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Golli proteins, products of the myelin basic protein gene, function as a new type of modulator of intracellular Ca++ levels in oligodendrocyte progenitor cells (OPCs). Because of this, they affect a number of Ca++ dependent functions such as OPC migration and process extension. To examine further the Ca++ channels regulated by golli, we studied the store operated Ca++ channels (SOCCs) in OPCs and acute brain slice preparations from golli-KO and golli-overexpressing mice. Our results showed that pharmacologically-induced Ca++ release from intracellular stores evoked a significant extracellular Ca++ entry after store depletion in OPCs. They also indicated that under these pharmacological conditions golli promoted activation of Ca++ influx by SOCCs in cultured OPCs as well as in tissue slices. The Canonical TRP (Transient Receptor Potential) family of Ca++ channels (TRPCs) has been postulated to be SOCC subunits in oligodendrocytes. Using a siRNA knock down approach, we provided direct evidence that TRPC1 is involved in store-operated Ca++ influx in OPCs, and that it is modulated by golli. Furthermore, our data indicated that golli is probably associated with TRPC1 at OPC processes. Additionally, we found that TRPC1 expression is essential for the effects of golli on OPC proliferation. In summary, our data indicate a key role for golli proteins in the regulation of TRPC mediated Ca++ influx, a finding that has profound consequences for the regulation of multiple biological processes in OPCs. More important, we have shown that extracellular Ca++ uptake through TRPC1 is an essential component in the mechanism of OPC proliferation.
The myelin basic protein (MBP) gene encodes two families of proteins: the classic MBP components of the myelin membrane, and a second set of products, the golli MBPs, whose function is only beginning to be understood (Campagnoni et al., 1993; Pribyl et al., 1993). Like the classic forms in the CNS, expression of golli proteins is developmentally regulated (Campagnoni et al., 1993; Landry et al., 1996). It tends to be high during embryonic development and declines with age (Landry et al., 1996, 1998). In the nervous system, golli proteins are expressed in oligodendrocytes (OLs) and in specific subsets of neurons, although golli protein expression has also been reported in activated microglia, macrophages and adult OL progenitors (Filipovic et al., 2002).
Previous work has shown golli to be an important regulator of OL development and myelination. Golli regulates oligodendrocyte progenitor cell (OPC) process extension and differentiation in vitro (Paez et al., 2009a; 2009b), and studies of golli-KO and golli-overexpressing (JOE) brains indicate this influence on OPC development has important consequences for myelination in vivo (Jacobs et al., 2005; 2009). The golli-KO animals exhibit widespread hypomyelination, with particularly striking myelin deficits in the visual and somatosensory cortex, and the optic nerve. This hypomyelination has clear functional effects since these animals exhibit alterations in visual evoked potentials (Jacobs et al., 2005). JOE animals express multiple copies of the J37 golli isoform under the control of the classic MBP promoter. These animals present severe intention tremors between ~P15 and ~P50 and exhibit generalized hypomyelination in the brain (Martin et al., 2007; Jacobs et al., 2009). At later ages the tremors abate and increased myelination is observed.
The cellular and molecular mechanisms governing the altered development of golli-KO and JOE OPCs have been partially elucidated. Recent findings have clearly established that golli protein plays a critical role in regulating Ca++ influx in OPCs (Paez et al., 2007; 2009a; 2009b; Fulton et al., 2010).
Calcium influx can occur via a number of different mechanisms within the cell including: (1) ligand-operated Ca++ channels (Simpson et al., 1997; Butt et al., 2006); (2) voltage-gated Ca++ channels (Paez et al., 2007) and (3) by the opening of store-operated Ca++ channels (SOCC) in the membrane in response to Ca++ depletion in the endoplasmic reticulum (Belachew et al., 2000; Alberdi et al., 2005). There are a number of Ca++ selective store-operated currents found in different cells (Parekh and Putney, 2005) indicating that multiple types of SOCCs probably exist. The TRP (Transient Receptor Potential) superfamily consists of six subfamilies, including the TRPC subfamily (the “canonical” TRPs). TRPCs have been postulated to be SOCC subunits and are a heterogeneous family of Ca++ channels (Zitt et al., 2002; Smyth et al., 2006; Liao et al., 2007). Expression of TRPCs has been described in nervous tissue (Putney, 1999; Riccio et al., 2002), but most store-operated cation conductances have been demonstrated in excitable cells (Putney, 1993). Up to now, a role for TRPC channels in OLs has not been defined.
The goal of this study was to define the Ca++ channels through which golli modulates Ca++ homeostasis in OPCs and thereby modulates essential OPC functions such as proliferation. In all the experiments, pharmacological tools and imaging techniques were used to compare the physiology of OPCs in genotype control mice to golli-KO and golli-overexpressing (JOE) mice, both in vitro (cultured OPCs) and in situ (slice preparation). We show that changes in golli expression alter the activity of TRPC channels in OPCs a finding that has profound consequences on multiple aspects of OPC maturation and survival.
We previously generated a golli knockout (KO) mouse in which the golli products of the MBP gene were selectively ablated while permitting normal expression of the classic MBPs (Jacobs et al., 20005). Through non-brother–sister crosses, a line was generated that is homozygous for the golli ablation on a background that is 50% 129S7/SvEvBrd and 50% C57BL6/J. A control line (KO Control) was established that was also 50% 129S7/SvEvBrd and 50% C57BL6/J but was negative for the golli ablation. The golli KO phenotype was observed before keeping the lines separate and then was studied over at least eight generations and remained stable.
We generated a transgenic mouse that overexpresses the golli isoform J37 in oligodendrocytes under the control of a classic MBP promoter (Martin et al., 2007; Jacobs et al., 20009). In this transgenic mouse, the J37 golli isoform is driven by a 1.9-kb region of the classic MBP promoter, thus directing overexpression of the protein specifically to OLs in the CNS. These mice are called JOE mice for golli J37 OverExpressing. A line was generated that is heterozygous for the MBP 1.9-J37 transgene on a background that is 50% BALB/cByJ, 37 to 50% C57BL/6, and 0 to12% C3H/He. A control line (JOE Control) was established that was also 50% BALB/cByJ, 37 to 50% C57BL/6, and 0 to12% C3H/He.
Enriched oligodendrocytes were prepared as described by Amur-Umarjee et al. (1993). First, cerebral hemispheres from 1 day old mice were mechanically dissociated and were plated on poly-D-lysine-coated flasks in Dulbecco's modified Eagle's medium and Ham's F12 (1:1 vol/vol) (Invitrogen), containing 100μg/ml gentamycin and supplemented with 4mg/ml dextrose anhydrous, 3.75mg/ml HEPES buffer, 2.4mg/ml sodium bicarbonate and 10% fetal bovine serum (FBS) (Omega Scientific). After 24 hours the medium was changed and the cells were grown in DMEM/F-12 supplemented with insulin (5μg/ml), transferring (50μg/ml), sodium selenite (30nM), T3 (15nM), d-Biotin (10mM), hydrocortisone (10nM), 0.1% BSA (Sigma), 1% horse serum and 1% FBS (Omega Scientific). After 9 days, OPCs were purified from the mixed glial culture by the differential shaking and adhesion procedure by Suzumura et al. (1984) and allowed to grow on polylysine-coated coverslips in defined culture media (Agresti et al., 1996) plus PDGF (10ng/ml) and bFGF (10ng/ml) (Peprotech).
Cells were stained with antibodies against golli, TRPC1 and Phospo-Histone H3 (Ser10) and examined by confocal microscopy. For golli and Phospo-Histone H3 (Ser10) immunostaining, the cells were rinsed briefly in PBS and fixed in 4% buffered paraformaldehyde for 30 min at room temperature. After rinsing in PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature and then processed for immunocytochemistry following the protocol as outlined by Reyes et al. (2002). Essentially, fixed cells were incubated in a blocking solution (5% goat serum in PBS) followed by an overnight incubation at 4°C with a polyclonal antibody for golli (1:500) and Phospo-Histone H3 (Ser10) (1:100; Millipore). Staining with TRPC1 (1:100; Alomone Labs) was performed on live cells without permeabilization for 1h at room temperature before fixation. Cells were then incubated with the appropriate secondary antibodies (1:200; Jackson), mounted onto slides with Aquamount (Lerner Laboratories), and fluorescent images were obtained using a Olympus spinning disc confocal microscope. Nuclei were stained with the fluorescent dye Hoechst 33342 (5μg/ml in 1% DMSO), in order to determine the total number of cells. Quantitative analysis of the results was done counting the antigen-positive and Hoechst-positive cells in 20 randomly selected fields, which resulted in counts of >2,000 cells for each experimental condition. Counts of antigen-positive cells were normalized to the counts of total Hoechst-positive cells for each condition.
Petri dishes containing approximately 1.5×106 cells/dish, under the different experimental conditions, were washed twice with PBS. The cells were collected by centrifugation (600×g for 10min at 4°C) and extracted with 20mM Tris-HCl, pH7.4, 150mM NaCl, 10mM EDTA, 0.5% Triton X-100 for 1h at 4°C. The insoluble material was cleared out by centrifugation (100.000×g for 20min at 4°C) and the resulting protein extracts (50μg/lane) were subjected to SDS-PAGE (12.5% gels). After transfer, samples were analyzed by Western blot using an specific antibody for TRPC1 (1:500; Alomone Labs). Antibody binding was visualized with horseradish peroxidase-conjugated secondary antibody (used at a 1:1000 dilution) and the ECL Plus substrate kit (Amershan Pharmacia Biotech).
Total RNA was isolated from 35mm cell culture dishes containing approximately 1.5×106 cells/dish, using TRIZOL reagent (Invitrogen) according to the instructions of the manufacturer. RNA content was estimated by measuring the absorbance at 260nm and the purity was assessed by measuring A260/A280 ratios. PCR primers for TRPC1-6 were designed based on published sequences by Wang et al. (2004) with some modifications (Table I). First-strand cDNA was prepared from 1μg of total RNA using SuperScriptTM III RNase H- reverse transcriptase (Invitrogen) and 1μg of oligo(dT). The mRNA samples were denaturated at 65°C for 5min. Reverse transcription was performed at 50°C for 55min and was stopped by heating samples at 85°C for 5min. The cDNA was amplified by PCR using the TRPC isoform-specific primers listed in Table I and PCR Platinum Supermix reagent (Invitrogen). PCR conditions were as follows: 94°C for 2min, 40 cycles of 94°C for 30s, 58°C for 30s followed by 68°C for 2min. After completion of the 40 cycles, samples were incubated at 72°C for 10min. A β-tubulin positive control was performed alongside the experimental samples, as well as a negative control with no reverse transcriptase. The PCR products were visualized on an ethidium bromide-stained agarose gel and the bands digitized using a UVP BioImaging Analysis System with VisionWorksLS™ 5.5 software.
OPCs were isolated and cultured in defined culture media (Agresti et al., 1996) plus PDGF (10ng/ml) and bFGF (10ng/ml). Twenty four hour after plating, the cells were transiently transfected with a combination of three different Stealth RNAi™ siRNA duplexes (Invitrogen) specific for TRPC1 (see Table II for siRNA duplex sequences). Briefly, 6pmol of each siRNA duplex were mixed with Lipofectamine™ RNAiMAX (Invitrogen) and the mixture was placed on 35-mm Petri dishes containing 80% confluent OPCs. OPCs were then further cultured for 48h in defined culture media (Agresti et al., 1996) plus PDGF (10ng/ml) and bFGF (10ng/ml) before used for mRNA and protein determinations and Ca++ imaging experiments. Control cells were exposed to Lipofectamine™ RNAiMAX alone using the same protocol.
Twenty four hour pulses of 10μM bromo-deoxyuridine (BrdU) (BD Pharmingen) were performed under different experimental conditions. After each BrdU pulse, cells were fixed in 4% paraformaldehyde and immunostained in order to determine the number of positive cells. After treatment with 6N HCl and 1% Triton X-100 to denature nuclear DNA, the cells were incubated in 0.1M sodium borate (in PBS and 1% Triton X-100) for 10min. Immunocytochemistry was done using an anti-BrdU antibody (1/1000; BD Pharmingen) and an anti-NG2 (1/100; Chemicon International Inc.) with the corresponding fluorescent secondary antibodies. The percentage of BrdU+ cells was estimated on the basis of the total number of NG2+ cells.
To examine cell cycle time (Tc), mixed glial culture of GFP labeled OPCs were used. These experiments were performed using a double transgenic mouse created by breeding the JOE mouse with a line expressing GFP under control of the PLP promoter (Mallon et al., 2002). Mixed glial cultures were prepared as described above (Primary Cultures of Cortical Oligodendrocytes). These cultures were incubated in a stage top chamber with 5% CO2 at 37°C, which was placed on the stage of a spinning disc confocal inverted microscope equipped with a motorized stage, an atmosphere regulator, and shutter control. Fluorescent field images were obtained with a specific GFP filter at 6min intervals. Individual clones of OPC~GFP were followed for a period of 48h beginning at 4 and 6 days in vitro before the shake-off (div). In these time-lapse experiments, ~30 clones were analyzed per experimental condition.
SlideBook™ 4.1 (SlideBook™ 4.1, Intelligent Imaging Innovations) was used in the analysis of videomicroscopic image sequences. The SlideBook software allow an investigator to cycle back-and-forth through the movie files frame-by-frame (minute-by-minute), facilitating the accurate determination of event time. To quantitatively analyse the dynamics of cell division, 120 cytokinetic events were randomly selected from movies at 4 and 6div. Cell proliferation was assessed by calculating the average cell cycle time (Tc) (time between birth cytokinesis and division cytokinesis) in different OPC~GFP clones. Tracking of cells was performed by visual observation of image sequences as described above. In some cases the cells were semiautomatically followed in SlideBook by attaching a number to the cell, which was propagated from frame to frame. Tracking was performed forwards and backwards in time from each identified cytokinesis events to maximize the number of lineally related cytokineses identified.
Calcium imaging acquisitions of GFP-labeled OPCs were performed on living slices cut in coronal orientation at postnatal day four (P4), as described elsewhere (Kakita and Goldman, 1999). Briefly, mice were anesthetized with isofurane, after which brains were rapidly removed and stored in ice cold bicarbonate buffered solution gassed with 95% O2 and 5% CO2 (Fulton et al., 2010). Coronal and sagittal slices were cut at 300μm thickness on a vibratome. Brain tissue was kept in ice-cold bicarbonate soltion during these procedures. The slices were then cultured with Eagle's Basal Medium with Earle's salts (BME; Invitrogen) supplemented with 18.6mM NaHCO3, 1% BSA (fraction 5; Sigma), 5μg/ml insulin, 5μg/ml transferrin, 5μg/ml sodium selenite (Sigma), 20U/ml penicillin–streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen) and 27mM glucose. After this, brain slices were ready for calcium imaging studies.
Methods were similar to those described previously (Colwell, 2000; Michel et al., 2002; Paz Soldan et al., 2003). Briefly, a cooled CCD camera (Hamamatsu ORCA-ER) was added to the Olympus spinning disc confocal microscope to measure fluorescence. To load the dye into cells, primary cultures of oligodendrocytes and brain slices were washed in serum and phenol red-free DMEM and incubated for 45 min at 37°C, 5% CO2 in the same media containing a final concentration of 4μM Fura-2 (AM) (TefLabs) plus 0.08% Pluronic F-127 (Molecular Probes), then washed four times in DMEM and stored in DMEM for 0-1 hours before being imaged (Paz Soldan et al., 2003). Resting calcium levels were made in serum-free HBSS containing 2mM Ca++ but no Mg++. Other measurements were made in HBSS. Calcium influx and resting Ca++ levels were measured on individual cells, and the results were pooled from five separate cells preparations for each condition. The fluorescence of Fura-2 was excited alternatively at wavelengths of 340 and 380 nm by means of a high-speed wavelength-switching device (Lambda DG-4; Sutter Instruments). Image analysis software (SlideBook™ 4.1, Intelligent Imaging Innovations) allowed the selection of several “regions of interest” within the field from which measurements are taken. To minimize bleaching, the intensity of excitation light and sampling frequency was kept as low as possible. In these experiments, measurements were normally made once every 2s.
Free [Ca++] was estimated from the ratio (R) of fluorescence at 340 and 380 nm, using the following equation: [Ca++] = Kd × slope factor × (R - Rmin)/(Rmax - R) (Grynkiewicz et al., 1985). The Kd was assumed to be 140nM, whereas values for Rmin and Rmax were all determined via calibration methods. An in vitro method (Fura-2 Ca++ imaging calibration kit, Molecular Probes) was used to make estimate values. With this method, glass coverslips were filled with a high-Ca++ (Fura-2 plus 10mM Ca++), a low-Ca++ (Fura-2 plus 10mM EGTA), and a control solution without Fura-2. Each solution also contains a dilute suspension of 15μm polystyrene microspheres to ensure uniform coverslip/slide separation and facilitate microscope focusing. The fluorescence (F) at 380 nm excitation of the low Ca++ solution was imaged, and the exposure of the camera adjusted to maximize the signal. These camera settings were then fixed, and measurements were made with 380 and 340 nm excitation of the three solutions. Rmin = F340nm in low Ca++/F380 in low Ca++; Rmax = F340 in high Ca++/F380 in high Ca++; Sf = F380 in low Ca++/F380 in high Ca++.
Enriched oligodendrocytes were prepared as described by Amur-Umarjee et al. (1993). Cells plated onto poly-D-lysine-coated 12mm glass coverslips were transfected using the Lipofectamine 2000 (Invitrogen). Briefly, 1μg of plasmid DNA (full length golli-J37 clone in pEGFP-N3) (Reyes and Campagnoni; 2002), was used to transfect 4×104 cells/coverslip. While the DNA was complexing, the cells were washed for 5min with serum free media. The complexed DNA mixture was then applied to the coverslips and incubated at 37°C, 5%CO2 for 6 hours. The samples were washed with media supplemented with 10% FBS and subsequently incubated in defined culture media (Agresti et al., 1996) plus PDGF (10ng/ml) and bFGF (10ng/ml) for 2 days prior to use. Correlation studies of golli-GFP with Ca++ influx kinetics were done by confocal microscopy using an Olympus spinning disc confocal equipment. Time lapse digital images were analyzed using the Pearson's correlation coefficient (Rr) facility, which is provided by Image analysis software (SlideBook™ 4.1, Intelligent Imaging Innovations). Rr is a well-defined and commonly accepted means for describing the extent of correlation between image pairs. It is a value ranging between -1.0 and 1.0, a -1.0 value signifies no correlation, while a value of 1.0 signifies perfect correlation (Manders et al., 1993).
Data is presented as mean ± S.E.M. unless otherwise noted. For Fura-2 and cell cycle time experiments statistical comparison between different experimental groups was performed by analysis of covariance ANCOVA. Measurements of the percentage of positive cells (Phospo-Histone H3 and BrdU) were carried out using Student's paired t-test, in which p<0.05 was defined as statistically significant.
We have found that the golli proteins function as new and novel modulators of intracellular Ca++ levels in oligodendrocyte progenitor cells (OPCs), T-cells and probably neurons (Feng et al., 2006; Paez et al., 2007, 2009a, b). To characterize further the Ca++ channels regulated by golli we examined the contribution of store-operated Ca++ entry in OPCs cultured from golli KO and JOE mice. Primary cultures of OPCs were first loaded with a membrane-permeable form of the Ca++ indicator dye Fura-2, before treatment with several agonists to activate different mechanisms that generate Ca++ signaling in OPCs. Initially we sought to examine the effect of golli proteins on Ca++ efflux from intracellular stores. Ca++ responses to caffeine and thapsigargin were studied in golli KO and JOE OPCs. Caffeine (2mM) and thapsigargin (100nM) both elicited a large and persistent increase in intracellular Ca++ in the presence of normal extracellular Ca++ (2mM) (Figure 1A). This response was significantly higher in JOE OPCs and lower in golli KO cells. The effect of extracellular Ca++ on caffeine and thapsigargin responses was examined in parallel cultures by perfusion with caffeine or thapsigargin in Ca++ free medium. This evoked a slow-onset transient elevation of intracellular Ca++ in all the OPC genotypes. This response was typically small in amplitude and under this experimental condition we did not find any appreciable difference between golli KO and JOE OPCs compared to genotype control cells (Figure 1A). Subtraction of responses in Ca++ free medium from those in normal extracellular Ca++ revealed a slow-onset extracellular Ca++ dependent response to caffeine and thapsigargin which began after most of the store depletion (Ca++ response in Ca++ free medium) had occurred. The usual explanation for this phenomenon is activation of Ca++ entry across store-operated Ca++ channels (SOCCs) in the plasma membrane, subsequent to store depletion (Putney, 1993; Simpson et al., 1997). To examine the contribution of SOCC entry, Fura-2 loaded OPCs were first pre-treated with thapsigargin in the presence of zero Ca++ to deplete intracellular stores, and then re-exposed to a Ca++ containing medium to trigger Ca++ influx via SOCCs. Re-exposure to Ca++ containing medium triggered a significantly larger increase in the Fura-2 signal in JOE cells in comparison to responses in genotype controls (Figure 1B). Interestingly, opposite effects were observed in golli-deficient OPCs (Figure 1B). Further pharmacological experiments in JOE cells treated with thapsigargin in zero Ca++ medium demonstrated that the increased Ca++ response was insensitive to Cd++ and verapamil (Figure 2D) and was abolished in the presence of the SOCCs inhibitors La++ and 2-APB (Figure 2B and D) confirming that this Ca++ influx is not carried by voltage-operated Ca++ channels, and more likely reflects influx via SOCCs. Similar results were found using MRS-1845, a specific blocker of store-operated Ca++ entry that does not activate intracellular Ca++ release (Figure 2C and D).
Taken together these data indicate an important role for golli proteins in the regulation of SOCC mediated Ca++ influx. As a whole, these results suggest that Ca++ release from ER stores might not be affected by golli, but rather, that golli regulates Ca++ influx after store depletion, probably mediated by SOCCs.
We performed in situ experiment using live tissue sections to examine Ca++ influx in GFP labeled OPCs in golli KO and JOE mice. These experiments were performed using double transgenic mice created by breeding the golli KO and JOE mice with a line expressing GFP under control of the PLP promoter (Mallon et al., 2002). In these mice GFP expression provides a convenient marker for cells in the oligodendroglial lineage, thus facilitating experiments aimed at imaging OPCs in live tissue slices. Initially, we focused our in situ measurements of OPC in slice preparations containing the lateral ventricle subventricular zone (SVZ) since this region have been well studied as a source of OPCs. Our goal in these experiments was to confirm the in vitro data in live preparations with respect to SOCCs activity in OPCs lacking golli or overexpressing golli. Recordings were made at postnatal day 4 (P4). To examine in situ SOCC Ca++ entry Fura-2 loaded tissue slices were first pre-treated with thapsigargin (Tg) in the presence of zero Ca++ to deplete intracellular stores, and then re-exposed to a Ca++ containing medium to trigger Ca++ influx via SOCCs. Re-exposure to Ca++ containing medium triggered a significantly larger increase in the [Ca++]int in JOE SVZ cells in comparison to controls OPCs (Figure 3A, B and C). Furthermore, pharmacological experiments demonstrated that the increased Ca++ response seen in JOE cells was abolished in the presence of 2-APB and MRS-1845, specific SOCCs blockers (Figure 3D). As expected, golli KO cells exhibited Ca++ signaling following stimulation with thapsigargin in the presence of zero Ca++, but these Ca++ signals were significantly weaker than that of the control cells (Figure 3E). Additional experiments were made at postnatal day 7, 14 and 21 (P7, P14 and P21) allowing analysis of the developmental regulation of store-operated Ca++ influx in OPCs. In agreement with our previous findings, Ca++ uptake after store depletion was significantly greater in golli-overexpressing cells located in the SVZ at all time points tested (Figure 3F). However, no significant differences between JOE and control cells were found in the corpus callosum (CC) of 21 day old mice (Figure 3F), suggesting that golli modulation of SOCCs may play a role during the early stages of OPC maturation. Taken together, and in agreement with our previous data from cultured OPCs, these in situ results uncover a key role for golli protein in the regulation of SOCC mediated Ca++ influx in SVZ and CC OPCs.
Oligodendrocytes express canonical transient receptor potential (TRPC) channels that have been implicated in mediating store-operated Ca++ entry (Fusco et al., 2004: Weerth et al., 2007). Using an antibody against TRPC1 we were able to block the function of TRPC1 channels and demonstrate their involvement in golli-evoked Ca++ entry in cultured OPCs. We used an antibody against TRPC1 that was designed to bind to amino acid residues 557–571 in the proposed pore forming region of human/mouse TRPC1 and block the functioning of the channel (Wang et al., 1999). This antibody has been used to block TRPC1 current (Kim et al., 2003) and TRPC1 mediated Ca++ influx (Antoniotti et al., 2002; Xu and Beech, 2001). Therefore, this antibody can specifically block the function of TRPC1 containing channels and determine the extent of their involvement in SOC entry. Cultured OPCs were pre-treated with thapsigargin in the presence of zero Ca++ to deplete intracellular stores, but in this case cells were pre-incubated with TRPC1 antibody (10 μg/ml for 1h) prior to Ca++ re-addition to allow the antibody sufficient time to bind and block the channels. Pre-incubation with the antibody caused a significant decrease in the measured amount of store-operated Ca++ entry compared to JOE cells that were not incubated with TRPC1 antibody (Figure 4A and B). This decrease in Ca++ entry was of a similar proportion to that seen using SOCC blockers (2-APB and MRS-1845) indicating that TRPC1 is involved in mediating store-operated Ca++ uptake in golli-overexpressing OPCs. It also represents the first time this channel has been shown to be functional in OPCs. Furthermore, the effect of anti-TRPC1 was abolished by preincubation of the antibody with the antigen peptide (+Pept), underlying the specificity of the primary antibody (Figure 4C and D).
To determine which TRPC homologs are expressed in OPCs, semi-quantitative RT-PCR reactions were run utilizing primers specific for individual TRPC homologs (Table I). As shown in Figure 5A, OPCs express mRNA for TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC6. Importantly, no significant differences in TRPC expression were observed between control and golli-overexpressing OPCs (Figure 5A). To confirm the role of TRPC1 in mediating SOC uptake in golli-overexpressing OPCs, we used an siRNA approach to selectively suppress mRNA levels for TRPC1. Twenty four hours after plating, OPCs were transiently transfected with a combination of three different siRNA duplexes specific for TRPC1 (Table II). Two days later, the mRNA levels were monitored using semiquantitative RT-PCR methods. The data in Figure 5B illustrate the selective suppression of TRPC1 mRNA by expression of siRNA specific for that TRPC homolog. Thus, levels of TRPC1 mRNA could be selectively suppressed by ~90%. Western blots were performed to confirm that TRPC1 protein levels were also suppressed by siRNA expression. The Western blots in Figure 5C illustrate that the specific reduction in mRNA for TRPC1 is accompanied by a reduction of ~95% in TRPC1 protein levels (Figure 5C). The effect of suppression of TRPC1 homolog on the level of SOC influx was then determined by monitoring thapsigargin stimulation of Ca++ entry in JOE cells at 4 and 5 days in vitro (4/5DIV). The suppression of TRPC1 protein levels produced an ~80% inhibition of thapsigargin-stimulated Ca++ influx at both 4 and 5DIV (Figure 6), indicating that TRPC1 is heavily involved in mediating SOC entry in golli-overexpressing OPCs.
Proliferation of OPCs is clearly important for normal myelination as well as for remyelination in demyelinating diseases. Our recently published data (Paez et al., 2009a) indicate that golli enhances the proliferative activity of PDGF, a potent mitogen for OPCs (Noble et al., 1988; Raff et al., 1988; Calver et al., 1998).
To investigate the physiological relevance of TRPC1 expression in OPCs, we used an siRNA approach to knock down the expression of TRPC1 channel and measure proliferation and cell cycle time to determine if TRPC channels are involved in golli-induced OPC proliferation. Twenty four hours after plating OPCs were transiently transfected with a combination of three different siRNA duplexes specific for TRPC1 (Table II). Two days later, the medium was changed and the cells were grown in the presence of PDGF (20ng/ml) for 48h. Since actively proliferating cells duplicate their DNA content we labeled proliferating OPCs from control and JOE mice with the thymidine analogue bromodeoxyuridine (BrdU). Twenty four hour pulses of 10μM BrdU were performed at 3 and 4 days in vitro (3/4DIV) (Figure 7A). After each BrdU pulse proliferating progenitors were identified by double immunofluorescence for BrdU and NG2 and the relative number of NG2+/BrdU+ cells was quantified in each cell population (Figure 7B). In agreement with our previous findings (Paez et al., 2009a), at 4DIV and 5DIV the average number of proliferating cells in the JOE population was significantly higher than that of the control group (Figure 7C). However, after TRPC1 knockdown the average percentage of proliferating OPCs in the golli-overexpressing cell population was found to be significant lower compare to non-transfected JOE cells and additionally no significant differences between JOE and control cells were found (Figure 7C).
The BrdU cell proliferation results in the JOE OPC population were confirmed using an anti Phospho-Histone-H3 (Ser28) antibody as a marker of mitotic cells. Phosphorylation of histone H3 at Ser10 and Ser28 is very important for chromosome condensation and segregation during mitosis (Wei et al., 1999). The phosphorylation of histone H3 at Ser28 occurs in chromosomes predominantly during early mitosis and coincides with the initiation of mitotic chromosome condensation in various mammalian cell lines (Goto et al., 1999). At 4DIV and 5DIV, in the presence of 20ng/ml PDGF, there were more H3+ cells in the golli overexpressing cell population than in the control cells (Figure 7D), but 24 and 48h after TRPC1 knockdown (4/5DIV) no significant differences between groups were found (Figure 7D).
Additionally, we measured the cell cycle times (Tc) and the percentage of mitotic OPCs of the JOE and control cells by performing time lapse imaging in mixed glial cultures using GFP labeled OPCs as we have done previously (Paez et al., 2009a). We imaged individual clones of GFP-labeled OPCs (OPC~GFP) in the presence of an astrocyte monolayer, a known source of growth factors such as PDGF and bFGF (Hicks and Franklin, 1999). These experiments were performed for a period of 48 hours at 4 and 6 days in vitro before the shake-off (div) (Figure 8A). In these time-lapse experiments the role of TRPC1 on cell proliferation was assessed by calculating the average cell cycle time (Tc) in different OPC~GFP clones using an anti-TRPC antibody to block the functioning of TRPC1 channels. Cell cycle time was defined as the period between the cytokinesis at which a cell was generated and when the cell divided, giving birth to two daughter cells. The time at which cytokinesis occurred was considered to be the first appearance of a distinct border between two daughter cells in a videomicroscopic image sequence (see Materials and Methods). Examples of cytokinetic events in GFP-labeled OPCs from JOE mice grown for 4 days on an astrocyte monolayer are shown in Figure 8B. At 4div control OPCs showed a Tc of ~24h whereas the JOE cells showed a Tc of ~18h. In contrast, the proliferation rate of JOE OPCs was increased to a Tc ~31hr at 4div and ~34hr at 6div in the presence of TRPC1 antibody used to block TRPC1 mediated Ca++ influx (Figure 8C). Furthermore, as shown in Figure 8D, the percentage of cycling OPCs during the first 12 hours of the time-lapse experiment was significantly higher in the JOE cell population suggesting that the fraction of proliferating OPCs is increased by golli overexpression.
These results indicated, by several independent measures, that golli promoted OPC proliferation in the presence of growth factors, i.e. an increase in the number of BrdU+ cells, a decrease in the cell cycle time and an increase in the number of cells in S phase. This effect disappeared when TRPC1 expression was suppressed by specific siRNAs, or when the channel was blocked by specific antibodies, indicating that golli influences OPC proliferation by modulating Ca++ influx through TRPC1. Additionally, these experiments are the first to demonstrate that extracellular Ca++ uptake through TRPC1 is an important component in the mechanism of OPC proliferation.
Using digital confocal analysis we found TRPC1 expression throughout the cell body and processes of OPCs (Figure 9A). A number of circular “hot-spots” were found in the cell body and along OPC processes. TRPC1 expression was highly varied such that certain sites along the processes were found to possess markedly higher levels of TRPC1 than surrounding regions (Figure 9A). We then examined whether these patches of TRPC1 expression along OPC processes were associated with golli. Double immunofluorescence for golli and TRPC1 revealed that cell regions with TRPC1 accumulations tended to co-localize with high levels of golli expression along OPC processes (Figure 9B). To examine whether the sites of high density golli protein distribution were related to the sites of high SOC influx kinetics, we performed correlation experiments between golli-GFP expression and Ca++ levels in OPC processes. We stimulated OPCs overexpressing golli-GFP with thapsigargin in the presence of zero Ca++ to deplete intracellular stores, and then re-exposed to a Ca++ containing medium to trigger Ca++ influx via SOCCs. The kinetics of the resulting Ca++ influx was measured in serial z sections along cell processes. The pattern of golli-GFP fluorescence was then imaged, and the intensities were measured within the same serial sections of the cell in which the Ca++ influx kinetics were measured. The resultant profile was then compared with the profile of Ca++ influx kinetics using correlation analysis (see Materials and Methods). In the OPC processes the Pearson's Rr was 0.912 ± 0.034 (n = 25), indicating that there is a significant overlap of golli-GFP and SOC influx sites in OPCs processes. A plot of the local Ca++ amplitudes against the length of the process, together with the intensity of golli-GFP measured in the same cellular sites, showed that the regions with high intensity golli-GFP corresponded closely with the regions of the process where the local peak Ca++ amplitudes were highest (Figure 9C and D). These studies indicated that golli is probably associated with TRPC1 at discrete cellular regions. Such high-density golli and TRPC1 expression at these sites suggest that these proteins will cooperatively to regulate Ca++ uptake in these cellular microdomains.
In many cells, including OLs, an important mechanism for stimulating Ca++ influx is through SOCCs, located in the plasma membrane (Simpson and Russell, 1997). The SOCCs are stimulated to take up Ca++ from increases in internal Ca++ concentrations through depletion of the internal stores, primarily from the ER. Release occurs via IP3 (inositol 1,4,5-triphosphate) or ryanodine receptors, and Ca++ is subsequently re-sequestered into stores via sarcoplasmic-endoplasmic reticulum Ca++ ATPases (SERCAs) (Berridge, 1993; Simpson et al., 1995). In many cell types, SERCA inhibition leads to elevation of cytosolic Ca++ secondary to leakage of Ca++ from stores. Thapsigargin is used to inhibit SERCA pumps in cultured cells, and it has been demonstrated that SERCA-mediated store depletion activates store-operated Ca++ entry in glial cells (Simpson and Russell, 1997). The results presented here show that inhibition of SERCA pumps by thapsigargin or ryanodine receptor activation by caffeine evokes significant Ca++ release from intracellular stores and Ca++ entry after store depletion in OPCs. Our data clearly indicated that under these pharmacological treatments golli promoted a significant activation of SOCCs and Ca++ uptake both in vitro (cultured OPCs) and in situ (slice preparation). Importantly this increased Ca++ influx in JOE cells was totally abolished by the specific SOCC inhibitors 2-APB and MRS-1845 confirming that this influx is carried by SOCCs.
In addition to entry through voltage-gated calcium channels and release from intracellular stores, members of the transient receptor potential (TRP) channel superfamily present an alternative mechanism for Ca++ entry and regulate multiple processes in the developing and mature nervous system. TRP channels are a family of 28 nonselective cation channels, and all except TRPM4 and TRPM5 display varying degrees of calcium permeability (Nilius et al., 2007). Members of the canonical TRP (TRPC) family are involved in SOC entry (Zitt et al., 2002; Smyth et al., 2006; Liao et al., 2007), which is thought to be an essential component in establishing intracellular Ca++ concentrations (Foskett and Wong, 1994; Woods et al., 1986). The expression of TRPC1 and TRPC3 proteins have been reported in OLs (Fusco et al., 2004: Weerth et al., 2007), and we have found by RT-PCR that TRPC1-6 are expressed in cultured OPCs.
In the mature nervous system TRP channels play important roles in the processing of sensory information (Clapham, 2003) and fear-related learning and memory (Riccio et al., 2009), and defects in particular channels underlie models of neurodegeneration such as cerebellar ataxia (Becker et al., 2009). During early development members of the TRP channel families modulate neural progenitor proliferation (Fiorio Pla et al., 2005), whereas at later stages specific members of the TRPC family have been shown to both positively and negatively regulate neurite extension (Greka et al., 2003; Wu et al., 2008), likely because of the activation of Ca++-dependent processes (Gomez and Zheng, 2006).
In this study we demonstrated that a large portion of SOC entry in cultured OPCs is likely mediated through TRPC1-containing channels in the plasma membrane. We demonstrated that Ca++ entry via TRPC1 plays an important role in oligodendrocy Ca++ dynamics. We found that after thapsigargin treatment in the presence of zero Ca++ re-exposure to a Ca++ containing medium caused an increase in cytoplasmic Ca++, which was reduced by ~80% when TRPC1 was blocked by specific antibodies or siRNA expression. This may indicate that Ca++ channels containing TRPC1 are dynamically refilling the intracellular stores during transient cytoplasmic Ca++ increases in OPCs. On the other hand, when TRPC1 was blocked in cells overexpressing-golli, a significant reduction in Ca++ uptake occurred after store depletion suggesting an involvement of TRPC1 channels in the SOC influx induced by golli proteins. Taken together the data presented here provide the first evidence for an involvement of a channel belonging to the TRPC family in store-operated Ca++ influx in oligodendroglial cells.
A previous study has shown that TRPC1 can hetero-tetramerize with other TRPCs expressed during brain development to form complexes containing TRPC1/TRPC3/TRPC6 and TRPC1/TRPC4/TRPC5, which give rise to channels with different properties (Strubing et al., 2003). We used RT-PCR to detect the presence of TRPC channels other than TRPC1 in primary culture of OPCs and found the presence of mRNAs for TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC6. Interestingly, no significant differences in the TRPC profiles were observed between control and golli-overexpressing OPCs. We found an 80% reduction in OPC SOC uptake when TRPC1 was knocked-down indicating that TRPC1 is the most important SOCC isoform in OPCs, but our results do not exclude a possible involvement of other TRPC homologs during store-operated Ca++ uptake in OPCs. Future studies will address whether other TRPC homologs are involved in OPC Ca++ dynamics.
Simpson and coworkers (1997) have been able to localize microdomains of Ca++ activity within OL processes. They have identified microdomains within OPC processes indicating the infiltration of the ER into the OPC processes. In many instances the ER lies in very close proximity to the plasma membrane of the processes where Ca++ channels are located. Thus, components of the Ca++ release mechanism from internal stores can be in close proximity to SOCCs located on the plasma membrane surface. Such a structural arrangement can facilitate the Ca++ waves along processes as well as initiate local Ca++ responses in the processes. Recently Weerth et al (2007) have been able to identify signaling microdomains associated with lipid raft-like membrane specializations in glial cells. They also identified putative SOCC components and IP3R2 receptors suggesting close interaction between Ca++ signaling sites on the ER with those on the plasma membrane. Thus, Ca++ signaling components are located within OPC processes and they may play an important role in process elongation, retraction and migration.
Using high resolution spatiotemporal analysis we showed that SOC influx in OPCs overexpressing golli initiated with different latencies at discrete cellular locations. Specifically, regions of the OL process with elevated golli levels consistently displayed higher amplitude Ca++ signals than were found in surrounding areas. We hypothesize that golli proteins are needed at wave-amplification sites along OPC processes to regulate the activity of SOCCs located in close apposition to ER. The principal role of high golli levels may be to maintain locally high levels of Ca++ within the ER and thereby contribute to elevated Ca++ influx at wave-amplification sites.
A recent independent study has shown binding of golli proteins to STIM1, the master regulator of SOCCs (Walsh et al., 2010). Walsh and colleagues propose that golli functions to regulate SOC influx via a direct interaction with STIM1, but the exact molecular mechanism underlying this interaction has yet to be defined.
Proliferation of OPCs is clearly important for normal myelination as well as for remyelination in demyelinating diseases. This study indicates that golli overexpression increases the proliferation rate of OPCs as assessed by several parameters including a decrease in the cell cycle time. Our results indicated that endogenous golli expression can modulate OPC proliferation through SOCCs. More importantly, this effect disappeared when TRPC1 expression was suppressed by specific siRNAs or blocked by antibodies, indicating a key role for TRPC1 in OPC proliferation.
Jacobs and colleagues (2009) have previously shown that golli overexpression causes a significant delay in OPC maturation, with accumulation of significantly greater numbers of premyelinating OLs that fail to myelinate axons during the normal myelinating period. Although an increase in proliferating OPCs was not detect in this study, a greater number of OPCs were found in the JOE brain around the second postnatal week suggesting that increased proliferation in the JOE mice may occur at early developmental stages.
Our recently published data indicate that golli enhances the proliferative activity of PDGF and that PDGF induced a biphasic transient increase in OPC intracellular Ca++ (Paez et al., 2009a). The first phase occurred in the absence of external Ca++ and was completely abolished by thapsigargin pretreatment, indicating that the first phase was due to Ca++ release from intracellular stores. The second phase was completely abolished by store-operated Ca++ channel inhibitors indicating that these channels were involved in maintaining the elevated intracellular Ca++ concentration characteristic of this phase. The store-operated Ca++ influx was higher in JOE cells suggesting that the mechanism responsible for the effect of golli on OPC proliferation was mediated through an increase in store-operated Ca++ influx. In this paper, we identified the channel responsible for this Ca++ uptake increase in golli-overexpressing cells and we revealed for the first time that extracellular Ca++ uptake through TRPC1 is an important component in the mechanism of OPC proliferation. Recently, Cuddon et al. (2008) reported that PDGF activates store-operated Ca++ entry in neuronal precursor cells, a finding that supports our data indicating that SOCCs are essential for neural cell division. Furthermore, several studies have shown that TRPC1 plays a role in Ca++ influx and smooth muscle cell proliferation (Golovina et al., 2001; Sweeney et al., 2002a, b) and mediates Ca++ influx activated by basic fibroblast growth factor (bFGF; FGF-2) in endothelial cells (Antoniotti et al., 2002).
We have found that golli regulates different aspects of OPC function through distinct routes of Ca++ entry. Golli regulation of SOCCs influences OPC proliferation, and its modulation of voltage-operated Ca++ channels (VOCCs) influences OPC process extension/migration (Paez et al., 2007; 2009b). How can these different routes of Ca++ influx modulates different aspects of OPC development? The answer must lie in the pattern of Ca++ signals in time and space evoked by the activation of different Ca++ channels. Ca++-sensitive sites that evoke process outgrowth and migration are located close to VOCCs (Bolsover, 2005), but TRPC1 activation, as occurs during store depletion in OPCs, will cause a significant increase of Ca++ concentration within the ER activating cell proliferation.
In summary, the findings described in this work suggest a key role for golli proteins in the regulation of OPC Ca++ homeostasis. This involvement in Ca++ signaling in the myelin forming cells of the CNS indicates that golli proteins can have a significant effect on the ability of OPCs to respond to remyelination cues in clinical situations resulting in demyelination. As such golli proteins represent important molecules that may be of direct relevance to demyelinating diseases.
This investigation was supported (in part) by a Postdoctoral Fellowship from the National Multiple Sclerosis Society FG1723A1/1 (PMP) and National Multiple Sclerosis Society Grant RG4205-A-8 (ATC).
This paper is dedicated to the memory of Celia W. Campagnoni who passed away on June 17th, 2010.