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Interleukin-3 prevents neuronal death induced by amyloid peptide.

Zambrano A, Otth C, Mujica L, Concha II, Maccioni RB - BMC Neurosci (2007)

Bottom Line: Its effects are mediated via interaction with cell surface receptors.It is of interest to note that our results suggest that cell survival induced by IL-3 required PI 3-kinase and Jak/STAT pathway activation, but not MAP kinase.Altogether these data strongly suggest that IL-3 neuroprotects neuronal cells against neurodegenerative agents like Abeta.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Chile. angarazambrano@uach.cl

ABSTRACT

Background: Interleukin-3 (IL-3) is an important glycoprotein involved in regulating biological responses such as cell proliferation, survival and differentiation. Its effects are mediated via interaction with cell surface receptors. Several studies have demonstrated the expression of IL-3 in neurons and astrocytes of the hippocampus and cortices in normal mouse brain, suggesting a physiological role of IL-3 in the central nervous system. Although there is evidence indicating that IL-3 is expressed in some neuronal populations, its physiological role in these cells is poorly known.

Results: In this study, we demonstrated the expression of IL-3 receptor in cortical neurons, and analyzed its influence on amyloid beta (Abeta)-treated cells. In these cells, IL-3 can activate at least three classical signalling pathways, Jak/STAT, Ras/MAP kinase and the PI 3-kinase. Viability assays indicated that IL-3 might play a neuroprotective role in cells treated with Abeta fibrils. It is of interest to note that our results suggest that cell survival induced by IL-3 required PI 3-kinase and Jak/STAT pathway activation, but not MAP kinase. In addition, IL-3 induced an increase of the anti-apoptotic protein Bcl-2.

Conclusion: Altogether these data strongly suggest that IL-3 neuroprotects neuronal cells against neurodegenerative agents like Abeta.

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Related in: MedlinePlus

Inhibition of PI 3-kinase blocked Akt phosphorylation and IL-3 mediated neuroprotection against Aβ toxicity. Primary cortical neurons were pre-treated with 50 μM LY2940002 for 30 min before addition of 5 nM IL-3. One hour after addition of growth factor, cells were then exposed to 10 μM Aβ and incubated for an additional 24 h. Cells incubated with vehicle (PBS containing ≤ 0.1% DMSO v/v) and not exposed to IL-3 or Aβ were defined as control cells. Then the cells were used for Western blot and viability analysis. (A) Western blot analysis using phosphorylation-specific antibodies (p-Jak2, p-Akt, and p-BAD), and total anti-Akt1 antibodies. (B). Normalized densitometry scans of proteins (mean ± SEM, *, #, p < 0.05). The student's t-test was used for the statistical analysis of significance of difference. (C). Neuronal death was determined by MTT colorimetric assay and Tripan blue exclusion. Data represent mean ± SEM for three independent experiments (with a minimum of 4–5 wells per group for each experiment).
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Figure 3: Inhibition of PI 3-kinase blocked Akt phosphorylation and IL-3 mediated neuroprotection against Aβ toxicity. Primary cortical neurons were pre-treated with 50 μM LY2940002 for 30 min before addition of 5 nM IL-3. One hour after addition of growth factor, cells were then exposed to 10 μM Aβ and incubated for an additional 24 h. Cells incubated with vehicle (PBS containing ≤ 0.1% DMSO v/v) and not exposed to IL-3 or Aβ were defined as control cells. Then the cells were used for Western blot and viability analysis. (A) Western blot analysis using phosphorylation-specific antibodies (p-Jak2, p-Akt, and p-BAD), and total anti-Akt1 antibodies. (B). Normalized densitometry scans of proteins (mean ± SEM, *, #, p < 0.05). The student's t-test was used for the statistical analysis of significance of difference. (C). Neuronal death was determined by MTT colorimetric assay and Tripan blue exclusion. Data represent mean ± SEM for three independent experiments (with a minimum of 4–5 wells per group for each experiment).

Mentions: Activation of Akt requires the phosphorylation of Thr-308 and Ser-473 in the Aktα molecule. In this study, phosphorylation of Ser-473 was used to evaluate the activation of Akt. In order to determine whether Akt activation participates in IL-3-induced neuroprotection we used a specific inhibitor of PI 3-kinase, LY2940002, which is highly selective for PI 3-kinase inhibition. Cortical neurons were pre-treated with 50 μM LY2940002 for 30 min prior to addition of 5 nM IL-3. Cells were then exposed to 10 μM Aβ and incubated for an additional 24 h. These cells were used for Western blot, Tripan blue exclusion and MTT analysis. As shown by Western blot analysis (Figs. 3A and 3B), pre-treatment with LY2940002 blocked the IL-3-evoked Akt activation. Also, LY2940002 blocked the BAD phosphorylation, a downstream effector of Akt, but had no effect on Jak 2 phosphorylation, which is a receptor-associated kinase upstream to Akt. As shown in Fig. 3C, pre-treatment of cells with LY2940002, totally abolished the protective effect of IL-3. The same results were obtained using 100 nM Wortmannin (data not shown). These results suggest that IL-3-induced activation of Akt, by PI 3-kinase, was necessary for protection from Aβ-induced cell death.


Interleukin-3 prevents neuronal death induced by amyloid peptide.

Zambrano A, Otth C, Mujica L, Concha II, Maccioni RB - BMC Neurosci (2007)

Inhibition of PI 3-kinase blocked Akt phosphorylation and IL-3 mediated neuroprotection against Aβ toxicity. Primary cortical neurons were pre-treated with 50 μM LY2940002 for 30 min before addition of 5 nM IL-3. One hour after addition of growth factor, cells were then exposed to 10 μM Aβ and incubated for an additional 24 h. Cells incubated with vehicle (PBS containing ≤ 0.1% DMSO v/v) and not exposed to IL-3 or Aβ were defined as control cells. Then the cells were used for Western blot and viability analysis. (A) Western blot analysis using phosphorylation-specific antibodies (p-Jak2, p-Akt, and p-BAD), and total anti-Akt1 antibodies. (B). Normalized densitometry scans of proteins (mean ± SEM, *, #, p < 0.05). The student's t-test was used for the statistical analysis of significance of difference. (C). Neuronal death was determined by MTT colorimetric assay and Tripan blue exclusion. Data represent mean ± SEM for three independent experiments (with a minimum of 4–5 wells per group for each experiment).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2089076&req=5

Figure 3: Inhibition of PI 3-kinase blocked Akt phosphorylation and IL-3 mediated neuroprotection against Aβ toxicity. Primary cortical neurons were pre-treated with 50 μM LY2940002 for 30 min before addition of 5 nM IL-3. One hour after addition of growth factor, cells were then exposed to 10 μM Aβ and incubated for an additional 24 h. Cells incubated with vehicle (PBS containing ≤ 0.1% DMSO v/v) and not exposed to IL-3 or Aβ were defined as control cells. Then the cells were used for Western blot and viability analysis. (A) Western blot analysis using phosphorylation-specific antibodies (p-Jak2, p-Akt, and p-BAD), and total anti-Akt1 antibodies. (B). Normalized densitometry scans of proteins (mean ± SEM, *, #, p < 0.05). The student's t-test was used for the statistical analysis of significance of difference. (C). Neuronal death was determined by MTT colorimetric assay and Tripan blue exclusion. Data represent mean ± SEM for three independent experiments (with a minimum of 4–5 wells per group for each experiment).
Mentions: Activation of Akt requires the phosphorylation of Thr-308 and Ser-473 in the Aktα molecule. In this study, phosphorylation of Ser-473 was used to evaluate the activation of Akt. In order to determine whether Akt activation participates in IL-3-induced neuroprotection we used a specific inhibitor of PI 3-kinase, LY2940002, which is highly selective for PI 3-kinase inhibition. Cortical neurons were pre-treated with 50 μM LY2940002 for 30 min prior to addition of 5 nM IL-3. Cells were then exposed to 10 μM Aβ and incubated for an additional 24 h. These cells were used for Western blot, Tripan blue exclusion and MTT analysis. As shown by Western blot analysis (Figs. 3A and 3B), pre-treatment with LY2940002 blocked the IL-3-evoked Akt activation. Also, LY2940002 blocked the BAD phosphorylation, a downstream effector of Akt, but had no effect on Jak 2 phosphorylation, which is a receptor-associated kinase upstream to Akt. As shown in Fig. 3C, pre-treatment of cells with LY2940002, totally abolished the protective effect of IL-3. The same results were obtained using 100 nM Wortmannin (data not shown). These results suggest that IL-3-induced activation of Akt, by PI 3-kinase, was necessary for protection from Aβ-induced cell death.

Bottom Line: Its effects are mediated via interaction with cell surface receptors.It is of interest to note that our results suggest that cell survival induced by IL-3 required PI 3-kinase and Jak/STAT pathway activation, but not MAP kinase.Altogether these data strongly suggest that IL-3 neuroprotects neuronal cells against neurodegenerative agents like Abeta.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Chile. angarazambrano@uach.cl

ABSTRACT

Background: Interleukin-3 (IL-3) is an important glycoprotein involved in regulating biological responses such as cell proliferation, survival and differentiation. Its effects are mediated via interaction with cell surface receptors. Several studies have demonstrated the expression of IL-3 in neurons and astrocytes of the hippocampus and cortices in normal mouse brain, suggesting a physiological role of IL-3 in the central nervous system. Although there is evidence indicating that IL-3 is expressed in some neuronal populations, its physiological role in these cells is poorly known.

Results: In this study, we demonstrated the expression of IL-3 receptor in cortical neurons, and analyzed its influence on amyloid beta (Abeta)-treated cells. In these cells, IL-3 can activate at least three classical signalling pathways, Jak/STAT, Ras/MAP kinase and the PI 3-kinase. Viability assays indicated that IL-3 might play a neuroprotective role in cells treated with Abeta fibrils. It is of interest to note that our results suggest that cell survival induced by IL-3 required PI 3-kinase and Jak/STAT pathway activation, but not MAP kinase. In addition, IL-3 induced an increase of the anti-apoptotic protein Bcl-2.

Conclusion: Altogether these data strongly suggest that IL-3 neuroprotects neuronal cells against neurodegenerative agents like Abeta.

Show MeSH
Related in: MedlinePlus