Polyglutamine aggregation in Huntington's disease and spinocerebellar ataxia type 3: similar mechanisms in aggregate formation.

AIMS: Polyglutamine (polyQ) diseases are characterized by the expansion of a polymorphic glutamine sequence in disease-specific proteins and exhibit aggregation of these proteins. This is combated by the cellular protein quality control (PQC) system, consisting of chaperone-mediated refolding as well as proteasomal and lysosomal degradation pathways. Our recent study in the polyQ disease spinocerebellar ataxia type 3 (SCA3) suggested a distinct pattern of protein aggregation and PQC dysregulation. METHODS: To corroborate these findings we have investigated immunohistochemically stained 5 µm sections from different brain areas of Huntington's disease (HD) and SCA3 patients. RESULTS: Irrespective of disease and brain region, we observed peri- and intranuclear polyQ protein aggregates. A subset of neurones with intranuclear inclusions bodies exhibited signs of proteasomal dysfunction, up-regulation of HSPA1A and re-distribution of DNAJB1. The extent of the observed effects varied depending on brain area and disease protein. CONCLUSIONS: Our results suggest a common sequence, in which formation of cytoplasmic and nuclear inclusions precede proteasomal impairment and induction of the cellular stress response. Clearly, impairment of the PQC is not the primary cause for inclusion formation, but rather a consequence that might contribute to neuronal dysfunction and death. Notably, the inclusion pathology is not directly correlated to the severity of the degeneration in different areas, implying that different populations of neurones respond to polyQ aggregation with varying efficacy and that protein aggregation outside the neuronal perikaryon (e.g. axonal aggregates) or other effects of polyQ aggregation, which are more difficult to visualize, may contribute to neurodegeneration.

Involvement of the cholinergic basal forebrain nuclei in spinocerebellar ataxia type 2 (SCA2).

AIMS: Spinocerebellar ataxia type 2 (SCA2) belongs to the CAG repeat or polyglutamine diseases. Along with a large variety of motor, behavioural and neuropsychological symptoms the clinical picture of patients suffering from this autosomal dominantly inherited ataxia may also include deficits of attention, impairments of memory, as well as frontal-executive and visuospatial dysfunctions. As the possible morphological correlates of these cognitive SCA2 deficits are unclear we examined the cholinergic basal forebrain nuclei, which are believed to be crucial for several aspects of normal cognition and may contribute to impairments of cognitive functions under pathological conditions. METHODS: We studied pigment-Nissl-stained thick tissue sections through the cholinergic basal forebrain nuclei (that is, medial septal nucleus, nuclei of the diagonal band of Broca, basal nucleus of Meynert) of four clinically diagnosed and genetically confirmed SCA2 patients and of 13 control individuals according to the pathoanatomical approach. The pathoanatomical results were confirmed by additional quantitative investigations of these nuclei in the SCA2 patients and four age- and gender-matched controls. RESULTS: Our study revealed a severe and consistent neuronal loss in all of the cholinergic basal forebrain nuclei (medial septal nucleus: 72%; vertical nucleus of the diagonal band of Broca: 74%; horizontal limb of the diagonal band of Broca: 72%; basal nucleus of Meynert: 86%) of the SCA2 patients studied. Damage to the basal forebrain nuclei was associated with everyday relevant cognitive deficits only in our SCA2 patient with an additional Braak and Braak stage V Alzheimer's disease (AD)-related tau pathology. CONCLUSIONS: The findings of the present study: (1) indicate that the mutation and pathological process underlying SCA2 play a causative role for this severe degeneration of the cholinergic basal forebrain nuclei and (2) may suggest that degeneration of the cholinergic basal forebrain nuclei per se is not sufficient to cause profound and global dementia detrimental to everyday practice and activities of daily living.

Spinocerebellar ataxia type 1 (SCA1): new pathoanatomical and clinico-pathological insights.

AIMS: Spinocerebellar ataxia type 1 (SCA1) represents the first molecular genetically characterized autosomal dominantly inherited cerebellar ataxia and is assigned to the CAG-repeat or polyglutamine diseases. Owing to limited knowledge about SCA1 neuropathology, appropriate pathoanatomical correlates of a large variety of SCA1 disease symptoms are missing and the neuropathological basis for further morphological and experimental SCA1 studies is still fragmentary. METHODS: In the present study, we investigated for the first time serial tissue sections through the complete brains of clinically diagnosed and genetically confirmed SCA1 patients. RESULTS: Brain damage in the three SCA1 patients studied went beyond the well-known brain predilection sites of the underlying pathological process. Along with neuronal loss in the primary motor cortex, it included widespread degeneration of gray components of the basal forebrain, thalamus, brainstem and cerebellum, as well as of white matter components in the cerebellum and brainstem. It involved the motor cerebellothalamocortical and basal ganglia-thalamocortical circuits, the visual, auditory, somatosensory, oculomotor, vestibular, ingestion-related, precerebellar, basal forebrain cholinergic and midbrain dopaminergic systems. CONCLUSIONS: These findings show for the first time that the extent and severity of brain damage in SCA1 is very similar to that of clinically closely related spinocerebellar ataxias (that is, SCA2, SCA3 and SCA7). They offer suitable explanations for poorly understood SCA1 disease symptoms and will facilitate the interpretation of further morphological and experimental SCA1 studies.

Pathoanatomy of cerebellar degeneration in spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3).

The cerebellum is one of the well-known targets of the pathological processes underlying spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3). Despite its pivotal role for the clinical pictures of these polyglutamine ataxias, no pathoanatomical studies of serial tissue sections through the cerebellum have been performed in SCA2 and SCA3 so far. Detailed pathoanatomical data are an important prerequisite for the identification of the initial events of the underlying disease processes of SCA2 and SCA3 and the reconstruction of its spread through the brain. In the present study, we performed a pathoanatomical investigation of serial thick tissue sections through the cerebellum of clinically diagnosed and genetically confirmed SCA2 and SCA3 patients. This study demonstrates that the cerebellar Purkinje cell layer and all four deep cerebellar nuclei consistently undergo considerable neuronal loss in SCA2 and SCA3. These cerebellar findings contribute substantially to the pathogenesis of clinical symptoms (i.e., dysarthria, intention tremor, oculomotor dysfunctions) of SCA2 and SCA3 patients and may facilitate the identification of the initial pathological alterations of the pathological processes of SCA2 and SCA3 and reconstruction of its spread through the brain.

Molecular cellular mechanisms of peptide regulation of melatonin synthesis in pinealocyte culture.

The effects of epithalone and vilone peptides on the synthesis of melatonin and factors involved in this process, arylalkylamine-N-acetyltransferase (AANAT) enzyme and pCREB transcription protein, were studied in rat pinealocyte culture. Epithalone stimulated AANAT and pCREB synthesis and increased melatonin level in culture medium. Simultaneous addition of norepinephrine and peptides into the culture potentiated the expression of AANAT and pCREB.

Inhibition of microglial and astrocytic inflammatory responses by the immunosuppressant mycophenolate mofetil.

AIMS: Nucleotide depletion induced by the immunosuppressant mycophenolate mofetil (MMF) has been shown to exert neuroprotective effects. It remains unclear whether nucleotide depletion directly counteracts neuronal demise or whether it inhibits microglial or astrocytic activation, thereby resulting in indirect neuroprotection. METHODS: Effects of MMF on isolated microglial cells, astrocyte/microglial cell co-cultures and isolated hippocampal neurones were analysed by immunocytochemistry, quantitative morphometry, and elisa. RESULTS: We found that: (i) MMF suppressed lipopolysaccharide-induced microglial secretion of interleukin-1ß, tumour necrosis factor-α and nitric oxide; (ii) MMF suppressed lipopolysaccharide-induced astrocytic production of tumour necrosis factor-α but not of nitric oxide; (iii) MMF strongly inhibited proliferation of both microglial cells and astrocytes; (iv) MMF did not protect isolated hippocampal neurones from excitotoxic injury; and (v) effects of MMF on glial cells were reversed after treatment with guanosine. CONCLUSIONS: Nucleotide depletion induced by MMF inhibits microglial and astrocytic activation. Microglial and astrocytic proliferation is suppressed by MMF-induced inhibition of the salvage pathway enzyme inosine monophosphate dehydrogenase. The previously observed neuroprotection after MMF treatment seems to be indirectly mediated, making this compound an interesting immunosuppressant in the treatment of acute central nervous system lesions.

Pinealocyte projections into the mammalian brain revealed with S-antigen antiserum.

Neural processes from mammalian pinealocytes have been discovered in several brain areas. These processes were visualized immunocytochemically in the Djungarian hamster, Phodopus sungorus, with an antiserum against bovine retinal S-antigen and traced as far as the region of the posterior commissure and habenular nuclei. This result indicates that pineal-to-brain connections exist in the mammal, and that the mammalian pineal gland, currently thought of only as a neuroendocrine organ, may communicate directly with select brain regions by way of these projections. The existence of mammalian pinealocyte projections is consistent with the view that these cells are not of glial origin but are derivatives of photoreceptor cells of the pineal complex of lower vertebrates that transmit signals to the brain by neural projections.

Dynamics of core body temperature cycles in long-term measurements under real life conditions in women.

Studies under real life conditions become more and more relevant in chronobiological and chronomedical research. The present study aims to analyze one of the most prominent biological rhythms: the core body temperature (CBT) rhythm in the real world outside the laboratory. CBT was recorded continuously in 37 healthy women (age between 21 and 44 years, median 29 years) with a newly developed intravaginal temperature sensor for up to 102 days. Sleep logs were available from 23 participants. To quantify the daily dynamics of each individual CBT-curve, novel measurement parameters are introduced which permit the quantification of the phase and shape of the CBT rhythms as well as their relation to the sleep-wake cycle. In addition to the classical phase markers (i.e. nadir and peak), the daily curves were segmented into quartiles by introducing the t25/t50/t75-values which can be used as phase and shape markers. At variance to previous studies, a conspicuous day-to-day variation was shown not only for the time point of the peak, but also for the time point of the nadir. However, the t-values, particularly the t75-value were relatively closely locked to external time and thus represent more reliable phase markers than the nadir. The (variable) time point of the nadir determined the period length, phase and shape of the subsequent CBT cycle. If a nadir occurred close to the wake-up time, the following cycle was considerably shorter than 24 hours, while a nadir distant from the wake-up time was followed by a longer cycle. Thus, the period lengths of the daily CBT cycles of each individual were characterized by an "expand/contract" rhythm. The analyses of the novel phase markers (t25/t50/t75) of the CBT curves allowed to identify "early" and "late" participants who may differ in their phase-response curves with regard to the entraining effect of light. In addition, the novel phase markers mirrored the different social entrainment conditions on weekends and workdays.

Mice, melatonin and the circadian system.

Melatonin effects are discussed by reviewing results from mice with intact or disrupted melatonin signaling. Melatonin, the neuroendocrine hand of the clock produced in the pineal gland during night, acts upon two receptor subtypes. Melatonin receptors are found in the suprachiasmatic nuclei (SCN), hypophysial pars tuberalis (PT) and adrenal gland. In SCN, melatonin interacts with PACAP, a neuropeptide of the retinohypothalamic tract. Moreover, melatonin acts on the SCN to modulate the activity of the sympathetic nervous system. Melatonin is not required to maintain rhythmic clock gene expression in SCN. By contrast, the rhythmic clock gene expression in PT depends on a melatonin signal interacting with adenosine. Melatonin may also affect clock gene protein levels in the adrenal cortex and influence adrenal functions. In conclusion, melatonin may serve the synchronization of peripheral oscillators by interacting with other neuroactive substances. A stress-reducing potency of melatonin needs to be explored in further studies.

Huntington's disease (HD): the neuropathology of a multisystem neurodegenerative disorder of the human brain.

Huntington's disease (HD) is an autosomal dominantly inherited, and currently untreatable, neuropsychiatric disorder. This progressive and ultimately fatal disease is named after the American physician George Huntington and according to the underlying molecular biological mechanisms is assigned to the human polyglutamine or CAG-repeat diseases. In the present article we give an overview of the currently known neurodegenerative hallmarks of the brains of HD patients. Subsequent to recent pathoanatomical studies the prevailing reductionistic concept of HD as a human neurodegenerative disease, which is primarily and more or less exclusively confined to the striatum (ie, caudate nucleus and putamen) has been abandoned. Many recent studies have improved our neuropathological knowledge of HD; many of the early groundbreaking findings of neuropathological HD research have been rediscovered and confirmed. The results of this investigation have led to the stepwise revision of the simplified pathoanatomical and pathophysiological HD concept and culminated in the implementation of the current concept of HD as a multisystem degenerative disease of the human brain. The multisystem character of the neuropathology of HD is emphasized by a brain distribution pattern of neurodegeneration (i) which apart from the striatum includes the cerebral neo-and allocortex, thalamus, pallidum, brainstem and cerebellum, and which (ii) therefore, shares more similarities with polyglutamine spinocerebellar ataxias than previously thought.

Tumorigenesis and eye abnormalities in transgenic mice expressing MSV-SV40 large T-antigen.

Transgenic mice which expressed SV40 large T-antigen under the control of the MSV enhancer and the SV40 promoter were generated. In animals containing an intact MSV enhancer, total lens cataracts and neuroectodermal brain tumors, originating in the pineal organ were observed. In contrast, 5' deletion of the MSV enhancer to a residual 53 bp resulted in a different spectrum of pathologies. Whilst lens cataracts still occurred, no brain tumors could be detected. Instead, fibrosarcomas and adenocarcinomas of the kidneys were induced. In addition, tumors of the endocrine pancreas were observed with both transgene constructs. We conclude that the MSV enhancer element is sufficient to direct the expression of the viral reporter gene to the lens and the pineal organ in transgenic mice. Deletion of the MSV enhancer correlates with the loss of DNA elements responsible for the pineal cell specific expression of SV40 large T-antigen.

Of rodents and ungulates and melatonin: creating a uniform code for darkness by different signaling mechanisms.

Melatonin synthesis in the mammalian pineal gland is one of the best investigated output pathways of the circadian clock because it can be readily measured and is tightly regulated by a clearly defined input, the neurotransmitter norepinephrine. In this system, a regulatory scenario was deciphered that is centered around the cyclic AMP pathway but shows peculiar species-specific differences. In rodents, the cyclic AMP-mediated, temporally sequential up-regulation of two transcription factors, the activator CREB (cyclic AMP-responsive element-binding protein) and the inhibitor ICER (inducible cyclic AMP-dependent early repressor), is the core mechanism to determine rhythmic accumulation of the mRNA encoding for the rate-limiting enzyme in melatonin synthesis, the arylalkylamine N-acetyltransferase (AA-NAT). Thus, in rodents, the regulation of melatonin synthesis bears an essential transcriptional component, which, however, is flanked by posttranscriptional mechanisms. In contrast, in ungulates, and possibly also in primates, AA-NAT appears to be regulated exclusively on the posttranscriptional level. Here, increasing cyclic AMP levels inhibit the breakdown of constitutively synthesized AA-NAT protein by proteasomal proteolysis, leading to an elevated enzyme activity. Thus, self-restriction of cellular responses, as a reaction to external cues, is accomplished by different mechanisms in pinealocytes of different mammalian species. In such a temporally gated cellular adaptation, transcriptionally active products of clock genes may play a supplementary role. Their recent detection in the endogenously oscillating nonmammalian pineal organ and, notably, also in the slave oscillator of the mammalian pineal gland underlines that the mammalian pineal gland will continue to serve as an excellent model system to understand mechanisms of biological timing.

S-antigen and rod-opsin immunoreactions in midline brain neoplasms of transgenic mice: similarities to pineal cell tumors and certain medulloblastomas in man.

Transgenic mice expressing the large T-antigen of the simian virus 40 (SV 40) under the control of 1) the enhancer of Moloney murine sarcoma virus (MSV) and 2) the SV 40 promoter develop undifferentiated neuroectodermal tumors located in the midline of the dorsal brain surface, abnormalities in lens fiber differentiation and retinal dysplasia. In this study the brain neoplasms of six adult animals and the brain of one 11-day old mouse were examined by conventional histology and immunocytochemical demonstration of S-antigen, rod-opsin, neuron-specific enolase, neurofilaments (160 and 200 kDa) and glial fibrillary acidic protein. According to histologic criteria the neoplasms were characterized as "primitive" neuroectodermal tumors composed mainly of small cells with scanty and ill-defined cytoplasm. Neoplastic cells displaying immunoreactive S-antigen were found in five brain tumors; three of these tumors also contained a limited number of rod-opsin immunoreactive neoplastic cells. Some tumor cells had neurite-like processes containing immunoreactive neurofilament (200 kDa). No pathologic lesions were found in the brain of the 11-day old animal. Tumors in transgenic mice may resemble pineal cell tumors and a special subtype of medulloblastoma in man. These neoplasms contain S-antigen immunoreactive and also rod-opsin immunoreactive tumors cells in certain cases. The findings suggest that transgenic mice expressing the large T-antigen of SV 40 may become a valuable animal model for analysing the origin, histogenesis and development of primitive neuroectodermal tumors with photoreceptor-like features (pineal cell tumors and certain medulloblastomas).

Antibodies against retinal photoreceptor-specific proteins reveal axonal projections from the photosensory pineal organ in teleosts.

With the aid of specific antisera to the retinal proteins S-antigen and alpha-transducin and to the rhodopsin apoprotein opsin, we have labeled various cell populations in the pineal organ, parapineal organ, habenular nucleus, and subcommissural organ in two teleost species: the rainbow trout and the European minnow. Although these proteins are associated with photoreceptor functions, not only photoreceptor cells but also the majority of parenchymal cells in the pineal organ were immunoreactive. Immunoreactive cells with dendrite- and axonlike processes were observed also in the parapineal organ and the habenular nucleus. Furthermore, S-antigen-immunoreactive, long, axonal processes were observed in the pineal organ and could be traced from the pineal organ to the habenular nucleus and to the pretectal area. In the light of recent HRP electron microscopical and immunocytochemical studies we propose (1) that not only the classical pineal photoreceptor cells of poikilothermic vertebrates but also other types of CSF-contacting neurons may be the phylogenetic ancestors of mammalian pinealocytes, and (2) a close interrelationship between the pineal organ and the limbic system, effectuated by the direct projections from pineal photoreceptors/CSF-contacting neurons/pinealocytes to the habenular nucleus, and by displaced "pinealocytelike" elements scattered in the habenular nucleus.

A semiquantitative image-analytical method for the recording of dose-response curves in immunocytochemical preparations.

Knowledge about intracellular signal transduction cascades is largely based on investigations of cultured cells whose responses to different stimuli are typically quantified via RIA, ELISA, or immunoblots. These techniques, which require relatively large amounts of biological material, are performed with homogenized cells and therefore do not allow localization of the molecules under investigation. We describe a protocol for recording dose-response curves directly from immunocytochemical preparations using rat pinealocytes as a model system. The cells were exposed to beta-adrenergic stimuli inducing the phosphorylation of the transcription factor CREB (mediated by PKA), an increase in ICER protein levels, and synthesis and release of melatonin. Melatonin concentrations were determined by ELISA. cPKA, phosphorylated CREB, and ICER were demonstrated by immunocytochemistry and immunoblots. Dose-response curves were recorded by measuring the integrated density of the immunoreactive sites with an image analysis program. Dose-response curves from immunoblots and immunocytochemical preparations showed almost identical dynamics, validating the immunocytochemical approach, which minimizes the amount of biological material needed for such studies, allows combined quantification and localization of biomolecules, and may even be more sensitive than immunoblotting.

Opsin- and S-antigen-like immunoreactions in photoreceptors of the tree shrew retina.

In the tree shrew retina individual rod and cone photoreceptors can be readily identified and quantified because their perikarya are arranged in a single layer. This retina is therefore an ideal system for testing the specificity of photoreceptor-directed antibodies. Here we describe the staining properties of polyclonal antibodies against (rhod)opsin and retinal S-antigen in the tree shrew retina. The (rhod)opsin antibody exclusively and completely labelled the rod population. The antibody against S-antigen also labelled all rods and, in addition, a regularly arrayed subpopulation of cones, which we argue to be the blue-sensitive cones. In the context of our findings, the labelling of pinealocytes with these antibodies is discussed.

Pineal nitric oxide synthase, but not heme oxygenase, mRNA is suppressed by continuous exposure to light.

We have previously shown that exposure of rats to constant light (LL) induced a decrease in NO synthase (NOS) activity in the pineal gland. We report here that the use of the sensitive technique of RT-PCR has demonstrated that mRNA for neuronal NOS is present in the pineal, and that it is photoneurally regulated. There was a marked decrease in pineal neuronal NOS mRNA levels in continuous light conditions, similar to the changes seen in NOS enzyme activity. Inducible NOS was not present in the pineal, and there was evidence that the photoregulatable form was not endothelial NOS. The mRNA for two isoforms of heme oxygenase, the enzyme responsible for the generation of the putative neuromodulator carbon monoxide, was also present in the pineal, but neither isoform was photoregulated. Using immunodetection, it was not possible to identify the presence of NOS protein, other than to a minimal extent, even though NOS activity was clearly present. NADPH-diaphorase staining and in situ hybridization were carried out in an attempt to identify the precise location of neuronal NOS message. A strong NADPH-diaphorase reaction was present in sympathetic nerve fibers of the pineal, but pinealocytes showed no or only very weak labelling. In situ hybridization was also unable to identify neuronal NOS message in pinealocytes. These data thus also suggest the possible presence of a pineal-specific NOS isoenzyme.

The pineal organ, its hormone melatonin, and the photoneuroendocrine system.

The vertebrate pineal organ rhythmically synthesizes and secretes melatonin during nighttime and forms an essential component of the photoneuroendocrine system which allows humans and animals to measure and keep the time. Regulation of the melatonin biosynthesis depends on signals from photoreceptors perceiving and transmitting environmental light stimuli and endogenous oscillators generating a circadian rhythm which is independent from any environmental time cue (zeitgeber). In nonmammalian species the photoreceptors responsible for regulating melatonin biosynthesis reside within the pineal organ itself. In several nonmammalian species (e.g., lamprey, zebra fish, house sparrow, chicken) the pineal organ is also capable of generating circadian rhythms and thus serves all key functions of the photoneuroendocrine system: photoreception, endogenous rhythm generation, and production of neurohormones. These may even be accomplished by a single "photoneuroendocrine" cell. In mammals the pineal organ has lost both the direct light sensitivity and the capacity of generating circadian rhythms, and melatonin biosynthesis is regulated by retinal photoreceptors and a circadian oscillator located in the suprachiasmatic nucleus of the hypothalamus. Due to this spatial separation the photoneuroendocrine system of mammals comprises neuronal and neuroendocrine pathways which interconnect its components. The neuronal pathways involve circuits of both the central and the peripheral nervous systems, and as an important final link noradrenergic sympathetic nerve fibers. The suprachiasmatic nucleus appears as a major target of melatonin in mammals. The pineal hormone may thus be involved in a feedback loop of the mammalian photoneuroendocrine system. The present comparative contribution considers, after a short survey of classical findings on the phylogenetic development and the gross anatomy of the pineal complex, cytoevolutionary and cell biological aspects of the various types of pinealocytes as well as the afferent and efferent innervation of the pineal organ (pinealofugal and pinealopetal neuronal pathways). Moreover, emphasis is placed on receptor mechanisms, second messenger systems (Ca2+ and cyclic AMP), transcription factors (e.g, CREB and ICER), and their roles for regulation of melatonin biosynthesis. Finally, the action, targets, and receptors of melatonin are dealt with. The synoptic approach of this contribution, which combines anatomical and ultrastructural findings with cell and molecular biological results, confirms the functional significance of the melatonin-synthesizing pineal organ as an important component of the photoneuroendocrine system and stresses the importance of this organ as a model to study signal transduction mechanisms both in photoreceptors and in neuroendocrine cells.

Activation of arylalkylamine N-acetyltransferase by phorbol esters in bovine pinealocytes suggests a novel regulatory pathway in melatonin synthesis.

In all mammalian species investigated, noradrenaline activates a beta-adrenoceptor/cAMP/protein kinase A-dependent mechanism to switch on arylalkylamine N-acetyltransferase and melatonin biosynthesis in the pineal gland. Other compounds which are known to influence the melatonin-generating system are phorbol esters. The effect of phorbol esters on regulation of melatonin synthesis has been mainly investigated in rat pinealocytes. In these cells, phorbol esters do not increase cAMP levels and arylalkylamine N-acetyltransferase on their own; however, phorbol esters potentiate the effects on cAMP and AANAT activity induced upon beta-adrenoceptor stimulation. In the present study, we investigated the effect of phorbol esters on the regulation of melatonin synthesis in bovine pinealocytes. We show that, in these cells, the phorbol esters 4beta-phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate have a direct stimulatory effect and induced 4-10-fold increases in AANAT protein levels, AANAT activity and melatonin production. The extent of these effects was similar to those induced by noradrenaline. Notably, responses to PMA were not accompanied by increases in cAMP levels. Northern blot analysis showed that Aanat mRNA levels did not change upon PMA treatment indicating that phorbol esters control AANAT at a post-transcriptional level. The effects on AANAT and melatonin production were reduced by use of protein kinase C inhibitors, but not by blockade of the cyclic AMP/protein kinase A pathway. Our results point towards a novel mechanism in the regulation of melatonin production that is cAMP-independent and involves protein kinase C. The study is of particular interest because regulation of melatonin biosynthesis in bovines may resemble that in primates more closely than that in rodents.

Melatonin: a clock-output, a clock-input.

In mammals, the circadian system is comprised of three major components: the lateral eyes, the hypothalamic suprachiasmatic nucleus (SCN) and the pineal gland. The SCN harbours the endogenous oscillator that is entrained every day to the ambient lighting conditions via retinal input. Among the many circadian rhythms in the body that are driven by SCN output, the synthesis of melatonin in the pineal gland functions as a hormonal message encoding for the duration of darkness. Dissemination of this circadian information relies on the activation of melatonin receptors, which are most prominently expressed in the SCN, and the hypophyseal pars tuberalis (PT), but also in many other tissues. A deficiency in melatonin, or a lack in melatonin receptors should therefore have effects on circadian biology. However, our investigations of mice that are melatonin-proficient with mice that do not make melatonin, or alternatively cannot interpret the melatonin message, revealed that melatonin has only minor effects on signal transduction processes within the SCN and sets, at most, the gain for clock error signals mediated via the retino-hypothalamic tract. Melatonin deficiency has no effect on the rhythm generation, or on the maintenance of the oscillation. By contrast, melatonin is essential for rhythmic signalling in the PT. Here, melatonin acts in concert with adenosine to elicit rhythms in clock gene expression. By sensitizing adenylyl cyclase, melatonin opens a temporally-restricted gate and thus lowers the threshold for adenosine to induce cAMP-sensitive genes. This interaction, which determines a temporally precise regulation of gene expression, and by endocrine-endocrine interactions possibly also pituitary output, may reflect a general mechanism by which the master clock in the brain synchronizes clock cells in peripheral tissues that require unique phasing of output signals.