Jody Eisenberg

Human Blood—Brain Barrier Insulin Receptor

A new model system for characterizing the human brain capillary, which makes up the blood–brain barrier (BBB) in vivo, is described in these studies and is applied initially to the investigation of the human BBB insulin receptor. Autopsy brains were obtained from the pathologist between 22–36 h postmortem and were used to isolate human brain microvessels which appeared intact on both light and phase microscopy. The microvessels were positive for human factor 8 and for a BBB‐specific enzyme marker, γ‐glutamyl transpeptidase. The microvessels avidly bound insulin with a high‐affinity dissociation constant, KD= 1.2 > 0.5 nM. The human brain microvessels internalized insulin based on acid‐wash assay, and 75% of insulin was internalized at 37°C. The microvessels transported insulin to the medium at 37°C with a t1/2=

Human blood-brain barrier transferrin receptor☆☆☆

70 min. Little of the 125I‐insulin was metabolized by the microvessels under these conditions based on the elution profile of the medium extract over a Sephadex G‐50 column. Plasma membranes were obtained from the human brain microvessels and these membranes were enriched in membrane markers such as γ‐glutamyl transpeptidase or alkaline phosphatase. The plasma membranes bound 125I‐insulin with an ED50= 10 ng/ml, which was identical to the 50% binding point in intact microvessels. The human BBB plasma membranes were solubilized in Triton X‐100 and were adsorbed to a wheat germ agglutinin Sepharose affinity column, indicating the BBB insulin receptor is a glycoprotein. Affinity cross‐linking of insulin to the plasma membranes revealed a 127K protein that specifically binds insulin. These studies demonstrate (a) the biochemistry of the human BBB may be investigated using the human brain microvessel model system, and (b) the human BBB insulin receptor has structural characteristics typical of the insulin receptor in peripheral tissues and may be part of a combined endocytosis‐exocytosis system for the transport of the peptide through the BBB in man.

Rapid Sequestration and Degradation of Somatostatin Analogues by Isolated Brain Microvessels

The kinetics of binding and endocytosis of 125I-human holotransferrin by isolated human brain capillaries was examined using this system as a model of the human blood-brain barrier (BBB). Both binding and endocytosis of the peptide by human brain capillaries was temperature-dependent and the binding was saturated by holotransferrin, but not by insulin, somatostatin, or vasopressin. Scatchard analysis of the binding reaction revealed a dissociation constant of 448 +/- 110 ng/mL (5.6 +/- 1.4 nmol/L) and a maximal binding constant (Ro) of 8.0 +/- 1.5 ng/mg protein. Thus, the affinity and capacity of the BBB transferrin receptor is within the same order of magnitude as the affinity and capacity of the BBB receptors for insulin, insulinlike growth factor-I, or insulinlike growth factor-II. The human brain capillary transferrin receptor was also detected with a mouse monoclonal antibody to the receptor using the avidin/biotin/peroxidase technique. In conclusion, these studies characterize the human BBB transferrin receptor and support the hypothesis that this receptor acts as a transport system which mediates the transcytosis of transferrin-bound iron through the brain capillary endothelial cell in man.

Chimeric peptides as a vehicle for peptide pharmaceutical delivery through the blood-brain barrier.

Abstract: Somatostatin (SRIF) is a putative peptide neurotransmitter that may interact with brain capillaries following neurosecretion of the peptide. The present studies investigate the binding and metabolism of SRIF analogues in isolated bovine brain microvessels. 125I [Tyr1]SRIF was rapidly degraded by capillary aminopeptidase with a half‐time of approximately 3 min at 23°C. The microvessel aminopeptidase had a low affinity and high capacity for the peptide, Km= 76 μM and Vmax= 74 nmol min−1. 125I‐[Tyr11]SRIF was converted to free iodotyrosine at a much slower rate, presumably by a lower‐activity endopeptidase. 125I‐[Tyr 11]SRIF was rapidly bound by microvessels, whereas another basic peptide, [Tyr8]bradykinin, or an acidic peptide, CCK8, or a neutral peptide, leucine enkephalin, were bound to a considerably less extent. The binding of 125I‐[Tyr11]SRIF to the capillaries was nonsaturable up to a concentration of 1 μg/ml of unlabeled peptide, and the binding reaction was extremely rapid, reaching equilibrium within 5 s at either 0°C or 37°C. Approximately 20% of the SRIF bound by the microvessels was resistant to acid wash and presumably represented internalized peptide. In addition, the 125I‐[Tyr11]SRIF bound rapidly to the endothelial cytoskeleton remaining after a 1% Triton X‐100 extraction of the microvessels. The peptide‐cytoskeletal binding reaction was nonsaturable up to 1 μg/ml of unlabeled [Tyr11]SRIF, but it was inhibited by 0.5% polylysine or 0.8 M KC1 and was stimulated by 1 mM dithiothreiotol. These studies suggest that brain microvessels rapidly sequester and degrade SRIF analogues and that this may represent one mechanism for rapid inactivation of the neuropeptides subsequent to neurosecretion.

Amyloid Angiopathy of Alzheimer's Disease: Amino Acid Composition and Partial Sequence of a 4,200-Dalton Peptide Isolated from Cortical Microvessels

A new strategy for peptide delivery through the brain capillary wall, i.e., the blood-brain barrier (BBB), is the synthesis of chimeric peptides which are formed by the covalent coupling of a non-transportable peptide (e.g., beta-endorphin) to a transportable peptide that undergoes receptor- or absorptive-mediated transcytosis at the BBB. beta-endorphin was covalently coupled via disulfide linkage to cationized albumin (pI greater than or equal to 9) which, owing to its highly basic charge, undergoes rapid absorptive-mediated transport into brain from blood. The [3H]labeled beta-endorphin-cationized albumin chimera was rapidly taken up by isolated brain capillaries in vitro and by rat brain in vivo; conversely, the BBB uptake of native [3H]beta-endorphin was negligible. The synthesis of chimeric peptides is a new strategy for solving the problem of peptide delivery through the BBB.

Blood–Brain Barrier Protein and Phosphorylation and Dephosphorylation

Abstract: The cardinal lesions of Alzheimers disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid‐like, β‐pleated sheet fibrils. Recently, a 4,200‐dalton pep‐tide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N‐terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical micro‐vessels from Alzheimers disease have not been previously isolated. The present studies describe the isolation of a mi‐crovessel fraction from Alzheimers disease and control fresh autopsy human brain. Alzheimers disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The arteriolc fraction from the Alzheimers subject with extensive amyloid angiopathy contained a unique 4,200‐dalton peptide. where as the arterioles or capillaries isolated from two controls and two Alzheimers disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200‐dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N‐terminal heterogeneity. The N‐terminal sequence was: Asp‐Ala‐Glu‐Phe‐Arg‐His‐Asp‐Ser‐Gly‐Tyr, which is identical to the N‐terminal sequence of the 4,200‐dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques. These studies report the first isolation of intracortical microvessels from Alzheimers disease brain and the finding of N‐terminal heterogeneity of the 4,200‐dalton peptide comprising the vascular amyloid.

Antibodies to Blood—Brain Barrier Bind Selectively to Brain Capillary Endothelial Lateral Membranes and to a 46K Protein

Abstract: Capillaries in vertebrate brain have unique permeability properties that make up the blood–brain barrier (BBB). Although it is known that capillaries are innervated by nerve endings of intracerebral origin and that brain capillary function is likely acutely regulated by neuronal inputs, the possible mechanisms of neuronal regulation of capillary function are at present unknown. One possible mode of regulation is via the phosphorylation of brain capillary proteins. The present studies characterize, for the first time, the major phosphoproteins in the bovine brain capillary using both intact bovine brain capillaries and plasma membrane fractions from bovine brain capillaries. The patterns of endogenous phosphorylation of capillary proteins are compared to similar patterns obtained with synaptosomal (P2) fractions from bovine brain. The major findings of this study are: (a) The activity of protein phosphorylation in brain capillaries is localized almost exclusively to the capillary plasma membrane, and is nearly comparable to the activity of protein phosphorylation in synaptosomal membranes. (b) A major phosphoprotein doublet in the capillary fraction comigrates on a sodium dodecyl sulfate gel with a major phosphoprotein doublet of approximate molecular weight of 80K in the synaptosomal fraction, and the latter is presumed to be synapsin I; in dephosphorylation assays the synaptosomal 80K phosphoprotein doublet is not subject to measurable dephosphorylation, whereas the capillary 80K doublet is subject to rapid dephosphorylation, and is essentially completely dephosphorylated within 5 s at 0°C. (c) A prominent triplet of phosphoproteins with molecular weight of 50–55K is present in the capillary fraction, and is not present in the synaptosomal fraction; thus, this 50—55K triplet of phosphoproteins appears specific for brain capillaries. In summary, these studies provide the basis for future investigations of a protein phosphorylation paradigm in regard to the rapid control of brain capillary function by brain.

High molecular weight Alzheimer's disease amyloid peptide immunoreactivity in human serum and CSF is an immunoglobulin G

To begin elucidating the biochemical basis of the polarized membrane features of the blood–brain barrier (BBB), a series of immunochemical and immunoperoxidase studies were initiated with bovine brain microvessels that make up the BBB in vivo. A rabbit antiserum was prepared against isolated bovine brain BBB plasma membranes. The bovine microvessel plasma membranes were radioiodinated with chloramine-T, and the antiserum selectively immunoprecipitated a 46K protein. The antibodies directed against the 46K protein were quantitatively absorbed with bovine brain capillaries but not with rat kidney or liver powder. Only the capillaries of brain reacted with the rat kidney-absorbed antiserum in immunoperoxidase studies of ethanol-fixed, 8-μm sections of bovine brain cortex, whereas the capillaries in heart, liver, and kidney did not react. This antiserum also strongly illuminated the lateral membranes of isolated bovine brain capillary endothelial cells grown in primary tissue culture. These studies provide evidence for a polarized distribution of a surface antigen in bovine brain capillary endothelial cells that is not present in capillary endothelia of liver, heart, or kidney. The correlation of the immunoperoxidase and immunoprecipitation techniques suggests that a candidate for the asymmetrically distributed surface antigen in the BBB is the 46K protein. The relationship between the 46K protein and the composition of BBB tight junctions remains to be determined.

Developmental changes in brain and serum binding of testosterone and in brain capillary uptake of testosterone-binding serum proteins in the rabbit

A radioimmunoassay (RIA) was developed to detect the 4200 Dalton amyloid (A4) peptide or its precursor (A4P) in human serum or cerebrospinal fluid (CSF). A synthetic peptide containing the first 28 amino acids of the 43 amino acid A4 peptide was covalently coupled to bovine thyroglobulin and a polyclonal antiserum in rabbits was prepared. This antiserum was specific for vascular amyloid and neuritic plaques in Alzheimers disease brain as detected by immunoperoxidase. The synthetic peptide, which has a tyrosine at residue 10, was iodinated with chloramine T and [125I]iodine and was purified to homogeneity by C4 reverse phase high performance liquid chromatography (HPLC). Extraction of human serum over a C18 Sep Pak cartridge indicated immunoreactive A4 peptide was not detectable in human serum. Conversely, high molecular weight A4 peptide immunoreactivity was detectable in human serum, at a concentration of 8.9 +/- 1.2 pmol-eq./ml, and in human CSF, at a concentration of 0.25 +/- 0.01 pmol-eq./ml, giving a CSF/serum ratio of 3.2%. The immunoreactivity in human serum was nearly completely removed by affinity deletion of serum immunoglobulin G (IgG), but not by affinity removal of IgA or IgM. Serum immunoreactivity was decreased 90% in hypogammaglobulinemia, and was increased 83% in human cord serum. There was no statistical difference in serum A4 immunoreactivity in Alzheimers serum or CSF. Serum immunoreactivity in Downs syndrome was increased 50%. These studies indicate the high molecular weight A4P immunoreactivity in human serum or CSF is an IgG. Whether the A4 precursor in Alzheimers disease is, in fact, an IgG, or whether there is an antibody in human serum and CSF that cross reacts with the A4 precursor cannot be determined until the serum immunoreactivity is purified and structurally characterized.

Absorptive-mediated endocytosis of cationized albumin and a beta-endorphin-cationized albumin chimeric peptide by isolated brain capillaries. Model system of blood-brain barrier transport

Developmental changes in the brain uptake of circulating testosterone and of testosterone-binding proteins, such as testosterone-binding globulin (TeBG) or albumin, may play a role in the sexually dimorphic changes in brain structure that are mediated by circulating testosterone. The present studies examine developmental changes in binding of testosterone in both the serum and brain compartments in postnatal rabbits in vivo and developmental changes in the uptake of [3H]TeBG or [3H]albumin by capillaries isolated from developing rabbit brain. The results show that between 10 and 15 days postnatally both the brain sequestration of testosterone and rabbit serum binding of the hormone are markedly increased relative to the newborn period. In addition, both [3H]TeBG and [3H]albumin were taken up by microvessels isolated from 28-day-old rabbit brain, and this process for [3H]TeBG was more active in capillaries obtained from neonatal rats as opposed to adult rats. In summary, these studies show that the binding systems for testosterone are modulated in a parallel fashion in both the serum and brain compartments. In addition, uptake mechanisms for serum testosterone-binding proteins such as TeBG and, to a lesser extent, albumin exist in the capillaries of developing rabbits. These brain capillary plasma protein uptake systems may allow for the distribution into brain of circulating serum proteins such as TeBG and, to a lesser extent, albumin, in developing rabbits.