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

Dunning CJ, McGauran G, Willén K, Gouras GK, O'Connell DJ, Linse S - ACS Chem Neurosci (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Structural Biology, Chemical Centre, Lund University , P O Box 124, SE22100 Lund, Sweden.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Principles of protein array screening to find interactionpartners of on-pathway Aβ42 oligomers. (A) Amino acid sequenceof Aβ(MC1–42) with the Alexa546 chromophore attachedto the cysteine residue added at the N-terminus. Hydrophobic residuesare on yellow background, while t, + , and – indicate residuesthat are titrating (t) and positively (+) and negatively (−)charged around neutral pH. (B) Fibril formation as a function of timestarting from 5 μM monomeric Aβ42 in physiological saltbuffer at pH 8.0. The fibril concentration is shown in blue and monomerconcentration in green. (C) Nucleation rates during the same aggregationreaction, calculated from concentrations and rate constants determinedin physiological salt (unpublished experiment). The rate of primarynucleation is shown in black, and the rate of secondary nucleationin green. The time window during which the solution was incubatedwith the protein array is shown as a shaded green area in panels (B)and (C). (D) Example of a subarray with guiding spots in red, andthe spots of a putative interaction in green. (E) Z-scores of three duplicate spots that were found above the cutoffwhen the array was probed with on-pathway Aβ42 assemblies.
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fig1: Principles of protein array screening to find interactionpartners of on-pathway Aβ42 oligomers. (A) Amino acid sequenceof Aβ(MC1–42) with the Alexa546 chromophore attachedto the cysteine residue added at the N-terminus. Hydrophobic residuesare on yellow background, while t, + , and – indicate residuesthat are titrating (t) and positively (+) and negatively (−)charged around neutral pH. (B) Fibril formation as a function of timestarting from 5 μM monomeric Aβ42 in physiological saltbuffer at pH 8.0. The fibril concentration is shown in blue and monomerconcentration in green. (C) Nucleation rates during the same aggregationreaction, calculated from concentrations and rate constants determinedin physiological salt (unpublished experiment). The rate of primarynucleation is shown in black, and the rate of secondary nucleationin green. The time window during which the solution was incubatedwith the protein array is shown as a shaded green area in panels (B)and (C). (D) Example of a subarray with guiding spots in red, andthe spots of a putative interaction in green. (E) Z-scores of three duplicate spots that were found above the cutoffwhen the array was probed with on-pathway Aβ42 assemblies.

Mentions: In vivo, proteolysis of the amyloid precursor protein (APP) leads to severalAβ length variants, including the disease-linked Aβ42(Figure 1A). Mutationsin APP that affect Aβ42 production rate or aggregation processcause familial forms of early onset AD,5−7 and a genetic correlationbetween AD and the apoε4 allele for apolipoprotein E has beenfound.8 Still the majority of AD casesare sporadic.


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

Dunning CJ, McGauran G, Willén K, Gouras GK, O'Connell DJ, Linse S - ACS Chem Neurosci (2015)

Principles of protein array screening to find interactionpartners of on-pathway Aβ42 oligomers. (A) Amino acid sequenceof Aβ(MC1–42) with the Alexa546 chromophore attachedto the cysteine residue added at the N-terminus. Hydrophobic residuesare on yellow background, while t, + , and – indicate residuesthat are titrating (t) and positively (+) and negatively (−)charged around neutral pH. (B) Fibril formation as a function of timestarting from 5 μM monomeric Aβ42 in physiological saltbuffer at pH 8.0. The fibril concentration is shown in blue and monomerconcentration in green. (C) Nucleation rates during the same aggregationreaction, calculated from concentrations and rate constants determinedin physiological salt (unpublished experiment). The rate of primarynucleation is shown in black, and the rate of secondary nucleationin green. The time window during which the solution was incubatedwith the protein array is shown as a shaded green area in panels (B)and (C). (D) Example of a subarray with guiding spots in red, andthe spots of a putative interaction in green. (E) Z-scores of three duplicate spots that were found above the cutoffwhen the array was probed with on-pathway Aβ42 assemblies.
© Copyright Policy - editor-choice
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4759616&req=5

fig1: Principles of protein array screening to find interactionpartners of on-pathway Aβ42 oligomers. (A) Amino acid sequenceof Aβ(MC1–42) with the Alexa546 chromophore attachedto the cysteine residue added at the N-terminus. Hydrophobic residuesare on yellow background, while t, + , and – indicate residuesthat are titrating (t) and positively (+) and negatively (−)charged around neutral pH. (B) Fibril formation as a function of timestarting from 5 μM monomeric Aβ42 in physiological saltbuffer at pH 8.0. The fibril concentration is shown in blue and monomerconcentration in green. (C) Nucleation rates during the same aggregationreaction, calculated from concentrations and rate constants determinedin physiological salt (unpublished experiment). The rate of primarynucleation is shown in black, and the rate of secondary nucleationin green. The time window during which the solution was incubatedwith the protein array is shown as a shaded green area in panels (B)and (C). (D) Example of a subarray with guiding spots in red, andthe spots of a putative interaction in green. (E) Z-scores of three duplicate spots that were found above the cutoffwhen the array was probed with on-pathway Aβ42 assemblies.
Mentions: In vivo, proteolysis of the amyloid precursor protein (APP) leads to severalAβ length variants, including the disease-linked Aβ42(Figure 1A). Mutationsin APP that affect Aβ42 production rate or aggregation processcause familial forms of early onset AD,5−7 and a genetic correlationbetween AD and the apoε4 allele for apolipoprotein E has beenfound.8 Still the majority of AD casesare sporadic.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Structural Biology, Chemical Centre, Lund University , P O Box 124, SE22100 Lund, Sweden.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus