Mechanisms of Vitamin E Function Studied in Zebrafish

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Vitamin E was discovered in 1922 as a lipid soluble factor necessary for reproduction and named -tocopherol from the Greek words for “birth” and for “to carry”. To date, - tocopherol’s role in reproduction has been impossible to approach experimentally because - tocopherol-deficient (E-) mothers do not produce embryonic tissues due to failed implantation. To circumvent this barrier, we have chosen to use the premier vertebrate model for studying development, the zebrafish, because they lay eggs; thus, implantation is not necessary. The zebrafish model is ideal for our studies because it, like humans, has a preference for - tocopherol, expresses the ttp gene and requires vitamin C, an important determinant in - tocopherol antioxidant function in humans. An additional strength of this model over traditional rodent transgenic models is that any gene can be knocked down during embryogenesis, a feature we plan to exploit. We have succeeded in developing the first defined zebrafish diet. Using our E- defined diet, we are able to produce E- eggs. By 48 hours post-fertilization (hpf), many E- embryos exhibit severe developmental malformations, which establishes for the first time that -tocopherol is required for fetal neurological and cardiovascular development, not just implantation. We also found that the -tocopherol transfer protein -TTP) is abundantly expressed by the 48 hpf zebrafish embryo and its expression increases with oxidative stress. Severe vitamin E deficiency with progressive neurologic degeneration occurs in humans with ttp gene defects, emphasizing the critical importance of this protein. -TTP expression in the zebrafish embryo suggests that during development -TTP directs -tocopherol from the yolk sac to specific sites in the developing embryo where it is especially required. We propose that -tocopherol is required to protect key oxidized lipid mediators from further oxidative degradation during specific developmental steps, especially those in the nervous system. Specifically, glutathione peroxidase 4 (GPx4), a detoxifier of phospholipid hydroperoxides, translates oxidative stress into cell death that is mediated by both 12/15-lipoxygenase (12/15 LOX) and apoptosis-inducing factor (AIF); -tocopherol prevents these abnormalities in vitro. We hypothesize that α-tocopherol provides antioxidant protection for specific, key lipid mediators necessary for embryonic development, including cell loss via programmed cell death. To explain the key developmental effects of -tocopherol deficiency, we propose the following Aim 1. Define the roles of oxidant and antioxidant apoptosis regulators during embryonic development; Aim 2. Define the role of -TTP during embryonic development. Completion of these specific aims will allow us to determine specific -tocopherol molecular functions and key antioxidant/oxidant signaling mechanisms. Defining the molecular targets altered by - tocopherol-deficiency in the zebrafish will provide a solid basis for understanding why humans require -tocopherol for reproduction as well as to maintain a healthy nervous system