Journal: Cells

Article Title: Activation of Nrf2 in Astrocytes Suppressed PD-Like Phenotypes via Antioxidant and Autophagy Pathways in Rat and Drosophila Models

doi: 10.3390/cells10081850

Figure Lengend Snippet: Overexpression of Nrf2 in glial cells suppressed rotenone-induced PD-like phenotypes in flies. ( A ) survival curve and statistical analysis by Log-rank test and half survival time. Nrf2 overexpression in DMSO control drosophila did not significantly improve survival time in the DMSO/Elav;cncc or DMSO/Repo;cncc group compared with the DMSO/WT control group (NS p > 0.05). Rotenone treatment significantly shortened survival time compared with the corresponding DMSO control group ( ## p < 0.01). Nrf2 overexpression in neurons of PD model Drosophila significantly increased survival time in the rotenone/Elav;cncc group compared with the rotenone/WT group (* p < 0.05). Nrf2 overexpression in glial cells more significantly increased survival time in the rotenone/Repo;cncc group compared with the rotenone/Elav;cncc group and the rotenone/WT group (* p < 0.05 and ** p < 0.01). ( B ) The left graph shows climbing ability throughout lifetime. The right bar graph shows the climbing ability at ages of 3–5 weeks. Rotenone treatment significantly reduced climbing ability in the rotenone/WT group compared with the DMSO group ( ## p < 0.01). Nrf2 overexpression in glial cells in the rotenone/Repo;cncc group more significantly improved the motor ability than overexpression in neurons in the rotenone/Elav;cncc group (* p < 0.05 and ** p < 0.01). ( C ) Western blot analysis of Nrf2, GCLC, HO-1, and Ref(2)P proteins in Drosophila brain homogenates. The bar graph below is the quantitative analysis of Western blot bands. Nrf2 overexpression in neurons and glial cells increased GCLC and HO-1 in the Elav;cncc and Repo;cncc group compared with the WT control group (* p < 0.05 and ** p < 0.01). Nrf2 overexpression in glial cells further increased Nrf2, GCLC and HO-1 in the Repo;cncc group compared to overexpression in neurons in the Elav;cncc group (* p < 0.05). The Nrf2 overexpression significantly increased Ref(2)P in the rotenone/Elav;cncc and rotenone/Repo;cncc groups compared with the rotenone/WT group (* p < 0.05 and ** p < 0.01), but not in the DMSO control group. Nrf2 overexpression in glial cells enhanced autophagy more than in neurons (* p < 0.05). ( D ) Measure of redox state by DHE staining. Oxidative stress was relatively low in the DMSO control group. Rotenone treatment significantly increased oxidative stress in the rotenone/WT group compared with the DMSO control group (*** p < 0.001). Nrf2 overexpression slightly reduced oxidative stress in the DMSO control group and significantly decreased rotenone-induced oxidative stress in the rotenone/WT group (* p < 0.05 and ** p < 0.01). Nrf2 overexpression in glial cells in the rotenone/Repo;cncc group resulted in more DHE staining than such overexpression in neurons in the rotenone/Elav;cncc group (* p < 0.05).

Article Snippet: The primary antibodies used for the Western blot included anti-Nrf2 (1:1000, Assaybiotech), anti-GCLC (1:1000, BD), anti-HO-1 and anti-LC3I/II (Sigma), anti-Ref(2)P (1:1000, BD Pharmingen, San Jose, CA, USA), and anti-β-tubulin (1:5000, Sigma-Aldrich).

Techniques: Over Expression, Western Blot, Staining

Journal: Cells

Article Title: Activation of Nrf2 in Astrocytes Suppressed PD-Like Phenotypes via Antioxidant and Autophagy Pathways in Rat and Drosophila Models

doi: 10.3390/cells10081850

Figure Lengend Snippet: Activation of the Nrf2 signaling pathway enhanced autophagy in flies. ( A ) The graph shows the lifetime survival curve, the Log-rank test, and half survival time used to analyze the difference between groups ( # , compared with the rotenone/WT group). Nrf2 overexpression in glial cells effectively prolonged survival time in the Rot/Repo;cncc + DMSO group compared with the rotenone/WT group ( ## p < 0.01). The Nrf2 inhibitor ML385 significantly reduced the prolonged survival time in the Rot/Repo;cncc + ML385 group compared with the Rot/Repo;cncc + DMSO group (** p < 0.01). The autophagy inhibitor 3-MA partly reduced the prolonged survival time of the Rot/Repo;cncc + 3-MA group (* p < 0.05). The Nrf2 inducer CDDO-Me and autophagy inducer rapamycin did not significantly affect the survival time of the Rot/Repo;cncc + DMSO group. ( B ) Western blot analysis of antioxidant and autophagy proteins in fly brain. Nrf2 overexpression in glial cells significantly increased HO-1 and Ref(2)P proteins in the rotenone/Repo;cncc group compared with the rotenone/WT group ( ## p < 0.01). The Nrf2 inhibitor ML385 effectively decreased HO-1 and Ref(2)P levels in the rotenone/Repo;cncc + ML385 group compared with the rotenone/Repo;cncc + DMSO group (* p < 0.05). The autophagy inhibitor 3-MA effectively decreased Ref(2)P protein but not HO-1 protein, while the Nrf2 inducer CDDO-Me and autophagy inducer rapamycin did not significantly affect the expression of HO-1 and Ref(2)P in the rotenone/Repo;cncc + DMSO group.

Article Snippet: The primary antibodies used for the Western blot included anti-Nrf2 (1:1000, Assaybiotech), anti-GCLC (1:1000, BD), anti-HO-1 and anti-LC3I/II (Sigma), anti-Ref(2)P (1:1000, BD Pharmingen, San Jose, CA, USA), and anti-β-tubulin (1:5000, Sigma-Aldrich).

Techniques: Activation Assay, Over Expression, Western Blot, Expressing

Journal: Cells

Article Title: Activation of Nrf2 in Astrocytes Suppressed PD-Like Phenotypes via Antioxidant and Autophagy Pathways in Rat and Drosophila Models

doi: 10.3390/cells10081850

Figure Lengend Snippet: Knockdown of Nrf2 in fly glial cells worsens rotenone-induced PD-like phenotypes in flies. ( A ) The survival curve in Nrf2-knockdown Drosophila. The graph shows the survival curve, the Log-rank test, and half survival time used to analyze the difference between groups ( # , compared with the rotenone/WT group). Nrf2 knockdown in glial cells reduced survival time in the Rot/Repo;cnccRNAi + DMSO group compared with the rotenone/WT group ( # p < 0.05). CDDO-Me significantly extended survival time in the Rot/Repo;cnccRNAi + CDDO-Me group compared with the Rot/Repo;cnccRNAi + DMSO group (** p < 0.01). Rapamycin partially restored the shortened survival time of the Rot/Repo;cnccRNAi + DMSO group (* p < 0.05). ( B ) qRT-PCR result for drosophila Nrf2, the cncc level in Repo;cnccRNAi flies was about 40% compared with WT Control ( ## p < 0.01). ( C ) Western blot analysis of antioxidant and autophagy proteins in fly brain. Nrf2 knockdown in glial cells by RNAi significantly decreased protein expression of HO-1 and Ref(2)P in the rotenone/Repo;cnccRNAi group compared with the rotenone/WT group ( # p < 0.05 and ## p < 0.01). CDDO-Me partially restored this decrease in HO-1 and Ref(2)P expression in the rotenone/Repo;cnccRNAi + CDDO-Me group compared with the rotenone/Repo;cnccRNAi + DMSO group (* p < 0.05). Rapamycin partially restored the expression of the Ref(2)P autophagy protein, but did not alter HO-1 levels in the rotenone/Repo;cnccRNAi + Rapa group compared with the rotenone/Repo;cnccRNAi + DMSO group (* p < 0.05).

Article Snippet: The primary antibodies used for the Western blot included anti-Nrf2 (1:1000, Assaybiotech), anti-GCLC (1:1000, BD), anti-HO-1 and anti-LC3I/II (Sigma), anti-Ref(2)P (1:1000, BD Pharmingen, San Jose, CA, USA), and anti-β-tubulin (1:5000, Sigma-Aldrich).

Techniques: Quantitative RT-PCR, Western Blot, Expressing