Delayed-onset ataxia in mice lacking alpha -tocopherol transfer protein: model for neuronal degeneration caused by chronic oxidative stress

Abstract

alpha-Tocopherol transfer protein (alpha-TTP) maintains the concentration of serum alpha-tocopherol (vitamin E), one of the most potent fat-soluble antioxidants, by facilitating alpha-tocopherol export from the liver. Mutations of the alpha-TTP gene are linked to ataxia with isolated vitamin E deficiency (AVED). We produced a model mouse of AVED by deleting the alpha-TTP gene, which showed ataxia and retinal degeneration after 1 year of age. Because the brain alpha-TTP functions in maintaining alpha-tocopherol levels in the brain, alpha-tocopherol was completely depleted in the alpha-TTP(-/-) mouse brain, and the neurological phenotype of alpha-TTP(-/-) mice is much more severe than that of wild-type mice when maintained on an alpha-tocopherol-deficient diet. Lipid peroxidation in alpha-TTP(-/-) mice brains showed a significant increase, especially in degenerating neurons. alpha-Tocopherol supplementation suppressed lipid peroxidation and almost completely prevented the development of neurological symptoms. This therapy almost completely corrects the abnormalities in a mouse model of human neurodegenerative disease. Moreover, alpha-TTP(-/-) mice may prove to be excellent animal models of delayed onset, slowly progressive neuronal degeneration caused by chronic oxidative stress.

Figure 1

Analysis of motor performance. (A) Dystonic posture of hind limbs of an α-TTP^−/− mouse when the mouse was lifted by its tail. (B) Representative footprint patterns of a 14-month-old wild-type mouse (Left) and an α-TTP^−/− mouse on an α-tocopherol-deficient diet (Right). (C) Quantitative assessment of step length. (D) Performances on the accelerating rotating rod apparatus; four trials per day for 2 consecutive days. α- TTP^−/− mice had significantly shorter step length and poor performance on the rotating rod, which improved with α-tocopherol supplementation. Values are the mean and SEM. n = 10 for each group.

Figure 2

Electrophysiological analysis. (A) SEPs: Representative wave forms from wild-type and α-TTP^−/− mice on an α-tocopherol-deficient diet (Upper). Two averages of 100–300 sweeps recorded for each response are superimposed. In α-TTP^−/− mice, the potential from the lumbar root was similar to that of wild type (arrowhead), but the cortical potential almost disappeared. Sizes of the cortical potential of SEPs were measured from N1 to P1 (Lower). Values are the mean and SEM. n = 6 for each group; *, P < 0.01. (B) ERG analysis: Representative wave forms from wild-type and α-TTP^−/− mice on a normal diet at a stimulus intensity of −10 db (Upper). Sizes of the a-wave and b-wave were measured when the stimulus intensity was changed to −30, −10, and 0 db (Lower). Values are the mean and SEM. n = 10 for each group; *, P < 0.01.

Figure 3

Histological findings indicating neurodegeneration. (A–E) Toluidine blue-stained sections of the gracile fasciculus (posterior column for hind limb) at the fifth cervical segment of the spinal cord of a wild-type mouse on a normal diet (A), an α-TTP^−/− mouse on an α-tocopherol-deficient diet (B), an α-TTP^−/− mouse on a normal diet (C), an α-TTP^−/− mouse with α-tocopherol supplementation (D), and a wild-type mouse on an α-tocopherol-deficient diet (E). Marked reduction of myelinated fibers is seen in the gracile fasciculus of the α-TTP^−/− mice. The change was more prominent for those on a deficient diet (B), improved dramatically by supplementation of α-tocopherol (D), and mild in wild-type mice on an α-tocopherol-deficient diet (E). (F–H) Gliosis in the gracile fasciculus and nucleus at the medulla. (I–K) Gliosis in the anterior horn of the fifth cervical segment of the spinal cord. (L–N) Small angulated atrophic fibers (arrows) in the quadriceps femoris muscle. Wild-type mouse on a normal diet (F, I, and L), α-TTP^−/− mouse on a normal diet (G, J, and M), and α-TTP^−/− mouse with α-tocopherol supplementation (H, K, and N). Immunohistochemistry with anti-antiglial fibrillary acidic protein antibody (F–K) and stain with hematoxylin/eosin (L–N, O, and P). Shown also are retinal sections from wild-type mouse (O) and α-TTP^−/− mouse (P) on a normal diet stained with hematoxylin/eosin. The thicknesses of the outer nuclear layer (ONL) and the inner (IS) and outer (OS) segments of photoreceptor cells were reduced. GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer. (Scale bars: A–E, 25 μm; I–K, 100 μm; L–N, 50 μm.)

Figure 4

α-TTP expressed in the brain maintains α-tocopherol levels in the brain, and α-tocopherol supplementation still increases its concentration in the α-TTP^−/− mouse brain, which prevents symptoms. (A) Western blotting of liver, heart, cerebral cortex (Cx), cerebellum (Cll), and spinal cord from a wild-type mouse using anti-α-TTP antibody. (B) Plasma and tissue concentrations of α-tocopherol. In α-TTP^−/− mice on a normal or deficient diet of α-tocopherol, α-tocopherol was depleted in plasma and each tissue (data not shown in the figure). Values are the mean and SEM. n = 4 for each group; *, P < 0.001 compared with the corresponding value of wild-type.

Figure 5

Lipid peroxidation is increased in the brain of α-TTP^−/− mice. (A) TBARS in the brains. Values are mean and SEM. n = 4 for each group; **, P < 0.01; *, P < 0.01. (B) Western blotting of the spinal cord from wild-type and α-TTP^−/− mice using anti-HNE antibody. HNE-modified protein (21 kDa) is elevated significantly in α-TTP^−/− mice as compared with wild-type or α-TTP^−/− mice with α-tocopherol supplementation. Cx, cerebrum; Cll, Cerebellum.

Figure 6

Deposition of lipofuscin. (A and B) Dorsal root ganglions of wild-type (A) and α-TTP^−/− mice (B) stained with toluidine blue. Fine granular deposits of lipofuscin were present in ganglion cells in α-TTP^−/− mice. (C and D) Retinal sections of wild-type (C) and α-TTP^−/− (D) mice. Autofluorescence (green signal in D) was detected in the outer segment of photoreceptor cells (OS) and retinal pigmentary epithelium (RPE). (E–H) Transverse sections of the anterior horn at the seventh thoracic segment of the spinal cord stained with Fontana–Masson. Shown are wild-type mice on normal (E) and deficient (F) diets and α-TTP^−/− mice on a deficient diet (G) and supplemented with α-tocopherol (H). Black pigments (white arrow) show the massive accumulation of lipofuscin. (I) Quantitative analysis of lipofuscin in the anterior horn. The lipofuscin parameter was calculated by (area of lipofuscin, μm²) × (relative density of lipofuscin). Values are the mean and SEM. n = 3–4 for each group; **, P < 0.01; *, P < 0.01. (Scale bars: a–b, 50 μm; e–h, 100 μm.)