Non-24-Hour Sleep-Wake Disorder Revisited - A Case Study.

The human sleep-wake cycle is governed by two major factors: a homeostatic hourglass process (process S), which rises linearly during the day, and a circadian process C, which determines the timing of sleep in a ~24-h rhythm in accordance to the external light-dark (LD) cycle. While both individual processes are fairly well characterized, the exact nature of their interaction remains unclear. The circadian rhythm is generated by the suprachiasmatic nucleus ("master clock") of the anterior hypothalamus, through cell-autonomous feedback loops of DNA transcription and translation. While the phase length (tau) of the cycle is relatively stable and genetically determined, the phase of the clock is reset by external stimuli ("zeitgebers"), the most important being the LD cycle. Misalignments of the internal rhythm with the LD cycle can lead to various somatic complaints and to the development of circadian rhythm sleep disorders (CRSD). Non-24-hour sleep-wake disorders (N24HSWD) is a CRSD affecting up to 50% of totally blind patients and characterized by the inability to maintain a stable entrainment of the typically long circadian rhythm (tau > 24.5 h) to the LD cycle. The disease is rare in sighted individuals and the pathophysiology less well understood. Here, we present the case of a 40-year-old sighted male, who developed a misalignment of the internal clock with the external LD cycle following the treatment for Hodgkin's lymphoma (ABVD regimen, four cycles and AVD regimen, four cycles). A thorough clinical assessment, including actigraphy, melatonin profiles and polysomnography led to the diagnosis of non-24-hour sleep-wake disorders (N24HSWD) with a free-running rhythm of tau = 25.27 h. A therapeutic intervention with bright light therapy (30 min, 10,000 lux) in the morning and melatonin administration (0.5-0.75 mg) in the evening failed to entrain the free-running rhythm, although a longer treatment duration and more intense therapy might have been successful. The sudden onset and close timely connection led us to hypothesize that the chemotherapy might have caused a mutation of the molecular clock components leading to the observed elongation of the circadian period.

Serum factors in older individuals change cellular clock properties.

Human aging is accompanied by dramatic changes in daily sleep-wake behavior: Activity shifts to an earlier phase, and the consolidation of sleep and wake is disturbed. Although this daily circadian rhythm is brain-controlled, its mechanism is encoded by cell-autonomous circadian clocks functioning in nearly every cell of the body. In fact, human clock properties measured in peripheral cells such as fibroblasts closely mimic those measured physiologically and behaviorally in the same subjects. To understand better the molecular mechanisms by which human aging affects circadian clocks, we characterized the clock properties of fibroblasts cultivated from dermal biopsies of young and older subjects. Fibroblast period length, amplitude, and phase were identical in the two groups even though behavior was not, thereby suggesting that basic clock properties of peripheral cells do not change during aging. Interestingly, measurement of the same cells in the presence of human serum from older donors shortened period length and advanced the phase of cellular circadian rhythms compared with treatment with serum from young subjects, indicating that a circulating factor might alter human chronotype. Further experiments demonstrated that this effect is caused by a thermolabile factor present in serum of older individuals. Thus, even though the molecular machinery of peripheral circadian clocks does not change with age, some age-related circadian dysfunction observed in vivo might be of hormonal origin and therefore might be pharmacologically remediable.

The physiological period length of the human circadian clock in vivo is directly proportional to period in human fibroblasts.

BACKGROUND: Diurnal behavior in humans is governed by the period length of a circadian clock in the suprachiasmatic nuclei of the brain hypothalamus. Nevertheless, the cell-intrinsic mechanism of this clock is present in most cells of the body. We have shown previously that for individuals of extreme chronotype ("larks" and "owls"), clock properties measured in human fibroblasts correlated with extreme diurnal behavior. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have measured circadian period in human primary fibroblasts taken from normal individuals and, for the first time, compared it directly with physiological period measured in vivo in the same subjects. Human physiological period length was estimated via the secretion pattern of the hormone melatonin in two different groups of sighted subjects and one group of totally blind subjects, each using different methods. Fibroblast period length was measured via cyclical expression of a lentivirally delivered circadian reporter. Within each group, a positive linear correlation was observed between circadian period length in physiology and in fibroblast gene expression. Interestingly, although blind individuals showed on average the same fibroblast clock properties as sighted ones, their physiological periods were significantly longer. CONCLUSIONS/SIGNIFICANCE: We conclude that the period of human circadian behaviour is mostly driven by cellular clock properties in normal individuals and can be approximated by measurement in peripheral cells such as fibroblasts. Based upon differences among sighted and blind subjects, we also speculate that period can be modified by prolonged unusual conditions such as the total light deprivation of blindness.

Ginkgo biloba extract ameliorates oxidative phosphorylation performance and rescues abeta-induced failure.

BACKGROUND: Energy deficiency and mitochondrial failure have been recognized as a prominent, early event in Alzheimer's disease (AD). Recently, we demonstrated that chronic exposure to amyloid-beta (Abeta) in human neuroblastoma cells over-expressing human wild-type amyloid precursor protein (APP) resulted in (i) activity changes of complexes III and IV of the oxidative phosphorylation system (OXPHOS) and in (ii) a drop of ATP levels which may finally instigate loss of synapses and neuronal cell death in AD. Therefore, the aim of the present study was to investigate whether standardized Ginkgo biloba extract LI 1370 (GBE) is able to rescue Abeta-induced defects in energy metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We used a high-resolution respiratory protocol to evaluate OXPHOS respiratory capacity under physiological condition in control (stably transfected with the empty vector) and APP cells after treatment with GBE. In addition, oxygen consumption of isolated mitochondria, activities of mitochondrial respiratory enzymes, ATP and reactive oxygen species (ROS) levels as well as mitochondrial membrane mass and mitochondrial DNA content were determined. We observed a general antioxidant effect of GBE leading to an increase of the coupling state of mitochondria as well as energy homeostasis and a reduction of ROS levels in control cells and in APP cells. GBE effect on OXPHOS was even preserved in mitochondria after isolation from treated cells. Moreover, these functional data were paralleled by an up-regulation of mitochondrial DNA. Improvement of the OXPHOS efficiency was stronger in APP cells than in control cells. In APP cells, the GBE-induced amelioration of oxygen consumption most likely arose from the modulation and respective normalization of the Abeta-induced disturbance in the activity of mitochondrial complexes III and IV restoring impaired ATP levels possibly through decreasing Abeta and oxidative stress level. CONCLUSIONS/SIGNIFICANCE: Although the underlying molecular mechanisms of the mode of action of GBE remain to be determined, our study clearly highlights the beneficial effect of GBE on the cellular OXPHOS performance and restoration of Abeta-induced mitochondrial dysfunction.

Abeta and human amylin share a common toxicity pathway via mitochondrial dysfunction.

Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) are leading causes of morbidity and mortality in the elderly. Both diseases are characterized by amyloid deposition in target tissues: aggregation of amylin in T2DM is associated with loss of insulin-secreting beta-cells, while amyloid beta (A beta) aggregation in AD brain is associated with neuronal loss. Here, we used quantitative iTRAQ proteomics as a discovery tool to show that both A beta and human amylin (HA) deregulate identical proteins, a quarter of which are mitochondrial, supporting the notion that mitochondrial dysfunction is a common target in these two amyloidoses. A functional validation revealed that mitochondrial complex IV activity was significantly reduced after treatment with either HA or A beta, as was mitochondrial respiration. In comparison, complex I activity was reduced only after treatment with HA. A beta and HA, but not the non-amyloidogenic rat amylin, induced significant increases in the generation of ROS. Co-incubation of HA and A beta did not produce an augmented effect in ROS production, again suggesting common toxicity mechanisms. In conclusion, our data suggest that A beta and HA both exert toxicity, at least in part, via mitochondrial dysfunction, thus restoring their function may be beneficial for both AD and T2DM.

Amyloid-beta and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer's disease mice.

Alzheimer's disease (AD) is characterized by amyloid-beta (Abeta)-containing plaques, neurofibrillary tangles, and neuron and synapse loss. Tangle formation has been reproduced in P301L tau transgenic pR5 mice, whereas APP(sw)PS2(N141I) double-transgenic APP152 mice develop Abeta plaques. Cross-breeding generates triple transgenic ((triple)AD) mice that combine both pathologies in one model. To determine functional consequences of the combined Abeta and tau pathologies, we performed a proteomic analysis followed by functional validation. Specifically, we obtained vesicular preparations from (triple)AD mice, the parental strains, and nontransgenic mice, followed by the quantitative mass-tag labeling proteomic technique iTRAQ and mass spectrometry. Within 1,275 quantified proteins, we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Notably, deregulation of complex I was tau dependent, whereas deregulation of complex IV was Abeta dependent, both at the protein and activity levels. Synergistic effects of Abeta and tau were evident in 8-month-old (triple)AD mice as only they showed a reduction of the mitochondrial membrane potential at this early age. At the age of 12 months, the strongest defects on OXPHOS, synthesis of ATP, and reactive oxygen species were exhibited in the (triple)AD mice, again emphasizing synergistic, age-associated effects of Abeta and tau in perishing mitochondria. Our study establishes a molecular link between Abeta and tau protein in AD pathology in vivo, illustrating the potential of quantitative proteomics.

Immunohistochemical Localization of Fas-associated phosphatase-1 (FAP-1) in Alzheimer disease hippocampus.

Fas-associated phosphatase-1 (FAP-1) is a regulatory peptide inhibiting apoptotic signal transduction via the death receptor Fas. Because apoptosis is a common mechanism leading to neuronal death in neurodegenerative disorders, the authors investigated the immunohistochemical distribution of FAP-1 in the hippocampus of elderly control subjects and Alzheimer disease (AD) patients. The current study provides the first evidence that FAP-1 is localized in the human hippocampus in pyramidal neurons of the hippocampal subfields CA1-4 and in granular cells. Cellular and extracellular FAP-1 intensity was increased in some control subjects and AD patients, but was not related to the stage of the illness. Rather, these data may indicate a general role for FAP-1 in neuronal death both in adult CNS and during the course of neurodegenerative disorders.

Reduced hippocampal MT2 melatonin receptor expression in Alzheimer's disease.

The aim of the present study was to identify the distribution of the second melatonin receptor (MT2) in the human hippocampus of elderly controls and Alzheimer's disease (AD) patients. This is the first report of immunohistochemical MT2 localization in the human hippocampus both in control and AD cases. The specificity of the MT2 antibody was ascertained by fluorescence microscopy using the anti-MT2 antibody in HEK 293 cells expressing recombinant MT2, in immunoblot experiments on membranes from MT2 expressing cells, and, finally, by immunoprecipitation experiments of the native MT2. MT2 immunoreactivity was studied in the hippocampus of 16 elderly control and 16 AD cases. In controls, MT2 was localized in pyramidal neurons of the hippocampal subfields CA1-4 and in some granular neurons of the stratum granulosum. The overall intensity of the MT2 staining was distinctly decreased in AD cases. The results indicate that MT2 may be involved in mediating the effects of melatonin in the human hippocampus, and this mechanism may be heavily impaired in AD.

Orexins and their receptors in the human retina.

OBJECTIVES: Orexins A and B are neuropeptides involved in the regulation of feeding behavior, energy homeostasis and arousal. In the human retina, however, immunohistochemical localization of orexins and their receptors, OX-R1 and OX-R2, has not been ascertained. METHODS: We localized orexins A and B, OX-R1 and OX-R2 in the human retina using immunohistochemistry. Retinae from 2 Alzheimer's disease (AD) patients provided preliminary evidence for possible orexin alterations. RESULTS: Orexin A, orexin B and OX-R1 were localized in ganglion and amacrine cells, cellular processes in the inner and outer plexiform layer and in the inner segments of photoreceptor cells. There was no OX-R2 immunoreactivity in the retina. The staining intensity for both orexins was decreased in the AD patients. CONCLUSION: This immunohistochemical study provides the first evidence for the distribution of orexin A, orexin B and OX-R1 in the human retina. The localization pattern suggests a modulatory role for orexins in the interactions of those retinal cells which transmit light information to the suprachiasmatic nuclei, and thus may be involved in circadian rhythm entrainment.

Immunohistochemical localization of angiotensin-converting enzyme, angiotensin II and AT1 receptor in human ocular tissues.

We investigated the immunohistochemical distribution of 3 components of the renin-angiotensin system (RAS), angiotensin-converting enzyme (ACE), angiotensin II (AngII) and AT1 receptor (AT1), in the human eye. ACE and AngII were localized to nonpigmented epithelial cells of the ciliary body, to endothelial and epithelial cells of the cornea, to epithelial cells of the conjunctiva and to trabecular meshwork cells in the anterior part of the eye. In the posterior part of the eye, ACE and AngII were localized to ganglion cells, some cells in the inner nuclear layer, photoreceptor cells and to endothelial cells of the retinal and choroidal vessels. The overall intensity of AT1 immunoreactivity was weak in all ocular tissues, but the main localization was in ganglion cells. As a preliminary investigation, we were able to include 2 Alzheimer's disease (AD) cases. In AD, no differences from controls were found in the cellular distribution and staining intensity of all 3 antigens. The manifold localization sites of the observed antigens point to rather generalized functions of the RAS in human ocular tissues, such as regulatory effects on neuronal cells, vessels and vitreous humor homeostasis.

Red wine ingredient resveratrol protects from beta-amyloid neurotoxicity.

BACKGROUND: beta-Amyloid peptide (Abeta), a neutrotoxic substance, has been implicated to a great degree in cell death during the course of AD. Resveratrol, a natural polyphenol mainly found in red wine, has been shown to be cardioprotective and chemoprotective. Since a moderate wine intake correlates with a lower risk for Alzheimer disease (AD), an additional neuroprotective effect has been postulated for resveratrol. OBJECTIVE: The present study aimed at elucidating the possible neuroprotective effects of resveratrol against Abeta-induced neurotoxicity. METHODS: The neuroprotective capacity against Abeta-related oxidative stress was studied in a cell culture model suitable for studying such potentially neuroprotective substances. RESULTS: Resveratrol maintains cell viability and exerts an anti-oxidative action by enhancing the intracellular free-radical scavenger glutathione. CONCLUSION: Our findings suggest that red wine may be neuroprotective through the actions of resveratrol.

Increased melatonin 1a-receptor immunoreactivity in the hippocampus of Alzheimer's disease patients.

The pineal secretory product melatonin has, in addition to regulating retinal, circadian and vascular functions, neuroprotective effects. Blood melatonin levels are often decreased in Alzheimer's disease (AD), a progressively disabling neurodegenerative disorder. In this study we provide the first immunohistochemical evidence for the localization of melatonin 1a-receptor (MT(1)) in aged human hippocampus and a comparison of AD cases. MT(1) was localized to pyramidal neurons in the hippocampal cornu ammonis (CA)1-4 subfields. There was a distinct increase in staining intensity in all AD cases indicating an up-regulation of the receptor, possibly as a compensatory response to impaired melatonin levels in order to augment melatonin's neuroprotective effects.