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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

Tau phosphorylation assay.(A) Examples of tau gel bands from Western blot imaging after kinaseassay reaction in the absence (−) and presence of Aβ42at 5 nM (+), 50 nM (++), and 500 nM (+++), and absence (−)or presence of GSK3α (+). (B) Quantification of tau phosphorylationin the presence (+++) or absence (−) of 500 nM Aβ42,and absence (−) or presence of GSK3α (+). Levels ofpTau were compared to reactions performed in the absence of GSK3α.Data shown are Mean ± SEM (n = 3).
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fig6: Tau phosphorylation assay.(A) Examples of tau gel bands from Western blot imaging after kinaseassay reaction in the absence (−) and presence of Aβ42at 5 nM (+), 50 nM (++), and 500 nM (+++), and absence (−)or presence of GSK3α (+). (B) Quantification of tau phosphorylationin the presence (+++) or absence (−) of 500 nM Aβ42,and absence (−) or presence of GSK3α (+). Levels ofpTau were compared to reactions performed in the absence of GSK3α.Data shown are Mean ± SEM (n = 3).

Mentions: We next asked whether Aβ42 directly stimulates GSK3α.We used an in vitro kinase assay with purified GSK3αand tau in a buffer system with ATP. The assay was performed in theabsence and presence of 5–500 nM Aβ42, which had beenpreincubated at 5 μM for a short time to initiate the formationof transient oligomers before dilution into the reactions. Tau phosphorylationby GSK3α was detected by Western blot using a phospho-tau specificantibody recognizing phosphorylation of Ser396 (Figure 6). In the presence of Aβ42, we finda factor of 6.3(±2.4) stronger signal with GSK3α comparedto no enzyme, with no variation over the Aβ42 concentrationrange studied in line with the high affinity of the interaction. Inthe absence of Aβ42 we find a factor of 1.9(±0.8) strongersignal with GSK3α compared to no enzyme. Thus, Aβ42 wasfound to increase GSK3α activity in terms of tau phosphorylationby a factor of 3 under the conditions of the assay.


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)

Tau phosphorylation assay.(A) Examples of tau gel bands from Western blot imaging after kinaseassay reaction in the absence (−) and presence of Aβ42at 5 nM (+), 50 nM (++), and 500 nM (+++), and absence (−)or presence of GSK3α (+). (B) Quantification of tau phosphorylationin the presence (+++) or absence (−) of 500 nM Aβ42,and absence (−) or presence of GSK3α (+). Levels ofpTau were compared to reactions performed in the absence of GSK3α.Data shown are Mean ± SEM (n = 3).
© Copyright Policy - editor-choice
Related In: Results  -  Collection

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

fig6: Tau phosphorylation assay.(A) Examples of tau gel bands from Western blot imaging after kinaseassay reaction in the absence (−) and presence of Aβ42at 5 nM (+), 50 nM (++), and 500 nM (+++), and absence (−)or presence of GSK3α (+). (B) Quantification of tau phosphorylationin the presence (+++) or absence (−) of 500 nM Aβ42,and absence (−) or presence of GSK3α (+). Levels ofpTau were compared to reactions performed in the absence of GSK3α.Data shown are Mean ± SEM (n = 3).
Mentions: We next asked whether Aβ42 directly stimulates GSK3α.We used an in vitro kinase assay with purified GSK3αand tau in a buffer system with ATP. The assay was performed in theabsence and presence of 5–500 nM Aβ42, which had beenpreincubated at 5 μM for a short time to initiate the formationof transient oligomers before dilution into the reactions. Tau phosphorylationby GSK3α was detected by Western blot using a phospho-tau specificantibody recognizing phosphorylation of Ser396 (Figure 6). In the presence of Aβ42, we finda factor of 6.3(±2.4) stronger signal with GSK3α comparedto no enzyme, with no variation over the Aβ42 concentrationrange studied in line with the high affinity of the interaction. Inthe absence of Aβ42 we find a factor of 1.9(±0.8) strongersignal with GSK3α compared to no enzyme. Thus, Aβ42 wasfound to increase GSK3α activity in terms of tau phosphorylationby a factor of 3 under the conditions of the assay.

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