Oliver Rawashdeh

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.

Melatonin suppresses nighttime memory formation in zebrafish.

Memory processes are modulated by the biological clock, although the mechanisms are unknown. Here, we report that in the diurnal zebrafish both learning and memory formation of an operant conditioning paradigm occur better during the day than during the night. Melatonin treatment during the day mimics the nighttime suppression of memory formation. Training in constant light improves nighttime memory formation while reducing endogenous melatonin concentrations. Treatment with melatonin receptor antagonists at night dramatically improves memory. Pinealectomy also significantly improves nighttime memory formation. We adduce that melatonin is both sufficient and necessary for poor memory formation during the night.

Cycling Behavior and Memory Formation

Circadian research has spent considerable effort in the determining clock output pathways, including identifying both physiological and behavioral processes that demonstrate significant time-of-day variation. Memory formation and consolidation represent notable processes shaped by endogenous circadian oscillators. To date, very few studies on memory mechanisms have considered potential confounding effects of time-of-day and the organisms innate activity cycles (e.g., nocturnal, diurnal, or crepuscular). The following studies highlight recent work describing this interactive role of circadian rhythms and memory formation, and were presented at a mini-symposium at the 2009 annual meeting of the Society for Neuroscience. The studies illustrate these time-of-day observations in a variety of behavioral paradigms and model organisms, including olfactory avoidance conditioning in Drosophila, long-term sensitization in Aplysia, active-avoidance conditioning in Zebrafish, and classical fear conditioning in rodents, suggesting that the circadian influence on memory behavior is highly conserved across species. Evidence also exists for a conserved mechanistic relationship between specific cycling molecules and memory formation, and the extent to which proper circadian cycling of these molecules is necessary for optimal cognitive performance. Studies describe the involvement of the core clock gene period, as well as vasoactive intestinal peptide, melatonin, and the cAMP/MAPK (cAMP/mitogen-activated protein kinase) cascade. Finally, studies in humans describe evidence for alterations in cognitive performance based on an interaction between sleep–wake homeostasis and the internal circadian clock. Conservation of a functional relationship between circadian rhythms with learning and memory formation across species provides a critical framework for future analysis of molecular mechanisms underlying complex behavior.

The hormonal Zeitgeber melatonin: role as a circadian modulator in memory processing.

The neuroendocrine substance melatonin is a hormone synthesized rhythmically by the pineal gland under the influence of the circadian system and alternating light/dark cycles. Melatonin has been shown to have broad applications, and consequently becoming a molecule of great controversy. Undoubtedly, however, melatonin plays an important role as a time cue for the endogenous circadian system. This review focuses on melatonin as a regulator in the circadian modulation of memory processing. Memory processes (acquisition, consolidation, and retrieval) are modulated by the circadian system. However, the mechanism by which the biological clock is rhythmically influencing cognitive processes remains unknown. We also discuss, how the circadian system by generating cycling melatonin levels can implant information about daytime into memory processing, depicted as day and nighttime differences in acquisition, memory consolidation and/or retrieval.

Non-ocular circadian oscillators and photoreceptors modulate long term memory formation in Aplysia.

In Aplysia californica, memory formation for long-term sensitization (LTS) and for a more complex type of associative learning, learning that food is inedible (LFI), is modulated by a circadian clock. For both types of learning, formation of long-term memory occurs during the day and significantly less during the night. Aplysia eyes contain a well-characterized circadian oscillator that is strongly coupled to the locomotor activity rhythm. Thus, the authors hypothesized that the ocular circadian oscillator was responsible for the circadian modulation of LFI and LTS. To test this hypothesis, they investigated whether the eyes were necessary for circadian modulation of LFI and LTS. Eyeless animals trained during the subjective day and tested 24 h later demonstrated robust long-term memory for both LFI and LTS, while eyeless animals trained and tested during the subjective night showed little or no memory for LFI or LTS. The amplitude of the rhythm of modulation in eyeless animals was similar to that of intact Aplysia, suggesting that extraocular circadian oscillators were mainly responsible for the circadian rhythms in long-term memory formation. Next, the authors investigated whether the eyes played a role in photic entrainment for circadian regulation of long-term memory formation. Eyeless animals were exposed to a reversed LD cycle for 7 days and then trained and tested for long-term memory using the LFI paradigm. Eyeless Aplysia formed significant long-term memory when trained during the projected shifted day but not during the projected shifted night. Thus, the extraocular circadian oscillator responsible for the rhythmic modulation of long-term memory formation can be entrained by extraocular photoreceptors.

Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK.

Memory performance varies over a 24‐h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen‐activated protein kinase (MAPK) and cyclic adenosine monophosphate response element‐binding protein (CREB) are central to the circadian (~ 24 h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory‐relevant hippocampal signaling pathways. We found that in wild‐type mice (WT), spatial learning triggers CREB phosphorylation only during the daytime, and that this effect depends on the presence of PER1. The time‐of‐day‐dependent induction of CREB phosphorylation can be reproduced pharmacologically in acute hippocampal slices prepared from WT mice, but is absent in preparations made from Per1‐knockout (Per1−/−) mice. We showed that the PER1‐dependent CREB phosphorylation is regulated downstream of MAPK. Stimulation of WT hippocampal neurons triggered the co‐translocation of PER1 and the CREB kinase pP90RSK (pMAPK‐activated ribosomal S6 kinase) into the nucleus. In hippocampal neurons from Per1−/− mice, however, pP90RSK remained perinuclear. A co‐immunoprecipitation assay confirmed a high‐affinity interaction between PER1 and pP90RSK. Knocking down endogenous PER1 in hippocampal cells inhibited adenylyl cyclase‐dependent CREB activation. Taken together, the PER1‐dependent modulation of cytoplasmic‐to‐nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime‐dependent memory performance, and adds a novel facet to the versatility of the clock gene protein PER1.

Circadian periods of sensitivity for ramelteon on the onset of running-wheel activity and the peak of suprachiasmatic nucleus neuronal firing rhythms in C3H/HeN mice.

Ramelteon, an MT1/MT2 melatonin receptor agonist, is used for the treatment of sleep-onset insomnia and circadian sleep disorders. Ramelteon phase shifts circadian rhythms in rodents and humans when given at the end of the subjective day; however, its efficacy at other circadian times is not known. Here, the authors determined in C3H/HeN mice the maximal circadian sensitivity for ramelteon in vivo on the onset of circadian running-wheel activity rhythms, and in vitro on the peak of circadian rhythm of neuronal firing in suprachiasmatic nucleus (SCN) brain slices. The phase response curve (PRC) for ramelteon (90 µg/mouse, subcutaneous [sc]) on circadian wheel-activity rhythms shows maximal sensitivity during the late mid to end of the subjective day, between CT8 and CT12 (phase advance), and late subjective night and early subjective day, between CT20 and CT2 (phase delay), using a 3-day-pulse treatment regimen in C3H/HeN mice. The PRC for ramelteon resembles that for melatonin in C3H/HeN mice, showing the same magnitude of maximal shifts at CT10 and CT2, except that the range of sensitivity for ramelteon (CT8–CT12) during the subjective day is broader. Furthermore, in SCN brain slices in vitro, ramelteon (10 pM) administered at CT10 phase advances (5.6 ± 0.29 h, n = 3) and at CT2 phase delays (−3.2 ± 0.12 h, n = 6) the peak of circadian rhythm of neuronal firing, with the shifts being significantly larger than those induced by melatonin (10 pM) at the same circadian times (CT10: 2.7 ± 0.15 h, n = 4, p < .05; CT2: −1.13 ± 0.08 h, n = 6, p < .001, respectively). The phase shifts induced by both melatonin and ramelteon in the SCN brain slice at either CT10 or CT2 corresponded with the period of sensitivity observed in vivo. In conclusion, melatonin and ramelteon showed identical periods of circadian sensitivity at CT10 (advance) and CT2 (delay) to shift the onset of circadian activity rhythms in vivo and the peak of SCN neuronal firing rhythms in vitro. (Author correspondence: mdubo@buffalo.edu)

Long-term effects of maternal separation on the responsiveness of the circadian system to melatonin in the diurnal nonhuman primate (Macaca mulatta).

Depression is often linked to early‐life adversity and circadian disturbances. Here, we assessed the long‐term impact of early‐life adversity, particularly preweaning mother–infant separation, on the circadian systems responsiveness to a time giver or synchronizer (Zeitgeber). Mother‐reared (MR) and peer‐reared (PR) rhesus monkeys were subjected to chronic jet‐lag, a forced desynchrony protocol of 22 hr T‐cycles [11:11 hr light:dark (LD) cycles] to destabilize the central circadian organization. MR and PR monkeys subjected to the T‐cycles showed split locomotor activity rhythms with periods of ~22 hr (entrained) and ~24 hr (free‐running), simultaneously. Continuous melatonin treatment in the drinking water (20 μg/mL) gradually increased the amplitude of the entrained rhythm at the expense of the free‐running rhythm, reaching complete entrainment by 1 wk. Upon release into constant dim light, a rearing effect on anticipation for both the predicted light onset and food presentation was observed. In MR monkeys, melatonin did not affect the amplitude of anticipatory behavior. Interestingly, however, PR macaques showed light onset and food anticipatory activities in response to melatonin treatment. These results demonstrate for the first time a rearing‐dependent effect of maternal separation in macaques, imprinting long‐term plastic changes on the circadian system well into late adulthood. These effects could be counteracted by the synchronizer molecule melatonin. We conclude that the melatonergic system is targeted by early‐life adversity of maternal separation and that melatonin supplementation ameliorates the negative impact of stress on the circadian system.

Clocking In Time to Gate Memory Processes: The Circadian Clock Is Part of the Ins and Outs of Memory

Learning, memory consolidation, and retrieval are processes known to be modulated by the circadian (circa: about; dies: day) system. The circadian regulation of memory performance is evolutionarily conserved, independent of the type and complexity of the learning paradigm tested, and not specific to crepuscular, nocturnal, or diurnal organisms. In mammals, long-term memory (LTM) formation is tightly coupled to de novo gene expression of plasticity-related proteins and posttranslational modifications and relies on intact cAMP/protein kinase A (PKA)/protein kinase C (PKC)/mitogen-activated protein kinase (MAPK)/cyclic adenosine monophosphate response element-binding protein (CREB) signaling. These memory-essential signaling components cycle rhythmically in the hippocampus across the day and night and are clearly molded by an intricate interplay between the circadian system and memory. Important components of the circadian timing mechanism and its plasticity are members of the Period clock gene family (Per1, Per2). Interestingly, Per1 is rhythmically expressed in mouse hippocampus. Observations suggest important and largely unexplored roles of the clock gene protein PER1 in synaptic plasticity and in the daytime-dependent modulation of learning and memory. Here, we review the latest findings on the role of the clock gene Period 1 (Per1) as a candidate molecular and mechanistic blueprint for gating the daytime dependency of memory processing.

The Hippocampal Autophagic Machinery is Depressed in the Absence of the Circadian Clock Protein PER1 that may Lead to Vulnerability During Cerebral Ischemia

BACKGROUND Autophagy is an intracellular bulk self-degrading process in which cytoplasmic contents of abnormal proteins and excess or damaged organelles are sequestered into autophagosomes, and degraded upon fusion with lysosomes. Although autophagy is generally considered to be pro-survival, it also functions in cell death processes. We recently reported on the hippocampal, higher vulnerability to cerebral ischemia in mice lacking the circadian clock protein PERIOD1 (PER1), a phenomenon we found to be linked to a PER1-dependent modulation of the expression patterns of apoptotic/autophagic markers. METHODS To exclude the contribution of vascular or glial factors to the innate vulnerability of Per1 knockout-mice (Per1-/--mice) to cerebral ischemia in vivo, we compared the autophagic machinery between primary hippocampal cultures from wild-type (WT)- and Per1-/--mice, using the lipophilic macrolide antibiotic, Rapamycin to induce autophagy. RESULTS Development of autophagy in WT cells involved an increased LC3-II-to-LC3-I ratio (microtubule-associated protein 1 light chain 3) and an overall increase in the level of LC3-II. In addition, immunostaining of LC3 in WT cells revealed the typical transformation of LC3 localization from a diffused staining to a dot- and ring-like pattern. In contrast, Per1-/--hippocampal cells were resistant to Rapamycin induced alterations of autophagy hallmarks. CONCLUSION Our in vitro data suggests that basal activity of autophagy seems to be modulated by PER1, and confirms the in vivo data by showing that the autophagic machinery is depressed in Per1-/--hippocampal neurons.The implication of both autophagy and circadian dysfunction in the pathogenesis of cerebral ischemia suggests that a functional connection between the two processes may exist.