Angela R. Aldred

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.

The Acute Phase Response in the Rodenta

In the rodent, the general response to acute inflammation and tissue damage is characterized by a complex rearrangement in the pattern of concentrations of proteins in the plasma leading to an increase in the sedimentation rate of erythrocytes, an increase in leukocyte concentration in the bloodstream, and a decrease in the hematocrit. Body temperature changes only slightly or not at all. The reasons for the change in plasma concentrations of proteins are changes in their rates of synthesis in the liver. Degradation of plasma proteins is not affected. The details of the acute phase response evolved in the interaction of species with their environment. Therefore, it is not surprising to find differences in the details of the acute phase response among species. For example, alpha 2-macroglobulin is a strongly positive acute phase reactant in the rat, but not in the mouse; C-reactive protein is a strongly positive acute phase protein in the mouse, but is not found in the rat. An inducible acute phase cysteine proteinase inhibitor system, which has evolved from a primordial kininogen gene, has been observed so far only in the rat. The changes in the synthesis rates of acute phase proteins during inflammation are closely reflected by corresponding changes in intracellular mRNA levels. In the liver, the capacity to induce the acute phase pattern of synthesis and secretion of plasma proteins probably develops around birth. Changes in mRNA levels are brought about by changes in transcription rates or by changes in mRNA stability. Kinetics of mRNA changes during the acute phase response differ for individual proteins. The main signal compound for eliciting the acute phase response in liver seems to be interleukin-6/interferon-beta 2/hepatocyte stimulating factor, whereas interleukin-1 leads to typical acute phase changes in mRNA levels only for alpha 1-acid glycoprotein, albumin, and transthyretin. Plasma protein genes are expressed in various extrahepatic tissues, such as the choroid plexus, the yolk sac, the placenta, the seminal vesicles, and other sites. All these tissues are involved in maintaining protein homeostasis in associated extracellular compartments by synthesis and secretion of proteins. Synthesis and secretion of plasma proteins in paracompartmental organs other than the liver is not influenced by the acute phase stimuli.

The cerebral expression of plasma protein genes in different species.

The cerebrospinal fluid (CSF) contains the same proteins as blood plasma, but with a different pattern of concentrations. Protein concentrations in CSF are much lower than those in blood. CSF proteins are derived from blood or synthesized within the brain. The choroid plexus is an important source of CSF proteins. Transthyretin is the protein most abundantly synthesized and secreted by choroid plexus. It determines the distribution of thyroxine in the cerebral compartment. Synthesis of transthyretin first evolved in the brain, then later it became a plasma protein synthesized in the liver. Other proteins secreted by choroid plexus are serum retinol-binding protein, transferrin, caeruloplasmin, insulin-like growth factors, insulin-like growth factor binding proteins, cystatin C, alpha 1-antichymotrypsin, alpha 2-macroglobulin, prothrombin, beta 2-microglobulin and prostaglandin D synthetase. Species differences in expression of the genes for these proteins are outlined, and their developmental pattern, regulation and roles in the cerebral extracellular compartment are discussed.

Synthesis of Transthyretin (Pre-albumin) mRNA in Choroid Plexus Epithelial Cells, Localized by In Situ Hybridization in Rat Brain

The sites of synthesis of transthyretin in the brain were investigated using in situ hybridization with [35S]-labeled recombinant cDNA probes specific for transthyretin mRNA. Autoradiography of hybridized coronal sections of rat brain revealed specific cellular localization of transthyretin mRNA in choroid plexus epithelial cells of the lateral, third, and fourth ventricles. Transferrin mRNA was also investigated and, in contrast to transthyretin mRNA, was localized mainly in the lateral ventricles. Our results indicate that substantial synthesis of transthyretin and transferrin mRNA may occur in the choroid plexus.

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.

Levels of messenger ribonucleic acids for plasma proteins in rat liver during acute experimental inflammation

The levels of mRNA for plasma proteins and for metallothionein in rat liver during the acute-phase response were studied by hybridization to specific cDNA probes. The mRNA forα2-macroglobulin, theβ-chain of fibrinogen, α1,-acid glycoprotein (so-called acute-phase reactants) reached a maximum level between 18 and 36 h after inducing an acute inflammation. The level of mRNA for metallothionein-I peaked earlier, after 12 h. The mRNA for transferrin showed a delayed increase with a broad maximum for its relative level after 36–60 h. The mRNA levels for albumin and α2u-globulin (so-called negative acute-phase reactants) decreased, reaching a minimum of 25 % of the normal level after 36 h (albumin) and after 72 h (α2u-globulin). The ratios of the rates of incorporation of leucine into the proteins over the levels of their mRNA in liver changed only a little, indicating that the rates of synthesis of plasma proteins in the liver are regulated at the mRNA level during the acute-phase response to inflammation.

Transthyretin expression in the rat brain: effect of thyroid functional state and role in thyroxine transport

Rats were made hypo- or hyperthyroid to study the role of thyroid hormones on cerebral transthyretin (TTR) mRNA expression. TTR mRNA was detected by Northern blot in rat liver, choroid plexus and meninges but not in cultured astrocytes or cultured cerebral endothelial cells. No changes were found in the levels of TTR mRNA in liver, choroid plexus or meninges in hypo- or hyperthyroid rats compared with the controls. In order to investigate the main route of thyroxine transport from blood to brain, the distribution of [125I]thyroxine in the brain was studied after intravenous (i.v.) and intraventricular (i.v.c.) injection by both direct counting and autoradiography. While distribution of [125I]thyroxine could be seen throughout the brain parenchyma after i.v. injection, the labelling was confined to the CSF spaces after i.v.c. administration. When protein synthesis was inhibited by cycloheximide treatment and [125I]thyroxine was injected intravenously, the uptake of [125I]thyroxine in the choroid plexus decreased while the uptake in the cerebral cortex increased. This indicates that thyroxine is transported into the brain primarily through the blood-brain barrier and not via the choroid plexus and CSF. We discuss the possibility that TTR has a role in the distribution of thyroxine throughout the brain.

Synthesis of rat transferrin in Escherichia coli containing a recombinant bacteriophage

Using mRNA from rat liver a cDNA library was constructed in lambda gt11Amp3. Immunochemical screening identified 15 clones producing transferrin. The identity of two clones was confirmed by nucleotide sequencing, which also indicated a presegment rich in hydrophobic amino acids but lack of a prosegment in precursor transferrin. A 920 base pair insert in one clone corresponded to 84% of the N-terminal domain of transferrin, which was synthesized as a hybrid protein with bacterial beta-galactosidase. A 1540 base pair insert in another clone corresponded to the N-terminal plus 50% of the carboxy terminal domain of transferrin. The product of this clone possessed only antigenic properties of transferrin.

Transthyretin expression evolved more recently in liver than in brain

1. Transthyretin was found to be synthesized and secreted by choroid plexus from rats, echidnas, and lizards, but not toads. 2. Transthyretin was observed in blood from placental mammals, birds, and marsupials, but not reptiles and monotremes. 3. The obtained data suggest that transthyretin synthesis by the liver evolved independently in the lineage leading to the placental mammals and marsupials and in that leading to the birds. 4. It is proposed that transthyretin gene expression in mammalian liver appeared about 200 million years later than its first occurrence in the choroid plexus of the stem reptiles.

Abundant Synthesis of Transthyretin in the Brain, but not in the Liver, of Turtles

The binding of thyroxine to proteins in the blood plasma of the turtle, Trachemys scripta, was analyzed by incubation with radioactive thyroxine, electrophoresis and autoradiography. Albumin and an alpha-globulin were found to bind thyroxine; no thyroxine-binding transthyretin was detected in the prealbumin region. In contrast to blood plasma, a thyroxine-binding prealbumin was observed in medium from T. scripta choroid plexus incubated in vitro. RNA was extracted from brain tissue containing choroid plexus and from liver of T. scripta and Chelydra serpentina and analyzed by hybridization with transthyretin cDNA from the lizard Tiliqua rugosa. The brain RNAs contained substantial amounts of transthyretin mRNA, whereas only trace amounts of transthyretin mRNA were detected in RNA from liver. No transthyretin mRNA was observed in RNA from kidney. The results support the hypothesis that the expression of the transthyretin gene first evolved in the choroid plexus of the brain at the stage of the stem reptiles, whereas abundant transthyretin synthesis in liver evolved much later, and independently, in mammals and birds.