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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 oligomers induce loss of MAP2 positive processes in primary neurons coincident with BrdU incorporation. Cultured primary cortical neurons (21 DIV) were treated with vehicle (Veh, A and D), 4 μg/ml Aβ monomers (AβM, B and E) or 4 μg/ml Aβ oligomers (AβO, C and F) for 24 hours. Following fixation, cells were immunostained with antibodies against MAP2, revealing several long MAP2 positives processes per cell in the vehicle treatment group, a modest reduction in the number of MAP2 positive processes in the AβM treatment group and a dramatic reduction in the number and length of the MAP2 processes in the AβO treatment group. Scale bar, 10 μm. MAP2 positive neurons with shorter processes displaying BrdU incorporation (arrows in F). G-H. Quantification of MAP2 positive dendrites following exposure to Veh or different concentrations of AβM (G) and AβO (H) via automated image processing revealed a statistically significant decrease in MAP2 positive processes (p = 0.006; Veh versus AβO-4 μg/ml; unpaired t test; mean ± SEM; n = 3 independent experiments) when compared to either vehicle or AβM. I-J. Quantification of the total number (I) of MAP2 positive processes as well as the number of processes longer than 5 μm per cell (J) in both BrdU positive and BrdU negative neurons revealed a statistically significant reduction in both number of MAP2 + processes (p = 0.015; mean ± SEM; unpaired t test; n = 3) as well as the number of processes longer than 5 μm (p = 0.05; mean ± SEM; unpaired t test; n = 3) in BrdU positive cells when compared to BrdU negative cells.
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Figure 3: Aβ oligomers induce loss of MAP2 positive processes in primary neurons coincident with BrdU incorporation. Cultured primary cortical neurons (21 DIV) were treated with vehicle (Veh, A and D), 4 μg/ml Aβ monomers (AβM, B and E) or 4 μg/ml Aβ oligomers (AβO, C and F) for 24 hours. Following fixation, cells were immunostained with antibodies against MAP2, revealing several long MAP2 positives processes per cell in the vehicle treatment group, a modest reduction in the number of MAP2 positive processes in the AβM treatment group and a dramatic reduction in the number and length of the MAP2 processes in the AβO treatment group. Scale bar, 10 μm. MAP2 positive neurons with shorter processes displaying BrdU incorporation (arrows in F). G-H. Quantification of MAP2 positive dendrites following exposure to Veh or different concentrations of AβM (G) and AβO (H) via automated image processing revealed a statistically significant decrease in MAP2 positive processes (p = 0.006; Veh versus AβO-4 μg/ml; unpaired t test; mean ± SEM; n = 3 independent experiments) when compared to either vehicle or AβM. I-J. Quantification of the total number (I) of MAP2 positive processes as well as the number of processes longer than 5 μm per cell (J) in both BrdU positive and BrdU negative neurons revealed a statistically significant reduction in both number of MAP2 + processes (p = 0.015; mean ± SEM; unpaired t test; n = 3) as well as the number of processes longer than 5 μm (p = 0.05; mean ± SEM; unpaired t test; n = 3) in BrdU positive cells when compared to BrdU negative cells.

Mentions: In analyzing the effect of Aβ oligomers on cortical neurons, a decrease in the number of MAP2 staining neuronal processes was consistently observed (see Figure 2G–I and Figure 3A–F). In general, control neurons exhibited 4–5 MAP2+ processes per cell, while those exposed to Aβ oligomers exhibited an apparent concentration dependent decrease in the number of MAP2+ processes together with increased nuclear BrdU staining. Quantification of these results was performed by examining the total number of dendrites per field in 5 random fields per treatment using a macro ("Dendrites") in the ImagePro Plus™ image analysis software (Materials and Methods). The data revealed that the number of dendrites was significantly reduced upon exposure to increasing concentration of Aβ oligomers (Figure 3H) when compared to either vehicle or increasing concentrations of Aβ monomer treatment (Figure 3G) or Aβ fibrils (data not shown). These results are consistent with earlier reports demonstrating that Aβ oligomer exposure induced a loss of the spine marker, drebrin, as well as dramatic changes in spine morphology and density in mature hippocampal cultures [15]. Similar findings were obtained upon Aβ exposure of 7 DIV cortical neuronal cultures (data not shown). Interestingly, none of the Aβ treatments, even at the highest concentrations, altered the total number of MAP2 positive cells nor exhibited a significant alteration in neuronal cell number as determined by measuring the formation of formazan from reduced MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in an in vitro cell-death assay (data not shown). These results suggests that consistent with previous data, Aβ oligomer exposure at sub-nanomolar concentrations induces synaptic and dendrite loss in the absence of robust neurodegeneration [15,33].


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 oligomers induce loss of MAP2 positive processes in primary neurons coincident with BrdU incorporation. Cultured primary cortical neurons (21 DIV) were treated with vehicle (Veh, A and D), 4 μg/ml Aβ monomers (AβM, B and E) or 4 μg/ml Aβ oligomers (AβO, C and F) for 24 hours. Following fixation, cells were immunostained with antibodies against MAP2, revealing several long MAP2 positives processes per cell in the vehicle treatment group, a modest reduction in the number of MAP2 positive processes in the AβM treatment group and a dramatic reduction in the number and length of the MAP2 processes in the AβO treatment group. Scale bar, 10 μm. MAP2 positive neurons with shorter processes displaying BrdU incorporation (arrows in F). G-H. Quantification of MAP2 positive dendrites following exposure to Veh or different concentrations of AβM (G) and AβO (H) via automated image processing revealed a statistically significant decrease in MAP2 positive processes (p = 0.006; Veh versus AβO-4 μg/ml; unpaired t test; mean ± SEM; n = 3 independent experiments) when compared to either vehicle or AβM. I-J. Quantification of the total number (I) of MAP2 positive processes as well as the number of processes longer than 5 μm per cell (J) in both BrdU positive and BrdU negative neurons revealed a statistically significant reduction in both number of MAP2 + processes (p = 0.015; mean ± SEM; unpaired t test; n = 3) as well as the number of processes longer than 5 μm (p = 0.05; mean ± SEM; unpaired t test; n = 3) in BrdU positive cells when compared to BrdU negative cells.
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Related In: Results  -  Collection

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Figure 3: Aβ oligomers induce loss of MAP2 positive processes in primary neurons coincident with BrdU incorporation. Cultured primary cortical neurons (21 DIV) were treated with vehicle (Veh, A and D), 4 μg/ml Aβ monomers (AβM, B and E) or 4 μg/ml Aβ oligomers (AβO, C and F) for 24 hours. Following fixation, cells were immunostained with antibodies against MAP2, revealing several long MAP2 positives processes per cell in the vehicle treatment group, a modest reduction in the number of MAP2 positive processes in the AβM treatment group and a dramatic reduction in the number and length of the MAP2 processes in the AβO treatment group. Scale bar, 10 μm. MAP2 positive neurons with shorter processes displaying BrdU incorporation (arrows in F). G-H. Quantification of MAP2 positive dendrites following exposure to Veh or different concentrations of AβM (G) and AβO (H) via automated image processing revealed a statistically significant decrease in MAP2 positive processes (p = 0.006; Veh versus AβO-4 μg/ml; unpaired t test; mean ± SEM; n = 3 independent experiments) when compared to either vehicle or AβM. I-J. Quantification of the total number (I) of MAP2 positive processes as well as the number of processes longer than 5 μm per cell (J) in both BrdU positive and BrdU negative neurons revealed a statistically significant reduction in both number of MAP2 + processes (p = 0.015; mean ± SEM; unpaired t test; n = 3) as well as the number of processes longer than 5 μm (p = 0.05; mean ± SEM; unpaired t test; n = 3) in BrdU positive cells when compared to BrdU negative cells.
Mentions: In analyzing the effect of Aβ oligomers on cortical neurons, a decrease in the number of MAP2 staining neuronal processes was consistently observed (see Figure 2G–I and Figure 3A–F). In general, control neurons exhibited 4–5 MAP2+ processes per cell, while those exposed to Aβ oligomers exhibited an apparent concentration dependent decrease in the number of MAP2+ processes together with increased nuclear BrdU staining. Quantification of these results was performed by examining the total number of dendrites per field in 5 random fields per treatment using a macro ("Dendrites") in the ImagePro Plus™ image analysis software (Materials and Methods). The data revealed that the number of dendrites was significantly reduced upon exposure to increasing concentration of Aβ oligomers (Figure 3H) when compared to either vehicle or increasing concentrations of Aβ monomer treatment (Figure 3G) or Aβ fibrils (data not shown). These results are consistent with earlier reports demonstrating that Aβ oligomer exposure induced a loss of the spine marker, drebrin, as well as dramatic changes in spine morphology and density in mature hippocampal cultures [15]. Similar findings were obtained upon Aβ exposure of 7 DIV cortical neuronal cultures (data not shown). Interestingly, none of the Aβ treatments, even at the highest concentrations, altered the total number of MAP2 positive cells nor exhibited a significant alteration in neuronal cell number as determined by measuring the formation of formazan from reduced MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in an in vitro cell-death assay (data not shown). These results suggests that consistent with previous data, Aβ oligomer exposure at sub-nanomolar concentrations induces synaptic and dendrite loss in the absence of robust neurodegeneration [15,33].

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