The Journal of Nutrition Vol. 128 No. 8 August 1998, pp. 1276-1282

Copper Efflux from Murine Microvascular Cells Requires Expression of the Menkes Disease Cu-ATPase^1,2

Yongchang Qian^*, Evelyn Tiffany-Castiglioni, Jane Welsh, and Edward D. Harris^{*, **, 3}

Departments of ^* Biochemistry & Biophysics,  Veterinary Anatomy and Public Health and the ^** Faculty of Nutrition, Texas A&M University, College Station, TX 77843 

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Previously, we showed that the transport of Cu by PC12 pheochromocytoma cells and C6 glioma cells correlated with the expression of a Cu-transporting ATPase (Atp7a) that has been linked to Menkes disease. Here, we show that cerebrovascular endothelial (CVE) cells that comprise the blood-brain barrier (BBB) also express the gene for the Cu-ATPase. By using reverse transcription-polymerase chain reaction (RT-PCR) and primers designed from mouse Atp7a cDNA, we amplified a 925-bp and a 760-bp cDNA fragment from two extreme regions of Atp7a mRNA from murine CVE cells; 777 bp of the 925-bp fragment and 677 bp of the 760-bp fragment had a 99.7 and 100% sequence homology, respectively, with mouse Atp7a cDNA. The 777-bp sequences covered the heavy metal binding (Hmb) domain and the 677-bp fragment coded for residues at the COOH terminus of Atp7a. A functional analysis showed that Cu efflux was blocked by the sulfhydryl reagent p-chloromercuribenzoate (p-CMB), a potential inhibitor of Atp7a function. This study provides strong evidence that a Cu-ATPase in the BBB controls the penetration of Cu into the brain and that lesions to the Cu-ATPase in CVE cells are a primary cause of low brain Cu levels in Menkes disease.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Copper is a cofactor for some 30 enzymes, including those involved in catecholamine biosynthesis, ATP production and protection from oxygen free radicals (Danks 1988, Goode 1991, Harris 1991). Harmful effects of a Cu deficiency on central nervous system (CNS)⁴ functions are known or suspected in a number of neurodegenerative diseases such as Menkes disease and Parkinson's disease (Danks et al. 1972, Danks and Cartwright 1973, Dexter et al. 1989 and 1991). This has deepened the understanding of Cu as an important micronutrient for brain function.

Nutritional copper deficiency, although rare in humans, has been described in a number of conditions such as general nutritional deficiency in Peruvian children, pediatric ambulatory peritoneal dialysis and excess zinc consumption by adults (Simon et al. 1988). It has also been suggested that chronic low grade Cu deficiency may become a problem in the United States as a result of removing Cu from prepared foods to achieve extended shelf-life (Danks 1995). Many of the sequelae of Cu deficiency in humans are nonneurologic, including anemia, neutropenia and osteoporosis. However, severe neurologic consequences occur in Menkes disease, an X-linked condition in which Cu transport is disrupted in various organs and brain Cu levels are dangerously low. Infants with Menkes disease exhibit neuronal degeneration throughout the brain regions and die before the age of 2 y (Danks et al. 1972, Danks and Cartwright 1973, Menkes et al. 1962). Cu deficiency in various areas of the brain has also been associated with Parkinson's disease (Dexter et al. 1989 and 1991) and a Parkinson-like syndrome in rats (Sun and O'Dell 1992); neuronal Cu deficiency is suggested as an etiologic factor in neurodegenerative diseases such as Alzheimer's and amyotrophic lateral sclerosis (Hartmann and Evenson 1992).

The mechanisms of copper transport and homeostasis in the CNS are unknown. However, for Cu to enter the CNS from the blood stream, it must be transported across the blood-brain barrier (BBB). The BBB is formed by a continuous layer of cerebral endothelial cells. The abluminal side of the BBB is composed of basement membrane, pericytes and type 1 astrocyte end-feet (Janzer and Raff 1987). The cerebrovascular endothelium (CVE), unlike other endothelia, lacks extensive pinocytic vesicles and has a high resistance (1900 /cm²), features that contribute to the limited permeability function of the BBB (Joo and Klatzo 1989).

The discovery of a Cu-transporting ATPase as the product of the Menkes gene has allowed researchers to explore the molecular mechanism of Cu transport (Chelly et al. 1993, Mercer et al. 1993, Vulpe et al. 1993). This ATPase is a typical P-type with six heavy metal-binding (Hmb) motifs in the N-terminus and an ATPase domain designed to pump Cu ions across membrane barriers such as intestinal mucosa or endothelial capillaries. A defect of gene expression results in Menkes disease and the characteristic neurologic degeneration associated with low brain Cu (Danks et al. 1972, Danks and Cartwright 1973, Menkes et al. 1962). Expression of the gene encoding the Cu-ATPase has been shown by Northern blotting analysis to be organ or tissue specific (Vulpe et al. 1993); cell-type specificity has not been studied as closely.

In a previous study (Qian et al. 1997), we found evidence for a Cu-ATPase expressed in C6 rat glioma cells and the neuron-like PC12 pheochromocytoma cells. The finding of the ATPase in these particular brain cells was consistent with Kodama's observation that glial cells from the brain of brindled and macular mutant mice (two animal models of Menkes disease), when grown in culture, selectively accumulated more Cu than normal cells (Kodama et al. 1991, Kodama 1993). Moreover, excessive glia Cu accumulation could be the defect that led to low Cu in the neurons (Kodama et al. 1993, Yoshimura 1994). Thus, a Cu-ATPase was predicted to be involved in Cu transport from glia into the neurons. However, a defect within the brain proper cannot explain the overall low Cu status of Menkes patients (Danks and Cartwright 1971, Danks 1972). A notable observation by Yoshimura and colleagues (Kodama et al. 1993, Yoshimura 1994) was that Cu accumulated in capillaries of the BBB in brindled and macular mutant mice, suggesting that Cu transport from blood to brain via the BBB was obstructed in Menkes disease. This led us to study the gene expression of Cu-ATPase in CVE cells that form the BBB.

At present, there is no direct method to measure the function of a Cu-ATPase. However, we have shown p-chloromercuribenzoate (p-CMB) to be a potential probe of Cu-ATPase function (Qian et al. 1995, 1996a and 1996b). In this study, we have analyzed Cu transport in CVE cells and combined the analysis with reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing to detect Cu-ATPase gene expression.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

The treatment of animals in this study was approved by the Texas A&M Animal Care and use Committee in accordance with the PHS Animal Welfare Policy.

Cells.  Cloned CVE cells derived from the brain microvessels of SJL-J mice (Harlan Laboratories, Indianapolis IN) were cultured in 75-cm² flasks (Corning, Corning, NY) at 37°C under 5% CO₂ with Iscove's Modified Dulbecco's medium (IMDM; Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco BRL) as described by Sapatino et al. (1993). The medium was changed every 2 or 3 d. Cells were seeded into new flasks or culture dishes after detachment with 0.25% trypsin-EDTA solution (Sigma, St. Louis, MO). Cultures were used for experiments 3 d after passage.

RNA extraction.  Total RNA was extracted from 3-d-old cultures of mouse CVE cells using SDS-phenol according to Sambrook et al. (1989) with modifications (Qian et al. 1997). All buffers were prepared with diethyl pyrocarbonate (DEPC)-treated water and/or autoclaved. Attached cells in a 75-cm² flask were rinsed twice with PBS lacking calcium and magnesium, lysed with 2 mL of 0.5% SDS, 10 mmol/L EDTA (pH 8.0) buffer and transferred to a 15-mL sterile disposable centrifuge tube (Corning). The flask was rinsed with 2 mL of 0.1 mol/L sodium acetate (pH 5.2) containing 10 mmol/L EDTA (pH 8.0); the washings were transferred to the same tube containing the cell lysate. The DNA and protein were precipitated by adding 4 mL phenol (equilibrated with water), shaken for 2 min at room temperature and separated by centrifugation at 4000 × g for 10 min in a Jouan CR412 centrifuge (Jouan, Winchester, VA). The upper aqueous phase was transferred to a fresh sterile disposable tube containing 440 µL of ice-cold 1 mol/L Tris HCl (pH 8.0) and 180 µL of 5 mol/L NaCl. RNA was precipitated by adding 2 volumes of ice-cold ethanol and setting the tube in an ice bath for 30 min. The RNA precipitate was collected by centrifugation (4000 × g for 10 min) (Jouan CR412) and dissolved in 200 µL of ice-cold Tris-EDTA (10 mmol/L Tris·HCl, 1 mmol/L EDTA, pH 8.0) buffer. The RNA was transferred to a 1.5-mL sterile Eppendorf tube, precipitated by adding 500 µL of ice-cold ethanol and 4 µL of 5 mol/L NaCl, and pelleted by centrifugation at 11,400 × g for 5 min on an Eppendorf 5415C centrifuge (Brinkmann Instruments, Westbury, NY). The RNA pellet was suspended in DEPC-treated water, precipitated overnight with 3.5 mol/L LiCl and collected by centrifugation at 11,400 × g for 5 min. Finally, the pelleted RNA was washed twice with 70% ethanol and resuspended in DEPC-treated water after complete evaporation of the ethanol.

Reverse transcription-polymerase chain reaction.  The extracted RNA (4 µg) was reverse transcribed to cDNA with GibcoBRL SuperScript. The Preamplification System for First Strand cDNA Synthesis (Gibco BRL) was as described previously (Qian et al. 1996a). PCR was accomplished with a Taq DNA polymerase amplification system (Promega, Madison, WI). Primers were designed from mouse Atp7a cDNA sequence (Levinson et al. 1994) by using the GCG Wisconsin Package Version 8.1 (University of Wisconsin, Madison, WI) and Vector NTI (Informax, North Bethesda, MD; version 3.0) as described previously (Qian et al. 1997). A forward primer (23 mer, 2871-2893): 5'-GCTACTTTGTTCCTTTCATCGTC-3' and a reverse primer (22 mer, 3630-3609): 5'-CCATTTCTAATCATCCATTCCC-3' for a 760-bp fragment were synthesized at Ana-Gen Technologies (Palo Alto, CA). A forward primer (20 mer, 725-744): 5'-TTTCAACCTCATCTCATCAC-3' and a reverse primer (19 mer, 1649-1631): 5'-ATCTTACTTCTGCCTTGCC-3' for the 925-bp fragment were synthesized at Genosys Biotechnologies (Houston, TX). The amplified products were applied to a 1.0% agarose Tris-Borate-EDTA (TBE; FisherBioTech, Fairlawn, NJ) gel with Lamda EcoRI-HindIII as markers (Promega). Detection was based on ethidium bromide fluorescence.

DNA sequencing.  RT-PCR products were purified from agarose gels with a QIAEX Gel Extraction Kit (QIAGEN, Valencia, CA) as previously described (Qian et al. 1997). The purified DNAs were sequenced with the ABI Dye Terminator Kit with AmpliTaq DNA Polymerase (Amersham, Cleveland, OH) by using cycle fluorescent sequencing as performed at the Crop Biotechnology Center, Texas A&M University. Primers used for sequencing were the same as those used in the PCR reactions for 5'- and 3'-terminus products. The extension products were purified on a Sephadex G-50 (Pharmacia, Piscataway, NJ) to remove free dNTP, suspended in automated sequencing loading buffer and run on an Applied Biosystems 377 DNA Sequencer (Perkin-Elmer, Norwalk, CT). The first 400-600 bp of each sequencing reaction was used in the analysis. Sequencing data were analyzed with GCG Wisconsin Package Version 8.1 (University of Wisconsin).

⁶⁷Cu accumulation and efflux.  Cells cultured in 35-mm dishes were rinsed in situ with 2 mL Dulbecco's phosphate-buffered saline (DPBS; Irvine Scientific, Santa Ana, CA) at room temperature for 30 s after removal of the old medium. Then 2 mL of fresh serum-free IMDM medium containing 50 nmol/L ⁶⁷CuCl₂ (carrier-free, 9.47 × 10¹² TBq/mmol; Brookhaven National Laboratory, Upton, NY) was added. The radioactive medium was removed after 60 min at 37°C. The cells were rinsed twice with 2 mL DPBS at room temperature. Measurements of cell-retained ⁶⁷Cu followed a previously described procedure (Qian et al. 1995). The cells were harvested with a cell scraper (Falcon, Franklin Lakes, NJ) after the addition of 1 mL of 0.5 mol/L NaOH and transferred to separate 4-mL vials for counting. The amount of cell-retained ⁶⁷Cu was determined with a Beckman Gamma 5500 counter. Protein content was assayed with bicinchoninic acid (BCA, Pierce, Rockford, IL) according to Pierce's assay protocol. The absolute molar quantity of ⁶⁷Cu was deduced from a standard curve that plotted CPM vs. standard concentrations of ⁶⁷Cu (1, 2, 5, 10 and 20 pmol) that were analyzed concomitantly with the unknown. The retention of ⁶⁷Cu is expressed as pmol Cu/mg protein.

Treatment of cells with p-chloromercuribenzoate (p-CMB) was conducted as previously described (Qian et al. 1995). Cells in culture dishes were rinsed in situ with 2 mL DPBS and then incubated in 2 mL fresh serum-free IMDM medium containing 0.2 mmol/L p-CMB at 37°C for 30 min. The time was chosen to minimize toxic effects to the cells as determined by trypan blue exclusion and spontaneous detachment of cells from the plastic substrate. After incubation, the medium was removed and cells were rinsed twice with DPBS at room temperature. Cultures were then processed for ⁶⁷Cu accumulation or for ⁶⁷Cu efflux as before. p-CMB at the concentration used (0.2 mmol/L) did not interfere with the BCA protein assay.

View larger version (7K): [in this window] [in a new window]   Fig 1. Regions of amplification on Atp7a cDNA.There were two remote areas on a full-length cDNA that were selected for amplification by the polymerase chain reaction (PCR).The first covered 925 base pairs in the region betwen heavy metal-binding (Hmb) sites 2 and 3 to about Hmb 5. This region encodes a polypeptide in the copper-binding domain of the protein. A second site of 760 base pairs spans part of transmembrane domain 5 (Tm5) and all of Tm6. The region includes many of the key sites in the copper-transporting domains of the protein.

For the study of ⁶⁷Cu efflux, cells were incubated at 37°C with 2 mL of fresh serum-free medium containing 50 nmol/L ⁶⁷CuCl₂. After 60 min, the medium was removed and cells were rinsed quickly with 2 mL of DPBS. Fresh serum-free medium (2 mL) was then added to the ⁶⁷Cu loaded cells. The efflux was interrupted at 0, 3, 8, 18 and 43 min. Each time, the cells were rinsed quickly and then scraped off the dish with 0.5 mol/L NaOH as described above. ⁶⁷Cu retained in the cells at the start of the efflux (⁶⁷Cu_t=0) and at any time into the efflux period (⁶⁷Cu_t) is expressed as the percentage of the initial copper load as described previously (Qian et al. 1996b).

Data analysis.  Where indicated, results are expressed as means ± SD. Data were tested by a 2-column unpaired Student t test procedure (GraphPad InStat package, GraphPad Software version 2.04; GraphPad Software, San Diego, CA). A minimal significant difference between means was considered to be P < 0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

Atp7a gene expression.  Our first goal was to determine whether CVE cells express the gene for Atp7a. To measure gene expression, we used RT-PCR with two sets of primers that were designed specifically for sequences in the Menkes Cu-ATPase and no other Cu-ATPase. The primers were further designed to amplify two remote regions of the transcript. The strategy for PCR primer design is shown in Figure 1. The first set amplified bases 725-1649 and included sequences that coded for three of the six Hmb sites (3rd, 4th and 5th), each sharing the structural motif GMT/HCXSC. The second set amplified a 760-bp fragment from nucleotide positions 2871-3630 and included part of the fifth transmembrane domain (Tm5), the whole sixth Tm (Tm6) with the transduction motif CPC embedded in this sequence, the DKTGT phosphorylation site, and the SEHPL motif after Tm6. As seen in Figure 2, cDNA fragments corresponding to the 760 bp (lane 1) and 925 bp (lane 3) were the major products amplified by the PCR procedure.

View larger version (96K): [in this window] [in a new window]   Fig 2. Identification of the 925-bp product and 760-bp product on electrophoresis gels. Total RNA (4 µg) from cerebrovascular endothelial cells was reverse transcribed to cDNA. The product of the polymerase chain reaction (PCR) was applied to a 1% agarose gel prepared in Tris-Borate-EDTA buffer (TBE) run at 50 V for 1 h. The gels were stained in 5 g/L ethidium bromide for 10 min and destained in TBE. The markers (Lambda EcoR I-Hind III, 0.5 µg) used to determine the size of the fragments (k = kilobases) are denoted in lane M. Lane 1: PCR product of 760 bp (0.2 µg RNA); lane 2: purified 760 bp; lane 3: PCR product of 925 bp (0.2 µg RNA); lane 4: purified 925 bp. Samples that had no cDNA template did not show any bands.

cDNA sequence and homology analysis.  To confirm their identification and position in the full transcript, the 760-bp and 925-bp fragments from PCR amplification were excised and purified for sequence analysis. Figure 3A displays the 777 bp in the 925-bp fragment and compares this sequence with mouse Atp7a cDNA in GenBank (bottom line in Figure 3A). The lower numbers correspond to the position in the published full transcript. Of the 777 bases in sequence, only two did not match Atp7a. These occurred at nucleotide positions 1490 and 1510. Figure 4A shows the 259 amino acid residues coded by the 777 bp. The positions of the three Hmb sites, viz., Hmb 3 (GMHCKSC), Hmb 4 (GMTCNSC) and Hmb 5 (GMTCASC) are clearly shown. Because of the nucleotide mismatch at nucleotide position 1490 mentioned earlier, there would be a proline in place of a leucine at residue 466. In both rat C6 glioma cells and PC-12 cells, Atp7a at this residue position showed a proline (P) (Qian et al. 1997). The mismatch at nucleotide 1510 would retain serine 472 before Hmb 5. The 777 nucleotide sequence was determined to have a 99.7% sequence homology to mouse Atp7a cDNA and the coded peptide of 259 amino acids a 99.6% homology to mouse Atp7a (Table 1).

View larger version (77K): [in this window] [in a new window]   View larger version (84K): [in this window] [in a new window]   Fig 3. Comparison of cDNA sequences of cerebrovascular endothelial(CVE) cells with known sequence of mouse Atp7a. Top lines (denoted SJL) show cDNA sequences from CVE cells (this study); bottom line (denoted Bnk) displays the GenBank data previously reported for mouse Atp7a. Nucleotide differences are underlined in bold. Upper numbers refer to nucleotide positions in the CVE cDNA; lower numbers are the corresponding nucleotide positions in a full-length mouse Atp7a cDNA. (A):nucleotide base sequences obtained from the 760-bp fragment; (B): nucleotide base sequences from the 925-bp fragment. Underlined bases denote differences.

View larger version (38K): [in this window] [in a new window]   View larger version (33K): [in this window] [in a new window]   Fig 4. Comparison of amino acid sequences derived from cerebrovascular endothelial (CVE) cDNA with mouse Atp7a. Top lines (denoted SJL) are amino acid sequences derived from CVE cells (this study); bottom lines (denoted Bnk) display amino acid sequence data for mouse Atp7a filed in GenBank. Bold underlined letters show residues that do not agree. (A): the alignment of 254 amino acids deduced from the 925-bp cDNA fragment. Extended underlining shows sequences in the regions where copper binds. (B): the alignment of 225 amino acids deduced from the 760-bp cDNA fragment. Boxes show the location of two transmembrane domains in this region of the protein. Transduction motif CPC, phosphorylation site DKTGT and SEHPL motifs are underlined. Letters are standard abbreviations for amino acids.

In contrast to the two mismatches observed in the 777-bp cDNA fragment, the 677 fragment showed perfect identity with mouse Atp7a (Fig. 3B). The 677 bp covers a region of 225 amino acid residues that comprise part of Tm5 and the whole of Tm6, which include the critical CPC transduction motif as shown by the boxes in Figure 4B. The phosphorylation site DKTGT, and the SEHPL motif are underlined (Fig. 4B). This 677 bp and its corresponding peptide product have 100% homology to mouse Atp7a (Table 1). Thus, the data support expression of Menkes gene by cerebrovascular endothelial cells of the BBB.

Functional analysis of Atp7a.  As a further test of gene expression, we tested cerebrovascular endothelial cells for Cu transport. The proposed function of Atp7a is to pump Cu ions out of cells (Levinson et al. 1994, Vulpe et al. 1993). In our previous studies (Qian et al. 1995, 1996a and1996b), we showed that p-CMB, a reagent that binds to the sulfhydryl groups on proteins, blocked Cu release and produced an efflux defect analogous to the defect in Menkes cells. As shown in Figure 5, CVE cells briefly exposed to 0.2 mmol/L p-CMB for 30 min and then washed to removed excess reagent accumulated significantly more Cu than untreated cells (n = 8, P < 0.001). In the 60 min incubation of 50 nmol/L ⁶⁷CuCl₂, the amount of ⁶⁷Cu accumulated was twice the amount accumulated by untreated control cells. In terms of measured quantities, p-CMB-exposed cells amassed 65.9 ± 7.2 pmol Cu/mg protein (n = 8) compared with 30.9 ± 3.0 pmol Cu/mg protein (n = 8) in control cells. In a second test of Atp7a function, p-CMB-treated and control cells were exposed to 50 nmol/L ⁶⁷CuCl₂ in a 60-min incubation and tested for ⁶⁷Cu efflux. Treated cells showed a negligible release of ⁶⁷Cu during a subsequent 45-min incubation in fresh medium (Fig. 6). In contrast, cells not treated with p-CMB released significantly more ⁶⁷Cu (25 ± 9%; n = 4, P < 0.01) after the 45-min incubation. These data are consistent with the hypothesis that the function of Atp7a contributed to Cu release.

View larger version (40K): [in this window] [in a new window]   Fig 5. Effect of p-chloromercuribenzoate (p-CMB) treatment on Cu retention in cultured cerebrovascular endothelial cells. Cells were incubated in serum-free Iscove's modified Dulbecco's medium at 37°C for 30 min with and without 0.2 mmol/L p-CMB. The cells were washed twice with Dulbecco's PBS to remove excess p-CMB and incubated at 37°C for 60 min in fresh medium containing 50 nmol/L 67CuCl2. After harvesting and washing, cell-retained 67Cu was determined. Values are means ± SD, n = 8 (***P < 0.001).

View larger version (18K): [in this window] [in a new window]   Fig 6. Effect of p-chloromercuribenzoate (p-CMB) on Cu efflux from cerebrovascular endothelial cells. Cells were preloaded with 50 nmol/L 67CuCl2 in serum-free Iscove's modified Dulbecco's (IMDM) medium at 37°C for 60 min. Unbound radioactive Cu was washed out with Dulbecco's PBS (DPBS) and fresh serum-free IMDM medium was added to the 67Cu-laden cells. Before the addition of 67Cu, one group of cells was treated with p-CMB (0.2 mmol/L at 37°C for 30 min) and excess p-CMB was removed by washing the cells twice with DPBS. Values are means ± SD, n = 4 or 5 (**P < 0.01).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

In this study, we detected and sequenced two cDNA fragments from mouse CVE cells that represented segments of a mouse homologue of the Menkes gene. The primers were designed so as not to amplify any other transcript that may be present in the extract, e.g., the mRNA coding for ATP7B, the Cu-ATPase associated with Wilson's disease. The sequence data confirmed that specificity criteria had been met. The fragments corresponded to sequences that encode part of the Hmb and ATPase domains of Atp7a. Because they possessed identical homologies to mouse Atp7a cDNA from the literature (Levinson et al. 1994), there is clear evidence that the Atp7a gene is expressed in CVE cells in the BBB. We have also confirmed the conclusion by synthesizing and cloning a 4.7-kb cDNA fragment containing the whole open reading frame of Atp7a by using a mRNA from CVE cells as a template (Qian et al., unpublished results). In a second study in this report, we obtained evidence that efflux of Cu from CVE cells is sensitive to sulfhydryl-binding agents. Together, the detection of Atp7a gene expression by RT-PCR and the functional analysis with p-CMB suggest that Atp7a is a component of Cu efflux from CVE cells of the BBB.

With this discovery, it is now possible to rationalize why a shutdown of Atp7a gene expression or dysfunction of Atp7a in brindled and macular mice resulted in Cu accumulation in the capillaries of the BBB (Yoshimura 1994, Yoshimura et al. 1995). The data suggest that the CVE cells have the capacity to absorb Cu ions into the cytosol but fail to clear the Cu completely through the barrier. After Cu passes through the BBB, its transport or distribution is still controlled by Atp7a proteins in other cells such as the glia and neurons. This rather complex and intricate network of intercommunication eventually leads to the movement of Cu ions into the neurons and its availability to enzymes engaged in the biosynthesis of neurotransmitters, dopamine and norepinephrine. Such a network begins at the CVE as shown in Figure 7.

View larger version (20K): [in this window] [in a new window]   Fig 7. Model of Cu homeostasis in the central nervous system. Dark circles indicate the membrane location of Atp7a. We have previously reported evidence for the presence of Atp7a in the astroglia-like C6 glioma cell line, as well as the neuronal PC-12 cell line (Qian et al. 1997). This model incorporates the glial and neuronal pumps, as well as the cerebrovascular endothelial (CVE) cell pump described in this study. According to the model, Cu taken up by CVE cells that form the blood-brain barrier (BBB) is pumped to the neuropil by endothelial cell Atp7a. Astroglia surrounding the BBB then accumulate Cu, which they provide to neurons via their Atp7a. Cu in the neurons is transported back to the glia through an Atp7a in the neurons, thereby maintaining Cu homeostasis in the neurons. Neurons (N), glia (G) and capillaries (CVE).

The prevalence of lesions in discrete brain regions is believed to be a causative factor in many of the symptoms of Menkes disease. Low Cu content in brain and low Cu in neurons have been observed in Menkes patients, confirming and in some respects extending the data obtained from brindled mice and macular mice (Danks et al. 1972, Danks and Cartwright 1973, Kodama 1993, Kodama et al. 1993, Yoshimura 1994). The clinical manifestations of Menkes disease, therefore, are not caused by a Cu toxicity but instead by a lack of Cu in certain regions beyond the BBB. A toxic Cu buildup in the BBB, should it occur, may be ameliorated somewhat by the binding of Cu to metallothionein (Nishimura et al. 1992). The widespread pathologic lesions induced by a Cu deficiency give rise to seizures and other disturbances in mental function characteristic of a seriously impaired CNS. The Menkes gene in capillaries must play an important, but as yet undefined role in transporting Cu from the blood to the brain. This study, therefore, suggests that a Cu-ATPase is at the critical entry point of Cu into the brain and strengthens the concept that the BBB is a primary lesion site for Menkes disease in the CNS.

	FOOTNOTES

Manuscript received 30 October 1997. Initial reviews completed 2 February 1998. Revision accepted 3 April 1998.

	ACKNOWLEDGMENT

We thank Deepani Tennakoon in the Department of Veterinary Anatomy and Public Health for providing the cell cultures used in the study.

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