We have previously shown that the extracellular nucleoside triphosphate-hydrolyzing enzyme NTPDase2 is highly expressed in situ by stem/progenitor cells of the two neurogenic regions of the adult murine brain: the subventricular zone (type B cells) and the dentate gyrus of the hippocampus (residual radial glia). We explored the possibility that adult multipotent neural stem cells express nucleotide receptors and investigated their functional properties in vitro. Neurospheres cultured from the adult mouse SVZ in the presence of epidermal growth factor and fibroblast growth factor 2 expressed the ecto-nucleotidases NTPDase2 and the tissue nonspecific isoform of alkaline phosphatase, hydrolyzing extracellular ATP to adenosine. ATP, ADP and, to a lesser extent, UTP evoked rapid Ca 2+ transients in neurospheres that were exclusively mediated by the metabotropic P2Y 1 and P2Y 2 nucleotide receptors. In addition, agonists of these receptors and low concentrations of adenosine augmented cell proliferation in the presence of growth factors. Neurosphere cell proliferation was attenuated after application of the P2Y 1 -receptor antagonist MRS2179 and in neurospheres from P2Y 1 -receptor knockout mice. In situ hybridization identified P2Y 1 -receptor mRNA in clusters of SVZ cells. Our results infer nucleotide receptor-mediated synergism that augments growth factor-mediated cell proliferation. Together with the in situ data, this supports the notion that extracellular nucleotides contribute to the control of adult neurogenesis.

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Abstract:The molecular processes underlying neural transmission are central issues in neurobiology. Here we describe a novel mechanism through which noradrenaline (NA) activates its target cells, using the mammalian pineal organ as a model. In this neuroendocrine transducer, NA stimulates arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87), the key enzyme regulating the nocturnal melatonin production. In rodents, AANAT protein accumulates as a result of enhanced transcription, but in primates and ungulates, the AANAT mRNA level fluctuates only marginally, indicating that other mechanisms regulate AANAT protein and activity. These were investigated in cultured bovine pinealocytes. AANAT mRNA was readily detectable in unstimulated pinealocytes, and levels did not change following NA treatment. In contrast, NA increased AANAT protein levels in parallel with AANAT activity, apparently through a cyclic AMPmediated mechanism. Immunocytochemistry revealed that the changes in AANAT protein levels occurred in virtually all pinealocytes. Inhibition of AANAT degradation by proteasomal proteolysis alone was found to switch-on enzyme activity by increasing AANAT protein levels fiveto 10-fold. Accordingly, under unstimulated conditions AANAT protein is continually synthesized and immediately destroyed by proteasomal proteolysis. NA appears to act via cyclic AMP to protect AANAT from proteolytic destruction, resulting in accumulation of the protein. These findings show that tightly regulated control of proteasomal proteolysis of a specific protein alone can play a pivotal role in neural regulation. Key Words: Arylalkylamine N-acetyltransferase-Biological rhythms-Neural signaling-Noradrenaline -Pineal gland-Proteasomal proteolysis. J. Neurochem. 75, 2123Neurochem. 75, -2132Neurochem. 75, (2000.A central issue in neurobiology is the nature of mechanisms involved in neural regulation of intracellular processes, critical for specific functions of neuronal cells. An excellent model to study this is the mammalian pineal organ, which transduces a neuronal signal, the release of the neurotransmitter noradrenaline (NA), into a specific response, the production of the neurohormone for darkness, melatonin. NA is released from intrapineal sympathetic nerve endings at the onset of darkness (Drijfhout et al., 1996) in response to neural signals from the endogeneous oscillator in the hypothalamic suprachiasmatic nucleus (Klein and Moore, 1979). NA stimulates pinealocytes through ␣ 1 -and ␤-adrenergic receptors (Klein, 1985;Chik and Ho, 1989). The critical molecular target of NA is arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87), the key regulatory enzyme in melatonin biosynthesis . AANAT activity is low during day and high during night. The rhythm in AANAT activity gives rise to the daily rhythm in circulating melatonin. This role of AANAT as a molecular transducer-one that converts regulatory input into changes in melatonin-is highly conserved among vertebrates; however, remarkable species-to-species differences exist in the ...

The day/night rhythm in melatonin production is a characteristic feature in vertebrate physiology. This hormonal signal reliably reflects the environmental light conditions and is independent of behavioral aspects. In all mammalian species, melatonin production is regulated by norepinephrine, which is released from sympathetic nerve fibers exclusively at night. Norepinephrine elevates the intracellular cAMP concentration via beta-adrenergic receptors and activates the cAMP-dependent protein kinase A. This pathway is crucial for regulation of the penultimate enzyme in melatonin biosynthesis, the arylalkylamine N-acetyltransferase (AANAT); cAMP/protein kinase A may, however, act in different ways. In ungulates and primates, pinealocytes constantly synthesize AANAT protein from continually available Aanat mRNA. During the day-in the absence of noradrenergic stimulation-the protein is immediately destroyed by proteasomal proteolysis. At nighttime, elevated cAMP levels cause phosphorylation of AANAT by protein kinase A. This posttranslational modification leads to interaction of phosphorylated AANAT with regulatory 14-3-3 proteins, which protect AANAT from degradation. Increases in AANAT protein are paralleled by increases in enzyme activity. Stimulation of the cAMP/protein kinase A pathway may also activate pineal gene expression. In rodents, transcriptional activation of the Aanat gene is the primary mechanism for the induction of melatonin biosynthesis and results in marked day/night fluctuations in Aanat mRNA. It involves protein kinase A-dependent phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB) and binding of phosphorylated CREB in the promoter region of the Aanat gene. In conclusion, a common neuroendocrine principle, the nocturnal rise in melatonin, is controlled by strikingly diverse regulatory mechanisms. This diversity has emerged in the course of evolution and reflects the high adaptive plasticity of the melatonin-generating pineal organ.

In rodents, the nocturnal rise and fall of arylalkylamine N-acetyltransferase (AANAT) activity controls the rhythmic synthesis of melatonin, the hormone of the pineal gland. This rhythm involves the transcriptional regulation of the AANAT by two norepinephrine (NE)-inducible transcription factors, e.g. the activator pCREB (phosphorylated Ca2+/cAMP-response element binding protein) and the inhibitor ICER (inducible cAMP early repressor). Most inbred mouse strains do not produce melatonin under standard laboratory light/dark conditions. As melatonin-deficient mice are often the founders for transgenic animals used for chronobiological experimentations, molecular components of neuroendocrine signalling in the pineal gland as an integral part of clock entrainment mechanisms have to be deciphered. We therefore compared calcium signalling, transcriptional events and melatonin synthesis in the melatonin-deficient C57BL mouse and the melatonin-proficient C3H mouse. Pineal glands and primary pinealocytes were cultured and stimulated with NE or were collected at various times of the light/dark (LD) cycle. Changes in intracellular calcium concentrations, the phosphorylation of CREB, and ICER protein levels follow similar dynamics in the pineal glands of both mouse strains. pCREB levels are high during the early night and ICER protein shows elevated levels during the late night. In the C57BL pineal gland, a low but significant increase in melatonin synthesis could be observed upon NE stimulation, and, notably, also when animals were exposed to long nights. We conclude that the commonly used C57BL mouse is not completely melatonin-deficient and that this melatonin-deficiency does not affect molecular details involved in regulating transcriptional events of melatonin synthesis.