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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

Aβ oligomer induced neuronal CCEs are blocked with inhibitors of the PI3K/Akt/mTOR pathway. A. Cultured primary cortical neurons (21 DIV) were pretreated for 30 min with the PI3K inhibitor wortmannin (100 nM), an Akt inhibitor (100 nM) and the mTOR inhibitor rapamycin (1 μM), followed by exposure to 2.0 μg/ml of Aβ oligomers (AβO) for 24 hours in the presence of BrdU. Following fixation, cells were stained with specific antibodies against BrdU and MAP2. Quantification of the BrdU positive/MAP2 positive cells demonstrated a statistically significant decrease in the percentage of neurons positive for BrdU with all three inhibitors (p < 0.001 for AβO vs AβO+wortmannin, AβO+Akt inhibitor and AβO+rapamycin; one-way ANOVA with Tukey multiple comparison test for pair-wise comparisons; mean ± SEM; n = 4 independent treatments per group). B. Quantification of the loss of MAP2 positive processes upon treatment with AβO in the presence of the PI3K, Akt and mTOR inhibitors was determined via automated image processing and revealed a statistically significant increase in the number of MAP2 positive processes upon pre-treatment with the PI3K inhibitor (p = 0.0005; unpaired t test; mean ± SEM; n = 4 independent treatments), but not the Akt or mTOR inhibitors when compared to AβO treatment alone. C. Diagram outlining a potential pathway underlining the effects of Aβ oligomers on neuronal process retraction and CCEs. Our data suggest that Aβ oligomers activate the PI3K-Akt-mTOR signaling pathway. Activation of PI3K causes phosphorylation of Akt at Ser473, which in turn activates mTOR via phosphorylation at Ser2448. Activation of mTOR results in induction of cell proliferation by down regulation of 4E-BP1 via inhibitory phosphorylation at ser65. Interestingly, our data suggest that the dendritic alterations induced by Aβ oligomers is blocked via inhibition of PI3K via as yet to be identified downstream effectors.
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Figure 8: Aβ oligomer induced neuronal CCEs are blocked with inhibitors of the PI3K/Akt/mTOR pathway. A. Cultured primary cortical neurons (21 DIV) were pretreated for 30 min with the PI3K inhibitor wortmannin (100 nM), an Akt inhibitor (100 nM) and the mTOR inhibitor rapamycin (1 μM), followed by exposure to 2.0 μg/ml of Aβ oligomers (AβO) for 24 hours in the presence of BrdU. Following fixation, cells were stained with specific antibodies against BrdU and MAP2. Quantification of the BrdU positive/MAP2 positive cells demonstrated a statistically significant decrease in the percentage of neurons positive for BrdU with all three inhibitors (p < 0.001 for AβO vs AβO+wortmannin, AβO+Akt inhibitor and AβO+rapamycin; one-way ANOVA with Tukey multiple comparison test for pair-wise comparisons; mean ± SEM; n = 4 independent treatments per group). B. Quantification of the loss of MAP2 positive processes upon treatment with AβO in the presence of the PI3K, Akt and mTOR inhibitors was determined via automated image processing and revealed a statistically significant increase in the number of MAP2 positive processes upon pre-treatment with the PI3K inhibitor (p = 0.0005; unpaired t test; mean ± SEM; n = 4 independent treatments), but not the Akt or mTOR inhibitors when compared to AβO treatment alone. C. Diagram outlining a potential pathway underlining the effects of Aβ oligomers on neuronal process retraction and CCEs. Our data suggest that Aβ oligomers activate the PI3K-Akt-mTOR signaling pathway. Activation of PI3K causes phosphorylation of Akt at Ser473, which in turn activates mTOR via phosphorylation at Ser2448. Activation of mTOR results in induction of cell proliferation by down regulation of 4E-BP1 via inhibitory phosphorylation at ser65. Interestingly, our data suggest that the dendritic alterations induced by Aβ oligomers is blocked via inhibition of PI3K via as yet to be identified downstream effectors.

Mentions: To provide evidence that the PI3K-Akt-mTOR pathway is directly involved in the Aβ oligomer-induced neuronal CCEs, WT primary cortical neurons were incubated with specific, cell-permeable, irreversible pharmacological inhibitors (Wortmannin for PI3K, Akt-inhibitor for Akt and Rapamycin for mTOR) 30 min prior to addition of Aβ oligomers (2.0 μg/ml) in the presence of BrdU. The number of BrdU+ MAP2+ cells was quantified for each treatment group in triplicates. Strikingly, pre-treatment of primary cortical neurons with PI3K, Akt or mTOR inhibitors resulted in over 80% reduction in the number of BrdU positive neurons compared to Aβ oligomer exposure alone (Figure 8A). This decrease in BrdU incorporation was not merely due to fewer numbers of cells as indicated by similar numbers of MAP2+ cells and DAPI stained nuclei and also the inhibitors alone did not have any direct effect on BrdU incorporation (data not shown). This suggests that the inhibition of several steps within the PI3K/Akt/mTOR pathway blocks Aβ oligomer induced neuronal CCEs.


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)

Aβ oligomer induced neuronal CCEs are blocked with inhibitors of the PI3K/Akt/mTOR pathway. A. Cultured primary cortical neurons (21 DIV) were pretreated for 30 min with the PI3K inhibitor wortmannin (100 nM), an Akt inhibitor (100 nM) and the mTOR inhibitor rapamycin (1 μM), followed by exposure to 2.0 μg/ml of Aβ oligomers (AβO) for 24 hours in the presence of BrdU. Following fixation, cells were stained with specific antibodies against BrdU and MAP2. Quantification of the BrdU positive/MAP2 positive cells demonstrated a statistically significant decrease in the percentage of neurons positive for BrdU with all three inhibitors (p < 0.001 for AβO vs AβO+wortmannin, AβO+Akt inhibitor and AβO+rapamycin; one-way ANOVA with Tukey multiple comparison test for pair-wise comparisons; mean ± SEM; n = 4 independent treatments per group). B. Quantification of the loss of MAP2 positive processes upon treatment with AβO in the presence of the PI3K, Akt and mTOR inhibitors was determined via automated image processing and revealed a statistically significant increase in the number of MAP2 positive processes upon pre-treatment with the PI3K inhibitor (p = 0.0005; unpaired t test; mean ± SEM; n = 4 independent treatments), but not the Akt or mTOR inhibitors when compared to AβO treatment alone. C. Diagram outlining a potential pathway underlining the effects of Aβ oligomers on neuronal process retraction and CCEs. Our data suggest that Aβ oligomers activate the PI3K-Akt-mTOR signaling pathway. Activation of PI3K causes phosphorylation of Akt at Ser473, which in turn activates mTOR via phosphorylation at Ser2448. Activation of mTOR results in induction of cell proliferation by down regulation of 4E-BP1 via inhibitory phosphorylation at ser65. Interestingly, our data suggest that the dendritic alterations induced by Aβ oligomers is blocked via inhibition of PI3K via as yet to be identified downstream effectors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2663563&req=5

Figure 8: Aβ oligomer induced neuronal CCEs are blocked with inhibitors of the PI3K/Akt/mTOR pathway. A. Cultured primary cortical neurons (21 DIV) were pretreated for 30 min with the PI3K inhibitor wortmannin (100 nM), an Akt inhibitor (100 nM) and the mTOR inhibitor rapamycin (1 μM), followed by exposure to 2.0 μg/ml of Aβ oligomers (AβO) for 24 hours in the presence of BrdU. Following fixation, cells were stained with specific antibodies against BrdU and MAP2. Quantification of the BrdU positive/MAP2 positive cells demonstrated a statistically significant decrease in the percentage of neurons positive for BrdU with all three inhibitors (p < 0.001 for AβO vs AβO+wortmannin, AβO+Akt inhibitor and AβO+rapamycin; one-way ANOVA with Tukey multiple comparison test for pair-wise comparisons; mean ± SEM; n = 4 independent treatments per group). B. Quantification of the loss of MAP2 positive processes upon treatment with AβO in the presence of the PI3K, Akt and mTOR inhibitors was determined via automated image processing and revealed a statistically significant increase in the number of MAP2 positive processes upon pre-treatment with the PI3K inhibitor (p = 0.0005; unpaired t test; mean ± SEM; n = 4 independent treatments), but not the Akt or mTOR inhibitors when compared to AβO treatment alone. C. Diagram outlining a potential pathway underlining the effects of Aβ oligomers on neuronal process retraction and CCEs. Our data suggest that Aβ oligomers activate the PI3K-Akt-mTOR signaling pathway. Activation of PI3K causes phosphorylation of Akt at Ser473, which in turn activates mTOR via phosphorylation at Ser2448. Activation of mTOR results in induction of cell proliferation by down regulation of 4E-BP1 via inhibitory phosphorylation at ser65. Interestingly, our data suggest that the dendritic alterations induced by Aβ oligomers is blocked via inhibition of PI3K via as yet to be identified downstream effectors.
Mentions: To provide evidence that the PI3K-Akt-mTOR pathway is directly involved in the Aβ oligomer-induced neuronal CCEs, WT primary cortical neurons were incubated with specific, cell-permeable, irreversible pharmacological inhibitors (Wortmannin for PI3K, Akt-inhibitor for Akt and Rapamycin for mTOR) 30 min prior to addition of Aβ oligomers (2.0 μg/ml) in the presence of BrdU. The number of BrdU+ MAP2+ cells was quantified for each treatment group in triplicates. Strikingly, pre-treatment of primary cortical neurons with PI3K, Akt or mTOR inhibitors resulted in over 80% reduction in the number of BrdU positive neurons compared to Aβ oligomer exposure alone (Figure 8A). This decrease in BrdU incorporation was not merely due to fewer numbers of cells as indicated by similar numbers of MAP2+ cells and DAPI stained nuclei and also the inhibitors alone did not have any direct effect on BrdU incorporation (data not shown). This suggests that the inhibition of several steps within the PI3K/Akt/mTOR pathway blocks Aβ oligomer induced neuronal CCEs.

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