Xinhe Huang

Down-Regulating Sphingolipid Synthesis Increases Yeast Lifespan

Knowledge of the mechanisms for regulating lifespan is advancing rapidly, but lifespan is a complex phenotype and new features are likely to be identified. Here we reveal a novel approach for regulating lifespan. Using a genetic or a pharmacological strategy to lower the rate of sphingolipid synthesis, we show that Saccharomyces cerevisiae cells live longer. The longer lifespan is due in part to a reduction in Sch9 protein kinase activity and a consequent reduction in chromosomal mutations and rearrangements and increased stress resistance. Longer lifespan also arises in ways that are independent of Sch9 or caloric restriction, and we speculate on ways that sphingolipids might mediate these aspects of increased lifespan. Sch9 and its mammalian homolog S6 kinase work downstream of the target of rapamycin, TOR1, protein kinase, and play evolutionarily conserved roles in regulating lifespan. Our data establish Sch9 as a focal point for regulating lifespan by integrating nutrient signals from TOR1 with growth and stress signals from sphingolipids. Sphingolipids are found in all eukaryotes and our results suggest that pharmacological down-regulation of one or more sphingolipids may provide a means to reduce age-related diseases and increase lifespan in other eukaryotes.

Sphingolipids and lifespan regulation

Diseases including cancer, type 2 diabetes, cardiovascular and immune dysfunction and neurodegeneration become more prevalent as we age, and combined with the increase in average human lifespan, place an ever increasing burden on the health care system. In this chapter we focus on finding ways of modulating sphingolipids to prevent the development of age-associated diseases or delay their onset, both of which could improve health in elderly, fragile people. Reducing the incidence of or delaying the onset of diseases of aging has blossomed in the past decade because of advances in understanding signal transduction pathways and cellular processes, especially in model organisms, that are largely conserved in most eukaryotes and that can be modulated to reduce signs of aging and increase health span. In model organisms such interventions must also increase lifespan to be considered significant, but this is not a requirement for use in humans. The most encouraging interventions in model organisms involve lowering the concentration of one or more sphingolipids so as to reduce the activity of key signaling pathways, one of the most promising being the Target of Rapamycin Complex 1 (TORC1) protein kinase pathway. Other potential ways in which modulating sphingolipids may contribute to improving the health profile of the elderly is by reducing oxidative stresses, inflammatory responses and growth factor signaling. Lastly, perhaps the most interesting way to modulate sphingolipids and promote longevity is by lowering the activity of serine palmitoyltransferase, the first enzyme in the de novo sphingolipid biosynthesis pathway. Available data in yeasts and rodents are encouraging and as we gain insights into molecular mechanisms the strategies for improving human health by modulating sphingolipids will become more apparent. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Reducing sphingolipid synthesis orchestrates global changes to extend yeast lifespan

Studies of aging and longevity are revealing how diseases that shorten life can be controlled to improve the quality of life and lifespan itself. Two strategies under intense study to accomplish these goals are rapamycin treatment and calorie restriction. New strategies are being discovered including one that uses low‐dose myriocin treatment. Myriocin inhibits the first enzyme in sphingolipid synthesis in all eukaryotes, and we showed recently that low‐dose myriocin treatment increases yeast lifespan at least in part by down‐regulating the sphingolipid‐controlled Pkh1/2‐Sch9 (ortholog of mammalian S6 kinase) signaling pathway. Here we show that myriocin treatment induces global effects and changes expression of approximately forty percent of the yeast genome with 1252 genes up‐regulated and 1497 down‐regulated (P < 0.05) compared with untreated cells. These changes are due to modulation of evolutionarily conserved signaling pathways including activation of the Snf1/AMPK pathway and down‐regulation of the protein kinase A (PKA) and target of rapamycin complex 1 (TORC1) pathways. Many processes that enhance lifespan are regulated by these pathways in response to myriocin treatment including respiration, carbon metabolism, stress resistance, protein synthesis, and autophagy. These extensive effects of myriocin match those of rapamycin and calorie restriction. Our studies in yeast together with other studies in mammals reveal the potential of myriocin or related compounds to lower the incidence of age‐related diseases in humans and improve health span.

Sphingolipid Signaling Mediates Iron Toxicity

Iron constitutes a major source of toxicity due to its ability to generate reactive oxygen species that can damage cellular macromolecules. However, the precise mechanism by which exposure to high iron concentrations results in cellular toxicity remains unknown. Here we identify sphingolipid synthesis and signaling as a major mediator of iron toxicity in S. cerevisiae. Inhibition of sphingolipid synthesis by myriocin treatment or after overexpression of the negative regulator Orm2p confers resistance to high iron. High iron conditions upregulate sphingolipid synthesis, and increasing sphingolipid levels by inactivating Orm2p exacerbates sensitivity to iron. Toxicity is mediated by sphingolipid signaling, as inactivation of the sphingolipid-activated protein kinases Pkh1p and Ypk1p and of the transcription factor Smp1p also enhances resistance to high iron conditions. These results demonstrate an unexpected connection between sphingolipid flux and iron toxicity and show that activation of a signal transduction cascade contributes to iron-mediated cellular toxicity.

Reducing signs of aging and increasing lifespan by drug synergy

Disease incidence rises rapidly with age and increases both human suffering and economic hardship while shortening life. Advances in understanding the signaling pathways and cellular processes that influence aging support the possibility of reducing the incidence of age‐related diseases and increasing lifespan by pharmacological intervention. Here, we demonstrate a novel pharmacological strategy that both reduces signs of aging in the budding yeast Saccharomyces cerevisiae and generates a synergistic increase in lifespan. By combining a low dose of rapamycin, to reduce activity of the target of rapamycin complex 1 (TORC1) protein kinase, and myriocin, to reduce sphingolipid synthesis, we show enhancement of autophagy, genomic stability, mitochondrial function, and AMP kinase pathway activity. These processes are controlled by evolutionarily conserved signal transduction pathways that are vital for maintaining a healthy state and promoting a long life. Thus, our data show that it ought to be possible to find pharmacological approaches to generate a synergistic reduction in the incidence of human age‐related diseases to improve health quality in the elderly and enhance lifespan.

Zinc protects against cadmium-induced toxicity by regulating oxidative stress, ions homeostasis and protein synthesis

The widespread environmental toxin cadmium (Cd) is associated with numerous human diseases. The essential trace element zinc (Zn) strongly counteracts Cd-induced toxicity; however, the mechanism is incompletely understood. Here, we conducted RNA sequencing and bioinformatics analyses to determine the global gene expression profiles of yeast cells exposed to Cd or Cd plus Zn. We identified 912 Cd-induced and 627 Cd plus Zn-induced differentially expressed genes (DEGs). Adding Zn during Cd exposure efficiently reversed the expression of 92.1% of Cd-induced DEGs; that of 48.7% was entirely reversed. Gene Ontology, Cluster of Orthologous Group and KEGG Ontology analyses revealed that the response of yeasts to Cd or Cd plus Zn was mainly involved in metal-specific oxidative stress; energy production and conversion; ion homeostasis and ribosome biogenesis and translation. Exposure of yeasts to Cd plus Zn protected them from oxidative stress by efficiently inhibiting the expression of genes associated with Cd-triggered oxidative stress and preventing the disruption of Fe- and Zn-ion homeostasis and reduced glutathione and partially restored mitochondrial membrane potential. Moreover, Zn reduced the intracellular level of Cd to prevent the replacement by Cd of elements required for antioxidant enzyme activity and to protect protein sulphydryl groups against oxidation by free radicals. Further, Zn inhibited the synthesis alterations of Cd-induced ribosomal proteins, S-containing amino acids, S-rich proteins and antioxidant enzymes. Conversely, the investigation results of our study on the yeast model revealed that the Cd-treated protective effects of Zn on Cd-induced toxicity might be partially protective.

RNA-Seq Identifies Redox Balance Related Gene Expression Alterations under Acute Cadmium Exposure in Yeast.

The nonessential metal cadmium can cause cell toxicity and is associated with a range of human diseases including cardiovascular diseases, neurodegenerative diseases and cancers. In this study, cadmium-induced global gene expression profile of yeast was obtained using RNA Sequencing (RNA-Seq) and further analyzed by means of informatics and experiments. A total of 912 Differentially Expressed Genes (DEGs) (FDR of q < 0.01), including 415 Cd-inducible and 497 Cd-repressed genes were identified. Based on the DEGs, 25 cadmium responsive Clusters of Orthologous Group (COG) and three types of cadmium-induced Gene Ontology (GO) including cellular components, molecular functions and biological processes were analyzed in details. Thereafter, 79 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways under cadmium exposure were assigned. Collectively, 108 redox balance related genes were extracted under cadmium exposure. Meanwhile, cadmium exposure lowered cellular Mitochondrial Membrane Potential (MMP) and increased Reactive Oxygen Species (ROS) levels significantly in the context of mitochondrial dysfunction. Furthermore, cadmium exposure increased cellular GSH levels and decreased GSSG levels and also lowered GSSG/GSH ratio of cells, which supports experimentally our claim that the redox balance is the primary mechanism for cadmium toxicity. The results present in this study may provide new strategies for cadmium detoxification and prevention or therapies of cadmium-associated diseases.

Drug Synergy Drives Conserved Pathways to Increase Fission Yeast Lifespan

Aging occurs over time with gradual and progressive loss of physiological function. Strategies to reduce the rate of functional loss and mitigate the subsequent onset of deadly age-related diseases are being sought. We demonstrated previously that a combination of rapamycin and myriocin reduces age-related functional loss in the Baker’s yeast Saccharomyces cerevisiae and produces a synergistic increase in lifespan. Here we show that the same drug combination also produces a synergistic increase in the lifespan of the fission yeast Schizosaccharomyces pombe and does so by controlling signal transduction pathways conserved across a wide evolutionary time span ranging from yeasts to mammals. Pathways include the target of rapamycin complex 1 (TORC1) protein kinase, the protein kinase A (PKA) and a stress response pathway, which in fission yeasts contains the Sty1 protein kinase, an ortholog of the mammalian p38 MAP kinase, a type of Stress Activated Protein Kinase (SAPK). These results along with previous studies in S. cerevisiae support the premise that the combination of rapamycin and myriocin enhances lifespan by regulating signaling pathways that couple nutrient and environmental conditions to cellular processes that fine-tune growth and stress protection in ways that foster long term survival. The molecular mechanisms for fine-tuning are probably species-specific, but since they are driven by conserved nutrient and stress sensing pathways, the drug combination may enhance survival in other organisms.

Genetic association of rs1344706 in ZNF804A with bipolar disorder and schizophrenia susceptibility in Chinese populations

Rs1344706 in the the zinc finger protein 804A (ZNF804A) gene has been identified to be associated with schizophrenia and bipolar disorder (BD) in Europeans. However, whether rs1344706 is associated with schizophrenia in Chinese populations remains inconclusive; furthermore, the association between rs1344706 and BD in Chinese populations has been rarely explored. To explore the association between rs1344706 and schizophrenia/BD in Chinese populations, we genotyped rs1344706 among 1128 Chinese subjects (537 patients with BD and 591 controls) and found that rs1344706 showed marginal allelic association with BD (P = 0.028) with T-allele being more prevalent in cases than that in controls (OR = 1.19, 95% CI 1.03–1.37). Meta-analysis of rs1344706 by pooling all available data showed that rs1344706 was significantly associated with BD (P = 0.001). Besides, positive association of rs1344706 with schizophrenia was observed in Northern Chinese (P = 0.005). Furthermore, ZNF804A is highly expressed in human and mouse brains, especially in prenatal stage.

Selection and evaluation of reference genes for expression analysis of Cassi.

Cassia obtusifolia, belonging to legume family, is important in many fields with high pharmaceutical, economic, and ecological values. These interests of C. obtusifolia triggered in-depth and fundamental genetic and molecular research. Therefore, the stable reference gene is necessary for normalization of the gene expression studies. In this study, 10 candidate reference genes were subjected to expression analysis in 12 different tissues and under different stresses by qRT-PCR. The expression stability was evaluated using geNorm, NormFinder, and BestKeeper software. In conclusion, different suitable reference genes were selected in different tissues and under different stress. CYP1, EF1α2, ACT2, UBQ1 were the most stable reference genes in all samples. The relative expression levels of WRKY gene were detected to confirm the reliability of the selected reference genes. These results provided suitable reference genes that could be used for normalization in C. obtusifolia tissues and under different stress. Graphical abstract The relative transcript level of WRKY gene in 12 tissues and under different stress normalized by the selected reference genes