I. Metabolic State of Mouse Embryonic Stem Cells:
By surveying the abundance of common metabolites in mouse embryonic (ES) cells as a function of differentiation, it has been observed that metabolites associated with one carbon metabolism change radically when undifferentiated ES cells are cued to differentiate into embryoid bodies. These observations led to the discovery that ES cells are uniquely dependent upon threonine catabolism. Jian Wang and Peter Alexander have found that undifferentiated ES cells express the threonine dehydrogenase (TDH) enzyme at a 1,000-fold higher level than any other cell line or mouse tissue tested. TDH is a mitochondrial enzyme that catabolizes threonine into glycine and acetyl-CoA. The former metabolite is further catabolized by the mitochondrial glycine cleavage system in order to charge tetrahydrofolate (THF) into methyl-THF for use in purine biosynthesis and other forms of one carbon metabolism. The latter metabolite, acetyl-CoA, can be used to feed the tricarboxylic acid (TCA) cycle for the generation of ATP. The first chapter of this story was published in the summer of 2009 (Wang, Alexander, Wu, Hammer, Cleaver and McKnight, 2009, Science 325:435). Evolution of this project will now turn towards the possibility that the exceptionally high levels of acetyl-CoA in undifferentiated stem cells might facilitate acetylation of certain target proteins instrumental for the unusually rapid cell division cycle of ES cells. These cells divide once every 4-5 hours, which is a more rapid rate of proliferation than even the fastest growing of all cancer cells. Efforts have been made to tailor strains of mice wherein the TDH gene can be conditionally eliminated via Cre-mediated recombination, such that it might be possible to rigorously test the necessity of TDH enzyme requirement in the inner cell mass of the blastocyst embryo. Finally, a high throughput screen of the UTSWMC compound file of 250,000 drug-like chemicals have led to the discovery of potent and specific inhibitors of the mouse TDH enzyme. These inhibitors are selectively toxic to mouse ES cells, presumably due to their ability to block the activity of the TDH enzyme which is critical for maintaining the unusual metabolic state of mouse ES cells.
II. Discovery of Pro-Neurogenic Chemicals:
Mice bearing an inactivating mutation in the gene encoding neuronal PAS domain protein 3 (NPAS3) are incapable of supporting neurogenesis in the dentate gyrus of adult brain tissue (Pieper, Wu, Han, Estill, Dang, Wu, Reece-Fincanon, Dudley, Richardson, Brat and McKnight, 2005, Proc. Nat. Acad. Sci. USA 102:14,052). Knowing that NPAS3-deficient mice suffer numerous behavioral abnormalities, these observations prompted a laborious, in vivo drug screen in search of pro-neurogenic chemicals. This project, spearheaded by Andrew Pieper when he was a postdoctoral fellow in the McKnight lab, is now five years in the making. Briefly, 1,000 compounds were selected from the UTSWMC compound file with help from our synthetic organic chemistry colleagues. Pools of ten compounds were infused into the left ventricle of adult mice via an Alzet mini-pump. Animals were injected daily with bromodeoxyuridine (BrdU) throughout the seven day infusion period, and then sacrificed so that brain tissue could be fixed, embedded, sectioned and stained with antibodies to BrdU. By counting the number of BrdU-positive cells in the subgranular layer of the dentate gyrus, it was possible to score for compound pools that enhanced neurogenesis. Ten of the one hundred pools tested yielded a positive effect on BrdU incorporation. These ten pools were, in turn, broken down so that the individual compounds could be tested for pro-neurogenic activity on four mice per compound. Eight of the ten pools yielded a single, pro-neurogenic compound. One of these eight compounds, designated “pool seven, compound three” – or P7C3 – was found to have favorable pharmacological properties. It has a half life in mice of seven hours, is orally bioavailable and crosses the blood brain barrier – thereby facilitating all kinds of in vivo experiments critical to its study. Ongoing experiments are seeking to determine whether prolonged administration of P7C3 to NPAS3-deficient mice might be capable of correcting morphological and electrophysiological deficits of the dentate gyrus granular layer. Efforts are also being extended towards mechanism of action studies – for example, does P7C3 induce neuronal stem cells in the subgranular layer of the dentate gyrus to divide more rapidly? The laboratories of Dr. Pieper, who is now an independent Assistant Professor of Psychiatry, and Dr. McKnight continue to study P7C3 and the other seven pro-neurogenic compounds on a close, collaborative basis.
III. Metabolic Cycles:
During the 2004-2008 timeframe the McKnight lab, in efforts primarily led by Benjamin Tu and Jake Chen, studied the growth of a prototrophic yeast strain under nutrient-limiting conditions in a chemostat. Quite remarkably, the cells enter into a self-synchronized metabolic cycle 4-5 hours in length. During this metabolic cycle yeast cells fluctuate rhythmically back and forth between respirative and glycolytic metabolism. DNA microarray studies have shown that over half of the genes in the yeast genome are selectively expressed during specific windows of this yeast metabolic cycle (YMC). Studies of the grouping of genes that become activated at specific times of the YMC have offered a simple logic to explain how and why the cells are able to perform specific functions at specific times (Tu, Kudlicki, Rowicka and McKnight, 2005, Science 310:1152). Moving beyond gene expression studies, Benjamin Tu – in collaboration with Ted Young and his colleagues at the University of Washington – studied fluctuation in the abundance of hundreds of metabolites as a function of the YMC by use of various forms of liquid and gas chromatography and mass spectrometry. These observed patterns of metabolite fluctuation largely conformed to predictions coming from patterns of gene expression (Tu, Mohler, Liu, Dombek, Young, Synovec and McKnight, Proc. Nat. Acad. Sci. USA 104:16,886).
One of the observations made by Tu and Chen was that yeast cells are only able to divide during one of the three defined phases of the YMC. This phase, designated the reductive building (RB) phase, is when cells cease mitochondrial respiration and enhance the rate of glycolytic metabolism. It was hypothesized that DNA synthesis is restricted to the RB phase as a means of protecting genome integrity. This idea was prosecuted in studies of yeast strains bearing mutations that impede progress through the cell division cycle. Slow-growing strains were found to display correlative reductions in the length of the YMC. Wild-type yeast cells, when grown in rich culture medium, double in growth about once per hour and display a YMC lasting roughly 4 hours. Strains missing genes encoding proteins important for cell cycle progression, such as swi6 or sic1, double much more slowly and display YMC’s of roughly two and one hours respectively. It was found that the latter strains are forced to undergo DNA replication in all phases of the YMC, including the oxidative phase when mitochondrial respiration is taking place. Analysis of the swi6 and sic1 mutants led to the discovery that they suffer considerably higher rates of spontaneous mutation when grown in the chemostat, but not when grown in glucose-rich culture medium (Chen, Odstrcil, Tu and McKnight, Science 316:1916). Such observations are consistent with the hypothesis that wild type yeast know how to restrict DNA synthesis and cell division to the reductive phase of the YMC as a means of protecting genome integrity.
Benjamin Tu has inherited the YMC project as an independent Assistant Professor of Biochemistry here at UT Southwestern Medical Center. The McKnight lab – in efforts spearheaded by Jake Chen and Shanhai Xie – have turned to mammalian systems in search of phenomena related to the YMC. Ongoing experiments have given evidence of a metabolic cycle of circadian dimensions in the brain tissue of adult mice. Using liquid chromatography/mass spectrometry, Drs. Chen and Xie quantitated the abundance of dozens of metabolites surveyed at four hour intervals for mice exposed to running wheels and kept on a 12:12hr light:dark cycle. A number of metabolites involved in sulfur flux, including S-adenosylmethinonie (SAM), S-adneosylhomocysteine (SAH) and cystothione fluctuated in abundance as a function of the day:night cycle. Intriguingly, the metabolites that fluctuate in abundance most robustly in the yeast metabolic cycle correspond closely with those that fluctuate most robustly in the brain tissue of adult mice. By subjecting animals to sleep deprivation, it has been observed that metabolite fluctuation is driven by sleep, not by circadian rhythm. We do not yet understand the significance of these observations, but are encouraged to consider the idea that the metabolic state of neurons may be dynamic. If so, it is possible that neurons are able to perform certain tasks more optimally at certain times of the day:night and/or sleep:wake cycle than others.