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.

Pineal melatonin synthesis is altered in Period1 deficient mice.

Melatonin is an important endocrine signal for darkness in mammals. Transcriptional activation of the arylalkylamine-N-acetyltransferase gene encoding for the penultimate enzyme in melatonin synthesis drives the daily rhythm of the hormone in the pineal gland of rodents. Rhythmic arylalkylamine-N-acetyltransferase expression is controlled by the cAMP-signal transduction pathway and involves the activation of ß-adrenergic receptors and the inducible cAMP early repressor. In addition, the rat arylalkylamine-N-acetyltransferase promoter contains an E-box element which can interact with clock proteins. Moreover, the pineal gland of mice shows a circadian rhythm in clock proteins such as the transcriptional repressor Period1, which has been shown to control rhythmic gene expression in a variety of tissues. However, the role of Period1 in the regulation of pineal melatonin synthesis is still unknown. Therefore, circadian rhythms in arylalkylamine-N-acetyltransferase, ß-adrenergic receptor, and inducible cAMP early repressor mRNA levels (real time PCR), arylalkylamine-N-acetyltransferase enzyme activity (radiometric assay) and melatonin concentration radio immuno assay (RIA) were analyzed in the pineal gland of mice with a targeted deletion of the Period1 gene (Per1-/-) and the corresponding wildtype. In Per1-/- the amplitude in arylalkylamine-N-acetyltransferase expression was significantly elevated as compared to wildtype. In contrast, ß-adrenergic receptor and inducible cAMP early repressor mRNA levels were not affected by the Period1-deficiency. This indicates that the molecular clockwork alters the amplitude of arylalkylamine-N-acetyltransferase expression. In vitro, pineal glands of Per1-/- mice showed a day night difference in arylalkylamine-N-acetyltransferase expression with high levels at night. This suggests that a deficient in Period1 elicits similar effects as the activation of the cAMP-signal transduction pathway in wildtype mice.

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.

The circadian system and melatonin: lessons from rats and mice.

The circadian system (CS) comprises three key components: (1) endogenous oscillators (clocks) generating a circadian rhythm; (2) input pathways entraining the circadian rhythm to the astrophysical day; and (3) output pathways distributing signals from the oscillator to the periphery. This contribution briefly reviews some general aspects ofthe organization of the rodent CS and pays particular attention to recent results obtained with various mouse strains, related to molecular mechanisms involved in entraining the endogenous clock and the role of the pineal hormone melatonin as a hand of the endogenous clock.

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.

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.

Analysis of cell signalling in the rodent pineal gland deciphers regulators of dynamic transcription in neural/endocrine cells.

In neurons, a temporally restricted expression of cAMP-inducible genes is part of many developmental and adaptive processes. To understand such dynamics, the neuroendocrine rodent pineal gland provides an excellent model system as it has a clearly defined input, the neurotransmitter norepinephrine, and a measurable output, the hormone melatonin. In this system, a regulatory scenario has been deciphered, wherein cAMP-inducible genes are rapidly activated via the transcription factor phosphoCREB to induce transcriptional events necessary for an increase in hormone synthesis. However, among the activated genes is also the inhibitory transcription factor ICER. The increasing amount in ICER protein leads ultimately to the termination of mRNA accumulation of cAMP-inducible genes, including the gene for the Aa-nat that controls melatonin production. This shift in ratio of phosphoCREB and ICER levels that depends on the duration of stimulation can be interpreted as a self-restriction of cellular responses in neurons and has also been demonstrated to interfere with cellular plasticity in many non-neuronal systems.

Clock gene protein mPER1 is rhythmically synthesized and under cAMP control in the mouse pineal organ.

The mammalian clock gene Per1 is an important element of endogenous oscillators that control daily rhythms in central and peripheral tissues. Although such autonomous clock function is lost in the mammalian pineal gland during evolution, mPer1 mRNA and mPER1 protein were found to be strongly elevated in the mouse pineal organ during the dark period compared to daytime values. In vitro studies showed that mPer1 mRNA and mPER1 protein in mouse pineal gland are induced following the activation of a signalling pathway of fundamental importance for pineal physiology, the norepinephrine/cAMP/phosphoCREB cascade. mPER1 may function in the mouse pineal gland as a time-measuring molecule to participate in regulating rhythmic cellular responses in vivo.

Melatonin limits transcriptional impact of phosphoCREB in the mouse SCN via the Mel1a receptor.

In the mouse, activity phase-shifts of the endogenous clock in the suprachiasmatic nucleus (SCN) are associated with phosphorylation of the transcription factor Ca2+/cAMP responsive element binding protein (CREB). CREB phosphorylation is induced by the retino-hypothalamic transmitter pituitary adenylate cyclase-activating polypeptide (PACAP). As detected by immunohistochemistry in SCN slices from wild-type mice, melatonin completely blocked PACAP-stimulated CREB phosphorylation at low concentrations (1 nM). In Mel1a melatonin receptor-deficient mice, the PACAP-induced CREB phosphorylation was inhibited only at melatonin concentrations of 100 nM. This inhibition was, however, blunted by blocking the Mel1b melatonin receptor. Thus, melatonin modulates PACAP-mediated retinal stimuli for clock entrainment primarily via the Mel1a melatonin receptor through molecular interaction within the cAMP-signalling pathway.

Transcription factor dynamics and neuroendocrine signalling in the mouse pineal gland: a comparative analysis of melatonin-deficient C57BL mice and melatonin-proficient C3H mice.

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.

Signal transduction in the rodent pineal organ. From the membrane to the nucleus.

The rodent pineal organ transduces a photoneural input into a hormonal output. This photoneuroendocrine transduction leads to highly elevated levels of the hormone melatonin at night-time which serves as a message for darkness. The melatonin rhythm depends on transcriptional, translational and posttranslational regulation of the arylalkylamine-N-acetyltransferase, the key enzyme of melatonin biosynthesis. These regulatory mechanisms are fundamentally linked to two second messenger systems, namely the cAMP- and the Ca(2+)-signal transduction pathways. Our data gained by molecular biology, immunohistochemistry and single-cell imaging demonstrate a time- and substance-specific activation of these signaling pathways and provide a framework for the understanding of the complex signal transduction cascades in the rodent pineal gland which in concert not only regulate the basic profile but also fine-tune the circadian rhythm in melatonin synthesis.

CREB in the mouse SCN: a molecular interface coding the phase-adjusting stimuli light, glutamate, PACAP, and melatonin for clockwork access.

The suprachiasmatic nucleus (SCN) is a central pacemaker in mammals, driving many endogenous circadian rhythms. An important pacemaker target is the regulation of a hormonal message for darkness, the circadian rhythm in melatonin synthesis. The endogenous clock within the SCN is synchronized to environmental light/dark cycles by photic information conveyed via the retinohypothalamic tract (RHT) and by the nocturnal melatonin signal that acts within a feedback loop. We investigated how melatonin intersects with the temporally gated resetting actions of two RHT transmitters, pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate. We analyzed immunocytochemically the inducible phosphorylation of the transcription factor Ca2+/cAMP response element-binding protein (CREB) in the SCN of a melatonin-proficient (C3H) and a melatonin-deficient (C57BL) mouse strain. In vivo, light-induced phase shifts in locomotor activity were consistently accompanied by CREB phosphorylation in the SCN of both strains. However, in the middle of subjective nighttime, light induced larger phase delays in C57BL than in C3H mice. In vitro, PACAP and glutamate induced CREB phosphorylation in the SCN of both mouse strains, with PACAP being more effective during late subjective daytime and glutamate being more effective during subjective nighttime. Melatonin suppressed PACAP- but not glutamate-induced phosphorylation of CREB. The distinct temporal domains during which glutamate and PACAP induce CREB phosphorylation imply that during the light/dark transition the SCN switches sensitivity between these two RHT transmitters. Because these temporal domains are not different between C3H and C57BL mice, the sensitivity windows are set independently of the rhythmic melatonin signal.

Identification of placental cytokine-producing cells in term and preterm labor.

OBJECTIVE: To determine if the production of proinflammatory cytokines by placentally derived macrophages changes with term and preterm labor and to examine if changes in antigen expression of these cytokines can be detected by immunohistologic methods. METHODS: Enzymatically dispersed placental cell suspensions of the trophoblastic villi, obtained from 16 women with spontaneous term delivery, 16 women with elective cesarean delivery without any labor, and 22 preterm delivering women with labor unresponsive to tocolysis, were fractionated by magnetic-associated-cell-sorting, on the basis of CD11b-antigen expression. Positively and negatively sorted cell fractions were cultured and concentrations of interleukin-6, interleukin-1beta, and tumor-necrosis-factor-alpha were measured in the culture supernatants. Immunohistologic staining was used for identification of cytokine-producing cells within placental tissues. RESULTS: Positively sorted cells obtained from term (median 2027 pg/mL, P = .037) and preterm (median 3628 pg/mL, P = .001) laboring women produced significantly elevated amounts of tumor-necrosis-factor-alpha compared with nonlaboring (median 1088 pg/mL) women at term. Negatively sorted cell fractions obtained from term (median interleukin-1beta 162 pg/mL, P = .031, median interleukin-6 3134 pg/mL, P = .004) and preterm (median interleukin-1beta 934 pg/mL, P = .003, median interleukin-6 5695 pg/mL, P = .001) laboring women produced significantly elevated amounts of interleukin-1beta and interleukin-6 compared with nonlaboring (median interleukin-1beta 29 pg/mL, median interleukin-6 135 pg/mL) women at term. Immunohistologic staining revealed that tumor-necrosis-factor-alpha activity was localized in isolated stromal cells, whereas interleukin-1beta and interleukin-6 were predominantly found in endothelial cells within placental villi. CONCLUSION: The source of labor-associated release of tumor-necrosis-factor-alpha from placental tissues are macrophages, whereas interleukin-1beta and interleukin-6 are released from placental endothelial cells.