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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60:852-858 (2005)
© 2005 The Gerontological Society of America

Decrease of Transthyretin Synthesis at the Blood–Cerebrospinal Fluid Barrier of Old Sheep

Ruo L. Chen, Senarath B. P. Athauda, Nouhad A. Kassem, Yi Zhang, Malcolm B. Segal and Jane E. Preston

Institute of Gerontology, King's College, London University, United Kingdom.

Address correspondence to Dr. R. L. Chen, Institute of Gerontology, Franklin-Wilkins Building, King's College, 150 Stamford Street, London SE1 9NH. E-mail: ronnie.chen{at}kcl.ac.uk


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
Transthyretin (TTR), synthesized by the choroid plexus (CP) and secreted into cerebrospinal fluid (CSF), is involved in thyroxine (T4) transport and chelation of ß-amyloid peptide, attenuating neurotoxicity. To characterize age-related changes in TTR synthesis, CSF and CPs were collected from young adult (1–2 years) and old (>8 years) sheep anesthetized with thiopentone sodium. TTR in old sheep CSF was low compared to young (n = 4 each); however, CP messenger RNA (mRNA) for TTR did not change. CPs were perfused with Ringer containing 14C-leucine to assess de novo protein synthesis, or with 125I-T4 to assess T4 transport. Protein synthesis, including TTR, was reduced in old sheep CP and in newly secreted CSF. 125I-T4 Vmax and Kd (but not Km) were reduced in old sheep CP. These age-related changes suggest reduced capacity of CP to maintain CSF T4 homeostasis and could also reduce chelation of ß-amyloid and be an added risk for Alzheimer's disease.

THE choroid plexus (CP) epithelium together with the arachnoid membrane form the blood–cerebrospinal fluid (CSF) barrier (1). The CP also secretes CSF, transports crucial nutrients and metabolites to the brain, removes a range of xenobiotics from CSF, and has an exclusive or contributory role in synthesizing protein for CSF distribution (2). During normal aging, there are some significant morphological and functional changes in CP (3,4). The epithelial cell layer becomes flattened, losing about 11% in height between infancy and 88 years in humans (5) and 16% in length comparing 6- and 30-month-old rats (6). A decline in CSF secretion with age has been demonstrated by using a variety of techniques in a number of species such as humans (7), rats (8), and sheep (4). Correspondingly, there is a parallel increase in the proportion of the intracranial volume occupied by CSF in humans, therefore, a significant reduction in the overall rate of CSF turnover (9). It has been suggested that these alterations contribute to age-related degenerative diseases of the central nervous system (10). An area of current interest is the role of the protein transthyretin (TTR) which is synthesized by the CP.

TTR, formally called thyroxine-binding prealbumin, accounts for at least 20% of all protein synthesized by CPs in the rat (11) and constitutes approximately 50% of all secreted proteins from CP cells into the CSF (12). TTR is the major transporter of thyroid hormones in CSF (13) with a probable role in vitamin A transport (14). Unidirectional secretion of TTR by the CP epithelium into CSF has been suggested to play an important role in the transfer of T4 from the blood to the CSF (15). Inhibition of protein synthesis by cycloheximide prevents T4 transfer from blood to CSF sides in an in vitro epithelial cell culture model (15). In vivo, inhibition of T4 binding TTR using the flavonoid EMD 21388 reduces entry of radioactive T4 from blood into CP and CSF (16). In relation to the aging central nervous system, T4 deficiency has been suggested to contribute to age-related cognitive decline (17), but it is not clear whether this decline is secondary to reduced TTR availability. In addition, TTR also chelates neurotoxic ß-amyloid peptide (Aß), preventing Aß fibrillation and brain deposition (18). Recent studies suggest that estrogen reduces the risk of Alzheimer's disease by stimulating TTR gene expression in CP (19), leading to enhanced sequestration and decreasing aggregation of Aß.

The aim of this study was to investigate the effect of aging on TTR levels in CSF, rate of TTR synthesis, and messenger RNA (mRNA) expression levels in the CP using an established sheep model. On a functional level, we also examined the capacity of the CP to transport T4 from blood.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
All procedures fell within the guidelines of the Animals (Scientific Procedures) Act, 1986. Clun Forest strain sheep between 1 and 10 years old were used, and divided into two groups: aged 1–2 years (young adult, 25–35 kg) and 6–10 years (old sheep, {approx}60 kg). The sheep were anesthetized with i.v. thiopentone sodium (20 mg/kg) and received injection of heparin (1000 U/kg). Blood and CSF samples were collected from the carotid artery and cisterna magna, respectively, by a needle puncture. CSF samples were centrifuged at 13,000 g for 10 minutes to eliminate any cells and other insoluble material. CSF samples contaminated with blood were discarded. Thereafter the sheep were exsanguinated via the carotid artery, and the heads were removed. Lateral ventricle CPs from four young adults were quickly harvested and placed in liquid nitrogen for later extraction of mRNA to generate a complementary DNA (cDNA) template. Lateral ventricle CPs from five pairs of young and old sheep were taken for extraction of protein and RNA. In a further 15 pairs of sheep, the lateral ventricle CPs were perfused via the choroidal artery with Ringer; 9 pairs were used for 125I-T4 transport studies, and 6 pairs were used for protein synthesis studies following 14C-leucine perfusion.

mRNA Isolation and cDNA Synthesis
Total cellular mRNA was extracted from the lateral ventricle CPs of four adult young sheep (200–300 mg per sheep) using a Micro-FastTrack 2.0 Kit (Invitrogen, Paisley, U.K.) and pooled together. The mRNA concentration was determined spectrophotometrically, and 0.1 µg of mRNA was used as template to make cDNA using a Superscript First-Strand Synthesis kit (Invitrogen).

RNA and Protein Extractions and Quantification
Lateral ventricle CPs from another 5 young and 5 old sheep were taken for extraction of protein and RNA. One CP (100–150 mg) from each sheep was homogenized in buffer containing 20 mM Tris, 5 mM EGTA, 15 mM 2-mercaptoethanol, PMSF at 0.1 mg/ml, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail (Sigma, St. Louis, MO) (1:100). Protein concentration in homogenate was analyzed using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as the standard. The other CP from each sheep was prepared for RNA extraction using a QuickPrep Total RNA Extraction Kit (Amersham Biosciences U.K. Limited, Little Chalfont).

Primer Synthesis
The cDNA sequences of TTR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, as the housekeeping gene) were obtained from the National Center for Biotechnology Information (NCBI) sequence database Genebank by using the web-based tool (Locus link, http://www.ncbi.nlm.nih.gov/LocusLink). Oligonucleotide primers were designed to obtain primer pairs with an annealing temperature of 55°C (Table 1)<--?1-->. First-strand cDNA was synthesized using the Superscript First-Strand Synthesis System (Invitrogen) for polymerase chain reaction (PCR). The amplified PCR products were size-fractionated by agarose gel electrophoresis, purified using a Concert matrix gel extraction system (Invitrogen), and sequenced by The Department of Neurobiology, King's College London, University of London. All samples were aliquoted into silicone-coated Eppendorf vials and stored at –20°C until assayed (within 1–3 weeks) or at –80°C as part of our sample bank.


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  		Table 1. Primers Used for Northern Blot and Real-Time Polymerase Chain Reaction.

 
Northern Blotting
RNA (3 µg) from five young and five old CPs were denatured, separated by electrophoresis on a 1.5% agarose formaldehyde gel, and then transferred to a Hybond-N nylon membrane (Sigma) with 20 x sodium chloride–sodium citrate buffer (SSC) and cross-linked by ultraviolet radiation for 90 seconds. Radiolabeled probe was made with purified TTR1 (Table 1) labeled with 32P-2'-deoxycytidine 5'-triphosphate using a Rediprime II Random Prime Labeling Kit (Amersham Biosciences U.K. Limited) according to the manufacturer's protocol. Nylon membranes were prehybridized at 68°C for 2 hours in 0.5 M sodium phosphate, 7% SDS, 1 mM EDTA, and denatured salmon sperm DNA at 200 µg/ml. Hybridization was performed at 68°C overnight with the 32P-2'-deoxycytidine 5'-triphosphate TTR1 in the same buffer. Membranes were washed at 23°C with 1 x SSC, 0.1% SDS for 10 minutes; 2 x SSC, 0.1% SDS for 15 minutes; 0.5 x SSC, 0.1% SDS for 15 minutes; 0.1 x SSC, 0.1% SDS for 15 minutes; then with 0.1 x SSC, 1.0% SDS at 50°C for 15 minutes; and finally 3 times with 0.5 x SSC, 0.1% SDS at 68°C for 15 minutes. Autoradiography was performed using Kodak XAR 5 film (Sigma) after storing at –80°C for 48 hours.

Real-Time PCR
One microgram of total RNA from each sample was used to produce cDNA in a volume of 10 µl using a SuperScript First-Strand Synthesis kit (Invitrogen). The LightCycler-FastStart DNA Master SYBR Green I kit (Roche, Indianapolis, IN) was used for all reactions according to the manufacturer's instruction. The MgCl2 concentration was optimized for each primer pair to obtain a single-peak melting curve. After an initial 10-minute preincubation at 95°C, the mixture was subjected to 42 cycles of a three-step PCR: 15 seconds of denaturation at 95°C, a 4-second annealing phase at 58°C, and a 6-second elongation phase at 72°C.

TTR Detection by Western Blot
There are a number of commercial kits for detecting TTR. Most are antibody-based assays, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunoturbance. However as CSF has much less TTR than serum does (14.7 mg/L in CSF compared to 260 mg/L in serum) (1), these kits could not detect TTR in our CSF samples. Therefore, we used a western blot to semiquantify TTR in CSF.

CSF samples (equal volume 10 µl), CP homogenate (equal protein content 10 µg), and sera (equal volume 2.5 µl) of young and old sheep were loaded on to a 10% SDS polyacrylamide gel. The gel was then run in a mini gel electrophoretic unit (Bio-Rad Labs Ltd) at 60 mV constant voltage until the dye front was near the end. The electrophoresed protein was transferred onto 0.45 µm-thick nitrocellulose sheets. The nitrocellulose membrane was saturated for 1 hour with 5% bovine serum albumin in Tris-buffered saline containing 0.05% Tween 20. Goat antihuman TTR antibody (diluted 1/500) was then added, and this mixture was incubated for 1 hour at room temperature. After washing in Tris-buffered saline containing 0.05% Tween 20, the membrane was incubated for 1 hour with alkaline phosphatase-conjugated antigoat immunoglobulin G (diluted in 1/10,000; Sigma). After the final wash, immunostained proteins were revealed using Nitro Blue Tetrazo (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as enzyme substrates (Sigma). The reaction was stopped with distilled water.

CP Perfusion
The method used here has been previously described (20,21). In brief, after removal of the brain, the Circle of Willis supplying the choroidal arteries to each lateral ventricle CP was cannulated, then perfusion was commenced with Ringer-plasma substitute (in mM: NaCl 123, KCl 4.8, NaH2PO4 1.22, CaCl2 2.4, MgSO4 1.22, NaHCO3 25, glucose 5), containing 4% dextran at 37°C and gassed with 95% O2/5% CO2. The Ringer outflow was collected from the Great vein of Galen, into which the veins from each CP flow. The cerebral hemispheres were opened to gain access to the CSF side of the plexuses, and ventricles superfused with mock CSF (in mM: NaCl 148, KCl 2.9, NaH2PO4 0.25, CaCl2 2.5, MgCl2 1.8, NaHCO3 26) at 37°C and gassed with 95% O2/5% CO2. The plexuses were stable and secreted CSF typically up to 5 hours.

125I-T4 Transport
During steady-state experiments, 125I-T4 (0.555 MBq/100 ml; 34 pM) and extracellular marker 14C-mannitol (2.77 MBq/100 ml; 13.9 µM) were added to the Ringer perfusate for 1 hour. Arterial and venous Ringer samples were taken every 5 minutes, and tracer activities (in disintegrations per minute) in 100 µl of each sample were determined by liquid scintillation counting (Rackbeta Spectral 1219 counter; LKB Wallac, Turku, Finland) after the addition of 3.0 ml of liquid scintillation fluid (National Diagnostics, Yorkshire, U.K.). Extraction percent (E%) was calculated as previously described (20). Net cellular T4 extraction (%) was calculated from the difference between 125I-T4 and 14C-mannitol extraction.

To determine rapid unidirectional uptake of 125I-T4 the paired tracer, single-pass methods were used (21). A 100-µl bolus containing 18.5 kBq of 125T4 (1.13 pM) and 74 kBq of 14C-mannitol (370 nM) was added to the perfusion fluid, and 20 single-drop samples of venous effluent were collected over approximately 1 minute. Uptake of 125I-T4 relative to 14C-mannitol in each drop was calculated (21), and the maximal cellular uptake (Umax) was determined from samples in which the 14C and 125I activities had reached peak levels. The total average uptake (Uav) and any backflux were also calculated (21). For kinetic analysis of T4 uptake, plexuses were perfused with different concentrations of unlabeled T4 (15, 30, 50, 70, 100, 150, 200, 500, and 700 nM), and flux was calculated (21). Plots of concentration versus flux were fitted by nonlinear regression analysis (Enzifitter program; Biosoft, Cambridge, U.K.) to calculate the half-saturation constant Km (nM), the maximal flux Vmax (pmol min/g), and the diffusion coefficient Kd (ml/min/g).

Protein Synthesis Studies
In separate experiments, the perfused CPs were covered with light mineral oil. 14C-leucine (925 MBq; NEN, Hounslow, U.K.) was added to 250 ml of perfusion medium (final leucine concentration: 0.66 µM), and was continuously perfused for 2.5 hours. Droplets of newly synthesized CSF emerging from the surface of CP could be seen under the oil, and were collected every 30 minutes using glass micropipettes as described by Schreiber and colleagues (22). The CP was then perfused with isotope-free Ringer for 15 minutes, and then homogenized in buffer (20 mM Tris, 5 mM EGTA, 15 mM 2-mercaptoethanol, PMSF at 0.1 mg/ml, 1% Triton X-100, 0.1% SDS) and protease inhibitor cocktail (Sigma) (1:100). CP homogenate was centrifuged at 12,000 g for 10 minutes, and the supernatant was collected for analysis.

Newly synthesized CSF (20 µl) and 10 µg CP homogenate were loaded onto 10% SDS-polyacrylamide gel and were subjected to electrophoresis under constant voltage until the dye line went to the bottom of the gel. The gel was dried using a Bio-Rad gel dry system, and was inserted into BioMax TranScreen LE with Kodak X-Omat AR film (Sigma) in a cassette. After storing at –80°C for 8 weeks, the film was developed.

Data Analysis
The membranes and films of western blots, northern blots, and autoradiographs were scanned and densitometrically quantified into arbitrary units using Bioimage Intelligence Quotient IQ software (BioImage Systems, Inc., Jackson, MI) on an Apple Macintosh PC (Cupertino, CA). All values were expressed as mean ± standard error of the mean. Unpaired t tests as appropriate were used to compare means. Values of p <.05 were considered statistically significant.


   RESULTS
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 Methods
 Results
Discussion
 References
 
TTR in CSF and CP
Sigma goat antihuman TTR antibody reacted with human TTR (Sigma) on three bands (14, 32, and 55 kD) (Figure 1). The 14 kD band corresponds to a monomer of TTR. The 32 kD band may represent a dimer of TTR, whereas the 55 kD band is the tetramer of TTR. This antibody reacted with sheep CSF at a single band of 55 kD (Figure 1). Comparing the optical density of this 55 kD band between young (0.91 ± 0.14, arbitrary units, n = 4) and old ovine CSF (0.53 ± 0.08, n = 4), the latter showed significant reduction (tdf=6 = 2.761, p =.033) (Figure 1). Using CP homogenate or serum samples, the antibody cross-reacted with multiple bands, indicating some nonspecific binding in the high-protein samples (data not shown).



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  		Figure 1. Western blot of ovine cerebrospinal fluid (CSF). Sigma antihuman transthyretin (TTR) antibody detected three bands (14, 32, and 55 kD) on purified human TTR (Lane 2) and cross-reacted with a single band (55 kD) on both the young (Lanes 3–5) and old (Lanes 6–8) ovine CSF. The 55 kD band density for four young sheep was 0.91 ± 0.14 (optical density, arbitrary units) and for four old sheep was 0.53 ± 0.08. Lane 1: molecular weight; Lane 2: purified human TTR; Lanes 3–5: old ovine CSF samples; Lanes 6–8: young ovine CSF samples

 
TTR Gene Expression at CP
Amplified PCR products with designed primers—TTR1, TTR2, and GADPH—were purified, sequenced, and confirmed as parts of sheep TTR or GADPH genes (Table 1). Purified TTR1 was labeled with 32P, and a single band with 399 bp was seen on the northern blot (Figure 2). There was no significant difference in the optical density of the band between the young (0.98 ± 0.43, arbitrary units, n = 3) and old CPs (0.94 ± 0.29, n = 4) (t5 = 0.088, p =.934) (Figure 2). Real-time PCR using primers TTRS2 and TTRA2 showed that the TTR gene was highly expressed in ovine CP compared to GADPH (Figure 3), and that the ratio of TTR to GADPH did not differ between the two age groups (Figure 3).



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  		Figure 2. Northern blot of ovine choroid plexus (CP). A probe generated with primers TTRA1 and TTRS1 was labeled with 32P and detected a single band of 399 bp in both young and old ovine CP RNA. There was no significant difference in the density of this band between ages (t5 = 0.088, p =.934). Lanes 1 and 4: young ovine CP RNA; Lanes 2 and 3: old ovine CP RNA

 


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  		Figure 3. Real-time polymerase chain reaction of ovine choroid plexus. Primers TTRA2 and TTRS2 showed similar cycles in young (12.67 ± 1.7 cycles, n = 5) and old (13.2 ± 1.17 cycles, n = 5) ovine CP complementary DNA (A). There was no significant difference in the ratio of transthyretin to GADPH with age (B) (Young CP: 0.475 ± 0.03, n = 5; Old CP: 0.493 ± 0.025, n = 5; t8 = –0.46, p =.658)

 
CP Protein Synthesis
In six pairs of young and old sheep, each lateral ventricle CP was perfused in situ with Ringer containing 14C-leucine. Radioactive content of proteins in CSF and CP homogenate was assessed by SDS-polyacrylamide gel electrophoresis followed by autoradiography of the gels. The autoradiographs showed 14C-leucine incorporation into several bands in the newly secreted CSF in both age groups, including a 14 kD MW band (consistent with the TTR monomer). However, only CSF from young sheep showed the 55 kD band (corresponding to the TTR tetramer) (Figure 4). In comparison, protein from CP homogenates showed multiple bands including the 14 kD and 55 kD bands in both age groups. However, the young CP homogenate optical density (1.85 ± 0.17, arbitrary units, n = 6) showed a higher density of all bands compared to old CP (1.22 ± 0.23, n = 6, t10 = 2.296, p =.045) (Figure 4).



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  		Figure 4. Representative autoradiograph of ovine choroid plexus (CP) homogenates and newly synthesized cerebrospinal fluid (CSF) after perfusion with 14C-leucine. Autoradiographs of CP homogenates showed multiple radioactive bands. The band densities from young CPs (Lane 4) were higher than from old CPs (Lane 5) at all molecular weights. In newly synthesized CSF samples, a 14 kD band (transthyretin [TTR] monomer) was seen in both young (Lanes 2 and 3) and old samples (Lanes 6 and 7) but a 55 kD band (TTR tetramer) was seen only in young CSF. Lane 1: molecular weight; Lanes 2 and 3: young ovine newly synthesized CSF; Lane 4: young ovine CP homogenate; Lane 5: old ovine CP homogenate; Lanes 6 and 7: old ovine newly synthesized CSF

 
125I-T4 Uptake
The steady-state extraction 125I-T4 was greater than that of 14C-mannitol in both young and old CPs, indicating net cellular uptake of T4. There was no significant difference in net T4 extraction between these two groups (Figure 5), at the concentration of T4 used (34 pM). Using the paired tracer technique, rapid unidirectional uptake of 125I-T4 could be measured. Figure 6 shows a typical dilution curve for both 125I-T4 and 14C-mannitol. The Umax, Uav, and backflux were not different between the age groups (Table 2).



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  		Figure 5. T4 extraction in the perfused ovine choroid plexus. Net 125I T4 extraction (after subtraction of extracellular marker 14C-mannitol) was not significantly different between young (18.59 ± 6.97%, n = 4) and old ovine choroid plexus (16.85 ± 3.75%, n = 3) (t5 = 0.229, p = 0.237)

 


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  		Figure 6. A representative 125I T4 single-pass of uptake curve. A, Recovery of 14C-mannitol and 125I-T4 in venous samples over 1 minute. B, Uptake of 125I-T4 in each venous sample relative to 14C-mannitol. Samples containing the highest recovery of isotopes were joined by a line and averaged to estimate Umax

 

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  		Table 2. 125I-T4 Uptake at Basolateral Side of Sheep Choroid Plexus.

 
In the presence of excess unlabeled T4, Umax decreased as the concentration of unlabeled T4 increased, indicating carrier-mediated T4 transport in the ovine CP. Figure 7 shows a plot of total concentration of T4 (both labeled and unlabeled T4) versus flux, which could be described by Michaelis–Menten type kinetics. Vmax, the maximum T4 transport, was significantly reduced in old sheep. Km, the affinity for uptake, was not different between these two groups. Kd, the diffusion coefficient, was not significantly different from zero in old sheep, but was positive in young sheep (Table 2).



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  		Figure 7. A plot of T4 flux into the choroid plexus versus T4 concentration in both young and old sheep. Values are mean ± standard error of the mean

 

   DISCUSSION
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 Methods
 Results
 Discussion
References
 
CP contains 10 times more TTR mRNA per gram of tissue than does liver (the principle systemic source) and secretes TTR 13 times faster than does liver in rats (12). The relative abundance of TTR synthesized and secreted by CP into CSF makes it a suitable model molecule to study CP function. We found that TTR decreased in old ovine CSF sampled in vivo, that the rate of new protein synthesis declined in vitro (although TTR mRNA did not alter), and that there was reduced capacity for T4 uptake by the CP.

The western blot with an antihuman TTR polyclonal antibody showed a clear single band (55 kD) in ovine CSF, rather than the multiple bands seen in serum and CP homogenates with high protein content. This 55 kD band corresponds to the tetramer of TTR. This finding is different than those from previous studies in rats (22,23) where CSF TTR is predominantly represented as the 14 kD monomer. This may be due to species difference or use of different antibodies in other laboratories. The overall level of TTR, as semiquantified by the western blot analysis, was decreased in CSF sampled from old sheep. Whereas CSF levels of TTR in healthy older humans have been variously described as decreased (14), increased (24,25), or unchanged (26), levels are consistently reported to decrease in Alzheimer's disease (25,27) and in patients with depression (28). Interpretation of TTR content in CSF has to be made in the context of reduced CSF turnover, which would have a concentrating effect on multiple CSF proteins and may therefore lead to overestimates of the capacity of the CP to synthesize TTR (4). For example, Serot and colleagues (25) showed that CSF albumin levels rose approximately 90% in aging humans from 113 mg/L to 217 mg/L (between 10 and 76 years), whereas the TTR levels did not increase in the same proportion, increasing by only 29%, from 15.5 to 20 mg/L. This finding suggests an actual reduction in relative TTR addition to CSF. Indeed, Reiber (29) suggested that protein measurements should be corrected for the albumin ratio to normalize the CSF turnover effect. In our model, we have previously reported that total CSF protein increased with age in sheep (30) by approximately 40%, indicating stagnation of CSF. Although it is not possible to fully quantify TTR levels in this study, the band densities suggest a substantial reduction in CSF TTR by around 40%, despite the apparent CSF stagnation.

To understand the reason for the decrease of TTR in the old ovine CSF, we compared TTR mRNA in CP from young and old sheep. TTR mRNA was found at high levels, compared to the housekeeping gene GADPH, in both young and old sheep, consistent with other studies (11,12). Also, no significant difference in TTR mRNA between young and old animals was seen. Nevertheless, expression level of mRNA does not necessarily translate into amount of functional protein. For instance, in aging mice and rats, the rate of protein synthesis decreases as translation slows down (31,32).

To study de novo protein synthesis, we perfused with 14C-leucine because this is an essential amino acid for TTR synthesis and has been used previously to study TTR synthesis in vivo and in vitro (12,22,33). We found a significant decrease in radiolabeled bands in both newly secreted CSF and in the CP in old sheep after 2.5 hours of 14C-leucine perfusion. This finding may be due in part to age-related reduction of amino acid uptake into CP (34). However, the concentration of leucine used was only 0.66 µM, far less than its Km of 3.3 ± 0.7 µM (35), and unlikely to be affected by altered transport kinetics. It is more likely that the decrease in radiolabeled bands is due to reduction of de novo protein synthesis in the old CP and is consistent with the lower level of TTR in in vivo CSF samples.

TTR is a binding protein for T4, and is suggested to be an important component in the transfer of T4 from the bloodstream across the CP, and from CSF to the brain (15,22). Steady-state T4 extraction showed no significant change with age, although these experiments were carried out at 34 pM T4, which is lower than the physiological concentration [100 nM in lamb (36) and 83 nM in sheep (37)]. However, differences were seen using the single-pass method when uptake was challenged with excess T4, up to 700 nM. In old sheep, the Vmax for T4 transport was half that of the young, although the Km did not alter. This suggests a change in the quantity of available T4 transporters, of which several have been identified (38), and the possible lack of cellular TTR, rather than a change in transporter affinity. The diffusion coefficient, Kd, was also reduced significantly and was close to zero in old CP, suggesting a change of lipid solubility of T4 in aged plasma membrane. This is consistent with age-related changes in liver and aortic endothelial cell plasma membrane fluidity in rats (39,40), and has consequences for transmembrane transport (41).

Conclusion
TTR levels decrease in old ovine CSF, most likely due to decline in TTR synthesis by the CP rather than to a reduction in TTR mRNA expression. Vmax and Kd for T4 transport from blood also decline, consistent with reduction in available transporters and availability of TTR to facilitate CP uptake. Decline in TTR synthesis parallels other known declines in CP function with age, such as CSF secretion (8), ion transport (42), and reduced amyloid degradation (4). These data suggest that age-related changes in the CP negatively affect of the capacity of the blood–CSF barrier to maintain CSF T4 homeostasis and to protect against ß-amyloid toxicity by chelation to TTR.


   Acknowledgments
 
We are grateful for the generous financial support of The Biotechnology and Biological Sciences Research Council (BBSRC), U.K.


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received September 27, 2004

Accepted January 31, 2005


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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