Protein Aggregation

Question 1

Pathological hall marks are all from protein aggregates - Examples

Answer 1

• Senile plaques (Aβ) & NFTs (Tau) in AD
• Lewy bodies in PD (α-synuclein)
• NIIs in polyQ disorders (+ neuritic aggregates in HD)
• UBIs in ALS (TDP-43; FUS in FUS cases, SOD1 inclusions in SOD1 FALS & DPRs in C9ORF72 cases)

Question 2

Are aggregates toxic ?

Answer 2

• Indirect evidence:
- Proteins that are major constituents of aggregates are mutated in disease (APP, Tau, -Syn, Htt, TDP-43, etc.)
- In HD, threshold for in vitro aggregation correlates closely with threshold for disease
• But, multiple studies imply toxicity in absence of aggregates

Question 3

Who tested whether inclusion bodies may themselves be protective ?

Answer 3

• Arrasate et al

Question 4

Arrasate et al- experiment

Answer 4

• Automated microscopy to monitor large number individual cells over time
• Rat striatal neurones co-transfected with Httex1-GFP & mRFP
- monitored inclusion formation (GFP)
- loss of mRFP as measure cell death

Note: 2 cells with inclusions live longer, loss of diffuse GFP, neurite retraction
• Repeats get longer risk of death gets higher

Question 5

Arrasate et al. results summary

Answer 5

•	Many neurones died without forming IBs
•	Levels diffuse mutant Htt correlated cell death
•	IB formation associated ↓ diffuse protein and ↑ survival
•	Conclusion - IB formation protective
•	Caveats-
-	Simple cell culture system 
-	Over expression 
-	Short time scale

Question 6

Evidence for toxi beta sheet species

Answer 6

Nagai et all
- SDS PAGE gel
- WB – anti- thio ot antipolyQ
- Anti poly Q stronger the signal depending on length
-Thio-Q62 undergoes transition to β-sheet structure & forms amyloid-like fibrils
Over time as it is stored it goes from an alpha helical conformation to a beta sheet as it forms a protein aggregate

Soluble Thio-Q62 Monomeric

Question 7

What is/are the toxic polyQ species?

Answer 7

• Most, but not all, think unlikely to be macromolecular aggregates
• Micro-aggregates? – detected (TEM) before/around symptom onset HD mice
• Homo-oligomers?
• Mis-folded monomer? – number of different structures reported

Question 8

Mechanisms :

Answer 8

• Accumulation – slower turnover may overwhelm protein homeostasis
• Sequestrations of other proteins
• Interreference with transport processes

Question 9

Protein homeostasis:

Answer 9

• Conditional HD mouse model suggested polyQ aggregation may be partly reversible
• 2 main protein removal pathways, both implicated suppressor screens worms/flies
- ubiquitin-proteasome system (UPS)
- autophagy (KdV lecture)
• Both implicated neurodegenerative pathology

Question 10

Tau Transmission

Answer 10

• Injected brain extracts from P301S tau-expressing mice into brains of transgenic WT human tau-expressing mice
• Observed assembly WT human tau into filaments & spread of pathology from site of injection to neighbouring brain regions

Question 11

Summary

Answer 11

• Aggregation prone proteins are toxic
• Inclusions - harmful or protective?
• Mis-folded/oligomeric species can be toxic
• Sequestration one mechanism
• Localisation is important: cytoplasm → n/c transport defects; nucleus → transcription
• “Prion-like” spread of pathology

Question 12

Prion-like transmission of pathology

Answer 12

• Prions - infectious protein particles, convert normal form of PrP to pathogenic form
• No evidence that Aβ, α-synuclein or mutant polyQ oligomers are infectious
• However, there is evidence that pathology may spread through the brain

Question 13

Research- mouse model over expression APP

Answer 13

Abeta immunostaining APP transgenic brains inject with human brain extracts
- Pathology due to the injection

Question 14

Study looking at protein aggregate - Microinjected with fluorescent dextran

Answer 14

Monomeric Beta sheet and amyloid fibrils are toxic

• • Alpha wasn’t
• Concentration dependence
• cell survival and GFP labelled

Question 15

Model for polyglutamine toxicity

Answer 15

-Expanded ply Q protein
Begins as a native monomer + QBP1&raquo_space; Soluble beta sheet monomer > Beta sheet oligomer > Amyloid Fribrils

> > CYTOTOXICITY

Question 16

Comparison with Aβ toxicity:

Answer 16

• Aβ oligomers secreted mutant APP-expressing cells, found human brain & brains APP transgenic mice
• Injection SDS-stable oligomers (not monomers or large fibrillar structures) blocks hippocampal LTP in vivo (Walsh et al. Nature 2002) and ↓ cognitive function (Cleary et al. Nature Neurosci. 2004)

Question 17

Solid-state NMR structure of amyloid fibrils

Answer 17

Beta amyloid plaques - 42 and 40 long but 42 more toxic

Question 18

Ubiquitin-proteasome system

Answer 18

• Ubiquitin - 76 aa protein. Ubn>4 chains signal for proteasomal degradation
• Proteasome - multi-catalytic multi-subunit protease with chaperone activity
• Molecular chaperones, e.g. Hsp40 & Hsp70 bind unfolded proteins & up-regulated by cell stress

Question 19

E1 - (See diagram ))

Answer 19

ubiquitin-activating

enzyme

Question 20

E2

Answer 20

– ubiquitin-conjugating

enzyme (~40

Question 21

E3

Answer 21

ubiquitin ligase (>650, 
•	3 main families)
•	+ De-ubiquitinating enzymes
•	(~100, 4 main families, USPs
largest)
Also ubiquitin-like proteins &
different linkages (K48, K63,
etc.)

Question 22

20S proteasome staining in SCA1

Answer 22

patient tissue and transgenic mice

- String staining for nuclear inclusions

Question 23

Study- PolyGA aggregates

Answer 23

Poly-GA aggregates impair proteasome function – stalled conformation
- Overwhelms proteasome
Effect of modulation on polyQ toxicity -
• Proteasome inhibition exacerbates polyQ toxicity
• Over-expression HSP40/HSP70/CHIP etc. ameliorates polyQ toxicity in cell, worm, fly and mouse models

Question 24

What do cytoplasmic aggregates do ??

Answer 24

Block nucleocytoplasmic transport

Question 25

Woerner et al - what were they testing ?

Answer 25

Transport : Cytoplasmic aggregates block nucleocytoplasmic transport

Question 26

Woerner et al experiment

Answer 26

• Expressed artificial (exclude RNA toxicity) aggregate-prone protein + NES or NLS
• Cytoplasmic forms more toxic
• Sequester low-complexity proteins – many involved in nuclear import/export e.g. THOC2

Question 27

Woerner et al summary/ Extension

Answer 27

Cytoplasmic aggregates interfere with nuclear protein transport.
Expression in cytoplasm is affecting them but expression in the nucleus isn’t
Nuclear transport Assay- Shuttling GFP protein
Leptomycin B inhibits Nuclear export
NES Vs NLS

• Protein aggregates in the cytoplasm inhibit mRNA export
• Cytoplasmic aggregates cause mislocalization of nuclear transport factors