80 research outputs found
Metal ions and amyloid fiber formation in neurodegenerative diseases. Copper, zinc and iron in Alzheimer's, Parkinson's and prion diseases
Imaging Amyloid-beta Membrane Interactions: Ion-Channel Pores and Lipid-Bilayer Permeability in Alzheimer's Disease
Imaging Amyloid‐β Membrane Interactions: Ion‐Channel Pores and Lipid‐Bilayer Permeability in Alzheimer's Disease
A Comparison of Three Fluorophores for the Detection of Amyloid Fibers and Prefibrillar Oligomeric Assemblies. ThT (Thioflavin T); ANS (1-Anilinonaphthalene-8-sulfonic Acid); and bisANS (4,4 '-Dianilino-1,1 '-binaphthyl-5,5 '-disulfonic Acid)
Amyloid
fiber formation is a key event in many misfolding disorders.
The ability to monitor the kinetics of fiber formation and other prefibrillar
assemblies is therefore crucial for understanding these diseases.
Here we compare three fluorescent probes for their ability to monitor
fiber formation, ANS (1-anilinonaphthalene-8-sulfonic acid) and bis-ANS
(4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic
acid) along with the more widely used thioflavin T (ThT). For this,
we have used two highly amyloidogenic peptides: amyloid-β (Aβ)
from Alzheimer’s disease and islet amyloid polypeptide (IAPP)
associated with type II diabetes. Using a well-plate reader, we show
all three fluorophores can report the kinetics of fiber formation.
Indeed, bis-ANS is markedly more sensitive to fiber detection than
ThT and has a submicromolar affinity for Aβ fibers. Furthermore,
we show that fluorescence detection is very sensitive to the presence
of excess fluorophore. In particular, beyond a 1:1 stoichiometry these
probes demonstrate marked fluorescence quenching, for both Aβ
and IAPP. Indeed, the fiber-associated fluorescence signal is almost
completely quenched in the presence of excess ThT. There is also intense
interest in the detection of prefibrillar amyloid assemblies, as oligomers
and protofibrils are believed to be highly cytotoxic. We generate
stable, fiber-free, prefibrillar assemblies of Aβ and survey
their fluorescence with ANS and bis-ANS. Fluorescence from ANS has
often been used as a marker for oligomers; however, we show ANS can
fluoresce more strongly in the presence of fibers and should therefore
be used as a probe for oligomers with caution
pH Dependence of Amyloid-β Fibril Assembly Kinetics: Unravelling the Microscopic Molecular Processes.
Central to Alzheimer's disease (AD) is the assembly of the amyloid-beta peptide (Aβ) into fibrils. A reduction in pH accompanying inflammation or subcellular compartments, may accelerate fibril formation as the pH approaches Aβ's isoelectric point (pI). Using global fitting of thioflavin-T to monitor fibril formation kinetics over a range of pHs, we identify the impact net charge has on individual microscopic rate constants which are associated with fibril assembly. We show that the primary nucleation has a strong pH dependence. The titration behaviour exhibits a mid-point or pKa of 7.0, close to the pKa of Aβ histidine imidazoles. Surprisingly, both the secondary nucleation and elongation rate constants are pH independent. This indicates the charge of Aβ, in particular histidine protonation, has little impact on this stage of Aβ assembly. These fundamental processes are key to understanding the forces that drive the assembly of Aβ into toxic oligomers and fibrils
Truncated Amyloid-beta((11-40/42)) from Alzheimer Disease Binds Cu2+ with a Femtomolar Affinity and Influences Fiber Assembly
pH Dependence of Amyloid‐β Fibril Assembly Kinetics: Unravelling the Microscopic Molecular Processes
Impact of Membrane Phospholipids and Exosomes on the Kinetics of Amyloid-β Fibril Assembly.
Alzheimer's disease (AD) is linked with the self-association of the amyloid-β peptide (Aβ) into oligomers and fibrils. The brain is a lipid rich environment for Aβ to assemble, while the brain membrane composition varies in an age dependent manner, we have therefore monitored the influence of lipid bilayer composition on the kinetics of Aβ40 fibril assembly. Using global-fitting models of fibril formation kinetics, we show that the microscopic rate constant for primary nucleation is influenced by variations in phospholipid composition. Anionic phospholipids and particularly those with smaller headgroups shorten fibril formation lag-times, while zwitterionic phospholipids tend to extend them. Using a physiological vesicle model, we show cellular derived exosomes accelerate Aβ40 and Aβ42 fibril formation. Two distinct effects are observed, the presence of even small amounts of any phospholipid will impact the slope of the fibril growth curve. While subsequent additions of phospholipids only affect primary nucleation with the associated change in lag-times. Heightened anionic phospholipids and cholesterol levels are associated with aging and AD respectively, both these membrane components strongly accelerate primary nucleation during Aβ assembly, making a link between disrupted lipid metabolism and Alzheimer's disease
N-Terminally Truncated Amyloid-beta((11-40/42)) Cofibrillizes with its Full-Length Counterpart: Implications for Alzheimer's Disease
Biotechnology and Biological Sciences Research Council. Grant Number: BB/M023877/
Copper(II) Can Kinetically Trap Arctic and Italian Amyloid‑β40 as Toxic Oligomers, Mimicking Cu(II) Binding to Wild-Type Amyloid‑β42: Implications for Familial Alzheimer’s Disease
The self-association of amyloid-β (Aβ) peptide into neurotoxic oligomers is believed to be central to Alzheimer’s disease (AD). Copper is known to impact Aβ assembly, while disrupted copper homeostasis impacts phenotype in Alzheimer’s models. Here we show the presence of substoichiometric Cu(II) has very different impacts on the assembly of Aβ40 and Aβ42 isoforms. Globally fitting microscopic rate constants for fibril assembly indicates copper will accelerate fibril formation of Aβ40 by increasing primary nucleation, while seeding experiments confirm that elongation and secondary nucleation rates are unaffected by Cu(II). In marked contrast, Cu(II) traps Aβ42 as prefibrillar oligomers and curvilinear protofibrils. Remarkably, the Cu(II) addition to preformed Aβ42 fibrils causes the disassembly of fibrils back to protofibrils and oligomers. The very different behaviors of the two Aβ isoforms are centered around differences in their fibril structures, as highlighted by studies of C-terminally amidated Aβ42. Arctic and Italian familiar mutations also support a key role for fibril structure in the interplay of Cu(II) with Aβ40/42 isoforms. The Cu(II) dependent switch in behavior between nonpathogenic Aβ40 wild-type and Aβ40 Arctic or Italian mutants suggests heightened neurotoxicity may be linked to the impact of physiological Cu(II), which traps these familial mutants as oligomers and curvilinear protofibrils, which cause membrane permeability and Ca(II) cellular influx
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