Limits...
Lithium protects against paraquat neurotoxicity by NRF2 activation and miR-34a inhibition in SH-SY5Y cells.

Alural B, Ozerdem A, Allmer J, Genc K, Genc S - Front Cell Neurosci (2015)

Bottom Line: Furthermore, lithium significantly decreased both basal and PQ-induced expression of miR-34a.Transfection of miR-34a specific mimic reversed neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium against PQ-toxicity.Our results revealed two novel mechanisms of lithium neuroprotection, namely NRF2 activation and miR-34a suppression.

View Article: PubMed Central - PubMed

Affiliation: Izmir Biomedicine and Genome Center, Dokuz Eylul University Izmir, Turkey ; Department of Neuroscience, Health Science Institute, Dokuz Eylul University Izmir, Turkey.

ABSTRACT
Lithium is a mood stabilizing agent commonly used for the treatment of bipolar disorder. Here, we investigated the potential neuroprotective effect of lithium against paraquat toxicity and its underlying mechanisms in vitro. SH-SY5Y human neuroblastoma cells were treated with paraquat (PQ) 0.5 mM concentration after lithium pretreatment to test lithium's capability in preventing cell toxicity. Cell death was evaluated by LDH, WST-8, and tryphan blue assays. Apoptosis was analyzed using DNA fragmentation, Annexin V immunostaining, Sub G1 cell cycle analysis, and caspase-3 activity assays. BCL2, BAX, and NRF2 protein expression were evaluated by Western-blotting and the BDNF protein level was determined with ELISA. mRNA levels of BCL2, BAX, BDNF, and NRF2 target genes (HO-1, GCS, NQO1), as well as miR-34a expression were analyzed by qPCR assay. Functional experiments were done via transfection with NRF2 siRNA and miR-34a mimic. Lithium treatment prevented paraquat induced cell death and apoptosis. Lithium treated cells showed increased anti-apoptotic protein BCL2 and decreased pro-apoptotic protein BAX expression. Lithium exerted a neurotrophic effect by increasing BDNF protein expression. It also diminished reactive oxygen species production and activated the redox sensitive transcription factor NRF2 and increased its target genes expression. Knockdown of NRF2 abolished neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium. Furthermore, lithium significantly decreased both basal and PQ-induced expression of miR-34a. Transfection of miR-34a specific mimic reversed neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium against PQ-toxicity. Our results revealed two novel mechanisms of lithium neuroprotection, namely NRF2 activation and miR-34a suppression.

No MeSH data available.


Related in: MedlinePlus

Lithium decreases paraquat toxicity in SH-SY5Y cells. Cells were treated with different doses of lithium (0.5–50 mM) for 24 h. Upon lithium treatment, (A) cell viability was quantified by WST-8 assay and (B,C) percentage of cell death was analyzed by LDH release assay. 10 mM and higher doses of lithium significantly reduced cell viability. PQ toxicity in SH-SY5Y cells is (D) dose and (E) time dependent. SH-SY5Y cells were treated with lithium (2–5 mM) for 24 h and then incubated with 0.5 mM PQ for a further 24 h. (F) Cell viability was quantified by WST-8 assay and (G) cell death was determined by LDH release assay. PQ treatment increased cell death and lithium reversed the toxic effect of PQ in SH-SY5Y cells. Representative light microscopy images (H,I) of trypan blue staining showed the neuroprotective effect of lithium against PQ-induced cell death. The data are presented as mean ± standard error (S.E), n = 5. (*p < 0.05 compared to control and #p < 0.05 compare to PQ treated cells).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4446540&req=5

Figure 1: Lithium decreases paraquat toxicity in SH-SY5Y cells. Cells were treated with different doses of lithium (0.5–50 mM) for 24 h. Upon lithium treatment, (A) cell viability was quantified by WST-8 assay and (B,C) percentage of cell death was analyzed by LDH release assay. 10 mM and higher doses of lithium significantly reduced cell viability. PQ toxicity in SH-SY5Y cells is (D) dose and (E) time dependent. SH-SY5Y cells were treated with lithium (2–5 mM) for 24 h and then incubated with 0.5 mM PQ for a further 24 h. (F) Cell viability was quantified by WST-8 assay and (G) cell death was determined by LDH release assay. PQ treatment increased cell death and lithium reversed the toxic effect of PQ in SH-SY5Y cells. Representative light microscopy images (H,I) of trypan blue staining showed the neuroprotective effect of lithium against PQ-induced cell death. The data are presented as mean ± standard error (S.E), n = 5. (*p < 0.05 compared to control and #p < 0.05 compare to PQ treated cells).

Mentions: First, we examined lithium cytotoxicity by LDH and WST-8 assays. Concentrations of lithium up to 10 mM did not result in an increased rate of cell death, but higher doses (> 10 mM) of lithium were toxic to SH-SY5Y cells (Figures 1A,B). Lithium treatment at 2 mM concentration did not decrease cell viability during 24–72 h incubation periods (Figure 1C). PQ alone increased cell death in SH-SY5Y cells in a dose and time dependent fashion (Figures 1D,E).


Lithium protects against paraquat neurotoxicity by NRF2 activation and miR-34a inhibition in SH-SY5Y cells.

Alural B, Ozerdem A, Allmer J, Genc K, Genc S - Front Cell Neurosci (2015)

Lithium decreases paraquat toxicity in SH-SY5Y cells. Cells were treated with different doses of lithium (0.5–50 mM) for 24 h. Upon lithium treatment, (A) cell viability was quantified by WST-8 assay and (B,C) percentage of cell death was analyzed by LDH release assay. 10 mM and higher doses of lithium significantly reduced cell viability. PQ toxicity in SH-SY5Y cells is (D) dose and (E) time dependent. SH-SY5Y cells were treated with lithium (2–5 mM) for 24 h and then incubated with 0.5 mM PQ for a further 24 h. (F) Cell viability was quantified by WST-8 assay and (G) cell death was determined by LDH release assay. PQ treatment increased cell death and lithium reversed the toxic effect of PQ in SH-SY5Y cells. Representative light microscopy images (H,I) of trypan blue staining showed the neuroprotective effect of lithium against PQ-induced cell death. The data are presented as mean ± standard error (S.E), n = 5. (*p < 0.05 compared to control and #p < 0.05 compare to PQ treated cells).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446540&req=5

Figure 1: Lithium decreases paraquat toxicity in SH-SY5Y cells. Cells were treated with different doses of lithium (0.5–50 mM) for 24 h. Upon lithium treatment, (A) cell viability was quantified by WST-8 assay and (B,C) percentage of cell death was analyzed by LDH release assay. 10 mM and higher doses of lithium significantly reduced cell viability. PQ toxicity in SH-SY5Y cells is (D) dose and (E) time dependent. SH-SY5Y cells were treated with lithium (2–5 mM) for 24 h and then incubated with 0.5 mM PQ for a further 24 h. (F) Cell viability was quantified by WST-8 assay and (G) cell death was determined by LDH release assay. PQ treatment increased cell death and lithium reversed the toxic effect of PQ in SH-SY5Y cells. Representative light microscopy images (H,I) of trypan blue staining showed the neuroprotective effect of lithium against PQ-induced cell death. The data are presented as mean ± standard error (S.E), n = 5. (*p < 0.05 compared to control and #p < 0.05 compare to PQ treated cells).
Mentions: First, we examined lithium cytotoxicity by LDH and WST-8 assays. Concentrations of lithium up to 10 mM did not result in an increased rate of cell death, but higher doses (> 10 mM) of lithium were toxic to SH-SY5Y cells (Figures 1A,B). Lithium treatment at 2 mM concentration did not decrease cell viability during 24–72 h incubation periods (Figure 1C). PQ alone increased cell death in SH-SY5Y cells in a dose and time dependent fashion (Figures 1D,E).

Bottom Line: Furthermore, lithium significantly decreased both basal and PQ-induced expression of miR-34a.Transfection of miR-34a specific mimic reversed neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium against PQ-toxicity.Our results revealed two novel mechanisms of lithium neuroprotection, namely NRF2 activation and miR-34a suppression.

View Article: PubMed Central - PubMed

Affiliation: Izmir Biomedicine and Genome Center, Dokuz Eylul University Izmir, Turkey ; Department of Neuroscience, Health Science Institute, Dokuz Eylul University Izmir, Turkey.

ABSTRACT
Lithium is a mood stabilizing agent commonly used for the treatment of bipolar disorder. Here, we investigated the potential neuroprotective effect of lithium against paraquat toxicity and its underlying mechanisms in vitro. SH-SY5Y human neuroblastoma cells were treated with paraquat (PQ) 0.5 mM concentration after lithium pretreatment to test lithium's capability in preventing cell toxicity. Cell death was evaluated by LDH, WST-8, and tryphan blue assays. Apoptosis was analyzed using DNA fragmentation, Annexin V immunostaining, Sub G1 cell cycle analysis, and caspase-3 activity assays. BCL2, BAX, and NRF2 protein expression were evaluated by Western-blotting and the BDNF protein level was determined with ELISA. mRNA levels of BCL2, BAX, BDNF, and NRF2 target genes (HO-1, GCS, NQO1), as well as miR-34a expression were analyzed by qPCR assay. Functional experiments were done via transfection with NRF2 siRNA and miR-34a mimic. Lithium treatment prevented paraquat induced cell death and apoptosis. Lithium treated cells showed increased anti-apoptotic protein BCL2 and decreased pro-apoptotic protein BAX expression. Lithium exerted a neurotrophic effect by increasing BDNF protein expression. It also diminished reactive oxygen species production and activated the redox sensitive transcription factor NRF2 and increased its target genes expression. Knockdown of NRF2 abolished neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium. Furthermore, lithium significantly decreased both basal and PQ-induced expression of miR-34a. Transfection of miR-34a specific mimic reversed neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium against PQ-toxicity. Our results revealed two novel mechanisms of lithium neuroprotection, namely NRF2 activation and miR-34a suppression.

No MeSH data available.


Related in: MedlinePlus