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 reduces apoptotic cell death induced by PQ in SH-SY5Y cells. (A) DNA fragmentation was increased with 0.5 mM PQ treatment, which was analyzed by Cell Death ELISA assay. (B) Lithium (2 mM and 5 mM) pretreatment reduces DNA fragmentation induced by PQ in SH-SY5Y cells. (C) Apoptotic cells were stained by Annexin-V-FITC dye and visualized using immunofluorescence microscopy. (D) Flow cytometric analysis of the sub G1 apoptotic population was assessed by using PI staining. Lithium attenuates PQ induced increase of sub G1 apoptotic population in SH-SY5Y cells (E). Caspase-3 activity was evaluated in lysates of treated cells by spectrophotometric detection of the chromophore p-nitroaniline (pNA) formed after cleavage from the labeled substrate DEVD-pNA. Lithium reduced PQ induced caspase-3 activity increase in SH-SY5Y cells. The data are presented as mean ± 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 2: Lithium reduces apoptotic cell death induced by PQ in SH-SY5Y cells. (A) DNA fragmentation was increased with 0.5 mM PQ treatment, which was analyzed by Cell Death ELISA assay. (B) Lithium (2 mM and 5 mM) pretreatment reduces DNA fragmentation induced by PQ in SH-SY5Y cells. (C) Apoptotic cells were stained by Annexin-V-FITC dye and visualized using immunofluorescence microscopy. (D) Flow cytometric analysis of the sub G1 apoptotic population was assessed by using PI staining. Lithium attenuates PQ induced increase of sub G1 apoptotic population in SH-SY5Y cells (E). Caspase-3 activity was evaluated in lysates of treated cells by spectrophotometric detection of the chromophore p-nitroaniline (pNA) formed after cleavage from the labeled substrate DEVD-pNA. Lithium reduced PQ induced caspase-3 activity increase in SH-SY5Y cells. The data are presented as mean ± S.E, n = 5. (*p < 0.05 compared to control and #p < 0.05 compare to PQ treated cells).

Mentions: Our results showed a significant 2.4-fold increase in DNA fragmentation upon 48 h of PQ treatment (Figure 2A). Pretreatment with lithium attenuated PQ induced DNA fragmentation significantly (Figure 2B).


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 reduces apoptotic cell death induced by PQ in SH-SY5Y cells. (A) DNA fragmentation was increased with 0.5 mM PQ treatment, which was analyzed by Cell Death ELISA assay. (B) Lithium (2 mM and 5 mM) pretreatment reduces DNA fragmentation induced by PQ in SH-SY5Y cells. (C) Apoptotic cells were stained by Annexin-V-FITC dye and visualized using immunofluorescence microscopy. (D) Flow cytometric analysis of the sub G1 apoptotic population was assessed by using PI staining. Lithium attenuates PQ induced increase of sub G1 apoptotic population in SH-SY5Y cells (E). Caspase-3 activity was evaluated in lysates of treated cells by spectrophotometric detection of the chromophore p-nitroaniline (pNA) formed after cleavage from the labeled substrate DEVD-pNA. Lithium reduced PQ induced caspase-3 activity increase in SH-SY5Y cells. The data are presented as mean ± 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 2: Lithium reduces apoptotic cell death induced by PQ in SH-SY5Y cells. (A) DNA fragmentation was increased with 0.5 mM PQ treatment, which was analyzed by Cell Death ELISA assay. (B) Lithium (2 mM and 5 mM) pretreatment reduces DNA fragmentation induced by PQ in SH-SY5Y cells. (C) Apoptotic cells were stained by Annexin-V-FITC dye and visualized using immunofluorescence microscopy. (D) Flow cytometric analysis of the sub G1 apoptotic population was assessed by using PI staining. Lithium attenuates PQ induced increase of sub G1 apoptotic population in SH-SY5Y cells (E). Caspase-3 activity was evaluated in lysates of treated cells by spectrophotometric detection of the chromophore p-nitroaniline (pNA) formed after cleavage from the labeled substrate DEVD-pNA. Lithium reduced PQ induced caspase-3 activity increase in SH-SY5Y cells. The data are presented as mean ± S.E, n = 5. (*p < 0.05 compared to control and #p < 0.05 compare to PQ treated cells).
Mentions: Our results showed a significant 2.4-fold increase in DNA fragmentation upon 48 h of PQ treatment (Figure 2A). Pretreatment with lithium attenuated PQ induced DNA fragmentation significantly (Figure 2B).

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