Transcriptional profile reveals altered hepatic lipid and cholesterol metabolism in hyposulfatemic NaS1 null mice

Sulfate plays an essential role in human growth and development, and its circulating levels are maintained by the renal Na+-SO42- cotransporter, NaS1. We previously generated a NaS1 knockout ( Nas1(-/-)) mouse, an animal model for hyposulfatemia, that exhibits reduced growth and liver abnormalities including hepatomegaly. In this study, we investigated the hepatic gene expression profile of Nas1(-/-) mice using oligonucleotide microarrays. The mRNA expression levels of 92 genes with known functional roles in metabolism, cell signaling, cell defense, immune response, cell structure, transcription, or protein synthesis were increased ( n = 51) or decreased ( n = 41) in Nas1(-/-) mice when compared with Nas1(-/-) mice. The most upregulated transcript levels in Nas1(-/-) mice were found for the sulfotransferase genes, Sult3a1 ( approximate to 500% increase) and Sult2a2 ( 100% increase), whereas the metallothionein-1 gene, Mt1, was among the most downregulated genes ( 70% decrease). Several genes involved in lipid and cholesterol metabolism, including Scd1, Acly, Gpam, Elov16, Acsl5, Mvd, Insig1, and Apoa4, were found to be upregulated ( >= 30% increase) in Nas1(+/+) mice. In addition, Nas1(+/+) mice exhibited increased levels of hepatic lipid ( approximate to 16% increase), serum cholesterol ( approximate to 20% increase), and low-density lipoprotein ( approximate to 100% increase) and reduced hepatic glycogen ( approximate to 50% decrease) levels. In conclusion, these data suggest an altered lipid and cholesterol metabolism in the hyposulfatemic Nas1(-/-) mouse and provide new insights into the metabolic state of the liver in Nas1(-/-) mice.

Effects of Variation at the ALDH2 Locus on Alcohol Metabolism, Sensitivity, Consumption, and Dependence in Europeans

Background: The low-activity variant of the aldehyde dehydrogenase 2 (ALDH2) gene found in East Asian populations leads to the alcohol flush reaction and reduces alcohol consumption and risk of alcohol dependence (AD). We have tested whether other polymorphisms in the ALDH2 gene have similar effects in people of European ancestry. Methods: Serial measurements of blood and breath alcohol, subjective intoxication, body sway, skin temperature, blood pressure, and pulse were obtained in 412 twins who took part in an alcohol challenge study. Participants provided data on alcohol reactions, alcohol consumption, and symptoms related to AD at the time of the study and subsequently. Haplotypes based on 5 single-nucleotide polymorphisms (SNPs) were used in tests of the effects of variation in the ALDH2 gene on alcohol metabolism and alcohol's effects. Results: The typed SNPs were in strong linkage disequilibrium and 2 complementary haplotypes comprised 83% of those observed. Significant effects of ALDH2 haplotype were observed for breath alcohol concentration, with similar but smaller and nonsignificant effects on blood alcohol. Haplotype-related variation in responses to alcohol, and reported alcohol consumption, was small and not consistently in the direction predicted by the effects on alcohol concentrations. Conclusions: Genetic variation in ALDH2 affects alcohol metabolism in Europeans. However, the data do not support the hypothesis that this leads to effects on alcohol sensitivity, consumption, or risk of dependence.

A golden age of human pigmentation genetics

The zebrafish golden mutation is characterized by the production of small and irregular-shaped melanin granules, resulting in a lightening of the pigmented lateral stripes of the animal. The recent positional cloning and localization of the golden gene, combined with genotype-phenotype correlations of alleles of its human orthologue (SLC24A5) in African-American and African-Caribbean populations, provide insights into the genetic and molecular basis of human skin colour. SLC24A5 promotes melanin deposition through maturation of the melanosome, highlighting the importance of ion-exchange in the function of this organelle.

The genetics of primary congenital glaucoma

One of the objectives of the molecular biological study of glaucoma is to establish how the disease develops as a result of the production of aberrant gene products. Many of the genes associated with glaucoma code for proteins which are likely to be directly or indirectly involved in the development and/or function of cells within the trabecular meshwork. The identification of specific defects in these genes is likely to lead to a better understanding of the mechanisms involved in PCG and glaucoma in general and to the development of alternative therapies to surgery. The CYP1B1 gene in particular, which is a linked to congenital glaucoma, and is expressed in the trabecular meshwork, codes for a member of the cytochrome P450 group of proteins. These iron binding proteins constitute a family of enzymes involved in the processes of xenobiotic metabolism, growth, and development. The discovery of the CYP1B1 gene in PCG emphases the importance of abnormalities in the molecular structure of proteins expressed in cells of the trabecular network as a cause of PCG. The identification of specific genetic defects leads to the possibility of more widespread screening for PCG especially in affected families and hence, the possibility of the identification of asymptomatic carriers of the disease. Early identification of 'at risk' parents may then enable earlier detection of PCG and intervention in the infant.

Arsenic transport by zebrafish aquaglyceroporins

Background Arsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport of AsIII and MAsIII in in any fish species has not been characterized. Results In this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII) and antimonite (SbIII) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were namedaqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from Xenopus leavis. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic AsIII, MAsIII and SbIII. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye, heart, intestine muscle and skin also exhibited significant ability to accumulate arsenic. The zebrafish larvae also accumulate considerable amounts of arsenic. Conclusion This is the first molecular identification of fish arsenite transport systems and we propose that the extensive expression of the fish aquaglyceroporins and their ability to transport metalloids suggests that aquaglyceroporins are the major pathways for arsenic accumulation in a variety of zebrafish tissues. Uptake is one important step of arsenic metabolism. Our results will contribute to a new understanding of aquatic arsenic metabolism and will support the use of zebrafish as a new model system to study arsenic associated human diseases.

Metabolism Regulates the Fate and Function of T Lymphocytes

Proper balancing of the activities of metabolic pathways to meet the challenge of providing necessary products for biosynthetic and energy demands of the cell is a key requirement for maintaining cell viability and allowing for cell proliferation. Cell metabolism has been found to play a crucial role in numerous cell settings, including in the cells of the immune system, where a successful immune response requires rapid proliferation and successful clearance of dangerous pathogens followed by resolution of the immune response. Additionally, it is now well known that cell metabolism is markedly altered from normal cells in the setting of cancer, where tumor cells rapidly and persistently proliferate. In both settings, alterations to the metabolic profile of the cells play important roles in promoting cell proliferation and survival.

It has long been known that many types of tumor cells and actively proliferating immune cells adopt a metabolic phenotype of aerobic glycolysis, whereby the cell, even under normoxic conditions, imports large amounts of glucose and fluxes it through the glycolytic pathway and produces lactate. However, the metabolic programs utilized by various immune cell subsets have only recently begun to be explored in detail, and the metabolic features and pathways influencing cell metabolism in tumor cells in vivo have not been studied in detail. The work presented here examines the role of metabolism in regulating the function of an important subset of the immune system, the regulatory T cell (Treg) and the role and regulation of metabolism in the context of malignant T cell acute lymphoblastic leukemia (T-ALL). We show that Treg cells, in order to properly function to suppress auto-inflammatory disease, adopt a metabolic program that is characterized by oxidative metabolism and active suppression of anabolic signaling and metabolic pathways. We found that the transcription factor FoxP3, which is highly expressed in Treg cells, drives this phenotype. Perturbing the metabolic phenotype of Treg cells by enforcing increased glycolysis or driving proliferation and anabolic signaling through inflammatory signaling pathways results in a reduction in suppressive function of Tregs.

In our studies focused on the metabolism of T-ALL, we observed that while T-ALL cells use and require aerobic glycolysis, the glycolytic metabolism of T-ALL is restrained compared to that of an antigen activated T cell. The metabolism of T-ALL is instead balanced, with mitochondrial metabolism also being increased. We observed that the pro-anabolic growth mTORC1 signaling pathway was limited in primary T-ALL cells as a result of AMPK pathway activity. AMPK pathway signaling was elevated as a result of oncogene induced metabolic stress. AMPK played a key role in the regulation of T-ALL cell metabolism, as genetic deletion of AMPK in an in vivo murine model of T-ALL resulted in increased glycolysis and anabolic metabolism, yet paradoxically increased cell death and increased mouse survival time. AMPK acts to promote mitochondrial oxidative metabolism in T-ALL through the regulation of Complex I activity, and loss of AMPK reduced mitochondrial oxidative metabolism and resulted in increased metabolic stress. Confirming a role for mitochondrial metabolism in T-ALL, we observed that the direct pharmacological inhibition of Complex I also resulted in a rapid loss of T-ALL cell viability in vitro and in vivo. Taken together, this work establishes an important role for AMPK to both balance the metabolic pathways utilized by T-ALL to allow for cell proliferation and to also promote tumor cell viability by controlling metabolic stress.

Overall, this work demonstrates the importance of the proper coupling of metabolic pathway activity with the function needs of particular types of immune cells. We show that Treg cells, which mainly act to keep immune responses well regulated, adopt a metabolic program where glycolytic metabolism is actively repressed, while oxidative metabolism is promoted. In the setting of malignant T-ALL cells, metabolic activity is surprisingly balanced, with both glycolysis and mitochondrial oxidative metabolism being utilized. In both cases, altering the metabolic balance towards glycolytic metabolism results in negative outcomes for the cell, with decreased Treg functionality and increased metabolic stress in T-ALL. In both cases, this work has generated a new understanding of how metabolism couples to immune cell function, and may allow for selective targeting of immune cell subsets by the specific targeting of metabolic pathways.

Isolation and Validation of Anti-B7-H4 scFvs from an Ovarian Cancer scFv Yeast-Display Library.

B7-H4 (VTCN1, B7x, B7s) is an inhibitory modulator of T-cell response implicated in antigen tolerization. As such, B7-H4 is an immune checkpoint of potential therapeutic interest. To generate anti-B7-H4 targeting reagents, we isolated antibodies by differential cell screening of a yeast-display library of recombinant antibodies (scFvs) derived from ovarian cancer patients and we screened for functional scFvs capable to interfere with B7-H4-mediated inhibition of antitumor responses. We found one antibody binding to B7-H4 that could restore antitumor T cell responses. This chapter gives an overview of the methods we developed to isolate a functional anti-B7-H4 antibody fragment.

iNOS-producing inflammatory dendritic cells constitute the major infected cell type during the chronic Leishmania major infection phase of C57BL/6 resistant mice.

Leishmania major parasites reside and multiply in late endosomal compartments of host phagocytic cells. Immune control of Leishmania growth absolutely requires expression of inducible Nitric Oxide Synthase (iNOS/NOS2) and subsequent production of NO. Here, we show that CD11b+ CD11c+ Ly-6C+ MHC-II+ cells are the main iNOS-producing cells in the footpad lesion and in the draining lymph node of Leishmania major-infected C57BL/6 mice. These cells are phenotypically similar to iNOS-producing inflammatory DC (iNOS-DC) observed in the mouse models of Listeria monocytogenes and Brucella melitensis infection. The use of DsRed-expressing parasites demonstrated that these iNOS-producing cells are the major infected population in the lesions and the draining lymph nodes. Analysis of various genetically deficient mouse strains revealed the requirement of CCR2 expression for the recruitment of iNOS-DC in the draining lymph nodes, whereas their activation is strongly dependent on CD40, IL-12, IFN-gamma and MyD88 molecules with a partial contribution of TNF-alpha and TLR9. In contrast, STAT-6 deficiency enhanced iNOS-DC recruitment and activation in susceptible BALB/c mice, demonstrating a key role for IL-4 and IL-13 as negative regulators. Taken together, our results suggest that iNOS-DC represent a major class of Th1-regulated effector cell population and constitute the most frequent infected cell type during chronic Leishmania major infection phase of C57BL/6 resistant mice.

FUNCTIONAL CHARACTERIZATION OF THE LINK BETWEEN CARBOHYDRATE METABOLISM AND THE PATHOGENESIS OF THE INVASIVE M1T1 GROUP A STREPTOCOCCUS

The Group A Streptococcus (GAS), or Streptococcus pyogenes, is a strict human pathogen that colonizes a variety of sites within the host. Infections can vary from minor and easily treatable, to life-threatening, invasive forms of disease. In order to adapt to niches, GAS utilizes environmental cues, such as carbohydrates, to coordinate the expression of virulence factors. Research efforts to date have focused on identifying how either components of the phosphoenolpyruvate-phosphotransferase system (PTS) or global transcriptional networks affect the regulation of virulence factors, but not the synergistic relationship between the two. The present study investigates the role of a putative PTS-fructose operon encoded by fruRBA and its role in virulence in the M1T1 strain 5448. Growth in fructose resulted in induction of fruRBA. RT-PCR showed that fruRBA formed an operon, which was repressed by FruR in the absence of fructose. Growth and carbon utilization profiles revealed that although the entire fruRBA operon was required for growth in fructose, FruA was the main fructose transporter. The ability of both ΔfruR and ΔfruB mutants to survive in whole human blood or neutrophils was impaired. However, the phenotypes were not reproduced in murine whole blood or in a mouse intraperitoneal infection, indicating a human-specific mechanism. While it is known that the PTS can affect activity of the Mga virulence regulator, further characterization of the mechanism by which sugars and its protein domains affect activity have not been studied. Transcriptional studies revealed that the core Mga regulon is activated more in a glucose-rich than a glucose-poor environment. This activation correlates with the differential phosphorylation of Mga at its PTS regulatory domains (PRDs). Using a 5448 mga mutant, transcriptome studies in THY or C media established that the Mga regulon reflects the media used. Interestingly, Mga regulates phage-encoded DNases in a low glucose environment. We also show that Mga activity is dependent on C-terminal amino acid interactions that aid in the formation of homodimers. Overall, the studies presented sought to define how external environmental cues, specifically carbohydrates, control complex regulatory networks used by GAS, contribute to pathogenesis, and aid in adaptation to various nutrient conditions encountered.

Two novel COLVI long chains in zebrafish that are essential for muscle development

Collagen VI (COLVI), a protein ubiquitously expressed in connective tissues, is crucial for structural integrity, cellular adhesion, migration and survival. Six different genes are recognized in mammalians, encoding six COLVI-chains that assemble as two ‘short’ (α1, α2) and one ‘long’ chain (theoretically any one of α3–6). In humans, defects in the most widely expressed heterotrimer (α123), due to mutations in the COL6A1-3 genes, cause a heterogeneous group of neuromuscular disorders, collectively termed COLVI-related muscle disorders. Little is known about the function(s) of the recently described α4-6 chains and no mutations have been detected yet. In this study, we characterized two novel COLVI long chains in zebrafish that are most homologous to the mammalian α4 chain; therefore, we named the corresponding genes col6a4a and col6a4b. These orthologues represent ancestors of the mammalian Col6a4-6 genes. By in situ hybridization and RT-qPCR, we unveiled a distinctive expression kinetics for col6a4b, compared with the other col6a genes. Using morpholino antisense oligonucleotides targeting col6a4a, col6a4b and col6a2, we modelled partial and complete COLVI deficiency, respectively. All morphant embryos presented altered muscle structure and impaired motility. While apoptosis was not drastically increased, autophagy induction was defective in all morphants. Furthermore, motoneuron axon growth was abnormal in these morphants. Importantly, some phenotypical differences emerged between col6a4a and col6a4b morphants, suggesting only partial functional redundancy. Overall, our results further confirm the importance of COLVI in zebrafish muscle development and may provide important clues for potential human phenotypes associated with deficiency of the recently described COLVI-chains.

A perfect day for zebrafish : neuromodulation of sleep in a diurnal vertebrate

Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal's well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish (Danio rerio), a diurnal vertebrate, has emerged as a promising model system for sleep and arousal research.

In this thesis, I describe two studies on sleep/arousal pathways that I conducted using zebrafish, and I discuss how the findings can be combined in future projects to advance our understanding of vertebrate sleep/arousal pathways. In the first study, I discovered a neuropeptide that regulates zebrafish sleep and arousal as a result of a large-scale effort to identify molecules that regulate behavioral states. Taking advantage of facile zebrafish genetics, I constructed mutants for the three known receptors of this peptide and identified the one receptor that exclusively mediates the observed behavioral effects. I further show that the peptide exerts its behavioral effects independently of signaling at a key module of a neuroendocrine signaling pathway. This finding contradicts the hypothesis put forth in mammalian systems that the peptide acts through the classical neuroendocrine pathway; our data further generate new testable hypotheses for determining the central nervous system or alternative neuroendocrine pathways involved.

Second, I will present the development of a chemigenetic method to non-invasively manipulate neurons in the behaving zebrafish. I validated this technique by expressing and inducing the chemigenetic tool in a restricted population of sleep-regulating neurons in the zebrafish. As predicted by established models of this vertebrate sleep regulator, chemigenetic activation of these neurons induced hyperactivity, whereas chemigenetic ablation of these neurons induced increased sleep behavior. Given that light is a potent modulator of behavior in zebrafish, our proof-of-principle data provide a springboard for future studies of sleep/arousal and other light-dependent behaviors to interrogate genetically-defined populations of neurons independently of optogenetic tools.

Tamoxifen metabolism in breast cancer treatment: Taking the focus off the CYP2D6 gene

Tamoxifen (TAM) has been widely used to treat estrogen receptor (ER)-positive breast cancer, and has led to reduction of 50% in the annual recurrence rate and 30% in breast cancer mortality after 5 years of treatment.1 The prodrug TAM is a selective ER modulator that antagonizes ERs in cancer cells. However, compared with its two active metabolites 4-hydroxy-TAM (4-OH-TAM) and endoxifen, it is a relatively weak antiestrogen.