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• W1998430636 abstract "Oligodendrocytes (OLGs) are generated by progenitor cells that are committed to differentiating into myelin-forming cells of the central nervous system. Rearrangement of the cytoskeleton leading to the extension of cellular processes is essential for the myelination of axons by OLGs. Here, we have characterized a new member of the Kelch-related protein family termed MRP2 (for Mayven-related protein 2) that is specifically expressed in brain. MRP2/KLHL1 is expressed in oligodendrocyte precursors and mature OLGs, and its expression is up-regulated during OLG differentiation. MRP2/KLHL1 expression was abundant during the specific stages of oligodendrocyte development, as identified by A2B5-, O4-, and O1-specific oligodendrocyte markers. MRP2/KLHL1 was localized in the cytoplasm and along the cell processes. Moreover, a direct endogenous association of MRP2/KLHL1 with actin was observed, which was significantly increased in differentiated OLGs compared with undifferentiated OLGs. Overexpression of MRP2/KLHL1 resulted in a significant increase in the process extension of rat OLGs, whereas MRP2/KLHL1 antisense reduced the process length of primary rat OLGs. Furthermore, murine OLGs isolated from MRP2/KLHL1 transgenic mice showed a significant increase in the process extension of OLGs compared with control wild-type murine OLGs. These studies provide insights into the role of MRP2/KLHL1, through its interaction with actin, in the process elongation of OLGs. Oligodendrocytes (OLGs) are generated by progenitor cells that are committed to differentiating into myelin-forming cells of the central nervous system. Rearrangement of the cytoskeleton leading to the extension of cellular processes is essential for the myelination of axons by OLGs. Here, we have characterized a new member of the Kelch-related protein family termed MRP2 (for Mayven-related protein 2) that is specifically expressed in brain. MRP2/KLHL1 is expressed in oligodendrocyte precursors and mature OLGs, and its expression is up-regulated during OLG differentiation. MRP2/KLHL1 expression was abundant during the specific stages of oligodendrocyte development, as identified by A2B5-, O4-, and O1-specific oligodendrocyte markers. MRP2/KLHL1 was localized in the cytoplasm and along the cell processes. Moreover, a direct endogenous association of MRP2/KLHL1 with actin was observed, which was significantly increased in differentiated OLGs compared with undifferentiated OLGs. Overexpression of MRP2/KLHL1 resulted in a significant increase in the process extension of rat OLGs, whereas MRP2/KLHL1 antisense reduced the process length of primary rat OLGs. Furthermore, murine OLGs isolated from MRP2/KLHL1 transgenic mice showed a significant increase in the process extension of OLGs compared with control wild-type murine OLGs. These studies provide insights into the role of MRP2/KLHL1, through its interaction with actin, in the process elongation of OLGs. Oligodendrocytes (OLGs) 3The abbreviations used are: OLG, oligodendrocyte; mAb, monoclonal antibodies; MRP2, Mayven-related protein 2; OPC, oligodendrocyte precursor cell; Tg, transgenic; WT, wild type; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; CMV, cytomegalovirus; RT, reverse transcriptase; as, antisense; SH3, Src homology domain 3.3The abbreviations used are: OLG, oligodendrocyte; mAb, monoclonal antibodies; MRP2, Mayven-related protein 2; OPC, oligodendrocyte precursor cell; Tg, transgenic; WT, wild type; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; CMV, cytomegalovirus; RT, reverse transcriptase; as, antisense; SH3, Src homology domain 3. are a major cell type in the central nervous system. Development of these cells is necessary for normal functioning of the brain, and injury to them is involved in the pathogenesis of important neurological disorders including cerebral palsy, multiple sclerosis, and periventricular leukomalacia (1Ludwin S.K. J. Neuropathol. Exp. Neurol. 1997; 56: 111-124Crossref PubMed Scopus (94) Google Scholar, 2Leviton A. Gilles F.H. J. Neurol. Sci. 1973; 18: 53-66Abstract Full Text PDF PubMed Scopus (62) Google Scholar). OLGs represent the myelin-forming cells of the central nervous system. They produce numerous membranous processes, which spirally enwrap neuronal axons, forming multilamellar myelin sheaths (3Richter-Landsberg C. J. Neurosci. Res. 2000; 59: 11-18Crossref PubMed Scopus (57) Google Scholar, 4Barry C. Pearson C. Barbarese E. Dev. Neurosci. 1996; 18: 233-242Crossref PubMed Scopus (36) Google Scholar). OLGs are metabolically the most active cells in the brain (5Wilson R. Brophy P.J. J. Neurosci. Res. 1989; 22: 439-448Crossref PubMed Scopus (158) Google Scholar). Before OLGs can remyelinate, they must first be able to extend their processes, and contact the demyelinated axons. However, the molecules involved in the mechanisms of OLG process extension are poorly defined. A new and unique family of actin-binding proteins with sequences and domains homologous with the Drosophila “Kelch” protein has emerged (6Xue F. Cooley L. Cell. 1993; 72: 681-693Abstract Full Text PDF PubMed Scopus (375) Google Scholar). Kelch protein is believed to be important for the maintenance of the ordered cytoskeleton (7Robinson D.N. Cant K. Cooley L. Development. 1994; 120: 2015-2025Crossref PubMed Google Scholar, 8Collins T. Stone J.R. Williams A.J. Mol. Cell. Biol. 2001; 21: 3609-3615Crossref PubMed Scopus (293) Google Scholar). The Kelch protein has two structural domains that are also found in other molecules. The first domain, which consists of about 115 amino acids, has been named the BTB (Bric-a-brac, Tramtrack, Broad-complex) domain (9Godt D. Couderc J.L. Cramton S.E. Laski F.A. Development. 1993; 119: 799-812Crossref PubMed Google Scholar) or POZ (Poxvirus zinc finger) domain (10Bardwell V.J. Treisman R. Genes Dev. 1994; 8: 1664-1677Crossref PubMed Scopus (657) Google Scholar). The second domain, composed of about 50 amino acids repeated in tandem, has been called the “Kelch repeats.” The BTB/POZ domain has been proposed to function as a protein-protein interaction interface, which organizes higher order structures involved in chromatin folding or cytoskeleton organization (11Albagli O. Dhordain P. Deweindt C. Lecocq G. Leprince D. Cell Growth Differ. 1995; 6: 1193-1198PubMed Google Scholar). The Kelch-related proteins are a superfamily of proteins conserved in a wide range of organisms, from viruses to mammals. At least 60 Kelch-related proteins have been identified, but their physiological and biochemical functions remain largely uncharacterized (12Adams J. Kelso R. Cooley L. Trends Cell Biol. 2000; 10: 17-24Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 13Avraham S. Avraham H. Jiang S. Bu X. Liang X.Q. Seng S. Kim T. Recent Res. Develop. Biol. Chem. 2003; 1: 231-254Google Scholar). The Drosophila Kelch proteins colocalize with actin filaments in a structure called the ring canal, which bridges 15 nurse cells and the oocyte. Drosophila Kelch protein plays an important role in maintaining actin organization during the development of ring canals (6Xue F. Cooley L. Cell. 1993; 72: 681-693Abstract Full Text PDF PubMed Scopus (375) Google Scholar, 8Collins T. Stone J.R. Williams A.J. Mol. Cell. Biol. 2001; 21: 3609-3615Crossref PubMed Scopus (293) Google Scholar). The Kelch-related proteins have diverse functions in cell morphology, cell organization, and gene expression, and function in multiprotein complexes through contact sites in their β-propeller domains (14Bork P. Doolittle R.F. J. Mol. Biol. 1994; 236: 1277-1282Crossref PubMed Scopus (162) Google Scholar). Recently, a new member of the BTB/Kelch repeat family, gigaxonin, was reported to be a pathological target for neurodegenerative disorders in which alterations were found to contain multiple mutations in the Kelch repeats in the neurofilament network (15Bomont P. Cavalier L. Blondeau F. Ben Hamida C. Belal S. Tazir M. Demir E. Topaloglu H. Korinthenberg R. Tuysuz B. Landrieu P. Hentati F. Koenig M. Nat. Genet. 2000; 26: 370-374Crossref PubMed Scopus (318) Google Scholar). We have previously identified and characterized two actinbinding proteins, termed NRP/B/ENC-1 (16Kim T.A. Lim J. Ota S. Raja S. Rogers R. Rivnay B. Avraham H. Avraham S. J. Cell Biol. 1998; 141: 553-566Crossref PubMed Scopus (96) Google Scholar, 17Kim T.A. Ota S. Jiang S. Pasztor L.M. White R.A. Avraham S. Gene (Amst.). 2000; 255: 105-116Crossref PubMed Scopus (37) Google Scholar, 18Kim T.A. Jiang S. Seng S. Cha K. Avraham H.K. Avraham S. J. Cell Sci. 2005; 118: 5537-5548Crossref PubMed Scopus (16) Google Scholar) and Mayven (19Soltysik-Espanola M. Rogers R.A. Jiang S. Kim T.A. Gaedigk R. White R.A. Avraham H. Avraham S. Mol. Biol. Cell. 1999; 10: 2361-2375Crossref PubMed Scopus (81) Google Scholar), predominantly expressed in brain. Mayven is an actin-binding protein that is co-localized with actin filaments in stress fibers and in the patchy cortical actin-rich regions of the cell margins and processes, including the process tips in primary neurons and U373-MG astrocytoma/glioblastoma cells (19Soltysik-Espanola M. Rogers R.A. Jiang S. Kim T.A. Gaedigk R. White R.A. Avraham H. Avraham S. Mol. Biol. Cell. 1999; 10: 2361-2375Crossref PubMed Scopus (81) Google Scholar). During our study of proteins that are related to Mayven, we identified and cloned a novel gene, which we termed: MRP2 (Mayven-related protein 2) that was found to be identical to KLHL1 (20Nemes J.P. Benzow K.A. Moseley M.L. Ranum L.P. Koob M.D. Hum. Mol. Genet. 2000; 9: 1543-1551Crossref PubMed Scopus (154) Google Scholar, 21Benzow K.A. Koob M.D. Mamm. Genome. 2002; 13: 134-141PubMed Google Scholar). In this study, we have investigated the expression of MRP2/KLHL1 in OLGs and its possible role in the dynamics of cytoskeletal rearrangement, leading to the elongation of OLG processes. Both the BLAST and Align Master programs use the crystal of the PLZF BTB/POZ domain as a three-dimensional template. The structure of the human PLZF BTB/POZ domain was obtained from the Protein Data Bank (Protein Data Bank codes 1BUO and 1CS3, chain A). Superimposition, model building, construction of insertion regions, structure validation, and calculation of structural properties were carried out using the sub-programs ProMod version 3.5, SPDBV version 3.5, Loop v2.60, Parameters version 3.5, and Topologies version 3.5, which are available in the Automated SwissModel Package Program. We analyzed the amino acids of the conserved region in the Kelch domain by the use of the BLAST2 and Cn3D programs (National Institutes of Health). The three-dimensional model of the Kelch domain was obtained as described above. Rabbit anti-peptide antibodies were raised for MRP2/KLHL1 (RRCSDLSML, residues 391-399 amino acids). These polyclonal antibodies have been characterized in our laboratory and tested for their specificity, and were found to be specific to MRP2/KLHL1. No cross-hybridization between these antibodies and other members of this family was observed using these antisera. Eukaryotic expression plasmids for full-length MRP2/KLHL1 were introduced into pFLAG-CMV4. The resulting expression constructs were designated as pCMV4-MRP2/KLHL1. pCMV4-MRP2/KLHL1 was also used as a template to generate the MRP2/KLHL1 antisense constructs. N-terminal MRP2 (574 bp) was amplified using a set of primers as follows: 5′-GCGGCCGCATGTCAGGCTCTGGGCG-3′ and 5′-TCTAGACAGCTTGATAGAATCTTC-3′. The PCR product was digested with NotI and XbaI, and the digested fragments were subcloned in a reverse manner into the pCMS-EGFP vector (BD Biosciences). The antisense constructs were designated as GFP-asMRP2, and GFP served as a reporter. pEGFP-KLHL1 was kindly provided by Dr. Michael D. Koob (University of Minnesota). Primary cultures of oligodendrocytes, microglial cells, and astrocytes were generated from the forebrains of 2-3-day-old Sprague-Dawley rats as described previously (22Vartanian T. Goodearl A. Viehover A. Fischbach G. J. Cell Biol. 1997; 137: 211-220Crossref PubMed Scopus (153) Google Scholar, 23Vartanian T. Fischbach G. Miller R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 731-735Crossref PubMed Scopus (141) Google Scholar). Briefly, brain tissue was dissociated with trypsin for 20 min at 37 °C. After mechanical dissociation, the cells were plated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Mixed glial cultures containing oligodendrocytes, microglia, and astrocytes were maintained for different periods of time. After 1 week in culture, mixed glial cultures were shaken for 30 min at 180 rpm. The supernatants containing >90% microglia were then plated. Fifteen minutes after plating, nonadherent cells (predominantly astrocytes and oligodendrocytes) were removed by three washes with PBS, leaving the culture with >98% microglia as shown by immunostaining. Microglia were maintained in Dulbecco's modified Eagle's medium with 5% fetal bovine serum. Oligodendrocyte progenitors were isolated from the remaining adherent cells by a second shaking of 12 h at 180 rpm. After this second shaking, the supernatant was preplated on tissue culture flasks for 1 h and passed successively through 20- and 10-μm mesh filters, removing most of the contaminating astrocytes and microglia. Enriched oligodendroglia were plated on poly-d-lysine-coated coverslips in serum-free Dulbecco's modified Eagle's medium with bovine serum albumin, N2 supplements, platelet-derived growth factor-AA (10 ng/ml), and basic fibro-blast growth factor (10 ng/ml). One-half of the media was exchanged with fresh media every 2 days. These enriched oligodendrocyte cultures were assayed for contamination by microglia and astrocytes for each preparation. Typically, cultures contained ∼0.5-2% microglial cells as assessed by the uptake of diacetyl low density lipoprotein. Cultures were switched to medium containing N2 supplements to allow the development of cells to the O4+/O1-stage (24Barres B.A. Hart I.K. Coles H.S. Burne J.F. Voyvodic J.T. Richardson W.D. Raff M.C. Cell. 1992; 70: 31-46Abstract Full Text PDF PubMed Scopus (1168) Google Scholar, 25Barres B.A. Hart I.K. Coles H.S. Burne J.F. Voyvodic J.T. Richardson W.D. Raff M.C. J. Neurobiol. 1992; 23: 1221-1230Crossref PubMed Scopus (143) Google Scholar, 26Barres B.A. Raff M.C. Nature. 1993; 361: 258-260Crossref PubMed Scopus (508) Google Scholar, 27Gard A.L. Pfeiffer S.E. Neuron. 1990; 5: 615-625Abstract Full Text PDF PubMed Scopus (234) Google Scholar). Under these conditions, >95% of the cells were O4+ and <5% were O1 positive. In this study, we use the term oligodendrocyte precursors to denote oligodendrocytes immunoreactive with A2B5 antibody. For A2B5 (28Kennedy P.G. Lisak R.P. Raff M.C. Lab. Investig. 1980; 43: 342-351PubMed Google Scholar), O1 and O4 (29Shi J. Marinovich A. Barres B.A. J. Neurosci. 1998; 18: 4627-4636Crossref PubMed Google Scholar) immunofluorescence, oligodendrocyte cultures were incubated for 15 min with the O4, O1 (29Shi J. Marinovich A. Barres B.A. J. Neurosci. 1998; 18: 4627-4636Crossref PubMed Google Scholar), A2B5, or control monoclonal antibodies, as described (28Kennedy P.G. Lisak R.P. Raff M.C. Lab. Investig. 1980; 43: 342-351PubMed Google Scholar, 29Shi J. Marinovich A. Barres B.A. J. Neurosci. 1998; 18: 4627-4636Crossref PubMed Google Scholar). The cultures were next washed with PBS, fixed in fresh 4% paraformaldehyde in PBS for 7 min at an ambient temperature, washed with PBS again, incubated with the relevant secondary antibody (Jackson ImmunoResearch Laboratories) and then visualized by fluorescent microscopy. For MRP2/KLHL1 immunofluorescence staining, cells were plated on coverslips 2 days before each experiment at a concentration of 3-5 × 104 cells/35-mm dish. Cells were fixed directly to the coverslips by the addition of 3% formaldehyde, and then placed at -20 °C for 15 min. After fixation, the cells were rinsed extensively with PBS (pH 7.4) and incubated for 1 h with PBS containing 2% bovine serum albumin, 2% normal goat serum, and 0.2% gelatin at room temperature to reduce nonspecific binding. The cells were then incubated with primary antibodies for 1 h at room temperature. After washing, cells were incubated with secondary antibodies for 30 min at room temperature. Following the labeling of the cells, the coverslips were mounted and viewed under a Zeiss fluorescent microscope. Confocal analyses were performed with an inverted confocal microscope. For the immunostaining of paraffin-embedded mouse brain sections, the brain sections were treated with xylene, and subsequently with 100, 95, 75, and 50% ethanol. The sections were rinsed in PBS and unmasked using Target Retrieval solution (DakoCytomation) at 99 °C for 30 min. The sections were then blocked with 10% normal goat serum and incubated with primary antibodies (MRP2/KLHL1 and O1) overnight at 4 °C. Following 3 washes in PBS, secondary antibodies conjugated with fluorescein isothiocyanate and Texas Red were applied to the slides for 30 min at room temperature. After 3 more washes in PBS, the slides were mounted with 4′, 6-diamidino-2-phenylindole solution and viewed under a Zeiss fluorescent microscope. 3-4 × 106 primary oligodendrocytes were transfected directly by electroporation (Bio-Rad). Fifteen micrograms of GFP-MRP2/KLHL1 construct was used in each electroporation experiment. Transfected cells were analyzed using an inverted fluorescent microscope with an attached digital camera. The number of processes per cell and process length longer than half the diameter of the cell soma were measured. In each experiment, 100 GFP-positive primary OLGs were analyzed. Three individual experiments per treatment were performed. The data were pooled, and a nonparametric Mann-Whitney rank sum test was applied for analysis of the results. Following a washing with cold PBS, 293T or primary rat OLG cells were lysed in RIPA buffer containing a protein inhibitor mixture, and cell extracts were solubilized for 30 min at 4 °C. After pre-clearing, 500 μg of protein extracts were immunoprecipitated with anti-MRP2 or anti-actin antibody. The precipitates were then blotted and probed with anti-actin or anti-MRP2 antibody, accordingly. For stripping, the membranes were rinsed twice in PBS and then submerged in membrane stripping buffer (BP-96, Boston BioProduct) at 60 °C for 20 min. The membranes were rinsed in PBS/Tween 20 three times for 5 min each and probed with the appropriate antibodies. Supernatant lysates were separated by SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. For the analysis of MRP2/KLHL1 transgene expression, brain tissues isolated from mice were homogenized in lysis buffer, rotated at 4 °C for 3 h, and then centrifuged at 14,000 × g for 1 h. Supernatants were loaded onto 4-12% SDS-PAGE gels, and the blots were blocked with 5% skim milk in PBS followed by probing with primary anti-GFP antibodies and horse-radish peroxidase-conjugated secondary antibodies. The membranes were washed and developed using chemiluminescent reagents (PerkinElmer Life Sciences) and exposed to x-ray film. DNA Microinjection—Plasmid pEGFP/KLHL1 (kindly provided by Dr. Michael D. Koob) was digested with AseI and MluI restriction enzymes. The fragment (3.8 kb) consisting of the CMV promoter, EGFP/KLHL1, and poly(A) was excised and purified from a 1% agarose gel by using the NucleoSpin Extraction Kit (catalog number 635960, BD Biosciences). Expression of the MRP2/KLHL1 transgene was confirmed by transfecting the 293T cells, and the transgene product was examined by Western blot analysis using anti-GFP antibody. The transgene was microinjected into fertilized eggs at the one-cell stage, cultured overnight in embryonic culture medium, and transferred to 0.5-day pseudo-pregnant recipient mice. Offspring were screened for transgene integration and expression. DNA Purification from Mouse Tails—Tails from offspring were cut and digested in tail digestion buffer (30Seng S. Yokoyama M. Suzuki R. Maki Y. Kato M. Lim C. Zayatiin B. Inoue N. Xuan X. Igarashi I. Nagasawa H. Fujisaki K. Mikami T. Suzuki N. Toyoda Y. Parasitol. Res. 2000; 86: 263-269Crossref PubMed Scopus (9) Google Scholar) containing proteinase K (Roche) at 60 °C. Following overnight digestion, tail samples were added together with 250 μl of NaCl-saturated buffer (6 m NaCl) and incubated on ice for 15 min. After centrifugation at 12,000 × g for 10 min at 4 °C, supernatants were transferred to new tubes, and DNA was precipitated with 650 μl of isopropyl alcohol. DNA pellets were washed with 70% ethanol, air dried, and resuspended with 200 μl of TE, at which point the DNA solution was ready for the PCR and Southern blot analyses. RNA Purification from Mouse Tissues—RNA isolation was performed using TRIzol Reagent (Invitrogen), according to the manufacturer's instructions. RNA concentration was measured and stored at -80 °C, and the TRIzol-isolated total RNA was subjected to RT-PCR analyses. Southern Analysis—Southern blotting was performed to screen founders of the MRP2/KLHL1 transgenic mice. Briefly, 20 μg of DNA from the mouse tail was digested with different restriction enzymes, run on a 1% agarose gel, and then sub-merged in two changes of denaturation solution (45 min/each change) and neutralization solution (45 min/each change). DNA was transferred onto Hybond-N+ (Amersham Biosciences) in 20× SSC. Membranes were cross-linked and pre-hybridized in pre-hybridization solution (5× SSPE, 2% SDS, 5× Denhardt's reagent, 100 μg/ml of DNA from denatured salmon testes) at 65 °C for 6 h. pEGFP-C2 (Clontech) was digested with NheI and EcoRI restriction enzymes, and purified GFP was labeled with α-32P using the NE blot kit (catalog N1500L, New England BioLabs). Membranes were incubated with hybridization solution containing the GFP-labeled probe overnight at 65 °C. The membranes were washed once in washing solution I (0.5× SSPE, 1% SDS) for 20 min, and twice in washing solution II (0.1 × SSPE, 1% SDS) for 20 min each. Membranes were then exposed to x-ray film at -80 °C. Polymerase Chain Reaction—DNA from transgenic founders was used to optimize the PCR conditions. PCR was performed using the BD Advantage 2 PCR kit (catalog number 639207, Clontech). The amplification was performed in an automated thermocycler (iCycle, Bio-Rad) using a set of specific primers as follows: 5′-TCAGGCTCTGGGCGAAAA-3′ and 5′-GCACACTTCCACCACCTG-3′ with the following temperature conditions: 95 °C for 1 min followed by 30 cycles of 95 °C for 30 s and 68 °C for 3 min. The cycling was followed by a final extension at 68 °C for 3 min. The amplified products (at the expected 903 bp in length) were visualized on a 1% agarose gel. The well established PCR conditions were used to genotype the MRP2 transgenic mice. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)—TRIzol-isolated RNA was treated with DNase and purified by using a phenol-chloroform procedure. Purified RNA was subjected to polymerase chain reaction. One-step RT-PCR was performed based on the manufacturer's instructions using a set of specific MRP2/KLHL1 primers: 5′-TCAGGCTCTGGGCGAAAA-3′ and 5′-GCACACTTCCACCACC TG-3′. For the internal RNA control, a set of mouse β-actin primers (5′-GTGGGCCGCTCTAG GCACCAA-3′ and 5′-CTCTTTGATGTCACGACACGATTTC-3′) was used. The temperature conditions were 50 °C for 1 h, 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 68 °C for 30 s, and 68 °C for 60 s and an extension of 68 °C for 2 min. The RT-PCR product of 903 bp was visualized on an ethidium bromide-stained agarose gel. Identification and Cloning of MRP2/KLHL1—During our search for proteins that are related to Mayven (19Soltysik-Espanola M. Rogers R.A. Jiang S. Kim T.A. Gaedigk R. White R.A. Avraham H. Avraham S. Mol. Biol. Cell. 1999; 10: 2361-2375Crossref PubMed Scopus (81) Google Scholar) and NRP/B (16Kim T.A. Lim J. Ota S. Raja S. Rogers R. Rivnay B. Avraham H. Avraham S. J. Cell Biol. 1998; 141: 553-566Crossref PubMed Scopus (96) Google Scholar), we have identified and cloned from the human brain cDNA library an additional novel Mayven-related protein (MRP), termed MRP2. MRP2 contains a BTB domain and six Kelch repeats (Fig. 1A). Sequence analysis of this Kelch protein confirmed that MRP2 is identical to the brain-specific KLHL1 transcript (20Nemes J.P. Benzow K.A. Moseley M.L. Ranum L.P. Koob M.D. Hum. Mol. Genet. 2000; 9: 1543-1551Crossref PubMed Scopus (154) Google Scholar), and therefore was termed MRP2/KLHL1. Alignment between Mayven and MRP2/KLHL1 indicates significant homology in the BTB/POZ domain and Kelch repeats (shown in Fig. 1B). The C-terminal domain of Mayven and MRP2/KLHL1, which consists of six tandem Kelch repeats, is predicted to be a β-sheet organized into propeller structures. The overall structures of the BTB/POZ domain and Kelch repeats of MRP2/KLHL1 are shown in Fig. 1C. Expression of MRP2/KLHL1 at Different Stages of OLG Differentiation—Development of OLGs capable of forming myelin internodes requires several distinct environmental cues, which include early, regional, and late axonally derived signals. The initial specification of OLG precursor cells (OPCs) is dependent on signals from the ventral structures, such as the notochord and floor plate (31Warf B.C. Fok-Seang J. Miller R.H. J. Neurosci. 1991; 11: 2477-2488Crossref PubMed Google Scholar, 32Timsit S. Martinez S. Allinquant B. Peyron F. Puelles L. Zalc B. J. Neurosci. 1995; 15: 1012-1024Crossref PubMed Google Scholar). An early stage of OPCs is characterized by A2B5 monoclonal antibody immunoreactive cells that are bipolar (33Raff M.C. Miller R.H. Noble M. Nature. 1983; 303: 390-396Crossref PubMed Scopus (1643) Google Scholar) and motile (13Avraham S. Avraham H. Jiang S. Bu X. Liang X.Q. Seng S. Kim T. Recent Res. Develop. Biol. Chem. 2003; 1: 231-254Google Scholar, 14Bork P. Doolittle R.F. J. Mol. Biol. 1994; 236: 1277-1282Crossref PubMed Scopus (162) Google Scholar, 34Small R.K. Riddle P. Noble M. Nature. 1987; 328: 155-157Crossref PubMed Scopus (400) Google Scholar, 35Armstrong R. Harvath L. Dubois-Dalcq M. Ann. N. Y. Acad. Sci. 1991; 633: 520-522Crossref PubMed Scopus (23) Google Scholar), with a mitogenic response to platelet-derived growth factor-AA and basic fibroblast growth factor (25Barres B.A. Hart I.K. Coles H.S. Burne J.F. Voyvodic J.T. Richardson W.D. Raff M.C. J. Neurobiol. 1992; 23: 1221-1230Crossref PubMed Scopus (143) Google Scholar, 27Gard A.L. Pfeiffer S.E. Neuron. 1990; 5: 615-625Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 36McKinnon R.D. Matsui T. Dubois-Dalcq M. Aaronson S.A. Neuron. 1990; 5: 603-614Abstract Full Text PDF PubMed Scopus (517) Google Scholar). These A2B5+ cells mature into pro-oligodendroblasts, less motile cells (37Warrington A.E. Barbarese E. Pfeiffer S.E. J. Neurosci. Res. 1993; 34: 1-13Crossref PubMed Scopus (234) Google Scholar) characterized by surface labeling with the O4 monoclonal antibody (27Gard A.L. Pfeiffer S.E. Neuron. 1990; 5: 615-625Abstract Full Text PDF PubMed Scopus (234) Google Scholar). Differentiation of pro-oligodendroblasts is accompanied by exit from the cell cycle (27Gard A.L. Pfeiffer S.E. Neuron. 1990; 5: 615-625Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 38Hart I.K. Richardson W.D. Bolsover S.R. Raff M.C. J. Cell Biol. 1989; 109: 3411-3417Crossref PubMed Scopus (124) Google Scholar) and acquisition of galactosyl-cerebroside expression (33Raff M.C. Miller R.H. Noble M. Nature. 1983; 303: 390-396Crossref PubMed Scopus (1643) Google Scholar, 39Sommer I. Schachner M. Dev. Biol. 1981; 83: 311-327Crossref PubMed Scopus (962) Google Scholar) identified by the O1 monoclonal antibody. Here, we examined the expression of MRP2/KLHL1 in OPCs at different stages of oligodendrocyte development. As shown in Fig. 2A, strong expression of MRP2/KLHL1 was seen in the OPCs. As a positive control, OPCs were stained with A2B5 antibody. Furthermore, we examined the staining of MRP2/KLHL1 in pro-oligodendroblasts. These cells showed positive staining for MRP2/KLHL1 with O4 (Fig. 2A). Mature OLGs immunostained with O1 antibody also abundantly expressed MRP2/KLHL1. Taken together, these results indicate that oligoden-drocytes at various stages of development highly express MRP2/KLHL1. Next, we demonstrated the co-localization of MRP2/KLHL1 with A2B5, O4, or O1 in O2A cells. The cells were stained with antibodies and 4′, 6-diamidino-2-phenylindole, as indicated in Fig. 2B. MRP2/KLHL1 was co-localized with specific developmental markers (A2B5, O4, and O1) of the OLGs. In addition, we examined the specific expression of MRP2/KLHL1 in mouse brain sections. The brain sections were analyzed using anti-MRP2 and anti-O1 antibodies. As shown in Fig. 2C, MRP2/KLHL1 was co-localized with O1 in the mouse brain section. These data indicate that MRP2/KLHL1 is developmentally expressed in OLGs. Up-regulation of MRP2/KLHL1 Expression in Differentiated Primary OLGs and Its Effects on the Process Extension of OLGs—OLGs obtained from the primary culture of neonatal rat cerebrum were grown in differentiating medium for 4 days by withdrawal of platelet-derived growth factor. Cells were then fixed and stained with affinity purified polyclonal anti-MRP2/KLHL1 antibody. MRP2/KLHL1 was expressed in the cell body of undifferentiated precursor cells, and no staining was observed with control antibody (Fig. 3A). To assess whether MRP2/KLHL1 plays a role in process formation during OLG differentiation, we immunolabeled primary OLG progenitor cells and mature OLGs with specific anti-MRP2/KLHL1 antibody. MRP2/KLHL1 showed intensive fibrous-like expression along the processes in differentiated OLGs as compared with the undifferentiated OLGs (Fig. 3A). These" @default.
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• W1998430636 date "2007-04-01" @default.
• W1998430636 modified "2023-10-02" @default.
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