EGCG-mediated autophagy flux has a neuroprotection effect via a class III histone deacetylase in primary neuron cells

Abstract

Prion diseases caused by aggregated misfolded prion protein (PrP) are transmissible neurodegenerative disorders that occur in both humans and animals. Epigallocatechin-3-gallate (EGCG) has preventive effects on prion disease; however, the mechanisms related to preventing prion diseases are unclear. We investigated whether EGCG, the main polyphenol in green tea, prevents neuron cell damage induced by the human prion protein. We also studied the neuroprotective mechanisms and proper signals mediated by EGCG. The results showed that EGCG protects the neuronal cells against human prion protein-induced damage through inhibiting Bax and cytochrome c translocation and autophagic pathways by increasing LC3-II and reducing and blocking p62 by using ATG5 small interfering (si) RNA and autophagy inhibitors. We further demonstrated that the neuroprotective effects of EGCG were exhibited by a class III histone deacetylase; sirt1 activation and the neuroprotective effects attenuated by sirt1 inactivation using sirt1 siRNA and sirtinol. We demonstrated that EGCG activated the autophagic pathways by inducing sirt1, and had protective effects against human prion protein-induced neuronal cell toxicity. These results suggest that EGCG may be a therapeutic agent for treatment of neurodegenerative disorders including prion diseases.

Keywords: EGCG; Sirt1; autophagy; gerotarget; neurotoxicity; prion.

Figure 1. EGCG prevents neuronal cells from PrP (106-126)-induced cell death

The primary neuron cells were pretreated with 10 μM of EGCG for 1 hr and then exposed to 50 μM PrP (106-126) or scrambled PrP (106-126) (scr-PrP) for 36 hr. Cell viability was measured by the Annexin V assay A, B. Western blot for cleaved caspases 3 protein was conducted with primary neuron cells. Beta-actin was used as the loading control C. SH-SY5Y cells were pretreated with 2.5, 5, or 10 μM of EGCG for 1 hr and then exposed to 50 μM PrP (106-126) or scrambled PrP (106-126) (scr-PrP) for 36 hr. Cell viability was measured by the Annexin V assay D, E. Lactate dehydrogenase (LDH) assay was used to quantify LDH released into the medium F. Western blot for cleaved caspases 3 protein was conducted with SH-SY5Y cells. Beta-actin was used as the loading control G. Bars indicate mean ± standard error (n = 4). The data were analyzed using ANOVA and Tukey multiple range tests (P < 0.01). Means sharing a common alphabetical symbol did not significantly differ. Cl-Cas-3, Cleaved caspase-3.

Figure 2. EGCG protects PrP (106-126)-induced mitochondrial damage

SH-SY5Y cells were pretreated with 2.5, 5, or 10 μM of EGCG for 1 hr and then exposed to 50 μM PrP (106-126) for 36 hr. The treated cells were measured for JC-1 mono form (green) by flow cytometry. Percent values in the histogram represent the population of JC-1 monomeric cells. A. The treated cells were measured for JC-1 aggregates form (red) and mono form (green) by confocal microscopy analysis. Scale bar = 50 μm B. Separation of the cytosol and mitochondrial extracts was analyzed by Western blot using antibodies, to cytochrome c and Bax protein D, E. Bars indicate mean ± standard error (n = 4). The data were analyzed using ANOVA and Tukey multiple range tests (P < 0.01). Means sharing a common alphabetical symbol did not significantly differ.

Figure 3. EGCG increases the induction of autophagy

The primary neuron cells were treated with 1, 2.5, 5, 10 μM of EGCG for 30 h. Western blot for LC3-II, and p62 proteins was analyzed from primary neuron cells. Beta-actin was used as a loading control A. The cells were immunostained with p62 antibody (green) and observed in fluorescent view. Primary neuron cells were pretreated with 10 μM of EGCG in presence of autophagy inhibitor (3MA and wortmannin) for 30 h and western blot for LC3-II and p62 proteins was analyzed B. SH-SY5Y cells were treated with 2.5, 5, or 10 μM EGCG for 30 hr. Western blot for LC3-II, and p62 proteins was conducted with SH-SY5Y cells. Beta-actin was used as the loading control C. SH-SY5Y cells were analyzed by immunocytochemistry for p62 D. The cells were immunostained with p62 antibody (green) and observed in fluorescent view. SH-SY5Y cells were pretreated with 10 μM of EGCG in the presence of autophagy inhibitors (3MA or wortmannin) for 1 hr and then exposed to 50 μM PrP (106-126) for 36 h. Cell viability was measured by the Annexin V assay E, F. SH-SY5Y cells were treated as described in Figure 3A and Western blot for the LC3-II and p62 proteins was analyzed G. Bars indicate mean ± standard error (n = 4). *p < 0.05, **p < 0.01, significant differences between control and each treatment group, and #p < 0.01; significantly different when compared with PrP (106-126)-treated group.

Figure 4. Inhibiting autophagy with ATG5 siRNA reduced the increase in autophagy caused by EGCG

ATG5 small interfering RNA (siATG5) or negative control siRNA (NC) transfected primary neuron cells were incubated with 50 μM PrP (106-126) for 36 h in the presence of EGCG. Cell viability was measured by annexin V assay A, B. siATG5 or NC transfected primary neuron cells were incubated with EGCG (10 μM) for 30 h. Western blot for ATG5, LC3 and p62 proteins was analyzed from SH-SY5Y cells. β-actin was used as loading control C. siATG5 or NC transfected SH-SY5Y cells were incubated with 50 μM PrP (106-126) for 36 hr in the presence of EGCG. Cell viability was measured by Annexin V assay D, E. siATG5 or NC transfected SH-SY5Y cells were incubated with EGCG (10 μM) for 30 hr. Western blot for ATG5, LC3-II and p62 proteins was analyzed from SH-SY5Y cells. Beta-actin was used as the loading control F. Bars indicate mean ± standard error (n = 4). *p < 0.05, **p < 0.01, significant differences between control and each treatment group, and #p < 0.01; significantly different when compared with PrP (106-126)-treated group.

Figure 5. EGCG upregulates sirt1 expression and activation

SH-SY5Y cells were treated with 2.5, 5, or 10 μM of EGCG for 30 hr. Western blot for sirt1 and acetyl-p53 proteins was analyzed from SH-SY5Y cells. Beta-actin was used as the loading control A. SH-SY5Y cells were analyzed by immunocytochemistry for sirt1 B. The cells were immunostained with sirt1 antibody (red) and observed in fluorescent view. SH-SY5Y cells were pretreated with 10 μM EGCG for 1 hr and then exposed to 50 μM PrP (106-126) for 36 hr. Sirt1 deacetylase activities in were analyzed in the nuclei of SH-SY5Y cells C. Bars indicate mean ± standard error (n = 4). *p < 0.05, **p < 0.01 significant differences between control and each treatment group.

Figure 6. EGCG prevents neuronal cells from PrP (106-126)-induced cell death through the sirt1 pathway

Sirt1 small interfering RNA (siSirt1) or negative control siRNA (NC) transfected SH-SY5Y cells were incubated with 50 μM PrP (106-126) for 36 hr in the presence of EGCG. Cell viability was measured by Annexin V assay A, B. SH-SY5Y cells were pretreated with sirtinol (10 μM) and EGCG (10 μM) for 1 h and then exposed to 50 μM PrP (106-126) for 36 hr. Cell viability was measured by Annexin V assay C. The cells were measured for JC-1 mono form (green) by flow cytometry. M1 represents the population of JC-1 monomeric cells D, E. Bars indicates mean ± standard error (n = 4). *p < 0.05, **p < 0.01, #p < 0.001 significant differences between control and each treatment group, and ##p < 0.01; significantly different when compared with PrP (106-126)-treated group.

Figure 7. Inhibiting sirt1 decreased the sirt1 increase caused by EGCG

Sirt1 small interfering RNA (siSirt1) or negative control siRNA (NC) transfected SH-SY5Y cells were incubated with EGCG (10 μM) for 30 hr. Western blot for sirt1 and acetyl-p53 proteins was conducted from SH-SY5Y cells. β-actin was used as the loading control A. Relative sirt1 mRNA expression levels were analyzed using quantitative real-time polymerase chain reaction. The indicated relative gene expression level shows expression levels that were normalized to β-actin expression as the standard B. siSirt1 or NC transfected SH-SY5Y cells were pre-incubated with EGCG (10 μM) for 1 hr and then exposed to PrP (106-126) for 30 hr. Sirt1 deacetylase activities were analyzed in SH-SY5Y cell nuclei. C. Bars indicate mean ± standard error (n = 4). *p < 0.05, **p < 0.01, significant differences between control and each treatment group, and #p < 0.01; significantly different when compared with PrP (106-126)-treated group.

Figure 8. Overexpression of sirt1 and EGCG increased neuroprotective effects in SH-SY5Y cells

SH-SY5Y cells were transfected by overexpressing adenovirus (Ad-Sirt1) or lacZ-bearing adenovirus (Ad-lacz). A Western blot for sirt1 and acetyl-p53 proteins was conducted from SH-SY5Y cells. Beta-actin was used as the loading control A. Ad-sirt1 or Ad-lacz transfected cells were incubated with or without EGCG and exposed to PrP (106-126) for 36 hr. Sirt1 deacetylase activities were analyzed in SH-SY5Y cell nuclei B. Cell viability was measured by the Annexin V assay C. Bars indicate mean ± standard error (n = 4). *p < 0.05, **p < 0.01, significant differences between control and each treatment group, and #p < 0.01; significantly different when compared with PrP(106-126)-treated group.

Figure 9. EGCG increases autophagy through the sirt1 pathway

The SH-SY5Y cells were transfected by overexpressing adenovirus (Ad-Sirt1) or lacZ-bearing adenovirus (Ad-lacz). A Western blot of the LC3-II, ATG5 and p62 proteins was conducted in SH-SY5Y cells. Beta-actin was used as the loading control A. Sirt1 small interfering RNA (siSirt1) or negative control siRNA (NC) transfected SH-SY5Y cells were incubated with EGCG (10 μM) for 30 hr. A Western blot for the LC3-II, ATG5, and p62 proteins was conducted with SH-SY5Y cells. Beta-actin was used as the loading control B. SH-SY5Y cells were treated as described in Figure 3A, and then ICC for p62 was analyzed C.