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Review Article |
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina (S.D., D.S.M.); and Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada (S.D., R.B.)
Abstract
Abstract I. Introduction II. The ATP-Binding Cassette Transporter Superfamily A. MRP1 B. MRP2 C. MRP3 D. MRP4 E. MRP5 F. MRP6 G. MRP7 H. MRP8 I. MRP9 III. Multidrug Resistance-Associated Protein Expression and Function in the Central Nervous System A. The Blood-Brain Barrier B. The Blood-Cerebrospinal Fluid Barrier C. Microglia D. Astrocytes E. Neurons and Oligodendrocytes IV. Clinical Relevance of Multidrug Resistance-Associated Proteins in the Central Nervous System A. Epilepsy B. Brain Cancer C. HIV/AIDS D. Parkinson's and Alzheimer's Diseases V. Concluding Remarks
Drug delivery to the brain is highly restricted, since compounds must cross a series of structural and metabolic barriers to reach their final destination, often a cellular compartment such as neurons, microglia, or astrocytes. The primary barriers to the central nervous system are the blood-brain and blood-cerebrospinal fluid barriers. Through structural modifications, including the presence of tight junctions that greatly limit paracellular transport, the cells that make up these barriers restrict diffusion of many pharmaceutically active compounds. In addition, the cells that comprise the blood-brain and blood-cerebrospinal fluid barriers express multiple ATP-dependent, membrane-bound, efflux transporters, such as members of the multidrug resistance-associated protein (MRP) family, which contribute to lowered drug accumulation. A relatively new concept in brain drug distribution just beginning to be explored is the possibility that cellular components of the brain parenchyma could act as a "second" barrier to brain permeation of pharmacological agents via expression of many of the same transporters. Indeed, efflux transporters expressed in brain parenchyma may facilitate the overall export of xenobiotics from the central nervous system, essentially handing themoff to the barrier tissues. We propose that these primary and secondary barriers work in tandem to limit overall accumulation and distribution of xenobiotics in the central nervous system. The present review summarizes recent knowledge in this area and emphasizes the clinical significance of MRP transporter expression in a variety of neurological disorders.
Neurologically based diseases remain difficult to treat, and are often refractory to currently available medications despite recent advances in our understanding of their underlying pathophysiological mechanisms. The reasons for the observed pharmacotherapeutic failures are multifactorial, but increasingly the presence of membrane-bound, active transport carriers has been implicated. Several ATP-dependent transport proteins have overwhelmingly emerged as the main culprits including P-glycoprotein (P-gp)1, the breast cancer resistance protein (BCRP), and multiple isoforms of multidrug resistance-associated proteins (MRPs). The importance of P-gp and BCRP within the central nervous system (CNS) have both been recently reviewed elsewhere (Doyle and Ross, 2003
; Lee and Bendayan, 2004; Bauer et al., 2005). Here we provide a comprehensive review of studies examining CNS expression, function, and localization of MRP transporters undertaken to date. Given the complexity of the MRP family, we first provide a summary of the larger superfamily to which MRP proteins belong, as well as an overview of MRP expression, function and localization in peripheral tissue compartments.
II. The ATP-Binding Cassette Transporter Superfamily
The ATP-binding cassette (ABC) superfamily of proteins contains a number of membrane-bound, ATP-driven transporters that pump drugs, drug metabolites, and endogenous metabolites out of cells. Members of the ABC family are classified as such according to the presence of several consensus sequences including two ATP binding motifs (Walker A and Walker B), as well as the ABC signature C motif (ALSGGQ) (Leslie et al., 2005). At present there are 49 known human ABC family members belonging to 7 different subfamilies. A comprehensive list of currently known human ABC transporters compiled by Dr. M. Müller (Wageningen University, The Netherlands) and divided by subfamilies can be found at http://nutrigene.4t.com/humanabc.htm. Mutations in some of the ABC genes result in the generation of genetic disorders such as cystic fibrosis, anemia, Dubin Johnson's syndrome, and retinal degeneration (Dean et al., 2001; Stefkova et al., 2004). Moreover, P-gp, BCRP, and several MRP isoforms in particular are important determinants of drug accumulation in target cells (e.g., tumor cells) and of overall drug uptake, distribution, and excretion. Collectively, they have all been implicated in the development of the multidrug resistance (MDR) phenotype. Underlying this type of MDR is active efflux from cells of a large number of structurally and functionally unrelated pharmacological agents. The development of MDR is associated with poor clinical outcome in several neurological disorders (Loscher and Potschka, 2005b).
At present the cystic fibrosis transmembrane conductance regulator/MRP family (ABC subfamily C) contains 13 members, including one ion channel (cystic fibrosis transmembrane regulator gene), two cell surface receptors [sulfonylurea 1 and 2 (SUR1 and 2)], and a truncated protein that does not mediate transport (ABCC13) (Haimeur et al., 2004). These proteins, which show no capacity for drug transport, will not be discussed further. The remaining nine MRP members can be further divided into two types based on putative membrane topology (Kruh and Belinsky, 2003). MRP1 to MRP3 and MRP6 and MRP7 contain three transmembrane domains, TMD0, TMD1, and TMD2, which show a 5 + 6 + 6 configuration in transmembrane helices (Fig. 1). Nucleotide binding domains 1 and 2 are located between TMD1 and TMD2 and between TMD2 and the carboxyl terminus, respectively. A cytoplasmic linker (L0) located between the first two TMDs is essential for a functional protein (Bakos et al., 1998). MRP4, MRP5, MRP8, and possibly MRP9 are considered to be "short" MRPs, as they do not contain TMD0 but do retain the cytoplasmic linker. Not surprisingly, the many MRP isoforms show differences with respect to tissue distribution, substrate specificity, and proposed physiological function. Table 1 provides a brief summary of the nomenclature and general substrate specificities of MRP1 through MRP9.
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The MRP12 gene was first cloned in 1992 from a human small cell lung cancer cell line (H69AR) that demonstrated an MDR phenotype without concomitant expression of P-gp (Cole et al., 1992). Mrp1 was subsequently cloned and sequenced in several other species including mouse, rat, dog, monkey, and cow (Stride et al., 1996; Ma et al., 2002; Taguchi et al., 2002; Yang et al., 2002; Godinot et al., 2003). In humans, MRP1 is widely expressed, with highest levels observed in the kidney, lung, testes, and peripheral blood mononuclear cells (Haimeur et al., 2004). Similar tissue distribution of Mrp1 has been noted in rats, dogs, mice, and cows (Stride et al., 1996; Conrad et al., 2001; Taguchi et al., 2002; Nunoya et al., 2003). In polarized epithelia of the intestine, kidney, liver, and lung, MRP1/Mrp1 is localized to basolateral plasma membranes of all species tested (Mayer et al., 1995; Peng et al., 1999; Pei et al., 2002; Scheffer et al., 2002b).
Cells that highly express MRP1 confer resistance to a variety of natural product anticancer drugs including vinca alkaloids, anthracyclines, and epipodophyllotoxins (Cole et al., 1994; Zaman et al., 1994; Breuninger et al., 1995). In contrast to P-gp, MRP1 shows preferential transport of anionic compounds such as glucuronide, glutathione (GSH), and sulfate conjugates (Leier et al., 1994, 1996; Jedlitschky et al., 1996, 1997; Loe et al., 1996). Typical conjugated substrates include 2,4-dinitrophenyl-S-glutathione (DNP-SG), estradiol-17
-glucuronide (E217G), and estrone 3-sulfate. MRP1 also has the capacity to transport metalloids as oxyanions, such as antimony and arsenic, acetanalide pesticides, and various dietary constituents including bioflavanoids and tobacco-derived carcinogens (Leslie et al., 2005). MRP1 transports certain cationic compounds such as vincristine and etoposide, but only in the presence of the antioxidant GSH, probably via cotransport (Rappa et al., 1997; Loe et al., 1998). Interestingly, the expression patterns of MRP1 and 
-glutamyl cysteine synthetase, the rate-limiting enzyme of GSH synthesis, may be coordinately regulated by oxidative stress and heavy metals (Ishikawa et al., 1996; Yamane et al., 1998; Lin-Lee et al., 2001). Transport of conjugated compounds and the oxidized form of GSH (GSSG), in addition to possible up-regulation of GSH-synthesizing enzymes, strongly suggests a role for MRP1 in detoxification and "phase III" elimination of toxic endogenous metabolites (Leslie et al., 2001). In addition, MRP1 shows high affinity (Km 
100 nM) for the inflammatory mediator leukotriene C4 (LTC4) and probably also plays a significant role in mediating immune responses (Haimeur et al., 2004). Indeed, Mrp1-deficient mice are viable and fertile but show decreased response to inflammatory stimuli, presumably due to decreased LTC4 efflux (Lorico et al., 1997; Wijnholds et al., 1997).
Recently, Nunoya et al. (2003) directly compared the resistance profiles and transport characteristics of three species of MRP1 commonly used in transport studies (i.e., rat, mice, and humans) and found substantial differences. In MRP1/Mrp1-transfected human embryonic kidney cell lines (HEK293), resistance conferred to vincristine by rat Mrp1 (7-fold) was slightly lower than for mouse Mrp1 (12-fold) and human MRP1 (11-fold). Rat and mouse Mrp1-expressing cells also showed little or no resistance to doxorubicin, daunorubicin, or epirubicin, compared with human MRP1. Bovine Mrp1 does not seem to transport doxorubicin efficiently either (Taguchi et al., 2002). Although LTC4 affinity is similar for rat Mrp1, mouse Mrp1, and human MRP1 (Km 
50–100 nM), human MRP1 transports E217G with greater than 10-fold efficiency compared with the mouse and rat homologs (Nunoya et al., 2003). These results clearly highlight the importance of using caution when making cross-species comparisons.
MRP2/Mrp2 has been cloned from various species including humans, dogs, mice, rats, and rabbits (Buchler et al., 1996; Taniguchi et al., 1996; van Kuijck et al., 1996; Fritz et al., 2000; Conrad et al., 2001). MRP2/Mrp2 protein is localized to the apical membrane of polarized cells from a variety of human and rat tissues including enterocytes of the small intestine (Mottino et al., 2000; Rost et al., 2002), hepatocytes (Buchler et al., 1996; Keppler et al., 1997a), and renal proximal tubules (Schaub et al., 1997, 1999). In this respect, MRP2 colocalizes with P-gp and BCRP (Schinkel and Jonker, 2003). High expression of MRP2 is found in liver, intestine, and kidney, with little or no expression observed in other tissues (Kool et al., 1997). The tissue distribution of Mrp2 in dogs, mice, and rats is very similar to that in humans with one notable exception: dogs show lower levels of Mrp2 in the liver compared with the kidney (Conrad et al., 2001; Cherrington et al., 2002; Ninomiya et al., 2005).
The substrate specificity and resistance profile of MRP2 are similar to those for MRP1 and include various conjugated and unconjugated organic anions and cations, i.e., methotrexate, LTC4, DNP-SG, E217G, vincristine, etoposide, and bilirubin glucuronosides (Jedlitschky et al., 1997; Evers et al., 1998; Cui et al., 1999; Hooijberg et al., 1999). Unlike MRP1, MRP2 also confers resistance to cisplatin (Koike et al., 1997; Cui et al., 1999; Borst et al., 2000). In several instances, MRP2 transports these compounds with lower affinity than MRP1 (Konig et al., 1999a). For example, compared with MRP1, MRP2 has exhibited 10- and 4-fold lower affinities for LTC4 and E217G, respectively (Cui et al., 1999). No difference was observed in the affinity of human and rat MRP2/Mrp2 for both LTC4 and E217G (Cui et al., 1999). Given its location and substrate specificity, MRP2/Mrp2 probably plays an important role in excreting metabolites into the bile. This conclusion is supported by the observation that absence of MRP2 from the canalicular membrane results in impaired efflux of bilirubin glucuronide into the bile and manifests clinically as Dubin-Johnson syndrome (Leslie et al., 2001).
The MRP3/Mrp3 gene has been cloned from human, rat and mouse (Hirohashi et al., 1998; Kiuchi et al., 1998; Belinsky et al., 2005). MRP3/Mrp3 is highly expressed in the intestine and kidney (Uchiumi et al., 1998; Cherrington et al., 2002; Maher et al., 2005). It is localized to the basolateral side of hepatocytes (Konig et al., 1999b; Kool et al., 1999b), cholangiocytes (Kool et al., 1999b; Soroka et al., 2001), and intestinal epithelial cells (Hirohashi et al., 2000; Rost et al., 2002). Only low levels of MRP3 are found normally in the liver. However, MRP3 expression in liver is higher in patients with Dubin-Johnson syndrome, probably as compensation for the absence of MRP2. This has been demonstrated in rodent models of Dubin-Johnson syndrome (e.g., the Eisai hyperbilirubinemic rat), in which Mrp3 mRNA and protein expression in liver and kidney are increased significantly (Kuroda et al., 2004). Furthermore, induction of MRP3/Mrp3 also occurs in cholestatic rat (Hirohashi et al., 1998) and human livers (Kool et al., 1999b; Konig et al., 1999b), which further supports up-regulation of MRP3/Mrp3 as a protective mechanism (i.e., bilirubin and metabolite removal) when MRP2/Mrp2 is either absent or nonfunctional.
MRP1, MRP2, and MRP3 have similar substrate profiles with some notable differences. Murine fibroblast cells transfected with MRP3 show high levels of resistance to etoposide and teniposide but not to doxorubicin, vincristine, or cisplatin (Zelcer et al., 2001). Although all three proteins transport etoposide, MRP3 does so in a GSH-independent manner (Zelcer et al., 2001). Unlike MRP1 (Loe et al., 1998) and MRP2 (Evers et al., 2000), MRP3-overexpressing cells do not transport GSH (Zelcer et al., 2001). Glucuronide and sulfate conjugates of bile salts are substrates of both MRP1 and MRP3, but MRP3 also mediates transfer of monovalent bile salts including glycocholate (Zeng et al., 2000). Glucuronide conjugates (i.e., E217G) seem to be preferentially transported by MRP3/Mrp3 compared with GSH conjugates such as DNP-SG and LTC4 (Hirohashi et al., 1999; Zeng et al., 2000).
The human and rat MRP3/Mrp3 orthologs generally have similar substrate specificities, although some kinetic differences have been described. In Sf9 transfected vesicles, the affinities of E217G by human and rat MRP3/Mrp3 are comparable, i.e., 42.9 and 33.4 µM, respectively (Akita et al., 2002). However, uptake of E217G by MRP3 is inhibited by methotrexate at concentrations 11-fold lower than those for rat Mrp3 (Akita et al., 2002). Conversely, a 4-fold higher concentration of DNP-SG is required to inhibit uptake of E217G by human MRP3 versus rat Mrp3 (Akita et al., 2002). Finally, the monovalent bile salt taurocholate is only transported by rat Mrp3 (Akita et al., 2002). Given its location and substrate profile, MRP3/Mrp3 is proposed to play an important role in enterohepatic circulation of endogenous compounds such as bile salts (Rost et al., 2002).
MRP4 is expressed at low levels in a variety of human tissues, with high levels occurring in the prostate and kidney (Kool et al., 1997; Lee et al., 1998). The rat and murine Mrp4 orthologs show greater than 83% amino acid identity with human MRP4 (Chen and Klaassen, 2004). Membrane localization of MRP4/Mrp4 in polarized cells remains unresolved. In human and rat kidney proximal tubule epithelia, MRP4/Mrp4 was located in the apical membrane (van Aubel et al., 2002; Leggas et al., 2004). However, when transfected into Madin-Darby canine kidney II (MDCKII) cells, MRP4 routed to the basolateral membrane (Lai and Tan, 2002). Immunocytochemical studies by Lee et al. (2000) localized MRP4 to the basolateral membrane of tubuloacinar cells of the prostate. Finally, MRP4/Mrp4 was localized to the sinusoidal membrane of human, rat, and mouse hepatocytes, as well as in the human hepatoma cell line HepG2 (Rius et al., 2003; Zelcer et al., 2003).
Unlike MRP1–3, MRP4 contains only two membrane-spanning domains (Fig. 1). MRP family members displaying this topology seem to show a unique capacity to transport and confer resistance to a variety of monophosphorylated compounds. The ability of MRP4 to transport cyclic nucleotides (cAMP and cGMP), nucleotide analogs such as 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and azidothymidine monophosphate and purine analogs (6-mercaptopurine and 6-thioguanine) has been well described (Schuetz et al., 1999; Chen et al., 2001; Lai and Tan, 2002; Wielinga et al., 2002, 2003; Reid et al., 2003a). MRP4 does not confer resistance to typical unconjugated substrates of MRP1 and MRP2 such as doxorubicin, etoposide, vincristine, or taxol (Lee et al., 2000) but does retain the capacity to efflux conjugated compounds including E217G (Zelcer et al., 2003).
As expected, MRP4-transfected cells show increased efflux of monophosphorylated nucleotides and nucleotide analogs such as cAMP, cGMP, and PMEA. However, the full substrate spectrum of MRP4 seems to be much broader than initially presumed. Recently Reid et al. (2003b) showed transport of prostaglandins E1 (Km = 2.4 µM) and E2 (Km = 3.4 µM), in MRP4-transfected HEK293 cells, but not in cells expressing MRP1, MRP2, MRP3, or MRP5. Low-affinity transport of methotrexate (Km = 0.22–1 mM) by MRP4 has also been reported (Chen et al., 2002; van Aubel et al., 2002). In membrane vesicles prepared from MRP4-transfected Sf9 insect cells, ATP-dependent uptake was observed for E217G (Km = 30 µM) and dehydroepiandrosterone 3-sulfate (Km = 2 µM), but not LTC4 or DNP-SG (Chen et al., 2001; van Aubel et al., 2002; Zelcer et al., 2003). In contrast to MRP1, MRP4-mediated transport of dehydroepiandrosterone 3-sulfate is GSH-independent (van Aubel et al., 2002). Interestingly, GSH stimulates transport of unconjugated bile acids (cholyltaurine, cholylglycine, and choline) from hepatocytes, suggesting that some MRP4 substrates may require GSH for efficient transport (Rius et al., 2003).
The MRP5 gene is ubiquitously expressed with the highest levels of expression found in skeletal muscle and the brain (Kool et al., 1997; Belinsky et al., 1998). Mouse and rat Mrp5 orthologs show high levels of amino acid identity with MRP5, i.e., >94% (GenBank accession no. AB020209; Suzuki et al., 2000). The tissue expression of mouse Mrp5 is reported to be similar to that in human and rat (Suzuki et al., 2000; Maher et al., 2005, 2006); Mrp5 knockout mice are healthy and fertile and do not show any observable physiological dysfunctions (Wijnholds et al., 2000b).
Similar to MRP4, MRP5 lacks the TMD0 domain and does not interact with typical substrates of MRP1, MRP2, or MRP3 such as vincristine, LTC4, etoposide, or daunorubicin (McAleer et al., 1999; Jedlitschky et al., 2000). However, the substrate profile of MRP5 seems to be much narrower than MRP4. Both MRP4 and MRP5 transport monophosphorylated compounds, such as PMEA (Wijnholds et al., 2000b; Reid et al., 2003a; Weilinga et al., 2003), but differences do exist with respect to sensitivities to MRP inhibitors. For example, the inhibitory concentration for probenecid is 10-fold higher for MRP4-mediated PMEA transport than for MRP5 (Reid et al., 2003a). In contrast, MK571 inhibits MRP4-mediated transport of PMEA at 4-fold lower concentrations than it does for MRP5 (Reid et al., 2003a). Studies in MRP4- and MRP5-overexpressing cells have shown that the anti-HIV agent stavudine monophosphate is one monophosphorylated compound transported by MRP5 but not by MRP4 (Reid et al., 2003a).
Membrane localization of MRP5 in polarized cells, including MRP5-transfected MDCKII cells, seems to be basolateral (Wijnholds et al., 2000b). Interestingly, in nonpolarized MRP5-transfected HEK293 cells, much of the MRP5 protein is located intracellularly, with little expression occurring in the plasma membrane. Whether this intracellular expression is an artifact of the transfection process or endogenous nonpolarized cells show a similar pattern of intracellular MRP5 expression is unknown.
Mrp6 was initially cloned from rat liver (Hirohashi et al., 1998) and has been subsequently cloned in humans and mice (Belinsky and Kruh, 1999; Madon et al., 2000; Beck et al., 2003). The mouse and rat Mrp6 orthologs show greater than 78% amino acid identity with human MRP6. Mutations in the MRP6 gene have been implicated in the etiology of pseudoxanthoma elasticum, a hereditary connective tissue disorder characterized by loss of tissue elasticity (Bergen et al., 2000; Hu et al., 2003).
Human, rat, and mouse MRP6/Mrp6 is predominantly expressed in the liver and kidney, with low levels detected in most other tissues (Kool et al., 1999a; Madon et al., 2000; Maher et al., 2005, 2006). Initial immunocytochemical studies localized rat Mrp6 to both the basolateral (strong staining) and canalicular (weaker staining) plasma membranes of hepatocytes (Madon et al., 2000). In a subsequent study, MRP6 was only present in the basolateral membrane of human hepatocytes (Scheffer et al., 2002a). A basolateral orientation was also demonstrated in human and mouse kidney proximal tubules and in MRP6-transfected MDCKII epithelial cells (Beck et al., 2003; Sinko et al., 2003).
Chinese hamster ovary cells transfected with MRP6 cDNA show increased resistance to a variety of anticancer agents including etoposide, doxorubicin, daunorubicin, and cisplatin but not to vincristine or vinblastine (Belinsky et al., 2002). In these same cells, MRP6 mediated transport of GSH conjugates (LTC4 and DNP-SG) and the cyclic pentapeptide endothelin receptor inhibitor BQ123 but not of glucuronide conjugates (i.e., E217G), methotrexate, or cyclic nucleotides (Belinsky et al., 2002). Likewise, in MRP6 transfected Sf9 insect cells, MRP6 transported LTC4 well (Km = 600 nM) but showed only low level transport of N-ethylmaleimide S-glutathione (Km = 282 µM) (Ilias et al., 2002). In contrast, under different conditions, Madon et al. (2000) reported that rat Mrp6 transports the endothelin receptor antagonist BQ-123 (Km 17 µM) but not LTC4, or DNP-SG. Little or no MRP6 expression is detectable in human tumor specimens (e.g., intestine, testis, prostate, lung, adrenal gland, cervix, ovary, kidney, and melanoma) or human tumor cell lines, which suggests that MRP6 does not play an important role in tumor MDR (Kool et al., 1999a; Scheffer et al., 2002a).
The MRP7 protein exhibits a membrane topology similar to that of MRP1, MRP2, MRP3, and MRP6 (Hopper et al., 2001). MRP7 mRNA was detected in a variety of tissues, with relatively higher levels reported in colon, skin, and testes (Hopper et al., 2001). Two Mrp7 genes have also been identified in mice (Mrp7A and Mrp7B), and these show >80% amino acid similarity with their human counterparts (Kao et al., 2003).
The substrate specificity and resistance profile of MRP7 have been examined in MRP7-transfected HEK293 cells (Chen et al., 2003; Hopper-Borge et al., 2004). Drug resistance to docetaxel, and to a lesser degree paclitaxel, vincristine, and vinblastine, was reported (Hopper-Borge et al., 2004). MRP7 did not transport methotrexate, DNP-SG, monovalent bile salts (glycocholic acid and taurocholate) or cyclic nucleotides (cAMP and cGMP) (Chen et al., 2003); only modest LTC4 transport was noted. Likewise, MRP7 mediated low-affinity transport of E217G (Km = 58 µM) (Chen et al., 2003). Further studies examining substrate specificity, subcellular localization, and physiological function are needed to clarify the role, if any, that MRP7 may play in development of the MDR phenotype.
MRP8 is the third MRP isoform without a third transmembrane domain in the amino-terminal portion of the protein. MRP8 mRNA transcript is highly expressed in breast cancer but also shows a low level of expression in a variety of other human tissues including breast, testes, and the brain (Bera et al., 2001; Tammur et al., 2001; Yabuchi et al., 2001). In transfected MDCKII and HepG2 cells, MRP8 is localized at the apical pole (Bortfeld et al., 2006). Despite extensive searches within the mouse genome, a murine Mrp8 ortholog was not found (Shimizu et al., 2003), and MRP8 has yet to be identified in any other species. In MRP8-transfected LLC-PK1 cells, MRP8 confers resistance to the pyrimidine analogs 5'-fluoro-5'-deoxyuridine, 5'-fluorouracil, and 5'-fluoro-2'-deoxyuridine, but not to typical MRP1 substrates such as vincristine, doxorubicin, or etoposide. MRP8-transfected cells also showed increased resistance to PMEA, but not to other purine analogs such as 6-thioguanine (Guo et al., 2003).
The ability of MRP8 to actively extrude compounds and contribute to MDR was examined in human MRP8-transfected cells. Compared with non-MRP8-expressing controls, efflux of PMEA and cAMP was significantly higher in MRP8-overexpressing cells (Guo et al., 2003). The ability of MRP8 to mediate transport of the monophosphorylated metabolite of 5'-deoxy-5'-fluorouridine might represent a general mechanism for MRP8-mediated resistance to fluoropyrimidines (Guo et al., 2003). Because MRP8-transfected cells show no resistance to vincristine, doxorubicin, etoposide, or taxol, it is unlikely these compounds are MRP8 substrates. In contrast, MRP8 mediates transport of E217G, dehydroepiandrosterone 3-sulfate, as well as LTC4 and the monoanionic bile acids taurocholate and glycocholate, but not prostaglandin E1 or E2 (Chen et al., 2005b; Bortfeld et al., 2006). These studies indicate that the resistance profiles of MRP4, MRP5, and MRP8 are certainly similar, but not identical.
Little is known about the newest member of the MRP family. Multiple transcript variants of the human MRP9 gene have been independently described, ranging in size from 1.3 to 4.5 kilobases (Tammur et al., 2001; Yabuuchi et al., 2001; Bera et al., 2002). MRP9 mRNA is expressed in a variety of adult tissues including brain, testes, and primary breast tumors, as well as the breast carcinoma cell line, GI-101 (Yabuuchi et al., 2001; Bera et al., 2002). MRP9 mRNA is also widely expressed in fetal tissues such as liver, spleen, kidney, and lung (Yabuuchi et al., 2001). Recently Mrp9 was cloned in the mouse, and low levels of mouse Mrp9 mRNA were detected in the brain, prostate, uterus, and stomach (Shimizu et al., 2003). Results from Northern blotting studies indicate that only testes shows significant expression of the mouse ortholog (Shimizu et al., 2003). Rats also seem to express Mrp9 mRNA; however, functional characteristics of the rat ortholog remain to be examined (GenBank accession no. NM_199377).
The function and substrates of MRP9 have yet to be studied. Based on chromosomal location (16q12.1), both MRP8 and MRP9 are proposed to play a role in the pathogenesis of paroxysmal kinesigenic choreoathetosis (Tammur et al., 2001; Yabuuchi et al., 2001), a movement disorder characterized by abnormal involuntary movements (Bhatia, 2001). With the recent identification of the murine ortholog, generation of a knockout mouse is probably underway (Shimizu et al., 2003). This model should provide valuable information regarding the physiological significance of MRP9.
III. Multidrug Resistance-Associated Protein Expression and Function in the Central Nervous System
Over the last decade, studies examining the expression of MRPs in the brain have produced contradictory and often controversial results. Overall transporter function reflects several factors: location (within a tissue, as well as within cells), level of expression, substrate and inhibitor specificity, and functional kinetics. At first glance, the first two factors would seem to be cell-dependent and the second two transporter-dependent. However, molecular-level interactions with lipids, proteins, and small signaling molecules can influence transporter specificity and kinetics, making those aspects of function at least partially cell-dependent.
Ideally, one would want to assess all aspects of transporter expression and function in the intact tissue in situ. This is rarely practical, because of limited access and experimental tools that can be less than optimal for the job at hand (below). Simpler experimental systems, i.e., isolated tissue, cultured cells (primaries and cell lines), can help to overcome some of the limitations. However, as one moves farther away from the in situ situation one may acquire a false picture of transporter function as a result of altered expression levels and changed membrane environment.
For MRPs, functional assessment is further confounded by overlapping specificities and similar tissue expression profiles among MRP family members and between MRPs and members of other transporter families, particularly organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs). Moreover, we lack inhibitors and substrates that are specific for certain MRPs or even for multiple family members. Thus, although one can establish expression of an MRP isoform within a tissue and localize the protein to certain cells and to a region of the plasma membrane, the contribution the transporter makes to transport of a specific substrate may be difficult to determine. In contrast, one research tool can provide a means to overcome this problem. That is, generation and use of animal models with altered expression of single MRP family members, e.g., Mrp1-null mice, Mrp4-null mice, and TR- rats (natural mutation; these animals do not express Mrp2). In the absence of specific compensation, these can provide unambiguous evidence of loss of transport function. But, it is important to remember that in many tissues, transport of MRP substrates may be also distributed over multiple transporters, including some OAT and OATP members.
Despite these difficulties, a large body of evidence does indicate that all of the functionally characterized MRP isoforms (1–8) are expressed in at least one CNS compartment and that they probably play a role in transport of drugs and metabolites (Table 2). However, it should not be surprising that there is substantial controversy in the field about the localization and function of certain family members. The following sections provide a comprehensive summary of current knowledge with respect to MRP expression and function in the different cellular compartments of the CNS.
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The cells of the CNS are particularly sensitive to chemical injury and thus require a protected and highly regulated extracellular environment. It has been known for over a century that exchange of solutes between blood and brain is restricted, and known for approximately 40 years that the site of the barrier is in the brain capillary endothelium. It has also been long appreciated that the functional unit of the blood-brain barrier (BBB) includes more than just capillary endothelial cells. Several other cell types are in constant and intimate contact with the endothelium and development and maintenance of the brain capillary phenotype (electrically tight, nonfenestrated endothelium with characteristic tight junctions and high expression of xenobiotic transporters) seems to be critically dependent on interactions with other cells found closely associated with brain capillaries, e.g., pericytes and astrocytes.
Two elements have been traditionally considered responsible for the barrier function of the brain capillary endothelium: very tight, tight-junctions (nonfenestrated endothelium), which form an effective seal to paracellular diffusion, and the cells themselves, which exhibit a low rate of endocytosis. Over the past decade it has become increasingly evident that superimposed upon this passive barrier is a selective, metabolism-driven barrier that largely reflects expression and function of ABC transporters (Begley, 2004). Among these transporters, P-gp is the best studied example. High levels of expression, luminal membrane localization, high transport potency, and affinity for a large number of commonly prescribed drugs make this ABC transporter a formidable element of the selective BBB. Consistent with these findings, experiments with P-gp-null mice show order of magnitude or larger increases in brain accumulation of a large number of drugs (Schinkel et al., 1996).
P-gp is not the only important contributor to the selective barrier. As discussed below, there is certainly evidence for participation of several MRP isoforms. MRPs are clearly expressed at the BBB (Fig. 2). However, for most MRP isoforms, there is still considerable discussion about at least one of the following: mRNA and protein expression levels, subcellular localization of the protein and its involvement in transport of specific drugs (Begley, 2004; Fricker and Miller, 2004; Graff and Pollack, 2004; Loscher and Potschka, 2005a). The lack of consistent data may reflect species differences in substrate specificity, metabolism and/or transporter expression, use of insensitive or less than specific antibodies, and substantial differences in expression levels between brain capillaries in in situ and in vitro systems, e.g., isolated brain capillaries, endothelial cells in primary culture, and endothelial cell lines (Regina et al., 1998; Torok et al., 2003).
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Because MRPs pump substrates out of cells, i.e., they are ATP-driven efflux transporters, only those MRP proteins localized to the luminal plasma membrane of brain capillary endothelial cells will contribute to the barrier and excretory functions of the tissue. Thus, although a case can be made for expression of mRNA and perhaps protein for MRP1 through MRP6 in brain capillary endothelial cells from at least one species (see Graff and Pollack, 2004; Loscher and Potschka, 2005b), firm evidence for luminal membrane localization has been obtained only for MRP1/Mrp1 (in cow and human), Mrp2 (in rat, but not detected in cow, human, or rats which do not express Mrp2), MRP4/Mrp4 (in mouse, cow, and human), and MRP5/Mrp5 (in human and cow) (Miller et al., 2000; Zhang et al., 2000, 2004; Leggas et al., 2004; Nies et al., 2004; Bronger et al., 2005).
Functional evidence for involvement of MRPs in the barrier and excretory functions of the BBB is hard to come by, and this certainly reflects the lack of isoform-specific substrates and inhibitors. Organic anion transport inhibitors that are also MRP inhibitors, e.g., probenecid and MK571, have been shown to increase drug accumulation in brain or to inhibit efflux from endothelial cell monolayers (Gutmann et al., 1999; Potschka et al., 2001; Potschka and Loscher, 2001; Sun et al., 2001). Consistent with this finding, Sugiyama et al. (2003) found reduced efflux of the endogenous metabolite E217G in Mrp1 knockout mice after intracerebral microinjection. However, other experiments with Mrp1 knockout mice show no increase in brain penetration of the organic anion fluorescein (Sun et al., 2001) or the efflux of etoposide (Cisternino et al., 2003) or morphine-6 -D-glucuronide (Bourasset et al., 2003), all transported by Mrp1, although some poorly.
For Mrp2 there is a naturally occurring knockout in two strains of rats (TR- and Esai hyperbilirubinemic rats), which are models for human Dubin-Johnson syndrome. Potschka et al. (2003b) found increased brain accumulation of the antiepileptic agent, phenytoin, in TR- rats compared with normal rats. In those experiments, plasma phenytoin levels were the same in both groups of animals, so it seems that Mrp2 is an important determinant of phenytoin penetration into the brain. From these findings, it is clear that further studies are needed to clarify the location and functional significance of the various MRP isoforms in the BBB.
In addition to MRPs, several drug-metabolizing enzymes have been shown to be expressed in the barrier including epoxide hydrolase, glutathione S-transferase, and various isoforms of the cytochrome P450 and UDP-glucuronosyltransferase families (Ghersi-Egea et al., 1994; Lawrenson et al., 1999; Miksys and Tyndale, 2002; Granberg et al., 2003). Biotransformation of foreign compounds via these phase I and II enzymes might result in metabolites that can then be removed from the brain by efflux transporters such as MRPs. In this way, metabolic enzymes and active transporters can act in concert as a biochemical barrier to remove potentially harmful compounds from the brain environment. The pharmacological significance of the metabolic barrier remains to be determined.
B. The Blood-Cerebrospinal Fluid Barrier
The choroid plexuses (CPs) are highly vascularized, leaf-like structures that protrude into the lateral, third, and fourth ventricles and form the major interface between cerebrospinal fluid (CSF) and the blood (Segal, 2000). The CP epithelium is composed of fenestrated capillaries, surrounded by a single layer of epithelial cells joined by tight junctions. Like the BBB, tight junctions between the epithelial cells restrict movement through the paracellular route, although these are not nearly as tight as those in brain capillaries. The CPs secrete CSF into the ventricles, thereby providing a fluid "cushion" for the brain (Segal, 2000). Additional functions of the CSF include nutrient supply, regulation of osmolarity, provision of neuroactive peptides, and metabolic waste removal (Strazielle et al., 2004). The composition of the CSF is rigorously maintained, and thus entry and exit of substances into the CSF are tightly regulated. Given its role in maintaining CSF homeostasis, it is not surprising that a variety of transport proteins are present in CP epithelium including ion channels, carriers of nonelectrolytes, nutrients, and neurotransmitters (Lee et al., 2001a; Graff and Pollack, 2004).
The CPs also express several drug transporters. CP expression of P-gp has been confirmed (Fig. 3), but this transporter has been localized mainly to intracellular compartments (Rao et al., 1999). An assortment of transporters belonging to the solute-carrier superfamily are also expressed in the apical and basolateral membranes of the CP epithelium including oatp/OATPs, OATs, and organic cation-organic cation/carnitine transporters (Ghersi-Egea and Strazielle, 2002; Graff and Pollack, 2004; Kusuhara and Sugiyama, 2004). Like the BBB, various drug-metabolizing enzymes have been identified in the CP including glutathione S-transferase, UDP-glucuronosyltransferase, and several isoforms of the cytochrome P450 family (Ghersi-Egea et al., 1994; Miksys and Tyndale, 2002). The activity of these enzymes is very high, so the CP has been postulated to be a major site of xenobiotic metabolism in the brain (Ghersi-Egea et al., 1995). As with the BBB, the extent to which metabolism contributes to the blood-CSF barrier remains to be established.
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MRP1/Mrp1 gene expression has been detected in human, mouse, and rat CP (Nishino et al., 1999; Rao et al., 1999; Sisodiya et al., 2001; Wijnholds et al., 2000a; Choudhuri et al., 2003; Mercier et al., 2004; Soontornmalai et al., 2006). By using immunocytochemical studies, Mrp1 was localized to the basolateral membranes of cultured rat CP cells and in mouse brain slices (Rao et al., 1999; Wijnholds et al., 2000a; Soontornmalai et al., 2006). When grown in vitro as confluent monolayers, rat CP cells show increased basal-to-apical transepithelial flux of 99mTc-sestamibi (a nonspecific MRP1 substrate) and accumulate more probe in the presence of the general MRP inhibitor MK571 (Rao et al., 1999). After intravenous dosing, triple knockout mice (Mrp1-/-/Mdr1a-/-/Mdr1b-/-) show a 10-fold increase in etoposide CSF concentrations, compared with double knockout mice (Mdr1a-/-/Mdr1b-/-) (Wijnholds et al., 2000a), further demonstrating the presence of a functional Mrp1 isoform on the basolateral side, which drives organic anion efflux into the blood.
After intracerebroventricular administration, CSF concentrations of DNP-GS and E217G were comparable in Mrp1 knockout (Mrp1-/-) and wild-type mice (Mrp1+/+) (Lee et al., 2004). Elimination of both substrates from the CSF was, however, probenecid-sensitive, suggesting that organic anion transporters other than Mrp1 mediate their efflux from the CSF (Lee et al., 2004). Mrp2, Mrp3, and Mrp6 were not considered candidates since expression of these MRP isoforms in rodent CP is negligible (Choudhuri et al., 2003; Lee et al., 2004). On the other hand, CP Mrp4 and Mrp5 mRNA levels are high (Choudhuri et al., 2003; Lee et al., 2004), and both isoforms have shown some ability to transport conjugated organic anions (Wijnholds et al., 2000b; Zelcer et al., 2003). One likely explanation for the lack of effect of knocking out Mrp1 is that the substrates examined are primarily handled by Oat and Oatp family members, e.g., apical Oat3 and Oatp3, and basolateral Oatp2. Another possibility is specific compensation. The ability of one MRP isoform to compensate for the absence of another has been reported previously in the liver and kidney for human, mice, and rats deficient in MRP2/Mrp2, i.e., up-regulation of MRP3/Mrp3 and/or Mrp4 (Konig et al., 1999b; Kuroda et al., 2004; Chen et al., 2005a; Chu et al., 2006).
Although expression of MRP4/Mrp4 in CP is certain, the exact location of the proteins has yet to be conclusively established. Recently MRP4/Mrp4 was localized to the basolateral (blood) side of intact mouse, rat, and human CP using a monoclonal antibody that recognizes amino acids 372 through 431 of the human MRP4 protein (Leggas et al., 2004). Since MRP4 has been localized to the apical pole of human brain endothelial cells and human, rat, and mouse kidney proximal tubules (van Aubel et al., 2002; Leggas et al., 2004; Nies et al., 2004), but the basolateral side of tubuloacinar cells (Lee et al., 2000), these studies would seem to support the hypothesis that the polarity of MRP4/Mrp4 expression is cell-specific. The expression of MRP4/Mrp4 in the apical and basolateral membranes of the BBB and CP, respectively, indicates a role for this transporter in limiting organic anion influx from blood and in driving organic anion efflux from the brain to blood.
First described by the Spanish neuroanatomist del Rio-Hortega (1932), microglia represent up to 20% of the total CNS glial population (Lawson et al., 1990; Raivich et al., 1999). Microglia exist in the CNS in several morphologically distinct forms, including ramified (resting), spheroid (activated), and phagocytic (reactive) types (Thomas, 1992). Normally, microglia are in a quiescent resting state, acting as a "sensor" of the brain microenvironment. Ramified microglia possess a small cell body and are highly branched. After injury or infection, microglia are activated, which results in retraction of processes, proliferation, and up-regulation of several cell surface factors (Hanisch, 2002; Liu and Hong, 2003). Progression of activated microglia to a phagocytic state is dependent on the severity of brain injury. In this way, microglia show remarkable functional plasticity (Streit et al., 1988). For example, following reversible axotomy (crushing of the nerve), microglia proliferate and surround the nerves while secreting soluble trophic factors such as basic fibroblast growth factor and nerve growth factor (Heumann et al., 1987; Gomez-Pinilla et al., 1990; Araujo and Cotman, 1992). Increased expression of various integrins and major histocompatibility complex class I markers also occurs. In this scenario microglia seem to play a neuroprotective role in the spheroid or activated stage and aid in the recovery of reversibly damaged neurons. Conversely, ricin-induced degeneration of neurons (an irreversible and lethal event) results in microglia becoming fully activated phagocytes. This stage of activation is characterized by a significant increase in the expression of phagocytic stage markers including several integrins (
51, 61, and M2), and major histocompatibility complex class I and II antigens. Excessive microglial activation is implicated in the pathogenesis of a variety of neurodegenerative diseases including HIV-associated dementia (HAD), Parkinson's disease, and Alzheimer's disease (McGeer and McGeer, 1998; Akiyama et al., 2000; Xiong et al., 2000; Liu and Hong, 2003; Block and Hong, 2005).
The ability of microglia to metabolize and transport compounds is not well characterized. Compared with cultured astrocytes and neurons, cultured microglia express higher intracellular levels of GSH, higher specific activity of GSH reductase and peroxidase, but limited catalase activity (Hirrlinger et al., 2000). Microglia also express a variety of ion channels (e.g., potassium and sodium) and receptors (Gottlieb and Matute, 1997; Eder, 1998; Noda et al., 2000). Microglial gene expression of Mrps (Fig. 2) was first demonstrated in 2002 (Ballerini et al., 2002; Hirrlinger et al., 2002a). Subsequent studies by our group confirmed expression of Mrp1, Mrp4, and Mrp5 mRNA and protein within a cultured rat microglia cell line, MLS-9 (Dallas et al., 2003, 2004b). These cells also express a functional P-gp protein (Lee et al., 2001b). In agreement with Hirrlinger et al. (2002a), we could not detect mRNA or protein for Mrp2 or Mrp6 in primary cultures of rat microglia or in the microglial cell line.
By using immunogold cytochemistry at the electron microscope level, Mrp1 protein was found primarily in the plasma membrane of the MLS-9 cells (Dallas et al., 2004a). The novel finding that Mrp1 also localized in smooth membrane caveolae and clathrin-coated vesicles in the plasma membrane of cultured microglia cells is particularly interesting (Dallas et al., 2004a). In a variety of cell types, caveolae and clathrin-coated vesicles are associated with endocytotic and pinocytotic transport as well as cell signaling (Gonzalez-Gaitan and Stenmark, 2003). Caveolins (1, 2, and 3) are the structural proteins associated with caveolae. In rat brain capillaries and the rat brain endothelial cell line RBE4, we previously reported P-gp localization in caveolae and clathrin-coated vesicles using immunogold cytochemistry and electron microscopy (Bendayan et al., 2002). Applying confocal microscopy, Virgintino et al. (2002) also observed colocalization of P-gp and caveolin-1 in the luminal side of isolated human microvessels. Likewise, Demeule et al. (2000) and Jodoin et al. (2003) reported colocalization of P-gp and caveolin-1 in isolated rat brain microvessels and in a bovine brain microvessel endothelial cell/astrocyte coculture system. Furthermore, the expression of caveolin-1 directly modulated the functional activity of P-gp (Jodoin et al., 2003). Recently, we have confirmed the expression of caveolin-1 and colocalization with P-gp in primary cultures of rat astrocytes, suggesting that caveolae represent an important membrane domain for transporter localization in brain parenchyma (Ronaldson et al., 2004a). Additional studies are needed to establish whether caveolin-1 also colocalizes with Mrp1 and other Mrps in brain cellular compartments including microglial cells. The functional relevance of these findings remains to be determined.
The Mrp proteins present in cultured rat microglia seem to be functional and display transport properties comparable with those reported previously in other cells types (Dallas et al., 2003, 2004b). That is, the cells displayed saturable, ATP-dependent uptake of vincristine, which was stimulated by a variety of MRP/organic anion inhibitors (i.e., MK571, genistein, probenecid, and sulfinpyrazone). As expected, intracellular depletion of GSH using an irreversible inhibitor of -glutamyl cysteine synthetase, i.e., 25 µM buthionine sulfoximine, resulted in a significant increase in vincristine accumulation by the cells. In addition, efflux of the acyclic nucleoside analog PMEA by microglia was rapid, ATP-dependent, but GSH-independent. PMEA efflux was also inhibited significantly by genistein, indomethacin, probenecid, sulfinpyrazone, and zidovudine monophosphate, suggesting the involvement of Mrp4/Mrp5 (Dallas et al., 2004b). Taken together, these studies support functional expression of at least four different ABC transporters in microglia (i.e., Mrp1, Mrp4, Mrp5, and P-gp), cells that are particularly important in pharmacoresistance development in HIV patients (discussed below).
First named for their star-shaped appearance, astrocytes display a variety of morphologies and account for 40% of the cells present in the brain. These cells are generally divided into two distinct groups based on morphology and location within the CNS (Privat and Rasaboul, 1986). Protoplasmic astrocytes, found in gray matter regions, are spheroid in shape, contain clumped chromatin, and have many highly branched processes. Fibrous astrocytes, found in the white matter, are characterized by oval nuclei with evenly dispersed chromatin and have a less complex branching pattern. Astrocytes perform a number of essential "housekeeping" functions in the brain including glutamate uptake and release, free radical scavenging, water transport, and ion buffering (Chen and Swanson, 2003). In situ, astrocyte foot processes are in close contact with endothelial cells that form the BBB and provide both functional and structural support to these cells (Goldstein, 1988). Like microglia, astrocytes can become reactive (reactive astrogliosis) following brain injury or infection (Eddleston and Mucke, 1993). Reactive astrocytes show morphological changes, increased production of soluble factors (e.g., growth factors, proteases, inflammatory cytokines, and metabolic enzymes), and increased proliferation. Reactive astrocytes may contribute to the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's, epilepsy, and HAD (D'Ambrosio, 2004; Kramer-Hammerle et al., 2005; Mrak and Griffin, 2005). Metabolically, astrocytes are very active, expressing a number of enzymes, including glutathione S-transferase, catalase, and several cytochrome P450 isoforms (Moreno et al., 1995; Sagara and Sugita, 2001; Miksys and Tyndale, 2002).
Several studies have demonstrated expression of Mrp1 mRNA transcript and protein in primary rat astrocyte cultures (Decleves et al., 2000; Hirrlinger et al., 2001, 2002a). Immunocytochemical studies confirmed that in many cases cells that were immunoreactive for Mrp1 protein also expressed the astrocyte marker glial fibrillary acidic protein (GFAP) (Hirrlinger et al., 2001). GFAP is an astrocyte-specific marker commonly used for identification in mixed glial cultures (Eng et al., 2000). Mrp1 staining of GFAP-negative cells probably represents contaminating microglial cells (up to 10%), generally found in astrocyte cultures. The Mrp protein(s) present in rat astrocytes may be functional since fluorescein accumulation increased considerably in the presence of the nonspecific organic anion transport inhibitors, indomethacin, probenecid, and sulfinpyrazone (Decleves et al., 2000).
Following generation of a hydrogen-peroxide induced oxidative stress, astrocytes release high levels of GSSG, a known MRP1 substrate (Hirrlinger et al., 2001). Addition of 20 µM MK571 to astrocyte cultures decreases GSSG efflux by 50%, suggesting the involvement of Mrp1 and/or another Mrp isoform. Efflux of reduced GSH by astrocytes is also inhibited by MK571 at concentrations >10 µM (Hirrlinger et al., 2002c). Given these results, Mrp1 may play an essential role in maintaining GSH concentrations and redox balance of astrocytes during oxidative stress (Hirrlinger et al., 2001, 2002c). Incomplete inhibition of GSH and GSSG efflux from primary astrocyte cultures in the presence of high concentrations of MK571 (50 µM) suggests that other transporters (e.g., Mrp4) might also contribute to thiol removal (Hirrlinger et al., 2002c).
Recently Gennuso et al. (2004) have implicated regulation of astroglial Mrp1 protein expression in the etiology of neonatal bilirubin encephalopathy. Modest levels of hyperbilirubinemia seem to be neuroprotective for infants; however, severely jaundiced newborns accumulate high levels of unconjugated bilirubin in astrocytes and neurons, leading to disruption of cellular functions and neuronal cell death (Ostrow et al., 2004). In cultured mouse astrocytes, concentrations of unconjugated bilirubin below the compound's aqueous saturation point (i.e., <70 nM) up-regulates Mrp1 protein expression and promotes trafficking of the protein from intracellular compartments to the plasma membrane (Gennuso et al., 2004). Presumably this trafficking prevents accumulation of unconjugated bilirubin within the astrocytes; MRP1 is confirmed to mediate transport of unconjugated bilirubin by MRP1-overexpressing cells and in Mrp1-/- knockout mice (Rigato et al., 2004; Calligaris et al., 2006). In contrast, bilirubin concentrations modestly above the solubility cutoff (i.e., 145 nM), resulted in the absence of Mrp1 trafficking and promoted a loss in astrocyte plasma membrane integrity, decreased mitochondrial function, and increased cell apoptosis (Gennuso et al., 2004).
Studies examining expression of the remaining Mrp isoforms in cultured astrocytes have been somewhat contradictory. Hirrlinger et al. (2002a) detected expression of Mrp1, Mrp3, Mrp4, and Mrp5, but not Mrp2 mRNA in astrocytes from neonatal Wistar rat pups. In contrast, Ballerini et al. (2002) reported detection of Mrp1 through Mrp6 mRNA in astrocytes isolated from rat fetuses. Differences in prenatal versus postnatal expression of some Mrp isoforms might explain these discrepancies (Kao et al., 2002; Yabuuchi et al., 2002). Human astrocytes maintained in culture show considerable amounts of MRP1 protein expression when probed with the MRP1-specific monoclonal antibody MRPr1 (Spiegl-Kreinecker et al., 2002). However, MRP1 expression in resting astrocytes in situ has yet to be established (Aronica et al., 2003; Nies et al., 2004). Recently Nies et al. (2004) showed strong MRP4 and MRP5 staining in both resting and reactive astrocytes present in resected perilesional glioma and cerebral hemorrhage biopsy samples. In contrast, no MRP2, MRP3, or MRP6 staining was noted in the same tissue samples (Nies et al., 2004). Further studies are needed to clarify the expression patterns and physiological function of MRPs in normal, healthy astrocytes and how these transporters may be regulated in various disease states.
E. Neurons and Oligodendrocytes
Neurons form the basic structural and functional component of the CNS. The primary function of neurons is to respond to stimuli by conducting electrical signals along conductive processes, i.e., the axon. The conduction of electrical impulses results in the release of neurotransmitters that further regulate (positively and negatively) nearby neuronal responses (Ludwig and Pittman, 2003). In this way, the brain maintains a complex communication network. In the CNS, oligodendrocytes are responsible for the formation of myelin around the axons of neurons, which aid in the propagation of neuronal impulses and maintain this communications array (Jessen, 2004). Neurodegenerative diseases are characterized by a progressive loss of neurons due to apoptosis, often as a direct result of inflammatory events mediated by astrocyte or microglial act
