Vitamin E suppression of microglial activation is neuroprotective

Abstract

Neurotoxic microglial-neuronal interactions have been implicated in the pathogenesis of various neurodegenerative diseases such as Alzheimer's disease, and vitamin E has been shown to have direct neuroprotective effects. To determine whether vitamin E also has indirect neuroprotective effects through suppression of microglial activation, we used a microglial-neuronal coculture. Lipopolysaccharide (LPS) treatment of a microglial cell line (N9) induced a time-dependent activation of both p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappaB (NFkappaB), with consequent increases in interleukin-1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production. Differentiated neuronal cells (PC12 cells treated with nerve growth factor) exhibited marked loss of processes and decreased survival when cocultured with LPS-activated microglia. Preincubation of microglia with vitamin E diminished this neurotoxic effect, independently of direct effects of the antioxidant on the neuronal cells. Microglial NO production and the induction of IL-1alpha and TNFalpha expression also were attenuated by vitamin E. Such antiinflammatory effects of vitamin E were correlated with suppression of p38 MAPK and NFkappaB activation and were mimicked by an inhibition of either p38 MAPK (by SB203580) or NFkappaB (by decoy oligonucleotides). These results suggest that, in addition to the beneficial effects of providing direct antioxidant protection to neurons reported by others, vitamin E may provide neuroprotection in vivo through suppression of signaling events necessary for microglial activation.

Fig. 1

Vitamin E inhibits microglial NO release. Microglia were preincubated with vitamin E (50 μM) for 1 hr and then treated with LPS (30 ng/ml) for the indicated time periods. The amount of NO was determined by measuring the amount of nitrite converted from NO in the media as described in Materials and Methods. Values are expressed as mean ± SEM of four to six samples. **P < 0.01.

Fig. 2

Vitamin E inhibits microglial expression of IL-1α, TNF-α, and iNOS. Illustration (A) and quantification (B) of transcriptional levels of IL-1α, TNF-α, and iNOS in LPS-stimulated microglia, with or without vitamin E pretreatment. RNAs were extracted from these cultures, and RT-PCR was performed to detect the mRNA levels of IL-1α, TNF-α, and iNOS. Values are expressed as mean ± SEM of four to six samples. **P < 0.01.

Fig. 3

Vitamin E decreases phosphorylation of p38 MAPK. Illustrations of time course of LPS-induced microglial p38 MAPK activation (Phos-p38) and the level of total p38 MAPK (Total-p38) (A) and vitamin E inhibition of p38 MAPK phosphorylation in microglia stimulated by LPS for 1 hr (B) (typical of three separate experiments).

Fig. 4

Vitamin E inhibits NFκB activation. Illustrations of time course of LPS-induced microglial NFκB activation (A) and inhibition of NFκB binding activity by vitamin E pretreatment of microglia stimulated with LPS for 2 hr (B) (typical of three separate experiments).

Fig. 5

Inhibition of p38 MAPK reduces IL-1α, TNF-α, and iNOS expression. Microglia were incubated for 1 hr with a specific inhibitor of p38 MAPK (SB 203580, 10 μM) and then treated with LPS (10 ng/ml) for 24 hr (typical of three separate experiments).

Fig. 6

NFκB decoy DNA suppresses IL-1α, TNF-α, and iNOS expression. Microglia were treated for 3 hr with either NFκB DNA decoy (Dec DNA) or scrambled control DNA (Con DNA) and then stimulated with LPS (30 ng/ml) for 24 hr (typical of three separate experiments).

Fig. 7

p38 MAPK inhibitor does not alter NFκB binding activity. LPS activation of microglia (2 hr) was unchanged by treatment with SB203580 (1 hr). Nuclear protein was isolated from these cultures, and EMSA was performed as described in Materials and Methods (typical of four separate experiments).

Fig. 8

Vitamin E reduces toxic effects of activated microglia on neuronal cells. Phase-contrast photomicrographs of differentiated PC12 cells before (A,C,E) or after 24 hr of (B,D,F) coculture with N9 microglia. Before coculture, N9 cells were pretreated for 2 hr with LPS (30 ng/ml) alone (LPS) or LPS plus vitamin E (50 μM) pretreatment (LPS + Vit E) or without treatment (CON), and then the N9 culture inserts were completely washed with fresh medium and cocultured with differentiated PC12 cells. Scale bar = 50 μm.

Fig. 9

Vitamin E promotes survival of PC 12 cells cocultured with N9 microglia. N9 microglia were preincubated with LPS (30 ng/ml), vitamin E (50 μM), or LPS plus vitamin E pretreatment for 2 hr, and then the N9 culture inserts were completely washed with fresh medium and cocultured with differentiated PC12 cells for 24 hr. MTT was added to the PC12 cell culture 2 hr before harvesting. Values are expressed as mean ± SEM of 6–12 samples. **P < 0.001.