Protein Aggregation Flashcards
Pathological hall marks are all from protein aggregates - Examples
- 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)
Are aggregates toxic ?
• 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
Who tested whether inclusion bodies may themselves be protective ?
• Arrasate et al
Arrasate et al- experiment
• 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
Arrasate et al. results summary
• 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
Evidence for toxi beta sheet species
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
What is/are the toxic polyQ species?
- 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
Mechanisms :
- Accumulation – slower turnover may overwhelm protein homeostasis
- Sequestrations of other proteins
- Interreference with transport processes
Protein homeostasis:
• 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
Tau Transmission
- 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
Summary
- 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
Prion-like transmission of pathology
- 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
Research- mouse model over expression APP
Abeta immunostaining APP transgenic brains inject with human brain extracts
- Pathology due to the injection
Study looking at protein aggregate - Microinjected with fluorescent dextran
Monomeric Beta sheet and amyloid fibrils are toxic
- Alpha wasn’t
- Concentration dependence
- cell survival and GFP labelled
Model for polyglutamine toxicity
-Expanded ply Q protein
Begins as a native monomer + QBP1»_space; Soluble beta sheet monomer > Beta sheet oligomer > Amyloid Fribrils
> > CYTOTOXICITY
