Week 8.2 AD Flashcards
Alzheimer’s Disease (AD)
Ab structure an fibril assembly
Lecture 2
i) Ab structure
ii) Fibril Assembly -Intermediates
iii) Detection amyloids
iv) Structure of amyloids
i) Structure of soluble Ab
What has been used to study Ab? under various solvents, including:
What strucute was observed in monomeric Ab
What structue was observed in a membrane-mimicking environment?
•Ab(1-42) and fragments have been studied by NMR, CD and IR.
- Under various conditions
- solvents (water, DMSO, THF, SDS) and pH’s
•In water monomeric Ab has a predominantly irregular structure (largely random), little define strucutre to Ab in water as monomer
- In a membrane-mimicking environment
- e.g. SDS (Sodium-dodecyl-sulphate)
- In these conditions, the peptide is largely helical
Conformational Switch
Amyloidogenesis involves ______ (__ _____) to b-sheet conversion of Ab
What is the conversion dependent on?
Conformational Switch
Amyloidogenesis involves random (or a-helical) to b-sheet conversion of Ab
Bellow CD trace, spectra shows over time changes from random coil to one rich in beta sheet
This spontaneous conversion is dependent on pH, salt, and shaking
**ii) Assembly of amyloid fibrils **
Assembly and Structure of Amyloid fibrils: Various meta-stable intermediates have been characterized
What is the possibly
- Fibers can pact together and elongate and fibers will stack together more and more
•Possible assembly process:
•Step 1: random Ab, self associates or/and converts to b-sheet
•Step 2: Ab forms: dimers, trimers, tetramers
•Step 3: 6 or 5 mers are formed –paranucleus
•Step 4: ~12 mer is formed (Globule / Ab56*)
•Step 5: ~18 mer is formed (micelle/ Ab-O)
(Bold = amyloid-beta derived diffusible ligands ADDL’s)
•Sept 6:protofibers >> 100 mers, can be long but “wiggly” 5 nm in diameter
•Step 7: Long straight protofilaments form 5 nm in diameter
•Step 8: Five or more protofilaments pack together to form a mature fiber, 10-20 nm in diameter, often with a twist
•Step 8: Mature fibres clump together to form plaques
Misfolding pathway to Amyloid fibres
Monomers
V
Oligomers
V
Protobrils
V
Mature fibrils
Anything that alters the kinetics or thermodynamics of the Ab forms will influence the disease
Fibril growth
and detection
By looking at the inherited forms of AD we can look for mutation in the test tube…
What two parts do fibril growth undergo i.e what does the test tube show?
What does ‘seeding show’?
Kinetic curve that we see is fundamental in understanding AD
Occurs in two parts, lag phase and then the elongation
‘Seed’ – nucleating structure that then rapidly forms fibers, until it plateaus which is because 99.9% of monomers have converted into fibers – you are at equilibrium
- *What are the methods of detection of Amyloids?
- *
Why is Thioflavin T (ThT) Fluorescence used?
CD Spectroscopy b-sheet
IR spectroscopy for b-sheet
Congo Red Dye
(Green-Red Bifringence)
Electron Microscopy
and Atomic Force Microscopy
Thioflavin T (ThT) Fluorescence: Specific ThT binding to cross beta-sheet of amyloids produces a fluorescence's signal. Twist in the two aromatic rings that change the way the delocalised e- on the two rings interact and causes it to fluorescence. (change in e- orbitals).
The process of fiber formation is called nucleated polymerisation reaction
What is the reaction anaglogus to?
The nucleating fibril formation is analogus to making copper sulphate crystals
The nuclei is formed (aggregation of oligomers)
This is known as the seeding process
The nucleation leads to rapid fibre formation - due to the seeding process
Key element of this process is fragmentation: fibre growth much quicker
Fragmentation can be induced by aggitation (shaking)
**What are the triggers that cause fibril formation? **
1) Elevated levels of Ab
2) Changes in the ratio of Ab40 to Ab42
(inherited AD)
2) Ab primary sequence mutations
(inherited AD)
3) Seed of misfolded Ab (b-form)
5) Drop in pH to the pI of Ab (Ab pI 5.3)
6) Elevated copper
7) Oxidation (Met-0X)
8) Dimerisation (di-Tyr or di-His)
9) Pyroglutamate Ab(3-42)
Concentration of Ab increases rate of fiber formation
How does change in pH affect fiber formation?
Concentration of Ab increases rate of fiber formation
Ab pl (solubility) is 5.3
Change in pH
pH 8 slow fiber formation
pH 6 faster formation
because: Ab becomes more neutrally charged as you drop the pH (drop in solubility) and nuteralised the Ab molecule and is much more prone to association causing the nucleation processes
Protination and deprotonation of Ab, correlates with histidine charge i.e pH 6 = more protonated and so you get more fiber formation
What metal ion changes the rate of fiber growth? Why?
What are the energetics of fiber formation?
Copper(II) increases rate of fiber growth
In the prescence of copper, fiber formation is accelerated, Cu changes the structure of Ab which might promote fiber formation
Energetics of fiber formation
Monomer more stable, need nucleating species so its less stable and harder to form and once its fomred we get rapid formation.
What is the structural motif of Ab?
What sort of NMR can you do?
Cross-beta motif: Ab not possible to cystalise, inter-molecular beta-sheet at right angeles to long axis of fibre
Solid state NMR is conducted
Special fetaure of the cross-beta motif is that you have hydrgen bonding across the molecules (NOT WITHIN THE SAME MOLECULE)
Within fibers H-bonding is between adjacent molecules - the reason the fiber is so stable is because the interaction of hydrogen bonds as the fiber assembles
**Fibril morphology **
What influences the morphology of these fibers?
In vetro-The morphology of the mature fiber is dependant on conditions; Agitation Concentration pH NaCl
Spheroids found in Plaques
Single fibres in the brain come together and aggregate and you get spheroids in plaques
Summary Lectures 1+2
Summary Lectures 1+2
Plaques in AD brain contain A-beta peptide 42 aa long
Ab peptide is derived from the larger APP protein
Ab peptide assembles together to form amyloids
