Myoclonus dystonia (M-D) is a hereditary movement disorder caused by a maternally imprinted gene that is often associated with psychiatric symptoms. Most cases of M-D are believed to result from mutations of the epsilon-sarcoglycan protein. The neuroanatomical distribution of epsilon-sarcoglycan-like immunoreactivity in mouse was investigated by using an antiserum against the epsilon-sarcoglycan protein. The expression of epsilon-sarcoglycan mRNA was studied by a sensitive fluorescence in situ hybridization (FISH) method. Immunohistochemistry and FISH revealed a wide distribution of epsilon-sarcoglycan protein and mRNA throughout the mouse brain. High expression levels of epsilon-sarcoglycan mRNA and immunoreactivity were found in the mitral cell layer of the olfactory bulb, the Purkinje cell layer in cerebellum, and the monoaminergic neurons in the mouse midbrain. Immunohistochemistry revealed a similar distribution of epsilon-sarcoglycan protein. Double-labeling FISH showed colocalization of tyrosine hydroxylase and epsilon-sarcoglycan mRNAs within all the midbrain dopaminergic (DAergic) cell groups. By combining FISH with fluorescence immunohistochemistry, coexpression of epsilon-sarcoglycan mRNA and tryptophan hydroxylase immunoreactivity was found in the serotonergic (5-HTergic) neurons within the dorsal raphe nucleus. The distribution of epsilon-sarcoglycan in the mouse brain suggests that the symptom complex of M-D may be related to the effects of decreased epsilon-sarcoglycan activity on the development or function of monoaminergic neurons.

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Mammalian reproduction requires gonadotropin-releasing hormone (GnRH)-mediated signaling from brain neurons to pituitary gonadotropes. Because the pulses of released GnRH vary greatly in amplitude, we studied the biosynthetic response of the gonadotrope to varying GnRH concentrations, focusing on extracellular-regulated kinase (ERK) phosphorylation and egr1 mRNA and protein production. The overall average level of ERK activation in populations of cells increased non-cooperatively with increasing GnRH and did not show evidence of either ultrasensitivity or bistability. However, automated image analysis of single-cell responses showed that whereas individual gonadotropes exhibited two response states, inactive and active, both the probability of activation and the average response in activated cells increased with increasing GnRH concentration. These data indicate a hybrid single-cell response having both digital (switch-like) and analog (graded) features. Mathematical modeling suggests that the hybrid response can be explained by indirect thresholding of ERK activation resulting from the distributed structure of the GnRH-modulated network. The hybrid response mechanism improves the reliability of noisy reproductive signal transmission from the brain to the pituitary.Mammalian reproduction and the survival of a species rely on a precise orchestration of temporally and spatially distributed molecular events. The control of reproduction represents a difficult engineering problem, because noisy molecular processes within cells that occur on time scales of minutes must regulate brain, pituitary, and gonadal activity in a process with an overall periodicity of days to weeks, depending on the species. At the center of this coordinated reproductive activity lies the pituitary gonadotrope, which converts hormone signals secreted by the brain into the biosynthesis and secretion of pituitary hormones controlling gonadal responses.The hypothalamus secretes discrete pulses of gonadotropinreleasing hormone (GnRH) 2 (for a review, see Refs. 1-3). GnRH interacts with high affinity GnRH receptors on the gonadotrope membrane to modulate the biosynthesis and release of the gonadotropins luteinizing hormone and follicle-stimulating hormone (4 -6). The function of the reproductive axis depends on appropriate responses of the gonadotrope to GnRH despite the high interpulse variability in the amplitude of GnRH secreted by the brain (3,(7)(8)(9)(10)(11)(12). Elucidating the mechanisms underlying the response of the gonadotrope to varying concentrations of GnRH is important for understanding the design principles of this key response locus for mammalian physiology.GnRH directs two distinguishable gonadotrope activities, the biosynthesis of gonadotropins and their secretion. We focus here on the biosynthetic response. The GnRH receptor is a heptahelical G protein-coupled receptor that modulates a signaling network leading to activation of protein kinases and regulation of both transcription and translation (13). The gene network responses...

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The initial waves of gene induction caused by GnRH in the LbetaT2 gonadotrope cell line have recently been identified using microarrays. We now investigate the relationship of the concentration of GnRH to the level of biosynthesis induced. Using an optimized custom cDNA microarray, we show that a large number of genes are induced in a concentration-dependent fashion. Detailed time course studies of the induction of six induced transcripts using quantitative real-time PCR suggest that the amplitude, but not the temporal pattern, depends on the concentration of GnRH. The early genes appear to show a delay in gene induction, followed by a linear phase of increase. The relationship of rate of synthesis and GnRH concentration was studied by mathematical modeling of the induction of two genes, gly96 and tis11. In both cases, only the rates of increase, but not the lag times, are influenced by the concentration of GnRH exposure. Western blot analyses for c-Jun and Egr1 show that the levels of nuclear protein for these transcription factors also depend on the concentration of GnRH. These studies indicate that, despite the complex signaling network connecting the receptor to the activated genes, the biosynthetic rate of RNA polymerase at induced genes is correlated with the concentration of GnRH at the GnRH receptor.

SummaryGonadotropin-releasing hormone (GnRH) regulates biosythesis in the pituitary gonadotrope via a complex signaling and gene network. Small non-coding microRNAs (miRNA) can play important roles in gene expression. We investigated the microtranscriptome in the mouse LβT2 gonadotrope cell line using microarray, single molecule coincidence detection assays, hairpin real time PCR and LNA (locked nucleic acid) primer-extension PCR. Expression of nearly 200 miRNAs were detected by array and a panel of 101 hairpin real-time PCR assays. Within this broad family of expressed miRNAs, GnRH induced upregulation of two miRNA products of the same primary transcript, miR-132 and miR-212, a result confirmed by single molecule, hairpin and LNA assays. Induction peaked 6 hours after GnRH exposure and showed no significant frequency sensitivity. Bioinformatics analysis was used to predict potential targets of each of these GnRH-regulated miRNAs. These findings suggest the importance of the microtranscriptome in gene control in the gonadotrope and implicate miR-132 and miR-212 in the regulation of GnRH-stimulated biosynthetic response.

Reproductive physiology depends on the control of biosynthesis in the pituitary gonadotrope by hypothalamic gonadotropin-releasing hormone (GnRH). The responses to GnRH include activation of extracellular signal-regulated kinase (ERK) and induction of Egr1. Using population and single cell signaling assays, we investigated the signal and noise transmission through this signaling and gene circuit, analyzing data obtained from 43,775 individual cells in 40 experiments. After exposure to GnRH, phosphorylated ERK (pERK) is elevated in 50% of the cells at 1.7 (SD = 0.3) min. Studies of the cell-to-cell response showed that for both pERK and for Egr1 protein production the mean response (mu) and standard deviation (sigma) within individual cells were linearly related (sigma = kmu) and had similar values of k. To understand the basis for the scaling observed for noise propagation through this system, we determined the relationship between pERK and egr1 mRNA levels induced at varying concentration of GnRH. While both pERK and egr1 mRNA show a saturating sigmoidal relationship to the concentration of GnRH exposure, egr1 mRNA is linearly related to the levels of pERK. These results explain the basis for variation in cellular responses in an important mammalian signaling pathway leading to gene induction.