Molecular basis of vitamin E action: tocotrienol modulates 12-lipoxygenase, a key mediator of glutamate-induced neurodegeneration

Abstract

Vitamin E is a generic term for tocopherols and tocotrienols. This work is based on our striking evidence that, in neuronal cells, nanomolar concentrations of alpha-tocotrienol, but not alpha-tocopherol, block glutamate-induced death by suppressing early activation of c-Src kinase (Sen, C. K., Khanna, S., Roy, S., and Packer, L. (2000) J. Biol. Chem. 275, 13049-13055). This study on HT4 and immature primary cortical neurons suggests a central role of 12-lipoxygenase (12-LOX) in executing glutamate-induced neurodegeneration. BL15, an inhibitor of 12-LOX, prevented glutamate-induced neurotoxicity. Moreover, neurons isolated from 12-LOX-deficient mice were observed to be resistant to glutamate-induced death. In the presence of nanomolar alpha-tocotrienol, neurons were resistant to glutamate-, homocysteine-, and l-buthionine sulfoximine-induced toxicity. Long-term time-lapse imaging studies revealed that neurons and their axo-dendritic network are fairly motile under standard culture conditions. Such motility was arrested in response to glutamate challenge. Tocotrienol-treated primary neurons maintained healthy growth and motility even in the presence of excess glutamate. The study of 12-LOX activity and metabolism revealed that this key mediator of glutamate-induced neurodegeneration is subject to control by the nutrient alpha-tocotrienol. In silico docking studies indicated that alpha-tocotrienol may hinder the access of arachidonic acid to the catalytic site of 12-LOX by binding to the opening of a solvent cavity close to the active site. These findings lend further support to alpha-tocotrienol as a potent neuroprotective form of vitamin E.

Figures

Figure 1. Protection against loss of neuronal viability by α-tocotrienol

Primary rat immature cortical neurons (A-C) or HT4 (D) were either treated or not with α-tocotrienol (as indicated) for 5 min and challenged with either glutamate (10 mM; A); L-homocysteic acid (1 mM; B); or buthionine sulfoximine (0.15 mM; BSO) for 24 h. Arachidonic acid (0.05 mM, C) potentiated BSO-induced cell death. α-Tocotrienol conferred total protection against all of the above neurotoxins. D, 100 nM tocotrienol not only prevented glutamate-induced toxicity but allowed glutamate-treated cells to proliferate at a rate comparable to cells not treated with glutamate. Cells were counted at 12, 24 and 36 h after glutamate challenge. A: †, lower compared to control glutamate non-treated group; *, higher compared to glutamate-treated group. B: †, lower compared to control L-homocysteic acid non-treated group; *, higher compared to L-homocysteic acid-treated group. C: †, lower compared to corresponding control; *, higher compared to the corresponding group challenged with toxin(s). D: †, lower compared to the corresponding control non-treated group; *, higher compared to the corresponding glutamate-treated group. P<0.05.

Figure 2. Imaging of glutamate-induced degeneration of rat primary cortical neurons and protection by α-tocotrienol and BL15

After 24h of seeding, cells were challenged with glutamate. Where indicated, neurons were pre-treated with either α-tocotrienol (250 nM) or BL15 (2.5 μM) for 5 min prior to glutamate treatment. a-h, Neuron specific Class III β-tubulin in the cultured neural network (for phase contrast microscopy see i-p). After 24h of glutamate treatment, cells were fixed and stained. a, control; b, glutamate; c, α-tocotrienol + glutamate; d, BL15+glutamate. e-h, Neurofilament staining in the cultured neural network (for phase contrast microscopy see i-p). e, control; f, glutamate; g, α-tocotrienol + glutamate; h, BL15+glutamate. i-p, Live cell imaging of glutamate treated neurons under standard ( not glass cover-slip) culture conditions. Phase contrast images were collected once every 15 mins for 18h from 8h after glutamate treatment. Frames illustrate time-dependent disintegration of the neural network. i, 8h; j, 12h; k, 16h; and l, 26h after glutamate treatment. Glutamate-challenged neurons pre-treated with α-tocotrienol (250 nM) resisted degeneration and continued to grow. m, 28h; n, 30h; o, 32h; and p, 34h after glutamate treatment. Two (i-l and m-p) .avi video micrographs have been supplemented for online publication. 200X magnification.

Figure 2. Imaging of glutamate-induced degeneration of rat primary cortical neurons and protection by α-tocotrienol and BL15

After 24h of seeding, cells were challenged with glutamate. Where indicated, neurons were pre-treated with either α-tocotrienol (250 nM) or BL15 (2.5 μM) for 5 min prior to glutamate treatment. a-h, Neuron specific Class III β-tubulin in the cultured neural network (for phase contrast microscopy see i-p). After 24h of glutamate treatment, cells were fixed and stained. a, control; b, glutamate; c, α-tocotrienol + glutamate; d, BL15+glutamate. e-h, Neurofilament staining in the cultured neural network (for phase contrast microscopy see i-p). e, control; f, glutamate; g, α-tocotrienol + glutamate; h, BL15+glutamate. i-p, Live cell imaging of glutamate treated neurons under standard ( not glass cover-slip) culture conditions. Phase contrast images were collected once every 15 mins for 18h from 8h after glutamate treatment. Frames illustrate time-dependent disintegration of the neural network. i, 8h; j, 12h; k, 16h; and l, 26h after glutamate treatment. Glutamate-challenged neurons pre-treated with α-tocotrienol (250 nM) resisted degeneration and continued to grow. m, 28h; n, 30h; o, 32h; and p, 34h after glutamate treatment. Two (i-l and m-p) .avi video micrographs have been supplemented for online publication. 200X magnification.

Figure 2. Imaging of glutamate-induced degeneration of rat primary cortical neurons and protection by α-tocotrienol and BL15

After 24h of seeding, cells were challenged with glutamate. Where indicated, neurons were pre-treated with either α-tocotrienol (250 nM) or BL15 (2.5 μM) for 5 min prior to glutamate treatment. a-h, Neuron specific Class III β-tubulin in the cultured neural network (for phase contrast microscopy see i-p). After 24h of glutamate treatment, cells were fixed and stained. a, control; b, glutamate; c, α-tocotrienol + glutamate; d, BL15+glutamate. e-h, Neurofilament staining in the cultured neural network (for phase contrast microscopy see i-p). e, control; f, glutamate; g, α-tocotrienol + glutamate; h, BL15+glutamate. i-p, Live cell imaging of glutamate treated neurons under standard ( not glass cover-slip) culture conditions. Phase contrast images were collected once every 15 mins for 18h from 8h after glutamate treatment. Frames illustrate time-dependent disintegration of the neural network. i, 8h; j, 12h; k, 16h; and l, 26h after glutamate treatment. Glutamate-challenged neurons pre-treated with α-tocotrienol (250 nM) resisted degeneration and continued to grow. m, 28h; n, 30h; o, 32h; and p, 34h after glutamate treatment. Two (i-l and m-p) .avi video micrographs have been supplemented for online publication. 200X magnification.

Figure 2. Imaging of glutamate-induced degeneration of rat primary cortical neurons and protection by α-tocotrienol and BL15

After 24h of seeding, cells were challenged with glutamate. Where indicated, neurons were pre-treated with either α-tocotrienol (250 nM) or BL15 (2.5 μM) for 5 min prior to glutamate treatment. a-h, Neuron specific Class III β-tubulin in the cultured neural network (for phase contrast microscopy see i-p). After 24h of glutamate treatment, cells were fixed and stained. a, control; b, glutamate; c, α-tocotrienol + glutamate; d, BL15+glutamate. e-h, Neurofilament staining in the cultured neural network (for phase contrast microscopy see i-p). e, control; f, glutamate; g, α-tocotrienol + glutamate; h, BL15+glutamate. i-p, Live cell imaging of glutamate treated neurons under standard ( not glass cover-slip) culture conditions. Phase contrast images were collected once every 15 mins for 18h from 8h after glutamate treatment. Frames illustrate time-dependent disintegration of the neural network. i, 8h; j, 12h; k, 16h; and l, 26h after glutamate treatment. Glutamate-challenged neurons pre-treated with α-tocotrienol (250 nM) resisted degeneration and continued to grow. m, 28h; n, 30h; o, 32h; and p, 34h after glutamate treatment. Two (i-l and m-p) .avi video micrographs have been supplemented for online publication. 200X magnification.

Figure 3. Pharmacologic inhibition of 12-lipoxygenase confers protection against glutamate-induced death of HT4 as well as primary immature cortical neurons (B-D)

HT4 neurons (A) were either treated or not with α−tocotrienol (250 nM) or BL15 (2.5 μM, 12-lipoxygenase inhibitor) for 5 min and then challenged with glutamate (10 mM). Cell viability was determined using propidium iodide (PI) exclusion flow cytometry assay. PI- = live; PI+ = dead. Rat primary immature cortical neurons (B-D) were either treated or not with α-tocotrienol (100 nM) or BL15 (2.5 μM) for 5 min and challenged either with glutamate (10 mM; B); L-homocysteic acid (1 mM; C) or buthionine sulfoximine (0.15 mM; BSO; D) for 24 h. Arachidonic acid, 50 μM for 24h. BL15, Baicalein 5,6,7-trihydroxy-flavone. Both α-tocotrienol and BL15 protected neurons against glutamate challenge despite loss of cellular glutathione (GSH; E). B-E: †, lower compared to the corresponding control non-treated group; *, higher compared to the corresponding toxin-treated group. P<0.05.

Figure 4. Primary immature cortical neurons isolated from 12-lipoxygenase knock out mice are resistant to glutamate-induced death

Murine primary immature cortical neuronal cells (C57BL/6, A; B6.129S2-Alox15 tm1Fun, B) were challenged with glutamate (10mM) for 24 h. Cell viability was assessed by lactate dehydrogenase assay. Treatment specifications are described in legend of Figure 1. α-tocotrienol, 100 nM. †, lower compared to the corresponding control non-treated group, also lower compared to corresponding group in 12-lipoxygenase deficient neurons; *, higher compared to the corresponding toxin-treated group. P<0.05.

Figure 5. Products of 12-lipoxygenase activity in glutamate-treated neurons

A, representative chromatogram for HETE, a key by-product of lipoxygenase activity. B, glutamate treatment for 12h resulted in elevation of 12(S)-HETE levels, a product of 12-lipoxygenase activity, in HT4 neurons. ND, not detectable.

Figure 6. 12-Lipoxygenase: over-expression, localization and sensitivity to α-tocotrienol

In HT4 cells, glutamate treatment for 2h resulted in diminished presence of 12-lipoxygenase in the cytosol (A) and increased presence in the membrane (B) suggesting mobilization of the enzyme from the cytosol to the membrane. C, successful over-expression of 12-lipoxygenase in HT4 cells. D, dose-dependent inhibition of pure 12-lipoxygenase activity by α-tocotrienol. Purified 12-lipoxygenase (porcine leukocyte; 10 units) was incubated with [14C]-arachidonic acid (25 μM) for 30 min at 37°C. Arachidonic acid and 12-HETE were resolved using thin layer chromatography as described in Materials & Methods.

Figure 7. Three-dimensional modeling of 12-lipoxygenase and α-tocotrienol docking analysis

A, three-dimensionsal structure of 12-lipoxygenase. Homology model construction was carried out on a Silicon Graphics O2 with 300MHz MIPS R5000, OS IRIX release 6.5. The theoretical model of 12-lipoxygenase was built using the Sybyl GeneFold module (v6.8, Tripos, Inc., St. Louis, MO). B & C, Theoretical model and α-tocotrienol dockings (two positions B & C shown with 10 different docking positions). Amino acid residues in red are His-360, His-365, His-540 and Ile-663 flanking the iron atom are visualized in bold typeface. D, Autodock calculated binding free energies for 10 different docking positions and sorted in increasing order energy of binding. RMSD, root mean square deviation.