Identification of ATP8B1 as a Blood–Brain Barrier-Enriched Protein

Abstract

In order to define the molecular anatomy of the blood–brain barrier (BBB) that may be relevant to either barrier or transport function, proteins that are overexpressed in the cerebral microvessels should be identified. We used differential display to identify novel proteins that are overexpressed or unique to the BBB. DNA sequence analysis is one of the differentially expressed transcripts showed that it is highly homologous with the ATPase class I, type 8B, and member 1 (ATP8B1) protein and contains an ATPase domain and a phospholipid-binding domain. ATP8B1 is expressed in the BBB microvessels but not brain tissue lacking microvessels. Likewise, ATP8B1 was enriched in BBB microvessels similar to glucose transporter 1. Immunohistochemistry using an ATP8B1-specific antibody demonstrated preferential staining of the microvessels within the cerebral tissue. These results suggest that ATP8B1, a P-type aminophospholipid translocase, is enriched in cerebral microvessels and may have a role in plasma membrane lipid transport.

Introduction

The blood–brain barrier (BBB) protects the brain from environmental toxins and assures the delivery of essential nutrients and hormones for the brain. A host of proteins has been localized to the BBB, either through their enzymatic activity or their immunologic reactivity to available antisera (Chehade et al. 2002; Engelhardt and Sorokin 2009; Liu et al. 2012; Mooradian 1997; Mooradian and Bastani 1993; Mooradian et al. 1993, 1994a, b). Although none of these proteins are unique to the BBB, some are postulated to contribute to the BBB properties of the cerebral endothelium. Because of the conceptual importance of understanding the biology of the BBB, rapid, and sensitive screening approaches are needed to identify novel constituents of the BBB. Some of these proteins may be components of the tight junctions, while others may have unique BBB-specific functions which have not been hitherto known.

Both genomic and proteomic approaches have been used to isolate and characterize BBB-enriched molecules. In addition to subtractive hybridization and differential display methodologies (reviewed by Partridge 2005), recent mRNA expression profiling and proteomic analysis of brain microvascular endothelial cells has been combined with laser-capture microdissection to examine expression of BBB-specific proteins (Demarest et al. 2012; Lu et al. 2008). In addition, serial analysis of gene expression (SAGE) analysis of the rat BBB transcriptome showed that most of the BBB-enriched genes encode transporters, receptors, vesicle trafficking proteins, structural proteins, and signal transduction pathway components (Enerson and Drewes 2006). However, each of these technologies has inherent limitations due to the fact that they each identify novel BBB-enriched proteins.

Therefore, to identify new BBB proteins, we utilized the technique of differential display of mRNA (Liang et al. 1993; Liang and Pardee 1992) using RNA isolated from purified cerebral microvessels and cerebral tissue devoid of microvessels. One of the clones, we identified encodes a novel BBB-enriched protein, ATPase class I, type 8B, and member 1 (ATP8B1) (Tang et al. 1996; Williamson and Schlegel 2002).

Materials and Methods

Preparation of Cerebral Microvessels

Cerebral microvessels were isolated from frozen cerebral tissue obtained from 3-month-old Fischer 344 rats by filtration as described previously (Mooradian and Bastani 1993; Mooradian et al. 1994a, b). Purity of the preparations was determined by light microscopic examination of microvessels stained with 0.1 % Toluidine blue and was determined to have <5 % contamination with nuclei and/or cells. γ-Glutamyl transpeptidase activity (Orlowski and Meister 1965) and alkaline phosphatase activity (Bessey et al. 1946) indicated a 20- to 21-fold enrichment in the microvessel preparations. Protein content was measured as described by (Lowry et al. 1951). All of the animals were maintained in accordance with NIH guidelines established in the Guide for the Care and Use of Laboratory Animals, 1996. All animal experiments were approved by the Animal Use and Care Committee at Saint Louis University.

Differential Display of mRNA

Total cellular RNA was isolated as previously described (Chomczynski and Sacchi 1987) from cerebral tissue, cerebral tissue stripped of microvessels, and cerebral microvessels, heart, lung, liver, kidney, and testicles. DNase1-treated total cellular RNA (250 μg) was used for reverse transcription and subsequent amplification by polymerase chain reaction (PCR) as described previously (Liang et al. 1993; Liang and Pardee 1992). A 3′ primer, T₁₁ CA (5′-TTTTTTTTTTTCA-3′), and three arbitrary 5′ primers were used. The 5′ primers were AP-1 (5′-CTGATCCATG-3), AP-11 (5′-CTTGATTGCC-3′), and AP-III (5′-CTGCTCTCA-3). The amplified cDNAs were separated on a denaturing 6 % polyacrylamide DNA sequencing gel. Three cDNAs of interest were identified, re-amplified, and tested by Northern blotting (Shah et al. 1998). One band (Fig. 1, arrow) was sub-cloned in the PCRII vector (Invitrogen, San Diego, CA) and sequenced. Using this 132 bp cDNA probe, a rat brain unidirectional cDNA library Uni-Zap XR (Agilent Technologies, Inc., Santa Clara, CA, USA) was screened and a 1,042 bp partial cDNA clone was sequenced with the M13 phage universal and reverse and synthetic oligonucleotide primers (Sanger et al. 1997). The sequence of the full-length cDNA was submitted to GenBank (EP1.sqn EP1 KC333996). A search for conserved domains (Marchler-Bauer et al. 2009) indicated that it has a phospholipid-binding ATPase domain and has significant homology to the ATP8B1 protein (Tang et al. 1996; Williamson and Schlegel 2002).

Fig. 1.

RT-PCR differential display of mRNA from the rat cerebrum. The result of amplification with a 3′ primer (5′-T11CA-3′) and 5′ primer (AP-1; 5′-CTGATCCATG-3′) is shown. Lanes 1, 2 are total cerebral RNA from different rats, whereas lanes 3 and 5 are RNA depleted of cerebral microvessels. Lane 4 is RNA from cerebral microvessels. Three bands are clearly absent from the cerebral tissue depleted of microvessels. The arrow shows the band that was sub-cloned and sequenced

Northern Blotting

ATP8B1 mRNA levels were measured by Northern blot (Green and Sambrook 2012). Total RNA was isolated from the indicated tissues and 15 μg of RNA was fractionated by electrophoresis on a 1.2 % formaldehyde/agarose gel. Probes for hybridization were prepared by oligolabeling with [α-³²P]-dCTP as described (Feinberg and Vogelstein 1983). After transfer to a nylon hybridization membrane, the blot washed under high-stringency conditions (0.1 × saline-sodium citrate (SSC; 1 × SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 0.1 % sodium dodecyl sulfate (SDS) at 65 °C, two-times, 30 min each) and exposed to autoradiography film.

Western Blotting

Cerebral microvessel homogenates (30 μg of protein) were separated by SDS–polyacrylamide gel electrophoresis under reducing conditions (Laemlli 1970) and transferred to nitrocellulose membranes (Towbin et al. 1979). The membranes were blocked with 10 % normal calf serum in phosphate buffered saline (4.3 mM Na₂HPO₄, 1.47 KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4) (PBS) containing 0.1 % Tween-20 for one hour at room temperature. The membranes were then incubated with either anti-Glut- 1 (1:1000) (#07-1401, Millipore, Temecula, CA, USA) or anti-ATP8B1 antiserum (1:100) (#sc-67715, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60-min. Membranes were next incubated with peroxidase-labeled goat-anti-rabbit IgG (1:5000) for 45-min. All membranes were visualized using Pierce ECL Western Blotting Substrate, exposed to autoradiography film, and quantified from scanned images using ImageJ.

Immunohistochemistry

Paraffin-embedded cerebral tissue sections were de-paraffinized with xylene, then rehydrated in a series of graded ethanol washes and deionized H₂O. The sections were then placed in 3 % hydrogen peroxide for 30-min at room temperature and Vectastain protein block (Vector Laboratories, Inc., Burlingame, CA, USA) was added to each tissue section for one hour. After washing in PBS, the sections were incubated with anti-ATP8B1 (1:50) or Glut-1 (1:100) for 60-min at room temperature. The slides were counterstained with Gills Hematoxylin II for 1 min and rinsed in deionized H₂O. The slides were dehydrated and mounted with Shandon Xylene Substitute Mountant (Thermo Fisher Scientific) and photographed with a Nikon Eclipse E400 microscope and QImaging Micropublisher 32-0032A-185 camera.

Results

The differential display PCR-amplified cDNA fragments were separated on a denaturing polyacrylamide gel (Fig. 1). Lanes 1 and 2 are total cerebral RNA from different rats, and lanes 3 and 5 are cerebral RNA depleted of microvessels. Lane 4 is RNA from cerebral microvessels. One band that was enriched in the microvessel fraction (lane 4, arrow) was sub-cloned. This DNA fragment was then used to isolate and sequence a full-length cDNA from a rat brain cDNA library. Northern blot analysis (Fig. 2a) demonstrated that ATP8B1 mRNA was enriched in cerebral microvessels (lanes 5, 6) but absent from cerebral tissue depleted of microvessels (lanes 3, 4), similar to Glut-l mRNA (Fig. 2b). G3PDH mRNA levels were similar in each sample suggesting that there was no sample degradation. The enrichment of ATP8B1 mRNA and Glut-1 mRNA in the cerebral microvessels is clearly evident. We did not detect any ATP8B1 mRNA in heart, lung, liver, kidney, and testicles by Northern blot analysis (data not shown).

Fig. 2.

ATP8B1 expression in cerebral tissue. A Northern blot with 5 μg of total RNA from rat cerebral tissue (lanes 1 and 2), cerebral tissue depleted of microvessels (lanes 3 and 4), and cerebral microvessels (lanes 5 and 6). The blot was hybridized sequentially with probes for a ATP8B1 and G3PDH and b Glut-1. The enrichment of cerebral microvessels with ATP8B1 and Glut-1 mRNA is evident. TC total cerebrum, −CMV minus cerebral microvessels, CMV cerebral microvessels. The paucity of Glut-1 and ATP8B1 mRNA in nonvascular cerebral tissue is shown in lanes 3 and 4. G3PDH was used to exclude the possibility that the RNA was degraded in the capillary-depleted samples. Glut-1 (c) and ATP8B1 (d) expressions were measured by Western blot in microvessel-depleted cerebrum and cerebral microvessels. −CMV minus cerebral microvessels, CMV cerebral microvessels. ATP8B1 was enriched in cerebral microvessels (8.0-fold) similar to Glut-1 (7.3-fold)

DNA sequence analysis suggested that the microvessel-enriched cDNA is highly homologous with the mammalian ATP8B1 protein. Therefore, we measured ATP8B1 levels in cerebral microvessels and in cerebrum depleted of microvessels by Western blot using a commercially available antibody. The results showed that Glut-1) (Fig. 2c) and ATP8B1 (Fig. 2d) were enriched 7.3- and 8.0-fold, respectively, in microvessels relative to cerebrum-depleted microvessels. The multiple species of Glut-1 are likely due to its glycosylation (Birnbaum et al. 1986) or phosphorylation (Devraj et al. 2011). The higher molecular weight bands observed with the anti-ATP8B1 antisera may be due to either non-specific interaction of the primary antibody or to the presence of glycosylated/phosphorylated ATP8B1 variants, while the band beneath ATP8B1 may be a non-specific interaction of the primary antibody or a degradation product of ATP8B1. These results suggest that ATP8B1 is preferentially expressed in cerebral microvessels.

To further determine if ATP8B1 expression is localized to the BBB, ATP8B1 expression was examined in cerebral tissue by immunohistochemistry. In the absence of ATP8B1 primary antibody, no staining was observed (Fig. 3a). ATP8B1 expression was observed primarily in cerebral microvessels (Fig. 3b), however, there was also intense staining in cells resembling pericytes (arrows). Glut-1, a marker of cerebral microvessels, stained cells in a similar manner (Fig. 3c).

Fig. 3.

Immunohistochemical localization of ATP8B1and Glut-1 in mouse cerebrum. Mouse cerebral tissue sections were immunostained with a commercially available antibody to ATP8B1 and Glut-1. a No primary antibody. Original magnification ×100. b Anti-ATP8B1 (1:50). Original magnification ×400. c Anti-Glut-1 (1:100). Original magnification ×400. ATP8B1 expression is observed primarily in the endothelial cells and potentially in the pericytes (arrow)

Discussion

Previous studies of the protein composition of the BBB have used enzymatic, immunologic, and protein electrophoresis to identify the specific proteins that are enriched in cerebral microvessels (Chehade et al. 2002; Engelhardt and Sorokin 2009; Liu et al. 2012; Mooradian 1997; Mooradian and Bastani 1993; Mooradian et al. 1993, 1994a, b). Our approach identified a novel transcript that codes for the P-type aminophospholipid translocase (APLT), ATP8B1 (Tang et al. 1996; Williamson and Schlegel 2002). To our knowledge, this is the first time that this protein or family of proteins has been identified in a screen for BBB-enriched proteins.

Previous studies by Harris and Arias (2003) demonstrated that ATP8B1 mRNA (measured by Northern blotting) is expressed at extremely low levels in multiple tissues, with high-level expression observed in the colon, while RNA expression array analysis by the same investigators showed no ATP8B1 expression in multiple tissues, but high expression in the colon samples and the aorta. ATP8B1 expression was not observed by tissue expression arrays in multiple regions of the brain, while Northern blotting showed barely detectable levels. These results suggest that ATP8B1 expression may be localized to specific cells within each tissue, such as brain microvascular cells.

ATP8B1 is a flippase that transports phosphatidylserine (PS) from the outer plasma membrane leaflet to the inner plasma membrane leaflet in an ATP-dependent fashion, maintaining the asymmetry of the plasma membrane (Tang et al. 1996; Williamson and Schlegel 2002). ATP8B1 is mutated in progressive familial intra-hepatic cholestasis type 1 (FIC1) (Bull et al. 1998; Harris and Arias 2003) and its mRNA is up-regulated in patients with severe Alzheimer’s disease relative to unaffected individuals (Kong et al. 2009). Changes in ATP8B1 expression or activity may also be associated with the loss of membrane PS asymmetry seen in cerebral tissue of individuals with mild cognitive impairment and Alzheimer’s disease (Lange et al. 2008). The transmembrane domain protein 30A (TMEM 30A), also known as CDC50A, is a receptor for the Llama single domain antibody trans-vascular cerebral delivery system (Muruganandam et al. 2002). TMEM 30A is an essential chaperone protein for ATP8B1 (Bryde et al. 2010; Van der Velden et al. 2010) suggesting TMEM 30A/ATP8B1 may have therapeutic potential for the development of compounds capable of crossing the BBB.

In conclusion, we identified ATP8B1 as a protein that is preferentially expressed in cerebral microvessels and is an integral component of the BBB.