Glut1 deficiency syndrome: New and emerging insights into a prototypical brain energy failure disorder.

Considering its small size relative to the rest of the body, the mammalian brain has a disproportionately high energy requirement. This energy is supplied to the brain mainly in the form of glucose through the principal cerebral glucose transporter, Glut1. Inactivation of even a single copy of the Glut1 gene, SLC2A1, has dire consequences for the brain, starving cerebral neurons of energy and triggering the debilitating neurodevelopmental disorder, Glut1 deficiency syndrome (Glut1 DS). Considering the monogenic nature of Glut1 DS, the disease serves as an excellent paradigm to study the larger family of brain energy failure syndromes. Here we review how studies of Glut1 DS are proving instructive to the brain's energy needs, focusing first on the requirements, both spatial and temporal of the transporter, second, on proposed mechanisms linking low Glut1 to brain dysfunction and, finally on efforts to treat the disease and thus restore nutritional support to the brain. These studies promise not only to inform mechanisms and treatments for the relatively rare Glut1 DS but also the myriad other conditions involving the Glut1 protein.

An early endothelial cell-specific requirement for Glut1 is revealed in Glut1 deficiency syndrome model mice.

Paucity of the glucose transporter-1 (Glut1) protein resulting from haploinsufficiency of the SLC2A1 gene arrests cerebral angiogenesis and disrupts brain function to cause Glut1 deficiency syndrome (Glut1 DS). Restoring Glut1 to Glut1 DS model mice prevents disease, but the precise cellular sites of action of the transporter, its temporal requirements, and the mechanisms linking scarcity of the protein to brain cell dysfunction remain poorly understood. Here, we show that Glut1 functions in a cell-autonomous manner in the cerebral microvasculature to affect endothelial tip cells and, thus, brain angiogenesis. Moreover, brain endothelial cell-specific Glut1 depletion not only triggers a severe neuroinflammatory response in the Glut1 DS brain, but also reduces levels of brain-derived neurotrophic factor (BDNF) and causes overt disease. Reduced BDNF correlated with fewer neurons in the Glut1 DS brain. Controlled depletion of the protein demonstrated that brain pathology and disease severity was greatest when Glut1 scarcity was induced neonatally, during brain angiogenesis. Reducing Glut1 at later stages had mild or little effect. Our results suggest that targeting brain endothelial cells during early development is important to ensure proper brain angiogenesis, prevent neuroinflammation, maintain BDNF levels, and preserve neuron numbers. This requirement will be essential for any disease-modifying therapeutic strategy for Glut1 DS.

Therapeutic strategies for glucose transporter 1 deficiency syndrome.

Proper development and function of the mammalian brain is critically dependent on a steady supply of its chief energy source, glucose. Such supply is mediated by the glucose transporter 1 (Glut1) protein. Paucity of the protein stemming from mutations in the associated SLC2A1 gene deprives the brain of glucose and triggers the infantile-onset neurodevelopmental disorder, Glut1 deficiency syndrome (Glut1 DS). Considering the monogenic nature of Glut1 DS, the disease is relatively straightforward to model and thus study. Accordingly, Glut1 DS serves as a convenient paradigm to investigate the more general cellular and molecular consequences of brain energy failure. Here, we review how Glut1 DS models have informed the biology of a prototypical brain energy failure syndrome, how these models are facilitating the development of promising new treatments for the human disease, and how important insights might emerge from the study of Glut1 DS to illuminate the myriad conditions involving the Glut1 protein.

Brain microvasculature defects and Glut1 deficiency syndrome averted by early repletion of the glucose transporter-1 protein.

Haploinsufficiency of the SLC2A1 gene and paucity of its translated product, the glucose transporter-1 (Glut1) protein, disrupt brain function and cause the neurodevelopmental disorder, Glut1 deficiency syndrome (Glut1 DS). There is little to suggest how reduced Glut1 causes cognitive dysfunction and no optimal treatment for Glut1 DS. We used model mice to demonstrate that low Glut1 protein arrests cerebral angiogenesis, resulting in a profound diminution of the brain microvasculature without compromising the blood-brain barrier. Studies to define the temporal requirements for Glut1 reveal that pre-symptomatic, AAV9-mediated repletion of the protein averts brain microvasculature defects and prevents disease, whereas augmenting the protein late, during adulthood, is devoid of benefit. Still, treatment following symptom onset can be effective; Glut1 repletion in early-symptomatic mutants that have experienced sustained periods of low brain glucose nevertheless restores the cerebral microvasculature and ameliorates disease. Timely Glut1 repletion may thus constitute an effective treatment for Glut1 DS.

Safety Evaluation of Neo Transgenic Pigs by Studying Changes in Gut Microbiota Using High-Throughput Sequencing Technology.

The neo (neomycin phosphotransferase) gene is widely used as a selection marker in the production of genetically engineered animals and plants. Recent attention has been focused on safety concerns regarding neo transgene expression. In this study, neo transgenic and non-transgenic piglets were randomly assigned into Group A and Group B to evaluate effects of neo transgene by studying changes in gut microbiota using high-throughput sequencing. Group A pigs were fed a standard diet supplemented with antibiotic neomycin; Group B pigs were fed a standard diet. We examined horizontal transfer of exogenous neo gene using multiplex PCR; and investigated if the presence of secreted NPT II (neo expression product) in the intestine could lead to some protection against neomycin in transgenic pigs by monitoring different patterns of changes in gut microbiota in Group A animals. The unintended effects of neo transgene on gut microbiota were studied in Group B animals. Horizontal gene transfer was not detected in gut microbiota of any transgenic pigs. In Group A, a significant difference was observed between transgenic pigs and non-transgenic pigs in pattern of changes in Proteobacteria populations in fecal samples during and post neomycin feeding. In Group B, there were significant differences in the relative abundance of phyla Firmicutes, Bacteroidetes and Proteobacteria, and genera Lactobacillus and Escherichia-Shigella-Hafnia between transgenic pigs and non-transgenic pigs. We speculate that the secretion of NPT II from transgenic tissues/cells into gut microbiota results in the inhibition of neomycin activity and the different patterns of changes in bacterial populations. Furthermore, the neo gene also leads to unintended effects on gut microbiota in transgenic pigs that were fed with basic diet (not supplemented with neomycin). Thus, our data in this study caution that wide use of the neo transgene in genetically engineered animals should be carefully considered and fully assessed.

CoQ10 Deficiency Is Not a Common Finding in GLUT1 Deficiency Syndrome.

CoQ10 deficiency has been recently described in tissues of a patient with GLUT1 deficiency syndrome. Here, we investigated patients and mice with GLUT1 deficiency in order to determine whether low CoQ is a recurrent biochemical feature of this disorder, to justify CoQ10 supplementation as therapeutic option.CoQ10 levels were investigated in plasma, white blood cells, and skin fibroblasts of 16 patients and healthy controls and in the brain, cerebellum, liver, kidney, muscle, and plasma of 4-month-old GLUT1 mutant and control mice.CoQ10 levels in plasma did not show any difference compared with controls. Since most of the patients studied were on a ketogenic diet, which can alter CoQ10 content in plasma, we also analyzed white blood cells and cultured skin fibroblasts. Again, we found no differences. In mice, we found slightly reduced CoQ in the cerebellum, likely an epiphenomenon, and activity of the mitochondrial respiratory chain enzymes was normal.Our data from GLUT1 deficiency patients and from GLUT1 model mice fail to support CoQ10 deficiency as a common finding in GLUT1 deficiency, suggesting that CoQ deficiency is not a direct biochemical consequence of defective glucose transport caused by molecular defects in the SLC2A1 gene.

Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs.

Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN(-/-)) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN(+/+)) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.

Functional and safety evaluation of transgenic pork rich in omega-3 fatty acids.

Genetically modified animals rich in omega-3 unsaturated fatty acid offer a new strategy to improve the human health, but at the same time present a challenge in terms of food safety assessment. In this study, we evaluated the function and safety of sFat-1 transgenic pork rich in omega-3 fatty acids in mice by feeding basic diet and diets that contain wild type pork and sFat-1 transgenic pork. Blood biochemistry, haematology, peripheral T cell distributions, bacterial counts, gross necropsy, histopathology and organ weights were performed in mice fed with different doses of wild type and transgenic pork. Results indicated that both low and high dose of wild type and transgenic pork had no significant effect on blood biochemistry, T cell distribution, immunoglobulins and bacterial counts in intestine and feces. However, it was noted that both low and high dose of transgenic pork improved the liver immune system in mice, which is probably due to the beneficial contribution of high level of the "good" fatty acids in transgenic pork. There is no significant effect of transgenic pork on all other organs in mice. In summary, our study clearly demonstrated that feeding transgenic pork rich in omega-3 fatty acids did not cause any harm to mice, and in fact, improved the liver immune system.

A 90-day safety study of genetically modified rice expressing rhIGF-1 protein in C57BL/6J rats.

Genetically modified plants expressing disease resistance traits offer new treatment strategies for human diseases, but at the same time present a challenge in terms of food safety assessment. The present 90-day feeding study was designed to assess the safety of transgenic rice expressing the recombinant human insulin-like growth factor-1 (rhIGF-1) compared to its parental wild rice. Male and female C57BL/6J rats were given a nutritionally balanced purified diet with 20% transgenic rhIGF-1 rice or 20% parental rice for 90 days. This corresponds to a mean daily rhIGF-1 protein intake of approximately 217.6 mg/kg body weight based on the average feed consumption. In the animal study a range of biological, biochemical, clinical, microbiological and pathological parameters were examined and several significant differences were observed between groups, but none of the effects were considered to be adverse. In conclusion, no adverse or toxic effects on C57BL/6J rats were observed in the design used in this 90-day study. These results will provide valuable information for the safety assessment of genetically modified food crops.

Safety assessment of sFat-1 transgenic pigs by detecting their co-habitant microbe in intestinal tract.

Transgenic pigs containing sFat-1 (synthesized fatty acid desaturase-1) gene were produced by DNA microinjection. The meat of these pigs contain ω-3 unsaturated fatty acid which is beneficial to the health of human being. The aim of this study is to assess the effects of expression of sFat-1 in pig on the health of animal themselves and on the safety of environment from the angle of the changes in microbe population of pig intestinal tract. Four F1 male semi-sibling of sFat-1 transgenic pigs and four F1 female semi-sibling of sFat-1 transgenic pigs were used as experimental animals, together with their none transgenic siblings as control animals. For inspection of any change in populations of microbial flora in various parts of intestinal tract and feces of sFat-1 transgenic pigs total aerobe, total anaerobe, main beneficial bacteria and main harmful bacteria were cultured and analyzed. At the same time foreign gene drift was assessed by PCR amplifying foreign fragment in samples of total aerobe and total anaerobe. Results indicated that in comparison with control pig microbe population of various species in different parts of intestinal tract and feces of sFat-1 transgenic pig remained unchanged and foreign gene sequence could not be detected in DNA of total aerobe and total anaerobe. In conclusion, the data of this study suggest that microbe population in intestinal tract and feces of transgenic pig was not influenced by the expression of transferred foreign gene sFat-1 and also foreign gene drifting from animal genome to microbial genome must be a rare incident.