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
Comparison with Aβ toxicity:
- 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)
Solid-state NMR structure of amyloid fibrils
Beta amyloid plaques - 42 and 40 long but 42 more toxic
Ubiquitin-proteasome system
- 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
E1 - (See diagram ))
ubiquitin-activating
enzyme
E2
– ubiquitin-conjugating
enzyme (~40
E3
ubiquitin ligase (>650, • 3 main families) • + De-ubiquitinating enzymes • (~100, 4 main families, USPs largest) Also ubiquitin-like proteins & different linkages (K48, K63, etc.)
20S proteasome staining in SCA1
patient tissue and transgenic mice
- String staining for nuclear inclusions
Study- PolyGA aggregates
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
What do cytoplasmic aggregates do ??
Block nucleocytoplasmic transport
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Lecture 115
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Lecture 331
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Lecture 223
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Huntingtons Disease - OB21
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Parkinsons Disease - OB19
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Multiple Sclerosis26
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Motor Neurone Disease25
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Stem Cells27
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Patterns and overlaps32
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Neuropathology of Dementia18
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Post Mortem Tissue24
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Autophagy and Neurodegeneration26
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Mitochondria in Neurodegeneration26
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Gene Therapy20
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Drug Discovery28
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In the News6
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Protein Aggregation27
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Axonal Transport44
