Calpain and calpastatin expression in primary oligodendrocyte culture ...

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Primary Oligodendrocyte Culture: Preferential Localization of Membrane. Calpain in Cell Processes. Swapan K. Ray,1 Timothy J. Neuberger,2 Gail Deadwyler,3 ...
Journal of Neuroscience Research 70:561–569 (2002)

Calpain and Calpastatin Expression in Primary Oligodendrocyte Culture: Preferential Localization of Membrane Calpain in Cell Processes Swapan K. Ray,1 Timothy J. Neuberger,2 Gail Deadwyler,3 Gloria Wilford,1 George H. DeVries,3 and Naren L. Banik1* 1

Department of Neurology, Medical University of South Carolina, Charleston, South Carolina Aurora Biosciences, San Diego, California 3 Department of Anatomy and Cell Physiology, Loyola University, Chicago, Illinois 2

The cellular localization of calpain is important in understanding the roles that calpain may play in physiological function. We, therefore, examined calpain expression, activity, and immunofluorescent localization in primary cultures of rat oligodendrocytes. The mRNA expression of m-calpain was 64.8% (P ⫽ 0.0033) and 50.5% (P ⫽ 0.0254) higher than that of ␮-calpain and calpastatin, respectively, in primary culture oligodendrocytes. The levels of mRNA expression of ␮-calpain and calpastatin were not significantly different. As revealed by Western blotting, cultured oligodendrocytes contained a 70 kD major band identified by membrane m-calpain antibody, a 80 kD band recognized by cytosolic m-calpain antibody, and calpastatin bands ranging from 45 to 100 kD detected by a calpastatin antibody. Calpain activity in oligodendrocytes was determined by Ca2⫹-dependent 71.2% degradation of endogenous myelin basic protein compared with control; this activity was inhibited significantly (P ⫽ 0.0111) by EGTA and also substantially by calpeptin. Localization of calpain in cultured oligodendrocytes revealed strong membrane m-calpain immunofluorescence in the oligodendrocyte cell body and its processes. In contrast, the cytosolic antibody stained primarily the oligodendrocyte cell body, whereas the processes were stained very weakly or not at all. These results indicate that the major form of calpain in glial cells is myelin (membrane) m-calpain. The dissimilar localization of cytosolic and membrane m-calpain may indicate that each isoform has a unique role in oligodendrocyte function. © 2002 Wiley-Liss, Inc. Key words: membrane calpain; myelination; demyelinating disease; degeneration

The brains of different mammalian species contain ubiquitous calpain that exists in two isoforms, ␮-calpain and m-calpain, requiring micromolar and millimolar calcium (Ca2⫹) concentrations, respectively, for activation. In contrast, tissue-specific calpain (p94) has been found in © 2002 Wiley-Liss, Inc.

muscle and lens (Shearer and David, 1990; Suzuki et al., 1995). Intracellularly, ubiquitous calpains are tightly regulated by calpastatin, a specific endogenous inhibitor. Calpains may have important roles in normal cell functions, such as turnover of intracellular proteins and modification of cell membrane by limited proteolysis, as well as in cellular pathophysiology (Shearer and David, 1990; Suzuki et al., 1995; Banik et al., 1999). Cellular and subcellular studies have provided important information about localization of calpains in membrane, including synaptic and myelin, indicating their roles in synaptic modification and in myelination and myelin metabolism (Baudry et al., 1987; Li and Banik, 1995). The demonstration that m-calpain is present in myelin and the increased activity of calpain at the peak period of myelination in developing rat brain are consistent with a role for calpain in myelination (Domanska-Janik et al., 1992; Chakrabarti et al., 1993). In contrast, recent studies imply an important role for calpain in cell death (Nath et al., 1996; Squier and Cohen, 1997; Pike et al., 1998; Ray et al., 1999b) and in the pathophysiology of CNS injury (Banik et al., 1999; Hayes et al., 1999; Ray et al., 1999a), Alzheimer’s disease (AD; Saitoh et al., 1993), brain ischemia (Bartus et al., 1995), and demyelinating diseases, such as multiple sclerosis (MS) and optic neuritis (Shields and Banik, 1999a,b). Increased calContract grant sponsor: National Institutes of Health; Contract grant number: NS-31622; Contract grant number: NS-38146; Contract grant number: NS-41088; Contract grant sponsor: National Multiple Sclerosis Society; Contract grant number: RG-2130B; Contract grant number: RG2076B6/IT; Contract grant sponsor: American Health Assistance Foundation. *Correspondence to: Naren L. Banik, PhD, Department of Neurology, Medical University of South Carolina (MUSC), 96 Jonathan Lucas Street, Suite 309, P.O. Box 250606, Charleston, SC 29425. E-mail: [email protected] Received 24 January 2002; Revised 26 June 2002; Accepted 28 June 2002 Published online 7 October 2002 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.10414

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pain expression and activity have been found in neurons and glial cells in these diseases. Moreover, calpain has recently been implicated in the activation of T cells (Schaecher et al., 2001). Although the roles of calpain isoforms in physiological function are not clear, studies showing their cellular and subcellular localization may provide some valuable information on their biological activity. In brain, more than 95% of total calpain activity is due to m-calpain, and only 5% of the total calpain activity is derived from ␮-calpain (Murachi, 1990). Previous studies regarding immunocytochemical localization of calpain revealed that ␮-calpain was predominantly localized in the neurons and cytosol, whereas major amounts of m-calpain were found in glial cells (Siman et al., 1985; Hamakubo et al., 1986; Li and Banik, unpublished), with a portion of m-calpain in the membranebound form. The preferential localization of calpain isoforms in neuronal and glial cells suggests that different calpains may have different roles in cell function. The finding of m-calpain in myelin also suggests that a substantial amount of m-calpain may be present in myelinforming oligodendroglial and Schwann cells (Li and Banik, 1995). Importantly, m-calpain is also distributed in different subcellular compartments (cytosol and membrane), suggesting that it may have discreet functional roles. Our previous findings of calpain activity in myelin and its overexpression in demyelinating diseases suggest an important role for this protease in the function of myelinforming oligodendroglial cells. In particular, expression of calpain isoforms at normal levels in oligodendrocytes may be very crucial for sustaining cell viability. Therefore, we used rat primary culture oligodendrocytes to determine the mRNA expression of ␮-calpain, m-calpain, and calpastatin relative to ␤-actin by reverse transcriptasepolymerse chain reaction (RT-PCR). We also examined calpain expression by immunofluorescence. Our investigation revealed more mRNA expression of m-calpain than of ␮-calpain in primary culture oligodendrocytes. Substantial calpain activity is also present in these cells. The findings of intense membrane m-calpain immunofluorescence in the cell body, nuclear membrane, and processes and the weaker cytosolic m-calpain immunofluorescence in these cell compartments suggest that membrane m-calpain may play a role in oligodendrocyte function. A preliminary report of this study has been presented elsewhere (Neuberger et al., 1999). MATERIALS AND METHODS Antibodies Antibodies to myelin m-calpain and cytosolic m-calpain and calpastatin were prepared in our laboratory, as reported previously (Chakrabarti et al., 1988). Antibody to myelin basic protein (MBP) was also raised in our laboratory and reported earlier (Greenfield et al., 1982). Preparation of Rat Primary Oligodendrocyte Cultures Rat pups on postnatal day 2 (P2) were used to obtain cerebral cortices for the preparation of oligodendrocyte cultures.

Neonatal oligodendrocyte cultures were prepared by the method of deVellis and McCarthy, with modifications as previously described (Raabe et al., 1997). Briefly, the cerebral cortices were isolated and the meninges removed. The cortices were minced, digested with acetylated trypsin (Sigma Chemical Co., St. Louis, MO), and filtered through Nitex 130 and 35 filters. The filtrate was seeded and maintained in OLG medium (Chen and DeVries, 1989) with 10% fetal calf serum for 2 days, then switched to OLG medium with 10% Nu-Serum I (Collaborative Research, Bedford, MA). After 7 days, the cultures were shaken overnight at 350 rpm, and the cell suspension was collected. The suspension containing oligodendrocytes was subjected to differential adhesion to remove contaminating astrocytes, resulting in ⬎95% purity as assessed by ⬍5% glial fibrillary acidic protein (GFAP)-positive cells (data not shown). Isolation of total RNA and RT-PCR TRI Reagent (Molecular Research Center, Cincinnati, OH) was used to isolate total RNA. We performed RT-PCR experiments following a procedure recently reported in detail (Ray et al., 2001). Briefly, cDNA was synthesized using heatdenatured total RNA (1 ␮g), oligo-d(T)16 primer, dNTPs (Perkin Elmer, Norwalk, CT), Superscript II reverse transcriptase (RT), and other reagents (Gibco/BRL, Grand Island, NY) and used for PCR amplification of one message at a time using rat-specific primers (Table I) for ␤-actin (Nudel et al., 1983), ␮-calpain (Sorimachi et al., 1996), m-calpain (DeLuca et al., 1993), or calpastatin (Ishida et al., 1991) and AmpliTaq Gold DNA polymerase (Perkin Elmer Applied Biosystems, Foster City, CA). All primers were designed using Oligo software (National Biosciences, Plymouth, MN) and were custom made by the manufacturer (Operon Technologies, Alameda, CA). Amplification of the target cDNA fragment was performed in a DNA Thermal Cycler (Perkin Elmer) using the programs in the following order: one cycle of activation of AmpliTaq Gold DNA polymerase at 95°C for 9 min; 35 cycles of PCR (denaturation of template at 94°C for 1 min, annealing of primers at 58°C for 1 min, and extension of primers at 72°C for 2 min); one cycle of extension of all nascent strands at 72°C for 7 min; and one cycle of soaking at 4°C for at least 20 min. The number of cycles for PCR amplification was predetermined to produce specific gene products in the exponential range. Use of AmpliTaq Gold DNA polymerase in PCR amplification produced highly specific products. Each RT-PCR product (10 ␮l) was resolved by electrophoresis on a 1.2% agarose gel, stained with ethidium bromide (1 ␮g/ml), and photographed using Polaroid (positive/negative) film type 665. The negative was scanned on a Umax PowerLook Scanner using Photoshop 5.0 (Adobe Systems, Mountain View, CA), and analyzed with Quantity One software (PDI, Huntington Station, NY). The mRNA expression of a target gene was normalized to that of ␤-actin. Results were expressed as percent change ⫾ SEM. Protein Extraction and Western Blotting We used the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting methods (Ray et al., 2001), with necessary modifications. Briefly, oligodendrocyte cells were homogenized in ice-cold (4°C) protein homogenization solution [50 mM Tris-HCl, pH 7.4, 320 mM

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TABLE I. Rat Primers* Employed in RT-PCR Experiments for Amplification of mRNA of Specific Genes Gene ␤-Actin

␮-Calpain

m-Calpain

Calpastatin

Primer sequences

Product size

Reference

F: 5⬘-TAC AAC CTC CTT GCA GCT CC-3⬘ (262–281 bases) R: 5⬘-GGA TCT TCA TGA GGT AGT CTG TC-3⬘ (2,348–2,371 bases) F: 5⬘-CAC TTG AAG CGT G AC TTC TTC CTG GCC AAT GC-3⬘ (1,413–1,444 bases) R: 5⬘-GCA CTC ATG CTG CCC GAC TTG TCC AGG TCA AAC TT-3⬘ (1,914–1,948 bases) F: 5⬘-GGG CAG ACC AAC ATC CAC CTC AGC AAA AAC-3⬘ (1,436–1,465 bases) R: 5⬘-GTC TCG ATG CTG AAG CCA TCT GAC TTG AT-3⬘ (1,811–1,839 bases) F: 5⬘-AGT AGT TCT GGA CCC AAT G-3⬘ (341–359 bases) R: 5⬘-CCC CAG TAG ACT TCT CTT TC-3⬘ (551–570 bases)

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Nudel et al., 1983

536

Sorimachi et al., 1996

404

DeLuca et al., 1993

230

Ishida et al., 1991

*F, forward; R, reverse.

sucrose, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM EDTA]. Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL) was used for determination of protein concentration by measuring color density at 595 nm in a spectrophotometer (Spectronic Instruments, Rochester, NY). Protein samples were mixed with an equal volume of 2⫻ Laemmli loading buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10 mM ␤-mercaptoethanol, 0.02% bromophenol blue); heat denatured at 95°C for 5 min; loaded onto the SDSpolyacrylamide gradient (4 –20%) gels (Bio-Rad Laboratories, Hercules, CA); and resolved by SDS-PAGE in 25 mM Tris, pH 8.3, 192 mM glycine, 0.1% SDS (Laemmli, 1970). For monitoring equal amounts of protein loading, one set of resolved proteins on gels after SDS-PAGE was stained with 0.025% Coomassie brilliant blue R-250, 40% methanol, 7% acetic acid, followed by destaining of background first in 40% methanol, 7% acetic acid, and then in 5% methanol, 7% acetic acid (Wilson, 1983). Similarly resolved proteins on sister gels were transferred by electroblotting to Immobilon-P membranes (Millipore, Bedford, MA) in Towbin transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine, 20% methanol, 0.1% SDS; Towbin et al., 1979). Incubations of blots in primary IgG antibody were followed by incubation in alkaline horseradish peroxidase (HRP)conjugated IgG secondary antibody (ICN Pharmaceuticals, Aurora, OH) and subsequent detection of specific protein bands by alkaline HRP-catalyzed oxidation of luminol in the presence of H2O2 using the enhanced chemiluminescence system (ECL) according to the instruction of the manufacturer (Amersham Pharmacia Biotech, Piscataway, NJ). The blots were immediately exposed to X-Omat XAR-2 film (Eastman Kodak, Rochester, NY) for autoradiography. Film exposure times were calibrated for analyzing the optical density (OD) values of specific protein bands within the linear range (Schumacher et al., 2000). The ECL autoradiograms were placed on a PowerLook Scanner (Umax, Fremont, CA) to image and digitize the bands using PhotoShop software (Adobe Systems). The OD values of the bands were estimated by using Quantity One software (Bio-Rad Laboratories). The extent of loss of protein was calculated as

percentage degradation compared with control and was taken as a measure of calpain activity. Results were expressed as percent loss ⫾ SEM. Separation of Membrane and Cytosolic Fractions for Western Blotting Primary oligodendrocyte cells were homogenized in 50 mM Tris-HCl, pH 7.4, 5 mM EGTA, and 1 mM PMSF. The homogenate was spun at 100,000g for 1 hr using an SW 60Ti rotor in the ultracentrifuge (Beckman Coulter, Fullerton, CA). The supernatant (cytosolic fraction) was separated from the pellet (membrane fraction). Membrane and cytosolic fractions were used for determination of protein concentration. Equivalent amounts of protein were loaded on 4 –20% gradient SDSpolyacrylamide gels for electrophoresis, followed by transfer to Immobilon-P membranes (Millipore) and Western blotting (as described above) with appropriate antibody to membrane or cytosolic m-calpain. Calpain Activity in Oligodendrocytes Oligodendrocyte homogenates were incubated at 37°C for 1 hr with and without 1 mM Ca2⫹ and in the presence or absence of 5 mM EGTA (a Ca2⫹-chelating agent) or 10 ␮M calpeptin (a specific inhibitor of calpain). We considered endogenous MBP as a substrate of calpain. The amount of calpain activity was determined by quantification of the extent of degradation of MBP on the Western blots, as described above. In Situ Immunofluorescence Technique Oligodendrocyte cells were washed thoroughly with Dulbecco’s phosphate-buffered saline (D-PBS) to remove all serum components. The intracellular localization of m-calpain was examined by fixing the cells with 4% paraformaldehyde (freshly prepared in D-PBS) for 5 min, followed by membrane permeabilization with 1% Triton X-100. Cells were incubated in 5% (w/v) nonfat powdered milk (prepared in cold D-PBS) for 1 hr to block nonspecific binding to the antibodies. Primary IgG antibodies were diluted in D-PBS, layered onto the cells, and

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incubated for 1 hr at room temperature. In contrast, surface or extracellular localization of m-calpain was carried out by adding diluted primary IgG antibody directly to the cells for 1 hr at 4°C. The primary IgG antibody–antigen complex was visualized with rhodamine-conjugated goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA). After incubation with the secondary antibody, cells were washed three times with D-PBS for 30 min each. Cells were overlaid with 5% n-propyl gallate in 1:9 PBS:glycerol to diminish photobleaching and covered with a glass coverslip. Stained oligodendrocyte cultures were examined under a Nikon phase-contrast microscope (for localization of surface and intracellular immunostaining) and also a fluorescence microscope (for localization of extracellular and intracellular immunostaining) equipped with an FX-35WA Nikon camera. After all the fine adjustments, photographs of stained oligodendrocytes were taken using Ilford HP-5 film, ASA 400, and printed to obtain the sharpest image possible.

RESULTS Levels of mRNA Expression of Calpain and Calpastatin in Oligodendrocytes Levels of mRNA expression of calpain and calpastatin were determined in cultured rat primary oligodendrocyte cells. We employed semiquantitative RT-PCR for analyzing the mRNA levels of ␮-calpain, m-calpain, and calpastatin relative to ␤-actin. The use of selectively designed rat primers and AmpliTaq Gold DNA polymerase in RT-PCR experiments helped us to generate the highly specific products (Fig. 1A) that, we predetermined, were produced in the exponential range of amplification. The mRNA expression of m-calpain was 64.8% (P ⫽ 0.0033) and 50.5% (P ⫽ 0.0254) higher than that of ␮-calpain and calpastatin, respectively (Fig. 1B). The mRNA levels of ␮-calpain and calpastatin did not differ greatly. Primary culture oligodendrocytes expressed mRNAs of ␮-calpain, m-calpain, and calpastatin in the proportion of 1.0:1.6:1.1. Relatively, increased mRNA expression of m-calpain suggests that m-calpain may be involved in many functions of rat oligodendocytes. We subsequently studied m-calpain contents in the membrane and cytosolic fractions of cultured oligodendrocyte cells. m-Calpain in Membrane and Cytosol of Oligodendrocytes First, we employed whole-homogenate proteins of rat primary oligodendrocytes for identification of the m-calpains in membrane and cytosol by Western blot analysis using specific antibodies to each isoform. m-Calpain antibodies were prepared using purified m-calpain of myelin and cytosolic fractions and were raised in rabbits. Western blot analysis of wholehomogenate proteins of oligodendrocytes revealed a major band at 70 kD and a minor band at around 60 kD with the use of membrane m-calpain antibody (lane 1, Fig. 2A) and only a major 80 kD band with cytosolic m-calpain antibody (lane 2, Fig. 2A). The 80 kD m-calpain in the cytosol of oligodendrocytes is likely to be similar to that found in

Fig. 1. RT-PCR analysis for mRNA levels of calpain and calpastatin in cultured oligodendrocytes. A: Representative gel showing RT-PCR products of different genes. M, marker (1 kb DNA ladder from Gibco/ BRL). Genes and their amplified cDNA segment sizes are labeled on the gel. B: Densitometric analysis of the band intensities for mRNA levels of ␮-calpain, m-calpain, and calpastatin genes relative to ␤-actin. Data are presented with histograms as mean ⫾ SEM (n ⫽ 3).

the brain cytosolic fraction. However, the significance of the major 70 kD m-calpain in the membrane of oligodendrocytes is not clear. It could be an interesting isoform of membrane-associated calpain in oligodendrocytes. Also, it

Calpain and Calpastatin in Cultured Oligodendrocytes

Fig. 2. Western blots showing m-calpain and calpastatin expression at the protein level in cultured oligodendrocytes. A: Membrane and cytosolic m-calpain bands were detected by using membrane and cytosolic m-calpain antibodies, respectively. M, molecular size standards; lane 1, m-calpain bands using an antibody to myelin m-calpain; lane 2, m-calpain band using an antibody to cytosolic m-calpain. B: Detection of different calpastatin bands. M, molecular size standards; lane 1, calpastatin isoforms. The molecular sizes are labeled at left of a representative Western blot.

could be a degradation product of calpain in the membrane. However, both membrane and cytosolic m-calpains, as identified on the Western blots (Fig. 2A), may have essential functions in rat oligodendrocytes. Further Western blot analysis of whole-homogenate proteins of primary oligodendrocytes (Fig. 2B) identified several calpastatin bands ranging from 45 to 100 kD. It should be noted that the 100 kD band of calpastatin is in the expected range for molecular size, and the multiple, more intense bands (the major one at 60 kD) of calpastatin may reflect proteolytic degradation or alternate splicing. These smaller molecular sizes of calpastatin are still large enough and may contain inhibitory regions capable of regulating calpain activity in rat oligodendrocytes. Second, we separated primary oligodendroglial proteins into membrane and cytosolic fractions for use in Western blot analysis to see whether m-calpain bands of different molecular sizes do segregate into membrane and cytosolic fractions (Fig. 3). As a control, we ran wholehomogenate proteins of rat spinal cord (lane 1, Fig. 3) on the Western blots. Membrane m-calpain antibody was used to probe the Western blots (Fig. 3A) containing oligodendrogial membrane-fraction proteins (lane 2, Fig. 3A). Identification of a major 70 kD band and a minor 60 kD band of m-calpain (lane 2, Fig. 3A) indicated that these isoforms indeed belonged to the oligodendrogial membrane fraction. Similarly, we used cytosolic m-calpain antibody to probe the Western blots (Fig. 3B) containing oligodendrogial cytosolic fraction proteins, and we identified a major 80 kD band of m-calpain (Fig. 3B, lane 2). These results further confirmed that primary oligodendroglial cells contained a major 70 kD m-calpain isoform in the membrane and a major 80 kD m-calpain isoform in the cytosol. Assessment of Calpain Activity in Oligodendrocytes The calpain activity in oligodendrocytes (Fig. 4) was determined by assessing the extent of Ca2⫹-induced deg-

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Fig. 3. Western blottings with the membrane and cytosolic fractions of oligodendrocytes. The whole homogenate of oligodendrocytes was fractionated into membrane and cytosolic fractions by ultracentrifugation at 100,000g. A: Western blot containing rat spinal cord homogenate proteins (lane 1) and oligodendroglial membrane fraction proteins (lane 2) was probed with membrane m-calpain antibody. B: Western blot containing rat spinal cord homogenate proteins (lane 1) and oligodendroglial cytosolic fraction proteins (lane 2) was probed with cytosolic m-calpain antibody. Results indicated that 60 and 70 kD m-calpain bands segregated into membrane fraction and 80 kD m-calpain band into cytosolic fraction.

Fig. 4. Determination of MBP degradation as a measure of calpain activity in cultured oligodendrocytes. An antibody to MBP was used to probe the Western blots containing oligodendrocyte homogenate samples after different treatments at 37°C for 1 hr: control (without Ca2⫹), 1 mM Ca2⫹, 1 mM Ca2⫹ ⫹ 5 mM EGTA, and 1 mM Ca2⫹ ⫹ 10 ␮M calpeptin. Optical density units of MBP bands were quantitated in three representative experiments. The results were presented as percentage degradation of MBP compared with control. The extent of inhibition of Ca2⫹-mediated degradation of MBP by EGTA was highly significant (P ⫽ 0.0111). Calpeptin also substantially inhibited Ca2⫹mediated degradation of MBP.

radation of endogenous MBP compared with control (no addition of Ca2⫹). Inhibitors such as EGTA and calpeptin were used in the assay to ascertain the specificity of calpain activity in oligodendrocytes. Incubation of oligodendrocyte homogenate proteins with Ca2⫹ caused 71.2% degradation of MBP, which was significantly (P ⫽ 0.0111) prevented in the presence of the Ca2⫹ chelator EGTA resulting in only 15.3% degradation of MBP compared with control (Fig. 4). Thus, more than 4.5-fold inhibition of calpain activity was observed with EGTA compared

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Fig. 5. Immunofluorescent labeling for localization of myelin and cytosolic m-calpain in cultured oligodendrocytes. Phase-contrast microscopy showing oligodendroglia after surface or extracellular immunostaining with myelin m-calpain antibody (A) and after intracellular

immunostaining with cytosolic m-calpain antibody (B). Fluorescence microscopy showing oligodendroglia after surface or extracellular immunostaining with myelin m-calpain antibody (C) and after intracellular immunostaining with cytosolic m-calpain antibody (D).

with Ca2⫹-induced degradation. The presence of calpeptin, a specific inhibitor of calpain, also substantially (P ⫽ 0.0789) inhibited calpain activity with reduction of MBP degradation compared with Ca2⫹-induced degradation. These results suggested that primary oligodendrocytes possessed calpain activity for cellular functions.

nuclei following surface or extracellular immunostaining with membrane m-calpain antibody (Fig. 5A). In contrast, phase-contrast microscopy of oligodendrocytes after intracellular immunostaining with the cytosolic m-calpain antibody (Fig. 5B) did not show cells and their processes as well defined as those seen with membrane m-calpain antibody (Fig. 5A). Also, fluorescent microsopy was used for both surface and intracellular localization of m-calpain in cultured oligodendrocytes. Fluorescent microscopic results indicated strong myelin m-calpain immunofluorescence in the oligodendrocyte cell body, nuclear membrane, and its processes (Fig. 5C), whereas the cytosolic m-calpain antibody stained primarily the cell body and very weakly the processes (Fig. 5D). Thus, results showed staining of oligodendrocyte cell bodies by both the membrane and the cytoplasmic anticalpain antibodies, whereas only the membrane anticalpain antibody stained the pro-

Immunofluorescent Localization of m-Calpain Expression in Oligodendrocytes Immunofluorescent localization of membrane and cytosolic m-calpains was carried out with the use of respective antibodies in cultured oligodendrocytes. Phasecontrast microscopy was performed for both surface and intracellular localization of m-calpain in cultured oligodendrocytes. Phase-contrast microscopy of mature oligodendrocytes revealed substantial extension of very-welldefined processes and well-preserved cell bodies and

Calpain and Calpastatin in Cultured Oligodendrocytes

cesses. However, sufficient characterization of antibody specificities would make a meaningful interpretation of such findings. The different staining patterns could just be because the cytosolic anticalpain antibody was less sensitive for detecting low levels of calpain in the processes. It should be noted that we had negative controls (without primary IgG antibodies) to document the specificity of the antibody staining. The negative controls did not show any immunostaining of oligodendrocytes (data not shown). DISCUSSION The physiological function of calpain is unknown even though it degrades many cellular and cytoskeletal proteins and is involved in processing hormones and enzymes (Carafoli and Molinari, 1998). Increased calpain activation is thought to be involved in the pathophysiology of degenerative diseases (Saitoh et al., 1993; Bartus, 1997), demyelination (Shields et al., 1999b), trauma (Saatman et al., 1996; Springer et al., 1997; Hayes et al., 1999; Ray et al., 1999a), and cell death (Nath et al., 1996; Squier et al., 1997; Ray et al., 1999b). To understand its role in cell function, it is essential to determine the cellular and subcellular localization of calpain. Both calpain isoforms are present in each neural cell type to a variable degree. Immunocytochemical studies have revealed that ␮-calpain is present predominantly in neurons, whereas m-calpain is localized primarily in glial cells (e.g., astrocytes, oligodendrocytes; Siman et al., 1985; Hamakubo et al., 1986; Li and Banik, unpublished). This suggests that different calpain isoforms may have discrete roles to play in neuronal and glial cell function. Subcellular distribution studies have indicated m-calpain localization in cytosol as well as in myelin, nuclear, and synaptic membrane (Banik et al., 1987, 1991; Baudry et al., 1987). In fact, a substantial amount of m-calpain has been shown to be associated with myelin (Sato et al., 1982; Banik et al., 1987, 1991; Kolehmainen and Kaisto, 1989; Domanska-Janik et al., 1992; Li and Banik, 1995). The extent to which m-calpains localized in the cytosol or the membrane are functionally the same or whether the m-calpains in different cellular locations represent different isozymes is not known. Earlier findings of m-calpain localization in myelin suggest that components of this membrane should be present in the myelin-forming oligodendrocytes of central nervous system (CNS) and Schwann cells of peripheral nervous system (PNS). Calpain expression and activity in Schwann cells have been previously reported (Mata et al., 1991; Chakrabarti et al., 1997; Neuberger et al., 1997). Here we show that primary cultures of oligodendrocytes express ␮-calpain and m-calpain as well as the endogenous inhibitor calpastatin at the mRNA level. The demonstration of greater expression of m-calpain mRNA compared with ␮-calpain in these cells confirms the earlier report of immunocytochemical localization of m-calpain primarily in glial cells (Hamakubo et al., 1986). This also lends support to the finding that m-calpain in brain constitutes more than 95% of total calpain (Murachi, 1990), because glial cells in brain outnumber neurons by at least 10 to 1. Although translational expression of m-calpain was evi-

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dent in these cells, it is important to note that the m-calpain polyclonal antibody used in this study would recognize both ␮-calpain and m-calpain isoforms. The antibodies prepared from both cytosolic and membrane (myelin) m-calpains recognized calpains of oligodendrocytes. Interestingly, the cytosolic m-calpain was found to be slightly larger (80 kD) than the major membrane m-calpain band (70 kD). Whether these are different isoforms or the membrane m-calpain is degraded to a smaller size (60 and 70 kD) during the myelin isolation procedure is not clear. In contrast, our previous studies using antibody prepared from whole-brain calpain did not show significant differences in size between these two sources of calpain when tested in myelin and cytosolic fractions isolated from brain (Chakrabarti et al., 1989). Although it is not known whether any differences exist between the properties of cytosolic and of membrane m-calpain, the immunofluorescent localization of m-calpains in primary culture oligodendrocytes has raised the possibility of functional differences for this enzyme in two distinct cellular locations. The demonstration of intense immunofluorescence of membrane (myelin) m-calpain in the oligodendrocyte cell body and particularly in the processes compared with weak or absent fluorescence of cytosolic m-calpain in the cell processes suggests that membrane m-calpain may be involved in CNS myelination/ myelin compaction. The findings of increased m-calpain expression and activity in rat brain during development and its association with myelin following maturation support this hypothesis (Chakrabarti et al., 1993). Furthermore, the demonstration of calpain activity and immunocytochemical localization in adult myelin also suggest a role for calpain in myelination (Domanska-Janik et al., 1992; Li and Banik, 1995). In addition to immunofluorescent localization, calpain activity in oligodendroglial cells was shown by Ca2⫹-dependent degradation of endogenous MBP (Fig. 3). This activity of calpain was inhibited significantly by EGTA (a Ca2⫹-chelating agent) and also by calpeptin (a specific inhibitor of calpain). Recent studies have shown an important role for calpain in apoptotic cell death (Ray et al., 1999b). Our findings of calpain activity in oligodendrocytes also suggest that, in the pathophysiology of diseases, increased calpain activation may contribute to apoptotic death of these cells. Stress (e.g., free radicals, Ca2⫹ influx)-induced cell death in lymphocytes and C6 glial cells could be attenuated by calpain inhibitors, indicating calpain involvement in cell death (Sarin et al., 1994; Squier et al., 1994; Ray et al., 1999b). Calpain-mediated apoptotic death of cultured neuronal cells also has been demonstrated (Nath et al., 1996; Pike et al., 1998; Ray et al., 2000; Boggan et al., 2001). Our findings of various stress-related vulnerabilities of C6 glial cells to apoptosis mediated by calpain (Ray et al., 1999b) suggest that glial cells (e.g., astrocytes, oligodendrocytes) as a result of increased calpain activity in inflammatory or demyelinating diseases (e.g., MS, optic neuritis) and in optic nerve degeneration in glaucoma may die by apoptosis. Such increased activity of calpain in

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oligodendrocytes has been demonstrated in MS and its experimental model experimental allergic encephalomyelitis (EAE; Shields et al., 1998). Apoptosis of oligodendrocytes in MS and EAE has been implicated (Bonetti et al., 1999; Hisahara et al., 2000). Whether calpain is one of the proteases involved in this process is not known. It is possible that, in the pathophysiology of demyelinating diseases or CNS injury, increased calpain in oligodendrocytes or calpain released from inflammatory cells (e.g., macrophages, T lymphocytes) and reactive endogenous cells (e.g., microglia, astrocytes; Shields et al., 1998) contributes to oligodendroglial death. The activity is substantially increased in reactive astroglial and microglial cells, suggesting a role for calpain activation in glial cells in demyelinating diseases (Shields et al., 1998) and perhaps in glaucoma, in which reactive gliosis predominates in the optic nerve head (Varela and Hernandez 1997; Liu and Neufeld, 2000). In this study, we demonstrated m-calpain activity and expression in primary cultures of oligodendrocytes. The membrane (myelin) m-calpain is preferentially localized in the oligodendroglial processes, whereas the cytosolic m-calpain is weak or absent, suggesting functional differences between the two isoforms of this enzyme. The membrane m-calpain localization in oligodendrocyte processes may be indicative of a role for this enzyme in myelination. Understanding the role of this enzyme in myelination may also yield clues concerning the pathophysiological role of upregulated calpain in oligodendroglial death and myelin breakdown in demyelinating diseases. Ultimately, therapeutic agents and calpain inhibitors may be targeted to block calpain-mediated cell death and thus protect oligodendrocytes in demyelinating and inflammatory diseases. ACKNOWLEDGMENT We thank Mrs. Denise D. Matzelle for her valuable assistance in preparation of the manuscript. REFERENCES Banik NL, Chakrabarti AK, Hogan EL. 1987. Distribution of calciumactivated neutral proteinase (CANP) in myelin and cytosolic fractions in bovine brain white matter. Life Sci 41:1089 –1095. Banik NL, DeVries GH, Neuberger T, Russell T, Chakrabarti AK, Hogan EL. 1991. Calcium-activated neutral proteinase (CANP) activity in Schwann cells: immunofluorescence localization and compartmentation of ␮ and mCANP. J Neurosci Res 29:346 –354. Banik NL, Shields DC, Ray S, Hogan EL. 1999. The pathophysiological role of calpain in spinal cord injury. In: Wang KKW, Yuen P-W, editors. Calpain: pharmacology and toxicology of calcium-dependent protease. Washington, DC: Taylor and Francis Publishers. p 211–227. Bartus RT. 1997. The calpain hypothesis of neurodegeneration: evidence for a common cytotoxic pathway. Neuroscientist 3:314 –327. Bartus R, Heyward NJ, Elliott PJ, Sawyer SD, Baker KL, Dean RL, Akiyuama A, Straub JA, Harbeson SL, Li Z, Powers J. 1995. Calpain inhibitor AK295 protects neurons from focal brain ischemia: effects of post-occlusion intra-arterial administration. Stroke 25:2265–2270. Baudry M, Dubrin R, Lynch G. 1987. Subcellular compartmentalization of calcium-dependent and calcium-independent neutral proteases in brain. Synapse 1:506 –511. Boggan WO, Ray SK, Nowak MW, Banik NL. 2001. Calpain and

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