Autophagy and Neurodegeneration

Question 1

What plays a major role in neurodegenerative disorders ??

Answer 1

Autophagy

Question 2

How was Autophagy discovered ??

Answer 2

Began with the discovery of lysosomes
• Intracellular organelle with an acid interior
• Specific membrane proteins
• Lack manose 6 phosphate R – therefore not

Question 3

What is autophagy ??

Answer 3

Self eating

• Lysosomal degradation pathway
• • Basal turnover – long lived proteins and organelles
• Physiological stress response
• Starvation
• Clearance
• Defence

Question 4

What are the 3 types of Autophagy??

Answer 4

1. Micro autophagy
2. Chaperone mediated autophagy
3. macro autophagy

Question 5

Micro Autophagy

Answer 5

Simplest from
No intermediates or adaptors required
Lysosomal membrane invaginates

Question 6

Chaperone mediated autophagy

Answer 6

Substrate protein bearing a KFERQ-like motif (Lys-Phe-Glu-Arg-Gln) is recognized by a chaperone/co-chaperone complex in the cytosol
The complex transfers the protein to the surface of the lysosomal membrane, where it binds to the cytosolic tail of the LAMP2A receptor
This promotes LAMP2A multimerization and substrate unfolding with the aid of the lysosomal Hsc-70 (Heat shock cognate protein 70kD)
This promotes the direct translocation of the protein across the lysosomal membrane and its subsequent degradation by the lysosomal proteases
Following substrate translocation, lys-Hsc70 enables disassembly of the LAMP2A complex which is then available for the binding of other substrates.

Question 7

Step 1 of Macropautophagy

Answer 7

1. Initiation/Translocation to the Phagophore
   - phagophore formation mediated by SNAREs
   - Can be mTOR dependent autophagy or mTOR - independent autophagy

Question 8

Step 2 Macroautophagy

Answer 8

2. ELonagtion and completion
   - Phagophore expansion is Poorly defined
   - LC3 – tags autophagosome from beginning to the end
   - LC3 is preforem > LC3-1
   - LC3 -2 recruited to the autophagasome with attached lipids

Question 9

STep 3 of Macroautophagy

Answer 9

3. Cargo Selection- Ubiquitination
   Misfolded proteins damaged organelles are labelled for degradation by adding ubiquitin chains
   Each kind of cargo different phagy name
   - Selective or unselective
   -Selective> Cargo is being recognized by receptors that interact with ubiquitin on the cargo (UBA domain) and LC3 (LIR domain) => bring cargo to forming autophagosome

Question 10

Agrosome

Answer 10

blob of ubiquitnated protein cant go through proteasome but recognised and brought to phagosome

Question 11

Step 4 in Macroautophagy

Answer 11

Fusion and Degradation
Autophagosome–lysosome fusion is mediated by SNAREs
During starvation-induced autophagy, Stx17, a Qa-SNARE protein (target-SNARE), is recruited to completed autophagosomes.
Stx17 interacts with the cytosolic Qbc-SNARE SNAP-29
SNAP-29 interacts with the R-SNARE (vesicle-SNARE) protein VAMP7 or VAMP8, which is located on lysosomal membrane.
This process drives fusion between the outer autophagosomal membrane and the lysosomal membrane.

Question 12

Regulation of Autophagy - Basal Turnover

Answer 12

-long-lived proteins & organelles

Question 13

Regulation of Autophagy -Physiological stress response

Answer 13

• Starvation - generate carbon sources and reduce unneeded structures
• Clearance of damaged constituents including protein aggregates
• Defense against intracellular pathogens

Question 14

What is the master regulator of autophagy ?

Answer 14

mTOR 
Active mTOr> No autophagy 
mTOr dependent reg – regulated by multiple signals 
-	Nutrient sensing 
-	Growth factors 
-	Stress and energy sensing

Question 15

mTOR pathway

Answer 15

acts via ULK1 initiation Complex
Mammalian Target of Rapamycin (mTOR) is the master regulator of autophagy
Active mTOR interacts with the ULK1/FIP200/ATG13 complex and phosphorylates and inactivates ULK1 - > no autophagy
mTOR pathways inactivate mTOR
mTOR dissociates from the ULK1 complex, ULK1 is activated and autophosphorylates,
Active ULK1 phosphorylatesFIP200/ATG13 -> autophagy

Question 16

mTOR independent Pathways

Answer 16

works directly on the Class 3 PI3 kinase complex
– Ionositol signalling pathway
- cAMP/EPAc/PLC inosotiol pathway
- ca/ calpain pathway
- JNK1/Beclin/PI3KC3 pathway
The downstream components of mTOR-independent pathways regulating autophagosome biogenesis are not clear.

Question 17

What is the link between autophagy and neurodegeneration ?

Answer 17

• Protein aggregation – evidence for autophagy not working well

Question 18

ALS and FTD Pathology

Answer 18

• TDp43 inclusions

- C9ORF72 P62 pathology – aggregation of the receptor/ not being cleared

Question 19

Disease

Answer 19

Lack of autophagy/ clearance hindered

Question 20

Pink and Parkin regulators of microautophagy

Answer 20

implicated in Parkinson’s LOF gives Parkinson’s

Question 21

Parkinsons

Answer 21

Mutant in LRRK2 inhibits autophagy of aggregated protein by aggrephagy

Lysosomal dysfunction also associated with Parkinson’s
Lysosome – Ph 4
Cytoplasm – Ph 7.2

Question 22

Alzeheimers

Answer 22

PSEN deficiency > Lysosomal dysfunction

Question 23

Treatments based on Autophagy

Answer 23

• Stimulation of autophagy
• Small molecule autophagy enhances
  Be wary – involved in lots of things – balance
  PI3K inhibitors
  mTOr inhibitors
  AMPK activators

Question 24

Genetics links disease to autophagy: Parkinson’s disease: lysosomal dysfunction

Answer 24

Mutations in lysosomal‐associated genes directly cause lysosomal impairment and can be associated with PD‐like neurodegeneration

Question 25

Huntington’s disease

Answer 25

mHTT inhibits cargo loading

Question 26

Study

Answer 26

Inhibition of Basal autophagy causes neurodegeneration
- Protein aggregation
- Direct evidence
- Spontaneous Neurodegeneration
Mutations and polymorphisms in autophagy genes linked to human diseases