Granule neurons in cerebellum express distinct splice variants of the inositol trisphosphate receptor that are modulated by calcium

Joseph Y. Choi, Carol M. Beaman-Hall, and Mary L. Vallano

Department of Neuroscience and Physiology, State University of New York Upstate Medical University, Syracuse, New York 13210

Submitted 15 December 2003 ; accepted in final form 27 May 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Primary cultures of granule cells (GC) from rat cerebellar cortex were used to determine whether bioelectric activity, via a Ca2+/calmodulin-dependent kinase (CaMK) signaling cascade, modulates expression and exon selection in the inositol trisphosphate receptor type 1 (IP3R1). IP3R1 contains or lacks three exons (S1, S2, and S3) that are regulated in a regionally and temporally specific manner. The neuronal, or long, form of IP3R1 is distinguished from peripheral tissues by inclusion of the S2 exon. Although previous studies indicated that IP3R1 are undetectable in the cerebellar granular layer in vivo, receptor protein and mRNA are induced in cultured GC grown in medium supplemented with 25 mM KCl or NMDA, two trophic agents that promote long-term survival, compared with GC grown in 5 mM KCl. IP3R1 induction in response to 25 mM KCl or NMDA is attenuated by coaddition of voltage-sensitive calcium channel or NMDA receptor antagonists, respectively. Actinomycin D, CaMK, and calcineurin antagonists likewise suppress induction. Unlike the major variants of IP3R1 in Purkinje neurons, which lack S1 and S3, GC grown with trophic agents express mRNA containing these exons. Both neuronal types contain S2. Evidence obtained using mutant mice with Purkinje cell lesions, laser-microdissected GC neurons from slices, and explant cultures indicates that GC predominantly express the S1-containing variant of IP3R1 in vivo.

calmodulin; exon selection


INOSITOL TRISPHOSPHATE RECEPTORS (IP3R) are encoded by three genes (IP3R1, IP3R2, and IP3R3) that differ in their affinities for IP3 (33) and mediate the release of Ca2+ from intracellular stores in response to ligands that stimulate the hydrolysis of membrane phospholipids (for review, see Ref. 43). IP3R1 predominates in neurons and can include sequences encoded by three alternatively spliced exons (S1, S2, and S3) that are regulated in a regionally and temporally specific manner (30), although the unique functions of the splice variants are still under investigation. The receptor is most enriched in Purkinje neurons of the cerebellar cortex, but significant amounts of mRNA and protein are also present in hippocampus, cerebral cortex, nucleus accumbens, caudate putamen, and deep cerebellar nuclei (9, 32). Although previously available methods of assay rendered IP3R1 undetectable in the granular layer of cerebellar cortex (24, 30, 32, 40), work done at our laboratory (35) previously demonstrated that primary cultures of granule cells (GC) grown in medium supplemented with elevated KCl (to promote long-term survival; see Ref. 11) express significant amounts of immunoreactive protein compared with those grown in medium containing 5 mM KCl. Genazzani et al. (12) verified this observation and further showed an increase in mRNA encoding the long, i.e., S2-containing, form and attenuation of depolarization-dependent IP3R1 induction by FK506 (also called tacrolimus) and cyclosporin A (CsA), agents that inhibit calcineurin (also called protein phosphatase 2B). Studies in hippocampal neurons also point to regulatory roles for depolarization and calcineurin in the induction of IP3R1 mRNA and suggest that the transcription factor nuclear factor of activated T cells (NF-ATc) mediates this effect (13). Taken together, these data suggest that depolarization-dependent activation of calcineurin leads to hypophosphorylation and nuclear translocation of NF-ATc and subsequent induction of IP3R1 in GC and other neurons. However, the IP3R gene is regulated by multiple transcription factors, including those that synergize with NF-ATc as well as those that are NF-ATc independent (7, 16). In particular, the effects of Ca2+/calmodulin-dependent kinase (CaMK)-dependent signaling cascades on IP3R1 expression and splicing have not been examined. Perhaps more important, the significance of IP3R1 expression in cultured GC, given its apparent paucity in vivo, has not been considered. These questions prompted the present investigation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Sprague-Dawley neonatal rats were purchased from Taconic Farms (Germantown, NY). Purkinje cell degeneration (pcd) mice and normal littermates were purchased from Jackson Laboratories (Bar Harbor, ME). All procedures involving the use of animals were approved by the institutional review committee in accordance with government guidelines. KN-62, CsA, and (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-iminemal-eate (MK-801) were purchased from Calbiochem (La Jolla, CA). D,L-2-Amino-5-phosphonopentanoic acid (AP5) was purchased from Sigma (St. Louis, MO). FK506 was purchased from A. G. Scientific (San Diego, CA). Texas red-conjugated secondary antibody of multiple-labeling grade was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Antibodies specific for type I IP3R, raised in rabbit and affinity purified (49), were generously provided by Dr. R. J. H. Wojcikiewicz (SUNY Upstate Medical University). The RNeasy Mini Kit was purchased from Qiagen (Valencia, CA). Reagents for RT, including Moloney murine leukemia virus (MMLV), were purchased from Promega (Madison, WI). Thermus aquaticus polymerase (AmpliTaq Gold or AmpliTaq), 10x PCR buffer, and MgCl2 were purchased from Applied Biosystems (Foster City, CA). [32P]dCTP was obtained from NEN (Wilmington, DE). Oligonucleotide primers were purchased from Genset (La Jolla, CA). PCR products were imaged with a Molecular Dynamics PhosphorImager obtained from Amersham Pharmacia Biotechnology (Piscataway, NJ) or on a Fluor-S from Bio-Rad (Hercules, CA). All other reagents were tissue culture or molecular biology grade, as required, and were obtained from commercial sources.

Cerebellar GC cultures. Cerebellar cortices from postnatal day 8 (P8) Sprague-Dawley rats were minced, trypsinized, and triturated to dissociate cells as described previously by investigators at our laboratory (23). Cells were plated on poly-L-lysine-coated (10 µg/ml) 35-mm dishes at a density of 2.5 x 106 cells/2 ml of medium (for RNA and protein extraction studies) or 6–6.3 x 105/0.5 ml of medium in 24-well Corning dishes containing glass coverslips (for immunocytochemical studies) and then incubated at 37°C in a humidified atmosphere of 5% CO2-air. Cultures were supplemented with 10 µM cytosine {beta}-D-arabinofuranoside 24 h after plating to inhibit nonneuronal cell, i.e., glial, proliferation. The growth medium consisted of basal Eagle's medium with Earle's salts, supplemented with final concentration (f.c.) heat-inactivated fetal calf serum (10%), gentamicin sulfate (100 µg/ml), and L-glutamine (2 mM). Where indicated, standard growth medium (containing 5 mM KCl) was augmented with 20 mM KCl or 140 µM N-methyl-D-aspartate (NMDA) plus 5 mM KCl [Note: 10 mM KCl alone does not enhance long-term survival, but when used in conjunction with NMDA, it relieves the Mg2+-dependent voltage block of NMDA receptors (NR), thereby permitting activation of NR by NMDA].

Laser microdissection. Whole cell RNA was extracted from microdissected samples of the granular and medullary layers of slices from cerebellar cortex and compared for expression of mRNA encoding IP3R1 and CaMKIV. Actin was used as a loading control. Briefly, 10-µm sections were cut on a cryostat onto Leica polyethylene terephthalate foil slides at –18°C and returned to –80°C by placement on dry ice. Individual slides were then removed from dry ice and immediately placed upside down into the slideholder platform of the Leica AS LMD application solution laser microdissection instrument (Leica Microsystems, Bannockburn, IL). At x20 magnification, unstained sections provided adequate morphology to readily distinguish the cellular laminae of the cerebellar cortex. The specific cell layer of interest was outlined (by hand) using a PC mouse. Next, with the laser beam apertures set between 9 and 13 µm, the software was used to follow the predetermined path and dissect the layer of interest directly into the RNase-free microfuge tubes containing RLT lysis buffer (Qiagen RNeasy Mini kit) previously inserted into the carrier beneath the microscope platform. Approximately three sections per animal were used to isolate the cell layers within the cerebellum, followed by extraction of RNA and analysis by RT-PCR.

Cerebellar explant cultures. With the use of the method described by Connor et al. (5), cerebella from P3 or P4 Sprague-Dawley rats were isolated, pooled, and minced into 0.3- to 1-mm pieces while bathed in basal Eagle's medium with Earle's salts, supplemented with (f.c.) heat-inactivated fetal calf serum (10%), gentamicin sulfate (100 µg/ml), and L-glutamine (2 mM) (see Ref. 5). Pieces (n = 10–20) were transferred to coverslips coated with 10 µg/ml poly-L-lysine and incubated at 37°C in a humidified atmosphere of 5% CO2-air.

Neuron survival. GC viability was examined using an assay in which 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) is converted to a blue formazan product by the mitochondria of viable cells as described previously (29). MTT was added directly to the culture medium at a final concentration of 0.5 mg/ml, and cultures were incubated at 37°C for 10 min. The solution was then aspirated and replaced with an equal volume of dimethyl sulfoxide. The absorbance of samples was quantified using a Hitachi U-2000 spectrophotometer at a wavelength of 540 nm. Our laboratory previously showed that the color yield was directly proportional to the number of viable cells, assessed by phase-contrast microscopy in conjunction with fluorescein diacetate/propidium iodide (FDA/PI; live/dead assay) (45).

Immunofluorescence/Western immunoblotting. GC or explants attached to coverslips were washed with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 min. Afterward, cultures were washed three times for 5 min each with PBS, permeabilized with 0.3% Triton-X in 2% donkey serum, 1% bovine serum albumin (BSA) in PBS for 20 min at RT, rinsed three times for 5 min each with PBS, blocked with 2% donkey serum, 1% BSA in PBS for 30 min, and then incubated overnight with antibody specific for IP3R1 protein (1:10). Subsequently, the coverslips were washed three times for 5 min each in PBS, incubated with Texas red-labeled donkey anti-rabbit IgG (1:100 dilution, 1 h at RT), washed three times for 5 min each in PBS, and then mounted with Vectashield antifade on glass slides. Photomicrographs were produced using a Zeiss microscope.

For Western immunoblotting analysis, GC were rinsed with PBS, and then protein was collected in 100 µl of homogenization buffer (63 mM Tris·HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 5 mM {beta}-mercaptoethanol, 100 µM PMSF). For IP3R1 protein detection, 15 µg/lane of whole cell homogenate was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 5% gels. After electrophoresis, proteins were transferred to nitrocellulose and incubated with 1:100 dilution of polyclonal antibody specific for IP3R1 protein (49) and then peroxidase-conjugated secondary antibodies (1:500). Immunoreactivity was detected using enhanced chemiluminescence.

RT-PCR. Whole cell RNA from GC, whole cerebellum, and hippocampus were isolated using the guanidinium isothiocyanate method (4). After the final ethanol precipitation, the RNA pellet was resuspended in sterile water. The concentration and purity of RNA were assessed with a spectrophotometer using nucleotide absorption at 260 nm and ratio at 260/280 nm (nucleotide/protein), respectively. Where indicated, RNA was isolated using a Qiagen RNeasy Mini kit. Harvested RNA was used in a RT reaction to produce cDNA for use as a template in PCR. RNA (0.1–0.2 µg) in 4 µl of sterile water was heated to 94°C for 1 min and then cooled on ice. Six microliters of a mixture containing (f.c.) MMLV RT (100 U), RT buffer (in mM: 50 Tris·HCl, pH 8.3, 75 KCl, 3 MgCl2, 10 dithiothreitol), the four dNTPs (0.5 mM each), RNasin ribonuclease inhibitor (20 U), and hexamer random primers (5 µM) were added to each RNA sample (final volume 10 µl). The RT reaction was initiated by incubation at 37°C for 60 min to promote synthesis of cDNA and terminated by heating to 95°C for 5 min and then placing the tubes on ice and diluting to 0.1–5.0 ng/µl using sterile H2O. After RT, PCR was performed in a final volume of 100 µl containing Taq buffer (10 mM Tris·HCl, pH 8.8, 50 mM KCl), 2 mM MgCl2, 0.17 mg/ml BSA, 2.5 units of Taq polymerase (AmpliTaq Gold), 2 µCi [32P]dCTP, and 0.05 mM of each dNTP, as well as 25 pmol of each oligonucleotide primer as follows: S1 upstream (5'-GGTCAACTCCGTCAACTGTAA-3': sense) and S1 downstream (5'-AGCAGGAGAAACGGGGACTAT-3': antisense); S2 upstream (5'-GGTTCATCTGCAAGCTAATAAAAC-3': sense) and S2 downstream (5'-AATGCTTTCATGGAATACTCGGTC-3': antisense); and S3 upstream (5'-TGACAACCATCTTCCCCATTAG-3': sense) and S3 downstream (5'- TGGATAGATCTCATCACGTTGC-3': antisense). Before PCR, samples were incubated at 94°C (S1 and S2) or at 96°C (S3) for 7 min. PCR was performed for 35 cycles in a PerkinElmer Tempcycler model 480 as follows: S1, 94°C for 30 s, 58°C for 30 s, and 72°C for 45 s; S2, 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min; and S3, 96°C for 1 min, 55°C for 1 min, and 72°C for 1 min. After PCR, the samples were heated to 72°C for 5 min. The amplified products were resolved on 8% (S1 and S2) or 20% (S3) polyacrylamide gels, and radioactivity was exposed on a PhosphorImager. Where indicated, variants containing or lacking the S1 exon were distinguished by restriction analysis with BpmI.

PCR of the region flanking the mouse IP3R1 S1 splice site was amplified in a final volume of 100 µl containing Taq buffer (10 mM Tris·HCl, pH 8.8, 50 mM KCl), 2 mM MgCl2, 0.17 mg/ml BSA, 2.5 units of Taq polymerase (AmpliTaq Gold), and 0.05 mM of each dNTP, as well as 25 pmol of each oligonucleotide primer: upstream (5'-GGAAAAGAAGCAGAATGAGAC-3': sense) and downstream (5'-CAGGGGAAACAGGAACTATGG-3': antisense). Before PCR, the samples were incubated at 94°C for 9 min. PCR was performed for 40 cycles in a PerkinElmer model 480 Tempcycler as follows: 94°C for 30 s, 59°C for 30 s, and 72°C for 65 s. After PCR, the samples were heated to 72°C for 5 min. The amplified products were resolved on 1.5% agarose gels and photographed under UV illumination.

PCR for actin was performed using the primers specified by Kudo et al. (22). PCR for CaMKIV was performed in a final volume of 100 µl containing Taq buffer (10 mM Tris·HCl, pH 8.8, 50 mM KCl), 2 mM MgCl2, 0.17 mg/ml BSA, 2.5 units of Taq polymerase (AmpliTaq Gold), and 0.05 mM of each dNTP, as well as 25 pmol of each oligonucleotide primer: upstream (5'-TGCAAGGTAGAAGGGACTCG-3': sense) and downstream (5'-GTACTGGAGGTGACCGAGGA-3': antisense). Before PCR, the samples were incubated at 94°C for 7 min. PCR was performed for 40 cycles in a PerkinElmer model 480 Tempcycler as follows: 95°C for 30 s, 56°C for 30 s, and 72°C for 45 s. After PCR, the samples were heated to 72°C for 5 min. The amplified products were resolved on 8% polyacrylamide gels and photographed under UV illumination.

Statistical analysis. Values are means ± SE and were analyzed for significance using Student's t-test or ANOVA followed by Tukey's test as appropriate. P < 0.05 was considered significant.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Survival-promoting agents that increase intracellular Ca2+ upregulate IP3R1. In vivo, IP3R1 expression is particularly robust in Purkinje neurons of the cerebellar cortex but is undetectable in the granular layer. Similarly, when GC are cultured in vitro in medium containing 5 mM KCl, a growth condition that does not sustain long-term survival of the majority of neurons (e.g., Fig. 1A), immunoreactive IP3R1 is low or undetectable (Fig. 1B). However, IP3R1 is readily detected in GC neurons when the growth medium is supplemented with 20 mM KCl (f.c. 25 mM) or 140 µM NMDA plus 5 mM KCl (Fig. 1B), two agents that promote long-term survival by activating Ca2+ influx through voltage-sensitive calcium channels (VSCC; inhibited by nimodipine or nifedipine) or NR (inhibited by MK-801 or AP5), respectively (e.g., Fig. 1A). Consistent with this, upregulation of IP3R1 by elevated KCl is attenuated when the VSCC antagonist nifedipine (5 µM) is added (Fig. 1C; similar results are observed using NR antagonists in GC grown in medium supplemented with NMDA; data not shown). Note that brief, overnight addition of VSCC or NR antagonists to the growth medium does not compromise GC viability, unlike longer term exposure. Note also that the observed increase in IP3R1 (per mg of homogenate protein) in GC is well below the amount observed in lysates of whole cerebellar cortex, which include Purkinje neurons (Fig. 1B).



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Fig. 1. Survival-promoting agents that increase intracellular Ca2+ upregulate inositol trisphosphate receptor type 1 (IP3R1). A: viability of granule cells (GC) grown in medium containing 5 mM KCl, 25 mM KCl ± 5 µM nimodipine (Nimo), or 140 µM N-methyl-D-aspartate (NMDA) + 5 mM KCl ± 10 µM (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-iminemaleate (MK-801). Drugs were added at 2 days in vitro (2 DIV) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was done at 7–9 DIV (n = 4). *Significantly different vs. 25 mM KCl. **Significantly different vs. NMDA. B, top: cultures were grown for 8 DIV in medium supplemented with 20 mM KCl (final concentration, 25 mM), whole cell homogenates were harvested, and 10–40 µg of homogenate protein were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose blotting membrane, and probed with an antibody against IP3R1. Bottom: adult cerebellum (AdCb), and GC cultured in medium containing 5 mM KCl, 25 mM KCl, or NMDA were grown for 5 DIV, and then whole cell homogenates were harvested for Western immunoblotting. Each well was loaded with 15 µg of protein and probed with an antibody against IP3R1. C: immunofluorescence photomicrograph comparing GC grown in 25 mM KCl ± overnight treatment with nifedipine (5 µM), added at 7 DIV. At 8 DIV, cultures were fixed and incubated with antibody against IP3R1. Similar results were observed in 3 separate cell preparations. Magnification, x63. Exposure time, 10 s.

 
Ca2+ influx through VSCCs and NR induces mRNA containing S1, S2, and S3 exons. In contrast to cerebella of adult rodents, cerebella from neonatal rodents express substantial amounts of a variant of IP3R1 mRNA that includes the S1 exon, i.e., IP3R1 S1+ (30), which is assumed to be localized in Purkinje neurons. Interestingly, the dominant expression of IP3R1 S1+ corresponds to the period of active GC migration and synaptogenesis. To explore the possibility that IP3R1 S1+ is expressed in developing GC, we examined whether exposure to 25 mM KCl or NMDA induces corresponding increases in IP3R1 mRNA containing S1+ and, if so, whether these require activation of VSCC and NR, respectively. The effects of these agents on selection of the S2 exon, as well as S3, which is not expressed in cerebellum in vivo (34), were also examined. GC were grown for 5 days in vitro (DIV) in media containing 140 µM NMDA plus 5 mM KCl, 5 or 25 mM KCl, in the presence or absence of the VSCC antagonist nimodipine (5 µM) or the NR antagonist MK-801 (10 µM), added at 4 DIV. Samples were then individually processed for RT-PCR using oligonucleotide primers (Fig. 2A) flanking the three exons and specifying amplicons of 691 bp (S1+) or 646 bp (S1), 517 bp (S2+) or 400 bp (S2), and 102 bp (S3+) or 75 bp (S3). As shown, amplicons were barely visible in GC grown in 5 mM KCl but were readily detectable after sustained growth in medium containing 25 mM KCl or NMDA (Fig. 2B). In all cases, amplicons were also detectable in GC grown overnight in 25 mM KCl, although to a lesser extent than chronic treatment. The KCl-mediated increases in IP3R1 mRNA were attenuated by overnight addition of nimodipine, and the NMDA-mediated increases were attenuated by overnight addition of MK-801. Volumetric analysis of S3 amplicons (sum total) was used to obtain estimates of the relative increases in IP3R1 mRNA, which were statistically robust (Fig. 2C). Taken together, these data indicate that 25 mM KCl and NMDA induce expression of IP3R1 mRNA by activating VSCC and NR, respectively.



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Fig. 2. IP3R1 S1, S2, and S3 mRNA variants are upregulated in a Ca2+-dependent manner. A: sequences of the primers used for PCR and the predicted sizes of the amplified fragments. B: RT-PCR (40 cycles; 5 ng of RNA) was performed on GC cultured for 5 DIV in medium containing 5 mM KCl, 25 mM KCl, or 140 µM NMDA + 5 mM KCl. As indicated, 20 mM KCl was added at 4 DIV to 5 mM cultures (5 {Delta} 25, 24 h); and nimodipine (5 µM) or MK-801 (10 µM) were added at 4 DIV to 25 mM KCl or NMDA cultures, respectively. (representative experiment; n = 5). Amplicons were resolved on 8% polyacrylamide gels and exposed on a PhosphorImager. C: volumetric analysis of radiolabeled samples from 3 cell preparations was performed to estimate differences in IP3R1 amplicon (sum of S3 variants). *Significant differences vs. 5 mM KCl-containing samples, set at 100%. **Significant differences vs. respective treatment (e.g., 5 {Delta} 25 or NMDA alone).

 
Interestingly, only amplicons corresponding to S1+ mRNA (691 bp) were detected in GC grown in elevated KCl or NMDA. In 12 of 12 cell preparations tested, GC exclusively expressed this variant, regardless of the developmental stage or culture condition used. This is in contrast to adult cerebellar cortex, in which the IP3R1 S1 variant predominates (646 bp) and is localized in Purkinje neurons (30). Low amounts of transcripts encoding both variants of S2 were detected in GC grown in 5 mM KCl, with proportionately more S2. Moreover, statistically significant increases in those containing the S2 exon were observed after sustained growth in medium containing 25 mM KCl or NMDA, compared with 5 mM KCl (%inclusion of S2: 5 mM, 41.7 ± 3.7; 25 mM, 81.9 ± 2.8; NMDA, 73.3 ± 5.7; n = 4). Likewise, statistically significant increases in transcripts containing the S3 exon were observed after growth in medium containing 25 mM KCl or NMDA, compared with 5 mM KCl (% inclusion of S3: 5 mM, 49.6 ± 2.8; 25 mM, 67.5 ± 1.6; NMDA, 61.8 ± 1.4; n = 5). Note that no effort was made to distinguish between the different subtypes of S2, i.e., S2ABC+ (520 bp) vs. S2B (517 bp, the major form in neurons) (30).

CaMK inhibitors attenuate Ca2+-dependent induction of IP3R1. To discern the possible role of CaMK in GC IP3R1 expression, cultures were grown in medium containing 5 or 25 mM KCl, and, where indicated, KN-62 (5 µM; IC50 in vitro 900 nM) was added at 4 DIV. The selection of agent for these studies was based on a rigorous analysis of the specificity of numerous kinase inhibitors in which the fidelity of KN-62 as a selective and potent inhibitor of CaMKII and CaMKIV was supported (8). Note that brief, overnight addition of KN-62 to the growth medium does not compromise GC viability, unlike longer-term exposure. GC were harvested at 5 DIV and processed for RT-PCR using the S1, S2, and S3 oligonucleotide primers (Fig. 2A). Consistent with the data in Fig. 2B, cultures grown in 25 mM KCl-containing media expressed amplicons corresponding to S1+, S2+, and S3+ IP3R1 mRNA. Importantly, overnight addition of KN-62 attenuated the KCl-mediated upregulation of all three variants (Fig. 3A) as well as attenuation of immunoreactive IP3R1 (Fig. 3B). Volumetric analysis of the S3 variants (sum total), readily measurable in the 5 mM samples, was performed to obtain estimates of the depolarization-induced increases in IP3R1 mRNA. These increases were statistically significant relative to 5 mM KCl or 25 mM KCl cultures receiving KN-62 (% of 5 mM KCl, set at 100; 25 mM + KN-62 = 273.3 ± 27.3; 25 mM KCl = 716.7 ± 112.0; n = 3). Note that attenuation of IP3R1 mRNA induction by 25 mM KCl was also observed after overnight treatment with KN-93, a less selective CaMK inhibitor that triggers some toxicity (not shown). These data indicate that activation of a target of KN-62, likely CaMKII or CaMKIV, is required for Ca2+-dependent induction of IP3R1 in GC.



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Fig. 3. CaMKs mediate IP3R1 variant expression. A: RT-PCR (S1, 40 cycles; S2, 35 cycles; S3, 35 cycles; actin, 26 cycles; 5 ng of RNA) was performed on GC cultured for 5 DIV in medium containing 5 or 25 mM KCl. As indicated, KN-62 (5 µM) was added overnight. Amplicons were resolved on 8% (S1, S2, and actin) or 20% (S3) polyacrylamide gels and exposed on a PhosphorImager (representative experiment; n = 3). B: immunofluorescence photomicrograph comparing GC grown in 25 mM KCl ± overnight treatment with KN-62 (5 µM) added at 7 DIV. At 8 DIV, cultures were fixed and incubated with antibody against IP3R1. Similar results were observed in 2 separate cell preparations. Magnification, x63. Exposure time, 10 s.

 
Actinomycin D and calcineurin inhibitors attenuate Ca2+-dependent induction of IP3R1. Upregulation of the IP3R1 mRNA by agents that increase Ca2+ influx could be due to increased stability and/or enhanced gene transcription. To distinguish between these possibilities, actinomycin D, a pleiotrophic transcription inhibitor, was used. GC were grown in medium containing 5 mM KCl, and, as indicated, some cultures were supplemented with 20 mM KCl alone or with 20 mM KCl plus actinomycin D (1 µg/ml), added at 4 DIV. At 5 DIV, RNA was harvested and S1-specific amplicons were compared. Overnight addition of 20 mM KCl alone induced IP3R1 S1+ mRNA, whereas simultaneous addition of actinomycin D (1 µg/ml) prevented this effect (Fig. 4A). This result indicates that depolarization-dependent upregulation of IP3R1 is due to enhanced gene transcription.



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Fig. 4. Actinomycin D and calcineurin inhibitors attenuate Ca2+-dependent induction of IP3R1. A: RT-PCR (S1, 40 cycles; actin, 26 cycles; 5 ng of RNA) was performed on GC cultured for 5 DIV in medium containing 5 mM KCl. As indicated, 20 mM KCl ± actinomycin D (A, 1.0 µg/ml) were added overnight to 5 mM KCl cultures. RNA was harvested at 5 DIV. RT-PCR analysis with oligonucleotide primers that specify mRNA corresponding to the S1+ variant or actin was performed. Amplicons were resolved on 8% polyacrylamide gels and exposed on a PhosphorImager. B: RT-PCR (S1, 40 cycles; actin, 26 cycles; 5 ng of RNA) was performed on GC cultured for 5 DIV in medium containing 5 or 25 mM KCl. As indicated, FK506 (FK, 0.2 µg/ml) or cyclosporin A (CsA, 1 µg/ml) was added overnight to 25 mM KCl cultures (and also to 5 mM cultures, although no effect was observed; data not shown). RNA was harvested at 5 DIV. RT-PCR analysis with oligonucleotide primers that specify mRNA corresponding to the S1+ variant or actin was performed. Amplicons were resolved on ethidium bromide-stained gels and photographed under UV illumination.

 
As discussed, FK506 or CsA, two calcineurin inhibitors, attenuates depolarization-dependent induction of IP3R1 in GC (12) and hippocampal neurons (13). To verify this effect in our cultures, GC were grown for 4 DIV and then vehicle, FK506 (0.2 µg/ml), or CsA (1 µg/ml) was added. After 24 h, whole cell RNA was harvested for RT-PCR analysis using oligonucleotide primers specifying the S1 variants of IP3R1. Figure 4B shows that these agents reduced the depolarization-dependent induction of IP3R1 S1+ mRNA in GC. Similar results were observed in 4 of 4 different cell preparations treated with FK506 and 2 of 2 preparations treated with CsA. Collectively, these data indicate that depolarization-dependent transcription of IP3R1 in GC requires both calcineurin and CaMK.

IP3R1 S1+ is the predominant variant in cerebella of mutant mice lacking Purkinje neurons. Investigators have largely ignored functional studies of IP3R1 in GC neurons in vivo because of the lack of detectable expression of mRNA and protein. As indicated, IP3R1 S1+ is prominent in the neonatal, but not adult, cerebellar cortex (30). After migration is complete (P21 in rat), there is a switch from prominent expression of IP3R1 S1+ to the adult form, S1. An intriguing possibility is that GC in vivo express IP3R S1+, and it contributes to the Ca2+-dependent process of migration and/or synaptic refinement. To explore this, oligonucleotide primers encoding the S1+ variant were used to compare the relative proportions of S1+ and S1 variants in the cerebella of mutant mice with specific Purkinje cell lesions (designated as pcd) vs. wild-type controls.

The two major neuronal types in cerebellar cortex are Purkinje and GC. In pcd mice, there is a rapid degeneration of nearly all Purkinje neurons beginning at approximately P15, without significant loss of GC (17). Thus, if GC in vivo predominately express IP3R1 S1+ mRNA (as observed in cultures under all conditions of assay), then an increase in the relative proportion of the S1+ variant should be observed in the cerebellar cortex of pcd–/– mice, compared with wild-type (+/+) littermates. In other words, by eliminating the major source of IP3R1 in cerebellum, namely, IP3R1 S1 in Purkinje neurons, a detectable amount of IP3R1 S1+ mRNA may be revealed in the remaining GC. Initially, a RT-PCR assay specifying S1+ and S1 variants in mice was tested. Despite a 99.6% similarity in the amino acid sequences between rat and mouse, different oligonucleotide primers were designed because of potential mismatches between the rat primers and the murine sequences flanking the S1 site. The predicted sizes of the amplicons are 158 and 113 bp (Fig. 5A). On the basis of in situ hybridization and RNase protection analyses, the major variant in adult murine hippocampus is S1+, whereas S1 predominates in adult cerebellum (30, 31). To establish the validity of the RT-PCR assay, adult murine hippocampus and cerebellum were compared (Fig. 5B). In agreement with previously published reports (30, 31), the S1+ variant predominated in hippocampus, and the major variant in cerebellum was S1. The identity of this S1+ variant was verified by restriction site analysis (with BpmI; data not shown). Next, RNA from the cerebella of 30-day-old wild-type and pcd–/– mice was compared (Fig. 5B). Again, the predominant variant in wild-type mice was S1. In contrast, a statistically significant increase in the relative proportion of amplicon corresponding to S1+ mRNA was observed in pcd mice (%S1 in wild-type vs. null mouse cerebellum: 82.5 ± 0.85 vs. 44.7 ± 2.02%; n = 4). In addition, the overall amount of IP3R1 amplicon was lower than that of wild type. These data verify that S1 is the major variant in Purkinje cells. Importantly, they suggest that GC predominately express IP3R1 S1+ in vivo.



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Fig. 5. IP3R1 S1 mRNA expression in Purkinje cell degeneration (pcd) vs. wild-type mice. A: schematic diagram of oligonucleotide primers flanking the S1 splice site. B, left: RT-PCR analysis of mRNA in adult murine hippocampus (Hip) and cerebellum (Cb). Hippocampus from normal mice expressed primarily the S1+ variant (158 bp), whereas the cerebella from normal mice expressed primarily the S1 variant (113 bp). Right: RT-PCR analysis of mRNA harvested from wild-type (+/+) and matched mutant/pcd–/– cerebella of 30-day-old mice. The (+/+) mice expressed primarily the S1 variant (113 bp). In the pcd–/– mouse, the S1+ variant predominated. Also, overall size of the cerebella and expression of the S1 mRNA were reduced compared with wild type. Amplicons were resolved on ethidium bromide-stained agarose gels (1.5%) and illuminated under UV light (representative experiment, n = 4). STD, standard markers.

 
GC in cerebellar explant cultures express IP3R1. If IP3R1 has a role in GC maturation, it may be possible to detect IP3R1 expression in GC grown in standard culture medium (e.g., 5 mM KCl) at an early stage of development. Connor et al. (5) reported that GC in explant cultures of rodent cerebellar cortex migrate outward along glial fibers that radiate from the explant core. As the GC migrate outward, they develop the biochemical and electrophysiological properties of maturing GC in vivo (5). We cultured explants derived from the cerebellar cortices at P3 and verified by phase-contrast microscopy the temporal migration of GC away from the core along radial glial fibers. To assess the expression of immunoreactive IP3R1 in GC, cultures were grown for 3 DIV and then fixed and processed for immunocytochemistry. As shown in Fig. 6, GC throughout the culture were positively stained with IP3R1 antibodies.



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Fig. 6. GC in cerebellar explant cultures express immunoreactive IP3R1. Explants were grown in culture medium containing 5 mM KCl for 3 DIV and were then fixed and processed for IP3R1 immunocytochemistry. GC (e.g., arrowheads) throughout the explant were immunopositive for IP3R1 (left). A phase-contrast micrograph of the same field is shown at right.

 
GC neurons in the granular laminae express IP3R1 S1+. The innovative technique of laser microdissection provides a means of obtaining highly enriched tissue from distinct laminae of the cerebellar cortex. If GC neurons express IP3R1 S1+ in vivo, then it should be enriched in the granular layer of slices from rat cerebellar cortex compared with the cell-sparse medullary layer (1). To test this hypothesis, slices were prepared from cerebellar cortices of rats at P13–P18, medullary and granular layers were microdissected (Fig. 7), and samples were subjected to RT-PCR analysis using oligonucleotide primers specifying mRNA corresponding to IP3R1 S1+/–. CaMKIV, which is highly enriched in GC (37), was also examined. Note that no effort was made to equalize RNA in these minute, microdissected samples before RT-PCR analysis, although actin was used to assess the relative yields of mRNA. Figure 7 shows that IP3R1 S1+ is readily detected in samples from the granular layer but not in samples from the medullary layer. Consistent with the enrichment of granular vs. medullary layers amplicon corresponding to CaMKIV mRNA was abundant in the granular layer but was undetectable in the medullary layer even when samples were subjected to a relatively high (e.g., saturating for CaMKIV in the granular layer) number of amplification cycles. To independently verify that the IP3R1 amplicon in the granular layer contained the S1 exon, restriction analysis was performed with BpmI, an enzyme that recognizes and cleaves a sequence only in amplicons containing the S1 exon. As shown in Fig. 7, the majority of amplicon obtained from the granular layer was sensitive to cleavage with BpmI, resulting in two fragments of ~377 and 314 bp. In contrast, only a modest amount of the amplicon from whole adult cerebellar cortex was sensitive to cleavage with BpmI. This result is consistent with the preponderance of IP3R1 S1 mRNA in the Purkinje neurons of adult cerebellar cortex. Importantly, it indicates that GC neurons in vivo express a form of IP3R1 containing the S1 exon.



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Fig. 7. GC in the granular laminae express IP3R1 S1+. Top: isolation of specific layers in the rat cerebellum by laser microdissection of cryostat sections. Top left: intact section showing laminae before dissection. Top right: section after dissection of medullary and granular layers. For illustrative purposes, a rapid Nissl stain was used to improve visualization of the laminae. Bottom left: RT-PCR of mRNA from laser microdissected granule and medullary layers. Whole rat brains were frozen and sliced. Specific areas were dissected and subjected to RT-PCR assay using primers corresponding to IP3R1 S1, CaMKIV, or actin. Amplicons were resolved on acrylamide gels and photographed under UV illumination. Bottom right: after IP3R1 S1 RT-PCR, equal aliquots of amplified samples (granular layer or whole adult cerebellum) were incubated in the absence (–) or presence (+) of BpmI, resolved on ethidium-stained gels, and photographed under UV illumination. The BpmI cleavage site is specific to the S1+ exonic sequence, and the predicted sizes of the products are 377 and 314 bp.

 

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It is well established that activity-dependent alterations in intracellular Ca2+ concentration and downstream Ca2+-sensitive enzymes translate changes in bioelectrical activity into distinct patterns of gene expression, ultimately affecting complex biological processes such as survival, differentiation, and plasticity (for review, see Refs. 2, 39, 41, and 48). In many central neurons, the excitatory transmitter glutamate initiates these processes by regulating the influx of Ca2+ through VSCC and NR, thereby activating downstream signaling pathways, in particular CaMK (e.g., CaMKIV) and phosphatases (e.g., calcineurin). These enzymes in turn decode and relay this highly specific information to the nucleus via coordinate activation of various transcription factors (for review, see Refs. 7, 18, and 27).

In the present study, evidence for activation of CaMK in IP3R1 induction in GC is based on the use of highly selective pharmacological agents. Accordingly, supplementation of the growth medium with elevated KCl or NMDA supported GC survival (11) and also induced IP3R1 protein and mRNA. Overnight addition of the L-type VSCC antagonists, nifedipine or nimodipine, effectively reversed 25 mM KCl-mediated induction of IP3R1 protein and mRNA (Figs. 1C and 2B). Likewise, the competitive and noncompetitive NR antagonists, AP5 and MK-801, respectively, effectively reversed NMDA-mediated induction of IP3R protein and mRNA (Fig. 2B). Thus, although the route of Ca2+ entry into neurons can differentially affect gene transcription, i.e., hippocampal neurons (28), both routes of Ca2+ entry induced IP3R1 in GC. The selective and potent cell-permeable CaMK inhibitor KN-62, which competes with calmodulin for binding to the regulatory domains of CaMKIV and CaMKII (6, 8, 44), also attenuates IP3R1 induction (Fig. 3). Similar effects were observed with the less selective agent, KN-93. On the basis of existing reports that these compounds attenuate transcriptional events mediated by CaMKIV in diverse cell types (e.g., Refs. 15, 38, 50), we suggest that nuclear CaMKIV is a component of this signaling cascade in GC. However, a role for CaMKII cannot formally be excluded.

Besides the present study and an earlier report (35), only one other group of investigators has examined depolarization-dependent induction of IP3R1 in GC (12). Those authors demonstrated that pharmacological blockade of calcineurin with FK506 or CsA attenuated depolarization-dependent expression of IP3R1 (independently verified herein, Fig. 4B). Depolarization- and calcineurin-dependent induction of IP3R1 was subsequently reported in hippocampal neurons, and a NF-ATc pathway was shown to be critical (13; see also Ref. 16). Taken together, these studies support roles for both calcineurin, acting through NF-ATc, and CaMK, probably CaMKIV, as regulators of IP3R1 expression in GC.

IP3R1 promoter activity appears to be regulated through several cis elements present in a short region within the 5'-flanking region, which contains a TATA box, and binding sites for the cAMP-responsive element, PU box motif, CCAAT-binding transcription factor/NF-1, AP2, basic helix-loop-helix factor, TPA-responsive element, AP3, B sequence, CCAGG-containing sequence, and POU/homeobox transcription factor binding sequence. These cis elements confer widespread and differential expression of the IP3R1 gene in brain and peripheral tissues (10). An interesting possibility in GC is that the basic helix-loop-helix promoter site in the IP3R1 gene is synergistically activated by calcineurin and CaMKIV. Along these lines, the myocyte enhancing factor-2 (MEF-2) can dimerize with basic helix-loop-helix proteins, and synergism between CaMKIV and calcineurin in transactivation through MEF-2 has been described in T cells, ventricular myocytes, and skeletal muscle (for review, see Refs. 18 and 39). Indeed, MEF-2 is expressed in newly generated GC and is activated by depolarization and Ca2+, where it contributes to survival (25). Moreover, MEF-2 activity in GC appears to be enhanced by calcineurin under depolarizing conditions because this process is sensitive to inhibition by CsA and FK506 (26). More recently, Groth and Mermelstein (16) reported that inhibition of NF-AT signaling only partially reduced expression of IP3R1 in response to brain-derived neurotropic factor (BDNF), indicating that multiple transcription factors can control the IP3R1 gene, some independently of NF-AT. In contrast, inhibition of NF-AT signaling completely abolished BDNF expression, indicating that although multiple transcription factors may regulate BDNF, NF-AT is required. Further studies are needed to determine whether a synergistic link between CaMKIV and calcineurin regulates Ca2+-dependent IP3R1 induction in GC.

Ca2+ and CaM kinases induce selection of a distinct IP3R1 variant in GC. In the present study, depolarizing agents that increase Ca2+ influx in GC produced splice variants that differed from those that predominate in Purkinje neurons or in GC grown in 5 mM KCl. GC grown in NMDA or in 25 mM KCl-containing medium exhibited significantly higher levels of amplicons that included all three exons. In contrast, Purkinje neurons predominantly express variants containing S2 but lacking the S1 and S3 exons (30, 34), while GC grown in 5 mM KCl predominantly express variants containing S1 with proportionately less S2+ and similar amounts of S3+/– (Figs. 2 and 3). These data suggest that Ca2+, calcineurin, and/or CaMK influence exon choice in IP3R1. Indeed, a handful of reports have indicated that Ca2+-dependent signaling cascades influence exon selection in excitable cells. For example, depolarization-dependent Ca2+ influx through VSCC or NR alters exon selection in pre-mRNA encoding Ca2+-ATPases (51), NR (46, 47), and the slo K+ channel (50). Using transfected cell lines, Xie and Black (50) specifically demonstrated that overexpression of CaMKIV regulates splicing of the STREX exon in slo K+, whereas overexpression of CaMKI, CaMKII, or dominant negative CaMKIV was ineffective. A cell-permeable CaMK inhibitor, KN-93, interfered with depolarization-dependent STREX exclusion, presumably by targeting CaMKIV, which is enriched in neuronal nuclei. Overexpression of CaMKIV produced similar effects on exon selection at three other sites: an alternative exon (SloII87) in the slo K+ channel and two exons (5 and 21) in NR1, hinting that CaMKIV may have a broad role in the regulation of alternative splicing. To identify specific features of the STREX exon needed for CaMKIV repression, deletions in the STREX exonic sequences that were similar to either purine-rich enhancer or pyrimidine-rich repressor elements, previously identified in other pre-mRNA, were mutated. The pyrimidine-rich repressor deletion increased exon inclusion with almost no repression by CaMKIV, leading to the conclusion that the pyrimidine-rich region is required for CaMKIV-mediated repression of the STREX exon (50). An additional repressor element in the upstream 3' splice site, termed the CaMKIV-responsive RNA element (CaRRE), that confers sensitivity to CaMKIV was also identified. However, a thorough sequence analysis of potential purine- and pyrimidine-rich sequences in and near the S2 and S3 exons in IP3R1 was unrevealing. Furthermore, we were unable to identify a CaRRE sequence in the 3' region of the S2 and S3 exons. In both cases, structurally distinct but functionally homologous sequences may underlie Ca2+-dependent exon selection, and GC should prove useful in studying such questions in the future.

Is IP3R1 physiologically relevant in cerebellar GC neurons in vivo? As indicated, previously available methods of assay did not reveal IP3R1 protein or mRNA in the cerebellar granular layer in vivo (24, 30, 32, 40). The inability to detect a low amount of immunoreactive IP3R1 may be further compounded by the fact that the adjacent Purkinje cell layer is by far the richest source known. Nevertheless, IP3R1-specific mRNA can be detected in GC grown in standard medium (5 mM KCl) when a highly sensitive RT-PCR assay is used (e.g., Figs. 24). Moreover, immunoreactive IP3R1 is detectable in migrating GC in explant cultures grown in culture medium containing standard concentrations of KCl (5 mM) (Fig. 6). In all conditions tested, we observed only the S1+ variant of IP3R1, the form that predominates in neonatal, but not adult, cerebellar cortex (30). These data suggest that IP3R1 S1+ may have a role in developing, but not mature, GC. Along these lines, Kirischuk et al. (19) compared agonist-induced Ca2+ mobilization in GC from acutely prepared slices from murine cerebella at two ages. IP3-mediated Ca2+ mobilization was observed in GC in slices from neonates (P6) but not adults (P30).

Herein we provide the first evidence that IP3R1 S1+ is expressed in GC in vivo. First, cerebella from pcd mice express proportionally more amplicon corresponding to the S1+ variant than do their wild-type littermates (Fig. 5). This result is consistent with the selective loss of the S1 variant due to degeneration of Purkinje cells relative to the S1+-expressing GC, which are spared. Second, microdissected tissue from the granular layer of cerebellar slices from neonatal rats specifically express amplicons corresponding to IP3R1 S1+ (Fig. 7). Although previous investigators failed to detect IP3R1 mRNA or immunoreactive protein in GC, there is evidence for IP3R1 promoter activity. Thus moderate expression of a transgene containing a strong IP3R1 promoter was detected in cerebellar GC neurons (10), and Ohkawa et al. (36) reported weak but significant AP2-dependent IP3R1 promoter activity in GC. Possibly, IP3R1 contributes to migration or synaptic refinement in developing GC, two Ca2+-dependent processes that depend critically on the concerted action of CaM kinases and phosphatases (for review, see Refs. 7, 20, 21). In this regard, emerging evidence suggests an important link between IP3R1 activity and neuronal morphogenesis (3, 9, 14, 16, 42). Notably, functional inactivation of IP3R1 in growth cones in dorsal root ganglia results in growth arrest and neurite retraction (42), and axonal outgrowth is compromised in NF-ATc null mice (14). Thus neurons may use IP3R1 in conjunction with other pathways known to integrate Ca2+ signals to strengthen maturing synapses. The data presented herein provide a strong rationale for pursuing functional studies of IP3R1 S1+ in developing GC.


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This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-40582 (to M. L. Vallano) and a predoctoral fellowship from the Pharmaceutical Research and Manufacturers of America Foundation (to J. Y. Choi).


    ACKNOWLEDGMENTS
 
We gratefully acknowledge the advice of Dr. Frank Middleton and the assistance of Jennifer Jamieson with the laser microdissection experiments.

Current address of J. Y. Choi: Dept. of Orthopedic Surgery, State Univ. of New York Upstate Medical Univ., Syracuse, NY 13210.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. L. Vallano, Dept. of Neuroscience and Physiology, State Univ. of New York Upstate Medical Univ., Syracuse, NY 13210 (E-mail: vallanom{at}upstate.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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