Spermatogenesis and testis development are normal in mice lacking testicular orphan nuclear receptor 2

Early in vitro cell culture studies suggested that testicular orphan nuclear receptor 2 (TR2), a member of the nuclear receptor superfamily, may play important roles in the control of several pathways including retinoic acids, vitamin D, thyroid hormones, and ciliary neurotrophic factor. Here we report the surprising results showing that mice lacking TR2 are viable and have no serious developmental defects. Male mice lacking TR2 have functional testes, including normal sperm number and motility, and both male and female mice lacking TR2 are fertile. In heterozygous TR2(+/-) male mice we found that beta-galactosidase, the indicator of TR2 protein expression, was first detected at the age of 3 weeks and its expression pattern was restricted mainly in the spermatocytes and round spermatids. These protein expression patterns were further confirmed with Northern blot analysis of TR2 mRNA expression. Together, results from TR2-knockout mice suggest that TR2 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. Alternatively, the roles of TR2 may be redundant and could be played by other close members of the nuclear receptor superfamily such as testicular orphan receptor 4 (TR4) or unidentified orphan receptors that share many similar functions with TR2. Further studies with double knockouts of both orphan nuclear receptors, TR2 and TR4, may reveal their real physiological roles.     

Neuronal polarization is impaired in mice lacking RhoE expression

Proper development of neuronal networks relies on the polarization of the neurons, thus the establishment of two compartments, axons and dendrites, whose formation depends on cytoskeletal rearrangements. Rnd proteins are regulators of actin organization and they are important players in several aspects of brain development as neurite formation, axon guidance and neuron migration. We have recently demonstrated that mice lacking RhoE/Rnd3 expression die shortly after birth and have neuromotor impairment and neuromuscular alterations, indicating an abnormal development of the nervous system. In this study, we have further investigated the specific role played by RhoE in several aspects of neuronal development by using hippocampal neuron cultures. Our findings show that neurons from a mice lacking RhoE expression exhibit a decrease in the number and the total length of the neurites. We also show that RhoE-deficient neurons display a reduction in axon outgrowth and a delay in the process of neuronal polarization. In addition, our results suggest an involvement of the RHOA/ROCK/LIMK/COFILIN signaling pathway in the neuronal alterations induced by the lack of RhoE. These findings support our previous report revealing the important role of RhoE in the normal development of the nervous system and may provide novel therapeutic targets in neurodegenerative disorders.     

Reduced fat mass in mice lacking orphan nuclear receptor estrogen-related receptor alpha

The estrogen-related receptor alpha (ERRalpha) is an orphan member of the superfamily of nuclear hormone receptors expressed in tissues that preferentially metabolize fatty acids. Despite the molecular characterization of ERRalpha and identification of target genes, determination of its physiological function has been hampered by the lack of a natural ligand. To further understand the in vivo function of ERRalpha, we generated and analyzed Estrra-null (ERRalpha-/-) mutant mice. Here we show that ERRalpha-/- mice are viable, fertile and display no gross anatomical alterations, with the exception of reduced body weight and peripheral fat deposits. No significant changes in food consumption and energy expenditure or serum biochemistry parameters were observed in the mutant animals. However, the mutant animals are resistant to a high-fat diet-induced obesity. Importantly, DNA microarray analysis of gene expression in adipose tissue demonstrates altered regulation of several enzymes involved in lipid, eicosanoid, and steroid synthesis, suggesting that the loss of ERRalpha might interfere with other nuclear receptor signaling pathways. In addition, the microarray study shows alteration in the expression of genes regulating adipogenesis as well as energy metabolism. In agreement with these findings, metabolic studies showed reduced lipogenesis in adipose tissues. This study suggests that ERRalpha functions as a metabolic regulator and that the ERRalpha-/- mice provide a novel model for the investigation of metabolic regulation by nuclear receptors.     

Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3

The expression of unconventional vesicular glutamate transporter VGLUT3 by neurons known to release a different classical transmitter has suggested novel roles for signaling by glutamate, but this distribution has raised questions about whether the protein actually contributes to glutamate release. We now report that mice lacking VGLUT3 are profoundly deaf due to the absence of glutamate release from hair cells at the first synapse in the auditory pathway. The early degeneration of some cochlear ganglion neurons in knockout mice also indicates an important developmental role for the glutamate released by hair cells before the onset of hearing. In addition, the mice exhibit primary, generalized epilepsy that is accompanied by remarkably little change in ongoing motor behavior. The glutamate release conferred by expression of VGLUT3 thus has an essential role in both function and development of the auditory pathway, as well as in the control of cortical excitability.     

Subfertility with defective folliculogenesis in female mice lacking testicular orphan nuclear receptor 4

Testicular orphan nuclear receptor 4 (TR4) plays essential roles for normal spermatogenesis in male mice. However, its roles in female fertility and ovarian function remain largely unknown. Here we found female mice lacking TR4 (TR4-/-) displayed subfertility and irregular estrous cycles. TR4-/- female mice ovaries were smaller with fewer or no preovulatory follicles and corpora lutea. After superovulation, TR4-/- female mice produced fewer oocytes, preovulatory follicles, and corpora lutea. In addition, more intensive granulosa apoptosis was found in TR4-/- ovaries. Functional analyses suggest that subfertility in TR4-/- female mice can be due to an ovarian defect with impaired folliculogenesis rather than a deficiency in pituitary gonadotropins. Molecular mechanism dissection of defective folliculogenesis found TR4 might induce LH receptor (LHR) gene expression via direct binding to its 5' promoter. The consequence of reduced LHR expression in TR4-/- female mice might then result in reduced gonadal sex hormones via reduced expression of enzymes involved in steroidogenesis. Together, our results showed TR4 might play essential roles in normal folliculogenesis by influencing LHR signals. Modulation of TR4 expression and/or activation via its upstream signals or unidentified ligand(s) might allow us to develop small molecule(s) to control folliculogenesis.     

Dissociation of diabetes and obesity in mice lacking orphan nuclear receptor small heterodimer partner

Mixed background SHP(-/-) mice are resistant to diet-induced obesity due to increased energy expenditure caused by enhanced PGC-1α expression in brown adipocytes. However, congenic SHP(-/-) mice on the C57BL/6 background showed normal expression of PGC-1α and other genes involved in brown adipose tissue thermogenesis. Thus, we reinvestigated the impact of small heterodimer partner (SHP) deletion on diet-induced obesity and insulin resistance using congenic SHP(-/-) mice. Compared with their C57BL/6 wild-type counterparts, SHP(-/-) mice subjected to a 6 month challenge with a Western diet (WestD) were leaner but more glucose intolerant, showed hepatic insulin resistance despite decreased triglyceride accumulation and increased β-oxidation, exhibited alterations in peripheral tissue uptake of dietary lipids, maintained a higher respiratory quotient, which did not decrease even after WestD feeding, and displayed islet dysfunction. Hepatic mRNA expression analysis revealed that many genes expressed higher in SHP(-/-) mice fed WestD were direct peroxisome proliferator-activated receptor alpha (PPARα) targets. Indeed, transient transfection and chromatin immunoprecipitation verified that SHP strongly repressed PPARα-mediated transactivation. SHP is a pivotal metabolic sensor controlling lipid homeostasis in response to an energy-laden diet through regulating PPARα-mediated transactivation. The resultant hepatic fatty acid oxidation enhancement and dietary fat redistribution protect the mice from diet-induced obesity and hepatic steatosis but accelerate development of type 2 diabetes.     

Generation and characterization of mice lacking the zinc uptake transporter ZIP3

The mouse ZIP3 (SLC39A3) gene encodes an eight-transmembrane-domain protein that has been conserved in mammals and can function to transport zinc. To analyze the expression of ZIP3 in the early embryo and neonate and to determine its in vivo function, we generated ZIP3 null mice in which the ZIP3 open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP) reporter. EGFP fluorescence revealed that ZIP3 was expressed in the inner cell mass of the blastocyst and later during embryonic development in many tissues. Elevated expression was apparent in the embryonic brain and neurotube and neonatal gonads. Homozygous knockout mice were viable and fertile and under normal growth conditions exhibited no obvious phenotypic abnormalities. Deletion of ZIP3 did not alter zinc homeostasis at the molecular level as assessed by essential metal levels and the expression of zinc-responsive genes. In knockout mice stressed with a zinc-deficient diet during pregnancy or at weaning, a subtle increase in the sensitivity to abnormal morphogenesis of the embryo and to depletion of thymic pre-T cells, respectively, was noted. These results suggest that this protein plays an ancillary role in zinc homeostasis in mice.     

Urea-selective concentrating defect in transgenic mice lacking urea transporter UT-B.

Urea transporter UT-B has been proposed to be the major urea transporter in erythrocytes and kidney-descending vasa recta. The mouse UT-B cDNA was isolated and encodes a 384-amino acid urea-transporting glycoprotein expressed in kidney, spleen, brain, ureter, and urinary bladder. The mouse UT-B gene was analyzed, and UT-B knockout mice were generated by targeted gene deletion of exons 3-6. The survival and growth of UT-B knockout mice were not different from wild-type littermates. Urea permeability was 45-fold lower in erythrocytes from knockout mice than from those in wild-type mice. Daily urine output was 1.5-fold greater in UT-B- deficient mice (p < 0.01), and urine osmolality (U(osm)) was lower (1532 +/- 71 versus 2056 +/- 83 mosM/kg H(2)O, mean +/- S.E., p < 0.001). After 24 h of water deprivation, U(osm) (in mosM/kg H(2)O) was 2403 +/- 38 in UT-B null mice and 3438 +/- 98 in wild-type mice (p < 0.001). Plasma urea concentration (P(urea)) was 30% higher, and urine urea concentration (U(urea)) was 35% lower in knockout mice than in wild-type mice, resulting in a much lower U(urea)/P(urea) ratio (61 +/- 5 versus 124 +/- 9, p < 0.001). Thus, the capacity to concentrate urea in the urine is more severely impaired than the capacity to concentrate other solutes. Together with data showing a disproportionate reduction in the concentration of urea compared with salt in homogenized renal inner medullas of UT-B null mice, these data define a novel "urea-selective" urinary concentrating defect in UT-B null mice. The UT-B null mice generated for these studies should also be useful in establishing the role of facilitated urea transport in extrarenal organs expressing UT-B.     

N-Arachidonyl-glycine inhibits the glycine transporter, GLYT2a

N-arachidonyl-glycine is one of a series of N-arachidonyl-amino acids that are derived from arachidonic acid. N-arachidonyl-glycine is produced in a wide range of tissues with greatest abundance in the spinal cord. Here we report that N-arachidonyl-glycine is a reversible and non-competitive inhibitor of glycine transport by GLYT2a, but has little effect on glycine transport by GLYT1b or gamma-amino butyric acid transport by GAT1. It has previously been reported that the activity of GLYT2a is down-regulated by protein kinase C and therefore we investigated whether the actions of N-arachidonyl-glycine on GLYT2a are mediated by second messenger systems that lead to the activation of protein kinase C. However, the protein kinase C inhibitor, staurosporine, had no effect on the actions of N-arachidonyl-glycine on GLYT2a. Thus, the actions of N-arachidonyl-glycine are likely to be mediated by a direct interaction with the transporter. We have further defined the pharmacophore by investigating the actions of other N-arachidonyl amino acids as well as the closely related compounds arachidonic acid, anandamide and R1-methanandamide. Arachidonic acid, anandamide and R1-methanandamide have no effect on glycine transport, but N-arachidonyl-l-alanine has similar efficacy at GLYT2a to N-arachidonyl-glycine, and N-arachidonyl-gamma-amino butyric acid is less efficacious. These observations define a novel recognition site for the N-arachidonyl amino acids.     

Aberrant expression of myosin isoforms in skeletal muscles from mice lacking the rev-erbAalpha orphan receptor gene

The rev-erbAalpha orphan protein belongs to the steroid nuclear receptor superfamily. No ligand has been identified for this protein, and little is known of its function in development or physiology. In this study, we focus on 1) the distribution of the rev-erbAalpha protein in adult fast- and slow-twitch skeletal muscles and muscle fibers and 2) how the rev-erbAalpha protein influences myosin heavy chain (MyHC) isoform expression in mice heterozygous (+/-) and homozygous (-/-) for a rev-erbAalpha protein null allele. In the fast-twitch extensor digitorum longus muscle, rev-erbAalpha protein expression was linked to muscle fiber type; however, MyHC isoform expression did not differ between wild-type, +/-, or -/- mice. In the slow-twitch soleus muscle, the link between rev-erbAalpha protein and MyHC isoform expression was more complex than in the extensor digitorum longus. Here, a significantly higher relative amount of the beta/slow (type I) MyHC isoform was observed in both rev-erbAalpha -/- and +/- mice vs. that shown in wild-type controls. A role for the ratio of thyroid hormone receptor proteins alpha1 to alpha2 in modulating MyHC isoform expression can be ruled out because no differences were seen in MyHC isoform expression between thyroid hormone receptor alpha2-deficient mice (heterozygous and homozygous) and wild-type mice. Therefore, our data are compatible with the rev-erbAalpha protein playing an important role in the regulation of skeletal muscle MyHC isoform expression.     

Zn2+ inhibits glycine transport by glycine transporter subtype 1b

In the central nervous system, glycine is a co-agonist with glutamate at the N-methyl-D-aspartate subtype of glutamate receptors and also an agonist at inhibitory, strychnine-sensitive glycine receptors. The GLYT1 subtypes of glycine transporters (GLYTs) are responsible for regulation of glycine at excitatory synapses, whereas a combination of GLYT1 and GLYT2 subtypes of glycine transporters are used at inhibitory glycinergic synapses. Zn2+ is stored in synaptic vesicles with glutamate in a number of regions of the brain and is believed to play a role in modulation of excitatory neurotransmission. In this study we have investigated the actions of Zn2+ on the glycine transporters, GLYT1b and GLYT2a, expressed in Xenopus laevis oocytes and we demonstrate that Zn2+ is a noncompetitive inhibitor of GLYT1 but has no effect on GLYT2. We have also investigated the molecular basis for these differences and the relationship between the Zn2+ and proton binding sites on GLYT1. Using site-directed mutagenesis, we identified 2 histidine residues, His-243 in the large second extracellular loop (ECL2) and His-410 in the fourth extracellular loop (ECL4), as two coordinates in the Zn2+ binding site of GLYT1b. In addition, our study suggests that the molecular determinants of proton regulation of GLYT1b are localized to the 2 histidine residues (His-410 and His-421) of ECL4. The ability of Zn2+ and protons to regulate the rate of glycine transport by interacting with residues situated in ECL4 of GLYT1b suggests that this region may influence the substrate translocation mechanism.     

Deficits in motor coordination with aberrant cerebellar development in mice lacking testicular orphan nuclear receptor 4

Since testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological function has remained largely unknown. Throughout postnatal development, TR4-knockout (TR4-/-) mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal TR4-/- cerebellum revealed gross abnormalities in foliation; specifically, lobule VII in the anterior vermis was missing. Further analyses demonstrated that the laminations of the TR4-/- cerebellar cortex were changed, including reductions in the thickness of the molecular layer and the internal granule layer, as well as delayed disappearance of the external granule cell layer (EGL). These lamination irregularities may result from interference with granule cell proliferation within the EGL, delayed inward migration of postmitotic granule cells, and a higher incidence of apoptotis. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4-/- cerebellum, as evidenced by aberrant dendritic arborization and reduced calbindin staining intensity. Expression of Pax-6, Sonic Hedgehog (Shh), astrotactin (Astn), reelin, and Cdk-5, genes correlated with the morphological development of the cerebellum, is reduced in the developing TR4-/- cerebellum. Together, our findings suggest that TR4 is required for normal cerebellar development.     

Calpain-mediated proteolytic cleavage of the neuronal glycine transporter, GlyT2

The glycine transporter 2 (GlyT2) belongs to the family of Na+/CL--dependent plasma membrane transporters and is localized on the presynaptic terminals of glycinergic neurons. GlyT2 differs from other family members by its extended N-terminal cytoplasmic region. We report that activation of a Ca2+-dependent protease, most likely calpain, in spinal cord synaptosomes or cultured spinal cord neurons, results in partial proteolysis of GlyT2. Regions sensitive to calpain cleavage in vivo are located in the N-terminal and, to a lesser extent, C-terminal regions of the transporter protein. Incubation of a GlyT2 N-terminal fusion protein with spinal cord extract in the presence of calcium followed by protein sequence analysis localized the major N-terminal cleavage site after methionine 156, with a second cleavage site being situated after glycine 164. Interestingly, the size of the N-terminally truncated GlyT2 protein (70 kDa) is similar to that of most other transporter family members, and truncated GlyT2 displayed full transport activity upon expression in HEK293 cells. Our data suggest that Ca2+-triggered proteolysis may contribute to the regulation of GlyT2 trafficking and/or function in the neuronal plasma membrane.