Dopamine production in the caudate putamen restores feeding in dopamine-deficient mice.

Dopamine-deficient (DD) mice cannot synthesize dopamine (DA) in dopaminergic neurons due to selective inactivation of the tyrosine hydroxylase gene in those neurons. These mice become hypoactive and hypophagic and die of starvation by 4 weeks of age. We used gene therapy to ascertain where DA replacement in the brain restores feeding and other behaviors in DD mice. Restoration of DA production within the caudate putamen restores feeding on regular chow and nest-building behavior, whereas restoration of DA production in the nucleus accumbens restores exploratory behavior. Replacement of DA to either region restores preference for sucrose or a palatable diet without fully rescuing coordination or initiation of movement. These data suggest that a fundamental difference exists between feeding for sustenance and the ability to prefer rewarding substances.

Dopamine-deficient mice are hypersensitive to dopamine receptor agonists.

Dopamine-deficient (DA-/-) mice were created by targeted inactivation of the tyrosine hydroxylase gene in dopaminergic neurons. The locomotor activity response of these mutants to dopamine D1 or D2 receptor agonists and l-3,4-dihydroxyphenylalanine (l-DOPA) was 3- to 13-fold greater than the response elicited from wild-type mice. The enhanced sensitivity of DA-/- mice to agonists was independent of changes in steady-state levels of dopamine receptors and the presynaptic dopamine transporter as measured by ligand binding. The acute behavioral response of DA-/- mice to a dopamine D1 receptor agonist was correlated with c-fos induction in the striatum, a brain nucleus that receives dense dopaminergic input. Chronic replacement of dopamine to DA-/- mice by repeated l-DOPA administration over 4 d relieved the hypersensitivity of DA-/- mutants in terms of induction of both locomotion and striatal c-fos expression. The results suggest that the chronic presence of dopaminergic neurotransmission is required to dampen the intracellular signaling response of striatal neurons.

Dopamine is required for hyperphagia in Lep(ob/ob) mice.

Feeding is a complex process responsive to sensory information related to sight and smell of food, previous feeding experiences, satiety signals elicited by ingestion and hormonal signals related to energy balance. Dopamine released in specific brain regions is associated with pleasurable and rewarding events and may reinforce positive aspects of feeding. Dopamine also influences initiation and coordination of motor activity and is required for sensorimotor functions. Thus, dopamine may facilitate integration of sensory cues related to hunger, initiating the search for food and its consumption. Dopaminergic neurons in the substantia nigra and ventral tegmental area project to the caudate putamen and nucleus accumbens, where they modulate movement and reward. There are projections from the nucleus accumbens to the lateral hypothalamus that regulate feeding. Dopamine-deficient mice (Dbh(Th/+), Th-/-; hereafter DD mice) cannot synthesize dopamine in dopaminergic neurons. They gradually become aphagic and die of starvation. Daily treatment of DD mice with L-3,4-dihydroxyphenylalanine (L-DOPA) transiently restores brain dopamine, locomotion and feeding. Leptin-null (Lep(ob/ob)) mice exhibit obesity, decreased energy expenditure and hyperphagia. As the hypothalamic leptin-melanocortin pathway appears to regulate appetite and metabolism, we generated mice lacking both dopamine and leptin (DD x Lep(ob/ob)) to determine if leptin deficiency overcomes the aphagia of DD mice. DD x Lep(ob/ob) mice became obese when treated daily with L-DOPA, but when L-DOPA treatment was terminated the double mutants were capable of movement, but did not feed. Our data show that dopamine is required for feeding in leptin-null mice.

Feeding behavior in dopamine-deficient mice.

Mice that cannot make dopamine (DA), a condition caused by the selective inactivation of tyrosine hydroxylase in dopaminergic neurons, are born normal but gradually become hypoactive and hypophagic, and die at 3 weeks of age. We characterized the feeding and locomotor responses of these DA-deficient (DA-/-) mice to 3, 4-dihyroxy-L-phenylalanine (L-DOPA) to investigate the relationship between brain DA levels and these complex behaviors. Daily administration of L-DOPA to DA-/- mice stimulated locomotor activity that lasted 6 to 9 hr; during that time the mice consumed most of their daily food and water. The minimal dose of L-DOPA that was sufficient to elicit normal feeding behavior in the DA-/- mice also restored their striatal DA to 9.1% of that in the wild-type (WT) mice at 3 hr; then DA content declined to <1% of WT levels by 24 hr. This dose of L-DOPA induced locomotor activity that exceeded that of treated WT mice by 5- to 7-fold, suggesting that DA-/- mice are supersensitive to DA. Unexpectedly, DA-/- mice manifested a second wave of activity 24 to 48 hr after L-DOPA treatment that was equivalent in magnitude to that of WT mice and independent of DA receptor activation. The DA-/- mice approached, sniffed, and chewed food during this second period of activity, but they ate <10% of that required for sustenance. Therefore, DA-/- mice can execute behaviors necessary to seek and ingest food, but they do not eat enough to survive.

Viral gene delivery selectively restores feeding and prevents lethality of dopamine-deficient mice.

Dopamine-deficient mice (DA-/- ), lacking tyrosine hydroxylase (TH) in dopaminergic neurons, become hypoactive and aphagic and die by 4 weeks of age. They are rescued by daily treatment with L-3,4-dihydroxyphenylalanine (L-DOPA); each dose restores dopamine (DA) and feeding for less than 24 hr. Recombinant adeno-associated viruses expressing human TH or GTP cyclohydrolase 1 (GTPCH1) were injected into the striatum of DA-/- mice. Bilateral coinjection of both viruses restored feeding behavior for several months. However, locomotor activity and coordination were partially improved. A virus expressing only TH was less effective, and one expressing GTPCH1 alone was ineffective. TH immunoreactivity and DA were detected in the ventral striatum and adjacent posterior regions of rescued mice, suggesting that these regions mediate a critical DA-dependent aspect of feeding behavior.

Dopamine-stimulated sexual behavior is testosterone dependent in mice.

Mice unable to synthesize dopamine (DA) in dopaminergic neurons were generated by gene-targeting techniques (Q.-Y. Zhou & R. D. Palmiter, 1995). These dopamine-deficient (DA-/-) mice required daily administration of 3,4-dihydroxyphenylalanine (L-DOPA) for survival beyond 2 to 3 weeks of age. This treatment stimulated mounting and aggressive behavior of adult DA-/- males toward both male and female mice. Both a nonspecific DA agonist (apomorphine) and a specific D1 agonist (SKF81297) stimulated aggression and mounting behavior; however, a D2 agonist (quinpirole) was less effective. Castration of male DA-/- mice demonstrated that these L-DOPA-stimulated behaviors depend on testosterone. In addition, replacement of testosterone to castrated males showed that the testosterone-responsive pathways of DA-/- males were more sensitive to testosterone than wild-type mice.

Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription.

The metal ions, Cu2+/+ and Fe3+/2+, are essential co-factors for a wide variety of enzymatic reactions. However, both metal ions are toxic when hyper-accumulated or maldistributed within cells due to their ability to generate damaging free radicals or through the displacement of other physiological metal ions from metalloproteins. Although copper transport into yeast cells is apparently independent of iron, the known dependence on Cu2+ for high affinity transport of Fe2+ into yeast cells has established a physiological link between these two trace metal ions. In this study we demonstrate that proteins encoded by genes previously demonstrated to play critical roles in vacuole assembly for acidification, PEP3, PEP5 and VMA3, are also required for normal copper and iron metal ion homeostasis. Yeast cells lacking a functional PEP3 or PEP5 gene are hypersensitive to copper and render the normally iron-repressible FET3 gene, encoding a multi-copper Fe(II) oxidase involved in Fe2+ transport, also repressible by exogenous copper ions. The inability of these same vacuolar mutant strains to repress FET3 mRNA levels in the presence of an iron-unresponsive allele of the AFT1 regulatory gene are consistent with alterations in the intracellular distribution of redox states of Fe3+/2+ in the presence of elevated extracellular concentrations of copper ions. Therefore, the yeast vacuole is an important organelle for maintaining the homeostatic convergence of the essential yet toxic copper and iron ions.

Cadmium tolerance mediated by the yeast AP-1 protein requires the presence of an ATP-binding cassette transporter-encoding gene, YCF1.

Elevations in gene dosage of the transcriptional regulatory protein yAP-1 in Saccharomyces cerevisiae can elicit pronounced phenotypic increases in tolerance of a variety of drugs including the toxic heavy metal cadmium. While a large elevation in cadmium tolerance occurs in response to overproduction of yAP-1, the target genes under yAP-1 control have not yet been identified that are responsible for this increase. We show here that the YCF1 gene, encoding a likely integral membrane protein, is required for yAP-1 to exert its normal effects on cadmium tolerance. Mutant strains of yeast that lack the YCF1 gene are hypersensitive to cadmium and this hypersensitivity is epistatic to yAP-1 overexpression. YCF1 mRNA levels and the expression of a YCF1-lacZ reporter construct positively correlates with changes in YAP1 gene dosage. A set of 5' truncation derivatives of the YCF1-lacZ fusion gene identified the region from -201 to +47 as being sufficient for the yAP-1-dependent increase in expression. DNase I footprinting using a probe from this segment of the YCF1 promoter showed that bacterially-produced yAP-1 protein was capable of binding a novel DNA element we have designated the yAP-1 response element. Insertion of the yAP-1 response element upstream of a CYC1-lacZ gene fusion led to the production of beta-galactosidase in a yAP-1-dependent fashion. These data establish that an important physiological target of yAP-1 transcriptional regulation is the YCF1 structural gene.

A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein.

Members of the ATP binding cassette (ABC) protein superfamily transport a variety of substances across biological membranes, including drugs, ions, and peptides. The yeast cadmium factor (YCF1) gene from Saccharomyces cerevisiae is required for cadmium resistance and encodes a 1,515 amino acid protein with extensive homology to both the human multidrug resistance-associated protein (MRP1) and the cystic fibrosis transmembrane conductance regulator (hCFTR). S. cerevisiae cells harboring a deletion of the YCF1 gene are hypersensitive to cadmium compared with wild type cells. Mutagenesis experiments demonstrate that conserved amino acid residues, functionally critical in hCFTR, play a vital role in YCF1-mediated cadmium resistance. Mutagenesis of phenylalanine 713 in the YCF1 nucleotide binding fold 1, which correlates with the delta F508 mutation found in the most common form of cystic fibrosis, completely abolished YCF1 function in cadmium detoxification. Furthermore, substitution of a serine to alanine residue in a potential protein kinase A phosphorylation site in a central region of YCF1, which displays sequence similarity to the central regulatory domain of hCFTR, also rendered YCF1 nonfunctional. These results suggest that YCF1 is composed of modular domains found in human proteins which function in drug and ion transport.

A system for gene cloning and manipulation in the yeast Candida glabrata.

The opportunistic pathogenic yeast, Candida (Torulopsis) glabrata, is an asexual imperfect fungus that exists largely as a haploid. Besides being a clinically important pathogen, this yeast also provides a model system for understanding basic biological mechanisms such as metal-activated metallothionein-encoding gene transcription. To facilitate molecular genetic studies in C. glabrata, we isolated a strain auxotrophic for uracil biosynthesis. The ura- mutation could be functionally complemented by the URA3 gene of Saccharomyces cerevisiae, consistent with a defect in the C. glabrata URA3 gene in this strain. We also found that the centromere-based S. cerevisiae plasmid pRS316 could stably transform and replicate in multiple copies in C. glabrata. In contrast, high-copy-number S. cerevisiae plasmids containing the 2 mu circle autonomous replication sequence were not able to replicate productively in C. glabrata. We cloned the C. glabrata URA3 gene, encoding orotidine-5'-phosphate decarboxylase, by complementation of a ura3- strain of S. cerevisiae. The deduced amino-acid sequence is highly similar to that of the URA3 protein from S. cerevisiae. C. glabrata URA3 provides a genetic locus for targeted gene integration in C. glabrata. Integrative plasmids were constructed based on the cloned C. glabrata URA3 and are applicable for directed insertions of genes of interest at the ura3 locus through homologous recombination.

Expression of a yeast metallothionein gene family is activated by a single metalloregulatory transcription factor.

The opportunistic pathogenic yeast Candida glabrata elicits at least two major responses in the presence of high environmental metal levels: transcriptional induction of the metallothionein gene family by copper and the appearance of small (gamma-Glu-Cys)nGly peptides in the presence of cadmium. On the basis of a trans-activation selection scheme in the baker's yeast Saccharomyces cerevisiae, we previously isolated a C. glabrata gene which encodes a copper-activated DNA-binding protein designated AMT1. AMT1 forms multiple specific DNA-protein complexes with both C. glabrata MT-I and MT-IIa promoter DNA fragments. In this report, we localize and define the AMT1-binding sites in the MT-I and MT-IIa promoters and characterize the mode of AMT1 binding. Furthermore, we demonstrate that the AMT1 protein trans activates both the MT-I and MT-IIa genes in vivo in response to copper and that this activation is essential for high-level copper resistance in C. glabrata. Although AMT1-mediated trans activation of the C. glabrata metallothionein genes is essential for copper resistance, AMT1 is completely dispensable for cadmium tolerance. The distinct function that metallothionein genes have in copper but not cadmium detoxification in C. glabrata is in contrast to the role that metallothionein genes play in tolerance to multiple metals in higher organisms.

A cysteine-rich nuclear protein activates yeast metallothionein gene transcription.

The ACE1 gene of the yeast Saccharomyces cerevisiae is required for copper-inducible transcription of the metallothionein gene (CUP1). The sequence of the cloned ACE1 gene predicted an open reading frame for translation of a 225-amino-acid polypeptide. This polypeptide was characterized by an amino-terminal half rich in cysteine residues and positively charged amino acids. The arrangement of many of the 12 cysteines in the configuration Cys-X-Cys or Cys-X-X-Cys suggested that the ACE1 protein may bind metal ions. The carboxyl-terminal half of the ACE1 protein was devoid of cysteines but was highly acidic in nature. The ability of a bifunctional ACE1-beta-galactosidase fusion protein to accumulate in yeast cell nuclei was consistent with the possibility that ACE1 plays a direct role in the regulation of copper-inducible transcription of the yeast metallothionein gene.