytosolic E3 ubiquitin ligase that protects cells against death and dopaminergic degeneration

Question 28

What sort of mutation occurs in PINK1?

Answer 28

Loss of function in 5-10% sporadic earl-onset Pakrinsons

Question 29

What mutation occurs in PARKIN?

Answer 29

Mutated in 50% of autosomal recessive | 10-15% sporadic early-onset Parkinson’s

Question 30

Describe the normal pathway in mitophagy?

Answer 30

1. Depolarised mitochondria 2. PINK1 and so PARKIN recruited 3. Ubiquitination of mitochondria 4. Degradation by mitophagy

Question 31

Mutation in PINK1/PARKIN effect?

Answer 31

* Less ubiquitination * Accumlation of damaged mitochondira * Lots of ROS made * Oxidative damage * Protein misfolding * Damaged organelles * Increase in DNA damage and damage of other proteins and mitochondria * Vicous cycle

Question 32

How many mechanisms are there for neurodegeneration?

Answer 32

Many

Question 33

What are the mechanisms for neurodegeneration?

Answer 33

Anything affecting trafficking/ability of phagosome to fuse/make a lysosome can predispose you to these conditions.

Question 34

Do you need a mutation in the protein aggregate for it to accumulate?

Answer 34

No

Question 35

Examples of the mechanisms involved in neurodegeration:

Answer 35

``` Impaired autophagosome formation Disrupted lysosomal function Secretion Inhibited autolysosome formation Autophagic cargo Disruption of cargo recognition ```

Question 36

How is cancer caused? Plus examples

Answer 36

Accumulation of DNA damage | • cellular metabolism, UV light, Ionising radiation, chemical exposure, replication errors

Question 37

Response to cancer?

Answer 37

• Response: Cell cycle checkpoint activation, transcriptional programme activation, DNA repair (direct removal, base excision repair, nucleotide excision repair, ds break repair (HR/NHEJ), apoptosis

Question 38

Is autophagy tumour suppressive or pro-oncogenic?

Answer 38

Both

Question 39

Describe how autophagy is tumours suppressive?

Answer 39

Autophagy ↓ in damaged organelles ↓ protein toxicity ↓ ROS | And so autophagy inhibits oxidative stress/DNA damage/tumourigenesis

Question 40

What causes DNA damage accumulation?

Answer 40

Beclin1 (Atg6)

Question 41

How often is beclin1 mutated?

Answer 41

Monoallelically deleted in 40-75% of ovarian, breast and prostate carcinomas

Question 42

What occurs in Beclin+/- mice?

Answer 42

They cannot do as much autophagy so accumulate damage and tumours

Question 43

How is autophagy pro-oncgenic?

Answer 43

• Autophagy is unregulated in hypoxic, nutrient-poor tumour regions.

Question 44

What would blocking autophagy occur in the pro-oncogenic autophagy?

Answer 44

Necrosis or apoptosis suggesting autophagy keeps the cells alive in this tumour region

Question 45

Does autophagy inhibit apoptosis?

Answer 45

yes

Question 46

How does autophagy inhibit or induce apoptosis?

Answer 46

* Pro-survival: The complex compromising of Beclin1 makes PIP3 on the subdomain of the ER which is used to make the autophagosome. Activity of Beclin1 will promote autophagosome formation. Autophagy is pro-survival. * Pro-death: In contrast apoptosis can be pro-death due to Bcl2 etc. which localise on the surface of mitochondria and cause mitochondria permeabilization and initiate caspases. This causes apoptosis.

Question 47

Pro-survival

Answer 47

Beclin1 PIP3 Autophagosomes

Question 48

Pro-death

Answer 48

Bcl2 surface of mitochondria Permeabilisation Caspases Apoptosis

Question 49

How do Bcl2 and Beclin1 interact?

Answer 49

Directly e.g. Pro-survival pathways will sequester Bcl2 from the mitochondria to repress apoptosis, hence activating autophagy decreases apoptosis and vice versa

Question 50

What does inhibiting apoptosis drive?

Answer 50

Tumour survival and chemotherapy resistance

Question 51

When do you want to increase autophagy?

Answer 51

Neurodegenerative diseases

Question 52

When and why do you want to decrease autophagy?

Answer 52

Cancer – increasing autophagy means less likely to apoptose, so drive tumour survival and chemotherapy resistance

Question 53

Name the three strategies for autophagy therapeutics?

Answer 53

* Block survival to metabolic stress with autophagy inhibitors * Inhibit autophagy to increase apoptosis during chemotherapy * Elevate autophagy to remove damage and prevent cancer

Question 54

How is autophagy anti-oncogenic?

Answer 54

- Cell homeostasis - Damage removal - Reduced ROS/genotoxicity - Reducing inflammation

Question 55

How is autophagy pro-oncogenic?

Answer 55

- Survival during oxygen of nutrient hortage - Prevention of apoptosis - Survival during chemotherapy