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Male and female rat pups were injected with β-endorphin, naloxone or a saline control solution during days 2-7 postnatally. At 90 days of age the rats were tested for analgesia with the tail flick test. Testing was conducted during the first 2 hours of the light and the dark cycle. In both sexes and during both phases of the light cycle rats treated with β-endorphin as infants evidenced a significant elevation in threshold for painful thermal stimuli. Early treatment with naloxone also resulted in elevated threshold for thermal stimuli. Administration of naloxone to these rats as adults did not reverse the analgesic effect. It was concluded that early exposure to β-endorphin results in permanent changes in behavior perhaps by altering the interaction of endogenous opiates with their binding sites during a ciritcal period of opiate receptor development. © 1979.

beta-endorphin-like immunoreactivity was measured by radioimmunoassay in the brains of adult rats treated neonatally with beta-endorphin, naloxone, or vehicle. After treatment with beta-endorphin, the decreases observed in beta-endorphin-like immunoreactivity in the hypothalamus, pineal, midbrain, pons-medulla, hippocampus, striatum, frontal cortex, occipital cortex, and posterior cortex were highly significant but the 23% decrease in the thalamus was not significantly different from that of control rats. Neonatal administration of naloxone only resulted in a significant decrease in beta-endorphin-like immunoreactivity in the hypothalamus. In contrast, no differences were discernible in content of either beta-endorphin-like immunoreactivity or ACTH-like immunoreactivity in the pituitary of rats treated with beta-endorphin, naloxone, or vehicle in the neonatal period. These same rats had shown an increased threshold to painful thermal stimulation by the tail-flick test after administration of either beta-endorphin or naloxone at birth. The results suggest that neonatally injected beta-endorphin may alter the levels of beta-endorphin-like immunoreactivity in rat brain as well as the response to pain.

In a completely crossed, double blind designed study, six rats received intraventricular injections of 0.1, 1.0 and 10 micrograms of alpha-MSH and a placebo. The rats were tested for response to painful thermal stimuli with the tail-flick test. All of the doses of alpha-MSH produced hyperalgesia during the first 20 min of testing. Only the 1.0 microgram dose of alpha-MSH produced hyperalgesia throughout the 80 min course of the experiment. This study, coupled with previous reports that MSH/ACTH fragments may attenuate morphine-induced analgesia, suggest that MSH can have opposite actions from those of the endorphins. It is possible that alpha-MSH and related peptides may be endogenous anti-opiates.

Learning and attention were examined in rats after injections of one of several molecules related to adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH). The initial phase of the learning process was linearly related to the length of the peptide with the smallest fragment (MSH/ACTH 4-10) improving learning the most and the largest molecule (ACTH 1-24) exerting no effect. Later stages of the learning problem, which were sensitive to the attentional state of the organism, resulted in U-shaped relations with the length of the same peptides. Enhancement of attention was significant only for the MSH compounds. These data indicate that some behaviors may be influenced as a function of the redundant information contained in the molecule while other behaviors may be discretely related to the specific conformation of the molecule.

The possible role of astrocytes in the regulation of feeding has been overlooked. It is well-established that the endothelial cells constituting the blood-brain barrier transport leptin from blood to brain and that hypothalamic neurons respond to leptin to induce anorexic signaling. However, few studies have addressed the role of astrocytes in either leptin transport or cellular activation. We recently showed that the obese agouti viable yellow mouse has prominent astrocytic expression of the leptin receptor. In this study, we test the hypothesis that diet-induced obesity increases astrocytic leptin receptor expression and function in the hypothalamus. Double-labelling immunohistochemistry and confocal microscopic analysis showed that all astrocytes in the hypothalamus express leptin receptors. In adult obese mice, 2 months after being placed on a high-fat diet, there was a striking increase of leptin receptor (+) astrocytes, most prominent in the dorsomedial hypothalamus and arcuate nucleus. Agouti viable yellow mice with their adult-onset obesity showed similar changes, but the increase of leptin receptor (+) astrocytes was barely seen in ob/ob or db/db mice with their early-onset obesity and defective leptin systems. The marked leptin receptor protein expression in the astrocytes, shown with several antibodies against different receptor epitopes, was supported by RT-PCR detection of leptin receptor-a and -b mRNAs in primary hypothalamic astrocytes. Unexpectedly, the protein expression of GFAP, a marker of astrocytes, was also increased in adult-onset obesity. Real-time confocal imaging showed that leptin caused a robust increase of calcium signalling in primary astrocytes from the hypothalamus, confirming their functionality. The results indicate that metabolic changes in obese mice can rapidly alter leptin receptor expression and astrocytic activity, and that leptin receptor is responsible for leptin-induced calcium signalling in astrocytes. This novel and clinically relevant finding opens new avenues in astrocyte biology. [ABSTRACT FROM AUTHOR]

Our previous studies documented that interleukin-15 receptor α (IL-15Rα) knockout (KO) mice exhibited hyperactivity, memory impairment, and desperate behavior, which are core features of schizophrenia and depression. Due to the overlapping symptomology and pathogenesis observed for schizophrenia and depression, the present study attempted to determine whether IL-15Rα was associated with the risk of schizophrenia or depression. One hundred fifty-six participants, including 63 schizophrenia patients, 29 depressive patients, and 64 age-matched healthy controls, were enrolled in the study. We investigated the circulating levels of soluble IL-15Rα and analyzed potential links between the IL-15Rα levels and clinical symptoms present in schizophrenia or depressive patients. We observed reduced serum IL-15Rα levels in schizophrenia patients, but not depressive patients compared with controls. Moreover, a significant negative association was observed between the circulating IL-15Rα levels and excited phenotypes in the schizophrenia patients. The IL-15Rα KO mice displayed pronounced pre-pulse inhibition impairment, which was a typical symptom of schizophrenia. Interestingly, the IL-15Rα KO mice exhibited a remarkable elevation in the startle amplitude in the startle reflex test compared to wild type mice. These results demonstrated that serum levels of soluble IL-15Rα were reduced in schizophrenia and highlighted the relationship of IL-15Rα and the excited phenotype in schizophrenia patients and mice., Competing Interests: WP was employed by the company BioPotentials Consult. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 He, Bo, Mao, Yang, Liu, Wang, Kastin, Pan, Wang and Sun.)

Background: Previous studies of the functions of IL15R α have been limited to immune activities and skeletal muscle development. Immunological factors have been identified as one of the multiple causes of psychosis, and neurological symptoms have been described in IL15R α knockout (KO) mice. Seeking to explore possible mechanisms for this in the IL15R α -/- mouse brain, we analyzed gene expression patterns in the cortex and hippocampus using the RNA-seq technique., Methods: IL15R α KO mice were generated and littermate wildtype (WT) mice were used as a control group. A Y-maze was used to assess behavior differences between the two groups. The cortex and hippocampus of 3-month-old male mice were prepared and RNA-seq and transcriptome analysis were performed by gene set enrichment analysis (GSEA)., Results: Compared with the WT group, IL15R α KO animals showed higher speed in the novel arm and more entrance frequency in the old arm in the Y-maze experiment. GSEA indicated that 18 pathways were downregulated and 13 pathways upregulated in both cortex and hippocampus from the GO, KEGG, and Hallmark gene sets. The downregulated pathways formed three clusters: respiratory chain and electron transport, regulation of steroid process, and skeletal muscle development., Conclusion: IL15R α KO mice exhibit altered expression of multiple pathways, which could affect many functions of the brain. Lipid biosynthesis and metabolism in the central nervous system (CNS) should be investigated to provide insights into the effect of IL15R α on psychosis in this murine model., Competing Interests: WP was employed by the company BioPotentials Consult. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer XW declared a past co-authorship with several of the authors YH, ZS, JY, AK, and WP to the handling editor., (Copyright © 2021 He, Yu, Li, Duan, Sun, Yang, Kastin, Pan, Zhang and Wang.)

Brain aging and Alzheimer's disease both demonstrate the accumulation of beta-amyloid protein containing "plaques" and tau protein containing "tangles" that contribute to accelerated memory loss and cognitive decline. In the present investigation we identified a specific plant extract and its constituents as a potential alternative natural solution for preventing and reducing both brain "plaques and tangles". PTI-00703 cat's claw (Uncaria tomentosa from a specific Peruvian source), a specific and natural plant extract from the Amazon rain forest, was identified as a potent inhibitor and reducer of both beta-amyloid fibrils (the main component of "plaques") and tau protein paired helical filaments/fibrils (the main component of "tangles"). PTI-00703 cat's claw demonstrated both the ability to prevent formation/aggregation and disaggregate preformed Aβ fibrils (1-42 and 1-40) and tau protein tangles/filaments. The disaggregation/dissolution of Aβ fibrils occurred nearly instantly when PTI-00703 cat's claw and Aβ fibrils were mixed together as shown by a variety of methods including Thioflavin T fluorometry, Congo red staining, Thioflavin S fluorescence and electron microscopy. Sophisticated structural elucidation studies identified the major fractions and specific constituents within PTI-00703 cat's claw responsible for both the observed "plaque" and "tangle" inhibitory and reducing activity. Specific proanthocyanidins (i.e. epicatechin dimers and variants thereof) are newly identified polyphenolic components within Uncaria tomentosa that possess both "plaque and tangle" reducing and inhibitory activity. One major identified specific polyphenol within PTI-00703 cat's claw was epicatechin-4β-8-epicatechin (i.e. an epicatechin dimer known as proanthocyanidin B2) that markedly reduced brain plaque load and improved short-term memory in younger and older APP "plaque-producing" (TASD-41) transgenic mice (bearing London and Swedish mutations). Proanthocyanidin B2 was also a potent inhibitor of brain inflammation as shown by reduction in astrocytosis and gliosis in TASD-41 transgenic mice. Blood-brain-barrier studies in Sprague-Dawley rats and CD-1 mice indicated that the major components of PTI-00703 cat's claw crossed the blood-brain-barrier and entered the brain parenchyma within 2 minutes of being in the blood. The discovery of a natural plant extract from the Amazon rain forest plant (i.e. Uncaria tomentosa or cat's claw) as both a potent "plaque and tangle" inhibitor and disaggregator is postulated to represent a potential breakthrough for the natural treatment of both normal brain aging and Alzheimer's disease.

Background: Schizophrenia is a complex and debilitating mental disorder with strong heritability. Its pathogenesis involves immune dysregulation. Interleukin 15 and interleukin 15 receptor alpha(IL-15Rα) are classical immune molecules. They also help maintain normal brain function, leading to our hypothesis that IL-15Rα gene(IL- 15RA) variants contribute to the pathogenesis of schizophrenia., Objective: We determine whether the genetic variants of IL-15RA are associated with the development and progression of schizophrenia and whether IL-15RA single nucleotide polymorphism(SNP) plays a key role in downstream signaling transduction., Methods and Results: We sequenced IL-15RA exon from 132 Chinese schizophrenic patients and identified a rare variant(rs528238821) in a patient diagnosed with catatonic schizophrenia and ankylosing spondylitis(AS). We overexpressed this missense variant in cells driven by pBI-CMV vector. The cells showed attenuated STAT3 phosphorylation in response to interleukin15., Conclusion: IL-15RA mutation is rare in schizophrenic patients but interfered with IL- 15Rα intracellular signal transduction. Given the similarity of symptoms of catatonic schizophrenia and the known phenotype of IL-15Rα knockout mice, gene variation might offer diagnostic value for sub-types of schizophrenia., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Sleep and its disorders are known to affect the functions of essential organs and systems in the body. However, very little is known about how the blood-brain barrier (BBB) is regulated. A few years ago, we launched a project to determine the impact of sleep fragmentation and chronic sleep restriction on BBB functions, including permeability to fluorescent tracers, tight junction protein expression and distribution, glucose and other solute transporter activities, and mediation of cellular mechanisms. Recent publications and relevant literature allow us to summarize here the sleep-BBB interactions in five sections: (1) the structural basis enabling the BBB to serve as a huge regulatory interface; (2) BBB transport and permeation of substances participating in sleep-wake regulation; (3) the circadian rhythm of BBB function; (4) the effect of experimental sleep disruption maneuvers on BBB activities, including regional heterogeneity, possible threshold effect, and reversibility; and (5) implications of sleep disruption-induced BBB dysfunction in neurodegeneration and CNS autoimmune diseases. After reading the review, the general audience should be convinced that the BBB is an important mediating interface for sleep-wake regulation and a crucial relay station of mind-body crosstalk. The pharmaceutical industry should take into consideration that sleep disruption alters the pharmacokinetics of BBB permeation and CNS drug delivery, being attentive to the chrono timing and activation of co-transporters in subjects with sleep disorders.

Here we summarize three aspects of our understanding of the interactions of cytokines and neurotrophic peptides/proteins with the blood-brain and bloodspinal cord barriers (BBB): (a) pharmacokinetic analysis that has been reported for native cytokines and neurotrophic peptides/proteins; (b) landmark work on conjugated proteins to enhance their delivery across the normal BBB; and (c) regulatory changes under pathophysiological conditions in rodents, particularly after spinal cord injury (SCI). First, though the BBB restricts the permeation of large proteins, some cytokines and neurotrophic peptides/proteins in the periphery can reach the central nervous system (CNS) by specific transport systems. Moreover, SCI and some other disease processes may regulate these transport systems. The significance of studies of the transport systems is obvious because of the biological impact of these molecules on the CNS in health and disease. We have characterized the pharmacokinetic characteristics of some stable cytokines and neurotrophic peptides/proteins in mice after intravenous administration and also in the setting of in situ brain perfusion. In the particular case of SCI, there are time- and regionspecific changes of BBB permeability and transport systems. Tumor necrosis factor-α, a cytokine with dual actions in regeneration of the spinal cord, has a slow basal influx into the brain and spinal cord. After SCI, the increase in the entry of tumor necrosis factor-α to the CNS differs from leakage after BBB disruption and is related to upregulation of the transport system in a unique temporal and regional pattern. Overall, the permeation of cytokines across the BBB can be mediated by specific transport systems. The regulation of transport in pathophysiological conditions affects the extent of neuroinflammation and is implicated in neuroregeneration.

Autophagy is essential for normal cellular survival and activity. Circadian rhythms of autophagy have been studied in several peripheral organs but not yet reported in the brain. Here, we measured the circadian rhythm of autophagy-related proteins in mouse hippocampus and tested the effect of sleep fragmentation (SF). Expressions of the autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3) and beclin were determined by western blotting and immunohistochemistry. Both the hippocampal LC3 signal and the ratio of its lipid-conjugated form LC3-II to its cytosolic form LC3-I showed a 24 h rhythm. The peak was seen at ZT6 (1 pm) and the nadir at ZT16 (1 am). The LC3 immunoreactivity in hippocampal CA1 pyramidal neurons also distributed differently, with more diffuse cytoplasmic appearance at ZT16. Chronic SF had a mild effect to disrupt the 24 h rhythm of LC3 and beclin expression. Interestingly, a greater effect of SF was seen after 24 h of recovery sleep when LC3-II expression was attenuated at both the peak and trough of circadian activities. Overall, the results show for the first time that the hippocampus has a distinct rhythm of autophagy that can be altered by SF.

Pertinent to pandemic obesity, the discovery of endogenous peptides that affect the ingestion of food has led to the question of how these ingestive peptides exert their actions in the brain. Whereas peripheral sources provide a ready reserve, the availability of ingestive peptides to their central nervous system targets can be regulated by the blood-brain barrier (BBB). Some of the peptides/polypeptides are transported by saturable mechanisms from blood to brain. Examples include leptin, insulin, mahogany, and pancreatic polypeptide. Some enter the brain by passive diffusion, such as neuropeptide Y, orexin A, cocaine- and amphetamine-regulated transcript, cyclo His-Pro, and amylin. Some others may have essentially no penetration of the BBB; this class includes agouti-related protein, melanin-concentrating hormone, and urocortin. The regulatory function of the BBB can be seen in various physiological states. Hyperglycemia may upregulate transport systems for leptin, urocortin, and galanin-like peptide, whereas fasting can down-regulate those for leptin and galanin-like peptide. Thus, the BBB plays a dynamic role in modulating the passage of ingestive peptides from blood to brain.

To test the hypothesis that astrocytic leptin signaling induces an overall potentiation of the neuronal response to leptin, we generated a new line of astrocyte-specific leptin receptor knockout (ALKO-Δ1) mice in which no leptin receptor is expressed in astrocytes. Corresponding to cell-specific Cre recombinase expression in hypothalamic astrocytes but not neurons, this new strain of ALKO mice had attenuated pSTAT3 signaling in the arcuate nucleus of the hypothalamus 30 min after intracerebroventricular delivery of leptin. In response to high-fat diet for 2 months, the ALKO mice showed a greater increase of percent fat and blood leptin concentration. This coincided with a mild reactive gliosis in the hypothalamus. Overall, the absence of leptin receptors in astrocytes attenuated hypothalamic pSTAT3 signaling, induced a mild reactive morphology, and promoted the development of diet-induced obesity. We conclude that leptin signaling in astrocytes is essential for the homeostasis of neuroendocrine regulation in obesity.

Chronic sleep fragmentation (SF), common in patients with sleep apnea, correlates with the development of obesity. We hypothesized that SF differentially affects neurobehavior in lean wild-type (WT) and obese pan-leptin receptor knockout (POKO) mice fed the same normal diet. First, we established an SF paradigm by interrupting sleep every 2 min during the inactive light span. The maneuver was effective in decreasing sleep duration and bout length, and in increasing sleep state transition and waking, without significant rebound sleep in the dark span. Changes of sleep architecture were evident in the light span and consistent across days 1-10 of SF. There was reduced NREM, shortened sleep latency, and increased state transitions. During the light span of the first day of SF, there also was reduction of REM and increased delta power of slow-wave sleep. Potential effects of SF on thermal pain threshold, locomotor activity, and anxiety were then tested. POKO mice had a lower circadian amplitude of pain latency than WT mice in the hot plate test, and both groups had lowest tolerance at 4 pm (zeitgeber time (ZT) 10) and longest latency at 4 am (ZT 22). SF increased the pain threshold in WT but not in POKO mice when tested at 8 a.m. (ZT 2). Both the POKO mutation and SF resulted in reduced physical activity and increased anxiety, but there was no additive effect of these two factors. Overall, SF and the POKO mutation differentially regulate mouse behavior. The results suggest that obesity can blunt neurobehavioral responses to SF.

Background: We have recently shown that mice with experimental autoimmune encephalomyelitis (EAE) have increased sleep fragmentation (SF) and reduced sleep efficiency, and that the extent of SF correlates with the severity of disease. It is not yet clear whether and how sleep promotes recovery from autoimmune attacks. We hypothesized that SF promotes leukocyte infiltration across the blood-spinal cord barrier, impairs immune regulation, and thus worsens EAE., Methods: Three groups of C57 mice were studied: Resting EAE; SF EAE with the mice subjected to the SF maneuver 12 h /day during zeitgeber time (ZT) 0-12 h; and naïve controls with neither EAE nor SF. Besides monitoring of the incidence and severity of EAE, the immune profiles of leukocytes in the spinal cord as well as those in the spleen were determined., Results: When analyzed 16 days after EAE induction, at which time the SF was terminated, the SF group had a greater number of CD4(+) T cells and a higher percent of CD4(+) cells among all leukocytes in the spinal cord than the resting EAE group. When allowed to recover to 28 days after EAE induction, the SF mice had lower EAE scores than the resting EAE group. EAE induced splenomegaly and an increase of Gr1(+)CD11b(+) myeloid-derived suppressor cells in the splenocytes. However, SF treatment had no additional effect on either peripheral splenocytes or granulocytes that reached the spinal cord., Conclusion: The SF maneuver facilitated the migration of encephalopathic lymphocytes into the spinal cord. Paradoxically, these mice had a better EAE score after cessation of SF compared with mice without SF.

Both obstructive sleep apnea (OSA) and Alzheimer's disease (AD) are increasing health concerns. The objective of this study is to review systematically the effects of OSA on the development of AD. The search was conducted in PubMed and Cochrane CENTRAL, and followed by a manual search of references of published studies. Cross-sectional, cohorts, and randomized clinical trials were reviewed. Besides clinical studies, we also discuss neuroimaging data, experimental animal evidence, and molecular mechanisms. Although a causal relationship between OSA and AD is not yet established, OSA induces neurodegenerative changes as a result of two major contributing processes: sleep fragmentation and intermittent hypoxia. As such, inflammation and cellular stress are sufficient to impair cell-cell interactions, synaptic function, and neural circuitry, leading to a decline of cognitive behavior. Sustained OSA could promote cognitive dysfunction, overlapping with that in AD and other neurodegenerative diseases. Early treatment by positive airway pressure and other current standards of care should have a positive impact to alleviate structural and functional deterioration. With better understanding of the cellular and neurophysiological mechanisms by which OSA contributes to AD, we may identify novel molecular targets for intervention.

The blood-brain barrier (BBB) is a large regulatory and exchange interface between the brain and peripheral circulation. We propose that changes of the BBB contribute to many pathophysiological processes in the brain of subjects with chronic sleep restriction (CSR). To achieve CSR that mimics a common pattern of human sleep loss, we quantified a new procedure of sleep disruption in mice by a week of consecutive sleep recording. We then tested the hypothesis that CSR compromises microvascular function. CSR not only diminished endothelial and inducible nitric oxide synthase, endothelin1, and glucose transporter expression in cerebral microvessels of the BBB, but it also decreased 2-deoxy-glucose uptake by the brain. The expression of several tight junction proteins also was decreased, whereas the level of cyclooxygenase-2 increased. This coincided with an increase of paracellular permeability of the BBB to the small tracers sodium fluorescein and biotin. CSR for 6 d was sufficient to impair BBB structure and function, although the increase of paracellular permeability returned to baseline after 24 h of recovery sleep. This merits attention not only in neuroscience research but also in public health policy and clinical practice., (Copyright © 2014 the authors 0270-6474/14/3414697-10$15.00/0.)

Leptin, a pleiotropic adipokine, crosses the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) from the periphery and facilitates experimental autoimmune encephalomyelitis (EAE). EAE induces dynamic changes of leptin receptors in enriched brain and spinal cord microvessels, leading to further questions about the potential roles of endothelial leptin signaling in EAE progression. In endothelial leptin receptor specific knockout (ELKO) mice, there were lower EAE behavioral scores in the early phase of the disorder, better preserved BSCB function shown by reduced uptake of sodium fluorescein and leukocyte infiltration into the spinal cord. Flow cytometry showed that the ELKO mutation decreased the number of CD3 and CD45 cells in the spinal cord, although immune cell profiles in peripheral organs were unchanged. Not only were CD4(+) and CD8(+) T lymphocytes reduced, there were also lower numbers of CD11b(+)Gr1(+) granulocytes in the spinal cord of ELKO mice. In enriched microvessels from the spinal cord of the ELKO mice, the decreased expression of mRNAs for a few tight junction proteins was less pronounced in ELKO than WT mice, as was the elevation of mRNA for CCL5, CXCL9, IFN-γ, and TNF-α. Altogether, ELKO mice show reduced inflammation at the level of the BSCB, less leukocyte infiltration, and better preserved tight junction protein expression and BBB function than WT mice after EAE. Although leptin concentrations were high in ELKO mice and microvascular leptin receptors show an initial elevation before inhibition during the course of EAE, removal of leptin signaling helped to reduce disease burden. We conclude that endothelial leptin signaling exacerbates BBB dysfunction to worsen EAE., (Copyright © 2014. Published by Elsevier Inc.)

Leptin, a pleiotropic protein hormone produced mainly by fat cells, regulates metabolic activity and many other physiological functions. The intrinsic circadian rhythm of blood leptin is modulated by gender, development, feeding, fasting, sleep, obesity, and endocrine disorders. Hyperleptinemia is implicated in leptin resistance. To determine the specificity and sensitivity of leptin concentrations in sleep disorders, we summarize here the alterations of leptin in four conditions in animal and human studies: short duration of sleep, sleep fragmentation, obstructive sleep apnea (OSA), and after use of continuous positive airway pressure (CPAP) to treat OSA. The presence and causes of contradictory findings are discussed. Though sustained insufficient sleep lowers fasting blood leptin and therefore probably contributes to increased appetite, obesity and OSA independently result in hyperleptinemia. Successful treatment of OSA by CPAP is predicted to decrease hyperleptinemia, making leptin an ancillary biomarker for treatment efficacy. Current controversies also call for translational studies to determine how sleep disorders regulate leptin homeostasis and how the information can be used to improve sleep treatment., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Sleep disturbance in patients with multiple sclerosis is prevalent and has multifactorial causes. In mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, we determined the dynamic changes of sleep architecture and the interactions between sleep changes and EAE symptoms. The changes of sleep patterns were mainly reflected by altered sleep stage distribution and increased sleep fragmentation. Increased waking and decreased non-rapid eye movement sleep occurred after EAE onset and persisted through the symptomatic phase. There also was increased sleep state transition, indicating a reduction of sleep cohesiveness. Furthermore, the extent of sleep fragmentation correlated with the severity of disease. This is the first study of sleep characteristics in EAE mice demarcating specific changes related to the autoimmune disorder without confounding factors such as psychosocial impact and treatment effects. The reduction of sleep efficiency and cohesiveness supports the notion that enhancing sleep might facilitate the recovery of mice from EAE, pertinent to the multimodality treatment of multiple sclerosis., (Copyright © 2014. Published by Elsevier Inc.)

The blood-brain barrier (BBB) is a regulatory interface between the central nervous system and the rest of the body. However, BBB changes in obesity and metabolic syndrome have not been fully elucidated. We hypothesized that obesity reduces energy metabolism in the cerebral microvessels composing the BBB, reflected by downregulation of protein expression and function. We performed comparative proteomic analyses in enriched microvessels from the cerebral cortex of mice 2 months after ingestion of a high-fat diet or regular rodent chow. In mice with diet-induced obesity (DIO), there was downregulation of 47 proteins in the cerebral microvessels, including cytoskeletal proteins, chaperons, enzymes, transport-related proteins, and regulators for transcriptional and translational activities. Only two proteins, involved in messenger RNA (mRNA) transport and processing, were upregulated. The changes of these proteins were further validated by quantitative polymerase chain reaction (qPCR), western blotting, and immunofluorescent staining of freshly isolated microvessels, in samples obtained from different batches of mice. The predominant downregulation suggests that DIO suppresses metabolic activity of BBB microvessels. The finding of a hypometabolic state of the BBB in mice at the chronic stage of DIO is unexpected and unprecedented; it may provide novel mechanistic insight into how obesity influences CNS function via regulatory changes of the BBB.

Excessive obesity correlates with hypersomnolence and impaired cognitive function, presumably induced by metabolic factors and cytokines. Production of the adipokine leptin correlates with the amount of adiposity, and leptin has been shown to promote sleep. To determine whether leptin plays a major role in the hypersomnolence of obesity, we measured sleep architecture in pan-leptin receptor knockout (POKO) mice that do not respond to leptin because of the production of a mutant, non-signaling receptor. The obese POKO mice had more non-rapid eye movement (NREM) sleep and less waking time than their littermate controls. This was mainly seen during the light span, although increased bouts of rapid eye movement sleep were also seen in the dark span. The increase of NREM sleep correlated with the extent of obesity. The POKO mice also had decreased locomotor activity and more immobility in the open field test, but there was no increase of forced immobility nor reduction of sucrose intake as would be seen in depression. The increased NREM sleep and reduced locomotor activity in the POKO mice suggest that it was obesity, rather than leptin signaling, that played a predominant role in altering sleep architecture and activity.

Background: Fibroblast growth factor (FGF)-19, an endocrine FGF protein mainly produced by the ileum, stimulates metabolic activity and alleviates obesity. FGF19 modulates metabolism after either intravenous or intracerebroventricular injection, and its receptor FGFR4 is present in the hypothalamus. This led to the question whether blood-borne FGF19 crosses the blood-brain barrier (BBB) to exert its metabolic effects., Methods: We determined the pharmacokinetics of FGF19 permeation from blood to brain in comparison with its distribution in peripheral organs. Multiple-time regression analysis after intravenous bolus injection, in-situ brain perfusion, and HPLC assays were performed., Results: FGF19 was relatively stable in blood and in the brain compartment. Significant influx was seen in the presence of excess unlabeled FGF19 in blood. This coincided with a slower decline of 125I-FGF19 in blood which suggested there was decreased clearance or peripheral tissue uptake. In support of an altered pattern of peripheral processing of 125I-FGF19 by excess unlabeled FGF19, the high influx to liver was significantly attenuated, whereas the minimal renal uptake was linearly accelerated. In the present setting, we did not detect a saturable transport of FGF19 across the BBB, as the entry rate of 125I-FGF19 was not altered by excess unlabeled FGF19 or its mouse homologue FGF15 during in-situ brain perfusion., Conclusion: FGF19 remained stable in the blood and brain compartments for up to 10 min. Its influx to the brain was non-linear, non-saturable, and affected by its blood concentration and distribution in peripheral organs. Liver showed a robust and specific uptake of FGF19 that could be inhibited by the presence of excess unlabeled FGF19, whereas kidney clearance was dose-dependent.

Leptin is commonly thought to play a detrimental role in exacerbating experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis. Paradoxically, we show here that astrocytic leptin signaling has beneficial effects in reducing disease severity. In the astrocyte specific leptin receptor knockout (ALKO) mouse in which leptin signaling is absent in astrocytes, there were higher EAE scores (more locomotor deficits) than in the wildtype counterparts. The difference mainly occurred at a late stage of EAE when wildtype mice showed signs of recovery whereas ALKO mice continued to deteriorate. The more severe symptoms in ALKO mice coincided with more infiltrating cells in the spinal cord and perivascular brain parenchyma, more demyelination, more infiltrating CD4 cells, and a lower percent of neutrophils in the spinal cord 28 days after EAE induction. Cultured astrocytes from wildtype mice showed increased adenosine release in response to interleukin-6 and the hippocampus of wildtype mice had increased adenosine production 28 days after EAE induction, but the ALKO mutation abolished the increase in both conditions. This indicates a role of astrocytic leptin in normal gliotransmitter release and astrocyte functions. The worsening of EAE in the ALKO mice in the late stage suggests that astrocytic leptin signaling helps to clear infiltrating leukocytes and reduce autoimmune destruction of the CNS., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Leptin, a hormone mainly produced by fat cells, shows cell-specific effects to regulate feeding and metabolic activities. We propose that an important feature of metabolic dysregulation resulting in obesity is the loss of the circadian rhythm of biopotentials. This was tested in the pan-leptin receptor knockout (POKO) mice newly generated in our laboratory. In the POKO mice, leptin no longer induced pSTAT-3 signaling after intracerebroventricular injection. Three basic phenotypes were observed: the heterozygotes had similar weight and adiposity as the wild-type (WT) mice (>60% of the mice); the homozygotes were either fatter (∼30%), or rarely leaner (<5%) than the WT mice. By early adulthood, the POKO mice had higher average body weight and adiposity than their respective same-sex WT littermate controls, and this was consistent among different batches. The homozygote fat POKO showed significant reduction of midline estimating statistic of rhythm of circadian parameters, and shifts of ultradian rhythms. The blunted circadian rhythm of these extremely obese POKO mice was also seen in their physical inactivity, longer feeding bouts, and higher food intake. The extent of obesity correlated with the blunted circadian amplitude, accumulative metabolic and locomotor activities, and the severity of hyperphagia. This contrasts with the heterozygote POKO mice which showed little obesity and metabolic disturbance, and only subtle changes of the circadian rhythm of metabolic activity without alterations in feeding behavior. The results provide a novel aspect of leptin resistance, almost manifesting as an "all or none" phenomenon.

We have shown that mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, have upregulated leptin receptor expression in reactive astrocytes of the hippocampus, a region involved in sickness behavior. Leptin can exacerbate EAE when its serum concentration is high. Although leptin receptors in astrocytes modulate leptin transport across cultured endothelial cell monolayers, it is not known how leptin transport in EAE mice is regulated. Here, we determined brain and cervical spinal cord uptake of leptin in early and recovery stages of EAE, after either intravenous delivery or in situ brain perfusion of (125)I-leptin and the vascular marker (131)I-albumin. While increased vascular space and general blood-brain barrier (BBB) permeability after EAE were expected, the specific saturable transport system for leptin crossing the BBB also persisted. Moreover, there was upregulation of leptin transport in hippocampus and cervical spinal cord in the early stage of EAE, shown by higher leptin uptake in these regions and by competitive inhibition with coadministered excess unlabeled leptin. We conclude that EAE induced a time- and region-specific increase of leptin transport. The results provide a link between circulating leptin and enhanced leptin signaling that may play a crucial role in disease progression.

Interleukin (IL)-15 is a ubiquitously expressed cytokine that in the basal state is mainly localized intracellularly, including the nucleus. Unexpectedly, tumor necrosis factor-α (TNF) time-dependently induced nuclear export of IL-15Rα and IL15. This process was inhibited by leptomycine B (LMB), a specific inhibitor of nuclear export receptor chromosomal region maintenance 1 (CRM1). In the presence of TNF, LMB co-treatment led to accumulation of both IL-15Rα and IL-15 in the nucleus of HeLa cells, suggesting that CRM1 facilitates nuclear export and that TNF enhances CRM1 activity. Once in the cytoplasm, IL-15 showed partial co-localization with late endosomes but very little with other organelles tested 4 h after TNF treatment. IL-15Rα showed co-localization with both early and late endosomes, and to a lesser extent with endoplasmic reticulum and Golgi. This indicates different kinetics and possibly different trafficking routes of IL-15 from its specific receptor. The TNF-induced secretion of IL-15 was attenuated by pretreatment of cells by brefeldin A that inhibits ER-to-Golgi transport, or by use of domain negative ADP-ribosylation factor 6 (ARF6) that interferes with exocytotic sorting. We conclude that TNF abolishes nuclear localization of IL-15 and IL-15Rα by acting on CRM1, and it facilitates exocytosis of IL-15 with the involvement of ARF6.

Hyperleptinemia is usually associated with obesity and leptin resistance. Endothelial cell leptin receptor knockout (ELKO) mice without a signaling membrane-bound leptin receptor in endothelia, however, have profound hyperleptinemia without signs of leptin resistance. Leptin mRNA in adipose tissue was unchanged. To test the hypothesis that the ELKO mutation results in delayed degradation and slowed excretion, we determined the kinetics of leptin transfer in groups of ELKO and wildtype mice after intravenous bolus injection of (125) I-leptin and the reference substance (131) I-albumin. The degradation pattern of (125) I-leptin in serum and brain homogenates at different time points between 10 and 60 min was measured by HPLC and acid precipitation. Although ELKO mice had reduced uptake of (125) I-leptin uptake by the brain and several peripheral organs, leptin was more stable in blood and tissue. There was no change in the rate of renal excretion. ELISA showed that serum soluble leptin receptor, known to antagonize leptin transport, had a 400-fold increase, probably contributing to the hyperleptinemia and reduced tissue uptake. Thus, the ELKO mutation unexpectedly increased the stability of leptin but suppressed its tissue uptake. These changes probably contribute to the known partial resistance of the ELKO mice to diet-induced obesity., (Copyright © 2013 Wiley Periodicals, Inc.)

To determine how astrocytic leptin signaling regulates the physiological response of mice to diet-induced obesity (DIO), we performed metabolic analyses and hypothalamic leptin signaling assays on astrocytic leptin-receptor knockout (ALKO) mice in which astrocytes lack functional leptin receptor (ObR) signaling. ALKO mice and wild-type (WT) littermate controls were studied at different stages of DIO with measurement of body wt, percent fat, metabolic activity, and biochemical parameters. When fed regular chow, the ALKO mice had similar body wt, percent fat, food intake, heat dissipation, respiratory exchange ratio, and activity as their WT littermates. There was no change in blood concentrations of triglyceride, soluble leptin receptor (sObR), mRNA for leptin and uncoupling protein 1 (UCP1) in adipose tissue, and insulin sensitivity. Unexpectedly, in response to a high-fat diet the ALKO mice had attenuated hyperleptinemia and sObR, a lower level of leptin mRNA in subcutaneous fat, and a paradoxical increase in UCP1 mRNA. Thus, ALKO mice did not show the worsening of obesity that occurs with normal WT mice and the neuronal ObR mutation that results in morbid obesity. The findings are consistent with a competing, counterregulatory model between neuronal and astrocytic leptin signaling.

Both proconvulsive and anticonvulsive roles of leptin have been reported, suggesting cell-specific actions of leptin in different models of seizure and epilepsy. The goal of our study was to determine the regulation and function of astrocytic leptin receptors in a mouse model of epilepsy and glutamate-induced cytotoxicity. We show that in pilocarpine-challenged mice developing epilepsy with recurrent seizures after a latent period of 2 weeks, hippocampal leptin receptor (ObR) immunofluorescence was increased at 6 weeks. This was more pronounced in astrocytes than in neurons. In cultured astrocytes, glutamate increased ObRa and ObRb expression, whereas leptin pretreatment attenuated glial cytotoxicity by excess glutamate, reflected by better preserved adenosine triphosphate production. The protective role of astrocytic leptin signaling is further supported by the higher lethality of the astrocyte-specific leptin receptor knockout mice in the initial phase of seizure production. Thus, leptin signaling in astrocytes plays a protective role against seizure, and the effects are at least partially mediated by attenuation of glutamate toxicity. Astrocytic leptin signaling, therefore, may be a novel therapeutic target.

The detrimental role of leptin in experimental autoimmune encephalomyelitis (EAE) is opposite to its neuroprotective role in other neuropathologies. We hypothesize that a shifted cellular distribution of leptin receptors underlies the differential effects of leptin. A robust increase of ObR immunoreactivity was seen along glial fibrillary acidic protein (GFAP)(+) intermediate filaments in reactive astrocytes in the hippocampus and hypothalamus of mice with EAE. Although astrocyte-specific GFAP mRNA and protein were both increased, ObRa mRNA was elevated only after resolution of EAE symptoms, and ObRb mRNA was even decreased at the peak time of symptoms of EAE. A cell type-specific action of leptin may underlie its differential effects.

Interleukin (IL)-15 is a ubiquitously expressed cytokine existing in both intracellular and secretory forms. Here we review the expression, regulation, and functions of IL15 and its receptors in the brain. IL15 receptors show robust upregulation after neuroinflammation, suggesting a major role of IL15 signaling in cerebral function. Involvement of the IL15 system in neuropsychiatric behavior is reflected by the effects of IL15, IL15Rα, and IL2Rγ deletions on neurobehavior and neurotransmitters, the effects of IL15 treatment on neuronal activity, and the potential role of IL15 in neuroplasticity/neurogenesis. The results show that IL15 modulates GABA and serotonin transmission. This may underlie deficits in mood (depressive-like behavior and decreased normal anxiety) and memory, as well as activity level, sleep, and thermoregulation. Although IL15 has only a low level of permeation across the blood-brain barrier, peripheral IL15 is able to activate multiple signaling pathways in neurons widely distributed in CNS regions. The effects of IL15 in "preventing" neuropsychiatric symptoms in normal mice implicate a potential therapeutic role of this polypeptide cytokine., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Leptin, an adipocyte-derived cytokine, crosses the blood-brain barrier to act on many regions of the central nervous system (CNS). It participates in the regulation of energy balance, inflammatory processes, immune regulation, synaptic formation, memory condensation, and neurotrophic activities. This review focuses on the newly identified actions of leptin on astrocytes. We first summarize the distribution of leptin receptors in the brain, with a focus on the hypothalamus, where the leptin receptor is known to mediate essential feeding suppression activities, and on the hippocampus, where leptin facilitates memory, reduces neurodegeneration, and plays a dual role in seizures. We will then discuss regulation of the nonneuronal leptin system in obesity. Its relationship with neuronal leptin signaling is illustrated by in vitro assays in primary astrocyte culture and by in vivo studies on mice after pretreatment with a glial metabolic inhibitor or after cell-specific deletion of intracellular signaling leptin receptors. Overall, the glial leptin system shows robust regulation and plays an essential role in obesity. Strategies to manipulate this nonneuronal leptin signaling may have major clinical impact., (© 2012 New York Academy of Sciences.)

Reactive gliosis, a sign of neuroinflammation, has been observed in mice with adult-onset obesity as well as CNS injury. The hypothesis that obesity-derived metabolic factors exacerbate reactive gliosis in response to mechanical injury was tested here on cultured primary glial cells subjected to a well-established model of scratch wound injury. Cells treated with serum from mice with diet-induced obesity (DIO) showed higher immunoreactivity of CD11b (marker for microglia) and GFAP (marker for astrocytes), with morphological changes at both the injury border and areas away from the injury. The effect of DIO serum was greater than that of scratch injury alone. Leptin was almost as effective as DIO serum in inducing microgliosis and astrogliosis in a dose-response manner. By contrast, C-reactive protein (CRP) mainly induced microgliosis in noninjured cells; injury-induced factors appeared to attenuate this effect. The effect of CRP also differed from the effect of the antibiotic minocycline. Minocycline attenuated the microgliosis and to a lesser extent astrogliosis, particularly in CRP-treated cells, thus serving as a negative control. We conclude that blood-borne proinflammatory metabolic factors in obesity increase reactive gliosis and probably exacerbate CNS injury.

Leptin, a polypeptide hormone produced mainly by adipocytes, has diverse effects in both the brain and peripheral organs, including suppression of feeding. Other than mediating leptin transport across the blood-brain barrier, the role of the endothelial leptin receptor remains unclear. We recently generated a mutant mouse strain lacking endothelial leptin receptor signaling, and showed that there is an increased uptake of leptin by brain parenchyma after its delivery by in situ brain perfusion. Here, we tested the hypothesis that endothelial leptin receptor mutation confers partial resistance to diet-induced obesity. These ELKO mice had similar body weight and percent fat as their wild-type littermates when fed with rodent chow, but blood concentrations of leptin were significantly elevated. In response to a high-fat diet, wild-type mice had a greater gain of body weight and fat than ELKO mice. As shown by metabolic chamber measurement, the ELKO mice had higher oxygen consumption, carbon dioxide production, and heat dissipation, although food intake was similar to that of the wild-type mice and locomotor activity was even reduced. This indicates that the partial resistance to diet-induced obesity was mediated by higher metabolic activity in the ELKO mice. Since neuronal leptin receptor knockout mice show obesity and diabetes, the results suggest that endothelial leptin signaling shows opposite effects from that of neuronal leptin signaling, with a facilitatory role in diet-induced obesity.

Background/aims: Acute phase C-reactive protein (CRP), elevated in obesity and inflammation, is a major binding protein for leptin. It is thought that CRP contributes to leptin resistance by preventing leptin from crossing the blood-brain barrier (BBB). Here we determined how CRP interacts with the BBB and whether it deters leptin from reaching CNS targets., Methods: BBB permeability, compartmental distribution, tracer stability, and expression of tight junction protein and inflammatory marker were determined., Results: CRP was stable in blood, but did not permeate the BBB in trace amounts. However, it increased paracellular permeability at a higher dose. Agouti viable (A(vy)) mice with adult-onset obesity show higher CRP entry into the brain. CRP did not permeate hCMEC/D3 cells nor change zona occludin-1 or cyclooxygenase-2 expression. An intermediate dose of CRP had no effect on leptin transport across the BBB after co-treatment. Thus, acute interactions between CRP and leptin at the BBB level were negligible and did not explain the leptin resistance seen in obesity., Conclusions: The interactions of CRP and the BBB are a two-phase process, with increased paracellular permeability at a high dose that enables its entry into the CNS and serves to induce reactive gliosis and impair CNS function., (Copyright © 2012 S. Karger AG, Basel.)

The blood-brain barrier (BBB) provides a vast interface for cytokines to affect CNS function. The BBB is a target for therapeutic intervention. It is essential, therefore, to understand how cytokines interact with each other at the level of the BBB and how secondary signals modulate CNS functions beyond the BBB. The interactions between cytokines and lipids, however, have not been fully addressed at the level of the BBB. Here, we summarize current understanding of the localization of cytokine receptors and transporters in specific membrane microdomains, particularly lipid rafts, on the luminal (apical) surface of the microvascular endothelial cells composing the BBB. We then illustrate the clinical context of cytokine effects on the BBB by neuroendocrine regulation and amplification of inflammatory signals. Two unusual aspects discussed are signaling crosstalk by different classes of cytokines and genetic regulation of drug efflux transporters. We also introduce a novel area of focus on how cytokines may act through nuclear hormone receptors to modulate efflux transporters and other targets. A specific example discussed is the ATP-binding cassette transporter-1 (ABCA-1) that regulates lipid metabolism. Overall, cytokine signaling at the level of the BBB is a crucial feature of the dynamic regulation that can rapidly change BBB function and affect brain health and disease.

The functions of leptin receptors (LRs) are cell-type specific. At the blood-brain barrier, LRs mediate leptin transport that is essential for its CNS actions, and both endothelial and astrocytic LRs may be involved. To test this, we generated endothelia specific LR knockout (ELKO) and astrocyte specific LR knockout (ALKO) mice. ELKO mice were derived from a cross of Tie2-cre recombinase mice with LR-floxed mice, whereas ALKO mice were generated by a cross of GFAP-cre with LR-floxed mice, yielding mutant transmembrane LRs without signaling functions in endothelial cells and astrocytes, respectively. The ELKO mutation did not affect leptin half-life in blood or apparent influx rate to the brain and spinal cord, though there was an increase of brain parenchymal uptake of leptin after in situ brain perfusion. Similarly, the ALKO mutation did not affect blood-brain barrier permeation of leptin or its degradation in blood and brain. The results support our observation from cellular studies that membrane-bound truncated LRs are fully efficient in transporting leptin, and that basal levels of astrocytic LRs do not affect leptin transport across the endothelial monolayer. Nonetheless, the absence of leptin signaling at the BBB appears to enhance the availability of leptin to CNS parenchyma. The ELKO and ALKO mice provide new models to determine the dynamic regulation of leptin transport in metabolic and inflammatory disorders where cellular distribution of LRs is shifted., (Copyright © 2011 Elsevier Inc. All rights reserved.)