Immunohistochemical study on distribution of transthyretin in normal human brain tissue and tumors

Immunohistochemical examination of transthyretin (TTR), which is known to be synthesized in the epithelial cells of the choroid plexus as well as in the liver cells, was carried out on normal brain tissues and 84 human brain tumors, using a peroxidase-antiperoxidase (PAP) technique. TTR was demonstrated diffusely and strongly in the cytoplasm of normal choroid plexus cells, but not in ependyma and other tissues of normal brain. In all of 10 choroid plexus papillomas, TTR was found within the cytoplasm of tumor cells. In contrast, neither the two papillary ependymomas nor any other brain tumors contained TTR. Among the choroid plexus papillomas, some cases showed clear positive reactions in almost all tumor cells, while others had only a few TTR-positive cells. With these immunohistochemical findings, TTR proved a very useful marker of normal choroid plexus and choroid plexus papilloma.     

Receptor-mediated endocytosis of transthyretin by ependymoma cells

Transthyretin (TTR) is involved in the transport of thyroxine (T4) and retinol-binding protein (RBP) in cerebrospinal fluid (CSF) and serum. TTR is secreted in the CSF by the epithelial cells of choroid plexus. The binding of [¹²⁵I]TTR to cultured ependymoma cells which form the brain cerebrospinal barrier, was studied to determine whether these cells carry receptor(s) for TTR. TTR was bound by ependymoma cells in a time-dependent manner reaching equilibrium within 2 h. Scatchard analysis was consistent with a single class of high-affinity binding sites with a K_d of approximately 18 nM. Saturable high-affinity binding of human TTR has previously been described in rat primary hepatocytes and human renal adenocarcinoma, neuroblastoma, hepatoma and astrocytoma cells, and also transformed lung cells. Endocytosis of fluorescent or biotinylated TTR was observed in ependymoma cells in cytoplasmic vesicles but TTR did not colocalize with clathrin in endocytic coated vesicles. Endocytosis of TTR was inhibited by high sucrose concentration (0.45 M). Finally, ligand blotting and chemical-linking experiments revealed the presence of a 100 kDa putative TTR receptor on the ependymoma cell membrane. Receptor binding of TTR provides a potential mechanism for the delivery of T4 within the central nervous system.     

Transthyretin is not expressed by dorsal root ganglia cells

Several mutations in transthyretin (TTR) are related to familial amyloidotic polyneuropathy (FAP), a neurodegenerative disorder caused by extracellular deposition of TTR fibrils, particularly in the peripheral nervous system (PNS). TTR is mainly synthesized by the liver and choroid plexus of the brain that contribute to the plasma and cerebrospinal fluid (CSF) pools of the protein, respectively. It has recently been reported that TTR is additionally expressed in the PNS, namely by peripheral glial cells of dorsal root ganglia (DRG). This lead to the hypothesis that TTR synthesis in the DRG might contribute to the PNS involvement in FAP. In this report we clarify this issue by showing that TTR synthesis is absent in both human and mouse DRG. Moreover, by using TTR KO mouse DRG as controls, we demonstrate that TTR-like immunoreactivity in the perineurium is an artifact. As such, and similarly to what has been previously shown in the central nervous system (CNS), TTR amplification by RT-PCR in the DRG most probably results from contamination by the meninges. In conclusion, TTR deposited in the PNS of FAP patients should still be regarded as having blood and/or CSF origin.     

Estrogen increases brain expression of the mRNA encoding transthyretin, an amyloid beta scavenger protein

Estrogen replacement therapy in postmenopausal women is associated with a reduced risk of Alzheimer's Disease (AD). The multiple mechanisms by which estrogen protects against AD are still unknown. To conduct a broad screen for estrogen-regulated AD-related genes in the brain, we used cDNA array assays of brain mRNA samples from ovariectomized (ovx) adult female mice treated with either 17beta-estradiol or vehicle at 1 or 5 weeks post-ovx. The gene encoding transthyretin (TTR), which has been reported to scavenge amyloid beta peptides and reduce amyloid plaque formation, is increased by estradiol treatment at both 1 and 5 weeks post-ovx. Northern blot analyses and RNase protection assays performed on whole brain samples obtained from estradiol- or vehicle-treated mice confirmed the cDNA array assays showing a significant increase in TTR mRNA with estradiol treatment. Qualitative in situ hybridization or immunocytochemistry performed on brain sections demonstrated that TTR mRNA is expressed only in choroid plexus and leptomeninges, and that both estrogen receptor proteins, alpha and beta, are present in choroid plexus cells. These novel findings suggest that estrogen may reduce the risk of AD by acting on choroid plexus cells to increase TTR gene expression, leading to enhanced sequestration and reduced aggregation of amyloid beta peptides.     

Low cerebrospinal fluid transthyretin levels in depression: correlations with suicidal ideation and low serotonin function.

BACKGROUND: Transthyretin (TTR) is a thyroid hormone binding protein synthesized by choroid plexus and secreted into cerebrospinal fluid (CSF). We sought to replicate and extend our previous findings of lower CSF TTR in depression. METHODS: Cerebrospinal fluid TTR concentrations of 17 medication-free patients with major depressive disorder (MDD) and 15 healthy individuals were determined by a sensitive, specific radioimmunoassay newly developed in our laboratory (ESL, TBC). RESULTS: Cerebrospinal fluid TTR was lower in the MDD patients compared with healthy volunteers (mean +/- SD, 19.7 +/- 1.6 vs. 21.8 +/- 2.2 mg/L, p = .005). Age correlated positively with CSF TTR (r = .38, p < .05). The group difference remained significant (p < .005) after covariance for age. Within the MDD group, Scale for Suicide Ideation total score correlated inversely with CSF TTR (beta = -.58, p < .05). In the entire sample of depressed and healthy individuals, CSF 5-hydroxyindoleacetic acid (5-HIAA) correlated positively (beta = .34, p < .05) with CSF TTR. CONCLUSIONS: We replicated our finding of low CSF TTR levels in depression and newly identified two relationships that may explain reports linking thyroid axis dysfunction and suicidal behaviors. Serotonergic hypofunction in depression, reflected by low CSF 5-HIAA, may result in decreased choroid plexus TTR production, alterations in central thyroid hormone kinetics, and increased vulnerability to suicidal ideation and perhaps suicide.     

THE DEVELOPMENT OF THE HUMAN BLOOD-BRAIN AND BLOOD-CSF BARRIERS

The commonly held belief that the fetal blood-brain and blood-CSF barriers are immature is reviewed. Results obtained from carefully conducted experiments with horseradish peroxidase and optimal freeze-fracturing suggest that the chick, rat and monkey brain barrier systems to proteins are tight from the earliest stages of development. Previous studies are reviewed in the light of new information on retrograde axonal transport, circumventricular organs, the proper use of horseradish peroxidase, freeze-fracturing, immunocytochemistry and plasma protein gene expression in the developing human brain. Original data on the development of human brain barrier systems are included. Tight junctions between cerebral endothelial and choroid plexus epithelial cells form the morphological basis for these systems. CSF in the fetus contains a remarkably high concentration of protein in contrast to adult CSF which is characterized by a very low protein concentration. This has previously been interpreted as due to immaturity of barriers in the fetal brain. Tight junctions between cerebral endothelial cells and between choroid plexus epithelial cells have been investigated in human embryos and fetuses by freeze fracture and thin section electron microscopy. As soon as the choroid plexus and the brain capillaries differentiated they exhibited well formed tight junctions. These junctions were very complex at early stages of development. A new barrier consisting of 'strap junctions' was found in the developing germinal matrix. The very high concentration of protein in early human fetal CSF cannot be accounted for by a lack of tight junctions in the developing brain barrier systems. Some transfer of proteins from blood to CSF, possibly via an intracellular route, has been demonstrated in immature experimental animals, but it seems that an important contribution to CSF proteins in the fetus may be synthesis by the developing brain and choroid plexuses with subsequent release into the CSF.     

α-Synuclein in human cerebrospinal fluid is principally derived from neurons of the central nervous system

The source of Parkinson disease-linked α-synuclein (aSyn) in human cerebrospinal fluid (CSF) remains unknown. We decided to measure the concentration of aSyn and its gradient in human CSF specimens and compared it with serum to explore its origin. We correlated aSyn concentrations in CSF versus serum (Q(aSyn)) to the albumin quotient (Q(albumin)) to evaluate its relation to blood-CSF barrier function. We also compared aSyn with several other CSF constituents of either central or peripheral sources (or both) including albumin, neuron-specific enolase, β-trace protein and total protein content. Finally, we examined whether aSyn is present within the structures of the choroid plexus (CP). We observed that Q(aSyn) did not rise or fall with Q(albumin) values, a relative measure of blood-CSF barrier integrity. In our CSF gradient analyses, aSyn levels decreased slightly from rostral to caudal fractions, in parallel to the recorded changes for neuron-specific enolase; the opposite trend was recorded for total protein, albumin and β-trace protein. The latter showed higher concentrations in caudal CSF fractions due to the diffusion-mediated transfer of proteins from blood and leptomeninges into CSF in the lower regions of the spine. In postmortem sections of human brain, we detected highly variable aSyn reactivity within the epithelial cell layer of CP in patients diagnosed with a range of neurological diseases; however, in sections of mice that express only human SNCA alleles (and in those without any Snca gene expression), we detected no aSyn signal in the epithelial cells of the CP. We conclude from these complementary results that despite its higher levels in peripheral blood products, neurons of the brain and spinal cord represent the principal source of aSyn in human CSF.     

Stress and Glucocorticoids Increase Transthyretin Expression in Rat Choroid Plexus via Mineralocorticoid and Glucocorticoid Receptors

Transthyretin (TTR) is a carrier for thyroid hormones and retinol binding protein. Several mutated forms of TTR cause familial amyloidotic polyneuropathy, an inheritable lethal disease. On the other hand, wild-type TTR has a protective role against Alzheimer's disease. Despite its overall importance in normal animal physiology and in disease, few studies have focused on its regulation. An in silico analysis of the rat TTR gene revealed a glucocorticoid responsive element in the 3' region of the first intron. Thus, we hypothesised that TTR could be regulated by glucocorticoid hormones and investigated the regulation of TTR expression in response to hydrocortisone in a rat choroid plexus cell line (RCP) and in primary cultures of choroid plexus epithelial cells (CPEC). In addition, the effect of psychosocial stress on TTR expression was analysed in rat liver, choroid plexus (CP) and cerebrospinal fluid (CSF). In RCP and CPEC cultures hydrocortisone upregulated TTR expression, an effect suppressed by glucocorticoid receptor and mineralocorticoid receptor antagonists. Moreover, induction of psychosocial stress increased TTR expression in liver, CP and CSF of animals subjected to acute and chronic stress conditions. Overall, we conclude that stress upregulates TTR expression in CP.     

Modification of protein transfer across blood/cerebrospinal fluid barrier in response to altered plasma protein composition during development

A developmentally regulated protein-specific transfer mechanism across choroid plexus epithelial cells has previously been proposed to contribute to the characteristically high concentration of protein in cerebrospinal fluid (CSF) in the immature brain. Here we demonstrate that this mechanism is sensitive to protein variations in plasma resulting in changed numbers of transferring cells for individual proteins and altered transfer into the CSF. Pups of Monodelphis domestica at postnatal day (P)9, P65 and P110 were injected intraperitoneally with either adult Monodelphis plasma or exogenous bovine fetuin. Samples of CSF, blood and brain were collected from terminally anaesthetized animals 3-48 h later. The concentration of total protein was measured and levels of albumin, hemopexin, α-fetoprotein and bovine fetuin were estimated by western blotting. Numbers of lateral ventricular choroid plexus cells positive for total and individual plasma proteins were counted in paraffin sections of brains stained with appropriate antibodies. Following intraperitoneal injections, the content of proteins in the CSF increased at all three ages, but the concentration increased only in the CSF of older animals. The total numbers of plexus cells positive for plasma protein did not change significantly, but cells positive for individual proteins did. Fetuin was detected in all protein-positive cells, but apparently displaced α-fetoprotein and, to a lesser degree, hemopexin. The results indicate that protein transfer across the blood/CSF barrier appears to be regulated by a molecular recognition mechanism that is probably saturable but may not be as specific for individual proteins as previously suggested.     

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.     

Multiple isoforms of the tumor protein p73 are expressed in the adult human telencephalon and choroid plexus and present in the cerebrospinal fluid

p73, a homolog of the p53 tumor suppressor, codes for full-length transactivating (TA) and N-terminally truncated (DeltaN) isoforms, with pro- and anti-apoptotic activities, respectively. We examined the expression of the main p73 isoforms in adult human and mouse telencephalon and choroid plexus by immunohistochemistry on paraffin sections, and immunoblotting (IB) of tissue extracts and cerebrospinal fluid (CSF), using antibodies against different protein domains. Cortical neurons expressed TAp73 predominantly in the cytoplasm and DeltaNp73 mainly in the nucleus, with partial overlap in the cytoplasm. Highest expression was found in the hippocampus. IB showed an array of TAp73 variants in adult human cortex and hippocampus. IB of human choroid plexus and CSF using TAp73-specific antibodies revealed the presence of a approximately 90-kDa protein whose molecular weight was reduced after N-deglycosylation, suggesting that glycosylated TAp73 is exported into the CSF. In the mouse, high expression of TAp73 was also detected in the subcommissural organ (SCO), an ependymal gland absent in adult humans. TAp73 colocalized with anti-fibra-Reissner-antibody (AFRU), which is a marker of Reissner's fiber, the secreted SCO product. p73-deficient mice had generalized cortical hypoplasia and hydrocephalus; in addition, we observed a dramatic size reduction of the choroid plexus. However, the SCOs were apparently unaltered and continued to secrete Reissner's fiber. Our findings point to complex and widespread p73 activities in the maintenance of adult cortical neurons and in brain homeostasis. TAp73 in the CSF may play important roles in the maintenance of the adult ventricular wall as well as in the development of the proliferating neuroepithelium.     

Cellular transfer of macromolecules across the developing choroid plexus of Monodelphis domestica

Choroid plexus epithelial cells secrete cerebrospinal fluid (CSF) and transfer molecules from blood into CSF. Tight junctions between choroidal epithelial cells are functionally effective from early in development: the route of transfer is suggested to be transcellular. Routes of transfer for endogenous and exogenous plasma proteins and dextrans were studied in Monodelphis domestica (opossum). Pups at postnatal (P) days 1–65 and young adults were injected with biotinylated dextrans (3–70 kDa) and/or foetal protein fetuin. CSF, plasma and brain samples were collected from terminally anaesthetized animals. Choroid plexus cells containing plasma proteins were detected immunocytochemically. Numbers of plasma protein-positive epithelial cells increased to adult levels by P28, but their percentage of plexus cells declined. Numbers of cells positive for biotinylated probes increased with age, while their percentage remained constant. Colocalization studies showed specificity for individual proteins in some epithelial cells. Biotinylated probes and endogenous proteins colocalized in about 10% of cells in younger animals, increasing towards 100% by adulthood. Injections of markers into the ventricles demonstrated that protein is transferred only from blood into CSF, whereas dextrans pass in both directions. These results indicate that protein and lipid-insoluble markers are transferred by separate mechanisms present in choroid plexuses from the earliest stage of brain development, and transfer of proteins from plasma across choroid plexus epithelial cells contributes to the high protein concentration in CSF in the immature brain.     

Human choroid plexus growth factors: What are the implications for CSF dynamics in Alzheimer's disease?

The choroid plexus plays a key role in supporting neuronal function by secreting cerebrospinal fluid (CSF) and may be involved in the regulation of various soluble factors. Because the choroid plexus is involved in growth factor secretion as well as CSF dynamics, it is important to understand how growth factors in CSF interact with the brain parenchyma as well as with cells in direct contact with the flowing CSF, i.e., choroid plexus and arachnoid villi. While the existence of growth factors in the choroid plexus has been documented in several animal models, the presence and distribution of growth factors in the human choroid plexus has not been extensively examined. This study describes the general distribution and possible functions of a number of key proteins in the human choroid plexus and arachnoid villi, including basic fibroblast growth factor, FGF receptor, and vascular endothelial growth factor. FGF and VEGF could both be readily demonstrated in choroid plexus epithelial cells. The presence of FGF and VEGF within the choroid plexus was also confirmed by ELISA analysis. Since Alzheimer's disease (AD) is known to be associated with a number of growth factor abnormalities, we examined the choroid plexus and arachnoid villi from AD patients. Immunohistochemical studies revealed the presence of FGF and VEGF within the AD choroid plexus and an increased density of FGFr in both the choroid plexus and the arachnoid villi of AD patients. No qualitative changes in the distribution of FGF and VEGF were observed in the AD choroid plexus. The appearance of FGFr in AD arachnoid was associated with robust amyloid and vimentin immunoreactivity. These findings confirm the presence of FGF and VEGF within the normal and AD choroid plexus and suggest that the alteration of growth factors and their receptors may contribute to the pathogenesis of the hydrocephalus ex vacuo that is characteristically seen in AD.     

Uptake and concentrations of calcium in rat choroid plexus during chronic hypo- and hypercalcemia

The choroid plexus has been implicated in the regulation of cerebrospinal fluid (CSF) [Ca], but little information is available concerning Ca transport by this epithelium. We determined the transfer coefficients for 45Ca uptake into choroid plexus from blood, as well as tissue [Ca], in weanling Fischer-344 rats fed low, normal, or high Ca diets for 8 weeks. Plasma [Ca] decreased by 45% with low Ca diet and increased by 25% with high Ca diet. Choroid plexus 45Ca uptake varied inversely with plasma [Ca]. This relation was due largely to changes in extracellular Ca binding rather than to entry from blood, as the transfer coefficient was independent of plasma [Ca]. The extracellular Ca distribution in choroid plexus, the intercept of a plot of tissue 45Ca distribution against time, was reciprocally related to plasma [Ca]. Changes in total cell [Ca] during hypercalcemia were equivalent to those in plasma, and in hypocalcemia were 70% of those in plasma. These findings indicate that regulation of CSF [Ca] does not involve saturable transport of Ca into the choroid epithelium from blood, and that the apical membrane of the choroid epithelium is involved in homeostasis of CSF [Ca].     

Transport of 5-aminolevulinic acid between blood and brain

Little is known about the movement of 5-aminolevulinic acid (delta-aminolevulinic acid; ALA) between blood and brain. This is despite the fact that increases in brain ALA may be involved in generating the neuropsychiatric symptoms in porphyrias and that systemic administration of ALA is currently being used to delineate the borders of malignant gliomas. The current study examines the mechanisms involved in the movement of [(14)C]ALA across the blood-brain and blood-CSF barriers in the rat. In the adult rat, the influx rate constant (K(i)) for [(14)C]ALA movement into brain was low ( approximately 0.2 microl/g per min), was unaffected by increasing plasma concentrations of non-radioactive ALA or probenecid (an organic anion transport inhibitor) and, therefore, appears to be a diffusional process. The K(i) for [(14)C]ALA was 3-fold less than that for [(14)C]mannitol, a molecule of similar size. This difference appears to result from a lower lipid solubility rather than saturable [(14)C]ALA transport from brain to blood. The K(i) for [(14)C]ALA for uptake into the neonatal brain was 7-fold higher than in the adult. However, again, this was unaffected by increasing plasma ALA concentrations suggesting a diffusional process. In contrast, at the blood-CSF barrier, there was evidence of carrier-mediated [(14)C]ALA transport from blood to choroid plexus and blood to CSF. Both processes were inhibited by administration of non-radioactive ALA and probenecid. However, experiments in choroid plexus epithelial cell primary cultures indicated that transport in these cells was polarized with [(14)C]ALA uptake from the apical (CSF) side being about 7-fold greater than uptake from the basolateral (blood) side. In total, these results suggest that the brain is normally fairly well protected from changes in plasma ALA concentration by the very low blood-brain barrier permeability of this compound and by a saturable efflux mechanism present at the choroid plexus.     

The uptake of delta9-tetrahydrocannabinol in choroid plexus and brain cortex in vitro and in vivo.

[3H]delta9 Tetrahydrocannabinol (delta9-THC) was actively transported by the choroid plexus and cerebral cortical slices of the rabbit when incubated as a BSA-microsuspension in artificial rabbit CSF. The transport system for delta9-THC in choroid plexus had a V max of 174 nmoles/mg tissue/h, approximately 9-fold greater than that observed for cortical slices. In vivo experiments demonstrated a preferential distribution of delta9-THC in choroid plexus at 1 h after intravenous injection. These results indicate that delta9-THC is actively accumulated by choroidal epithelium and may also be transported across the epithelial stroma into the capillary circulation. This suggests that the choroid plexus participates in the regulation of delta9-THC concentration in CSF and indirectly in brain by means of the "sink" function of the CSF.     

Iron and transferrin uptake by brain and cerebrospinal fluid in the rat.

Iron and transferrin uptake into the brain, CSF and choroid plexus, and albumin uptake into the CSF and choroid plexus, were determined after the intravenous injection of [59Fe-125I]transferrin and [131I]albumin into control rats aged 15, 21 and 63 days and 21-day iron-deficient rats. Iron uptake by the brain was unidirectional, greatly exceeded that of transferrin and was equivalent to 39 and 36% of the plasma iron pool per day in the 15-day control and 21-day iron-deficient rats. The rate of transferrin catabolism in the rats was only about 20% of the plasma pool per day. Iron and transferrin uptake into the brain and CSF decreased with increasing age and was greater in the iron-deficient than in the control 21-day rats. The quantity of 125I-transferrin recovered in the CSF could account for only a small proportion of the iron taken up by the brain. Albumin transfer to the CSF also decreased with age but was lower than that of transferrin and was not affected by iron deficiency. Similarly, the plasma: CSF concentration ratios of transferrin and albumin, as determined immunologically, decreased with age and were greater for transferrin than albumin. It is concluded that iron uptake by the brain is dependent on iron release from transferrin at the cerebral capillary endothelial cells with recycling of transferrin to the plasma and transfer of the iron into the brain interstitium. Only a small fraction of the transferrin bound by brain capillaries is transcytosed into the brain and CSF, this being one source of CSF transferrin while other sources are local synthesis and transfer from the plasma by the choroid plexuses.