High prealbumin and transferrin mRNA levels in the choroid plexus of rat brain

Expression of plasma protein genes in various parts of the rat brain was studied by hybridizing radioactive cDNA to RNA in cytoplasmic extracts. No mRNA could be detected in brain for the beta subunit of fibrinogen, major acute phase alpha 1-protein, alpha 1-acid glycoprotein and albumin. However, per g tissue, the choroid plexus contained at least 100 times larger amounts of prealbumin mRNA than the liver and about the same amount of transferrin mRNA as liver. No prealbumin mRNA was found in other areas of the brain. The results obtained suggest very active synthesis of prealbumin in choroid plexus, which would be an important link in the transport of thyroid hormones from the blood to the brain via the cerebrospinal fluid.     

Identification of carriers of mutant prealbumin gene associated with familial amyloidotic polyneuropathy type I by Southern blot procedures: study of six pedigrees in the Arao district of Japan

Summary Fifty-six Japanese individuals from six pedigrees with familial amyloidotic polyneuropathy (FAP), together with 2 individuals with symptomatic FAP from an unknown pedigree were analyzed, using the Southern blot procedures for the prealbumin gene structure. A human prealbumin cDNA was used as the probe. Altogether, these individuals included 20 with symptomatic FAP, 30 who were asymptomatic, and 8 disease-free spouses. Twenty individuals with symptomatic FAP were all heterozygous for the prealbumin genes, carrying one normal and one mutant gene. We confirmed the direct linkage between the mutant prealbumin gene and the Japanese type of FAP. Moreover, 10 of the 30 asymptomatic individuals from pedigrees with FAP were also heterozygous for the prealbumin gene. The number of asymptomatic individuals with the mutant prealbumin gene showed age-related decreases, and none was over 40 years. A linkage between the mutant prealbumin gene and serum levels of the prealbumin variant was also evident.     

Molecular genetics of familial amyloidotic polyneuropathy

We established a diagnosis of familial amyloidotic polyneuropathy (FAP) based on DNA and demonstrated a direct link between prealbumin gene mutation and FAP. The individuals with FAP, so far examined, were heterozygous for the prealbumin gene, carrying one normal and one mutant gene. To investigate the molecular pathogenesis of FAP, we isolated a normal prealbumin gene (7 kb in length) and also a mutant prealbumin gene associated with FAP. We compared their nucleotide sequences and found that they matched except for a single-base substitution present in the second exon. In an effort to construct mouse model systems for the FAP, we developed strains of transgenic mice carrying human mutant prealbumin genes.     

The distribution of cerebral expression of the transferrin gene is species specific.

Various plasma proteins, for example, transferrin, are synthesized not only in the liver, but also in the brain. The proportion of transferrin mRNA in total RNA from different regions of brains from various mammalian species was studied by Northern blot analysis. Absolute amounts of transferrin mRNA were determined in brain, choroid plexus, and liver from rats, sheep, and pigs by hybridization in solution followed by ribonuclease protection assay. Corrections for differences in yields of RNA were made using internal RNA standards. Large proportions of transferrin mRNA in total RNA and high absolute levels of transferrin mRNA in choroid plexus were found only in rats. Small proportions of transferrin mRNA were observed in RNA from choroid plexus from mice, dogs, and rabbits, while no transferrin mRNA at all was detected in choroid plexus from humans, sheep, pigs, cows, and guinea pigs. In further analysis of sheep and pigs, various amounts of transferrin mRNA were found in many parts of the brain, in contrast to the absence of transferrin mRNA from choroid plexus. In conclusion, a striking species specificity was observed for the pattern of cerebral expression of the transferrin gene.     

Rat choroid plexus specializes in the synthesis and the secretion of transthyretin (prealbumin). Regulation of transthyretin synthesis in choroid plexus is independent from that in liver

Synthesis of total protein and of transthyretin in rat choroid plexus was studied by measuring the incorporation of radioactive leucine into proteins in choroid plexus tissue incubated in vitro. About 20% of the protein newly synthesized in choroid plexus and about 50% of the newly synthesized protein secreted into the medium was transthyretin. Evidently, the choroid plexus is very active in the biosynthesis of this carrier protein for thyroid hormones and could be an important link in the chemical communication between the body and the central nervous system. Acute inflammation, which leads to a profound rearrangement of the pattern of plasma protein synthesis rates in the liver, produced distinct changes in the levels for plasma protein mRNAs in the liver. The levels of the mRNAs for alpha 1-acid glycoprotein and major acute phase alpha 1-protein increased more than 30-fold, those for transthyretin and albumin decreased to 27 and 57% of normal, respectively. The pattern of the observed changes in the levels of mRNAs for plasma proteins in the liver was independent of whether the acute inflammation was produced by subcutaneous injection of turpentine or intraperitoneal injection of a suspension of talcum. However, levels of transthyretin mRNA in choroid plexus were affected only very slightly, or not at all. Apparently, transthyretin synthesis in liver and choroid plexus is regulated independently during the acute phase response. No mRNA was detected in choroid plexus for albumin, alpha 1-acid glycoprotein, and major acute phase alpha 1-protein under any conditions.     

Genetic basis for familial amyloidotic polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is an inherited systemic amyloidosis, characterized by the extracellular deposition of fibrillar amyloid protein, i.e. a variant type of prealbumin, and by prominent peripheral nerve involvement. We recently established the basis of FAP, using a cloned human prealbumin cDNA, restriction endonuclease(s) and Southern blot procedures. This approach clearly revealed a direct link between mutation in the prealbumin gene and FAP; individuals with FAP are heterozygous for the prealbumin gene, carrying one normal and one mutant gene. Molecular analysis of the prealbumin gene yielded pertinent data on the genetic basis for FAP.     

Species specificity and developmental patterns of expression of the beta amyloid precursor protein (APP) gene in brain, liver and choroid plexus in birds.

1. Human APP cDNA hybridized to a 3.5 kb mRNA in liver and brain RNA from chickens, pigeons, quail and ducks as well as in RNA from choroid plexus of chicken and quail. In contrast to all other species hitherto examined a 1.6 kb mRNA hybridizing to APP cDNA was found in abundant amounts in RNA from chicken and quail livers. 2. In the chicken, before hatching, the levels of APP mRNA in total RNA from liver and choroid plexus were higher than those in RNA from liver and choroid plexus of adults. However, RNA from the rest of the brain of chicken embryos contained less APP mRNA than RNA from brain of adults. 3. In the chicken, between 10 and 40 days after hatching, APP mRNA levels in RNA from liver were higher than adult levels, APP mRNA levels in RNA from choroid plexus were similar to adult levels and APP mRNA levels in RNA from the rest of brain were below the adult levels.     

Prealbumin gene expression during mouse development studied by in situ hybridization

Localization of prealbumin mRNA in tissues from mice at various stages of gestation was investigated using in situ hybridization procedures. Prealbumin mRNA was detected as early as the 10th day of gestation. It was specifically localized in endodermal cells of the visceral yolk sac, tela choroidea, and hepatocytes. In the adult mice, prealbumin mRNA was localized in the hepatocytes and choroid plexus epithelial cells. These observations indicate that synthesis of prealbumin mRNA is initiated in several different types of cells at early stages of fetal development.     

The cDNA structure and expression analysis of the genes for the cysteine proteinase inhibitor cystatin C and for beta 2-microglobulin in rat brain

Tissue patterns of gene expression were analyzed by measuring mRNA levels and incorporation of radioactive amino acids for cystatin C and beta 2-microglobulin, the two extracellular proteins in the brain with the highest ratio of concentration in cerebrospinal fluid over that in blood plasma. The primary structure of rat cystatin C mRNA from choroid plexus was determined by nucleotide sequencing of cloned cDNA and the tissue patterns of gene expression were analysed by RNA blot analysis and in situ hybridization. Cystatin C was found to be composed of 120 amino acids and to contain a potential site for N-linked glycosylation. The tissue with the highest cystatin C mRNA level was the choroid plexus of the brain. Cystatin C mRNA was also detected in lower levels in other areas of the brain, testis, epididymis, seminal vesicles, prostate, ovary, submandibular gland, and, in trace amounts, in liver. Choroid plexus pieces in culture secreted radioactive cystatin C when incubated with radioactive leucine. Rat beta 2-microglobulin cDNA was cloned and identified by nucleotide sequencing and comparison of the obtained sequence with that of mouse and human beta 2-microglobulin cDNA. Tissue levels of beta 2-microglobulin mRNA in the rat were measured by hybridization to rat beta 2-microglobulin cDNA. The highest levels of beta 2-microglobulin mRNA were observed in liver and choroid plexus. Other parts of the brain and testis contained lower levels of beta 2-microglobulin mRNA.     

Transthyretin (prealbumin) gene expression in choroid plexus is strongly conserved during evolution of vertebrates

1. The major protein synthesized and secreted by the choroid plexus from mammals, birds, reptiles and probably amphibians is similar in subunit structure to transthyretin. 2. In mammals and birds the proportion of transthyretin mRNA is much higher in choroid plexus RNA than in liver RNA. No transthyretin mRNA is found in brain outside the choroid plexus. 3. Transthyretin-like protein, such as that secreted by the choroid plexus, was not detected in amphibian serum and was present in very low levels in reptile serum. 4. It is proposed that transthyretin synthesis and secretion arose earlier in evolution in the choroid plexus than in the liver.     

Localization of organic anion transporting polypeptide 3 (oatp3) in mouse brain parenchymal and capillary endothelial cells

Organic anion transporting polypeptide 3 (oatp3) transports various CNS-acting endogenous compounds, including thyroid hormones and prostaglandin E2, between extra- and intracellular spaces, suggesting a possible role in CNS function. The purpose of this study was to clarify the expression and localization of oatp3 in the mouse brain. RT-PCR analysis revealed that oatp3 mRNA is expressed in brain capillary-rich fraction, conditionally immortalized brain capillary endothelial cells, choroid plexus, brain and lung, but not in liver or kidney, where oatp1, 2 and 5 mRNAs were detected. Immunohistochemical analysis with anti-oatp3 antibody suggests that oatp3 protein is localized at the brush-border membrane of mouse choroid plexus epithelial cells. Furthermore, intense immunoreactivity was detected in neural cells in the border region between hypothalamus and thalamus, and in the olfactory bulb. Immunoreactivity was also detected in brain capillary endothelial cells in the cerebral cortex. These localizations in the mouse brain suggest that oatp3 plays roles in blood-brain and -cerebrospinal fluid barrier transport of organic anions and signal mediators, and in hormone uptake by neural cells.     

Cellular gene expression in papillomas of the choroid plexus from transgenic mice that express the simian virus 40 large T antigen.

Transgenic mice that contain the simian virus 40 (SV40) enhancer-promoter and large tumor (T) antigen gene develop papillomas of the choroid plexus. The tumors remain well differentiated on histological examination and express normal levels of tissue-specific mRNAs for transthyretin (TTR) and the 5-HT1C serotonin receptor, two differentiated cell markers. Both Northern (RNA) blot analysis and in situ cytohybridization have been used to monitor the steady-state levels of the mRNAs from the viral oncogene (T antigen) and from several cellular oncogenes. In situ hybridization demonstrated, in serial sections, increased levels of both T antigen mRNA and p53 mRNA localized in the tumor tissue but not in the normal brain tissue. The ratios of the steady-state levels of mRNA for p53/TTR and p53/L32, a ribosomal protein gene, were 2- to 20-fold higher in the tumor tissue than in the normal choroid plexus tissue. Several other oncogenes did not show elevated levels of mRNA in these tumors. p53 protein levels were not detectable in normal brain tissue, but p53 levels were very high in tumor tissue in which all of the p53 was found in a complex with the SV40 large T antigen. These data continue to show a close relationship between SV40 T-antigen-mediated tumorigenesis and the role of p53 in these tumors.     

Presence of N‐glycosylated transthyretin in plasma of V30M carriers in familial amyloidotic polyneuropathy: an escape from ERAD

Familial amyloid polyneuropathy (FAP) is an autosomal dominant disease characterized by deposition of amyloid related to the presence of mutations in the transthyretin (TTR) gene. TTR is mainly synthesized in liver, choroid plexuses of brain and pancreas and secreted to plasma and cerebrospinal fluid (CSF). Although it possesses a sequon for N‐glycosylation N‐D‐S at position 98, it is not secreted as a glycoprotein. The most common FAP‐associated mutation is TTR V30M. In a screening for monoclonal antibodies developed against an amyloidogenic TTR form, we detected a distinct TTR with slower electrophoretic mobility in Western of plasma from carriers of the V30M mutation, not present in normal plasma. Mass spectrometry analyses of this slower migrating TTR (SMT) identified both wild‐type and mutant V30M; SMT was undetectable upon N‐glycosidase F treatment. Furthermore, SMT readily disappeared in the plasma of V30M ‐ FAP patients after liver transplantation and appeared in plasma of transplanted domino individuals that received a V30M liver. SMT was also detected in plasma, but not in CSF of transgenic mice for the human V30M mutation. A hepatoma cell line transduced to express human V30M did not present the SMT modification in secretion media. Glycosylated TTR was absent in fibrils extracted from human kidney V30M autopsy tissue or in TTR aggregates extracted from the intestine of human TTR transgenic mice. Studies on the metabolism of this novel, glycosylated TTR secreted from FAP liver are warranted to provide new mechanisms in protein quality control and etiopathogenesis of the disease.     

Ontogenesis of transthyretin gene expression in chicken choroid plexus and liver.

1. Chicken liver transthyretin cDNA hybridizes strongly with choroid plexus transthyretin mRNA from chickens, pigeons, quails and ducks. 2. In the chicken at hatching the choroid plexus has reached 70%, total brain 30%, and liver 5.8% of their organ masses in adults. 3. The proportion of transthyretin mRNA in total RNA is 0.45-times the adult value in the choroid plexus of the chicken at hatching. 4. In the liver at hatching, the proportion of transthyretin mRNA in total RNA is 1.1-times the value in adult chickens. 5. The pattern of maturation of transthyretin gene expression in chicken liver is comparable to that in precocial, but differs from that in altricial mammals.     

Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met 30) gene

To analyze the pathologic processes of amyloid deposition in type I familial amyloidotic polyneuropathy (FAP), mice were made transgenic by introducing the human mutant transthyretin (TTR) gene(MT-hMet 30). An inbred strain of mouse, C57 BL/6, was chosen. Transgenic mice were killed using ether anesthesia at 3-mo intervals up to 24 mo after birth. In these transgenic mice, amyloid deposition started in the gastrointestinal tract, cardiovascular system, and kidneys and extended to various other organs and tissues with advancing age. The pattern of amyloid deposition was similar to that observed in human autopsy cases of FAP, except for its absence in the choroid plexus and in the peripheral and autonomic nervous systems. We extracted the amyloid fibrils from kidneys of these mice with a human mutant TTR gene and analyzed them immunochemically and electronmicroscopically. Deposited amyloid was shown to be composed of human mutant TTR and mouse serum amyloid P component. Amyloid fibril from transgenic mice was morphologically and immunohistochemically similar to that of human FAP. The most striking pathologic feature of the transgenic mice was the absence of amyloid deposition in the peripheral and autonomic nervous tissues. Thus, other intrinsic factors may be involved in amyloid deposition in the nervous tissues of human FAP.     

Rat ceruloplasmin. Molecular cloning and gene expression in liver, choroid plexus, yolk sac, placenta, and testis

A rat ceruloplasmin cDNA clone was isolated from a rat liver cDNA library and identified by partial nucleotide sequence analysis. Rat liver ceruloplasmin mRNA levels were measured during the acute phase response to inflammation by cytoplasmic dot hybridization to ceruloplasmin cDNA. Regulation of ceruloplasmin synthesis appeared to be at the mRNA level, with the concentration of ceruloplasmin mRNA increasing significantly 12 h after induction of inflammation, reaching a maximum of 350% of normal at 36 h and returning to normal levels within 60 h. Using Northern blot analysis, extrahepatic ceruloplasmin gene expression was observed in choroid plexus, yolk sac, placenta, and testis. All these tissues are at the interface between, and possibly involved in maintaining homeostasis in, adjacent extracellular compartments. No ceruloplasmin mRNA was detected in RNA from stomach and small intestine.     

Splice variants of the OB receptor gene are differentially expressed in brain and peripheral tissues of mice

A high affinity receptor for OB protein was recently cloned from the choroid plexus of mice. At least six alternatively spliced forms of the OB receptor (OB-R) gene have been described, all of which encode proteins containing the OB-R extracellular domain. One splice variant encodes a receptor with a long intracellular domain, OB-RL, that has been implicated in OB-R signaling. Here, we have used in situ hybridization to examine the localization of OB-R splice variants in brain and peripheral tissues of adult and newborn mice. Using a probe hybridizing with all known splice variants, we confirmed that OB-R mRNA was widely distributed in the adult tissues. In the CNS, choroid plexus was the major site of expression. We now demonstrate that OB-R mRNA is expressed in peripheral tissues; primarily associated with connective tissues. In addition, OB-R mRNA was detected at higher levels in peripheral tissues of newborn mice than in adult mice. With a probe specific for OB-RL, we confirmed that high mRNA expression was detected in hypothalamic nuclei, while low levels were observed in choroid plexus. We now report that in peripheral tissues of adult mice, OB-RL mRNA expression was either very low or undetectable. In newborn mice, the pattern of OB-RL message expression in the CNS was similar to that of adult mice, while bone was the site of highest OB-RL message expression in the peripheral tissue. These data suggest different biological roles for OB-R splice variants encoding the short and long forms of OB-R. The localization of OB-RL to hypothalamic nuclei supports the idea that OB-RL is the brain receptor that mediates OB protein signaling and actions. In addition, the expression of OB-R message in newborn mice also suggests a biological role of OB-R during development in mice.