Sigma 1 receptor regulates ERK activation and promotes survival of optic nerve head astrocytes

<div><p>The sigma 1 receptor (S1R) is a unique transmembrane protein that has been shown to regulate neuronal differentiation and cellular survival. It is expressed within several cell types throughout the nervous system and visceral organs, including neurons and glia within the eye. S1R ligands are therapeutic targets for diseases ranging from neurodegenerative conditions to neoplastic disorders. However, effects of S1R activation and inhibition within glia cells are not well characterized. Within the eye, the astrocytes at the optic nerve head are crucial to the health and survival of the neurons that send visual information to the brain. In this study, we used the S1R-specific agonist, (+)-pentazocine, to evaluate S1R activation within optic nerve head-derived astrocytes (ONHAs). Treatment of ONHAs with (+)-pentazocine attenuated the level and duration of stress-induced ERK phosphorylation following oxidative stress exposure and promoted survival of ONHAs. These effects were specific to S1R activation because they were not observed in ONHAs that were depleted of S1R using siRNA-mediated knockdown. Collectively, our results suggest that S1R activation suppresses ERK1/2 phosphorylation and protects ONHAs from oxidative stress-induced death.</p></div

S1R knockdown in HeLa cells and ONHAs.

<p>HeLa cells were transfected with human scrambled siRNA or with human S1R siRNA. Western blot analysis of S1R levels at 3 days (A) and 6 days (B) following transfection is shown. Quantitation of western blot results shows S1R levels normalized to GAPDH as the internal control (C, D). Results are presented as fold change of S1R levels derived from the S1R siRNA-transfected cells compared to S1R levels derived from scrambled siRNA-transfected cells. Data were analyzed using t-test. Significantly different from control *p<0.05, **p<0.01. Experiments were repeated 3 times. MTT assay was performed to assess viability at 3 days (E) and 6 days (F) after scrambled or S1R siRNA transfection. Viability was significantly decreased in S1R siRNA-transfected HeLa cells compared to scrambled siRNA-transfected cells. Transfection with scrambled siRNA did not cause significant HeLa cell death compared to non-transfection control (G, H). Data were analyzed using t-test. Significantly different from control ****p<0.0001. Experiments were performed in quadruplicate and repeated 3 times. ONHAs were transfected with rat scrambled siRNA or with rat S1R siRNA. Western blot analysis of S1R levels at 3 days (I) and 6 days (J) following transfection is shown. Quantitation of western blot results shows S1R levels normalized to GAPDH as the internal control (K, L). Results are presented as fold change of S1R levels derived from the S1R siRNA-transfected cells compared to S1R levels derived from scrambled siRNA-transfected cells. Data were analyzed using t-test. Significantly different from control: **p<0.01, ***p<0.001. Experiments were repeated 3 times. MTT assay was performed to assess viability at 3 days (M) and 6 days (N) after scrambled or S1R siRNA transfection. Viability was not significantly changed in S1R siRNA-transfected ONHAs compared to scrambled siRNA-transfected cells. Transfection with scrambled siRNA did not cause significant ONHA death compared to non-transfection control (O, P). Experiments were performed in quadruplicate and repeated 3 times.</p

Effect of PTZ on ROS generation when ONHAs exposed to H₂O₂.

<p>(A) Representative images of ONHAs treated with 100μM H₂O₂ for 24 hours in the presence or absence of PTZ (10μM, 1 hour pretreatment followed by co-treatment). ROS generation was visualized using CellROX Green reagent. Scale bar: 100μm. (B) Quantitative analysis of intracellular ROS. For each group, three coverslips were quantified, and eight images were taken from each coverslip. Mean signal intensity was quantified by ImageJ. Intracellular ROS generation increased when ONHAs were exposed to H₂O₂. The ROS generation was inhibited by PTZ. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control ****P<0.0001. Significantly different between groups ####P<0.0001. Experiments were repeated 3 times.</p

Knockdown of S1R within ONHAs blocks the (+)-pentazocine-mediated suppression of ERK1/2 phosphorylation.

<p>Phophorylation of ERK was detected after 3 days (A) and 6 days (C) following transfection of scrambled or S1R siRNA in ONHA. At 3 days (A) and 6 days (C) following S1R siRNA transfection, pERK was increased compared to scrambled siRNA control. (B, D) Quantitative analysis of pERK levels represented as pERK normalized to total ERK. Results are presented as fold change of the pERK/total ERK ratio derived from S1R siRNA transfected cells versus scrambled siRNA transfected control cells. Data were analyzed using t-test. Significantly different from control: *p<0.05. Experiments were repeated 3 times. (E) Effect of PTZ on H₂O₂–exposed S1R siRNA transfected ONHA. 5 days after S1R siRNA transfection, ONHA were incubated with 100μM H₂O₂ at 37°C for 15 minutes, 30 minutes, 1 hour, 3 hours and 24 hours in the presence or absence of PTZ (10μM, 1 hour pretreatment followed by cotreatment). Western blot analysis is shown. (F) Quantitative analysis of pERK levels represented as pERK normalized to total ERK. Results are presented as fold change of the pERK/total ERK ratio derived from H₂O₂ exposed, S1R siRNA transfected cells versus control non-H₂O₂ exposed, S1R siRNA transfected cells. Data were analyzed using two-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons.</p

The effect of (+)-pentazocine and H₂O₂ on ONHAs viability.

<p>(A) ONHAs were treated with (+)-pentazocine (PTZ) at varying concentrations (1,3,10 or 50μM) for 24 hours. MTT assay was performed to assess viability. Treatment with PTZ did not cause a significant change in percentage viability compared to the untreated control cells. At the highest concentration of PTZ (50μM), there was a trend toward decrease in viability that was not significant. (B) ONHAs were exposed to various H₂O₂ concentrations (50,100,150, 200, 250, 500μM) for 24 hours. H₂O₂ induced ONHA death in a dose dependent manner. (C) ONHAs were treated with 100μM H₂O₂ for 24 hours in the presence or absence of PTZ (10μM, 1 hour pretreatment followed by co-treatment). Exposure to H₂O₂ significantly decreased viability compared to non-exposed cells. Compared to H₂O₂-exposure with no PTZ treatment, the H₂O₂-exposed, PTZ-treated ONHAs showed significantly increased viability. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Significantly different between groups ###P<0.001. Experiments were performed in quadruplicate and repeated 3 times.</p

Knockdown of S1R within ONHAs blocks (+)-pentazocine-mediated suppression of ROS generation.

<p>(A) Five days following transfection with S1R siRNA, ONHAs were treated with 100μM H₂O₂ for 24 hours in the presence or absence of PTZ (10μM, 1 hour pretreatment followed by co-treatment). MTT assay was performed to assess viability.100μM H₂O₂ induced 50% cell death, and PTZ treatment did not significantly increase cell viability. (B-C) Effect of S1R knockdown on ROS generation in ONHAs. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control: ***p<0.001, ****p<0.0001. (B) Representative images of ONHAs six days following transfection with either scrambled siRNA or S1R siRNA. ROS generation was visualized using CellROX Green reagent. Scale bar: 100μm. (C) Quantitative analysis of intracellular ROS. For each group, three coverslips were quantified, and eight images were taken from each coverslip. Mean signal intensity was quantified by ImageJ. Transfection with scrambled siRNA did not significantly increase ROS generation compared with non-transfected control cells. S1R siRNA-transfected ONHAs showed increased intracelluar ROS compared with scrambled siRNA-transfected ONHAs. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control: **p<0.01. Significantly different between groups: #p<0.05. (D) Representative images of S1R siRNA-transfected ONHAs treated with 100μM H₂O₂ with or without PTZ for 24 hours. Scale bar: 100μm. (E) Quantitative analysis of intracellular ROS. For each group, three coverslips were quantified, and eight images were taken from each coverslip. Mean signal intensity was quantified by ImageJ. ROS generation increased when cells were incubated with H₂O₂. The ROS generation was not inhibited by PTZ treatment. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control *p<0.05. Experiments were repeated 3 times.</p

Characterization of cultured primary rat optic nerve head astrocytes (ONHA).

<p>(A) ONHAs were fixed and probed with antibodies against GFAP (red) and NCAM (green), S1R (red), OSP (red), and Iba-1 (red). The cells were counterstained with DAPI to label DNA (blue) as a marker for nuclei. Scale bar: 50μm. (B) Quantitative analysis shows that more than 95% of the cells in culture express GFAP. (C) The cell lysates from ONHAs (lane 3) were positive for GFAP, a marker for astrocytes and S1R, but negative for Iba-1, a marker for microglial cells, and OSP, a marker for oligodendrocytes. The protein extract from rat brain (lane 1) and from rat optic nerve tissue (lane 2) were used as positive controls.</p

Inhibition of ERK1/2 phosphorylation in ONHAs.

<p>(A) ONHAs were incubated with 100μM H₂O₂ at 37°C for 15 minutes, 30 minutes, 1 hour, 3 hours and 24 hours in the presence or absence of PTZ (10μM, 1 hour pretreatment followed by cotreatment). Western blot analysis of cell lysates is shown. Phosphorylation of ERK was increased at the 15 minute time point following H₂O₂ application, and peaked between 30 minutes and 1 hour. Lysates derived from H₂O₂-exposed cells treated with PTZ showed decreased ERK phosphorylation at 1 hour, 3 hour and 24 hour time points. (B) Quantitative analysis of pERK levels represented as pERK normalized to total ERK. Results are presented as fold change of the pERK/total ERK ratio derived from H₂O₂ exposed cells versus non-H₂O₂ exposed cells. Experiments were repeated 3 times. Data were analyzed using two-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significance levels for pERK change in the untreated (no PTZ) control group (black columns) are as follows: *p<0.05 **p<0.01, ***p<0.001. Significance level for pERK change in the PTZ-treated group (gray columns) is as follows: <b>♮</b>p<0.05. Significance between the untreated control group (black columns) and PTZ-treated group (gray columns) at the 1 hour, 3 hour and 24 hour time points is represented as follows: #p<0.05 Note that the change in pERK between these groups was not significant at the 0 minute, 15 minute, and 30 minute time points. (C) ONHAs were exposed to 100μM H₂O₂ for 24 hours in the presence or absence of U0126 (10μM, 1hour pretreatment followed by co-treatment). Western blot showed decreased ERK phosphorylation levels under conditions of U0126 treatment compared with no treatment. (D) ONHAs were treated with 100μM H₂O₂ for 24 hours in the presence or absence of U0126 (10μM, 1hour pretreatment followed by co-treatment). MTT assay was performed to assess viability. Exposure to H₂O₂ significantly decreased ONHA viability and treatment with U0126 increased viability compared to the non-treated, H₂O₂-exposed cells. Combined treatment with PTZ and U0126 did not show a significant additive effect on cell viability. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Significantly different from control: ****p<0.0001. Significantly different between groups: ####p<0.0001. Experiments were performed in quadruplicate and repeated 3 times.</p

Diagram depicting the proposed role of proNGF/p75^NTR in diabetic retinopathy.

<p>Retinal neurodegeneration is thought to be induced via direct activation of RhoA kinase in RGC and paracrine inflammatory action in response to increases in proNGF.</p

Inhibiting Rho kinase blocked diabetes- and proNGF-induced apoptotic markers expression.

<p><b>A,C.</b> WB analysis showing 1.9- and 2.2-fold increase in the expression of cleaved PARP and caspase-3 in rats electroporated with proNGF as compared with the controls (n = 4–5). <b>B,D.</b> WB analysis showing 1.9- and 2.2-fold increase in the expression of cleaved PARP and caspase-3 in RGC-5 cells treated with proNGF as compared with the controls (n = 4). <b>E.</b> WB analysis showing 2.1- and 1.6-fold increase in the expression of cleaved PARP and caspase-3 in RGC-5 treated with proNGF as compared with the controls. Treatment of rats or RGC-5 with Y27632 blocked all these effects in rats and media treated with proNGF and did not affect the control groups. * = significant difference as compared with the rest of the groups at p<0.05. C, control.</p