ERK/MAPK Is Essential for Endogenous Neuroprotection in SCN2.2 Cells

Glutamate (Glu) is essential to central nervous system function; however excessive Glu release leads to neurodegenerative disease. Strategies to protect neurons are underdeveloped, in part due to a limited understanding of natural neuroprotective mechanisms, such as those present in the suprachiasmatic nucleus (SCN). This study tests the hypothesis that activation of ERK/MAPK provides essential protection to the SCN after exposure to excessive Glu using the SCN2.2 cells as a model.Immortalized SCN2.2 cells (derived from SCN) and GT1-7 cells (neurons from the neighboring hypothalamus) were treated with 10 mM Glu in the presence or absence of the ERK/MAPK inhibitor PD98059. Cell death was assessed by Live/Dead assay, MTS assay and TUNEL. Caspase 3 activity was also measured. Activation of MAPK family members was determined by immunoblot. Bcl2, neuritin and Bid mRNA (by quantitative-PCR) and protein levels (by immunoblot) were also measured.As expected Glu treatment increased caspase 3 activity and cell death in the GT1-7 cells, but Glu alone did not induce cell death or affect caspase 3 activity in the SCN2.2 cells. However, pretreatment with PD98059 increased caspase 3 activity and resulted in cell death after Glu treatment in SCN2.2 cells. This effect was dependent on NMDA receptor activation. Glu treatment in the SCN2.2 cells resulted in sustained activation of the anti-apoptotic pERK/MAPK, without affecting the pro-apoptotic p-p38/MAPK. In contrast, Glu exposure in GT1-7 cells caused an increase in p-p38/MAPK and a decrease in pERK/MAPK. Bcl2-protein increased in SCN2.2 cells following Glu treatment, but not in GT1-7 cells; bid mRNA and cleaved-Bid protein increased in GT1-7, but not SCN2.2 cells.Facilitation of ERK activation and inhibition of caspase activation promotes resistance to Glu excitotoxicity in SCN2.2 cells.Further research will explore ERK/MAPK as a key molecule in the prevention of neurodegenerative processes

Disruption of CLOCK-BMAL1 Transcriptional Activity Is Responsible for Aryl Hydrocarbon Receptor–Mediated Regulation of Period1 Gene

The aryl hydrocarbon receptor (AhR) is a period-aryl hydrocarbon receptor nuclear transporter-simple minded domain transcription factor that shares structural similarity with circadian clock genes and readily interacts with components of the molecular clock. Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters behavioral circadian rhythms and represses the Period1 (Per1) gene in murine hematopoietic stem and progenitor cells. Per1 expression is driven by circadian locomotor activity cycles kaput-brain muscle ARNT-like (CLOCK-BMAL1)–dependent activation of Eboxes in the Per1 promoter. We hypothesized that the effects of AhR activation on the circadian clock are mediated by disruption of CLOCK-BMAL1 function and subsequent Per1 gene suppression. Effects of AhR activation on rhythmic Per1 transcripts were examined in livers of mice after treatment with the AhR agonist, TCDD; the molecular mechanisms of Per1 repression by AhR were determined in hepatoma cells using TCDD and β-napthoflavone as AhR activators. This study reports, for the first time, that AhR activation by TCDD alters the Per1 rhythm in the mouse liver and that Per1 gene suppression depends upon the presence of AhR. Furthermore, AhR interaction with BMAL1 attenuates CLOCK-BMAL1 activity and decreases CLOCK binding at Ebox1 and Ebox3 in the Per1 promoter. Taken together, these data suggest that AhR activation represses Per1 through disrupting CLOCK-BMAL1 activity, producing dysregulation of rhythmic Per1 gene expression. These data define alteration of the Per1 rhythm as novel signaling events downstream of AhR activation. Downregulation of Per1 could contribute to metabolic disease, cancer, and other detrimental effects resulting from exposure to certain environmental pollutants

ERK/MAPK Inhibitor PD98059 Induces NMDAR-Mediated Cell Death in SCN2.2 Cells.

<p><b>A:</b> Live cells in GT1-7 and SCN2.2 cultures exposed to normal culture conditions (control); 10 µM PD98059 (PD); 10 mM Glu (Glu); or 10 mM Glu+10 µM PD98059 (Glu+PD) for 48 h. PD98059 was added 1 h prior to Glu, and continued throughout Glu treatment. Live cells were measured with the Live/Dead assay. Results are mean ± SD from 2 experiments with n = 18 in each. <b>B:</b> Dead cells as measured by Live/Dead assay for same samples as in A. <b>C:</b> % Metabolic activity in GT1-7 and SCN2.2 cultures exposed to normal culture conditions (control); 10 µM PD98059 (PD); 10 µM MK-801 (MK); 10 µM PD98059+10 µM MK-801 (PD+MK); 50 µM NMDA+10 µM MK-801 (NMDA+MK); 50 µM NMDA+10 µM MK-801+10 µM PD98059 (NMDA+MK+PD); 50 µM NMDA (NMDA); or 50 µM NMDA+10 µM PD98059 (NMDA+PD) for 48 h. Inhibitors were added 1 h prior to NMDA. Metabolic activity was measured with the MTS assay. Results are mean ± SD from 2 experiments with n = 12 each. <b>D:</b> % Metabolic activity in GT1-7 and SCN2.2 cultures exposed to normal culture conditions (control); 10 µM PD98059 (PD); 10 µM MK-801 (MK); 10 µM PD98059+10 µM MK-801 (PD+MK); 10 mM Glu+10 µM MK-801 (NMDA+MK); 10 mM Glu+10 µM MK-801+10 µM PD98059 (Glu+MK+PD); 10 mM Glu (Glu); or 10 mM Glu+10 µM PD98059 (NMDA+PD) for 48 h. Inhibitors were added 1 h prior to Glu. Metabolic activity was measured with the MTS assay. Results are mean ± SD from 2 experiments with n = 12 each. All data analyzed by two-way ANOVA with Bonferroni's post hoc test. Comparisons of GT1-7 vs. SCN2.2 within a treatment group are indicated by: * = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001. Comparisons of treatment groups vs. control within either GT1-7 or SCN2.2 cells are indicated by: b = p<0.01; c = p<0.001; d = p<0.0001.</p

Cell Signaling Downstream of ERK/MAPK Pathway.

<p><b>A:</b> mRNA for neuritin in GT1-7 and SCN2.2 cells treated with media change (control) or 10 mM Glu for 6, 12, 18, 24, 36 and 48 h. Immunoblots for neuritin protein in GT1-7 and SCN2.2 cells treated with Glu or control for 48 h are also shown, along with their quantification. <b>B:</b> mRNA for Bcl2 in GT1-7 and SCN2.2 cells treated in the same way as in A. Immunoblots for Bcl2 protein in GT1-7 and SCN2.2 cells treated with Glu or control for 48 h are also shown, along with their quantification. <b>C:</b> mRNA for Bid in GT1-7 and SCN2.2 cells treated in the same was as in A. Immunoblots for cleaved Bid protein in GT1-7 cells treated with Glu or control for 48 h are also shown, along with their quantification. No suitable antibody was found for cleaved Bid in SCN2.2. PCR data is the average of 2 experiments with n = 3 for each experiment. The Immunoblots are the average of 3 to 4 experiments with n = 1 each. mRNA data were analyzed by two-way ANOVA with Bonferroni's post hoc comparison; immunoblot data were compared using paired <i>t</i> test. For Bcl2 and Bid, where two-way ANOVA was significant for an interaction between cell type and treatment time, comparisons of GT1-7 mRNA vs. SCN2.2 mRNA within a time point are indicated by: * = p<0.05; ** = p<0.01; *** = p<0.001, **** = p<0.0001. Comparisons of time points vs. control (0 min) within either GT1-7 or SCN2.2 cells are indicated by: a = p<0.05; b = p<0.01; c = p<0.001; d = p<0.0001. For Neuritin, where two-way ANOVA did not show a significant interaction between cell type and treatment time, comparison between cell types as a whole (not broken down by treatment time) is indicated by **** = p<0.001, and comparison between time points as a whole (not broken down by cell type) is indicated by a = p<0.05. Protein data were analyzed by paired samples t test for control vs. 48 h; ** = p<0.01; *** = p<0.001.</p

Preferential Activation of ERK/MAPK in Response to Glu in SCN2.2 Cells.

<p><b>A:</b> Immunoblot for pERK/MAPK in GT1-7 and SCN2.2 cells exposed to 10 mM Glu for 0 min (control), 5 min, 10 min, 30 min, 1 h, 4 h and 12 h. Sample blots are shown on the left, quantification of blots are displayed on the right. <b>B:</b> Immunoblot for p-p38/MAPK in GT1-7 and SCN2.2 cells exposed to 10 mM Glu for the same time periods as in A. Sample blots are shown on the left, quantification of blots are displayed on the right. <b>C:</b> Immunoblot for pSAPK-JNK/MAPK in GT1-7 and SCN2.2 cells exposed to 10 mM Glu for the same time periods as in A. Sample blots are shown on the left, quantification of blots are displayed on the right. All experiments were repeated 3 or 4 times, with n = 1 per experiment. Data were analyzed by two-way ANOVA with post hoc Bonferroni's test. For pERK/MAPK and pSAPK-JNK/MAPK where two-way ANOVA showed a significant interaction between cell type and treatment time, comparisons of GT1-7 vs. SCN2.2 within a time point are indicated by: * = p<0.05; ** = p<0.01; *** = p<0.001, **** = p<0.0001. For pERK/MAPK and pSAPK-JNK/MAPK comparisons of time points vs. control (0 min) within either GT1-7 or SCN2.2 cells are indicated by: a = p<0.05; b = p<0.01; c = p<0.001; d = p<0.0001. For p-p38/MAPK, where two-way ANOVA did not show a significant interaction between cell type and treatment time, comparison between cell types as a whole (not broken down by treatment time) is indicated by **** = p<0.001.</p

Activation of pERK/MAPK is Sustained for 48 h in SCN2.2 Cells Exposed to Glu.

<p><b>A:</b> Immunoblot for pERK/MAPK in GT1-7 and SCN2.2 cells treated with media change (control) or 10 mM Glu for 48 h. Sample blots are shown on the left, quantification of blots are displayed on the right. <b>B:</b> Immunoblot for p-p38/MAPK in GT1-7 and SCN2.2 cells treated with media change (control) or 10 mM Glu for 48 h. Sample blots are shown on the left, quantification of blots are displayed on the right. <b>C:</b> Immunoblot for pSAPK-JNK/MAPK in GT1-7 and SCN2.2 cells treated with media change (control) or 10 mM Glu for 48 h. Sample blots are shown on the left, quantification of blots are displayed on the right. All experiments were repeated 3 or 4 times, with n = 1 per experiment. Data were analyzed by paired samples <i>t</i> test for control vs. 48 h; * = p<0.05.</p

qPCR Primers and Standard Curves.

<p>Primers for genes from rat (for the SCN2.2 cell line) and mouse (for the GT1-7 cell line) are shown. The sequences of all primers from Qiagen are proprietary; the sequences for the mouse Bid primers are given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023493#s4" target="_blank">Materials and Methods</a> section. Standard curves were prepared from serial dilutions of total RNA from each tissue. Efficiency is calculated as 2^−(1/slope). The values for R² and the equation for the line were derived from Excel.</p