Jian Yi Li

Blood—Brain Barrier Genomics

The blood–brain barrier (BBB) is formed by the brain microvascular endothelium, and the unique transport properties of the BBB are derived from tissue-specific gene expression within this cell. The current studies developed a gene microarray approach specific for the BBB by purifying the initial mRNA from isolated rat brain capillaries to generate tester cDNA. A polymerase chain reaction–based subtraction cloning method, suppression subtractive hybridization (SSH), was used, and the BBB cDNA was subtracted with driver cDNA produced from mRNA isolated from rat liver and kidney. Screening 5% of the subtracted tester cDNA resulted in identification of 50 gene products and more than 80% of those were selectively expressed at the BBB; these included novel gene sequences not found in existing databases, ESTs, and known genes that were not known to be selectively expressed at the BBB. Genes in the latter category include tissue plasminogen activator, insulin-like growth factor-2, PC-3 gene product, myelin basic protein, regulator of G protein signaling 5, utrophin, IκB, connexin-45, the class I major histocompatibility complex, the rat homologue of the transcription factors hbrm or EZH1, and organic anion transporting polypeptide type 2. Knowledge of tissue-specific gene expression at the BBB could lead to new targets for brain drug delivery and could elucidate mechanisms of brain pathology at the microvascular level.

Cloned blood-brain barrier adenosine transporter is identical to the rat concentrative Na^+ nucleoside cotransporter CNT2

Adenosine transport into brain is regulated by the activity of the adenosine transporter located at the brain capillary endothelial wall, which forms the blood–brain barrier (BBB) in vivo. To facilitate cloning of BBB adenosine transporters, poly A+ RNA was purified from isolated rat brain capillaries for production of a rat BBB cDNA library in the pSPORT vector. The cloned RNA (cRNA) generated from in vitro transcription of this library was injected into frog oocytes followed by measurement of [3H]-adenosine uptake. After dilutional cloning, a full-length, 2905-nucleotide adenosine transporter cDNA, designated clone A-11, was isolated. The A-11 clone yielded [3H]-adenosine flux ratios of 400 to 500 after injection of cRNA in oocytes. The adenosine uptake was sodium-dependent and insensitive to inhibition by S-(4-nitrobenzyl)-6-thioinosine (NBTI). The Km and Vmax of adenosine transport in the cRNA-injected oocytes were 23.1 ± 3.7 μmol/L and 10.8 ± 0.9 pmol/oocyte · min. The K0.5 for sodium was 2.4 ± 0.1 mmol/L, with a Hill coefficient (n) of 1.06 ± 0.07. DNA sequence analysis indicated the rat BBB A-11 adenosine cDNA was identical to rat concentrative nucleoside transporter type 2 (CNT2). The adenosine transporter activity of the rat BBB A-11 CNT2 clone is 50-fold more active than previously reported rat CNT2 clones. In summary, these studies describe the expression cloning of CNT2 from a rat BBB library and show that the pattern of sodium dependency and NBTI insensitivity of the cloned CNT2 are identical to patterns of adenosine transport across the BBB in vivo. These results suggest that BBB adenosine transport in vivo is mediated by CNT2, which would make CNT2 one of the few known sodium-dependent cotransporters that mediate substrate transport across the BBB in the blood to brain direction.

Rat Blood–Brain Barrier Genomics. II

The tissue-specific gene expression at the brain microvasculature, which forms the blood–brain barrier (BBB) in vivo, can be elucidated with a brain vascular genomics program, which starts with the isolation of gene products derived from purified brain microvessels. Genes commonly expressed in peripheral organs are subtracted with the suppression subtractive hybridization method using driver cDNA produced from a pool of rat liver/kidney–derived poly A+RNA. From a screen of 480 clones in the subtracted tester cDNA library, 156 clones were sequenced. The clones fell into 3 groups: known genes (51%), rat expressed sequence tags (31%), and novel rat genes not found in databases (18%). The known genes could be categorized into families of common function including vascular remodeling, signal transduction, transcription factors, biologic transport, vascular amyloid, hemostasis, myelin, lipids, secretion, cytoskeleton, and junctional complexes. Brain vascular genomics, or BBB genomics, allows for an accelerated discovery of the gene families that are differentially expressed at the microvasculature in brain.

Blood—Brain Barrier Genomics and Cloning of a Novel Organic Anion Transporter

A novel organic anion transporter selectively expressed at the blood—brain barrier (BBB), originally designated BBB-specific anion transporter type 1 (BSAT1), and now classified as Slco1c1, has been cloned from a BBB genomics program as a partial cDNA; this study describes the cloning and expression of the full-length cDNA from a rat brain capillary cDNA library. Northern analysis revealed the selective expression of the transporter at the BBB, and the transporter was expressed after permanent transfection of human 293 cells with cDNA encoding either the full length or open reading frame mRNA. The full-length transporter cDNA was 2.6 kb, and the mRNA was highly expressed at the rat brain microvasculature, but not in kidney, liver, heart, or lung, or in glial cells or brain glial tumors. Blood—brain barrier-specific anion transporter type 1 expression in 293 cells was poor after the transfection of the full-length cDNA, whereas transporter expression in 293 cells was high after transfection of the open reading frame. The transporter showed asymmetric kinetic properties in comparison of the influx and efflux of model substrates, thyroxine (T4), triiodothyronine (T3), and estradiol-glucuronide (E2G). Thyroxine and T3 inhibited the influx of E2G, but E2G did not inhibit thyroxine influx, and T3 only weakly inhibited the influx of T4. Extracellular E2G stimulated the transefflux of intracellular T4. Blood—brain barrier-specific anion transporter type 1 is a novel organic anion transporter that is a sodium-independent exchanger that may participate in the active efflux of iodothyronines and steroid conjugates at the BBB.

Organ-specific expression of the lacZ gene controlled by the opsin promoter after intravenous gene administration in adult mice

The tissue‐specific expression of an exogenous gene, under the influence of a tissue‐specific promoter, has been examined in the past with pro‐nuclear injections of the transgene and the development of transgenic mouse models. ‘Adult transgenics’ is possible with the acute expression of an exogenous gene that is administered to adult animals, providing the transgene can be effectively delivered to distant sites following an intravenous administration.

Site-directed mutagenesis of rabbit LAT1 at amino acids 219 and 234

The availability of amino acids in the brain is regulated by the blood–brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (low Km) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site‐directed mutagenesis of the BBB LAT1. The rabbit has a high Km LAT1 large neutral amino acid transporter, as compared to the low Km neutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on the Km and Vmax of tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced the Km by 64% and the Vmax by 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.

Hypoxia induces de-stabilization of the LAT1 large neutral amino acid transporter mRNA in brain capillary endothelial cells.

Blood–brain barrier (BBB) transport of large neutral amino acids is mediated by the large neutral amino acid transporter type 1 (LAT1 transporter). Although the gene encoding the Glut1 glucose transporter is up‐regulated in hypoxia, the response of the LAT1 gene to hypoxia is not known. The present study investigates the changes in the LAT1 mRNA in cultured bovine brain capillary endothelial cells exposed to 1% O2 for 24–48 h. The LAT1 mRNA was initially down‐regulated in hypoxia with reciprocal changes in the Glut1 mRNA. No changes in the 4F2hc mRNA in hypoxia were observed. Hypoxia caused an initial de‐stabilization of the LAT1 mRNA, and the t1/2 of the LAT1 mRNA in control and hypoxic cells was 6.4 ± 0.5 and 2.4 ± 0.1 h, respectively. To further explore post‐transcriptional regulation of LAT1 gene expression, the polysome and cytosol fractions of the control and hypoxic endothelial cells were isolated, and LAT1 mRNA binding proteins were detected by ultraviolet light cross‐linking. Whereas the cytosol contained no LAT1 mRNA binding proteins, the cell polysome fraction expressed several LAT1 mRNA binding proteins, including principal 40‐, 70‐ and 80‐kDa proteins. These studies are consistent with post‐transcriptional de‐stabilization of the LAT1 large neutral amino acid transporter in hypoxia.

Selective Lutheran glycoprotein gene expression at the blood-brain barrier in normal brain and in human brain tumors.

The Lutheran (LU) glycoprotein was shown to be a specific marker of brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo. A 1.5 kb partial cDNA encoding the bovine LU was isolated from a bovine brain capillary cDNA library. Sequence analysis showed that the bovine and human LU had a 75% and 79% identity in the amino acid and nucleotide sequences, respectively. Northern blot analysis demonstrated a very high level of gene expression of the LU transcript in freshly isolated bovine brain capillaries, but no measurable LU mRNA in whole bovine brain. The high level of LU gene expression was maintained when bovine brain capillary endothelium was grown in tissue culture. Because many BBB specific genes are downregulated in tissue culture and in brain tumors, the expression of the LU mRNA and immunoactive LU protein was investigated in primary and metastatic human brain tumors. Immunocytochemistry of fresh frozen human brain and human brain tumors showed abundant immunostaining of brain capillary endothelium. Northern blot analysis showed the presence of LU transcripts in a panel of primary and metastatic human brain tumors. These studies demonstrated that the LU glycoprotein was a novel new marker of the BBB, and unlike other BBB specific genes, there was a persistent gene expression of the LU glycoprotein both in brain capillary endothelial cells grown in culture and in the endothelium of capillaries perfusing human brain cancer.

Developmental regulation of the rabbit blood-brain barrier LAT1 large neutral amino acid transporter mRNA and protein.

The expression of the blood-brain barrier (BBB) LAT1 large neutral amino acid transporter mRNA and protein was investigated in development in rabbits. The BBB LAT1 mRNA was down-regulated with postnatal development. However, the BBB immunoreactive LAT1 protein was unchanged in postnatal development, despite an up-regulation of the BBB GLUT1 glucose transporter protein during this period. The dissociation between LAT1 protein and mRNA levels in development is consistent with posttranscriptional regulation of BBB LAT1 gene expression.