The cerebral expression of plasma protein genes in different species

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Comp. Biochem. Physiol. Vol. 111B, No, 1, pp. 1-15, 1995 Copyright © 1995 Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0305-0491/95 $29.00 + 0.00

REVIEW The cerebral expression of plasma protein genes in different species* Angela R. Aldred, Charlotte M. Brack and Gerhard Schreiber Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia

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, ~:antichymotrypsin, ~-macroglobulin, prothrombin, ~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. Key words: Plasma protein; Gene expression; Messenger RNA; Brain; Choroid plexus; Transthyretin; Serum retinol-binding protein; Transferrin; Caeruloplasmin; Insulin-like growth factors; Insulin-like growth factor binding proteins; Cystatin C; Ctl-Antichymotrypsin; 0~2-Macroglobulin; Prothrombin; flE-Microglobulin; Prostaglandin D synthetase.

Comp. Biochem. Physiol. l l l B , 1-15, 1995.

Introduction Plasma proteins are defined as proteins which can be isolated in relatively large amounts from blood plasma and for which the main site of function is the intravascular compartment and the interstitial space. Plasma proteins equilibrate between vascular and interstitial space in a process requiring hours to days. The proportion of total plasma protein in the interstitial compartment is larger than that in *Dedicated to the memory of H. Cserr, a pioneer of cerebrospinal fluid and choroid plexus research. Correspondence to: G. Schreiber, Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia. Tel. 61 3 344 5914; Fax 61 3 347 7730. Received 8 April 1994; revised 5 October 1994; accepted 15 October 1994.

the vascular compartment (Schreiber et al., 1982). Proteins which have their main function within cells, such as muscle or liver enzymes, and which are observed in plasma because they are released during cell turnover or from damaged cells with leaky membranes, are not regarded as plasma proteins. The main site of synthesis of plasma proteins is the liver. Typical features of a plasma protein are its synthesis in the rough endoplasmic reticulum as a precursor protein with a presegment rich in hydrophobic amino acids, modification in the Golgi apparatus and secretion via vesicles. The main function of plasma proteins is to provide an appropriate environment in the extracellular space for the cells of the body (homeostasis). Some of the extracellular

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A . R . Aldred et al.

proteins, such as the proteins of the clotting system and proteinase inhibitors, help to preserve the integrity of the extracellular compartment. Proteins binding components, which are insoluble or sparely soluble in aqueous phase, such as iron and fatty acids, can serve as transport proteins. Examples are transferrin and albumin. Other extracellular proteins can prevent compounds, which are more soluble in lipid membranes than in plasma, from disappearing into the lipid phase of cell membranes, by binding to specific sites on the protein. An example of such a protein is transthyretin, which binds thyroid hormones with a high affinity. A circulatory pool of thyroxine of a sufficient size is thus created. The distribution of thyroxine between cells and their environment ig dependent on the presence of the binding protein in the extraceUular environment (Southwell et al., 1993). Several extracellular compartments are found in the body. They are separated from each other by barrier systems. Thus, the blood-brain barrier and the blood-cerebrospinal fluid barrier separate the extracellular space of the central nervous system from that in the main part of the body. The blood-testis barrier and the placenta-fetal membrane system separate the bloodstream from the lumen of the androgenital tract and the extracellular spaces in the embryo, respectively. The composition of the extracellular fluid can vary considerably in different compartments, related to the different requirements for extracellular homeostasis in different tissues. However, within the same compartment, the variation in concentration of proteins and other compounds is usually kept within a narrow limit by various regulatory mechanisms. This would appear to be of particular importance for the central nervous system, since the synaptic cleft, through which communication occurs between nerves, is in open connection with the extracellular environment. Several characteristic features are found in cells whose function is the synthesis and secretion of proteins. A high rate of protein synthesis is suggested by an extensive rougk endoplasmic membrane system. A high rate of processing and intracellular transport of proteins is suggested by a very well developed Golgi/vesicle system. An extensive enlargement of the cellular surface by numerous microvilli creates the possibility for high rates of transfer, or flux, through the plasma membrane. All these features are observed in abundance in liver cells, Sertoli cells and in the ependymal epithelial cells of the choroid plexus. Furthermore, all these cells are found lining associated extracellular compartments. An extensive litera-

ture exists documenting the dominant role of the liver in the synthesis and secretion of plasma proteins (reviewed by Schreiber, 1987; Schreiber and Aldred, 1993b). The cells of the body-testis barrier and the mother-fetus barrier have also been shown to synthesize and secrete plasma proteins (reviewed by Schreiber, 1987; Aldred et al., 1992; Aldred and Schreiber, 1993; Schreiber and Aldred, 1993b). Based on the analogies in cell structures and the position between compartments, it seems reasonable to suspect a significant role of the choroid plexus in the production of proteins for the extracellular compartment in the brain. The choroid plexus is a villous organ, protruding into the lateral and the third and fourth ventricles (reviewed by Netsky and Shuangshoti, 1975). The interior of the villi is filled by relatively loose stroma. Blood flow through the choroid plexus is extremely rapid, surpassed only by that through the glomus caroticum. The fenestrations of capillaries in the choroid plexus allow rapid exchange of fluid between the stroma and the capillary lumen. The ependymal epithelial cells of the choroid plexus, linked by tight junctions, form the blood-cerebrospinal fluid barrier. These epithelial cells show an abundance of microvilli covered by ciliae at the brain side. Most of the cerebrospinal fluid is produced by the choroid plexus (reviewed by Cserr, 1971). The cerebrospinal fluid filling the ventricles and the space surrounding the brain is in free communication with the interstitial fluid of the brain (Cserr et al., 1986). The composition of the cerebrospinal fluid changes only a little under normal circumstances, providing a relatively constant environment for the cells of the central nervous system. Essentially the same proteins as in blood plasma are found in the cerebrospinal fluid (see Table 1). However, the total protein content in cerebrospinal fluid is much lower than that in blood plasma. Furthermore, the pattern of concentrations of proteins in the cerebrospinal fluid differs from that in the blood plasma (reviewed by Schreiber, 1987). The intracerebral synthesis and the selective transfer from blood to brain [transfer rates depending on hydrodynamic volumes (Felgenhauer, 1974)] are the two factors explaining this difference in patterns of protein concentrations in blood plasma and cerebrospinal fluid. A second type of cells assumed to provide an appropriate environment for neuronal cells is the glia cells. Some of the extraceUular proteins discussed here are synthesized by both choroid plexus and glia cells. This review summarizes the current knowledge of the origin of plasma proteins in the

Cerebral expression of plasma protein genes

cerebral extracellular compartment. The species differences in expression of the genes for these proteins are outlined, and their developmental pattern, regulation and roles within the central nervous system are also discussed.

Transthyretin Transthyretin (formerly called thyroxinebinding prealbumin) is the major protein

product of the choroid plexus (Dickson et al., 1986). It was discovered in 1942 in human cerebrospinal fluid (Kabat et al., 1942a, b) and serum (Seibert and Nelson, 1942) as a protein migrating faster than albumin in nondenaturing electrophoresis at a pH above 8. Transthyretin is a tetramer composed of four identical subunits (Blake et al., 1971; Gonzalez and Offord, 1971; Rask et al., 1971), whose amino acid sequence was first reported for

Table 1. Concentrations of homeostasis-maintaining proteins in cerebrospinal fluid and blood plasma in humans (modified from Schreiber et al., 1989a; values compiled from Diem, 1960; Hagen and Elliott, 1973; Lentmer, 1981; Grubb and L6fberg, 1985; Haselbacher and Humbel, 1982). Proteins for which gene expression has been demonstrated in the brain are indicated in bold Concentration in cerebrospinal fluid

Protein

Function

Cystatin C

Inhibition of proteinases with cysteine in their active center

II2-Microglobulin

Light chain of class I histocompatibility antigen, cell recognition

Transthyretin

Binding of thyroxine

Concentration In cerebrospinal In blood Concentration in blood fluid plasma or serum plasma or serum (mg/1) (mg/l) ( x 104) 7.3

1.4

52,000

I. 1

2

550

14.7

176

833

Insulin-like growth

factor-ll

Regulation

0.05

0.95

526

Regulation

0.002

0.15

133

9.9

133

In,~dlin=li~growth

factor-I Thyroxine-binding globulin

Binding of thyroxine

Transferrln

Transport of iron

Albumin

Binding of water, bilirubin, fatty acids, thyroid hormones, aldosterone and other compounds

~l-Acid glycoprotein

Unknown, perhaps influencing of immune reactions

3.5

980

36

~2-HS-glycoprotein

Unknown

1.7

600

28

Caernloplasmin

Binding of copper, ferroxidase, amine oxidase, superoxide dismutase

0.9

370

24

~rAntitrypsin

Inhibition of elastase

7

3000

23

Inhibition of a broad spectrum of proteinases by binding to "bait" region

4.6

3000

15

Haptoglobin

Scavenging of globulin

2.24

4800

5

Plasminogen

Precursor of plasmin, flbrinolysis

0.25

700

3.6

Fibrinogen

Blood coagulation

0.6

2600

2.3

-MacroglobuHn

Insulin-like growth

factor binding proteins

Regulation

~t-Antichymotrypsin

Inhibition of proteinase

Prothrombin

Precursor of thrombin, blood coagulation

Prostaglandin D synthetase

Unknown

0.132 14

2600

54

155

40,000

39

4

A.R. Aldred et al.

human transthyretin by Kanda et al. (1974). A molecular weight of 54,980 can be calculated from the amino acid composition. The threedimensional structure of transthyretin is known with a resolution of 1.8/~ (Blake et al., 1978; Hamilton et al., 1993). Although containing the sequence Asn-X-Ser, a recognition sequence for the attachment of carbohydrate moieties, transthyretin is not a glycoprotein (Putnam, 1975). Transthyretin is one of the plasma proteins binding thyroxine (Ingbar, 1958, 1963). It has two binding sites for thyroxine in a central channel formed by the four subunits. Negative cooperatively exists for the two binding sites with Kl = 1.0 x 10SM -~ and K2 = 1.0 x 106M -I (Ferguson et al., 1975; Somack et al., 1982). In addition to thyroxine, transthyretin binds retinol (Alvasker et al., 1967) via serum retinolbinding protein (Kanai et al., 1968). Due to the advances in the techniques for determination of the sequences of amino acids and nucleotides, the primary structure of transthyretin is known for a large number of species. These are the human (Kanda et al., 1974; Mita et al., 1984), sheep (Tu et al., 1989), rabbit (Sundelin et al., 1985), rat (Dickson et al., 1985b; Duan et al., 1989), mouse (Wakasugi et al., 1985), pig (unpublished data), sugar glider (Babon, 1993), dunnart (Babon, 1993), Tammar wallaby (Brack et al., 1994), short-tailed grey possum (Heyes, 1993), chicken (Duan et al., 1991) and the stumpy-tailed lizard (Achen et al., 1993). Very high levels of transthyretin mRNA are found in the choroid plexus (Dickson et al., 1985a, b; Dickson and Schreiber, 1986; Dickson et al., 1986; Herbert et al., 1986; Kato et al., 1986; Mita et al., 1986; Stauder et al., 1986). Molecular titration of transthyretin mRNA, using a calibrated rat transthyretin mRNA antisense probe for hybridization in a ribonuclease protection assay, showed that the concentration of transthyretin mRNA in rat choroid plexus was 11.3 times greater than in liver, with 4.4 mg mRNA in 1 g choroid plexus and 0.39mg mRNA in 1 g of liver (Schreiber et al., 1990). 1 g of RNA from rat choroid plexus contained 20 times more transthyretin mRNA than 1 g of RNA from rat liver. Similarly, for the chicken, 1 g of RNA from choroid plexus contained 47 times more transthyretin mRNA than 1 g of RNA from liver (Duan et al., 1991). All of the transthyretin mRNA observed in choroid plexus was located in the epithelial cells, i.e. the blood-cerebrospinal fluid barrier (Stauder et al., 1986). Very small amounts of transthyretin mRNA, much smaller than those found in the choroid plexus and in the liver, have been found in the meninges in rat brains (Stauder et al., 1986; Blay et al., 1993).

About 20% of the protein newly synthesized in choroid plexus and about 50% of newly synthesized protein secreted into the medium by in vitro incubated choroid plexus pieces was transthyretin (Dickson et al., 1986). In experiments using in vitro perfused sheep choroid plexus, transthyretin secretion by choroid plexus was found to be directed exclusively towards the brain (Schreiber et al., 1990). It was proposed that the transthyretin synthesized and secreted by choroid plexus may be involved in the distribution of thyroxine in the brain (Dickson et al., 1987a; Schreiber et al., 1990; Southwell et al., 1993). Transthyretin is known to be a negative acute phase protein, that is its concentration in the blood plasma and its mRNA level in the liver decrease during the acute phase response to trauma and inflammation (Dickson et al., 1982; Thomas and Schreiber, 1985; Dickson et al., 1985b; Birch and Schreiber, 1986; Dickson et al., 1986, 1987b; Schreiber et al., 1989b; MiUand et al., 1990). However, in contradistinction to the decrease of transthyretin mRNA in the liver during the acute phase response, the levels of transthyretin mRNA in the choroid plexus did not change during acute inflammation (Dickson et al., 1986, 1987b). Consistent with the importance of the thyroid hormones for the development of the brain in the embryo,, expression of the transthyretin gene occurs early during foetal development (Thomas et al., 1988; Fung et al., 1988; Thomas et al., 1989; CavaUaro et al., 1993). The rate of growth of the brain and the choroid plexus and the levels of transthyretin mRNA are correlated in a growing animal, the transthyretin gene being expressed in choroid plexus earlier, in relation to the length of pregnancy, in precocial than in altricial animals (Tu et al., 1990; Southwell et al., 1991; Schreiber and Aldred, 1993a, b). Thus, the timing of the onset of transthyretin gene expression in the choroid plexus is correlated with the formation of the blood-brain barrier and the functional maturation of the brain. During evolution, expression of the transthyretin gene occurred earlier in the choroid plexus than in the liver. The strong conservation of the structure and expression of the transthyretin gene in mammals and in the chicken suggests that transthyretin was already synthesized in the common ancestor of birds and mammals, i.e. the stem reptiles, living about 350 million years ago (Duan et al., 1991). Strong expression of transthyretin in the choroid plexus, but none in the liver, was found for the stumpy-tailed lizard (Achen et al., 1993). In the cane toad, an amphibian, the choroid plexus

Cerebral expressionof plasma protein genes was found to synthesize a lipocalin as the most abundant protein for secretion, but no expression of transthyretin was observed (Achen et al., 1992). Echidna choroid plexus, incubated in vitro, synthesized and secreted transthyretin into the surrounding medium (Schreiber et al., 1993). Transthyretin is not abundant in echidna blood plasma, nor is transthyretin mRNA in echidna liver (Schreiber et al., 1993). In the liver, transthyretin expression evolved much more recently than in the brain (Schreiber et aL, 1993). In Australian marsupials, the expression of the transthyretin gene in liver first occurred during the radiation of the diprotodont marsupials in Australia (Richardson et al., 1993).

Serum Retinol-binding protein Serum retinol-binding protein transports retinol from the liver to target tissues (reviewed by Blaner, 1989). It consists of one polypeptide chain of about 21,000 Da, with a single binding site for one molecule of retinol. In the bloodstream, retinol-binding protein circulates as a 1:1 complex with transthyretin. After cellular uptake of retinoid compounds, cellular retinolbinding proteins (which are not related to the retinol-binding proteins in the blood), bind retinol and retinoic acid for intraceUular transport and processing (reviewed by Blomhoff et al., 1990). Soprano et al. (1986) detected serum retinolbinding protein mRNA, by Northern blot analysis of polyadenylated RNA, in rat brain at 1-3% of the level found in liver. Using Northern blot analysis and reverse transcription coupled with a polymerase chain reaction, Duan and Schreiber (1992) found wide variation in the levels of serum retinol-binding protein mRNA in choroid plexus from rats, mice, sheep and cattle. Serum retinol-binding protein mRNA could not be detected by Northern blot analysis in total RNA from choroid plexus of mice and dogs (Duan and Schreiber, 1992), nor in polyadenylated RNA from choroid plexus of rats (Thomas et al., 1988). However, using reverse transcription coupled with a polymerase chain reaction (a much more sensitive method than Northern analysis of total RNA), serum retinol-binding protein mRNA was detected in RNA from choroid plexus and other parts of the brain from rats and mice (Duan and Schreiber, 1992). The wide variation of serum retinol-binding protein expression in choroid plexus among species suggests that serum retinol-binding protein is not of similar general importance in the transport of retinol within the cerebrospinal fluid compartment of all species.

Transferrin The major function of transferrin is the transport of iron, which is relatively insoluble in the bloodstream (reviewed by Morgan, 1981). Transferrin has bacteriostatic activity, probably related to its iron-binding capability (reviewed by Weinberg, 1984) and is required for the growth (reviewed by Morgan, 1981) and differentiation (reviewed by Bowman et al., 1988) of many cell types. Iron has important roles in motor function, mental development and behaviour (reviewed by Pollit and Leibel, 1982). Transferrin was observed in cerebrospinal fluid by several workers (Burtin, 1959; Parker and Beam, 1962; Frick, 1963). After incubation of rat choroid plexus pieces in vitro, transferrin represented 2% of newly synthesized cellular protein and 4% of secreted protein (Aldred et al., 1987a). Isolation of eDNA probes for transferrin enabled detection of transferrin mRNA in brains from chickens (McKnight et al., 1980) and rats (Levin et al., 1984; Idzerda et aL, 1986). High levels of transferrin mRNA were found in rat choroid plexus (Dickson et al., 1985a; Aldred et al., 1987a), localized in the epithelial cells (Bloch et al., 1987). Quantitation of transferrin mRNA by ribonuclease protection assay (Tu et al., 1991) showed that the amount of transferrin mRNA in rat choroid plexus (470 ng transferrin mRNA/g wet weight tissue) was one-eighth of that in liver (3700 ng/g) and 30-fold greater than in the rest of the brain (15ng/g). The observation of regulation of transferrin mRNA levels in cultured rat choroid plexus epithelial cells by serotonin (Tsutsumi et al., 1989; Tsutsumi and Sanders-Bush, 1990) is interesting, because serotonin is involved in regulation of the production of cerebrospinal fluid (Lindvall-Axelsson et al., 1988). However, because of the striking species-specificity in the pattern of transferrin gene expression in the brain (see below), extrapolation of the interpretation of these observations made with rat cells to other species, such as humans, should be avoided. Low levels of transferrin mRNA have been detected in many other areas of rat brain (Dziegielewska et al., 1985; Aldred et al., 1987a), mainly in oligodendrocytes (Bloch et al., 1985). Despite vigorous synthesis of transferrin in rat choroid plexus, due to the small size of this tissue, the choroid plexus might contribute only a fraction of overall cerebral transferrin synthesis. It has been proposed that oligodendrocytes are responsible for at least 80% of transferrin mRNA present in mouse brain (Bartlett et al., 1991). Transferrin mRNA has also been detected in cultured astrocytes (Espinosa de los Monteros et al., 1990; Zahs

6

A.R. Aldred et al.

1993) and neurons (Espinosa de los Monteros et al., 1990). In the brain of the growing rat, the levels of transferrin mRNA are not related to the net growth of the total brain (Levin et al., 1984; Thomas et al., 1989). Expression of the transferfin gene in rat brain begins only when the period of rapid brain growth is essentially over, i.e. after 12 postnatal days. This corresponds to the time of weaning. It suggests that the regulation of transferrin gene expression in rat brain may be related to a change in food composition. In a comparative study, Northern blot analysis showed a striking species specificity of the pattern of expression of transferrin mRNA in the brain (Tu et al., 1991). In contrast to the high proportions of transferrin mRNA in total RNA from rat choroid plexus, only small proportions of transferrin mRNA were found in choroid plexus from mice and dogs, while still smaller proportions were detected in rabbit choroid plexus. Transferrin mRNA was not detected at all in human, sheep, pig, cow or guinea-pig choroid plexus. For sheep, pig, rat, mouse, rabbit, dog and cow, various amounts of transferrin mRNA were found in other parts of the brain. Because of the wide variations in the pattern of transferrin gene expression among different species, caution is indicated in the comparative interpretation and extrapolation to other species of transferrin synthesis seen in rat choroid plexus cells (see above). It is unlikely that transferrin synthesis and secretion by the choroid plexus has a general role in iron transport through the bloodcerebrospinal fluid barrier. It has been proposed that plasma transferrin is responsible for iron delivery to the brain across the blood-brain barrier (Fishman et al., 1987; Pardridge et al., 1987), via transferrin receptors located on the brain capillary endothelium (Jefferies et al., 1984). However, in myelin-deficient rats with almost complete depletion of transferrin in the brain, iron uptake and distribution within the brain is not impaired, raising the possibility that transferrin synthesized by the choroid plexus can mediate iron transport within the brain (Gocht et al., 1993). et al.,

Caeruloplasmin Caeruloplasmin is a copper-containing protein with several functions (reviewed by Frieden, 1979; Gutteridge and Stocks, 1981; Cousins, 1985): (i) transport of copper, (ii) ferroxidase, amino oxidase and superoxide dismutaselike activities, (iii) deaminase activity, and (iv) possible anti-oxidant and protective roles in the inflammatory response. Caeruloplasmin

consists of a single polypeptide chain with a molecular weight of 134,000. Caeruloplasmin mRNA was detected in choroid plexus RNA from adult rats by Northern blot analysis (Aldred et al., 1987b). The levels of caeruloplasmin mRNA in foetal rat brain between 14.5 and 20.5 days of gestation, and in newborn rat brain were higher than the level in the brain of adult rats (Thomas et al., 1989). A 4.7 kb caeruloplasmin transcript was detected in low abundance in polyadenylated RNA from choroid plexus of newborn rats by Northern blot analysis (Fleming and Gitlin, 1990). However, the low sensitivity of detection did not allow detection of caeruloplasmin mRNA in total RNA from brain of foetal and newborn rats. Caeruloplasmin mRNA has also been detected in human brain by reverse transcriptasepolymerase chain reaction analysis (Yang et al., 1990). The function of caeruloplasmin synthesized in the brain is not known. It may be related to one or a number of the known functions of the caeruloplasmin molecule.

Insulin-like Growth Factors Insulin-like growth factors (IGFs) are members of the insulin gene family which stimulate cellular metabolism, differentiation and proliferation (reviewed by Sara and Hall, 1990). The IGF-I gene is expressed in several regions of the central nervous system and in cultured neuronal and glial ceils (reviewed by Sara et al., 1991). Multiple IGF-I mRNA species are expressed in varying abundance in a developmentally and regionally specific manner (reviewed by Sara and Hall, 1990). Greater abundance of IGF-I mRNA is found in foetal, compared with adult brain, in humans (Sandberg et al., 1988) and in rats (Rotwein et al., 1988). Post-translational modification of the IGF-I precursor results in two neuroactive products: a truncated IGF-I with autocrine or paracrine actions in promoting cell growth and differentiation; and a tripeptide which may modulate neurotransmission in the central nervous system (reviewed by Sara et al., 1991). The similar pattern of cerebral expression of IGF-1 and the IGF-1 receptor also suggests a local autocrine or paracrine mode of action for cerebral IGF-1 (Bondy et al., 1992). Studies with transgenic mice have demonstrated a role for IGF-I in stimulation of brain growth (Behringer et al., 1990). Proliferative effects of IGF-I on cultured neonatal rat oligodendrocytes (McMorris et al., 1986) and astroglial cells (Han et al., 1987) have also been observed.

Cerebral expressionof plasma protein genes The IGF-II gene is expressed in the choroid plexus of rats (Beck et al., .1987; Hynes et al., 1988; Ichimiya et al., 1988; Stylianopoulou et al., 1988), humans (McKelvie et al., 1992), pigs (Nilsson et al., 1992) and sheep (Delhanty and Han, 1993; Holm et al., 1994). It is also expressed in the leptomeninges of rat brain (Hynes et al., 1988; Stylianopoulou et al., 1988). IGF-II is present in human cerebrospinal fluid (Haselbacher and Humbel, 1982). Secretion of newly synthesized IGF-II from primary cultures of sheep choroid plexus epithelial cells has recently been demonstrated (Holm et aL, 1994). During development, the distribution of IGF-II mRNA has been reported to be widespread in human brain from mid-gestation (20-22 postconceptional weeks) through to the perinatal (37-42 postconceptional weeks) period (McKelvie et al., 1992). After two postnatal months, a dramatic decline in IGF-II mRNA levels was observed in human brain parenchyma, with persisting abundance of IGF-II mRNA in choroid plexus (McKelvie et al., 1992). However in foetal sheep brain, IGF-II mRNA was not detected in extrachoroidal regions (Delhanty and Han, 1993) and in rat brain, from the late embryonic stage through to adulthood, IGF-II mRNA was observed only in choroid plexus (Beck et al., 1987, 1988), indicating possible species differences in the distribution of IGF-II gene expression in foetal brain. IGF-II mRNA is first detected in foetal rat choroid plexus during the mid-gestational period when the choroid plexus Anlage first develops (Wood et al., 1990). IGF-II mRNA expression in primordial rat choroid plexus epithelium begins at day 13 and increases gradually (Cavallaro et al., 1993) to sustained high level expression throughout postnatal life and adulthood (Beck et al., 1988). In the primordial choroid plexus stroma, IGF-II mRNA is expressed in abundance prior to choroid plexus morphogenesis but decreases as embryogenesis proceeds and is absent by adulthood (Cavallaro et al., 1993). Multiple mRNA species are transcribed from the IGF-II gene (reviewed by Sara and Hall, 1990). In human choroid plexus, expression of a 6.0 kb IGF-II transcript predominates in early fetal life, and a 4.8kb transcript predominates by 35 postconceptional weeks and thereafter into infancy (McKelvie et al., 1992). Tissue-specific parental imprinting of the IGF-II gene was observed in mice with a targeted disruption of the IGF-II gene (DeChiara et al., 1991). Embryonic mice express the paternal IGF-II allele in most tissues, while both paternal and maternal IGF-II alleles are expressed in the choroid plexus and leptomeninges, tissues in which IGF-II expression persists in adult life.

A reduced growth rate of these mice also provided direct evidence for a physiological role of IGF-II in regulation of embryonic growth (DeChiara et al., 1990).

Insulin-like Proteins

Growth

Factor

Binding

IGFs are normally complexed with IGF binding proteins (reviewed by Rechler, 1993), specific, high affinity binding proteins which modulate the interaction of IGFs with the IGF-1 receptor. The role of IGF binding proteins in the nervous system is not well understood (reviewed by Ocrant, 1991). IGF binding protein-1 mRNA was detected at very low levels in the rat central nervous system by Northern blot analysis (Murphy et al., 1990; Ooi et al., 1990). IGF binding protein-2 mRNA has been detected in brain from fetal (Brown et al., 1989) and adult rats (Margot et al., 1989), and fetal (Agarwal et aL, 1991) and adult (Binkert et al., 1989) humans. Tseng et al. (1989) reported exclusive localization of IGF binding protein-2 mRNA in adult rat brain to the choroid plexus by in situ hybridization, although detection in brain and pituitary by Northern blot analysis has also been reported (Lamson et al., 1989). Sustained high levels of IGF binding protein-2 mRNA were detected in developing sheep choroid plexus throughout gestation and for nine postnatal weeks, with slightly lower levels detected in the adult (Delhanty and Han, 1993). In early to mid-gestation, IGF binding protein-2 mRNA was also observed in sheep cortex, hypothalamus, cerebellum and medulla (Delhanty and Han, 1993). Throughout the postnatal life of rats, IGF binding protein-2 and IGF-I/ mRNAs are expressed at high levels in choroid plexus (Lee et al., 1993). IGF binding protein-2 mRNA is also found in postnatal rat astroglial cells (Lee et al., 1993) and in cultured fetal rat neuronal and astroglial cells (Lamson et al., 1989). During postnatal rat brain development, expression of IGF binding protein-2 mRNA in astroglia is anatomically and temporally coordinated with neuronal IGF-I mRNA expression (Lee et al., 1993). In mid-gestational rat embryos, IGF binding protein-2 mRNA is expressed in choroid plexus epithelium, whereas IGF-II mRNA is present in the mesenchymal layer of choroid plexus (Wood et al., 1990), suggesting that IGF binding protein-2 might be involved in the transport of IGF-II to the cerebrospinal fluid. IGF binding protein-2 is secreted from cultured rat choroid plexus (Ocrant et al., 1990), astroglial (Hart et al., 1988; Ocrant et al., 1989) and neuronal (Ocrant et al.,

8

A.R. Aldredet al.

1989) cells and has been detected in human, rat and bovine cerebrospinal fluid (reviewed by Rechler, 1993). A complex involving IGF binding protein-3 is considered to be the storage form of the endocrine IGFs in blood (Blum, 1994). IGF binding protein-3 mRNA was not detected in rat hypothalamus and brain cortex by Northern blot analysis (Shimasaki et al., 1989), but secretion of IGF binding protein-3 by cultured astrocytes and neurons has been reported (Han et al., 1988; Ocrant et al., 1989). The pattern of expression of IGF binding protein-4 mRNA in rat brain differs markedly from that of IGF binding protein-2. IGF binding protein-4 mRNA is detected in rat choroid plexus and meninges at embryonic day 20 (Brar and Chernausek, 1993). With increasing embryonic and postnatal development, IGF binding protein-4 mRNA is detected in an increasing number of brain regions, resulting in widespread distribution of IGF binding protein-4 mRNA in adult brain (Brar and Chernausek, 1993). IGF binding protein-5 mRNA, like IGF binding protein-4 mRNA is abundant within several discrete regions of rat brain (Stenvers et al., 1994). However, the neuroanatomical distribution of IGF binding protein-5 mRNA is distinct from that observed for IGF binding protein-2 (Bondy and Lee, 1993) and IGF binding protein-4 (Stenvers et al., 1994). It is interesting that at least three IGF binding proteins are synthesized and secreted by the choroid plexus. Such proteohormone binding proteins might influence the activity of the proteohormone and may be part of a regulatory system in the cerebral compartment. Other possible functions of IGF-binding proteins in the central nervous system are the transport of IGFs between tissue compartments and effects independent of the binding of IGF.

Cystatin C Cystatin C (also known as v-trace or post-y-globulin) is one of a family of proteinase inhibitors which regulate the activities of cysteine proteinases (reviewed by Barrett, 1987). It is the extraceUular brain protein with the highest ratio of its concentration in cerebrospinal fluid over that in blood plasma. Cystatin C mRNA was abundant in rat choroid plexus and, compared with choroid plexus, was detected at lower levels in brain minus choroid plexus (Cole et al., 1989). Rat choroid plexus pieces, incubated with radioactive amino acids, synthesized and secreted cystatin C (Cole et al., 1989). The cerebral expression of the cystatin C gene was examined

in a comparative study of 11 mammalian and four avian species (Tu et al., 1992). Cystatin C expression in choroid plexus was conserved among all studied species. However, in brain from which choroid plexus had been removed, cystatin C mRNA levels varied. Relatively high cystatin C mRNA levels, comparable with those in choroid plexus, were detected in RNA from brain minus choroid plexus from rabbits, dogs and pigs. Cystatin C mRNA was detected in brain minus choroid plexus from foxes, guineapigs, cats, rats and sheep, but not from cattle, chickens, pigeons,, ducks and quail. The cystatin C gene was found to be expressed very early in ontogeny. In sheep (Tu et al., 1990) and chicken (Tu et al., 1992) choroid plexus, cystatin C mRNA levels increased throughout foetal development, reaching adult levels by the time of birth. In rat brain, cystatin C mRNA reached adult levels a little later, around 15 days after birth (Thomas et al., 1989). During development, cystatin C may function to protect cerebral tissues from inappropriate proteolytic degradation by proteinases released during growth and remodelling of tissues. The strong conservation and early ontogeny of expression of cystatin C in choroid plexus indicate an important function of cystatin C within the central nervous system. In patients with hereditary cerebral hemorrhage with amyloidosis, massive hemorrhage is caused by deposition of variant cystatin C as amyloid in cerebral arteries, with an associated abnormally low concentration of cystatin C in the cerebrospinal fluid (reviewed by Grubb et al., 1984).

Other Proteins During normal aging and in patients with Alzheimer's disease ~rantichymotrypsin, a serine protease inhibitor, is associated with deposits of flA4 amyloid protein found in the brain in plaque cores and in the walls of blood vessels. Using in situ hybridization, ~,-antichymotrypsin mRNA was found in astrocytes of human (Abraham and Potter, 1989; Pasternack et al., 1989) and monkey (Koo et al., 1991) brains. In human brain, levels of 0q-antichymotrypsin mRNA increased with age, from negligible levels in foetuses and young adults to slightly higher levels in aged individuals (Koo et al., 1991). Levels of 0t,-antichymotrypsin mRNA are greatly elevated in the brain of individuals with Alzheimer's disease (Pasternack et al., 1989). ~t2-Macroglobulin is a proteinase inhibitor which acts by binding proteinases to the "bait" region of the molecule. Astroglial cells from newborn rats have been shown to synthesize

Cerebral expressionof plasma protein genes and secrete ~2-macroglobulin (Gebicke-Haerter et aL, 1987). Synthesis of 0t2-maeroglobulin in rat brain is regulated independently from that in liver (Bauer et al., 1988). Cultured astroglia contained ~t2-macroglobulin mRNA, detected by Northern blot analysis (Saitoh et al., 1992). ct2-Macroglobulin mRNA was detected in fetal rat choroid plexus at much higher levels than in total brain (Thomas et al., 1989). The ~2-macroglobulin mRNA level in total brain increased during foetal development, approaching adult levels soon after birth. Messenger RNA for prothrombin, the proenzyme of thrombin was detected in rat brain tissues throughout development, by Northern blot, in situ hybridization and polymerase chain reaction studies (Dihanich et al., 1991). Prothrombin mRNA was present as early as after 13 embryonic days. It was also detected in cultured human astrocytes (Deschepper et al., 1991). Messenger RNA for antithrombin III, a proteinase inhibitor, was detected in rat brain tissues by Northern blot using a human antithrombin III cDNA probe (Deschepper et al., 1991). The increasing evidence for synthesis and release of proteases and protease inhibitors by cells within the central nervous system has led to the suggestion that a delicate balance between these types of proteins modulates brain cell growth and differentiation (Monard, 1988). //2-Microglobulin is the light chain of the class l histocompatibility antigens. High levels of fl~-microglobulin mRNA are detected in adult rat choroid plexus and lower levels are present in other parts of rat brain (Cole et al., 1989). Low levels of //2-microglobulin mRNA have also been detected in the brain of adult mice (Drezen et al., 1993). During fetal and neonatal development of rats, //2-microglobulin mRNA levels in brain increase in parallel to the growth of the brain (Thomas et al., 1989). Lipocalins are a family of proteins with a calyx or cup-like three-dimensional structure. Lipophilic molecules are bound within the hydrophobic interior pocket of the calyx. Beta-trace protein, a lipocalin, was reported at a higher concentration in choroid plexus than in other parts of the brain (Olsson and Link, 1973). The primary sequence of//-trace protein, isolated from human cerebrospinal fluid (Kuruvilla et al., 1991; Zahn et al., 1993), is very similar to the N-terminal sequence of human brain prostaglandin D synthetase (Nagata et al., 1991). Prostaglandin D synthetase mRNA is detected in choroid plexus, leptomeninges and oligodendrocytes of adult rat brain (Urade et al., 1993). The protein most abundantly synthesized and secreted by amphibian choroid plexus is a member of the lipocalin superfamily,

with a sequence similarity to prostaglandin D synthetase (Achen et aL, 1992). In contrast transthyretin, a carrier of the lipophilic thyroid hormones, is the major protein secreted by choroid plexus of mammals, birds and reptiles (Harms et al., 1991). Expression of brainspecific prostaglandin D synthetase mRNA is dependent on thyroid hormone during rat brain development (Garciafernandez et aL, 1993). One might speculate that the lipocalin synthesized and secreted by amphibian choroid plexus may be involved in the cerebral transport of small hydrophobic molecules. Messenger RNA encoding fetuin, a foetal plasma protein, has been detected by in situ hybridization in fetal sheep brain (Dziegielewska et al., 1993).

Conclusions A comparison of the concentrations of extracellular proteins in cerebrospinal fluid with those of blood plasma is given in Table 1. Three proteins stand out by the high ratio of their level in cerebrospinal fluid to that in blood plasma. They are cystatin C, //:-microglobulin and transthyretin. The intracerebral expression of the genes for these three proteins substantially contributes to their relative high levels in the cerebrospinal fluid. All three proteins are made in the choroid plexus. The position and structure of the choroid plexus are well suited to its role in synthesis and secretion of proteins for the extracellular compartment of the brain. The choroid plexus is a typical example of a paracompartmental organ, i.e. an organ whose cells are involved in homeostasis in an adjacent extracellular compartment (Schreiber and Aldred, 1993c). Transthyretin is the protein most abundantly synthesized and secreted by the choroid plexus. About 12% of all protein made by the choroid plexus and about one half of newly made protein secreted by the choroid plexus is transthyretin (Dickson et al., 1986). This makes the ependymal epithelial cells of the choroid plexus one of the cell types of the body most highly specialized for the synthesis of one particular protein. Consistent with its important role in the distribution of thyroxine in the brain, the structure of transthyretin is highly conserved (Duan et al., 1991; Richardson et al., 1994; Bracket al., 1994). It is expressed early in the embryonic development of the brain at about the same time as the appearance of a barrier between the blood and the brain (Thomas et al., 1988, 1989). This is understandable since the presence of appropriate amounts of thyroid hormones is critical for the early differentiation of neurons.

10

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During evolution, the expression of transthyretin occurs much earlier in the brain than in the liver (Achen et al., 1992, 1993; Schreiber et ai., 1993; Richardson et al., 1993, 1994). In fact, transthyretin first evolved as an extraceUular brain protein. Only later did it become a plasma protein synthesized in the liver due to a change in tissue specificity of transthyretin gene expression. The brain is the organ which increased in size most dramatically during the evolution of higher vertebrates (Wilson, 1991). The increase in lipid space into which transthyretin could partition might have been one of the factors contributing to the selection pressure for the evolution of a thyroxine-binding protein retaining thyroxine in the extracellular space of the brain. Due to the lipophilicity of thyroid hormones, and the reactive and sparely soluble nature of iron and copper, protein-bound forms of these molecules are required for their appropriate distribution to tissues. Synthesis of binding proteins for these molecules, transthyretin, transferrin and caeruloplasmin, respectively, by the choroid plexus allows fine control of the levels of their ligands in the cerebrospinal fluid and the extracellular fluid of the central nervous system. For example, the relative enrichment in cerebrospinal fluid of transthyretin compared with other thyroid hormone binding proteins, and the lower concentration of transthyretin in cerebrospinal fluid compared with blood plasma, results in a higher concentration of free (unbound) thyroxine in cerebrospinal fluid than in the main vascular compartment of the body (Schreiber et al., 1989a). In the case of transferrin, in addition to synthesis by the choroid plexus, synthesis by oligodendrocytes also contributes to the local requirements for iron homeostasis within the cerebral compartment. During development, the growth and differentiation of cells require tissue remodelling. Proteinases involved in these processes are synthesized in the cerebral compartment for local function. Prevention of inappropriate proteolytic degradation of cerebral tissues by proteases released from cells may be achieved by proteinase inhibitors locally synthesized by the choroid plexus and other cerebral tissues. Cystatin C, ~tE-macroglobulin, 0q-antichymotrypsin and antithrombin III are such proteinase inhibitors which may contribute to the homeostatic control of proteolysis within the brain. Recently, a new function for proteins in plasma has been discovered in addition to their well known roles in transport, inhibition of proteinases and blood clotting. This is the interaction of specific binding proteins with proteohormones. This binding leads to regulation of the activity of the hormone. Thus,

insulin-binding protein interacts with insulin and a specific growth hormone-binding protein, a part of the growth hormone receptor shed into the bloodstream, interacts with growth hormone (Baumann, 1994). The synthesis of insulin-like growth factor-binding proteins in the choroid plexus is well documented (see the section on insulin-like growth factor binding proteins). No data are yet available about the extent to which hormone activity in the cerebral extracellular space might be modulated by binding to the specific extracellular proteins synthesized in the brain. Finally, comparative studies of tissue-specific gene expression can give insight into the relative importance of the function of a protein. Such studies led to the discovery that transthyretin evolved first as a brain-specific protein, then later as a plasma protein (discussed above). The strong conservation of cerebral expression of transthyretin and cystatin C among species indicates the requirement for the functions of these proteins in the cerebral compartment. Demonstration of species-specificity of the pattern of cerebral synthesis of serum retinolbinding protein, transferrin, and IGF-II raises the question of whether these proteins are of general importance in the brain o f many species.

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