Inhibition of GluN2D-Containing NMDA Receptors Protects Dopaminergic Neurons against 6‑OHDA-Induced Neurotoxicity via Activating ERK/NRF2/HO‑1 Signaling

Abnormal glutamate signaling is implicated in the heightened vulnerability of dopaminergic neurons in Parkinson’s disease (PD). NMDA receptors are ion-gated glutamate receptors with high calcium permeability, and their GluN2D subunits are prominently distributed in the basal ganglia and brainstem nuclei. Previous studies have reported that dopamine depletion led to the dysfunctions of GluN2D-containing NMDA receptors in PD animal models. However, it remains unknown whether selective modulation of GluN2D could protect dopaminergic neurons against neurotoxicity in PD. In this study, we found that allosteric activation of GluN2D-containing NMDA receptors decreased the cell viability of MES23.5 dopaminergic cells and the GluN2D inhibitor, QNZ46, showed antioxidant effects and significantly relieved apoptosis in 6-OHDA-treated cells. Meanwhile, we demonstrated that QNZ46 might act via activation of the ERK/NRF2/HO-1 pathway. We also verified that QNZ46 could rescue abnormal behaviors and attenuate dopaminergic cell loss in a 6-OHDA-lesioned rat model of PD. Although the precise mechanisms underlying the efficacy of QNZ46 in vivo remain elusive, the inhibition of the GluN2D subunit should be a considerable way to treat PD. More GluN2D-selective drugs, which present minimal side effects and broad therapeutic windows, need to be developed for PD treatment in future studies

Effect of the P65/NF-κB signaling pathway on ox-LDL uptake in HUVECs.

<p>(A, B) Phosphorylation and degradation of IκBα analyzed by western blotting. *P < 0.05 versus control; **P < 0.01 versus control; ***P < 0.001 versus control; ##P < 0.01 versus ox-LDL group; NS = not significant compared with the control group. Data are expressed as mean ± SEM, n = 3–4. (C) Western blotting indicated that rapamycin time-dependently inhibited the phosphorylation of IκBα. *P < 0.05 versus control; ***P < 0.001 versus control; NS = not significant compared with the control group. Data are expressed as mean ± SEM, n = 3. (D) Translocation of NF-κB p65 was observed after ox-LDL treatment for 30 h under a fluorescence microscope. The experiment was repeated independently three times. (E-G) Flow cytometry showed that inhibition of NF-κB significantly reduced Dil-ox-LDL uptake in HUVECs. ***P < 0.001 versus control; ##P < 0.01 versus Dil-ox-LDL group; ###P < 0.001 versus Dil-ox-LDL group. Data are expressed as mean ± SEM, n = 3.</p

Upstream and downstream relationship between mTOR, NF-κB, LOX-1 and ox-LDL uptake in HUVECs.

<p>(A, B) Western blotting showed that inhibition of mTOR significantly reduced the increase in the expression of LOX-1 protein expression induced by ox-LDL. ***P < 0.001 versus control; ###P < 0.001 versus ox-LDL; NS = not significant. Data are expressed as mean ± SEM, n = 3. (C-E) Western blotting showed that inhibition of NF-κB significantly reduced the increase in the expression of LOX-1 protein expression induced by ox-LDL. ***P < 0.001 versus control; ###P < 0.001 versus ox-LDL; NS = not significant. Data are expressed as mean ± SEM, n = 3. (F) q-PCR indicated that mTOR and NF-κB knockdown reduced the upregulated expression of LOX-1 mRNA induced by ox-LDL. **P < 0.01 versus control; ###P < 0.001 versus ox-LDL. Data are expressed as mean ± SEM, n = 3. (G, H) IκBα phosphorylation and (I-K) mTOR phosphorylation were analyzed by western blotting, which showed that mTOR deficiency significantly reduced the IκBα phosphorylation triggered by ox-LDL, whereas inhibition of NF-κB did not reduce mTOR phosphorylation induced by ox-LDL. **P < 0.01 versus control; ***P < 0.001 versus control; ##P < 0.01 versus ox-LDL group; NS = not significant. Data are expressed as mean ± SEM, n = 3.</p

Effect of rapamycin on Dil-ox-LDL uptake in HUVECs.

<p>(A) MTT assay for cell viability after treatment with rapamycin by concentration course. NS = not significant. Data are expressed as mean ± SEM, n = 3. (B) Flow cytometry showed that pretreatment with at least 20 nM rapamycin for 1h significantly reduced Dil-ox-LDL accumulation in HUVECs. ***P < 0.001 versus control; ###P < 0.001 versus Dil-ox-LDL group; NS = not significant compared with the Dil-ox-LDL group. Data are expressed as mean ± SEM, n = 3. (C) Continuous images obtained from live cell imaging after 30 μg/mL Dil-ox-LDL treatment for 6 h. (D) Continuous images obtained from live cell imaging after 30 μg/mL Dil-ox-LDL treatment for 6 h following pretreatment with 20 nM rapamycin for 1 h. The experiment was repeated independently three times. (E)Western blot indicated that p62 was increased by bafilomycin A1, and was not reduced by simultaneous treatment with ox-LDL. NS = not significant. **P < 0.01 versus bafilomycin A1 group. Data are expressed as mean ± SEM, n = 3. (F)Flow cytometry indicated that after pretreatment with bafilomycin A1, rapamycin could still inhibit Dil-ox-LDL uptake in HUVECs. ***P < 0.001 versus control; #P < 0.05 versus Dil-ox-LDL group; ###P < 0.001 versus Dil-ox-LDL group; ##P < 0.01 versus Dil-ox-LDL group. Data are expressed as mean ± SEM, n = 3.</p

Effect of rapamycin on LOX-1 expression in HUVECs.

<p>(A, B) Knocking down LOX-1 significantly reduced the rate of Dil-ox-LDL uptake in HUVECs. ***P < 0.001 versus control; ###P < 0.001 versus ox-LDL group. Data are expressed as mean ± SEM, n = 3. (C, D) The expression of LOX-1 protein assessed by western blotting after treatment of HUVECs with ox-LDL by time and concentration course. (E) The q-PCR showed that at least 20 nM rapamycin reduced the increase in LOX-1 mRNA expression induced by ox-LDL. ***P < 0.001 versus control; ###P < 0.001 versus Dil-ox-LDL group; NS = not significant compared with the Dil-ox-LDL group. Data are expressed as mean ± SEM, n = 3. (F) Western blotting indicated that rapamycin dose-dependently reduced the increase in production of LOX-1 protein triggered by ox-LDL. GAPDH was used as the loading control. ***P < 0.001 versus control; #P < 0.05 versus ox-LDL group; ###P < 0.001 versus ox-LDL group; NS = not significant compared with the ox-LDL group. Data are expressed as mean ± SEM, n = 3. (G) Western blotting indicated that 20 nM rapamycin time-dependently reduced the production of LOX-1 protein. *P < 0.05 versus control; ***P < 0.001 versus control; NS = not significant compared with the control group. Data are expressed as mean ± SEM, n = 3.</p

CAR treatment increased the activation of Akt after MCAO.

<p>Mice were treated with CAR (50 mg/kg, i.p.) or saline 2 h before ischemia. After 30 min of ischemia and 6 h reperfusion, brain tissues were collected and protein levels were determined by Western blot. (A)The representative photographs show levels of p-Akt, t-Akt, and beta-tubulin. Beta-tubulin was used as a loading control. (B) Quantitative analysis of the ratio of p-Akt to t-Akt was performed. Bars represent mean ± SEM for samples from 4 brains in each group. *, <i>P</i><0.05 versus sham group; #, <i>P</i><0.05 I/R group versus I/R+CAR group.</p

A PI3K inhibitor LY-294002 blocked the neuroprotection of CAR on cerebral I/R injury.

<p>Mice were treated with CAR (50 mg/kg, i.p.) or saline 2 h before ischemia and 5 µl LY-294002 (10 mM) was administered (i.c.v.) at 15 min before ischemia. (A) After 30 min of ischemia and 6 h reperfusion, brain tissues were collected and p-Akt and t-Akt levels were determined by Western blot. The photographs show that LY-294002 treatment inhibited the activation of Akt by CAR. (B) After 75 min of ischemia and 24 h of reperfusion, cerebral infarct volume was determined by TTC staining. The representative TTC-stained coronal sections demonstrate that LY-294002 treatment abolished the protection of CAR on infarct volume. (C) Statistical analysis of cerebral infarct volume shows that PI3K inhibitor LY-294002 blocked the protective effects of CAR. I/R+CAR+LY: CAR and LY-294002-treated I/R group. Bars represent mean ± SEM of 6–9 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p

CAR post-treatment reduced infarct volume after cerebral I/R injury.

<p>CAR was administered (i.p. and i.c.v.) at different reperfusion times after 75 min of ischemia. TTC staining for cerebral infarct volume was performed after 24 h reperfusion. (A) Mice were administered CAR (50 mg/kg, i.p.) and saline at 0 h, 2 h, and 4 h after reperfusion. CAR had no protection on infarct volume when it was administered at 4 h after reperfusion. (B) Mice were administered with 10 µg CAR (i.c.v.) or saline at 2 h, 4 h, 6 h, and 7 h after reperfusion. CAR still had protective effect when mice treated with CAR after 6 h of reperfusion. Bars represent mean ± SEM of 6 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p

The protection of CAR on infarct volume was in a dose-dependent manner.

<p>Mice were administered (i.p.) with CAR at doses of 5, 25, and 50 mg/kg at 2 h before ischemia. Cerebral infarct volume was determined by TTC staining after 75 min of ischemia and 24 h of reperfusion. Bars represent mean ± SEM of 6 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p

Nrf2 Signaling Contributes to the Neuroprotective Effects of Urate against 6-OHDA Toxicity

<div><p>Background</p><p>Mounting evidence shows that urate may become a biomarker of Parkinson's disease (PD) diagnosis and prognosis and a neuroprotectant candidate for PD therapy. However, the cellular and molecular mechanisms underlying its neuroprotective actions remain poorly understood.</p><p>Results</p><p>In this study, we showed that urate pretreatment protected dopaminergic cell line (SH-SY5Y and MES23.5) against 6-hydroxydopamine (6-OHDA)- and hydrogen peroxide- induced cell damage. Urate was found to be accumulated into SH-SY5Y cells after 30 min treatment. Moreover, urate induced NF-E2-related factor 2 (Nrf2) accumulation by inhibiting its ubiquitinationa and degradation, and also promoted its nuclear translocation; however, it did not modulate Nrf2 mRNA level or Kelch-like ECH-associated protein 1 (Keap1) expression. In addition, urate markedly up-regulated the transcription and protein expression of γ-glutamate-cysteine ligase catalytic subunit (γ-GCLC) and heme oxygenase-1 (HO-1), both of which are controlled by Nrf2 activity. Furthermore, Nrf2 knockdown by siRNA abolished the intracellular glutathione augmentation and the protection exerted by urate pretreatment.</p><p>Conclusion</p><p>Our findings demonstrated that urate treatment may result in Nrf2-targeted anti-oxidant genes transcription and expression by reducing Nrf2 ubiquitination and degradation and promoting its nuclear translocation, and thus offer neuroprotection on dopaminergic cells against oxidative stresses.</p></div