Mice lacking alpha-tocopherol transfer protein gene have severe alpha-tocopherol deficiency in multiple regions of the central nervous system

Abstract

Ataxia with vitamin E deficiency is caused by mutations in alpha-tocopherol transfer protein (alpha-TTP) gene and it can be experimentally generated in mice by alpha-TTP gene inactivation (alpha-TTP-KO). This study compared alpha-tocopherol (alpha-T) concentrations of five brain regions and of four peripheral organs from 5 months old, male and female, wild-type (WT) and alpha-TTP-KO mice. All brain regions of female WT mice contained significantly higher alpha-T than those from WT males. alpha-T concentration in the cerebellum was significantly lower than that in other brain regions of WT mice. These sex and regional differences in brain alpha-T concentrations do not appear to be determined by alpha-TTP expression which was undetectable in all brain regions. All the brain regions of alpha-TTP-KO mice were severely depleted in alpha-T. The concentration of another endogenous antioxidant, total glutathione, was unaffected by gender but was decreased slightly but significantly in most brain regions of alpha-TTP-KO mice. The results show that both gender and the hepatic alpha-TTP, but not brain alpha-TTP gene expression are important in determining alpha-T concentrations within the brain. Interestingly, functional abnormality (ataxia) develops only very late in alpha-TTP-KO mice in spite of the severe alpha-tocopherol deficiency in the brain starting at an early age.

Figure 1. α-T concentrations in five brain regions of 5 months-old WT male and female brains

All brain regions from female mice (n = 5) contained significantly higher concentrations of α-T than that from the brains of male mice (n = 3). α-T concentration of the cerebellum was lower than that of the other brain regions. COR = cortex, CER = cerebellum, HIP = hippocampus, BS = brainstem, MB = midbrain.

Figure 2. Cholesterol concentrations in male and female brain regions

Cholesterol concentrations in five brain regions of 5 months-old WT male and female brains. Data show no significant effect of sex on the cholesterol concentrations in the five brain regions. COR = cortex, CER = cerebellum, HIP = hippocampus, BS = brainstem, MB = midbrain. The data show mean ± SEM, n = 3 for male mice and n = 5 for female mice.

Figure 3. α-TTP expression by immunoblot analysis

Immunoblot analysis of α-TTP expression in cerebellum, cortex and, liver homogenates from WT (n = 2, lanes 1 and 2) and α-TTP-KO (n = 2, lanes 3 and 4) mice. Forty µg of protein from each tissue homogenate were resolved by PAGE and electro transferred to membranes as described in the Methods. The immunoblots were probed with rabbit anti-α-TTP antiserum (a generous gift from Professor Maret G. Traber, Linus Pauling Institute, Oregon State University, Corvallis, OR, USA). A ~32kDa protein was detected in livers from WT mice. The protein was absent in livers from α-TTP-KO mice. The ~32 kDa anti-α-TTP antiserum reactive protein was not detectable in cortex or in the cerebellum of either the WT or the α-TTP-KO mice. MW= Molecular weight standards.

Figure 4. α-TTP mRNA expression by qRT-PCR analysis

The expression of α-TTP mRNA in cerebellum and cortex is undetectable by qRT-PCR assay. The data were normalized for the expression of GAPDH mRNA in the same samples. α-TTP mRNA is abundantly expressed in livers from wild type (WT) mice and its expression is near the limit of detection in the livers from α-TTP-KO mice. Its expression was not detectable (ND) in either the cerebellum or the cortex from the same mice. Data are mean ± SEM, n=5.

Figure 5. Scavenger receptor B1 expression is undetectable in brain regions

The data show abundant expression of the receptor (~82 kDa) in liver and adrenal glands but undetectable expression in the three brain regions. A ~57kDa immunoreactive protein was also detected in livers and adrenal gland, and it probably is an unglycosylated SRB1. Aliquots of homogenates from WT liver (lane 1, 60 µg protein), α-TTP-KO liver (lane 2, 60 µg protein), WT adrenal glands (lane 3, 60 µg and lane 4, 20 µg), WT cerebellum, WT medulla and WT cortex (lanes 5, 6 and 7, respectively, 60 µg protein each,) were processed for immunoblot detection of SR-B1 expression. The blots were over-exposed to enable visualization of the immunoreactive protein in the brain regions.

Figure 6. Brain regions of male a-TTP-KO mice are severely α-T depleted IN AT

α-T concentrations in the five regions of brains from five months-old male mice. The data show that α-T concentrations in all brain regions were significantly (p< 0.00001) lower than those in the brains of either the WT mice fed the same α-T diet (35 IU/kg diet) or in the brains of WT mice fed the α-T deficient diet. The data show mean ± SEM, n = 3. COR = cortex, CER = cerebellum, HIP = hippocampus, BS = brainstem, MB = midbrain.

Figure 7. Cholesterol concentrations are unaffected by the absence of α-TTP

Cholesterol concentrations in the five distinct anatomical regions of the female and male brains were unaffected by the absence of α-TTP gene. COR = cortex, CER = cerebellum, HIP = hippocampus, BS = brainstem, MB = midbrain. The data show mean ± SEM, n = 3 for male mice and n = 5 for female mice.

Figure 8. Differential effects of α-TTP gene deletion on non-CNS tissues

α-T concentrations in peripheral tissues of male and female mice of WT and α-TTP-KO mice. α-T concentrations in livers (LIV) and hearts (HRT) from WT female mice were significantly ( * , p<0.001) higher (43%) than those in the male mice. α-T in ovaries (OVA) was significantly higher than that in testes (TST). Absence of the α-TTP gene resulted in a significant (†, p< 0.0001) and a large (~90%) decrease in the α-T concentrations of heart, testes and ovaries. In contrast, the decrease in liver α-T of α-TTP-KO mice was smaller (~50%) than that of the other tissues. The data are mean ± SEM (n = 5–7)

Figure 9. Glutathione concentrations in tissues of WT and α-TTP-KO mice

Absence of α-TTP gene results in small but statistically significant decrease in total GSH of selected brain regions and in liver of female mice. The data are mean ± SEM, N = 3–5. Total GSH was significantly (p< 0.001–0.034) lower (~7–18%) in cerebellum (CER), hippocampus (HIP), brainstem (BS), midbrain (MB) and liver (LIV) from α-TTP-KO mice compared to that in the tissues from the WT mice. There was no significant difference (NS) between the WT and α-TTP-KO mice for the total GSH in cortex (COR), heart (HRT), ovaries (OVA).