Ethanol Suppresses PGC-1α Expression by Interfering with the cAMP-CREB Pathway in Neuronal Cells

<p>Alcohol intoxication results in neuronal apoptosis, neurodegeneration and manifest with impaired balance, loss of muscle coordination and behavioral changes. One of the early events of alcohol intoxication is mitochondrial (Mt) dysfunction and disruption of intracellular redox homeostasis. The mechanisms by which alcohol causes Mt dysfunction, disrupts cellular redox homeostasis and triggers neurodegeneration remains to be further investigated. Proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) plays critical roles in regulating Mt biogenesis and respiration, cellular antioxidant defense mechanism, and maintenance of neuronal integrity and function. In this study, we sought to investigate whether alcohol causes Mt dysfunction and triggers neurodegeneration by suppressing PGC-1α expression. We report that ethanol suppresses PGC-1α expression, and impairs mitochondrial function and enhances cellular toxicity in cultured neuronal cell line and also in human fetal brain neural stem cell-derived primary neurons. Moreover, we report that cells over-expressing exogenous PGC-1α or treated with Rolipram, a selective phosphodiesterase-4 inhibitor, ameliorate alcohol-induced cellular toxicity. Further analysis show that ethanol decreases steady-state intracellular cAMP levels, and thus depletes phosphorylation of cAMP-response element binding protein (p-CREB), the key transcription factor that regulates transcription of PGC-1a gene. Accordingly, we found PGC-1α promoter activity and transcription was dramatically repressed in neuronal cells when exposed to ethanol, suggesting that ethanol blunts cAMP→CREB signaling pathway to interfere with the transcription of PGC-1α. Ethanol-mediated decrease in PGC-1α activity results in the disruption of Mt respiration and function and higher cellular toxicity. This study might lead to potential therapeutic intervention to ameliorate alcohol-induced apoptosis and/or neurodegeneration by targeting PGC-1α.</p

Time- and dose-dependent effects of ethanol on cellular toxicity in SH-SY5Y cells.

<p>The confluent SH-SY5Y cells were treated with 300 mM ethanol for various times (<b>Panel A</b>), or various concentrations of ethanol for 24 hours (<b>Panel B</b>). The cell culture medium was collected, and the LDH release was measured to assess ethanol-induced cellular toxicity. Mean ± SE, n = 4; **<i>P</i><0.01 <i>vs.</i> control.</p

Concentration-dependent effect of ethanol on mitochondrial function, and expression of the <i>PGC-1α</i> in SH-SY5Y cells.

<p>The SH-SY5Y cells were treated with various concentration of ethanol for 24 hours, and MTS activity and intracellular ATP level were measured to assess mitochondrial function (<b>Panel A & B</b>). Total protein was isolated from the ethanol-treated and control cells and expression of <i>PGC-1α</i> was measured by Western blot analysis, and a representative Western blot is shown in Panel C. The protein density in each Western blot was quantitatively analyzed, and is shown in Panel D. Mean ± SE, n = 3, *<i>P</i><0.05 <i>vs.</i> control, **<i>P</i><0.01.</p

Ethanol Suppresses <i>PGC-1α - Figure 8 </i> Expression by Interfering with the cAMP-CREB Pathway in Neuronal Cells

<p>Dose-dependent effect of ethanol on the expression levels of PGC-1α, CREB, and p-CREB in human fetal brain neural stem cell-derived primary neurons. The primary neuronal cells were incubated with various concentrations of ethanol for 24 hrs, and after incubation with ethanol, the cells lysates were analyzed by Western blot to determine expression levels of PGC-1α, CREB, and p-CREB (Panel A). β-actin was used as loading control. The protein density in each Western blot was quantitatively analyzed, and is shown in Panels B and C. Mean ± SE, n = 3, *<i>P</i><0.05, **<i>P</i><0.01 <i>vs.</i> control.</p

Dose-dependent effects of ethanol on intracellular cAMP level in SH-SY5Y cells.

<p>The confluent SH-SY5Y cells were treated with various concentrations of ethanol for 24 hours. The cells were corrected and lysed, and cellular cAMP levels were measured by cAMP analysis kit. Mean ± SE, n = 4. *<i>P</i><0.05 vs. control, and **<i>P</i><0.01 <i>vs.</i> control.</p

The proposed mechanisms for ethanol-mediated regulation of PGC-1α expression and neurodegeneration.

<p>The proposed mechanisms for ethanol-mediated regulation of PGC-1α expression and neurodegeneration.</p

Over-expression of PGC-1α protect SH-SY5Y cells from ethanol-induced cellular toxicity.

<p>The SH-SY5Y cell line constitutively over-expressing PGC-1α was treated with various concentrations of alcohol and cellular toxicity (relative cell viability: panel A; LDH release: panel B) was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104247#pone-0104247-g001" target="_blank">Figure 1</a>. Mean ± SE, n = 4, *<i>P</i><0.05. **<i>P</i><0.01 <i>vs.</i> control.</p

Effect of ethanol on PGC-1α promoter activity and <i>PGC-1α</i> expression in SH-SY5Y cells.

<p>The SH-SY5Y cells were co-transfected with <i>PGC-1α</i> promoter plasmid and luciferase-expression plasmids, and after 24 hours the cells were treated with various concentrations of ethanol for 24 hours. The ethanol-treated and control cells were lysed, and luciferase activity was measured (Panel A). In a separated experiment, total RNA was extracted from the ethanol-treated (500 mM) and control cells, and the level of PGC-1α mRNA was measured by real time quantitative RT-PCR (Panel B). Mean ± SE, n = 4, **<i>P</i><0.01 <i>vs.</i> Control.</p

Dose-dependent effect of ethanol on the expression of CREB, P-CREB and PARIS in SH-SY5Y cells.

<p>The SH-SY5Y cells were treated with various concentrations of ethanol for 48 hours. After the ethanol treatment, the cells were harvested and the cell lysate was analyzed to determine expression of total CREB, p-CREB and PARIS by Western blot analysis. β-actin was used for loading control. The protein density in each Western blot was quantitatively analyzed, and is shown in Panels B and C. Mean ± SE, n = 3, *<i>P</i><0.05, **<i>P</i><0.01 <i>vs.</i> control.</p

Effect of a PDE-4 inhibitor on ethanol-induced cytotoxicity and the expression of PGC-1α in SH-SY5Y cells.

<p>The confluent SH-SY5Y cells were pretreated with 30 µM Rolipram (a selective PDE-4 inhibitor) for 2 hrs, and then continuously treated with same concentration of Rolipram in the presence of various concentrations of alcohol for 24 hrs or 300 mM ethanol for different times. LDH release in the medium was measured, and the expression of PGC-1α was analyzed by Western-blot as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104247#pone-0104247-g002" target="_blank">Figure 2</a>. Mean ± SE, n = 4. *<i>P</i><0.05 <i>vs.</i> control, and **<i>P</i><0.01 <i>vs.</i> control.</p