Chronic loss of ovarian function decreases transport of leptin into mouse brain.

Loss of ovarian function, such as occurs with menopause in human beings and ovariectomy in rodents, results in weight gain. Using multiple-time regression analysis, a sensitive technique for quantifying blood-to-brain transport of peptides and polypeptides, we found that mice ovariectomized for at least 5 weeks had markedly reduced entry of the satiety factor leptin into brain. The rate of entry of leptin into brain remained reduced half a year later. The results suggest that the weight gain resulting from loss of ovarian function could be explained by decreased transport of leptin into the brain.

In vitro demonstration of a saturable transport system for leptin across the blood-brain barrier.

A saturable blood-to-brain transport system for leptin across the blood-brain barrier (BBB) has been observed in vivo. Since the main component of the non-fenestrated microvessels of the BBB is the endothelial cell, we established an in vitro culture system of these cerebrovascular cells to study leptin transport and to determine whether the self-inhibition of leptin transport characteristic of a saturable system occurs at this level. The results show that 125I-leptin crossed from the luminal to abluminal side of a monolayer of cerebral microvessel cells significantly faster than the albumin and lactalbumin controls. This transport of 125I-leptin across an in vitro BBB was significantly faster than in the opposite direction and was dose-relatedly inhibited by the addition of unlabeled leptin. Thus, the results establish that the saturable transport system for leptin across the BBB occurs at the level of the endothelial cells of the BBB.

Differential transport of rat and human interleukin-1alpha across the blood-brain barrier and blood-testis barrier in rats.

Human interleukin-1alpha is transported across the murine blood-brain barrier (BBB) and blood-testis barrier (BTB) by a saturable transport system. Differences in the biological activity and binding of human IL-1 in mouse and rat brain raise the possibility of species differences in the transport of IL-1 across the BBB and BTB. We measured the transport of recombinant human 125I-IL-1alpha (I-huIL-1alpha) and rat 125I-IL-1alpha (I-ratIL-1alpha) across the rat BBB and BTB after intravenous injection using a sensitive in vivo technique and film autoradiography. I-ratIL-1alpha was found to cross the rat BBB and rat BTB at rates comparable to those reported previously for murine IL-1alpha in mice. Passage across the BBB was inhibited by the addition of unlabeled rat IL-1alpha, demonstrating saturable transport. In contrast, I-huIL-1alpha entered the brain of the rat much more slowly, and its entry was not inhibited by the addition of unlabeled human IL-1alpha. These results show that the rat interleukin-1 transporter, unlike the murine transporter, does not transport human IL-1alpha. This difference highlights the importance of species specificity in IL-1alpha transport and may partly explain the different physiological responses to exogenous human IL-1alpha among rodent species.

Persistence of blood-to-brain transport of leptin in obese leptin-deficient and leptin receptor-deficient mice.

In lean CD-1 mice, leptin is delivered into the brain by a saturable transport mechanism. Previous work has shown that obesity is associated with decreased leptin transport. Here, we investigated the transport of leptin across the blood-brain barrier (BBB) in two murine models of obesity. Radioiodinated leptin was intravenously injected into ob/ob (no leptin production) and db/db (high leptin levels, but no long-form leptin receptor) mutant mice and their lean controls. In all groups, the labeled polypeptide was transported across the BBB by a saturable mechanism. The rates of transport were not significantly different between the mutant strains and their lean controls. The results demonstrate that leptin transport persists in the absence of production of the endogenous polypeptide or its signal-transducing receptor and suggest that the impaired transport previously seen is not directly explained by only obesity or alterations in serum plasma levels.

Peptides crossing the blood-brain barrier: some unusual observations.

An interactive blood-brain barrier (BBB) helps regulate the passage of peptides from the periphery to the CNS and from the CNS to the periphery. Many peptides cross the BBB by simple diffusion, mainly explained by their lipophilicity and other physicochemical properties. Other peptides cross by saturable transport systems. The systems that transport peptides into or out of the CNS can be highly specific, transporting MIF-1 but not Tyr-MIF-1, PACAP38 but not PACAP27, IL-1 but not IL-2, and leptin but not the smaller ingestive peptides NPY, orexin A, orexin B, CART (55-102[Met(O)(67)]), MCH, or AgRP(83-132). Although the peptides EGF and TGF-alpha bind to the same receptor, only EGF enters by a rapid saturable transport system, suggesting that receptors and transporters can represent different proteins. Even the polypeptide NGF enters faster than its much smaller subunit beta-NGF. The saturable transport of some compounds can be upregulated, like TNF-alpha in EAE (an animal model of multiple sclerosis) and after spinal cord injury, emphasizing the regulatory role of the BBB. As has been shown for CRH, saturable transport from brain to blood can exert effects in the periphery. Thus, the BBB plays a dynamic role in the communication of peptides between the periphery and the CNS.

Saturable entry of ciliary neurotrophic factor into brain.

Ciliary neurotrophic factor (CNTF), like tumor necrosis factor-alpha (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), is a cytokine with neurotrophic properties. Since all three cytokines are found in the periphery as well as brain, and since TNF and GM-CSF cross the blood-brain barrier (BBB) by a saturable mechanism, we investigated whether CNTF also saturably enters the brain from the blood. We found that CNTF crosses the BBB rapidly, with a rate of entry (Ki) of 4.60 (+/-0.78) x 10(-4) ml/g min, considerably faster than that of the 99mTc-albumin control. The Ki was reduced more than 3-fold by addition of excess unlabeled CNTF. The results indicate that CNTF is saturably transported across the BBB from blood to brain.

Decreased transport of leptin across the blood-brain barrier in rats lacking the short form of the leptin receptor.

Leptin is produced in adipose tissue in the periphery, but its satiety effect is exerted in the CNS that it reaches by a saturable transport system across the blood-brain barrier (BBB). The short form of the leptin receptor has been hypothesized to be the transporter, with impaired transport of leptin being implicated in obesity. In Koletsky rats, the splice variant that gives rise to the short form of the leptin receptor contains a point mutation that results in marked obesity. We studied the transport of leptin across the BBB in Koletsky rats and found it to be significantly less than in their lean littermates. By contrast, Sprague-Dawley rats matched in weight to each of these two groups showed no difference in the blood-to-brain influx of leptin. HPLC showed that most of the leptin crossing the BBB in rats remained intact and capillary depletion showed that most of the leptin reached the parenchyma of the brain. The results indicate that the short form of the leptin receptor is involved in the transport of leptin across the BBB.

Fate of leptin after intracerebroventricular injection into the mouse brain.

The fate of the metabolic regulatory protein leptin was studied after intracerebroventricular (i.c.v.) administration into the lateral ventricle of the brain. In the brain, a mean of 72% of the recovered radioiodinated leptin was intact. Efflux from the brain for leptin occurred with the reabsorption of the cerebrospinal fluid into the blood. Leptin appearing in the blood was 71% intact over the course of the study. The amount of leptin in the blood rose slowly, and 20 min after i.c.v. injection equaled or exceeded levels previously seen 20 min after i.v. administration. Autoradiography showed the slow disappearance of leptin from the ventricular system over time. The degree of periventricular penetration of radiolabeled leptin also was determined. By 30 min, leptin was detected 600 microm from the midline, but computer-assisted image analysis showed that the amount of radioactivity had fallen to half the midline value by 300 microm. The concentration of leptin within the arcuate nucleus, previously observed after i.v. administration, was not seen after i.c.v. injection. High concentrations of leptin were found at the choroid plexus, suggesting the presence of leptin receptors on the brain side of the blood-cerebrospinal fluid barrier and within the lumen of the middle cerebral arteries.

Relative contributions of a CVO and the microvascular bed to delivery of blood-borne IL-1alpha to the brain.

Diffusion from brain regions lacking a blood-brain barrier (BBB) and saturable transport across capillaries are possible pathways for the entry of blood-borne interleukin-1alpha into the central nervous system (CNS). To assess the involvement of these putative routes, mice received intravenous injections of radioiodinated interleukin-1alpha, and their brains were subjected to emulsion autoradiography. The resulting patterns of silver grain distribution showed that diffusion of interleukin-1alpha from the choroid plexus and the subfornical organ was greatly restricted. These restrictive properties were quantified by the determination of D1/2 values, the distances needed for the concentration of silver grains to decrease by one-half. Within several brain regions, a subset of the microvasculature indicated transport of interleukin-1alpha across the BBB. Individual microvessels showed different patterns of transport ranging from robust to absent. The high degree of containment of blood-borne interleukin-1alpha within the regions lacking a BBB indicates that these sites cannot account for total delivery of the cytokine into the brain and suggests instead that the microvascular network may serve as the major route of entry into the CNS.

Interactions of beta-amyloids with the blood-brain barrier.

Blood-borne beta-amyloids (A beta s) could affect brain function by (1) crossing the BBB to directly interact with brain tissues or (2) altering BBB function by interacting with the brain capillaries that make up the BBB. Several radioactively labeled A beta s have been examined for such interactions. Blood-borne A beta 1-28 is hindered from accumulating in brain by a slow rate of passage across the BBB and by robust enzymatic degradation. A beta 1-40, but not A beta 40-1 or A beta 1-42, is sequestered by brain capillaries, raising the possibility that it could affect BBB function. Small amounts of circulating A beta 1-40 are recovered intact from CSF and brain. A beta 1-40 is degraded by aluminum-sensitive, calcium-dependent intracellular enzymes. Apo-J, which can bind A beta, has been shown with an in situ method to be transported by a saturable system across the BBB. However, our recent work has shown that this system is not operable in vivo, probably because the transporter is saturated at physiological blood levels. In conclusion, A beta s have been shown to interact with and to cross the BBB.

Aluminum-sensitive degradation of amyloid beta-protein 1-40 by murine and human intracellular enzymes.

Both amyloid beta protein (A beta) and aluminum (A1) have been implicated in Alzheimer's disease. Recently, A beta has been found to be produced by peripheral tissues as well as by the CNS and to cross and accumulate in the vascular bed of the brain, which comprises the blood-brain barrier (BBB). This raises the possibility that blood-borne A beta may be a source of A beta within the CNS. Al has been shown to alter the structure and function of A beta, to inhibit the class of enzymes (metalloproteases) associated with the processing and degradation of A beta, and to alter the permeability of the BBB to peptides of similar size to A beta. Therefore, Al could alter the access of blood-borne A beta to the CNS either by changing the permeability of the BBB or by affecting enzymatic degradation. We examined the effect of Al on both of these parameters and found that Al did not alter the permeability of the BBB to A beta radioactively labeled with 125I (I-A beta) ever after correction for in vivo degradation. However, Al did enhance clearance and degradation of I-A beta in the circulation but not in the brain. Alterations in clearance can indirectly affect the CNS accumulation of circulating substances by modifying their presentation to the brain. In vitro studies of intracellular enzymatic activity of lysates of mouse and human erythrocytes (RBC) showed that Al could inhibit degradation of I-A beta through a mechanism antagonized by calcium and dependent on the concentrations of RBC lysate and Al. Analysis by high performance liquid chromatography showed that Al acted primarily by inhibiting the initial degradation of I-A beta to a peptide intermediate without inducing the aggregation of I-A beta under the conditions of these studies. No difference was found in sensitivity to Al between RBCs from patients with Alzheimer's disease and age- and sex-matched controls. The ability of Al to alter the degradation of A beta suggests a way in which these two potentially neurotoxic substances might interact in conditions such as Alzheimer's disease.

Periventricular penetration and disappearance of ICV Tyr-MIF-1, DAMGO, tyrosine, and albumin.

The penetration of four radioiodinated materials-Tyr-MIF-1, DAMGO, tyrosine, and albumin-into the periventricular tissue after ICV injection was studied in rats by film autoradiography. Rates of disappearance from the CNS for the injected compounds were also determined by computer-assisted image analysis of the autoradiographic images. The four materials showed distinct patterns of dispersion from the ventricular system, with Tyr-MIF-1 moving farthest into the parenchyma of the brain and albumin primarily restricted to the ventricular space. The other two compounds, tyrosine and DAMGO, had intermediate values. Tyr-MIF-1 also displayed the fastest rate of removal from the brain, which may represent the ability of the peptide to gain access to sites of saturable transport. By contrast, the exit from the brain of DAMGO was minimal, whereas the efflux of albumin and tyrosine was intermediate. These results show the utility of these methods in the simultaneous measurement of both the patterns of distribution within the CNS and the rates of removal from the CNS of compounds injected into the brain.

Leptin enters the brain by a saturable system independent of insulin.

Leptin, or OB protein, is produced by fat cells and may regulate body weight by acting on the brain. To reach the brain, circulating leptin must cross the blood-brain barrier (BBB). Intravenously injected radioiodinated leptin (125I-leptin) had an influx constant (Ki) into brain of (5.87)10(-4) ml/g-min, a rate 20 times greater than that of labeled albumin. Unlabeled leptin inhibited the influx of 125I-leptin in a dose-dependent manner whereas unlabeled tyrosine and insulin, which have saturable transport systems, were without effect. HPLC and acid precipitation showed that the radioactivity in brain and serum represented intact 125I-leptin. About 75% of the extravascular 125I-leptin in brain completely crossed the BBB to reach brain parenchyma. Autoradiography detected uptake at the choroid plexus, arcuate nuclei of the hypothalamus, and the median eminence. Saturable transport did not occur out of the brain. The results show that leptin is transported intact from blood to brain by a saturable system.

Selective transport of blood-borne interleukin-1 alpha into the posterior division of the septum of the mouse brain.

Film autoradiography was used to demonstrate the transport and sites of accumulation of blood-borne radioiodinated interleukin-1 alpha (II-1 alpha) and other cytokines into the brain after intravenous administration. [125 I]Il-1 alpha, [125I]Il-1 beta, [125I]interleukin-1 receptor antagonist (II-1ra), and [125I]tumor necrosis factor-alpha (TNF alpha) labeled the choroid plexus and the capillary network 30 min after injection into the blood, suggesting that these areas may serve as sites of blood-to-brain transport. [125I]Il-1alpha, but not [125I]Il-1beta, [125I]Il-1ra, [125I]TNF alpha, or [125I]interleukin-2 (Il-2), was also found localized to the caudal region of the septal nuclei. Only unlabeled II-1 alpha was able to inhibit this accumulation. These findings provide further evidence for the passage of select cytokines across the blood-brain barrier (BBB) and are the first to identify a target site within the central nervous system (CNS) for a transported cytokine.

Permeability of the blood-brain barrier to amylin.

Amylin is co-secreted with insulin from the pancreas of patients with non-insulin dependent diabetes mellitus, and its deposition may contribute to the central nervous system (CNS) manifestations of this disease. Amylin, but not its mRNA, is found in brain, suggesting that CNS amylin is derived from the circulation. This would require amylin to cross the blood-brain barrier (BBB). We used multiple-time regression analysis to determine the unidirectional influx constant (Ki) of blood-borne, radioactively labeled amylin (I-Amy) into the brain of mice. The Ki was 8.99(10(-4)) ml/g-min and was not inhibited with doses up to 100 micrograms/kg, but it was inhibited by aluminum (Al). About 0.11 to 0.13 percent of the injected dose of I-Amy entered each gram of brain. Radioactivity recovered from brain and analyzed by HPLC showed that the majority of radioactivity taken up by the brain represented intact I-Amy. Capillary depletion confirmed that blood-borne I-Amy completely crossed the BBB to enter the parenchymal/interstitial fluid space of the cerebral cortex. Taken together, these results show that blood-borne amylin has access to brain tissue and may be involved in some of the CNS manifestations of diabetes mellitus.

Passage of human amyloid beta-protein 1-40 across the murine blood-brain barrier.

Previous studies have suggested that the amyloid beta-protein present in the brains of patients with Alzheimer's disease may be derived in part from peripheral blood. We determined that after IV injection of synthetic amyloid beta-protein 1-40 (A beta), labeled with radioactive 125I (I-A beta), radioactivity accumulated in the brains of mice by a nonsaturable mechanism. Radioactivity also accumulated in the brain after the i.v. injection of radioiodinated reverse amyloid beta-protein 40-1 (I-rA beta). Capillary depletion techniques, however, showed I-A beta to have a much greater degree of association with brain capillaries than I-rA beta. Acid precipitation of radioactivity in CSF samples and recovery from cortical homogenates suggested the presence of intact I-A beta within the CNS after peripheral administration. HPLC analysis of cortical homogenates confirmed the presence of intact I-A beta. Gel electrophoresis of the CSF acid precipitates and of the HPLC fractions further verified the presence of intact blood-derived I-A beta peptide in CNS. These results suggest that endogenous bloodborne A beta can enter the CNS after associating with the capillary endothelium to accumulate intact within the parenchymal and CSF spaces of the brain.

Saturable efflux of the peptides RC-160 and Tyr-MIF-1 by different parts of the blood-brain barrier.

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 neurotrophins and their receptors: structure, function, and neuropathology.

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.

Lack of saturable transport across the blood-brain barrier in either direction for beta-amyloid1-28 (Alzheimer's disease protein).

beta-Amyloid and related peptides are components of the neurofibrillary tangles found in the brains of patients with Alzheimer's disease and have been suggested to be directly involved in the pathophysiology of that condition. It is unclear whether the amyloid deposited in the brain arises from the peripheral circulation, which would require passage across the blood-brain barrier (BBB), or whether it is produced within the brain itself. We examined the ability of beta-amyloid1-28 (beta Am), a commercially available, biologically active fragment, radioactively labeled with 125I (I-beta Am), to cross the BBB. After IV injection of I-beta Am, radioactivity entered the brain slowly, but to a greater extent than could be attributed to its being trapped in the vascular space of the brain. Entry was not inhibited by an excess of unlabeled beta Am or by pretreatment with aluminum, indicating that entry was by the nonsaturable mechanism of transmembrane diffusion. After intraventricular injection of I-beta Am, radioactivity was cleared slowly from the brain and was not affected by excess unlabeled beta Am or by pretreatment with aluminum, indicating that clearance probably occurred with reabsorption of cerebrospinal fluid. The excess beta Am did not alter the brain/blood ratio or the clearance rate of radioactively labeled albumin, indicating that under the conditions of this experiment beta Am did not have measurable effects on BBB integrity or on the rate of reabsorption of cerebrospinal fluid. High performance liquid chromatography (HPLC) showed that I-beta Am was rapidly degraded, especially by the brain, to smaller peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)