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Glial cell line-derived neurotrophic factor (GDNF) is produced both in the central nervous system (CNS) and the periphery. Effective in ameliorating neurodegeneration in several animal models of CNS disease, its promise as a therapeutic agent would be greatly enhanced if it readily crossed the blood–brain barrier (BBB) in unmodified form. Here, we used the sensitive techniques of multiple-time regression analysis and ex-vivo perfusion in blood-free buffer to examine the entry of 125 I-GDNF into mouse brain. The integrity of GDNF in blood and brain was examined by high performance liquid chromatography and the physicochemical properties determining permeability were measured by octanol/buffer partition coefficient and hydrogen bonding. The efflux of 125 I-GDNF was determined to test for the presence of a bidirectional transport system. The results show that 125 I-GDNF differs from other peptides and polypeptides in that it does not enter brain any faster than 99m Tc-albumin, an effect that cannot be explained by degradation, rapid efflux, protein binding, or inadequate lipophilicity. Thus, GDNF shows a different type of interaction with the BBB. In normal mice, the BBB functions as a substantial physical barrier; in pathological or traumatic situations when the barrier is partially disrupted, the lack of restriction by a saturable transport system could make GDNF a suitable candidate for peripheral delivery in promoting neuroregeneration.

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.

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.BBB permeability, compartmental distribution, tracer stability, and expression of tight junction protein and inflammatory marker were determined.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.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.

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.

Brain-to-blood transport, or efflux, systems play important roles in brain functions and can affect the CNS uptake and activity of endogenous and exogenous blood-borne substances. Several efflux systems have been described for peptides. These efflux systems may play important roles in communication between the CNS and peripheral tissues and may be important in conditions such as alcoholism.

In this double-blind pilot study, 20 significantly depressed patients who all met the DSM-III R criteria for major depression were given a single subcutaneous injection of either 10 mg MIF-1 (Pro-Leu-Gly-NH2) or placebo on each of 5 consecutive days. Treatments were reversed for a second week of 5 consecutive daily injections. At the end of the first week, the group receiving MIF-1 was significantly improved on all rating scales as compared with the group receiving placebo. Eight out of 9 patients receiving MIF-1 showed marked improvement (scoreor = 7 on the Hamilton Scale) as compared with only 2 of 11 patients receiving saline (P0.01). Administration of MIF-1 during the second week to the patients who had received placebo during the first week resulted in substantial improvement so that by the end of the second week the two groups were indistinguishable.

The neurotrophins are a family of polypeptides that promote differentiation and survival of select peripheral and central neurons. Nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and neurotrophin-5 are included in this group. In recent years, tremendous advances have been made in the study of these factors. This has stimulated our review of the field, characterizing the neurotrophins from initial isolation to molecular analysis. The review also discusses their synthesis, localization, and responsive tissues, in both the periphery and CNS. The complex receptor interactions of the neurotrophins are also analyzed, as are putative signal transduction mechanisms. Discussion of the observed and postulated involvement in neuropathological disorders leads to the conclusion that the neurotrophins are involved in the function and dysfunction of the nervous system.

The metabolism of the endogenous brain peptides Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and MIF-1 (Pro-Leu-Gly-NH2) was determined by HPLC after incubation of the tritiated peptides in human and rat plasma. Degradation of Tyr-MIF-1 was rapid in the plasma from both species, in contrast to the slightly delayed degradation of MIF-1 in rat plasma and the extremely prolonged persistence of MIF-1 in human plasma. In rat plasma, more than half of the intact Tyr-MIF-1 and MIF-1 was degraded within 5 min, in contrast to the 5 days required for 50% degradation of MIF-1 in human plasma at 37 degrees. To slow the rapid rate of metabolism, studies were then performed at 0 degree. Incubation of Tyr-MIF-1 in human plasma at 0 degree for 2 hr resulted in HPLC identification of more Tyr-Pro than Tyr at all times. At 0 degree in rat plasma, however, more Tyr than Tyr-Pro was formed after the first 5 min of incubation of the Tyr-MIF-1 that was labeled on the Tyr. This raised the possibility that the tetrapeptide Tyr-MIF-1 might be serving as a precursor of the tripeptide MIF-1. Incubation of Tyr-MIF-1 tritiated at the Pro under the same conditions with and without Tyr-MIF-1 tritiated at the Tyr showed that Tyr-Pro, not MIF-1, was the predominant degradation product of Tyr-MIF-1. In addition to the metabolism of Tyr-MIF-1 being slower at lower temperatures, it was also slowed by some enzyme inhibitors. After 10 min of incubation at 37 degrees, EDTA appeared to be more effective than bestatin, p-chloromercuribenzoic acid (PCMB), pepstatin, or aprotinin, but after 30 min, bestatin was more effective. Intravenous injection of the tritiated peptides into rats showed short half-time disappearances; again, MIF-1 persisted in blood longer than Tyr-MIF-1. Thus, the results show the rapid metabolism of Tyr-MIF-1 in human and rat plasma, the slightly slower metabolism of MIF-1 in rat plasma, the predominant formation of Tyr-Pro rather than MIF-1 from Tyr-MIF-1, and the markedly delayed metabolism of MIF-1 in human plasma.

The relative binding to mu, delta, and kappa opiate receptors was characterized for the brain peptides Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2), and two fragments of Tyr-W-MIF-1 (Tyr-Pro-Trp and Tyr-Pro-Trp-Gly) previously shown to have antagonist as well as agonist activity in the guinea pig ileum. Tyr-MIF-1 had relatively low affinity (Ki = 1 microM at the mu site) but high selectivity (400- and 700-fold greater affinity for mu over delta and mu over kappa binding). Tyr-W-MIF-1 (Ki = 71 nM at the mu site) showed higher affinity binding to all three sites than Tyr-MIF-1 while retaining 200-fold selectivity for mu over delta and kappa receptors. The affinity of the fragments of Tyr-W-MIF-1 was lower for mu but higher for delta receptors. We also tested two cyclized analogs of Tyr-W-MIF-1 that were about 200-fold more active than the parent compound in producing analgesia. These analogs showed higher affinity binding to all three opiate receptors. One of the analogs showed binding affinity to mu sites (Ki = 1.3 nM) that was within 3-fold of that of the potent analog of enkephalin, DAMGO. Thus, brain peptides with an N-terminal Tyr-Pro, rather than the Tyr-Gly-Gly-Phe sequence typical of other endogenous opiates, can provide high selectivity for mu opiate receptors. Analogs based on one of them, Tyr-Pro-Trp-Gly-NH2, show high affinity as well as potent analgesic activity.

This paper is the sixteenth installment of our annual review of research concerning the opiate system. It is restricted to papers published during 1993 that concern the behavioral effects of the endogenous opiate peptides, and does not include papers dealing only with their analgesic properties. The specific topics this year include stress; tolerance and dependence; eating; drinking; gastrointestinal, renal, and hepatic function; mental illness and mood; learning, memory, and reward; cardiovascular responses; respiration and thermoregulation; seizures and other neurological disorders; electrical-related activity; general activity and locomotion; development; immunological responses; and other behaviors.

Peptides have been shown to be transported in the direction of both blood to brain and brain to blood. Although blood to brain transport is known to occur at both the choroid plexus and the capillary bed of the brain, comprising the two major components of the blood-brain barrier, the location of efflux systems for peptides remains largely unstudied. We adapted established methodologies to study this question for two peptides known to be transported out of the brain after injection into the cerebrospinal fluid (CSF): Tyr-MIF-1, transported by peptide transport system (PTS)-1 and RC-160, a somatostatin analog transported by PTS-5. Radioactive iodide, known to be transported out of the brain primarily by the capillaries, also was studied. We found that after injection into brain tissue, RC-160 and iodide were rapidly transported out of the brain by saturable mechanisms. By contrast, efflux of Tyr-MIF-1 was slow and nonsaturable after injection into brain tissue, but rapid and saturable after injection into the lateral ventricle of the brain. Autoradiography confirmed that peptide injected into brain tissue did not diffuse far from the site of injection during the study period. The results indicate that the efflux system for RC-160 is located at least partly at the capillaries and suggest that the major location for the efflux system of Tyr-MIF-1 is at the choroid plexus.

The presence in diverse species of a similar mode of communication, that of a soluble messenger binding to a receptor, raises the question as to whether the specific components of this system are equally widespread. Do invertebrates use the same hormones and receptors as vertebrates do? Invertebrates ranging from unicellular organisms to insects have been shown to contain opiate-like peptides and binding sites, and they exhibit biological responses to opiates. However, critical genetic data are lacking. It is not known how signal systems arise phylogenetically, but it is conceivable that signal molecules that are already present cause the formation of their own receptors from membrane proteins.

The centrally mediated effects of peptides as published in the journal Peptides from 1986 to 1993 are tabulated in two ways. In one table, the peptides are listed alphabetically. In another table, the effects are arranged alphabetically. Most of the effects observed after administration of peptides are grouped, wherever possible, into categories such as cardiovascular and gastrointestinal. The species used in most cases has been rats; where other animals were used, the species is noted. The route of administration of peptides and source of information also are included in the tables, with a complete listing provided at the end. Many peptides have been shown to exert a large number of centrally mediated effects.

Blood-borne interleukin-2 (IL-2), like other cytokines, is known to affect the central nervous system (CNS). One mechanism by which circulating substances can alter brain function is to directly cross the blood-brain barrier (BBB). We investigated the ability of IL-2 to cross the BBB, the interface between the periphery and the CNS. IL-2 labeled with 125I (I-IL-2) was injected into mice intravenously and its rate of entry into the brain determined by multiple-time regression analysis. I-IL-2 was found to enter the brain about 10 times faster than albumin. Neither morphine nor antibodies to IL-2, IL-1 alpha, or the IL-1 receptor affected the entry of I-IL-2. High performance liquid chromatography (HPLC) confirmed that the radioactivity entering the brain represented intact cytokine. However, excess unlabeled IL-2 was unable to impede the entry of I-IL-2, indicating that this transport is nonsaturable. This contrasts with saturable transport systems found for the cytokines IL-1 alpha and TNF-alpha, but still may explain how IL-2 can exert central effects.

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and MIF-1 (Pro-Leu-Gly-NH2) are endogenous peptides that can exert opiate-related actions on the CNS after peripheral administration. We found that Tyr-MIF-1 radioactively labeled at the tyrosine was transported across the blood-brain barrier (BBB) in the direction of brain to blood by a saturable system. Transport occurred equally well when the tetrapeptide was labeled with 125I or when it was labeled with 3H. [3H]MIF-1 and [3H]morphine were not transported out of the CNS but were retained by the brain after intracerebroventricular injection. Both [3H]MIF-1 and [3H]morphine entered the brain after i.v. injection, with [3H]MIF-1 crossing the BBB by a mechanism that was partially saturable. The entry rate and accumulation of radioactivity by the brain was 50-100 times greater after the i.v. injection of [3H]MIF-1 than after [3H]morphine. The results show that Tyr-MIF-1 labeled with either 3H or 125I can serve equally well for the measurement of transport across the BBB, that MIF-1 has relatively substantial and rapid access from the blood to the CNS by directly crossing the BBB, and that the BBB can differentially regulate the exchange of related substances between the CNS and blood.

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.

Interleukin-1 alpha (IL-1 alpha), an immunoregulatory protein secreted by the peripheral immune system, affects the central nervous system (CNS). IL-1 alpha could directly enter the parenchyma of the brain in intact form to alter brain function, or it could be blocked or sequestered by the capillary bed comprising the blood-brain barrier (BBB) that normally retards entry of circulating proteins to the brain and cerebrospinal fluid (CSF). We show here by use of the selective interleukin receptor antagonist (IL-1ra), capillary depletion method, high performance liquid chromatography (HPLC) and saturation with unlabeled IL-1 alpha that radioactively labeled IL-1 alpha injected iv directly enters the CNS in intact form. This also occurs in the brain cortex, an area devoid of circumventricular organs (CVOs), and in the CSF, an area devoid of capillaries. Capillaries can also sequester IL-1 alpha in a saturable manner, suggesting that they may be the site for the carrier-mediated entry of IL-1 alpha into the CNS. Thus, the results show that circulating IL-1 alpha has direct access to cortical brain cells behind the BBB through a saturable transport system that provides a major pathway by which the brain and immune system interact.

Morphine and endogenous peptides can alter developmental processes, inducing changes that can endure into adulthood. Morphiceptin binds to mu opiate receptors and to non-opiate sites labeled by Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), an endogenous brain peptide known to modulate opiate effects. Morphine, morphiceptin, Tyr-MIF-1, morphine + Tyr-MIF-1, and morphiceptin+Tyr-MIF-1 (50 micrograms, s.c.) were given to rats during their first week of life. Animals given morphine alone or in combination with Tyr-MIF-1 had significantly lower body weights for the first 3 weeks of life and delayed eye opening on day 16. Rats given morphine had hypersensitive tail flick responses on day 9 while those given morphine + Tyr-MIF-1 were hypersensitive on days 3, 8, and 9. Locomotor, passive avoidance, and rotorod behaviors were not altered by the neonatal treatments. Transport of [125I]Tyr-MIF-1 out of the brain was tested on day 23 and found to be increased by neonatal morphine, an effect that was significantly potentiated by neonatal Tyr-MIF-1. The results indicate that neonatal administration of peptides and opiates can affect later peptide transport across the blood-brain barrier as well as selected developmental characteristics.

Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) is a recently isolated peptide that belongs to a larger family that includes Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and MIF-1 (Pro-Leu-Gly-NH2). Despite similarities in structure, Tyr-MIF-1 and MIF-1 can act differently in behavioral, blood-brain barrier (BBB) transport, and receptor binding systems. Tyr-W-MIF-1, like Tyr-MIF-1, has both opiate and antiopiate activity, but may be more opiate-like than Tyr-MIF-1. Tyr-MIF-1, but not MIF-1, is transported from brain to blood by peptide transport system (PTS)-1. PTS-1 transports mainly Tyr-MIF-1 and methionine enkephalin, but does not transport amino acids, peptide fragments of Tyr-MIF-1, D-Tyr-MIF-1, or unrelated peptides and proteins. We tested whether Tyr-W-MIF-1 also was transported across the BBB and, if so, whether PTS-1 was involved. 125I-Tyr-W-MIF-1 had a half-time disappearance from the brain of 22.4 min. This is faster than the efflux occurring with non-saturable reabsorption of the cerebrospinal fluid and, therefore, is consistent with saturable transport, but it is slower than the efflux rate of Tyr-MIF-1, suggesting a less robust transport than for Tyr-MIF-1 Self-inhibition with excess unlabeled Tyr-W-MIF-1 confirmed a saturable component, with a dose of 4.2 nmol producing 50% inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

The known occurrence of opiates in the adrenals stimulated us to determine whether Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), an endogenous peptide that can act as an antiopiate, is present in these glands and whether adrenalectomy affects its concentrations in plasma. The presence of Tyr-MIF-1 in the adrenals of the rat was shown by radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). Binding sites for the tetrapeptide also were found in the adrenal. The concentrations and binding of Tyr-MIF-1 were higher in the adrenal medulla than in the adrenal cortex. The adrenals preferentially trapped 125I-Tyr-MIF-1 as compared with 99mTc-albumin after intravenous administration. However, after correction for tissue weight and vascular space, accumulation of 125I-Tyr-MIF-1 by the adrenals was in the general range seen for several other tissues. The concentrations of Tyr-MIF-1-like immunoreactivity determined by RIA in the plasma of adrenalectomized rats were increased in comparison with sham operated or unoperated controls. This approximate doubling was unchanged by the site of sampling: hepatic portal vein, renal vein, inferior vena cava, jugular vein, or truncal blood. Hypophysectomy, removal of the adrenal medulla, or treatment with corticosterone also did not block this increase. Tyr-MIF-1-like immunoreactivity in the plasma of adrenalectomized rats eluted at the same position by HPLC as did the synthetic Tyr-MIF-1 standard. Thus, the adrenal gland contains Tyr-MIF-1 and can affect its concentrations in plasma.

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.

The interactions between the cytokine interleukin (IL)-15 and the classical neurotransmitter GABA have been shown in IL15Rα receptor knockout mice by observations of memory deficits and reduction of GABA. To test the hypothesis that IL15 affects anxiety-like behavior, knockout mice without IL15, IL15Rα, or the co-receptor IL2Rγ were subjected to open-field and elevated plus maze tests. All three strains showed reduction of anxiety, with greater changes in the IL15Rα knockout mice than in the IL15 or IL2Rγ knockout mice. This unexpected observation is opposite to the reported increase of anxiety in mice lacking other proinflammatory cytokines or their receptors. The reduced anxiety was not associated with changes in associated serum cytokines. However, Western blotting, qPCR, and immunohistochemistry all showed that IL15Rα knockout mice had mild microgliosis and astrogliosis in the hippocampus. To determine whether this gliosis plays a role in decreasing anxiety, IL15Rα knockout mice were treated with minocycline, but this did not cause a change in open field performance. To determine whether IL15 plays a direct role in anxiety, wildtype mice were treated with IL15 by intraperitoneal injection. This also failed to cause a change in open field behavior under the experimental conditions tested. Thus, IL15Rα is essential for normal anxiety-like behavior, but inhibition of gliosis in the fearless IL15Rα knockout mice or IL15 treatment of normal mice did not acutely modulate behavioral performance as tested.

Interleukin (IL)-15 and its receptors in cerebral microvascular endothelial cells play an important role in mediating neuroinflammatory signaling across the blood-brain barrier. Although alternative splice variants of IL15Ralpha (the specific receptor) are seen in immune cells, the presence and functions of splice variants have not been studied in the cerebral endothelia that compose the blood-brain barrier. In this study, we identified five splice variants from mouse cerebral capillaries by RT-PCR, cloning, and DNA sequencing, and performed domain analysis. Four of these isoforms have never been described in any tissue. All isoforms were detected by qPCR in enriched mouse cerebral microvessels and their expression was increased by tumor necrosis factor treatment in vivo. To determine their functions, plasmids encoding individual isoforms were transfected into RBE4 cerebral endothelial cells. All of these predicted alkalinic proteins were expressed and most showed post-translational modifications. There were variations in their subcellular distribution. Only the full length IL15Ralpha and to a lesser degree isoform alpha1 were trafficked to the cell surface 24 h after over-expression. As shown by a luciferase reporter for signal transducer and activator of transcription (STAT)-3, over-expression of isoforms alpha2 and alpha4 reduced basal STAT3 activation. In comparison with the control, over-expression of the full length IL15Ralpha had a greater effect in increasing IL15-induced STAT3 transactivation than other isoforms. The results show that IL15 signaling in cerebral endothelia is probably an orchestrated effect of all IL15Ralpha splice variants that determine the eventual outcome by differential regulation.

Impairment in blood-to-brain transport of leptin is a major cause as well as consequence of obesity. Leptin crosses the blood-brain barrier by transcytosis rather than undergoing intracellular degradation. Results from previous studies have indicated that the membrane juxtapositional cytoplasmic sequence of the leptin receptor ObR is responsible for leptin transport. To identify the specific structural domains, we generated a series of ObR truncates with different lengths of the intracellular sequence, overexpressed them in 3 types of mammalian cells including cerebral endothelia, and quantified leptin binding and endocytosis. All mutant ObRs were able to bind and mediate the internalization of leptin. Surprisingly, ObR860, a construct with no cytoplasmic sequence, could act like the classical ObRa transporter in internalizing leptin. There were some cell type-dependent variations in the intracellular trafficking of Alexa-labeled leptin when mediated by ObR860 or ObRa because of differential involvement of membrane microdomains, as shown by use of the clathrin inhibitor chlorpromazine and the dynamin inhibitor Dynasore. The clathrin- and dynamin-mediated endocytosis of leptin contrasts with the lack of effect of the caveolae inhibitors nystatin and filipin. Thus, leptin-induced internalization of the ligand-receptor complex can occur without specific sorting signals in the cytoplasmic region of ObR. This novel finding may have significant implications for leptin transport.—Tu, H., Hsuchou, H., Kastin, A. J., Wu, X., Pan, W. Unique leptin trafficking by a tailless receptor.

MIF-1 (Pro-Leu-Gly-) is a tripeptide for which the therapeutic potential in Parkinson's disease and depression has been indicated by many studies. However, the cellular mechanisms of action of MIF-1 are not yet clear. Here, we show the specific brain regions responsive to MIF-1 treatment by c-Fos mapping, and determine the kinetics of cellular signaling by western blotting of pERK, pSTAT3, and c-Fos in cultured neurons. The immunoreactivity of c-Fos was increased 4 hours after MIF-1 treatment in brain regions critically involved in the regulation of mood, anxiety, depression, and memory. The number of cells activated was greater after peripheral treatment (intravenous delivery) than after intracerebroventricular injection. In cultured SH-SY5Y neuronal cells, c-Fos was induced time- and dose-dependently. The activation of cellular c-Fos was preceded by a transient increase of mitogen-activated protein kinase pERK but a reduction of phosphorylated Signal Transducer and Activator of Transcription (pSTAT3) initially. We conclude that MIF-1 can modulate multiple cellular signals including pERK, and pSTAT3 to activate c-Fos. The cellular activation in specific brain regions illustrates the biochemical and neuroanatomical basis underlying the therapeutic effect of MIF-1 in Parkinson's disease and depression.

This paper is the fourteenth installment of our annual review of research concerning the opiate system. It includes papers published during 1991 involving the behavioral, nonanalgesic, effects of the endogenous opiate peptides. The specific topics this year include stress; tolerance and dependence; eating; drinking; gastrointestinal and renal function; mental illness and mood; learning, memory, and reward; cardiovascular responses; respiration and thermoregulation; seizures and other neurological disorders; electrical-related activity; general activity and locomotion; sex, pregnancy, and development; immunological responses; and other behaviors.

Transport, binding, and metabolism of [phenylalanyl-3,4,5,-3H(N)]arginine vasopressin (AVP) by the blood-brain barrier (BBB) was studied in adult guinea-pigs by means of a novel vascular brain perfusion (VBP)/capillary depletion technique and HPLC. A time-dependent, progressive brain uptake of 3H-radioactivity was measured over the 10 min period of VBP both in brain homogenates and in brain tissue depleted of cerebral microvessels. The unidirectional blood-to-brain transport constant, K(IN), estimated by multiple-time tissue uptake analysis of the homogenate and postcapillary supernatant, indicated that the BBB transfer rate for [3H]AVP (K(IN) = 2.37 +/- 0.25 microliters min-1 per gram brain homogenate) was almost 10 times higher than for simultaneously perfused [14C]sucrose, a cerebrovascular space marker. In contrast to homogenate and postcapillary supernatant, the [3H]radioactivity determined in the vascular pellet after dextran density centrifugation of the brain homogenate was very low and only somewhat higher than for [14C]sucrose. HPLC analysis of the perfused brain tissue revealed time-dependent degradation of the blood-borne neuropeptide. The percentage of intact [3H]AVP as determined in the postcapillary supernatant progressively declined during brain perfusion, from 49% at 1 min to 11.9% at 10 min. The major detectable labeled metabolite was [3H]phenylalanine, the labeled amino acid residue of [3H]AVP. The aminopeptidase inhibitor bestatin (0.5 mM), perfused simultaneously with [3H]AVP by the VBP technique, did not alter tissue uptake of [3H]AVP, indicating that there was no significant hydrolysis of peptide by the luminal BBB surface.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel tetrapeptide, Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1), was purified from extracts of frontal cortex of human brain tissue by several consecutive reversed-phase high performance liquid chromatographic steps followed by a radioimmunoassay originally developed for Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1). Sequencing, mass spectrometric analysis, and comparison of its chromatographic behavior with that of the synthetic peptide confirmed the structure. Like Tyr-MIF-1, which was previously isolated from human brain tissue, Tyr-W-MIF-1 can inhibit the binding of 3H-DAMGO (selective for mu opiate receptors) to rat brain and can act as an opiate agonist as well as antagonist. Tyr-W-MIF-1 was a more potent opiate agonist than Tyr-MIF-1, the free acid of Tyr-W-MIF-1, and the structurally related hemoglobin-derived opiate peptide hemorphin-4 (Tyr-Pro-Trp-Thr) in the guinea pig ileum. Each of these peptides acted as opiate antagonists on the ileum from morphine-tolerant guinea pigs; the free acid of Tyr-W-MIF-1 was the most potent antagonist in inhibiting the activity of DAMGO. The results demonstrate the presence in human brain of a new member of the Tyr-MIF-1 family of biologically active peptides.

Previous studies have suggested that some of the central nervous system (CNS) effects of interleukin-2 (IL-2) and perhaps other cytokines might be mediated through disruption of the blood-brain barrier (BBB). We investigated the ability of human IL-2 and, in selected studies, human IL-1 alpha and human IL-1 beta to disrupt the BBB to radioiodinated bovine serum albumin (RISA) after intravenous (i.v.) and intracerebroventricular (i.c.v.) injection. No disruption of the BBB occurred for up to 2 h after the i.v. injection of 2 micrograms/mouse of IL-2 (10(5) U/kg of body weight), 2 micrograms of IL-1 alpha (10(7) U/kg), or 2 micrograms of IL-1 beta (10(7) U/kg). This dose of i.v. IL-2 also did not affect BBB permeability to RISA in the brain to blood direction. Damage to the BBB induced by hypertension elicited by i.v. epinephrine was not enhanced or prolonged by IL-2. When given directly into the CNS by the i.c.v. route, 100 ng of IL-2 (2.2 x 10(5) U/kg of brain), 100 ng of IL-1 alpha (2.2 x 10(7) U/kg of brain), or 100 ng of IL-1 beta (2.2 x 10(7) U/kg of brain) had no effect on BBB integrity in either the blood to brain or the brain to blood direction. We conclude that the effects of IL-1 alpha, IL-1 beta, and IL-2 on the CNS, as studied under these conditions, are not due to disruption of the BBB but are mediated by other mechanisms including the ability of some interleukins to cross the BBB by a saturable transport system described previously.

The brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH 2 ) was tested for its effects on electrically stimulated contractions in the guinea pig ileum assay. Tyr-MIF-1 acted as an opiate agonist in reducing these contractions. Its IC 50 was about 9 μM, and its effects were reversed by naloxone and CTOP. The ability of Tyr-MIF-1 also to antagonize the inhibitory effects of opiates on electrically stimulated contractions was more evident in the ileum removed from a guinea pig tolerant to morphine or after partial inactivation of opiate receptors with β-CNA. Similar results were observed with hemorphin. The endogenous peptide Tyr-MIF-1 and the blood-derived peptide hemorphin, therefore, can act as agonists as well as antagonists in the guinea pig ileum. The effects as antagonists are best observed in preparations of ileum with reduced receptor reserve (tolerant or β-CNA treated) and are consistent with the idea that properties of endogenous peptides as opiate antagonists are enhanced in the tolerant state.

Interleukin-15 (IL-15) is a cytokine produced in the normal brain that acts on its specific receptor IL-15Ralpha and co-receptors IL-2Rbeta and IL-2Rgamma in neuronal cells. The functions of the cerebral IL-15 system, however, are not yet clear. To test the hypothesis that IL-15Ralpha regulates metabolic activity and body temperature, we quantified the specific metabolic phenotype of IL-15Ralpha knockout mice. These normal-appearing mice were leaner with lower fat composition. During the entire circadian cycle, the knockout mice had a significantly higher acrophase in locomotor activity and heat dissipation. During the light phase, there was significantly greater food intake, oxygen consumption, and carbon dioxide production. The difference in the dark and light phases suggests that IL-15Ralpha participates in circadian rhythm regulation. The higher oxygen consumption in the light phase indicates adaptive thermogenesis in the knockout mice. The body temperature of the receptor knockout mice was significantly higher than the control in the light phase, and this was mainly caused by a large difference occurring between 0600 and 0900 h. In addition to the metabolic chamber studies and circadian rhythm analyses, qPCR of hypothalamic homogenates indicated higher mRNA expression of orexin and transient receptor potential vanilloid 4 cation channels. Consistent with a direct role of IL-15Ralpha in the hypothalamus, IL-15 treatment of the wild-type mice induced c-Fos expression in the preoptic area. We conclude that activation of hypothalamic neurons by IL-15 in mice contributes to thermoregulation and modifies the metabolic phenotype.

MIF-1 (Pro-Leu-Gly-NH(2)) is a tripeptide for which the therapeutic potential in Parkinson's disease and depression has been indicated by many studies. However, the cellular mechanisms of action of MIF-1 are not yet clear. Here, we show the specific brain regions responsive to MIF-1 treatment by c-Fos mapping, and determine the kinetics of cellular signaling by western blotting of pERK, pSTAT3, and c-Fos in cultured neurons. The immunoreactivity of c-Fos was increased 4 hours after MIF-1 treatment in brain regions critically involved in the regulation of mood, anxiety, depression, and memory. The number of cells activated was greater after peripheral treatment (intravenous delivery) than after intracerebroventricular injection. In cultured SH-SY5Y neuronal cells, c-Fos was induced time- and dose-dependently. The activation of cellular c-Fos was preceded by a transient increase of mitogen-activated protein kinase pERK but a reduction of phosphorylated Signal Transducer and Activator of Transcription (pSTAT3) initially. We conclude that MIF-1 can modulate multiple cellular signals including pERK, and pSTAT3 to activate c-Fos. The cellular activation in specific brain regions illustrates the biochemical and neuroanatomical basis underlying the therapeutic effect of MIF-1 in Parkinson's disease and depression.

The blood-brain barrier is a gatekeeper and modulatory interface for the CNS. Cerebral endothelial cells are the major component of the blood-brain barrier, and they modify inflammatory signals from the circulation to the CNS by production and secretion of secondary substances. The inflammatory mediators induced by tumor necrosis factor alpha (TNF) were determined by microarray analysis of RBE4 cerebral endothelial cells, at 0, 6, 12, or 24 h after TNF treatment. Interleukin (IL)-15 and its receptors were among the most robustly up-regulated genes. This was confirmed by real-time RT-PCR and western blotting. The three subunits of the IL15 receptor complex (IL15Ralpha, IL2Rbeta, and IL2Rgamma) showed differential regulation by TNF in their time course and amplitude of increased expression. Consistent with increased expression of the specific high affinity receptor IL15Ralpha, TNF increased cellular uptake of (125)I-IL15 and enhanced the fluorescent intensity of Alexa568-IL15 in RBE4 cells. TNF treatment in mice also increased the level of expression of IL15 receptors in enriched cerebral microvessels. We conclude that the cerebral microvascular IL15 system is a novel inflammatory mediator that transduces the actions of TNF.

The co-existence of receptors for leptin and melanocortin in cerebral microvessels suggests possible interactions between leptin and alpha-melanocyte stimulating hormone (MSH) signaling. In this study, we showed that ObRb and melanocortin receptor MC3R and MC4R were present in enriched cerebral microvessels. To test the interactions between ObRb and MC3R or MC4R-mediated cellular signaling, we over-expressed these plasmids in RBE4 cerebral microvascular endothelial cells and HEK293 cells in culture. Activation of signal transducers and activators of transcription-3 (STAT3) in response to leptin was determined by western blotting and luciferase reporter assays. Production of cAMP downstream to melanocortin receptors was determined with a chemiluminescent ELISA kit. alphaMSH, which increased intracellular cAMP, also potentiated leptin-induced STAT3 activation. This potentiation was abolished by a specific MEK inhibitor, indicating the involvement of the mitogen-activated kinase pathway. Reversely, the effect of leptin on alphaMSH-induced cAMP production was minimal. Thus, melanocortin specifically potentiated STAT3 signaling downstream to ObRb by cross-talk with mitogen-activated kinase. The cooperation of ObRb and G protein-coupled receptors in cellular signaling may have considerable biological implications not restricted to feeding and obesity.

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.

Cyclin-dependent kinase (Cdk) 5 is ubiquitously expressed in the brain and plays an essential role in central nervous system development and synaptic plasticity. The p35 kinase is a neuronal specific activator of Cdk5. Here, we show for the first time that Cdk5 activation modulates leptin signaling. P35 and its metabolite p25 were colocalized with the leptin receptor ObR in selective neurons in the hypothalamus. Overexpression of p35 alone was sufficient to induce the transcriptional activation of signal transducer and activator of transcription 3 (STAT3) in a cellular model. In retinoic acid-differentiated SH-SY5Y neuronal cells where ObRb was induced, leptin increased the expression of Cdk5, p35, and p25 kinases. The time course of induction coincided with that of phosphorylated (p)-STAT3. When Cdk5 activity was inhibited, either by roscovitine or overexpression of dominant negative Cdk5, there was a reduction of pSTAT3 activation. The results show that the activation of Cdk5 by p35 sustained leptin-induced pSTAT3 at 3-6 h. Thus, p35 is a novel modulator of leptin-induced STAT3 signaling.

Melanocyte-stimulating hormone (MSH) release-inhibiting factor (MIF)-1 is a tripeptide mainly produced by the hypothalamus. Since its discovery in 1968, MIF-1 has invoked a rich body of literature elucidating its biochemical properties, cellular actions, effects on animal behavior, and therapeutic potential in the human disorders Parkinson’s disease and mental depression. The chemical synthesis of MIF-1 analogs and isolation of naturally occurring peptides with potent biological activities have yielded a family of Tyr-MIF-1 peptides. Among these, endomorphin-1 and endomorphin-2 show selective agonistic activity for the µ-opiate receptor and therapeutic potential in pain and addiction. Overall, the structural-functional analyses of MIF-1 and other members in this peptide family during the past four decades clearly demonstrate the evolution of the concepts of peptides during our lifetime. This review will summarize some of the concepts, from their initial controversy to widely accepted facts nowadays.

The ability of naltrexone but not methyl naltrexone to cross the blood-brain barrier (BBB) was used to provide a different approach for the demonstration that opiates can enter the brain. Cortical electroencephalographic (EEG) measurements were made in rats receiving peripheral (IP) injections of naltrexone or methyl naltrexone and morphine or an enkephalin analog [Tyr-D-Ala-Gly-MePhe-Met(O)-ol]. Naltrexone significantly blocked the EEG effects of morphine and the enkephalin analog, but methyl naltrexone failed to do so. The results provide biological evidence that an opiate peptide can cross the BBB to affect the activity of the brain.

Interleukins (IL) are naturally occurring proteins that regulate, and thus link, both the immune system and the central nervous system (CNS). Since proteins are assumed not to be able to cross the blood-brain barrier (BBB), it is controversial how this linkage could occur. We show here that after iv injection of 125I-hIL-1 alpha, radioactivity in the brain eluted on HPLC in the position of the labeled cytokine. In addition, entry was inhibited by unlabeled hIL-1 alpha. Our demonstration of a saturable, carrier-mediated system that transports recombinant human IL-1 alpha in intact form from the blood into the CNS indicates a direct immune-CNS connection.

Peptides have been shown to cross the blood–brain barrier (BBB) as intact molecules so that they can influence the central nervous system. Peptides cross by saturable and nonsaturable mechanisms in the direction of both brain to blood and blood to brain. Passage of peptides, especially by saturable transport, has been shown to be influenced by pharmacological agents and physiological events. These findings support the view that peptides or their analogues could be useful as therapeutic agents for disorders of the central nervous system. They also suggest strategies in approaching therapeutic goals, including manipulating transport rates, targeting diseases due to altered BBB–peptide interactions, and designing analogues capable of taking advantage of such mechanisms of passage as paracellular trans-membrane diffusion and brain-to-blood transport.

The transport of 125I-oxytocin from brain to blood was investigated in mice after intraventricular injection of radioactively labeled oxytocin with or without unlabeled candidate inhibitors. Residual radioactivity in the brain detected after decapitation was the principal determinant of transport activity. The half-time disappearance from the central nervous system of labeled oxytocin was 19.1 min. Inhibition by 10 nmol/mouse of oxytocin showed a saturable component to transport. A 10-nmol dose of tyrosine-melanocyte-stimulating hormone release inhibiting factor (Tyr-MIF-1) and pressinamide also significantly inhibited transport of labeled oxytocin (p less than 0.05). There was no inhibition of the system by a 10-nmol dose of tyrosine, iodotyrosine, MIF-1, or arginine vasopressin. Studies performed with 125I-oxytocin injected simultaneously with 131I-Tyr-MIF-1 with or without unlabeled oxytocin or Tyr-MIF-1 were consistent with both peptides being transported by the previously described peptide transport system-1 (PTS-1). Pretreatment with aluminum (100 mg/kg of elemental aluminum given 60-90 min before intraventricular injection), previously shown to inhibit PTS-1 and some other transport systems, inhibited the transport of labeled oxytocin. Radioactivity collected from the blood after intraventricular injection of 125I-oxytocin eluted on HPLC at the same position as the labeled oxytocin standard and differently from tyrosine, Tyr-MIF-1, MIF-1 and tocinamide. It is concluded that a saturable system exists for the transport of intact oxytocin from brain to blood which appears to be the previously described PTS-1.

Biology is replete with examples of hormesis, the term introduced and developed by Calabrese. The corresponding concept in the field of peptide research has been characterized as the inverted U-shaped dose-response relationship. The articles by Calabrese in this issue summarize the notable progress occurring in the past three decades. In contrast to the skepticism encountered when we introduced this concept for peptides in the early 1970s, hormesis is now becoming recognized as characteristic of many actions of these small proteins. Calabrese is performing a considerable service by his strong advocacy and promotion of the concept to a more general readership. Hopefully, hormesis will be routinely considered in the design of research projects and the discovery of pharmaceutical agents.

Interleukin15 (IL 15) is a proinflammatory cytokine with elevated concentrations in autoimmune diseases involving the periphery (e.g. rheumatoid arthritis) and CNS (e.g. multiple sclerosis). Its interactions with the blood-brain barrier (BBB) were studied in normal and lipopolysaccharide (LPS)-treated mice. (125)I-IL15 remained intact for at least 10 min after i.v. injection and reached CNS parenchyma with regional differences between brain and spinal cord. Both in vivo and in situ brain perfusion of (125)I-IL15 showed that its permeation of the BBB was non-saturable. LPS induced a significant increase of IL15 uptake by the brain and spinal cord, partly related to a higher general permeability of the BBB. The results suggest that the BBB is an interface for blood-borne IL15 to interact with the CNS in the basal state and during inflammation.

Red blood cells (RBCs) harvested from mice were used to investigate the possible existence of an uptake system for peptides in these cells. The radioactively iodinated tetrapeptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-amide) was incubated with RBCs for varying lengths of time with or without inhibitors. The RBCs showed saturable uptake that could be inhibited by Tyr-Pro containing peptides. Uptake was also found in human RBCs, but was more robust in the mouse. Uptake by mouse RBCs was temperature dependent and magnesium sensitive but did not require sodium, potassium, or glucose. With the exception of some enkephalin- and dynorphin-related peptides that partially inhibited uptake, most substances tested were without effect. The results of HPLC showed internalization of the N-Tyr-Pro containing peptides, with accumulation of degradation products over time. The degradation products, however, did not inhibit transport, suggesting that peptides were transported intact into the RBCs with degradation occurring after internalization. This suggestion was strengthened by the finding that only the cytosol of the RBC, not its membranes, rapidly degraded Tyr-MIF-1 to free iodine and iodotyrosine. Nevertheless, the cytosol contained a large amount of immunoreactive material that eluted at the position of intact Tyr-MIF-1 on HPLC. These findings show that RBCs can take up, store, and degrade Tyr-Pro containing peptides.

The last decade has seen rapid growth in research with neuropeptides. During this time, we have been actively developing several concepts including the highly controversial one that peptides can cross the blood-brain barrier in intact form. One of the endogenous brain peptides used as a prototype for that concept, Tyr-MIF-1, also was used for the concept of the existence of endogenous antiopiate neuropeptides. As has been true for most novel developments in science, these concepts, as well as some older ones, were met with a great deal of skepticism when first suggested. Eventually, however, amnesia concerning the difficulties initially encountered with the introduction of new concepts occurs, with their subsequent "rediscovery" made easier.

Novel peptides with opiate activity, derived from endogenous sources (human and bovine casomorphins from milk, hemorphins from hemoglobin, and cytochrophins from mitochondrial cytochrome b), were tested for their ability to inhibit binding of the brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) to its high affinity sites in rat brain. The order of potency in inhibiting binding of 125I-Tyr-MIF-1 was: hemorphin and bovine casomorphins greater than Tyr-MIF-1 greater than cytochrophins greater than human casomorphins. Naloxone and DAMGO were ineffective at inhibiting Tyr-MIF-1 binding. The results provide evidence that, in addition to their ability to bind to mu opiate receptors, these novel endogenous peptides with opiate activity and a peptide (Tyr-MIF-1) with antiopiate properties also bind to a non-opiate site labeled by Tyr-MIF-1. These sites could be involved in a balance between opiate and antiopiate peptides.