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The PI3K-Akt-mTOR pathway regulates Abeta oligomer induced neuronal cell cycle events.

Bhaskar K, Miller M, Chludzinski A, Herrup K, Zagorski M, Lamb BT - Mol Neurodegener (2009)

Bottom Line: Retraction of neuronal processes correlated with the induction of CCEs and the Abeta monomer or Abeta fibrils showed only minimal effects.Finally, our results also demonstrate that Abeta oligomer treated neurons exhibit elevated levels of activated Akt and mTOR (mammalian Target Of Rapamycin) and that PI3K, Akt or mTOR inhibitors blocked Abeta oligomer-induced neuronal CCEs.Taken together, these results demonstrate that Abeta oligomer-based induction of neuronal CCEs involve the PI3K-Akt-mTOR pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurosciences, Cleveland Clinic Foundation, Cleveland, OH, USA. lambb@ccf.org.

ABSTRACT
Accumulating evidence suggests that neurons prone to degeneration in Alzheimer's Disease (AD) exhibit evidence of re-entry into an aberrant mitotic cell cycle. Our laboratory recently demonstrated that, in a genomic amyloid precursor protein (APP) mouse model of AD (R1.40), neuronal cell cycle events (CCEs) occur in the absence of beta-amyloid (Abeta) deposition and are still dependent upon the amyloidogenic processing of the amyloid precursor protein (APP). These data suggested that soluble Abeta species might play a direct role in the induction of neuronal CCEs. Here, we show that exposure of non-transgenic primary cortical neurons to Abeta oligomers, but not monomers or fibrils, results in the retraction of neuronal processes, and induction of CCEs in a concentration dependent manner. Retraction of neuronal processes correlated with the induction of CCEs and the Abeta monomer or Abeta fibrils showed only minimal effects. In addition, we provide evidence that induction of neuronal CCEs are autonomous to primary neurons cultured from the R1.40 mice. Finally, our results also demonstrate that Abeta oligomer treated neurons exhibit elevated levels of activated Akt and mTOR (mammalian Target Of Rapamycin) and that PI3K, Akt or mTOR inhibitors blocked Abeta oligomer-induced neuronal CCEs. Taken together, these results demonstrate that Abeta oligomer-based induction of neuronal CCEs involve the PI3K-Akt-mTOR pathway.

No MeSH data available.


Related in: MedlinePlus

Elevation of pAkt levels in primary neurons treated with Aβ oligomers and in neurons from the R1.40 transgenic mouse model of AD. A. Cultured primary cortical neurons (21 DIV) were treated with vehicle (Veh), 2.0 μg/ml Aβ monomers (AβM) or 2.0 μg/ml Aβ oligomers (AβO) for 24 hours. Western blot analysis of cell extracts with antibodies against pAktS473, total Akt, p4E-BP1S65 and GAPDH revealed a slight increase in the levels of pAkt and p4E-BP1 in AβO treated cultures when compared to vehicle or AβM treated cultures. B-C. Quantification of the ratios of pAkt/total Akt (B) and p4E-BP1/GAPDH (C) revealed statistically significant increases upon exposure to AβO (2.0 μg/ml) when compared to AβM treatment (2.0 μg/ml) or vehicle controls (p < 0.05 for AβO versus vehicle or AβM; unpaired t test; mean ± SEM; n = 3 independent treatments). D. Western blot analysis of detergent soluble cell extracts of from cultured primary cortical neurons (21 DIV) from non-transgenic (WT) and homozygous R1.40 (R/R) mice with antibodies against pAkt (pAktS473), p-mTOR (p-mTORS2448) and GAPDH revealed elevated levels of pAkt (pAktS473) but not p-mTOR in the R/R neurons when compared to WT neurons. E-F Quantification of the ratios of pAkt/GAPDH (E) and p-mTOR/GAPDH (F) revealed statistically significant increases in the levels of pAkt/GAPDH (p = 0.04; unpaired t test; mean ± SEM; n = 3 independent cultures) but not p-mTOR/GAPDH (p = 0.74; unpaired t test; mean ± SEM; n = 3 independent cultures) in cell lysates form the R/R cultures.
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Figure 7: Elevation of pAkt levels in primary neurons treated with Aβ oligomers and in neurons from the R1.40 transgenic mouse model of AD. A. Cultured primary cortical neurons (21 DIV) were treated with vehicle (Veh), 2.0 μg/ml Aβ monomers (AβM) or 2.0 μg/ml Aβ oligomers (AβO) for 24 hours. Western blot analysis of cell extracts with antibodies against pAktS473, total Akt, p4E-BP1S65 and GAPDH revealed a slight increase in the levels of pAkt and p4E-BP1 in AβO treated cultures when compared to vehicle or AβM treated cultures. B-C. Quantification of the ratios of pAkt/total Akt (B) and p4E-BP1/GAPDH (C) revealed statistically significant increases upon exposure to AβO (2.0 μg/ml) when compared to AβM treatment (2.0 μg/ml) or vehicle controls (p < 0.05 for AβO versus vehicle or AβM; unpaired t test; mean ± SEM; n = 3 independent treatments). D. Western blot analysis of detergent soluble cell extracts of from cultured primary cortical neurons (21 DIV) from non-transgenic (WT) and homozygous R1.40 (R/R) mice with antibodies against pAkt (pAktS473), p-mTOR (p-mTORS2448) and GAPDH revealed elevated levels of pAkt (pAktS473) but not p-mTOR in the R/R neurons when compared to WT neurons. E-F Quantification of the ratios of pAkt/GAPDH (E) and p-mTOR/GAPDH (F) revealed statistically significant increases in the levels of pAkt/GAPDH (p = 0.04; unpaired t test; mean ± SEM; n = 3 independent cultures) but not p-mTOR/GAPDH (p = 0.74; unpaired t test; mean ± SEM; n = 3 independent cultures) in cell lysates form the R/R cultures.

Mentions: To confirm the ICW analysis of pAkt levels, conventional Western blot analysis was performed on cell lysates of neurons exposed to 2.0 μg/ml of Aβ oligomers, a concentration that was sufficient for the induction of neuronal CCEs. Western blots were probed with antibodies against pAkt (Ser 473) and total Akt and quantified for the relative levels of pAkt/Akt (Figure 7A and 7B). Our results demonstrate a relatively modest, but statistically significant increase in the pAkt/Akt ratio upon exposure to 2.0 μg/ml Aβ oligomers, with a 13% increase in the pAkt/Akt ratio following Aβ oligomers treatment compared to vehicle (0.6 ± 0.0096 vs 0.504 ± 0.009) and a 23% increase when compared to Aβ monomer treatment (0.6 ± 0.0096 vs 0.44 ± 0.014). These results confirm our ICW results and together demonstrate that exposure of neurons to Aβ oligomers results in altered pAkt and p-mTOR levels that correlate with the presence of neuronal CCEs as well as MAP2+ process loss.


The PI3K-Akt-mTOR pathway regulates Abeta oligomer induced neuronal cell cycle events.

Bhaskar K, Miller M, Chludzinski A, Herrup K, Zagorski M, Lamb BT - Mol Neurodegener (2009)

Elevation of pAkt levels in primary neurons treated with Aβ oligomers and in neurons from the R1.40 transgenic mouse model of AD. A. Cultured primary cortical neurons (21 DIV) were treated with vehicle (Veh), 2.0 μg/ml Aβ monomers (AβM) or 2.0 μg/ml Aβ oligomers (AβO) for 24 hours. Western blot analysis of cell extracts with antibodies against pAktS473, total Akt, p4E-BP1S65 and GAPDH revealed a slight increase in the levels of pAkt and p4E-BP1 in AβO treated cultures when compared to vehicle or AβM treated cultures. B-C. Quantification of the ratios of pAkt/total Akt (B) and p4E-BP1/GAPDH (C) revealed statistically significant increases upon exposure to AβO (2.0 μg/ml) when compared to AβM treatment (2.0 μg/ml) or vehicle controls (p < 0.05 for AβO versus vehicle or AβM; unpaired t test; mean ± SEM; n = 3 independent treatments). D. Western blot analysis of detergent soluble cell extracts of from cultured primary cortical neurons (21 DIV) from non-transgenic (WT) and homozygous R1.40 (R/R) mice with antibodies against pAkt (pAktS473), p-mTOR (p-mTORS2448) and GAPDH revealed elevated levels of pAkt (pAktS473) but not p-mTOR in the R/R neurons when compared to WT neurons. E-F Quantification of the ratios of pAkt/GAPDH (E) and p-mTOR/GAPDH (F) revealed statistically significant increases in the levels of pAkt/GAPDH (p = 0.04; unpaired t test; mean ± SEM; n = 3 independent cultures) but not p-mTOR/GAPDH (p = 0.74; unpaired t test; mean ± SEM; n = 3 independent cultures) in cell lysates form the R/R cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: Elevation of pAkt levels in primary neurons treated with Aβ oligomers and in neurons from the R1.40 transgenic mouse model of AD. A. Cultured primary cortical neurons (21 DIV) were treated with vehicle (Veh), 2.0 μg/ml Aβ monomers (AβM) or 2.0 μg/ml Aβ oligomers (AβO) for 24 hours. Western blot analysis of cell extracts with antibodies against pAktS473, total Akt, p4E-BP1S65 and GAPDH revealed a slight increase in the levels of pAkt and p4E-BP1 in AβO treated cultures when compared to vehicle or AβM treated cultures. B-C. Quantification of the ratios of pAkt/total Akt (B) and p4E-BP1/GAPDH (C) revealed statistically significant increases upon exposure to AβO (2.0 μg/ml) when compared to AβM treatment (2.0 μg/ml) or vehicle controls (p < 0.05 for AβO versus vehicle or AβM; unpaired t test; mean ± SEM; n = 3 independent treatments). D. Western blot analysis of detergent soluble cell extracts of from cultured primary cortical neurons (21 DIV) from non-transgenic (WT) and homozygous R1.40 (R/R) mice with antibodies against pAkt (pAktS473), p-mTOR (p-mTORS2448) and GAPDH revealed elevated levels of pAkt (pAktS473) but not p-mTOR in the R/R neurons when compared to WT neurons. E-F Quantification of the ratios of pAkt/GAPDH (E) and p-mTOR/GAPDH (F) revealed statistically significant increases in the levels of pAkt/GAPDH (p = 0.04; unpaired t test; mean ± SEM; n = 3 independent cultures) but not p-mTOR/GAPDH (p = 0.74; unpaired t test; mean ± SEM; n = 3 independent cultures) in cell lysates form the R/R cultures.
Mentions: To confirm the ICW analysis of pAkt levels, conventional Western blot analysis was performed on cell lysates of neurons exposed to 2.0 μg/ml of Aβ oligomers, a concentration that was sufficient for the induction of neuronal CCEs. Western blots were probed with antibodies against pAkt (Ser 473) and total Akt and quantified for the relative levels of pAkt/Akt (Figure 7A and 7B). Our results demonstrate a relatively modest, but statistically significant increase in the pAkt/Akt ratio upon exposure to 2.0 μg/ml Aβ oligomers, with a 13% increase in the pAkt/Akt ratio following Aβ oligomers treatment compared to vehicle (0.6 ± 0.0096 vs 0.504 ± 0.009) and a 23% increase when compared to Aβ monomer treatment (0.6 ± 0.0096 vs 0.44 ± 0.014). These results confirm our ICW results and together demonstrate that exposure of neurons to Aβ oligomers results in altered pAkt and p-mTOR levels that correlate with the presence of neuronal CCEs as well as MAP2+ process loss.

Bottom Line: Retraction of neuronal processes correlated with the induction of CCEs and the Abeta monomer or Abeta fibrils showed only minimal effects.Finally, our results also demonstrate that Abeta oligomer treated neurons exhibit elevated levels of activated Akt and mTOR (mammalian Target Of Rapamycin) and that PI3K, Akt or mTOR inhibitors blocked Abeta oligomer-induced neuronal CCEs.Taken together, these results demonstrate that Abeta oligomer-based induction of neuronal CCEs involve the PI3K-Akt-mTOR pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurosciences, Cleveland Clinic Foundation, Cleveland, OH, USA. lambb@ccf.org.

ABSTRACT
Accumulating evidence suggests that neurons prone to degeneration in Alzheimer's Disease (AD) exhibit evidence of re-entry into an aberrant mitotic cell cycle. Our laboratory recently demonstrated that, in a genomic amyloid precursor protein (APP) mouse model of AD (R1.40), neuronal cell cycle events (CCEs) occur in the absence of beta-amyloid (Abeta) deposition and are still dependent upon the amyloidogenic processing of the amyloid precursor protein (APP). These data suggested that soluble Abeta species might play a direct role in the induction of neuronal CCEs. Here, we show that exposure of non-transgenic primary cortical neurons to Abeta oligomers, but not monomers or fibrils, results in the retraction of neuronal processes, and induction of CCEs in a concentration dependent manner. Retraction of neuronal processes correlated with the induction of CCEs and the Abeta monomer or Abeta fibrils showed only minimal effects. In addition, we provide evidence that induction of neuronal CCEs are autonomous to primary neurons cultured from the R1.40 mice. Finally, our results also demonstrate that Abeta oligomer treated neurons exhibit elevated levels of activated Akt and mTOR (mammalian Target Of Rapamycin) and that PI3K, Akt or mTOR inhibitors blocked Abeta oligomer-induced neuronal CCEs. Taken together, these results demonstrate that Abeta oligomer-based induction of neuronal CCEs involve the PI3K-Akt-mTOR pathway.

No MeSH data available.


Related in: MedlinePlus