Fluorescent Filter-Trap Assay for Amyloid Fibril Formation Kinetics in Complex Solutions

Nasir, Irem; Linse, Sara; Cabaleiro-Lago, Celia

doi:10.1021/acschemneuro.5b00104

Cited by 23 publications

(21 citation statements)

References 52 publications

(79 reference statements)

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“…The design of the study relies on detailed knowledge of the reaction mechanism (refs (9−12); Figure 1B,C), the high affinity of Aβ42 for many surfaces 13 requiring stringent surface blocking, and the recent finding of unperturbed reaction kinetics when samples are doped with a minor fraction of Aβ42 with N-terminally incorporated fluorophore. 22 …”

Section: Results and Discussionmentioning

confidence: 99%

Direct High Affinity Interaction between Aβ42 and GSK3α Stimulates Hyperphosphorylation of Tau. A New Molecular Link in Alzheimer’s Disease?

Dunning

McGauran

Willén

et al. 2015

ACS Chem. Neurosci.

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Amyloid β peptide (Aβ42) assemblies are considered central to the development of Alzheimer’s disease, but the mechanism of this toxicity remains unresolved. We screened protein microarrays with on-pathway oligomeric Aβ42 to identify candidate proteins interacting with toxic Aβ42 species. Samples prepared from Alexa546-Aβ42 and Aβ42 monomers at 1:5 molar ratio were incubated with the array during a time window of the amyloid fibril formation reaction during which the maximum number of transient oligomers exist in the reaction flux. A specific interaction was detected between Aβ42 and glycogen synthase kinase 3α (GSK3α), a kinase previously implicated in the disease pathology. This interaction was validated with anti-GSK3α immunoprecipitation assays in neuronal cell lysates. Confocal microscopy studies further identified colocalization of Aβ42 and GSK3α in neurites of mature primary mouse neurons. A high binding affinity (KD = 1 nM) was measured between Alexa488-Aβ42 and GSK3α in solution using thermophoresis. An even lower apparent KD was estimated between GSK3α and dextran-immobilized Aβ42 in surface plasmon resonance experiments. Parallel experiments with GSK3β also identified colocalization and high affinity binding to this isoform. GSK3α-mediated hyperphosphorylation of the protein tau was found to be stimulated by Aβ42 in in vitro phosphorylation assays and identified a functional relationship between the proteins. We uncover a direct and functional molecular link between Aβ42 and GSK3α, which opens an important avenue toward understanding the mechanism of Aβ42-mediated neuronal toxicity in Alzheimer’s disease.

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Section: Results and Discussionmentioning

confidence: 99%

Direct High Affinity Interaction between Aβ42 and GSK3α Stimulates Hyperphosphorylation of Tau. A New Molecular Link in Alzheimer’s Disease?

Dunning

McGauran

Willén

et al. 2015

ACS Chem. Neurosci.

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“…Aβ(MC1-42), a mutant with an extra Cys residue placed between the starting Met and Asp1 was used for fluorophore labelling (35). An aliquot of purified peptide monomer was dissolved in 6 M GuHCl, 10 mM DTT, pH 8.5, incubated 1 h and the monomer was isolated by gel filtration in 20 mM sodium phosphate buffer, pH 8.0.…”

Section: Alexa-647 Labelling Of Peptides and Antibodiesmentioning

confidence: 99%

Kinetic fingerprints differentiate anti-Aβ therapies

Linse

Scheidt

Bernfur

et al. 2019

Preprint

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Alzheimer's disease affects nearly 50 million people worldwide with an overall cost of over 1% of the global economy. The amyloid cascade hypothesis, according to which the misfolding and aggregation of the amyloid-β peptide (Aβ) triggers a series of pathological processes that eventually result in massive brain tissue loss (1,2), has driven many therapeutic efforts for the past 20 years. Repeated failures, however, have highlighted the challenges of characterizing the molecular mechanisms of therapeutic candidates targeting Aβ, and connecting them to the outcomes of clinical trials (3-7). Here, we determine the mechanism of action of four clinical stage antibodies (aducanumab, gantenerumab, bapineuzumab and solanezumab). We quantify the dramatic differences that these antibodies have on the aggregation kinetics and on the production of oligomeric aggregates, and link these effects to the affinity and stoichiometry of each antibody for the monomeric

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“…Filter trap assays have been used extensively in the past to assess inclusion formation in cells (Chang and Kuret 2008; cells following each treatment is displayed as mean ± SEM (n = 3) and was analysed via a one-way ANOVA with a Dunnett's post-test compared to the untreated EGFP control, where ***p < 0.001 Juenemann et al 2015;Nasir et al 2015). Typically, soluble protein moves through the membrane pores, whilst aggregated protein is retained on the membrane such that proteins in the aggregates can be detected by immunoblotting.…”

Section: α-Syna53t* Readily Aggregates To Form Inclusions In Cellsmentioning

confidence: 99%

The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein

Cox

Ecroyd

2017

Cell Stress and Chaperones

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Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. Proteostasis is preserved in the face of stress by a complex network of cellular machinery, including the small heat shock molecular chaperone proteins (sHsps), which act to inhibit the aggregation and deposition of misfolded protein intermediates. Despite this, the pathogenesis of several neurodegenerative diseases has been inextricably linked with the amyloid fibrillar aggregation and deposition of α-synuclein (α-syn). The sHsps are potent inhibitors of α-syn aggregation in vitro. However, the limited availability of a robust, cellbased model of α-syn aggregation has, thus far, restricted evaluation of sHsp efficacy in the cellular context. As such, this work sought to establish a robust model of intracellular α-syn aggregation using Neuro-2a cells. Aggregation of α-syn was found to be sensitive to inhibition of autophagy and the proteasome, resulting in a significant increase in the proportion of cells containing α-syn inclusions. This model was then used to evaluate the capacity of the sHsps, αB-c and Hsp27, to prevent α-syn aggregation in cells. To do so, we used bicistronic expression plasmids to express the sHsps. Unlike traditional fluorescent fusion constructs, these bicistronic expression plasmids enable only individual transfected cells expressing the sHsps (via expression of the fluorescent reporter) to be analysed, but without the need to tag the sHsp, which can affect its oligomeric structure and chaperone activity. Overexpression of both αB-c and Hsp27 significantly reduced the intracellular aggregation of α-syn. Thus, these findings suggest that overexpressing or boosting the activity of sHsps may be a way of preventing amyloid fibrillar aggregation of α-syn in the context of neurodegenerative disease.

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Fluorescent Filter-Trap Assay for Amyloid Fibril Formation Kinetics in Complex Solutions

Cited by 23 publications

References 52 publications

Direct High Affinity Interaction between Aβ42 and GSK3α Stimulates Hyperphosphorylation of Tau. A New Molecular Link in Alzheimer’s Disease?

Direct High Affinity Interaction between Aβ42 and GSK3α Stimulates Hyperphosphorylation of Tau. A New Molecular Link in Alzheimer’s Disease?

Kinetic fingerprints differentiate anti-Aβ therapies

The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein

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