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Originally published In Press as doi:10.1074/jbc.M701019200 on February 25, 2007

J. Biol. Chem., Vol. 282, Issue 16, 12319-12329, April 20, 2007
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Process Elongation of Oligodendrocytes Is Promoted by the Kelch-related Protein MRP2/KLHL1*Formula

Shuxian Jiang1, Seyha Seng1, Hava Karsenty Avraham, Yigong Fu, and Shalom Avraham2

From the Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, February 2, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Oligodendrocytes (OLGs)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 (1, 2). 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 (3, 4). OLGs are metabolically the most active cells in the brain (5). 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 (6). Kelch protein is believed to be important for the maintenance of the ordered cytoskeleton (7, 8). 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 (9) or POZ (Poxvirus zinc finger) domain (10). 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 (11).

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 (12, 13). 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 (6, 8). 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 beta-propeller domains (14). 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 (15).

We have previously identified and characterized two actinbinding proteins, termed NRP/B/ENC-1 (16-18) and Mayven (19), 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 (19). 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 (20, 21). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Three-dimensional Modeling of MRP2/KLHL1
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.

Generation of Specific MRP2/KLHL1 Antibodies
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.

Construction of MRP2/KLHL1 cDNA Constructs
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 Culture Preparation
Primary cultures of oligodendrocytes, microglial cells, and astrocytes were generated from the forebrains of 2-3-day-old Sprague-Dawley rats as described previously (22, 23). 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 (24-27). 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.

Immunofluorescence Microscopy
For A2B5 (28), O1 and O4 (29) immunofluorescence, oligodendrocyte cultures were incubated for 15 min with the O4, O1 (29), A2B5, or control monoclonal antibodies, as described (28, 29). 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 x 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.

Transfection Experiments
3-4 x 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.

Immunoprecipitation
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.

Western Blot Analysis
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 x 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.

Generation of MRP2 Transgenic Mice
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 (30) 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 x 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 20x SSC. Membranes were cross-linked and pre-hybridized in pre-hybridization solution (5x SSPE, 2% SDS, 5x 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 {alpha}-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 (0.5x SSPE, 1% SDS) for 20 min, and twice in washing solution II (0.1 x 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 beta-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.


Figure 1
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  		FIGURE 1.
Alignment of amino acid sequences of human MRP2/KLHL1 and Mayven. A, summary of the MRP2/KLHL1 domains and their possible functions. B, alignment of MRP2/KLHL1 and Mayven amino acid sequences. The various domains of MRP2/KLHL1 and Mayven are indicated. C, three-dimensional modeling of the BTB/POZ domain and the Kelch repeats of MRP2/KLHL1. 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 (PDB codes 1BUO and 1CS3, chain A). Superimposition, model building, construction of insertion regions, structure validation, and calculation of structural properties were carried out as detailed under "Experimental Procedures." Red indicates {alpha}-helix and yellow indicates the beta-sheet structure. The three-dimensional structure of the BTB/POZ domain forms potential pocket-like structures that can act as a docking site for the binding partners. The Kelch domain of MRP2/KLHL1 forms propeller structures.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification and Cloning of MRP2/KLHL1—During our search for proteins that are related to Mayven (19) and NRP/B (16), 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 (20), 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 beta-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 (31, 32). An early stage of OPCs is characterized by A2B5 monoclonal antibody immunoreactive cells that are bipolar (33) and motile (13, 14, 34, 35), with a mitogenic response to platelet-derived growth factor-AA and basic fibroblast growth factor (25, 27, 36). These A2B5+ cells mature into pro-oligodendroblasts, less motile cells (37) characterized by surface labeling with the O4 monoclonal antibody (27). Differentiation of pro-oligodendroblasts is accompanied by exit from the cell cycle (27, 38) and acquisition of galactosyl-cerebroside expression (33, 39) 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 confocal images clearly indicate that MRP2/KLHL1 may be involved in OLG differentiation.

To further determine the expression pattern of MRP2/KLHL1 in primary undifferentiated OLGs as well as in differentiated OLGs, total cell lysates were prepared from primary rat OLGs and differentiated OLGs and then 100 µg of the protein extracts were subjected to SDS-PAGE and Western blot analyses. As shown in Fig. 3B, expression of MRP2/KLHL1 was significantly up-regulated during OLG differentiation, strongly suggesting the potential involvement of MRP2/KLHL1 protein in the differentiation process of OLGs.

The confocal images (Fig. 3A) and Western blot analyses (Fig. 3B) revealed that MRP2/KLHL1 expression might be critical for OLG process elongation. Therefore, we assessed the effects of forced expression of MRP2/KLHL1 on process elongation in O2A cells. The O2A cells were seeded on slide glasses for 24 h and transfected with GFP or GFP-MRP2/KLHL1 constructs. Cells were incubated for 48 h with complete O2A culture medium. Cells were then fixed and labeled with the OLG markers (A2B5 or O1). As shown in Fig. 3C, process elongation was induced only in O2A cells transfected with GFP-KLHL1/MRP2, and not in cells transfected with GFP (control vector).


Figure 2
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  		FIGURE 2.
MRP2/KLHL1 expression in oligodendrocytes during specific stages of their development. A, OPCs and OLGs were purified from rat forebrain. For the A2B5, O4, and O1 staining, cells were immunostained on the 2nd, 4th, and 6th day of culture. Cells were next incubated for 20 min with A2B5, O4, and O1 monoclonal antibodies, washed with PBS three times, fixed in fresh 4% paraformaldehyde in PBS for 7 min at an ambient temperature, washed again three times with PBS, and incubated with Cy3-conjugated secondary antibody. A parallel experiment was performed by staining cells with a rabbit polyclonal antibody against MRP2, followed by incubation with fluorescein isothiocyanate-conjugated rabbit secondary antibody. B, colocalization of MRP2 with oligodendrocyte markers. O2A cells were stained with anti-MRP2 along with anti-A2B5, anti-O4, or anti-O1 antibodies. C, mouse brain section was stained with anti-MRP2 and anti-O1 antibodies. DAPI, 4', 6-diamidino-2-phenylindole.

 
To further support the contention that MRP2/KLHL1 plays a critical role in OLG process extension, we examined the effects of antisense MRP2/KLHL1 on the process extension of primary rat OLGs. Primary rat OLGs were transfected with GFP, with the GFP-KLHL1/MRP2 construct or with antisense GFP-MRP2/KLHL1 (GFP-asMRP2). After 24-48 h, primary rat OLGs that overexpressed MRP2/KLHL1 demonstrated longer processes than the GFP-transfected differentiated primary rat OLGs (Fig. 4A). Three individual experiments were then performed to analyze the effects of MRP2/KLHL1 on the process length of primary rat OLGs. GFP- or GFP-MRP2/KLHL1-expressing cells (100 cells in total) were selected and their process lengths were assessed. As shown in Fig. 4B, ~25% of the GFP-MRP2/KLHL1-expressing cells contained processes of 2-3 µm in length, whereas only 5% of the GFP-expressing cells exhibited a similar process length. Moreover, about 12 and 5% of the GFP-MRP2/KLHL1-expressing cells exhibited process lengths of 4 and 5 µm, respectively, but this effect was not observed in the GFP-expressing cells (Fig. 4B).

Next, we determined whether GFP-asMRP2 inhibits the extension of processes. Process lengths longer than half the diameter of the cell soma were measured to assess the effect of the antisense treatment. As shown in Fig. 4C, the GFP-asMRP2-transfected cells showed no process elongation as compared with cells transfected with GFP-MRP2/KLHL1. Cells were then transfected with GFP, GFP-MRP2/KLHL1, or GFP-asMRP2. Following 24 h of the transfection, 300 GFP-expressing cells from each group in the three individual experiments were measured for process length. Approximately 30 and 35% of cells transfected with GFP and GFP-asMRP2, respectively, showed processes longer than half the diameter of their cell somas, whereas 77% of cells transfected with GFP-MRP2/KLHL1 exhibited such processes (Fig. 4D). Taken together, these results demonstrated that MRP2/KLHL1 is essential in the process elongation of OLGs.


Figure 3
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  		FIGURE 3.
Analysis of MRP2 expression in undifferentiated and differentiated OLGs. A, confocal analysis of MRP2/KLHL1 expression in undifferentiated and differentiated OLGs. OLGs were obtained from primary cultures of neonatal rat cerebrum. Most of the proliferating cells were small, round, bipolar, or multipolar with several short processes (panel a). When cells were introduced into the MBP medium, differentiating OLGs were observed. Affinity purified polyclonal antibodies (MRP2/KLHL1) were used for immunostaining after cells were fixed in 4% paraformaldehyde. OLGs were also stained with preimmune serum as shown in the square area outlined in red. The boxed areas in the middle panels (panel b) were enlarged as shown in the right panels (panel c). The arrows indicate the expression of MRP2/KLHL1 in the processes. B, expression of MRP2/KLHL1 in undifferentiated and differentiated primary OLGs. OLGs were obtained from primary cultures of neonatal rat cerebrum. Cultures were grown for 8 days in polyornithine-coated plates in O4 medium containing platelet-derived growth factor (10 ng/ml) and fibroblast growth factor (10ng/ml). Cells under these conditions were 85% O4 positive. To obtain myelin basic protein-positive cells, the cells were placed in myelin basic protein medium containing ciliary neurotrophic factor (10 ng/ml), 5 µM forskolin and T3 (15 nM) 1 week after plating. 95% of the cells were myelin basic protein-positive after switching. Affinity purified polyclonal antibodies of MRP2/KLHL1 as well as actin were used for the Western blot (WB) analysis. C, effect of the forced expression of MRP2/KLHL1 on process elongation. O2A cells were transfected with GFP or GFP-MRP2/KLHL1 and cultured in appropriate conditions. Cells were next stained with anti-A2B5 or anti-O1 antibody and 4', 6-diamidino-2-phenylindole (DAPI). No Diff., undifferentiated OLGs; Diff., differentiated OLGs.

 
MRP2/KLHL1 Is an Actin-binding Protein—Several proteins possessing Kelch repeats have been shown to be either actin-binding proteins or associated with actin filaments. To examine the potential of MRP2/KLHL1 to bind actin, 293T cells were transfected with pEGFP vector (Fig. 5A, lane 1) or with the pEGFP-KLHL1/MRP2 construct (Fig. 5A, lane 2). After 48 h, total cell lysates were prepared and immunoprecipitated with MRP2/KLHL1 antibodies. The immune complexes were analyzed by SDS-PAGE and Western blotting with either MRP2/KLHL1 or actin antibody. As shown in Fig. 5A, the binding of MRP2/KLHL1 with actin was observed, strongly suggesting that MRP2/KLHL1 is an actin-binding protein. To examine the direct endogenous association of MRP2/KLHL1 with actin in undifferentiated and differentiated primary OLGs, total cell lysates were prepared from the OLGs, and 500 µg of protein extracts were immunoprecipitated with either anti-MRP2/KLHL1 antibody or anti-actin antibody. The immune complexes were then analyzed by SDS-PAGE and probed with either actin or MRP2/KLHL1 antibodies, respectively. As shown in Fig. 5, B and C, a direct endogenous association was observed between MRP2/KLHL1 and actin in both undifferentiated and differentiated OLGs, and this association was strongly increased in the differentiated OLGs. Furthermore, we examined co-localization of MRP2/KLHL1 and actin in undifferentiated and differentiated O2A cells. Cells were cultured in appropriate medium conditions for 48 h, and then fixed and stained with anti-MRP2 antibody and F-actin. As shown in Fig. 5D, the co-localization of MRP2/KLHL1 and actin was observed in both the cell body and along the processes, indicating that the upregulated association of MRP2/KLHL1 and actin is important for the rearrangement of the actin-based cytoskeleton.

Characterization of MRP2/KLHL1 Transgenic Mice—Our data showed that overexpression of MRP2/KLHL1 promoted process elongation in primary rat oligodendrocytes, whereas antisense MRP2/KLHL1 inhibited the process elongation of OLGs (Fig. 4). To further support these results, we generated a mouse model that carries the MRP2/KLHL1 transgene and elucidated whether overexpression of MRP2/KLHL1 affects the process elongation of oligodendrocytes in these transgenic mice. The circular plasmid, pEGFP-KLHL1/MRP2, was digested with AseI and BglII restriction enzymes, and the transgene consisting of CMV-IE, EGFP, MRP2/KLHL1, and poly(A) was purified and designated as the MRP2/KLHL1 transgene. The MRP2/KLHL1 transgene was then microinjected into fertilized mouse eggs at the one-cell stage, and the fertilized eggs were transferred to recipient 0.5-day pseudo-pregnant mice. After weaning, the offspring resulting from the microinjection were analyzed for transgene integration. As shown in the supplemental data (Fig. S1), the presence of a 1.4-kb fragment of the MRP2/KLHL1 transgene was observed in the mice, and these MRP2/KLHL1 transgenic founders were designated as TG mice. To confirm integration of the MRP2/KLHL1 transgene, we digested DNA from the MRP2/KLHL1 founder mice with a PstI restriction enzyme and subjected the DNA to Southern blotting using a 32P-labeled GFP probe. Southern blot analysis confirmed the integration of the MRP2/KLHL1 transgene (supplemental materials Fig. 1B).


Figure 4
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  		FIGURE 4.
Effect of MRP2/KLHL1 overexpression on the process elongation of primary rat OLGs. A, rat primary OLGs were transfected with GFP or the GFP-MRP2/KLHL1 construct, as indicated. The control cells showed small process formation, whereas the cells transfected with MRP2/KLHL1 demonstrated significant elongation of the OLG processes. B, quantitative analysis of the effects of MRP2/KLHL1 on process length in OLGs. Processes longer than half the diameter of the cell soma were included in the measurements. Three individual experiments were performed and the data were summarized and analyzed using the Mann-Whitney rank sum test. A total of 50 OLGs per treatment were analyzed for each assay. *, p < 0.001. C, effects of antisense of MRP2/KLHL1 on the process elongation of primary rat OLGs. Primary rat OLGs were transfected with GFP, GFP-MRP2/KLHL1, and GFP-asMRP2 constructs, and then the cell process length of the variously transfected OLGs was analyzed. D, primary rat OLGs were transfected with GFP, GFP-MRP2/KLHL1, or GFP-asMRP2. Following 24 h of transfection, the process lengths were assessed by measuring processes longer than half of their cell body diameters. Three hundred GFP-expressing cells from each group in the three individual experiments were measured for process length. *, p < 0.001.

 
Next, we optimized the PCR conditions for genotyping the MRP2/KLHL1 transgenic mice. Genomic DNA from the MRP2/KLHL1 transgenic founders and wild-type (WT) mice were amplified with specific primers for human MRP2/KLHL1. PCR analysis showed a specific band of 903 bp corresponding to the MRP2/KLHL1 insert (supplemental materials Fig. 1C). MRP2/KLHL1 founders were then bred and F1 offspring were analyzed for MRP2/KLHL1 expression. After genotyping the newborn mice, we purified RNA from mouse brains. DNase-treated RNA was amplified by RT-PCR using specific primers for MRP2/KLHL1. As shown (supplemental data Fig. S1D), MRP2/KLHL1 mRNA was detected in the TG mice. Additionally, brain tissue lysates were analyzed by Western blotting with anti-GFP (supplemental materials Fig. 1E) or anti-MRP2 (supplemental materials Fig. 1F) antibodies. A protein product of the MRP2/KLHL1 transgene (97 kDa) was detected, as expected. Endogenous MRP2/KLHL1 was also detected in the brain protein extracts when anti-MRP2 antibody was used. These data show that transgenic MRP2/KLHL1 mice were successfully generated and characterized as overexpressing MRP2/KLHL1 protein.

Effect of MRP2/KLHL1 Transgene Expression on OLG Process Extension—To examine the effect of overexpressing MRP2/KLHL1 in vivo, we isolated primary OLGs from MRP2/KLHL1 transgenic mice. One day after delivery, pieces of tails from the individual newborn mice were isolated and subjected to rapid PCR-based phenotyping (supplemental materials Fig. 1G). OLGs from PCR-positive litters were isolated and pooled together for comparison with the PCR-negative litters (control). Expression of the MRP2/KLHL1 transgene was also confirmed by RT-PCR analysis of RNA isolated from the brain tissues of the offspring (supplemental data Fig. 1H). The primary cultures of OLGs isolated from the MRP2/KLHL1 transgenic mice were incubated and subjected to immunohistochemistry with primary rabbit anti-MRP2/KLHL1 antibody as well as monoclonal anti-beta-actin antibody. Primary OLGs from the MRP2/KLHL1 TG mice showed a significant increase in process length, as compared with the WT control mice (Fig. 6A). Confocal analysis revealed that MRP2/KLHL1 is localized with actin along the processes of these OLGs (Fig. 6B). To determine the effect of MRP2/KLHL1 on process elongation, primary OLGs isolated from TG and WT mice were seeded on slide glasses and immunostained with anti-MRP2 antibody. One hundred cells were randomly counted from each group in the three individual experiments and the lengths of their processes were measured. The process elongation was significantly increased in OLGs isolated from the MRP2/KLHL1 transgenic mice (95.65 ± 4.68 µm), as compared with OLGs isolated from the wild-type control mice (44.7 ± 4.25 µm) (Fig. 6C).


Figure 5
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  		FIGURE 5.
MRP2/KLHL1 is an actin-binding protein. A, 293T cells were grown in 10-cm plates with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Lipofectamine was used for the transient transfections using the GFP (lane 1) or the GFP-MRP2/KLHL1 construct (lane 2). The cells were lysed in RIPA buffer containing protein inhibitors and MRP2/KLHL1 expression was analyzed by immunoprecipitation (IP) and Western blotting (WB) with anti-MRP2/KLHL1 antibody. Actin expression was determined by stripping and reprobing the same blot with anti-actin monoclonal antibody. B and C, undifferentiated and differentiated primary rat OLGs were lysed with RIPA buffer. Following pre-cleaning, the lysates were immunoprecipitated with anti-MRP2/KLHL1 or anti-actin antibodies. The immune complexes were then analyzed by Western blotting with anti-MRP2/KLHL1 or anti-actin antibodies, as indicated. D, undifferentiated and differentiated O2A cells were stained with anti-MRP2/KLHL1 antibody and F-actin. Merge indicates the co-localization of MRP2/KLHL1 and F-actin. No Diff., undifferentiated OLGs; Diff., differentiated OLGs.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The function of the Kelch-related protein family in mammalians is largely unknown. Here, we studied the function of MRP2/KLHL1 in OLGs, and showed that MRP2/KLHL1 is involved in process elongation in OLGs. MRP2/KLHL1 expression was up-regulated during OLG differentiation as well as during specific stages of OLG development. Furthermore, MRP2/KLHL1 was expressed and co-localized with the OLG marker (O1) in murine brain OLGs in vivo. Forced expression of MRP2/KLHL1 enhanced process elongation in the OLGs, whereas treatment with MRP2/KLHL1 antisense inhibited the process elongation. In addition, OLGs derived from MRP2/KLHL1 transgenic mice exhibited significantly enhanced process elongation as compared with OLGs derived from wild-type littermates. MRP2/KLHL1 directly associated and co-localized with actin along the elongated processes, and this interaction was up-regulated during OLG differentiation. Thus, increased MRP2/KLHL1 protein expression may regulate the actin-based cytoskeleton, leading to morphological changes and promotion of process elongation in OLGs.

The first Kelch-related protein was identified in Drosophila oocytes. It was co-localized with actin filaments in the ring canal bridging 15 nurse cells and oocytes, thus providing a path to the cytoplasm. This Kelch protein was found to play an important role in maintaining actin organization during the development of ring canals in Drosophila (6, 8). More than 60 Kelch-related proteins have been identified in various organisms from virus to mammals, and have been shown to possess diverse functions including the regulation of cell morphology and cell organization (14). Recently, MRP2/KLHL1 has been demonstrated to modulate glycogen synthase kinase 3beta-mediated neuronal outgrowth (40) and to play a crucial role in the biological functioning of Purkinje cells as well as in the pathophysiology of spinocerebellar ataxia type 8 (41). Here, we demonstrated that MRP2/KLHL1 is also involved in enhancing the process elongation of OLGs, which may provide important insights into its role in the process of myelination and remyelination in injured OLGs.

Myelination is essential for the normal functioning of the central nervous system. Prior to re-myelination or myelination, OLGs are required to extend their processes and contact demyelinated or normal axons. During myelination processes, cells of the OLG lineage have to alter the transcription rates of numerous genes in a highly coordinated manner. OLGs have a high rate of synthetic activity and produce vast amounts of myelin. The large membranous sheets of OLGs contain an elaborate cytoskeletal network of microtubules and microfilaments as well as actin-binding proteins. The present study demonstrates that the association of MRP2/KLHL1 with actin is up-regulated during differentiation. MRP2/KLHL1 has been shown to be involved in the rearrangement of actin networks, leading to the process extension of OLGs. Therefore, the presence of MRP2/KLHL1 in OLGs points to its possible role in the structural stability and plasticity of the OLG cytoskeleton in general and OLG processes in particular.

Advanced developments in biomedical research provide additional sophisticated tools to dissect molecular pathways in the nervous system by introducing transgenes into animal models. In this report, we generated MRP2/KLHL1 transgenic mice to support the finding that overexpression of MRP2/KLHL1 enhanced the process elongation of OLGs. MRP2/KLHL1 transgenic mice were successfully created as evidenced by MRP2/KLHL1 transgene integration and expression at the transcriptional and translational levels (supplemental materials Fig. S1). A significant increase in the process elongation of OLGs was observed in OLGs derived from MRP2/KLHL1 transgenic mice. In addition, MRP2/KLHL1 was co-localized with actin. Based on our results, we suggest that an increase in the protein levels of MRP2/KLHL1 is essential for the dynamics of cytoskeletal rearrangement through its binding to actin, leading to process elongation in the OLGs.


Figure 6
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  		FIGURE 6.
Effect of MRP2/KLHL1 expression on the process elongation of primary OLGs in transgenic mice. Primary OLGs were isolated from MRP2/KLHL1 TG and WT mice. Cells were then plated on poly-L- or nithine-coated coverslips in 12-well culture plates. After fixing the cells for 10 min with 4% paraformaldehyde, cells were permeabilized with 0.2% (v/v) Triton X-100 in PBS for 5 min, at which point blocking buffer (0.1% bovine serum albumin, 1% normal goat serum, 0.2% (v/v) Triton X-100 in PBS) was added. This was followed by incubation with primary rabbit anti-MRP2/KLHL1 antibody as well as monoclonal anti-actin antibody. Goat anti-rabbit IgG/fluorescein isothiocyanate was added as a secondary antibody to detect MRP2/KLHL1 expression (green color) and goat anti-mouse IgG/Texas Red was added as a secondary antibody to detect actin expression. Co-localization of MRP2/KLHL1 and actin is indicated in B by the yellow color. The samples were analyzed using a Zeiss LSM 510 Meta confocal microscope. A, images were obtained with a light microscope. B, images were obtained with a confocal microscope. DIC, differential interference contrast. C, comparison of the process length of OLGs isolated from MRP2/KLHL1 TG versus WT mice. Cells were immunostained with anti-MRP2/KLHL1 and anti-actin antibodies. The stained cells were counted in 10 randomly selected fields, and OLG process length was measured and expressed in micrometers. *, p < 0.05.

 
The current MRP2/KLHL1 transgenic model has some limitations for the in vivo study of MRP2/KLHL1 myelination in OLGs due to the fact that MRP2/KLHL1 transgene expression was driven ubiquitously under a CMV promoter. To pinpoint the role of MRP2/KLHL1 in the process elongation of OLGs, isolation of OLGs from MRP2/KLHL1 transgenic mice and their littermates may serve both as a model for process elongation and as a system to extend our insights into the role of MRP2/KLHL1 in myelination, re-myelination, and spinal injury. However, an additional transgenic mouse model should be constructed to direct the expression of MRP2/KLHL1 specifically in OLGs using an OLG-specific promoter.

Our previous report (42) demonstrated that Mayven, another member of the Kelch-related protein family, associated with the SH3 domain of Fyn kinase and with actin, which led to dynamic rearrangement of the cytoskeleton and to the process extension of OLGs. These observations were confirmed in a recent report (43), demonstrating that treatment with Mayven small interfering RNA or overexpressed Mayven lacking the SH3 domain reduced the process length of OLGs and resulted in shorter process formation. MRP2/KLHL1 is an additional member of the superfamily of Kelch-related proteins based on its structure, which consists of two main domains termed BTB and Kelch repeats. Analysis of the amino acid sequences of MRP2 showed the similarity of its basic domain structures to those of Mayven. However, MRP2/KLHL1 lacks an SH3 domain at its N terminus (Fig. 1). Whereas the effects of Mayven on process elongation in OLGs is mediated through its binding to the SH3 domain of Fyn kinase, MRP2/KLHL1-mediated effects on OLGs are Fyn kinase-independent, and probably are due to the direct association of MRP2/KLHL1 with actin during OLG differentiation. The up-regulation of Mayven and MRP2/KLHL1 in differentiated OLGs and their involvement in OLG process elongation indicate the importance of Kelch-related proteins in both the dynamics of cytoskeletal rearrangement and the process elongation of these cells (42).

The myelination of axons by OLGs involves the coordinated recognition of the axonal surface, ensheathment of the axonal process, and ultimately compaction of the wrappings of oligodendroglial membranes to generate the myelin sheath. The interplay of adhesion molecules expressed by OLGs and axons as well as the downstream signal transduction cascades mediating these complex cellular interactions are not well defined. Because the cytoskeleton has a central role in controlling the shape and motility of OLGs, we have focused on understanding the mechanism by which components of the OLG cytoskeleton might participate in the extension of OLG processes. Inherited spinocerebellar ataxia type 8 is caused by a CTG expansion mutation in the natural antisense RNA of KLHL1, whereas the normal function of KLHL1 is unknown (20, 21, 44). The expression of an antisense of MRP2/KLHL1 was identified in fetal and adult brains, specifically in the cerebellum, pons, medulla, and frontal lobe (45). Although the precise function of this antisense of MRP2/KLHL1 remains to be elucidated, it has been suggested to regulate MRP2/KLHL1 expression levels in the brain (45).

In summary, this study demonstrates a novel function of MRP2/KLHL1 and its role in the process elongation of primary rat OLGs. This study provides further insights into the function of Kelch-related proteins in the brain, and may contribute to a better understanding of OLG neuropathology.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants NS 39558 (to S. A.), CA 096805 (to H. A.), and NHLBI K18 PAR-02-069 (to H. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-667-0063; Fax: 617-975-6373 or 617-975-5240; E-mail: savraham{at}bidmc.harvard.edu.

3 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Wei Fu for typing assistance and Janet Delahanty for editing the manuscript. We also thank Drs. Tae-Aug Kim and Radoslaw Zagozdzon for help in this study, Dr. Timothy Kurt Vartanian (Beth Israel Deaconess Medical Center) for providing the primary rat oligodendrocyte cultures as well as specific oligoden-drocyte markers, and Dr. Kiweon Cha for help with computer modeling.



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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
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