Search

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.

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.

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.

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.

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.

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.

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 125I-leptin and the reference substance 131I-albumin. The degradation pattern of 125I-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 125I-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. J. Cell. Physiol. 228: 1610–1616, 2013. © 2013 Wiley Periodicals, Inc.

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.

Growth hormone (GH) therapy is associated with improved neurobehavior, leading to the question whether GH crosses the blood-brain barrier (BBB) from blood after its release there from the pituitary, or whether GH exerts CNS effects by secondary mediators, particularly insulin-like growth factor (IGF)-1. GH release from the pituitary is controlled not only by hypothalamic factors, but also by ghrelin that is mainly produced in the stomach and uses its unique structure to interact with the BBB. This review summarizes studies of the permeation of GH, IGF-1, and ghrelin across the BBB, and discusses them in the context of neuroendocrine regulation. Exogenous GH has a half-life of approximately 3.8-7.6 min in mouse blood and shows a slow non-saturable permeation across the BBB, with 26.8 % remaining intact in the brain 20 min after intravenous delivery. IGF-1 crosses the BBB by saturable transport mediated by its receptors, and its interactions with the BBB are greatly affected by serum binding proteins. The interactions of ghrelin with the BBB appear to be dependent on species and show directionality. The BBB provides regulatory compartmentalization to fine-tune the CNS actions of GH and its related hormones.

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.

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.

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.

Interleukin (IL)-15 and its receptors are induced by tumor necrosis factor α (TNF) in the cerebral endothelial cells composing the blood-brain barrier, but it is not yet clear how IL-15 modulates endothelial function. Contrary to the known induction of JAK/STAT3 signaling, here we found that nuclear factor (NF)- κB is mainly responsible for IL-15 actions on primary brain microvessel endothelial cells and cerebral endothelial cell lines. IL-15-induced transactivation of an NFκB luciferase reporter resulted in phosphorylation and degradation of the inhibitory subunit IκB that was followed by phosphorylation and nuclear translocation of the p65 subunit of NFκB. An IκB kinase inhibitor Bay 11-7082 only partially inhibited IL-15-induced NFκB luciferase activity. The effect of IL-15 was mediated by its specific receptor IL-15Rα, since endothelia from IL-15Rα knockout mice showed delayed nuclear translocation of p65, whereas those from knockout mice lacking a co-receptor IL-2Rγ did not show such changes. At the mRNA level, IL-15 and TNF showed similar effects in decreasing the tight junction protein claudin-2 and increasing the p65 subunit of NFκB but exerted different regulation on caveolin-1 and vimentin. Taken together, NFκB is a major signal transducer by which IL-15 affects cellular permeability, endocytosis, and intracellular trafficking at the level of the blood-brain barrier.

Interleukin (IL)-2Rgamma shows robust upregulation in neuroinflammatory states associated with clinical depression. We tested the hypothesis that mice lacking IL2Rgamma would have decreased depressive-like behavior. Contrary to this expectation, these knockout mice showed increased immobility in both the Porsolt forced swimming and Nomura water wheel tests. By comparison, the auditory fear conditioning test showed increased retention of contextual freezing. Thus, intact IL2Rgamma combats depressive-like behavior.

We tested the hypothesis that astrocytic activity modulates neuronal uptake and signaling of leptin in the adult-onset obese agouti viable yellow (A vy) mouse. In the immunohistochemical study, A vy mice were pretreated with the astrocyte metabolic inhibitor fluorocitrate or phosphate-buffered saline (PBS) vehicle intracerebroventricularly (icv) followed 1 h later by Alexa568-leptin. Confocal microscopy showed that fluorocitrate pretreatment reduced astrocytic uptake of Alexa568-leptin 30 min after icv while increasing neuronal uptake in the arcuate nucleus and dorsomedial hypothalamus. Fluorocitrate also induced mild astrogliosis and moderately increased pSTAT3 immunopositive neurons in response to Alexa568-leptin in the dorsomedial hypothalamus. In the Western blotting study, A vy mice were pretreated with either PBS or fluorocitrate, and received PBS or leptin 1 h later followed by determination of pSTAT3 and GFAP expression an additional 30 min afterward. The results show that fluorocitrate induced a mild pSTAT3 activation but attenuated leptin-induced pSTAT3 activation and decreased GFAP expression independently of leptin treatment. We conclude that inhibition of astrocytic activity resulted in enhanced neuronal leptin uptake and signaling. This suggests opposite roles of astrocytes and neurons in leptin's actions in the A vy mouse with adult-onset obesity.

We have shown that TNFα specifically activates the interleukin-15 (IL15) system in cerebral endothelial cells composing the blood-brain barrier. To determine the functions of cerebral IL15 signaling in inflammation, we first treated mice with lipopolysaccharide (LPS) and determined the expression of the three receptor subtypes of IL15. Robust time-dependent upregulation occurred in all subunits. We then tested whether IL15Rα knockout (KO) affected the maintenance of body temperature and activity level after a single dose of LPS. Circadian telemetry data were analyzed by the cosinor method. Both wild-type and KO mice had clear 24-h rhythms of basal temperature and activity. KO mice had a significantly higher midline estimating statistic of rhythm (MESOR; approximating 24 h mean) of temperature and delayed 24-h acrophase (peak) of activity than the wild-type mice. LPS disrupted the circadian rhythm of activity more severely in the KO group. Besides a decrease in MESOR and 24-h amplitude of activity after LPS, the KO mice showed a significant reduction of MESOR, amplitude, and changed acrophase for temperature on the second of 2 days. The disrupted circadian rhythm of temperature and activity in the KO mice after LPS suggests that upregulated IL15 receptors may serve a beneficial role to counteract the consequences of neuroinflammation.

Astrocytic leptin receptors (ObR) can be up-regulated in conditions such as adult-onset obesity. To determine whether the levels and subtypes of astrocytic ObR modulate leptin transport, we co-cultured hCMEC/D3 human brain endothelial cells and C6 astrocytoma cells in the Transwell system, and tested leptin permeation from apical to basolateral chambers. In comparison with hCMEC alone, co-culture of C6 cells reduced the permeability of paracellular markers and leptin. Unexpectedly, ObRb over-expression in C6 cells increased leptin permeation whereas ObRa over-expression showed no effect when compared with the control group of pcDNA-transfected C6 cells. By contrast, the paracellular permeability to the sodium fluorescein control was unchanged by over-expression of ObR subtypes. Leptin remained intact after crossing the monolayer as shown by HPLC and acid precipitation, and this was not affected by C6 cell co-culture or the over-expression of different ObR subtypes. Thus, increased expression of ObRb (and to a lesser extent ObRe) in C6 cells specifically increased the permeation of leptin across the hCMEC monolayer. Consistent with the evidence that the most apparent regulatory changes of ObR during obesity and inflammation occur in astrocytes, the results indicate that astrocytes actively regulate leptin transport across the blood-brain barrier, a mechanism independent of reduction of paracellular permeability.

Interleukin (IL)-15 can cross the blood-brain barrier to act on its specific brain receptor (IL15Ralpha) and co-receptors. The important roles of neuronal IL15 and IL15Ralpha in experimental autoimmune encephalomeylitis (EAE) are suggested by the upregulation of IL15Ralpha mRNA in different regions of the brain and spinal cord, and by double-labeling immunohistochemistry showing neuronal localization of IL15 and IL15Ralpha in different neurons. Contrary to expectations, IL15 treatment lessened EAE severity. IL15 knockout mice showed heightened susceptibility to EAE with significantly higher scores that were decreased by treatment with IL15. Thus, IL15 improves this CNS autoimmune disorder as a potential therapeutic agent.

Interleukin-15 (IL15) is a cytokine produced by normal brain, but the functions of the IL15 system in normal adults are not yet clear. The hypothesis that the hippocampal IL15 system is essential for memory consolidation was tested by use of IL15Rα knock-out mice in behavioral, biochemical, immunohistological, and electron microscopic analyses. The knock-out mice showed deficits in memory, determined by the Stone T-maze and fear conditioning. In their hippocampi, the concentration of GABA was significantly lower. There were region-specific changes of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), with increased GAD-67-immunopositive interneurons in the stratum oriens of the CA1 region of the hippocampus, accompanied by nonsignificant reduction of GAD-67 synapses in the CA3 region. Western blotting showed an increase of GAD-65, but not GAD-67, in the hippocampal homogenate. The ultrastructure of the hippocampus remained intact in the knock-out mice. To further test the hypothesis that IL15 directly modulates GABA turnover by reuptake mechanisms, the dose–response relationship of IL15 on3H-GABA uptake was determined in two neuronal cell lines. The effective and nontoxic dose was further used in the synaptosomal uptake studies. IL15 decreased the uptake of3H-GABA in synaptosomes from the forebrain of wild-type mice. Consistent with this, IL15Rα knock-out mice had increased synaptosomal uptake of3H-GABA. Overall, the results show novel functions of a unique cytokine in normal hippocampal activity by regulating GABA transmission.

The transcription factor NFkappaB is a major mediator of lipopolysaccharide (LPS) signaling. We determined the role of NFkappaB activation in regulatory changes of the P-glycoprotein (Pgp) drug efflux transporter at the blood-brain barrier (BBB) and proinflammatory cytokine receptors.We treated NFkappaB knockout and wildtype mice with LPS or vehicle, obtained enriched cerebral microvessels, and determined target mRNA by qPCR for MDR1a/b, IL15Ralpha, IL2 Ralpha, IL2Rgamma, LIFR, gp130, and TNFR1/2, and protein expression by western blotting for P-gp, IL15Ralpha, IL2Rgamma, LIFR, and gp130.The effects of LPS on the transporters and cytokine receptors showed differences between wildtype and NFkappaB knockout mice, and between mRNA and protein changes. NFkappaB not only mediated the LPS-induced increase of MDR1b, IL2Rgamma, and TNFR2 mRNA in the wildtype mice, but it showed opposite effects by elevating IL15Ralpha and TNFR1 mRNA and decreasing IL2Ralpha in the knockout mice. Although basal vinblastine uptake was unchanged in the NFkappaB knockout mice, LPS induced an increase of the uptake (depressed efflux transport) greater than that seen in the wildtype mice, indicating that NFkappaB helps to maintain Pgp efflux transporter function.The results show differential involvement of NFkappaB signaling in response to LPS at the BBB.

In this study we tested the hypothesis that receptor-mediated transport of urocortin across the blood-brain barrier (BBB) undergoes developmental changes. Urocortin is a peptide produced by both selective brain regions and peripheral organs, and it is involved in feeding, memory, mood, cardiovascular functions, and immune regulation. In BBB studies with multiple-time regression analysis, we found that neonatal mice had a significant influx of 125I-urocortin. By contrast, adult mice did not transport urocortin across the BBB. Quantitative RT-PCR showed that corticotropin-releasing hormone receptor (CRHR)-1 was developmentally regulated in enriched cerebral microvessels as well as hypothalamus, being significantly higher in neonatal than adult mice. This change was less dramatic in agouti viable yellow mice, a strain that develops adult-onset obesity. The level of expression of CRHR1 mRNA was 33-fold higher in the microvessels than in hypothalamic homogenates. The mRNA for CRHR2 was less abundant in both regions and less prone to changes with development or the agouti viable yellow mutation. Supported by previous findings of receptor-mediated endocytosis of urocortin, these results suggest that permeation of urocortin across the BBB is dependent on the level of CRHR1 expression in cerebral microvessels. These novel findings of differential regulation of CRH receptor subtypes help elucidate developmental processes in the brain, particularly for the urocortin system.

We recently reported that mouse astrocytes express leptin receptors (ObR), and that obesity induces upregulation of astrocytic ObR. To provide further evidence of the importance of astrocytic ObR expression, we performed double-labeling fluorescent in situ hybridization (FISH) and immunohistochemistry in the rat hypothalamus. Laser confocal microscopic image analysis showed that ObR mRNA was present in glial fibrillary acidic protein (+) cells that show distinctive astrocytic morphology as well as in neurons. In addition to the presence of ObR mRNA, ObR protein was shown in both astrocytes and neurons in the rat hypothalamus by double-labeling immunohistochemistry. In cultured rat C6 astrocytoma cells treated with different doses of lipopolysaccharide for 6h, the mRNA for ObRa or ObRb did not show significant changes, as measured by quantitative RT-PCR. However, the protein expression of both ObRa and ObRb, determined by Western blotting, was increased after the C6 cells were treated with either lipopolysaccharide or tumor necrosis factor-alpha. The results indicate that astrocytic ObR expression is present in rats as well as mice, and that it probably plays a role in the neuroinflammatory response.

MIF-1 (Pro-Leu-Gly-NH(2)) has potent therapeutic effects in depression and Parkinson's disease, but its CNS sites of production are not yet clear. In this study, the concentration of MIF-1 in different brain regions was measured by the multiple reaction monitoring technique on a 4000 QTRAP mass spectrometer. The limit of quantification was 300 fg of MIF-1, and limit of detection was 60 fg. The low molecular weight fractions of tissue homogenates from different regions of mouse brain were analyzed. The concentration of MIF-1 ranged from 22+/-3 fg/microg protein in cerebral cortex to 930+/-60 fg/microg protein in the hypothalamus. Moderate concentrations were also detected in all other regions tested, including the striatum, thalamus, and hippocampus. By incubation of stable isotope-labeled oxytocin with tissue preparations, it was also confirmed that oxytocin at least partially contributed to the production of MIF-1 in the hypothalamus by action of peptidases. Regional differences were also found. The results are the first to show the ultrasensitive quantification of MIF-1 in different brain regions, and support the neuromodulatory actions of MIF-1 in the striatum.

Regulatory changes in cytokine permeation across the blood—brain barrier (BBB) may have crucial roles in central nervous system (CNS) autoimmune disease. Accordingly, we examined the interactions of interleukin (IL)-15 with the cerebral vasculature after induction of experimental autoimmune encephalomyelitis (EAE). In contrast to the influx of 125I-IL15 from blood to the CNS in normal mice and the persistence of IL15 influx in the spinal cord of EAE mice, influx was reduced in the EAE brain. Analyses of disappearance kinetics, FITC (fluorescein isothiocyanate)-albumin space, and delivery of IL15 by in situ perfusion, all indicate that the changes were not caused by BBB disruption but by the rapid availability (high volume of distribution) of IL15 and albumin. Although there was no significant change in the BBB permeation of IL15 in either direction in EAE mice, there was an upregulation of its specific receptor, IL15Rα, and an increased in situ production of IL15 mRNA that showed regional variation in both basal and EAE states. Overall, for IL15, its increased cerebral vascular space in the brain was equally as important as its persistent influx across the blood—spinal cord barrier, indicating that it is fully capable of activating the upregulated IL15Rα in the brain along with the intrinsic CNS source of IL15 in EAE mice.

Spinal cord injury (SCI) induces dynamic changes of the blood-spinal cord barrier and even the more distant blood-brain barrier. Besides an immediate increase of paracellular permeability resulting from the direct impact of the injury, the transport systems for selective cytokines undergo regulatory changes. Since many of the transported molecules play essential roles in neuroregeneration, we propose that this altered peripheral tissue / CNS interaction benefits remodeling of the spinal cord and functional recovery after SCI. This review examines the transport of cytokines and neurotrophic factors into the spinal cord, emphasizing the upregulation of two cytokines--tumor necrosis factor alpha (TNF) and leukemia inhibitory factor (LIF)--during the course of SCI. The increased transport of TNF and LIF after SCI remains saturable and does not coincide with generalized BBB disruption, highlighting a pivotal regulatory role for the blood-spinal cord barrier.

Leukemia inhibitory factor (LIF) is a proinflammatory cytokine mediating a variety of central nervous system (CNS) responses to inflammatory stimuli. During lipopolysaccharide (LPS)-induced inflammation, blood concentrations of LIF increase, correlating with lethality of sepsis. Circulating LIF crosses the blood-brain barrier (BBB) by a saturable transport system. Here we determine how this transport system is regulated in neuroinflammation. Using transport assays that quantify the influx rate and volume of distribution of LIF in mice, we show that LPS facilitated the permeation of LIF from the blood to the brain without compromising the paracellular permeability of the BBB as determined by coadministration of fluorescein. Concurrently, gp130 (shared by the interleukin-6 family of cytokines), but not gp190 (the specific receptor for LIF) or cilliary neutrophic factor (CNTF-Rα, a unique receptor for cilliary neurotrophic factor that also uses gp130 and gp190), showed increased levels of mRNA and protein expression in cerebral microvessels from the LPS-treated mice. The upregulation of gp130 by LPS was at least partially mediated by vascular tumor necrosis factor receptor (TNFR)1 and TNFR2. This was shown by elevated TNFR1 and TNFR2 mRNA and protein in cerebral microvessels after LPS and by the absence of the LPS effect on gp130 in knockout mice lacking these receptors. The results show that neuroinflammation by LPS induces endothelial signaling and enhances cytokine transport across the BBB.

The two main ways for peptides in the peripheral body to enter the brain are by either saturable transport or passive diffusion across the blood-brain barrier (BBB). Saturable transport systems have the advantage of being responsive to physiological and pathological stimuli. Since saturable systems can regulate peptide entry into the brain, they have the potential to play controlling roles in feeding behavior. For therapeutic applications, however, saturable systems have the disadvantage of functioning as a threshold to limit access of large amounts of peptides into the brain. This pharmacological problem presumably would not be encountered for peptides crossing the BBB by passive diffusion, a process dependent on physicochemical properties. Thus, the gatekeeper function of the BBB can be expanded to a primary governing role, especially for entry of ingestive peptides subject to their respective saturable transport systems.

The agouti viable yellow (Avy) spontaneous mutation generates an unusual mouse phenotype of agouti-colored coat and adult-onset obesity with metabolic syndrome. Persistent production of agouti signaling protein in Avy mice antagonizes melanocortin receptors in the hypothalamus. To determine how this disruption of neuroendocrine circuits affects leptin transport across the blood-brain barrier (BBB), we measured leptin influx in Avy and B6 control mice after the development of obesity, hyperleptinemia, and increased adiposity. After iv bolus injection, 125I-leptin crossed the BBB significantly faster in young (2 month old) B6 mice than in young Avy mice or in older (8 month old) mice of either strain. This difference was not observed by in situ brain perfusion studies, indicating the cause being circulating factors, such as elevated leptin levels or soluble receptors. Thus, Avy mice showed peripheral leptin resistance. ObRa, the main transporting receptor for leptin at the BBB, showed no change in mRNA expression in the cerebral microvessels between the age-matched (2 month old) Avy and B6 mice. Higher ObRb mRNA was seen in the Avy microvasculature with unknown significance. Immunofluorescent staining unexpectedly revealed that many of the ObR(+) cells were astrocytes and that the Avy mice showed significantly more ObR(+) astrocytes in the hypothalamus than the B6 mice. Although leptin permeation from the circulation was slower in the Avy mice, the increased ObR expression in astrocytes and increased ObRb mRNA in microvessels suggest the possibility of heightened central nervous system sensitivity to circulating leptin.

Background/aims: Although it is known that drug delivery across the blood-brain barrier (BBB) may be hampered by efflux transport activity of the multidrug resistance (mdr) gene product P-glycoprotein, it is not clear how inflammation regulates efflux transporters. In rat brain endothelial (RBE4) cells of BBB origin, the proinflammatory cytokine TNF mainly induced transcriptional upregulation of mdr1b, and to a lesser extent mdr1a, resulting in greater efflux of the substrates. This study further determines the mechanisms by which TNF activates mdr1b promoter activity. Methods/Results: Luciferase reporter assays and DNA binding studies show that (1) maximal basal promoter activity was conferred by a 476 bp sequence upstream to the mdr1b transcriptional initiation site; (2) TNF induced upregulation of promoter activity by NFκB nuclear translocation; and (3) the NFκB binding site of the mdr1b promoter was solely responsible for basal and TNF-activated gene transcription, whereas the p53 binding site was not involved. Binding of the p65 subunit of NFκB to nuclear DNA from RBE4 cells was shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. Conclusion: NFκB mediates TNF-induced upregulation of mdr1b promoter activity, illustrating how inflammation activates BBB efflux transport.

FGF21 recently has been proposed as a missing link in the biology of fasting, raising the question of whether it directly reaches the brain. We used multiple time-regression analysis to quantify the influx rate of this polypeptide across the blood-brain barrier (BBB), size-exclusion chromatography to examine degradation, capillary depletion to differentiate entry into brain parenchyma from retention in the microvasculature, and measurement of efflux rate to determine a possible confounding effect on measurement of entry. FGF21 was 94% intact in serum and 75% in brain 10 min after intravenous bolus delivery. Its influx rate was 0.23+/-0.12 microl/g-min, nearly four times faster than that of the vascular marker albumin. At 10 min, about 0.5% of the administered FGF21 was present in a gram of brain tissue. Of this, 70% reached the parenchyma of the brain. Co-injection of excess FGF21 failed to inhibit the influx, showing a lack of saturation. Efflux, which occurred at the same rate as the bulk reabsorption of cerebrospinal fluid, also was not saturable. In summary, FGF21 shows significant, non-saturable, unidirectional influx across the BBB.

The adipokine leptin participates not only in the regulation of feeding and obesity in adults but also in neonatal development. It crosses the blood-brain barrier (BBB) by receptor-mediated transport. Leptin concentrations in blood differ between neonates and adults. We determined the developmental changes of leptin receptor subtypes in the cerebral microvessels composing the BBB and examined their expected correlation with leptin transport across the BBB. Total RNA was extracted from enriched cerebral microvessels of mice 1, 7, 14, and 60 d of age for real-time RT-PCR analysis of leptin receptor subtypes. In cerebral microvessels from neonates, ObRa, ObRb, ObRc, and ObRe mRNA were all higher than in adults, but ObRd was not detectable. Hypothalamus showed similar age-related changes except for ObRb, which was higher in adults. The homologous receptor gp130 did not show significant age-related changes in either region. Despite the increase of leptin receptors, leptin permeation across the BBB after iv injection was less in the neonates. In situ brain perfusion with blood-free buffer showed no significant difference in the brain uptake of leptin between neonates and adults, indicating an antagonistic role of leptin-binding proteins in the circulation, especially the soluble receptor ObRe. The results are consistent with our previous finding that ObRe antagonizes leptin endocytosis in cultured endothelia and transport from blood to brain in mice. Overall, the developmental changes observed for leptin receptors unexpectedly failed to correlate with the entry of leptin into brain, and this may indicate different functions of the receptors in neonates and adults.

Just as the blood-brain barrier (BBB) is not a static barrier, the adipocytes are not inert storage depots. Adipokines are peptides or polypeptides produced by white adipose tissue; they play important roles in normal physiology as well as in the metabolic syndrome. Adipokines secreted into the circulation can interact with the BBB and exert potent CNS effects. The specific transport systems for two important adipokines, leptin and tumor necrosis factor alpha, have been characterized during the past decade. By contrast, transforming growth factor beta-1 and adiponectin do not show specific permeation across the BBB, but modulate endothelial functions. Still others, like interleukin-6, may reach the brain but are rapidly degraded. This review summarizes current knowledge and recent findings of the rapidly growing family of adipokines and their interactions with the BBB.

Leptin, a polypeptide mainly produced in the periphery, crosses the blood-brain barrier (BBB) by receptor-mediated transport to exert multiple central nervous system actions including decreased food intake. The reciprocal interactions between leptin transport and alcohol drinking are not clear. In this study, we tested whether alcohol increases leptin entry into brain and, if this occurs, whether it is a consequence of a generalized increase in the permeability of the BBB. BBB permeability to albumin, the increased permeation of which indicates BBB disruption, as well as to leptin was measured after alcohol ingestion. CD1 and B6 mice ingested a 5% liquid alcohol diet or its isocaloric control for 2 weeks. Alcohol ingestion resulted in increased blood-alcohol levels, decreased blood-leptin concentrations, and increased permeation of radioactively labeled leptin across the BBB as shown by in situ perfusion. Although the increased influx of the vascular marker albumin into brain showed partial disruption of the BBB, the influx of (125)I-leptin still could be suppressed by excess unlabeled leptin, indicating persistence of its saturable transport system. When given a choice of either alcohol or control diet, even the alcohol-preferring B6 mice showed a significantly greater preference for the control liquid diet, and there was no evidence of BBB disruption or alterated leptin transport. Furthermore, acute alcohol intoxication induced by intraperitoneal injection of 20% alcohol did not result in BBB disruption or increased leptin permeation 4 h later. Thus, partial disruption of the BBB and increased permeation of leptin in both CD1 and B6 mice were only induced by chronic alcohol ingestion. The results showing increased leptin permeation across the BBB lead to the speculation that leptin may serve as a homeostatic feeding signal in these mice.

Spinal cord injury (SCI) has devastating physical and socioeconomical impact. However, some degree of functional recovery is frequently observed in patients after SCI. There is considerable evidence that functional plasticity occurs in cerebral cortical maps of the body, which may account for functional recovery after injury. Additionally, these plasticity changes also occur at multiple levels including the brainstem, spinal cord, and peripheral nervous system. Although the interaction of plasticity changes at each level has been less well studied, it is likely that changes in subcortical levels contribute to cortical reorganization. Since the permeability of the blood-brain barrier (BBB) is changed, SCI-induced factors, such as cytokines and growth factors, can be involved in the plasticity events, thus affecting the final functional recovery after SCI. The mechanism of plasticity probably differs depending on the time frame. The reorganization that is rapidly induced by acute injury is likely based on unmasking of latent synapses resulting from modulation of neurotransmitters, while the long-term changes after chronic injury involve changes of synaptic efficacy modulated by long-term potentiation and axonal regeneration and sprouting. The functional significance of neural plasticity after SCI remains unclear. It indicates that in some situations plasticity changes can result in functional improvement, while in other situations they may have harmful consequences. Thus, further understanding of the mechanisms of plasticity could lead to better ways of promoting useful reorganization and preventing undesirable consequences.

We have sought to identify a high-capacity transport system that mediates transcytosis of proteins from the blood to the brain. The 39 kDa receptor-associated protein (RAP) functions as a specialized endoplasmic reticulum chaperone assisting in the folding and trafficking of members of the low-density lipoprotein (LDL) receptor family. RAP efficiently binds to these receptors and antagonizes binding of other ligands. Previous studies have shown that two large members of the LDL receptor family, LDL receptor-related protein 1 (LRP1) and LDL receptor-related protein 2 (LRP2 or megalin), possess the ability to mediate transcytosis of ligands across the brain capillary endothelium. Here, we tested whether blood-borne RAP crosses the blood-brain barrier (BBB) by LRP1- or megalin-mediated transport by studying the pharmacokinetics of [125I]-RAP transport into the brain in intact mice and across cell monolayers in vitro. Our results show that [125I]-RAP is relatively stable in blood for 30 minutes and has a mean influx constant of 0.62±0.08 μl/g-minute from blood to brain. In situ brain perfusion in blood-free buffer shows that transport of [125I]-RAP across the BBB is a saturable process. Capillary depletion of brain homogenates indicates that 70% of [125I]-RAP is localized in the parenchyma rather than in the vasculature of the brain. Results of transport in stably transfected MDCK cells are consistent with the hypothesis that megalin mediates most of the apical-to-basolateral transport across polarized epithelial cells. The inhibition of [125I]-RAP influx by excess RAP and the involvement of megalin indicate the presence of a saturable transport system at the BBB. The higher permeability of RAP compared with that of melanotransferrin and transferrin show that the LRP receptor is a high capacity transport system. These studies suggest that RAP may provide a novel means of protein-based drug delivery to the brain.

The peptide urocortin is a member of the corticotropin-releasing factor (CRF) family and a potent satiety signal to the brain. Urocortin in blood does not reach the brain significantly by itself, but its permeation across the blood-brain barrier (BBB) can be enhanced by leptin. How leptin facilitates the influx of urocortin has not been elucidated. In this study, we tested the hypothesis that leptin activates receptor-mediated endocytosis of urocortin. We measured the kinetics of permeation of radioactively labeled urocortin across the mouse BBB and determined the specific effects of leptin and receptor antibodies. The results show that the influx transfer constant of urocortin was enhanced in the presence of leptin and mediated by CRF-2beta, the specific receptor for urocortin. To determine the specificity of this modulation, the effect of leptin was compared with that of TNFalpha. Both TNFalpha and leptin independently facilitated receptor-mediated transport of urocortin across the BBB. Even though TNFalpha and leptin have similar effects on urocortin transport, leptin did not significantly affect the influx of TNFalpha across the BBB. The results indicate that permeation of ingestive peptides and cytokines across the BBB can be acutely modulated, consistent with a role of BBB in regulating feeding behavior. Thus, sites of action of leptin, urocortin, and TNFalpha exist not only in the brain but also at the BBB where they each control the flow of other ingestive signals to CNS targets.

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two highly selective mu-opiate receptor agonists. We recently demonstrated that EM-1 and EM-2 have a saturable transport system from brain-to-blood in vivo. Since the endothelial cells are the main component of the non-fenestrated microvessels of the blood-brain barrier (BBB), we examined whether these endogenous tetrapeptides have a saturable transport system in cultured cerebral endothelial cells. EM-1 and EM-2 binding and transport were studied in a transwell system in which primary mouse endothelial cells were co-cultured with rat glioma cells. We found that binding of both endomorphins was greater on the basolateral than the apical cell surface. Flux of EM-1 and EM-2 occurred predominantly in the basolateral to apical direction, each showing self-inhibition with an excess of the respective endomorphin. Transport was not influenced by the addition of the P-glycoprotein inhibitor, cyclosporin A. Neither the mu-opiate receptor agonist DAMGO nor the delta-opiate receptor agonist DPDPE had any effect on the transport. Thus, the results show that a saturable transport system for EM-1 and EM-2 occurs at the level of endothelial cells of the BBB, and unlike beta-endorphin and morphine, P-glycoprotein is not needed for the brain-to-blood transport. Cross-inhibition of the transport of each endomorphin by the other suggests a shared transport system that is different from mu- or delta-opiate receptors. As endormorphins are mainly produced in the CNS, the presence of the efflux system at the BBB could play an important role in pain modulation and neuroendocrine control.

Proteins of the neuregulin (NRG) family play important regulatory roles in neuronal survival and synaptic activity. NRG-1-beta1 has particular potential as a therapeutic agent because it enhances myelination of neurites in spinal cord explants. In this study, we determined the permeation of NRG-1-beta1 across the blood-brain and blood-spinal cord barriers (BBB and BSCB respectively). Intact radioactively labeled NRG-1-beta1 had a saturable and relatively rapid influx rate from blood to the CNS in mice. Capillary depletion studies showed that NRG-1-beta1 entered the parenchyma of the brain and spinal cord rather than being trapped in the capillaries that compose the BBB. The possible mechanism of receptor-mediated transport was shown by the ability of antibodies to erbB3 and erbB4 receptors to inhibit the influx. Lipophilicity, less important for such saturable transport mechanisms, was measured by the octanol : buffer partition coefficient and found to be low. The results indicate that NRG-1-beta1 enters spinal cord and brain by a saturable receptor-mediated mechanism, which provides the opportunity for possible therapeutic manipulation at the BBB level.

Leptin is a 17-kDa protein, secreted by fat, that controls adiposity and has been proposed to have numerous effects on reproduction in the mouse. To assess whether the effects of leptin on testicular function are direct, we determined whether leptin can cross the murine blood-testis barrier. Multiple time regression analysis showed that a small amount of blood-borne leptin is able to enter the testis but does so by a nonsaturable process. In addition, no significant expression of leptin receptors was found at the Leydig cells or Sertoli cells of the testis. This compares with the presence of a saturable transport system for leptin at the blood-brain barrier and abundant receptors for leptin at the leptomeninges, neurons, and choroid plexus of the central nervous system (CNS). These results support the hypothesis that the effects of leptin on reproductive function are not mediated at the level of the testis but indirectly, probably through the CNS.

Endomorphin 1 (EM1) and endomorphin 2 (EM2), recently isolated from bovine brain, are tetrapeptides with C-terminal amidation (Zadina, Hackler, Ge & Kastin, 1997). EMs have been shown to have the highest affinity and specificity for μ-opioid receptors by ligand-binding assay (Zadina et al. 1997). EM-like immunoreactivity is found in thalamus, hypothalamus, striatum and frontal cortex (Zadina et al. 1997), where μ-opioid receptors are concentrated (George et al. 1994; Bunzow et al. 1995). The question arises whether EMs function as neurotransmitters to mediate analgesia, opioid dependence and neuroendocrine effects. If we assume that EMs are neurotransmitters, EMs should fulfill several criteria. A crucial criterion is that EMs should activate or inhibit G proteins and effector enzymes to produce second messengers, or alternatively, EMs should change ionic conductance after interacting with μ-receptors in the postsynaptic or presynaptic membranes. To pursue the above hypothesis, we used cultured NGMO-251 cells that overexpress cloned rat μ-opioid receptors (Morikawa, Fukuda, Kato, Mori & Higashida, 1995). The parental NG108-15 cells were transfected with cDNA encoding the μ-receptor (Fukuda, Kato, Mori, Nishi & Takeshima, 1993) so that NGMO-251 cells represent a transformant of NG108-15 mouse neuroblastoma × rat glioma hybrid cells (Nirenberg et al. 1983). NGMO-251 cells maintain low- and high-threshold Ca2+ currents (Morikawa et al. 1995), as described in the parental NG108-15 cells (Tsunoo, Yoshii & Narahashi, 1986; Hescheler, Rosenthal, Trautwein & Schultz, 1987; Brown, Docherty & McFadzean, 1989; Kasai & Neher, 1992). ω-Conotoxin-sensitive (N-type) Ca2+ channel currents of NGMO-251 cells containing both μ- and δ-receptors are inhibited by the μ-agonist [D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and the δ-agonist [D-Pen2,D-Pen5]-enkephalin (DPDPE), whereas the parental NG108-15 cells are only inhibited by DPDPE (Morikawa et al. 1995). Therefore, NGMO-251 cells are a good neuronal model for investigating agonist-induced conductance changes in ion channels. Here we demonstrate that EMs induce an inhibition of high-threshold Ca2+ channel currents in the μ-containing NGMO-251 cells, but not in the δ-containing NG108-15 cells (Evans, Keith, Morrison, Magendzo & Edwards, 1992), in a N-methylmaleimide (NMM)- and pertussis toxin (PTX)-sensitive manner.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), a glycoprotein with hormonal properties, is produced by several cell types, most of which exist outside the CNS. GM-CSF, however, affects the CNS. If capable of crossing from blood to CNS, GM-CSF might be an important signalling molecule between the CNS and periphery. We used an established in vivo method in mice and rats to study passage of radioactively labelled GM-CSF from blood to CNS. We found that GM-CSF crossed the blood-brain barrier and blood-spinal cord barrier significantly faster than the control substance, albumin. Labelled GM-CSF was recovered in intact form by high performance liquid chromatography from brain after peripheral injection, and passage was not significantly reduced by simultaneous injection of unlabelled L-tryptophan. Both findings indicate that the observed passage of radioactivity was intact protein. Capillary depletion experiments showed that most of the GM-CSF was deposited in brain parenchyma rather than cerebral capillary endothelium. Co-injection of unlabelled GM-CSF significantly reduced the passage rate of labelled cytokine across the blood-brain and blood-spinal cord barriers, demonstrating that passage was mediated by a saturable system. In summary, a saturable mechanism transports GM-CSF intact from blood to CNS.

Granulocyte macrophage-colony stimulating factor (GM-CSF) has hormone-like effects on female reproductive systems. Recent evidence has suggested that GM-CSF also might be important to male testicular function. It is possible, however, that most sources of GM-CSF might not be able to reach the testis, since testes are shielded from contact with the general circulation by vascular and Sertoli cell barriers, which together comprise the blood-testis barrier (BTB). We used a sensitive in vivo method to determine whether blood-borne GM-CSF crossed the BTB in mice. 125I-GM-CSF was found to cross the BTB, showing a unidirectional influx constant (Ki) of 1.45 x 10(-3) ml/g-min, nine times faster than the influx rate of the control substance, 99mTc-albumin. HPLC analysis confirmed the presence of intact 125I-GM-CSF in the testis after peripheral injection. More 125I-GMCSF than 99mTc-albumin crossed both the vascular barrier, to enter the testicular interstitial fluid, and the Sertoli cell barrier, to enter the seminiferous tubule fluid. Coinjection of unlabeled GM-CSF significantly reduced the passage rate of labeled cytokine across the BTB, demonstrating that passage was mediated by a saturable system and suggesting the presence of a transport or facilitated diffusion system for GM-CSF. In summary, GM-CSF passes intact from blood to testis by means of a saturable mechanism.

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.

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 (REM) 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.

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 (

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.

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.

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.