Katrin Ackermann

A survey of molecular details in the human pineal gland in the light of phylogeny, structure, function and chronobiological diseases

Abstract:  The human pineal gland is a neuroendocrine transducer that forms an integral part of the brain. Through the nocturnally elevated synthesis and release of the neurohormone melatonin, the pineal gland encodes and disseminates information on circadian time, thus coupling the outside world to the biochemical and physiological internal demands of the body. Approaches to better understand molecular details behind the rhythmic signalling in the human pineal gland are limited but implicitly warranted, as human chronobiological dysfunctions are often associated with alterations in melatonin synthesis. Current knowledge on melatonin synthesis in the human pineal gland is based on minimally invasive analyses, and by the comparison of signalling events between different vertebrate species, with emphasis put on data acquired in sheep and other primates. Together with investigations using autoptic pineal tissue, a remnant silhouette of premortem dynamics within the hormone’s biosynthesis pathway can be constructed. The detected biochemical scenario behind the generation of dynamics in melatonin synthesis positions the human pineal gland surprisingly isolated. In this neuroendocrine brain structure, protein‐protein interactions and nucleo‐cytoplasmic protein shuttling indicate furthermore a novel twist in the molecular dynamics in the cells of this neuroendocrine brain structure. These findings have to be seen in the light that an impaired melatonin synthesis is observed in elderly and/or demented patients, in individuals affected by Alzheimer’s disease, Smith–Magenis syndrome, autism spectrum disorder and sleep phase disorders. Already, recent advances in understanding signalling dynamics in the human pineal gland have significantly helped to counteract chronobiological dysfunctions through a proper restoration of the nocturnal melatonin surge.

Effect of sleep deprivation on the human metabolome

Significance Sleep restriction and circadian clock disruption are associated with metabolic disorders including obesity and diabetes; this association can be studied by using the powerful tool of metabolomics. By using liquid chromatography/MS metabolomics, we have characterized plasma metabolites that were significantly affected by acute sleep deprivation (mainly lipids and acylcarnitines), all increasing during sleep deprivation. Observed increased levels of serotonin, tryptophan, and taurine may explain the antidepressive effect of sleep deprivation and deserve further study. Clear daily rhythms were observed in most metabolites, with 24 h wakefulness mainly reducing the amplitude of these rhythms. Our results further the understanding of sleep/wake regulation and the associated metabolic processes, and will be vital when using metabolic profiling to identify robust biomarkers for disease states and drug efficacy. Sleep restriction and circadian clock disruption are associated with metabolic disorders such as obesity, insulin resistance, and diabetes. The metabolic pathways involved in human sleep, however, have yet to be investigated with the use of a metabolomics approach. Here we have used untargeted and targeted liquid chromatography (LC)/MS metabolomics to examine the effect of acute sleep deprivation on plasma metabolite rhythms. Twelve healthy young male subjects remained in controlled laboratory conditions with respect to environmental light, sleep, meals, and posture during a 24-h wake/sleep cycle, followed by 24 h of wakefulness. Two-hourly plasma samples collected over the 48 h period were analyzed by LC/MS. Principal component analysis revealed a clear time of day variation with a significant cosine fit during the wake/sleep cycle and during 24 h of wakefulness in untargeted and targeted analysis. Of 171 metabolites quantified, daily rhythms were observed in the majority (n = 109), with 78 of these maintaining their rhythmicity during 24 h of wakefulness, most with reduced amplitude (n = 66). During sleep deprivation, 27 metabolites (tryptophan, serotonin, taurine, 8 acylcarnitines, 13 glycerophospholipids, and 3 sphingolipids) exhibited significantly increased levels compared with during sleep. The increased levels of serotonin, tryptophan, and taurine may explain the antidepressive effect of acute sleep deprivation and deserve further study. This report, to our knowledge the first of metabolic profiling during sleep and sleep deprivation and characterization of 24 h rhythms under these conditions, offers a novel view of human sleep/wake regulation.

Diurnal rhythms in blood cell populations and the effect of acute sleep deprivation in healthy young men.

STUDY OBJECTIVES The sleep/wake cycle is accompanied by changes in circulating numbers of immune cells. The goal of this study was to provide an in-depth characterization of diurnal rhythms in different blood cell populations and to investigate the effect of acute sleep deprivation on the immune system, as an indicator of the bodys acute stress response. DESIGN Observational within-subject design. SETTING Home environment and Clinical Research Centre. PARTICIPANTS 15 healthy male participants aged 23.7 ± 5.4 (standard deviation) yr. INTERVENTIONS Total sleep deprivation. MEASUREMENTS AND RESULTS Diurnal rhythms of several blood cell populations were assessed under a normal sleep/wake cycle followed by 29 hr of extended wakefulness. The effect of condition (sleep versus sleep deprivation) on peak time and amplitude was investigated. Interindividual variation of, and the level of correlation between, the different cell populations was assessed. Comprehensive nonlinear curve fitting showed significant diurnal rhythms for all blood cell types investigated, with CD4 (naïve) cells exhibiting the most robust rhythms independent of condition. For those participants exhibiting significant diurnal rhythms in blood cell populations, only the amplitude of the granulocyte rhythm was significantly reduced by sleep deprivation. Granulocytes were the most diverse population, being most strongly affected by condition, and showed the lowest correlations with any other given cell type while exhibiting the largest interindividual variation in abundance. CONCLUSIONS Granulocyte levels and diurnal rhythmicity are directly affected by acute sleep deprivation; these changes mirror the bodys immediate immune response upon exposure to stress.

Effect of sleep deprivation on rhythms of clock gene expression and melatonin in humans

This study investigated the impact of sleep deprivation on the human circadian system. Plasma melatonin and cortisol levels and leukocyte expression levels of 12 genes were examined over 48 h (sleep vs. no-sleep nights) in 12 young males (mean ± SD: 23 ± 5 yrs). During one night of total sleep deprivation, BMAL1 expression was suppressed, the heat shock gene HSPA1B expression was induced, and the amplitude of the melatonin rhythm increased, whereas other high-amplitude clock gene rhythms (e.g., PER1-3, REV-ERBα) remained unaffected. These data suggest that the core clock mechanism in peripheral oscillators is compromised during acute sleep deprivation.

Melatonin Synthesis in the Human Pineal Gland: Advantages, Implications, and Difficulties

Rhythms in the mammalian pineal organ depend on afferent information that is derived from the endogenous clock residing in the hypothalamic suprachiasmatic nucleus (SCN). The best characterized function of the pineal gland is the nocturnally elevated synthesis of the hormone melatonin, which provides the body with the signal of the duration of the night period. The rate‐limiting enzyme for melatonin synthesis is arylalkylamine N‐acetyltransferase (AANAT). In contrast to the transcriptional regulation of the Aanat gene in rodents, a post‐translational shaping of the melatonin pattern is indicated in the human pineal gland. Despite the fact that melatonin levels can be determined easily in various body fluids, the molecular elements involved in shaping the rhythmic hormone synthesis cannot be analyzed experimentally in the living organism. However, the use of post‐mortem pineal material seems to constitute a valid approach to decipher the regulation of human melatonin synthesis.

Dynamics in enzymatic protein complexes offer a novel principle for the regulation of melatonin synthesis in the human pineal gland.

Abstract:  Time of day is communicated to the body through rhythmic cues, including pineal gland melatonin synthesis, which is restricted to nighttime. Whereas in most rodents transcriptional regulation of the arylalkylamine N‐acetyltransferase (Aanat) gene is essential for rhythmic melatonin synthesis, investigations into nonrodent mammalian species have shown post‐transcriptional regulation to be of central importance, with molecular mechanisms still elusive. Therefore, human pineal tissues, taken from routine autopsies were allocated to four time‐of‐death groups (night/dawn/day/dusk) and analyzed for daytime‐dependent changes in phosphorylated AANAT (p31T‐AANAT) and in acetyl‐serotonin‐methyltransferase (ASMT) expression and activity. Protein content, intracellular localization, and colocalization of p31T‐AANAT and ASMT were assessed, using immunoblotting, immunofluorescence, and immunoprecipitation techniques. Fresh sheep pineal gland preparations were used for comparative purposes. The amount of p31T‐AANAT and ASMT proteins as well as their intracellular localization showed no diurnal variation in autoptic human and fresh sheep pineal glands. Moreover, in human and sheep pineal extracts, AANAT could not be dephosphorylated, which was at variance to data derived from rat pineal extracts. P31T‐AANAT and ASMT were often found to colocalize in cellular rod‐like structures that were also partly immunoreactive for the pinealocyte process‐specific marker S‐antigen (arrestin) in both, human and sheep pinealocytes. Protein–protein interaction studies with p31T‐AANAT, ASMT, and S‐antigen demonstrated a direct association and formation of robust complexes, involving also 14‐3‐3. This work provides evidence for a regulation principle for AANAT activity in the human pineal gland, which may not be based on a p31T‐AANAT phosphorylation/dephosphorylation switch, as described for other mammalian species.

Day–night expression patterns of clock genes in the human pineal gland

Abstract:  Rhythm generation within the mammalian circadian system is achieved by clock genes and their protein products. As an integral part of this system, the pineal gland serves the need to tune the body to the temporal environment by the rhythmic synthesis and release of melatonin. A number of human disorders and syndromes are associated with alterations in circadian rhythms of clock genes and their protein products and/or a dysfunction in melatonin synthesis. In the human, little is known about the molecular signature of time management. Pineal tissue from regular autopsies was allocated to asserted time‐of‐death groups (dawn, day, dusk, night), and analyzed by RT‐PCR, immunoblotting, immunohistochemistry, and confocal laser scanning microscopy for expression of clock genes. Despite the observed diurnal rhythms in activity of the arylalkylamine N‐acetyltransferase and in melatonin content, mRNA levels for the clock genes Period1, Cryptochrome1, Clock, and Bmal1, and also amounts of corresponding clock gene proteins showed no differences between time‐ of‐death groups. In contrast, a time‐of‐day‐dependent nucleocytoplasmic shuttling of clock gene proteins was detected. These data confirm the minor importance of a transcriptional regulation for dynamics in the human pineal gland, and offer a novel twist in the molecular competence of clock gene proteins.

Clock gene expression in the human pituitary gland.

Pituitary function relies on strictly timed, yet plastic mechanisms, particularly with respect to the daytime-dependent coordination of hormone synthesis and release. In other systems, clock genes and their protein products are well-described candidates to anticipate the daily demands in neuroendocrine coupling and to manage cellular adaptation on changing internal or external circumstances. To elucidate possible mechanisms of time management, a total of 52 human autoptic pituitary glands were allocated to the 4 time-of-day groups, night, dawn, day, and dusk, according to reported time of death. The observed daytime-dependent dynamics in ACTH content supports a postmortem conservation of the premortem condition, and thus, principally validates the investigation of autoptic pituitary glands. Pituitary extracts were investigated for expression of clock genes Per1, Cry1, Clock, and Bmal1 and corresponding protein products. Only the clock gene Per1 showed daytime-dependent differences in quantitative real-time PCR analyses, with decreased levels observed during dusk. Although the overall amount in clock gene protein products PER1, CRY1, and CLOCK did not fluctuate with time of day in human pituitary, an indication for a temporally parallel intracellular translocation of PER1 and CRY1 was detected by immunofluorescence. Presented data suggest that the observed clock gene expression in human pituitary cells does not provide evidence for a functional intrinsic clockwork. It is suggested that clock genes and their protein products may be directly involved in the daytime-dependent regulation and adaptation of hormone synthesis and release and within homeostatic adaptive plasticity.

Accurate extraction of nanometer distances in multimers by pulse EPR

Abstract Pulse electron paramagnetic resonance (EPR) is gaining increasing importance in structural biology. The PELDOR (pulsed electron–electron double resonance) method allows extracting distance information on the nanometer scale. Here, we demonstrate the efficient extraction of distances from multimeric systems such as membrane‐embedded ion channels where data analysis is commonly hindered by multi‐spin effects.

Binding dynamics of a monomeric SSB protein to DNA: a single-molecule multi-process approach

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all organisms and are characterized by the presence of an OB (oligonucleotide/oligosaccharide/oligopeptide) binding motif to recognize single-stranded DNA (ssDNA). Despite their critical role in genome maintenance, our knowledge about SSB function is limited to proteins containing multiple OB-domains and little is known about single OB-folds interacting with ssDNA. Sulfolobus solfataricus SSB (SsoSSB) contains a single OB-fold and being the simplest representative of the SSB-family may serve as a model to understand fundamental aspects of SSB:DNA interactions. Here, we introduce a novel approach based on the competition between Förster resonance energy transfer (FRET), protein-induced fluorescence enhancement (PIFE) and quenching to dissect SsoSSB binding dynamics at single-monomer resolution. We demonstrate that SsoSSB follows a monomer-by-monomer binding mechanism that involves a positive-cooperativity component between adjacent monomers. We found that SsoSSB dynamic behaviour is closer to that of Replication Protein A than to Escherichia coli SSB; a feature that might be inherited from the structural analogies of their DNA-binding domains. We hypothesize that SsoSSB has developed a balance between high-density binding and a highly dynamic interaction with ssDNA to ensure efficient protection of the genome but still allow access to ssDNA during vital cellular processes.