Characterization of Highper, an ENU-induced mouse mutant with abnormal psychostimulant and stress responses.

RATIONALE: Chemical mutagenesis in the mouse is a forward genetics approach that introduces random mutations into the genome, thereby providing an opportunity to annotate gene function and characterize phenotypes that have not been previously linked to a given gene. OBJECTIVES: We report on the behavioral characterization of Highper, an N-ethyl-N-nitrosourea (ENU)-induced mutant mouse line. METHODS: Highper and B6 control mice were assessed for locomotor activity in the open field and home cage environments. Basal and acute restraint stress-induced corticosterone levels were measured. Mice were tested for locomotor response to cocaine (5, 20, 30, and 45 mg/kg), methylphenidate (30 mg/kg), and ethanol (0.75, 1.25, and 1.75 g/kg). The rewarding and reinforcing effects of cocaine were assessed using conditioned place preference and self-administration paradigms. RESULTS: Highper mice are hyperactive during behavioral tests but show normal home cage locomotor behavior. Highper mice also exhibit a twofold increase in locomotor response to cocaine, methylphenidate, and ethanol and prolonged activation of the hypothalamic-pituitary-adrenal axis in response to acute stress. Highper mice are more sensitive to the rewarding and reinforcing effects of cocaine, although place preference in Highper mice appears to be significantly influenced by the environment in which the drug is administered. CONCLUSIONS: Altogether, our findings indicate that Highper mice may provide important insights into the genetic, molecular, and biological mechanisms underlying stress and the drug reward pathway.

Hearing requires otoferlin-dependent efficient replenishment of synaptic vesicles in hair cells.

Inner hair cell ribbon synapses indefatigably transmit acoustic information. The proteins mediating their fast vesicle replenishment (hundreds of vesicles per s) are unknown. We found that an aspartate to glycine substitution in the C(2)F domain of the synaptic vesicle protein otoferlin impaired hearing by reducing vesicle replenishment in the pachanga mouse model of human deafness DFNB9. In vitro estimates of vesicle docking, the readily releasable vesicle pool (RRP), Ca(2+) signaling and vesicle fusion were normal. Moreover, we observed postsynaptic excitatory currents of variable size and spike generation. However, mutant active zones replenished vesicles at lower rates than wild-type ones and sound-evoked spiking in auditory neurons was sparse and only partially improved during longer interstimulus intervals. We conclude that replenishment does not match the release of vesicles at mutant active zones in vivo and a sufficient standing RRP therefore cannot be maintained. We propose that otoferlin is involved in replenishing synaptic vesicles.

NeuroD1 and Mash1 temporally regulate GnRH receptor gene expression in immortalized mouse gonadotrope cells.

Accurate spatial and temporal expression of gonadotrope-specific genes, such as the gonadotropin-releasing hormone receptor (GnRHR) gene, is critical for gonadotrope maturation. Herein, we show that a specific E-box in the mouse GnRHR promoter binds two group A basic-helix-loop-helix (bHLH) transcription factors. Mutation of this E-box decreases expression in mouse gonadotrope-derived alphaT3-1 and LbetaT2 cell lines. Microarray and western blots show that the bHLH transcription factor NeuroD1 is strongly expressed in the gonadotrope progenitor, alphaT3-1, whereas Mash1 is strongly expressed in the more mature gonadotrope, LbetaT2. Over-expression of NeuroD1 or Mash1 increases expression of the GnRHR gene or a multimer of the E-box and this increase is lost upon mutation of the E-box. Electrophoretic mobility shift assays reveal that the GnRHR E-box binds NeuroD1 from alphaT3-1 cells, but binds Mash1 from LbetaT2 cells. The sequential binding of different members of the group A bHLH transcription factor family to mouse GnRHR E-box 3 as the gonadotrope differentiates may represent a mechanism necessary for proper spatial and temporal expression of the GnRHR during gonadotrope development.

A forward genetics screen in mice identifies recessive deafness traits and reveals that pejvakin is essential for outer hair cell function.

Deafness is the most common form of sensory impairment in the human population and is frequently caused by recessive mutations. To obtain animal models for recessive forms of deafness and to identify genes that control the development and function of the auditory sense organs, we performed a forward genetics screen in mice. We identified 13 mouse lines with defects in auditory function and six lines with auditory and vestibular defects. We mapped several of the affected genetic loci and identified point mutations in four genes. Interestingly, all identified genes are expressed in mechanosensory hair cells and required for their function. One mutation maps to the pejvakin gene, which encodes a new member of the gasdermin protein family. Previous studies have described two missense mutations in the human pejvakin gene that cause nonsyndromic recessive deafness (DFNB59) by affecting the function of auditory neurons. In contrast, the pejvakin allele described here introduces a premature stop codon, causes outer hair cell defects, and leads to progressive hearing loss. We also identified a novel allele of the human pejvakin gene in an Iranian pedigree that is afflicted with progressive hearing loss. Our findings suggest that the mechanisms of pathogenesis associated with pejvakin mutations are more diverse than previously appreciated. More generally, our findings demonstrate that recessive screens in mice are powerful tools for identifying genes that control the development and function of mechanosensory hair cells and cause deafness in humans, as well as generating animal models for disease.

Activin modulates the transcriptional response of LbetaT2 cells to gonadotropin-releasing hormone and alters cellular proliferation.

Both GnRH and activin are crucial for the correct function of pituitary gonadotrope cells. GnRH regulates LH and FSH synthesis and secretion and gonadotrope proliferation, whereas activin is essential for expression of FSH. Little is known, however, about the interplay of signaling downstream of these two hormones. In this study, we undertook expression profiling to determine how activin pretreatment alters the transcriptional response of LbetaT2 gonadotrope cells to GnRH stimulation. Activin treatment alone altered the transcriptional profile of 303 genes including inducing that of the 17beta-hydroxysteroid dehydrogenase B1 gene that converts estrone to 17beta-estradiol, altering the sensitivity of the cells to estrone. Furthermore, activin had a dramatic effect on the response of LbetaT2 cells to GnRH. Hierarchical clustering of 2453 GnRH-responsive genes identified groups of genes the response of which to GnRH was either enhanced or blunted after activin treatment. Mapping of these genes to gene ontology classifications or signaling pathways highlighted significant differences in the classes of altered genes. In the presence of activin, GnRH regulates genes in pathways controlling cell energetics, cytoskeletal rearrangements, organelle organization, and mitosis in the absence of activin, but genes controlling protein processing, cell differentiation, and secretion. Therefore, we demonstrated that activin enhanced GnRH induction of p38MAPK activity, caused GnRH-dependent phosphorylation of p53, and reduced the ability of GnRH to cause G1 arrest. Thus, although activin alone changes a modest number of transcripts, activin pretreatment dramatically alters the response to GnRH from an antiproliferative response to a more differentiated, synthetic response appropriate for a secretory cell.

Mouse GnRH receptor gene expression is mediated by the LHX3 homeodomain protein.

Appropriate expression of GnRH receptor (GnRHR) is necessary for the correct regulation of the gonadotropins, LH and FSH, by GnRH. GnRHR is primarily expressed in the gonadotrope cells of the anterior pituitary, and a number of regulatory elements important for both basal and hormonal regulation of the gene have been identified. Using the gonadotrope-derived cell line, alpha T3-1, that endogenously expresses GnRHR, we have identified an ATTA element located at -298 relative to the transcriptional start site that is essential for basal expression of the GnRHR gene. LHX3, a member of the LIM homeodomain family, binds the -298 ATTA site in vitro as well as to the endogenous GnRHR promoter in vivo. Additionally, LHX3 specifically activates through this -298 ATTA site in transient transfection assays. LHX3 is essential for pituitary development and has been implicated in the regulation of a number of pituitary specific genes; however, this is the first report identifying its role in the regulation of GnRHR.

Activin regulation of the follicle-stimulating hormone beta-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1.

FSH is critical for normal reproductive function in both males and females. Activin, a member of the TGFbeta family of growth factors, is an important regulator of FSH expression, but little is known about the molecular mechanisms through which it acts. We used transient transfections into the immortalized gonadotrope cell line LbetaT2 to identify three regions (at -973/-962, -167, and -134) of the ovine FSH beta-subunit gene that are required for full activin response. All three regions contain homology to consensus binding sites for Smad proteins, the intracellular mediators of TGFbeta family signaling. Mutation of the distal site reduces activin responsiveness, whereas mutation of either proximal site profoundly disrupts activin regulation of the FSHbeta gene. These sites specifically bind LbetaT2 nuclear proteins in EMSAs, and the -973/-962 site binds Smad4 protein. Interestingly, the protein complex binding to the -134 site contains Smad4 in association with the homeodomain proteins Pbx1 and Prep1. Using glutathione S-transferase interaction assays, we demonstrate that Pbx1 and Prep1 interact with Smads 2 and 3 as well. The two proximal activin response elements are well conserved across species, and Pbx1 and Prep1 proteins bind to the mouse gene in vivo. Furthermore, mutation of either proximal site abrogates activin responsiveness of a mouse FSHbeta reporter gene as well, confirming their functional conservation. Our studies provide a basis for understanding activin regulation of FSHbeta gene expression and identify Pbx1 and Prep1 as Smad partners and novel mediators of activin action.

Androgen regulates follicle-stimulating hormone beta gene expression in an activin-dependent manner in immortalized gonadotropes.

Little is known about the molecular mechanisms of androgen regulation of the FSHbeta gene; however, studies suggest that it consists of a complex feedback loop that involves multiple mechanisms acting at both the level of the hypothalamus and the pituitary. In the present study, we address androgen regulation of the FSHbeta gene in immortalized gonadotrope cells and investigate the roles of activin and GnRH in androgen action. Using transient transfection assays in the FSHbeta-expressing mouse gonadotrope cell line, LbetaT2, we demonstrate that androgens stimulate expression of an ovine FSHbeta reporter gene in a dose-dependent manner. Mutation of either of two conserved androgen response elements at -245/-231 and -153/-139 within the proximal region of the ovine FSHbeta gene promoter abolishes this stimulation, and androgen receptor binds directly to the -244 ARE in vitro. Androgen induction of the FSHbeta reporter gene is also dependent upon the activin autocrine loop present in the LbetaT2 cells, as well as an activin-response element at -138/-124 of the FSHbeta gene. However, activin regulation of other genes remains unaffected by androgens. In addition, androgens stimulate expression of a mouse GnRH receptor reporter gene, and thus may indirectly augment the response of the FSHbeta gene to GnRH. Taken together, these data demonstrate that, in mouse gonadotropes, androgens act directly on the ovine FSHbeta gene to stimulate expression by a mechanism that is dependent upon activin, as well as acting indirectly, potentially through a second mechanism that may be dependent upon induction of GnRH receptor.