Transport pathways for clearance of human Alzheimer's amyloid beta-peptide and apolipoproteins E and J in the mouse central nervous system.

Amyloid beta-peptide (Abeta) clearance from the central nervous system (CNS) maintains its low levels in brain. In Alzheimer's disease, Abeta accumulates in brain possibly because of its faulty CNS clearance and a deficient efflux across the blood-brain barrier (BBB). By using human-specific enzyme-linked immunosorbent assays, we measured a rapid 30 mins efflux at the BBB and transport via the interstitial fluid (ISF) bulk flow of human-unlabeled Abeta and of Abeta transport proteins, apolipoprotein E (apoE) and apoJ in mice. We show (i) Abeta40 is cleared rapidly across the BBB via low-density lipoprotein receptor-related protein (LRP)1 at a rate of 0.21 pmol/min g ISF or 6-fold faster than via the ISF flow; (ii) Abeta42 is removed across the BBB at a rate 1.9-fold slower compared with Abeta40; (iii) apoE, lipid-poor isoform 3, is cleared slowly via the ISF flow and across the BBB (0.03-0.04 pmol/min g ISF), and after lipidation its transport at the BBB becomes barely detectable within 30 mins; (iv) apoJ is eliminated rapidly across the BBB (0.16 pmol/min g ISF) via LRP2. Clearance rates of unlabeled and corresponding 125I-labeled Abeta and apolipoproteins were almost identical, but could not be measured at low physiologic levels by mass spectrometry. Amyloid beta-peptide 40 binding to apoE3 reduced its efflux rate at the BBB by 5.7-fold, whereas Abeta42 binding to apoJ enhanced Abeta42 BBB clearance rate by 83%. Thus, Abeta, apoE, and apoJ are cleared from brain by different transport pathways, and apoE and apoJ may critically modify Abeta clearance at the BBB.     

Transthyretin: a choroid plexus-specific transport protein in human brain. The 1986 S. Weir Mitchell award

Plasma transthyretin (TTR, formerly called prealbumin) is a 55-kd protein that participates in the plasma transport of both thyroxine and retinol (vitamin A). TTR concentrations are disproportionately high in human ventricular CSF, suggesting that TTR is either selectively transported across or synthesized de novo within the blood-CSF barrier. To address this question, we adopted a molecular genetic approach; after isolating a cDNA clone encoding human TTR, we previously demonstrated specific TTR messenger RNA (mRNA) synthesis in rat choroid plexus. We have now extended these investigations to the human brain. Northern analysis of postmortem brain homogenates revealed abundant TTR mRNA in choroid plexus, but not in cerebellum or cerebral cortex. Choroid plexus mRNA was readily translated into TTR preprotein in an in vitro translation system. An immunocytochemical survey of human postmortem brain sections revealed the presence of TTR protein specifically and uniquely in the cytoplasm of choroid plexus epithelial cells; these results were corroborated at the mRNA level by an extensive survey of whole rat-brain sections by in situ hybridization. Therefore, within the mammalian CNS, TTR is the first known protein synthesized solely by the choroid plexus, suggesting a special role for TTR in the brain or CSF. Whether this function differs from its established plasma transport functions is presently unknown.     

Regional distribution of cyclooxygenase-3 mRNA in the rat central nervous system

We determined COX-3 mRNA expression in regions of the rat central nervous system (CNS). On a regional basis, levels were the highest in choroid plexus and spinal chord followed by pituitary gland, hypothalamus, hippocampus, medulla, cerebellum, and cortex. COX-3 mRNA levels were higher in major brain arteries, and dramatically higher in brain microvessels. Our results suggest that the expression pattern of COX-3 mRNA in the rat CNS primarily relates to the vascular density of a given region.     

Localization of zip1 and zip4 mRNA in the adult rat brain

The localization of two members of the Slc39a (zip1 and zip4) family of zinc transporters was examined in the brains of adult mice. Zip1 was highly enriched in brain regions with high densities of neuronal cell bodies, including the hippocampus, thalamus, and perifontal cortex. Zip1 was also expressed in commissural fiber tracts such as the corpus callosum and anterior commissure, but little was found in the internal and external capsules. Also, very low amounts of zip1 mRNA were detected in resting astrocytes and reactive astrocytes that were examined at 14 days after inflicting a stab wound. Zip1 mRNA was detected in ependymal cells lining the third and lateral ventricles and epithelium cells in the choroid plexus. Interestingly, zip4 mRNA was detected in the choroid plexus but not in the ependymal cells or other neural elements. Zip4 mRNA was also detected in brain capillaries, but zip1 mRNA was not. In zip4 knockout heterozygotes that express green fluorescent protein regulated by the zip4 promoter, green fluorescent protein was detected in brain capillaries. Because zip4 levels are regulated by dietary Zn, our studies suggest that the brain has the potential of adapting to changes in Zn status. © 2009 Wiley‐Liss, Inc.     

Evidence for ascorbic acid transport system in rat brain capillaries

Although ascorbic acid (AA) crosses the choroid plexus and may enter the brain at an appreciable rate, it is not clearly established that there exist transport system(s) carrying this vitamin from blood into the brain cells across the brain capillaries. Thus the rate of its uptake by choroid plexus and cerebral capillaries were evaluated in vitro in this study. Choroid plexus and brain capillaries were isolated from two-month-old male Sprague-Dawley rats. Time course of AA incorporation in micro vessels and choroid plexus was studied up to 30 min. After stopping the incorporation with the excess of cold isotonic saline, micro vessels were filtered and sonicated. The intracellular incorporated AA radioactivity was measured by liquid scintillation counting. AA uptake by micro vessel was tested for Na+-dependence and saturability. The time course studies showed linear increase in total uptake and accumulation of AA by choroid plexus and endothelial cells up to 30 min. Treatment with oubain or replacement with sodium chloride showed that uptake is an Na+- independent process. Transport of AA to cerebrospinal fluid and brain was also shown to be readily saturated by increasing the level of cold AA. These results document that the brain capillary endothelial cells are able to transport and accumulate AA, and may have a critical role in the homeostasis and regulation of cerebral ascorbic acid concentration.     

Transferrin gene expression in choroid plexus of the adult rat brain

Transferrin immunoreactivity and transferrin messenger RNA (mRNA) were recently found to be present in oligodendrocytes of the adult rat brain by using immunohistochemistry and in situ hybridization procedure. The present study demonstrates, in the same way, that epithelial cells of the choroid plexus also contain transferrin together with transferrin mRNA. Choroid plexus of the lateral and the third ventricle are rich in transferrin mRNA, while choroid plexus of the fourth ventricle contain few if any transferrin mRNA. These results demonstrate that epithelial cells of the choroid plexus as well as oligodendrocytes express the transferrin gene in the adult rat brain.     

Molecular mechanism of distorted iron regulation in the blood-CSF barrier and regional blood-brain barrier following in vivo subchronic manganese exposure

Previous studies in this laboratory indicated that manganese (Mn) exposure in vitro increases the expression of transferrin receptor (TfR) by enhancing the binding of iron regulatory proteins (IRPs) to iron responsive element-containing RNA. The current study further tested the hypothesis that in vivo exposure to Mn increased TfR expression at both blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barrier (BCB), which contributes to altered iron (Fe) homeostasis in the CSF. Groups of rats (10-11 each) received oral gavages at doses of 5 mg Mn/kg or 15 mg Mn/kg as MnCl(2) once daily for 30 days. Blood, CSF, and choroid plexus were collected and brain capillary fractions were separated from the regional parenchyma. Metal analyses showed that oral Mn exposure decreased concentrations of Fe in serum (-66%) but increased Fe in the CSF (+167%). Gel shift assay showed that Mn caused a dose-dependent increase of binding of IRP1 to iron responsive element-containing RNA in BCB in the choroid plexus (+70%), in regional BBB of capillaries of striatum (+39%), hippocampus (+56%), frontal cortex (+49%), and in brain parenchyma of striatum (+67%), hippocampus (+39%) and cerebellum (+28%). Real-time RT-PCR demonstrated that Mn exposure significantly increased the expression of TfR mRNA in choroid plexus and striatum with concomitant reduction in the expression of ferritin (Ft) mRNA. Collectively, these data indicate that in vivo Mn exposure results in Fe redistribution in body fluids through regulating the expression of TfR and ferritin at BCB and selected regional BBB. The disrupted Fe transport by brain barriers may underlie the distorted Fe homeostasis in the CSF.     

Distribution and phenotype of dendritic cells and resident tissue macrophages in the dura mater, leptomeninges, and choroid plexus of the rat brain as demonstrated in wholemount preparations

Dendritic cells (DC) are regarded as the 'sentinels' of the immune system. They play a crucial role in surveillance of peripheral tissues, trapping antigens encountered there, and migrating via the lymphatics to lymphoid organs where they interact with naive T cells thus generating antigen-specific primary immune responses. Until now it has been assumed DC are largely absent from the brain, meninges, and the choroid plexus within the ventricles. Such a situation was thought to partly explain the 'immune privileged' nature of the central nervous system (CNS). The present study of normal rat tissues using single and double immunohistochemistry reveals for the first time that extensive networks of major histocompatability (MHC) class II+/OX62+ DC are widely distributed in sites which may potentially encounter CNS antigens. These sites included the dura mater, leptomeninges, and the choroid plexus. These putative DC were negative when stained with the anti-resident tissue macrophage monoclonal antibody ED2. In addition to the rich networks of DC, dense populations of resident tissue macrophages (ED2+ and ED1+) were also demonstrated in the dura mater, leptomeninges and to a lesser extent in the choroid plexus. The presence of rich networks of DC and macrophages in the vascular and supporting tissues of the brain may play an important role in inflammatory and immune-mediated disorders affecting the CNS, including auto-immune demyelinating diseases such as multiple sclerosis.     

Expression of growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in the perinatal, adult, and injured rat brain

We and others have recently cloned a new member of the transforming growth factor-beta superfamily, growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF-15/MIC-1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF-15/MIC-1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF-15/MIC-1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF-15/MIC-1-producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Fluorescent microscopy revealed both intra- and extracellular GDF-15/MIC-1 ir. Up-regulation of GDF-15/MIC-1 in activated macrophages (Mstraight phi) is also supported by RT-PCR, ICC, and Western blot experiments showing pronounced induction of GDF-15/MIC-1 expression (mRNA and protein) in retinoic acid/phorbol ester-stimulated human M phi. Our data suggest that 1) GDF-15/MIC-1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme-epithelial interactions. Finally, GDF-15/MIC-1 may also act within the antiinflammatory cytokine network activated in CNS lesions.     

Association of apolipoprotein J-positive beta-amyloid plaques with dystrophic neurites in Alzheimer's disease brain

Apolipoprotein J (apoJ), also known as clusterin and SP-40,40, binds soluble beta-amyloid (Abeta and is up-regulated in the Alzheimer's disease (AD) brain. In the present study we classified apoJ-immunopositive Abeta deposits in AD temporal cortex, and found apoJ-immunoreactive plaques were often associated with dystrophic neurites. Quantitative immunohistochemical analysis of five AD brains showed that 29% of Abeta deposited in the parenchyma was associated with apoJ. Of Abeta deposits with apoJ immunopositivity, 71% were associated with phospho-tau-positive dystrophic neurites in the surrounding tissue. Conversely, 64% of phospho-tau-labeled neuritic deposits were labeled with apoJ. ApoJ was found at the core of these deposits, and co-localized with the amyloid staining agent thioflavine-S. To test the direct effects of apoJ on tau metabolism, we treated cells in culture with apoJ-containing conditioned media, and we injected apoJ-containing media into the rat hippocampus. Using both systems, we observed increases in levels of tau and phosphorylated tau. Our findings demonstrate that apoJ immunopositivity strongly correlates with the presence of amyloid and associated neuritic dystrophy in the neuropil of AD temporal cortex, and supports a model where extracellular apoJ facilitates the conversion of diffuse Abeta deposits into amyloid and enhances tau phosphorylation in neurites surrounding these of plaques.     

Changes in the Brain Accumulation of Glucocorticoids in abcb1a‐Deficient CF‐1 Mice

The multidrug resistance transporter, P‐glycoprotein (P‐gp), contributes to highly lipophilic molecules penetrating the brain from the blood at a much lower rate than expected, and has numerous substrates, inhibitors and modulators. The drug‐transporting isoform of P‐gp is coded by a single human gene, ABCB1, and shares 80% homology with the murine drug‐transporting isoforms, abcb1a and abcb1b, which share 92% homology with each other. Although these murine isoforms are highly similar, there are known affinity differences between the isoforms, and the localisation of the two isoforms in the brain is also disputed. Studies using mice genetically modified to be deficient in one or both isoforms of P‐gp have also resulted in conflicting data. The contribution of the abcb1a isoform, which is considered to contribute most to the central nervous system (CNS)‐protective role of P‐gp, is investigated in the present study using CF‐1‐abcb1a(?/?) mice and the well‐established brain/choroid plexus perfusion technique. Twenty‐minute in situ brain/choroid plexus perfusions in CF‐1‐abcb1a(?/?) mice indicated the increased accumulation of [³H]cortisol, [³H]corticosterone and [³H]dexamethasone in most of the brain regions examined compared to CF‐1‐abcb1a(+/+) mice. Taken together with our earlier published studies in abcb1a/b(?/?) mice, these data strongly suggest that the in vivo CNS accumulation of glucocorticoids obtained using single knockout strains [e.g. abcb1a(?/?)] cannot be directly compared with those obtained in double knockout strains [e.g. abcb1a/b(?/?)].     

Neuroendocrine regulatory mechanisms in the choroid plexus-cerebrospinal fluid system

The CSF is often regarded as merely a mechanical support for the brain, as well as an unspecific sink for waste products from the CNS. New methodology in receptor autoradiography, immunohistochemistry and molecular biology has revealed the presence of many different neuroendocrine substances or their corresponding receptors in the main CSF-forming structure, the choroid plexus. Both older research on the sympathetic nerves and recent studies of peptide neurotransmitters in the choroid plexus support a neurogenic regulation of choroid plexus CSF production and other transport functions. Among the endocrine substances present in blood and CSF, 5-HT, ANP, vasopressin and the IGFs have high receptor concentrations in the choroid plexus and have been shown to influence choroid plexus function. Finally, the choroid plexus produces the growth factor IGF-II and a number of transport proteins, most importantly transthyretin, that might regulate hormone transport from blood to brain. These studies suggest that the choroid plexus-CSF system could constitute an important pathway for neuroendocrine signalling in the brain, although clearcut evidence for such a role is still largely lacking.     

The choroid plexus-cerebrospinal lfuid interface in Alzheimer’s disease：more than just a barrier

The choroid plexus is a complex structure which hangs inside the ventricles of the brain and consists mainly of choroid plexus epithelial (CPE) cells surrounding fenestrated capillaries. These CPE cells not only form an anatomical barrier, called the blood-cerebrospinal lfuid barrier (BCSFB), but also present an active interface between blood and cerebrospinal lfuid (CSF). CPE cells perform indispensable functions for the development, maintenance and functioning of the brain. Indeed, the primary role of the choroid plexus in the brain is to maintain homeostasis by secreting CSF which contains different molecules, such as nutrients, neurotrophins, and growth factors, as well as by clearing toxic and undesirable molecules from CSF. The choroid plexus also acts as a selective entry gate for leukocytes into the brain. Recent ifndings have revealed distinct changes in CPE cells that are associated with aging and Alzheimer’s disease. In this review, we review some recent ifndings that highlight the importance of the CPE-CSF system in Alzheimer’s dis-ease and we summarize the recent advances in the regeneration of brain tissue through use of CPE cells as a new therapeutic strategy.     

Immunohistochemical localization of intraneuronal transferrin receptor immunoreactivity in the adult mouse central nervous system

Iron is essential for a variety of intracellular functions. Accordingly, the transfer of iron from blood to brain is vital for normal brain function. In the CNS, the receptor for iron-transferrin is generally accepted to be located in endothelial cells, whereas its occurrence in other cell types is less well established. I have investigated the distribution of the transferrin receptor in the adult mouse central nervous system by immunohistochemistry by using a monoclonal antibody raised against the transferrin receptor protein. Immunoreactive cell types comprised brain capillary endothelial cells, excluding those of circumventricular organs, and choroid plexus epithelial cells. Moreover, transferrin receptor immunoreactivity was detected intraneuronally in several brain regions without access to peripheral blood. The immunoreactive cell bodies were mainly confined to the cerebral cortex, hippocampus, habenular nucleus, red nucleus, substantia nigra, pontine nuclei, reticular formation, several cranial nerve nuclei, deep cerebellar nuclei, and cerebellar cortex. Transferrin receptor immunoreactivity was not detected in astrocytes, oligodendrocytes, or microglial cells. The occurrence of transferrin receptors at brain-barrier sites, i.e., the brain endothelium and choroid plexus epithelium, and the presence of the receptors intraneuronally are in accordance with the generally held belief that iron is released from liver transferrin and transported through capillaries and the choroid plexus into the brain interstitium. Subsequently, iron may be linked to brain transferrin synthesized within oligodendrocytes and choroid plexus epithelial cells followed by a concomitant uptake of iron-transferrin in neurons expressing transferrin receptors. The clinical importance of the intraneuronal transferrin receptor expression is discussed. © 1996 Wiley-Liss, Inc.     

The lateral,third, and fourth ventricle choroid plexus of the dog: A structural and ultrastructural study

The morphology of the choroid plexuses of the lateral, third, and fourth ventricles in dogs was studied by histological and ultrastructural techniques after in situ fixation. Most portions of the plexus in the lateral ventricle showed a parallel arrangement of capillaries, producing a fine corrugation of the overlying layer of epithelial cells. Relatively large amounts of connective tissue separated the capillaries from the epithelium. The fourth ventricle choroid plexus showed short capillary loops projecting into the ventricle and more intimately covered by epithelium. Smaller amounts of connective tissue separated the capillaries from the epithelium. The choroid plexus of the third ventricle showed characteristics seen in the plexuses of both the lateral and fourth ventricles. The total surface of the choroid plexuses in the dog averaged 10.7 cm², of which 55% was fourth ventricle choroid plexus.