Anti-Apoptotic and Anti-Inflammatory Role of Trans ε-Viniferin in a Neuron-Glia Co-Culture Cellular Model of Parkinson's Disease

Abstract

The polyphenol trans-ε-viniferin (viniferin) is a dimer of resveratrol, reported to hold antioxidant and anti-inflammatory properties. The aims of our study were to evaluate the neuroprotective potential of viniferin in the nerve growth factor (NGF)-differentiated PC12 cells, a dopaminergic cellular model of Parkinson's disease (PD) and assess its anti-inflammatory properties in a N9 microglia-neuronal PC12 cell co-culture system. The neuronal cells were pre-treated with viniferin, resveratrol or their mixture before the administration of 6-hydroxydopamine (6-OHDA), recognized to induce parkinsonism in rats. Furthermore, N9 microglia cells, in a co-culture system with neuronal PC12, were pre-treated with viniferin, resveratrol or their mixture to investigate whether these polyphenols could reduce lipopolysaccharide (LPS)-induced inflammation. Our results show that viniferin as well as a mixture of viniferin and resveratrol protects neuronal dopaminergic cells from 6-OHDA-induced cytotoxicity and apoptosis. Furthermore, when viniferin, resveratrol or their mixture was used to pre-treat microglia cells in our co-culture system, they reduced neuronal cytotoxicity induced by glial activation. Altogether, our data highlight a novel role for viniferin as a neuroprotective and anti-inflammatory molecule in a dopaminergic cellular model, paving the way for nutraceutical therapeutic avenues in the complementary treatments of PD.

Keywords: Parkinson’s disease; apotosis; dopamine; neuroinflammation; neuroprotection; oxidative stress; resveratrol; trans-ε-viniferin.

Figure 1

Representative microphotograph of nerve growth factor (NGF)-differentiated PC12 for 9 days by immunofluorescence. Nuclei are counterstained in blue with Dapi. NF: neurofilaments revealed with an anti-neurofilaments antibody (green). TH: tyrosine hydroxylase revealed with an anti-tyrosine antibody as a marker of dopamine (red). Scale bar = 10 µm.

Figure 2

Levels of cytotoxicity (A) and metabolic activity (B) of PC12 neurons pretreated for three hours with culture medium (control), resveratrol, viniferin or a mixture of both polyphenols (Mix), all at a final concentration of 10–9. Cells were then treated with 6-hydroxydopamine (6-OHDA) at 50 μM for 24 h, as described in the Material and Methods. The supernatants and the cells were used to perform cell death (lactate dehydrogenase (LDH) assay). Data are expressed as means ± SEM of five independent experiments. For each experiment, each condition was in assessed in sextuplicate. Asterisks (*) indicate statistical differences between the treatments and their respective controls (*** p < 0.001), plus signs (+) denote statistical differences between 6-OHDA and 6-OHDA in the presence of polyphenols (+++ p < 0.001, ++ p < 0.01,).

Figure 3

Detection of apoptosis by single stranded (ssDNA) fragmentation (A) and by caspase-3 and TUNEL double immunofluorescence (B). PC12 neurons pre-treated for three hours with culture medium (control), resveratrol, viniferin or a mixture of both polyphenols (Mix) all at a final concentration of 10–9 M. Cells were then treated with 6-OHDA at 50 μM for 24 h, as described in the Material and Methods. Apoptotic neuronal cells among 300 randomly chosen neuronal cells were counted on 10 different optical fields from 3 slides per group, as described in Material and Methods. Data are expressed as means ± SEM of three experiments. For each experiment, each condition was assessed in triplicate. Asterisks (*) indicate statistical differences between the treatments and their respective controls (*** p < 0.001, ** p < 0.01), plus signs (+) denote statistical differences between 6-OHDA and 6-OHDA in the presence of polyphenols (+++ p < 0.001, ++ p < 0.01), dollar sign ($) indicates statistical difference between 6-OHDA+resveratrol and 6-OHDA+mixture of the two polyphenols ($$$ < 0.001) and number sign (#) indicates statistical difference between 6-OHDA+viniferin and 6-OHDA + mixture of the two polyphenols (### p < 0.001).

Figure 4

Expression of apoptotic cleaved caspase-3 (A) and cleaved Parp-1 (B) in PC12 neurons pretreated for three hours with the culture medium (control), resveratrol, viniferin or a mixture of both polyphenols (Mix) at a final concentration of 10--9 M. Cells were then treated with 6-OHDA at 50 μM for 24 h, as described in the Material and Methods. Top panel: Data are expressed as the ratio of cleaved caspase-3 (A) or cleaved Parp-1 (B) to β-actin. Bottom panel: representative Western blot bands of cleaved caspase-3, cleaved Parp-1 or β-actin. Optical densities were measured on the same membrane. Data are expressed as means ± SEM of three experiments performed in triplicate. Asterisks (*) indicate statistical differences between the treatments and their respective controls (*** p < 0.001, ** p < 0.01), plus signs (+) denote statistical differences between 6-OHDA and 6-OHDA in the presence of polyphenols (+++ p < 0.001, + p < 0.05).

Figure 5

Co-culture of N9 microglial cells on neuronal PC12 cells. (A) N9 cells were pre-treated with polyphenols followed incubation with lipopolysaccharide (LPS), as described in the Materials and Methods. (B) N9 cells were treated with LPS and neuronal PC12 were pre-incubated with polyphenols as described in the Materials and Methods. Data are expressed as means ± SEM of three experiments. Asterisks (*) indicate statistical differences between the treatments and their respective controls (*** p < 0.001), plus signs (+) denote statistical differences between LPS and LPS in the presence of polyphenols. (+++ p < 0.001, ++ p < 0.01), dollar sign ($) indicates statistical difference between LPS + resveratrol and LPS + mixture of the two polyphenols ($$$ < 0.001, $ < 0.05) and number sign (#) indicates statistical difference between LPS + viniferin and LPS + mixture of the two polyphenols (## p < 0.01).

Figure 6

Measurements of cytokine secretion from LPS-activated microglial N9 cells by ELISA specific kit for IL-1α (A) and TNFα (B). Data are expressed as means ± SEM of four experiments. Asterisks (*) indicate statistical differences between the treatments and their respective controls (*** p < 0.001, ** p < 0.01).