Journal of Neurocytology 33, 297–307 (2004)
Specific differential expression of phospholipase A2 subtypes in rat cerebellum Y O S H I N O R I S H I R A I ∗ and M A S A O I T O Laboratory for Memory and Learning, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan
[email protected] Received 31 March 2004; revised 3 May 2004; accepted 3 May 2004
Abstract Phospholipase A2 (PLA2 ) is a family of enzymes playing diverse roles in lipid signaling in neurons and glia cells. In this study, we examined the expression of subtypes of PLA2 in the cerebellum using immunolabeling and in situ hybridization methods. Two Ca2+ -dependent cytosolic subtypes (cPLA2 α and cPLA2 β), one Ca2+ -independent cytosolic subtype (iPLA2 ), and two secretory subtypes (sPLA2 IIA and sPLA2 V) were detected in the cerebellum. cPLA2 α is present in somata and dendrites of Purkinje cells, while sPLA2 IIA is associated with the endoplasmic reticulum in perinuclear regions of Purkinje cell somata. iPLA2 is present in granule cells, stellate cells and also in the nucleus of Purkinje cells. In addition, cPLA2 β is localized in granule cells, and sPLA2 V in Bergmann glia cells. These results provide an important basis for identifying functional roles of PLA2 s in the cerebellum.
Introduction Phospholipases A2 (PLA2 s) constitute a large family of enzymes that act to break down phospholipids to arachidonic acid (AA) and other unsaturated fatty acids. AA in turn acts to produce lipid mediators, such as prostaglandins, leukotrienes, or hydroxyeicosatetraenoic acids, which play roles in various biological processes including inflammation. PLA2 has been suggested to play a role in the induction of long-term depression (LTD) in cerebellar Purkinje cells (PCs) (Linden, 1995), which is thought to be a basis of motor learning (Ito, 2001). This is because, in cultured PCs, inhibitors of PLA2 block the induction of a reduced form of LTD and this effect is compensated by exogenously applied AA (Linden, 1995). It has also been shown that rat PCs express PLA2 (Kataoka et al., 1997; Kishimoto et al., 1999). PLA2 s are grossly subdivided into three groups, cytosolic Ca2+ -dependent (cPLA2 ), Ca2+ -independent (iPLA2 ), and secretory (sPLA2 ). cPLA2 s have a number of subtypes such as cPLA2 α, cPLA2 β, and cPLA2 γ . There are several splicing variants of iPLA2 . Altogether, 20 subtypes of PLA2 have been identified in mammals (see Murakami & Kudo, 2002; Ho et al., 2001). Of these, the activity of four subtypes (cPLA2 α, iPLA2 , sPLA2 IIA, and sPLA2 V) has been detected in rat cerebellum lysate (Yang et al., 1999a, 1999b). In this study, using immunolabeling and in situ hybridization methods, we con∗ To
whom correspondence should be addressed.
0300–4864
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2004 Kluwer Academic Publishers
firmed the presence of these four subtypes and cPLA2 β in the rat cerebellum, of which three (cPLA2 α, sPLA2 IIA and iPLA2 ) are located in PCs. The differential localization of these PLA2 subtypes in PCs implies their distinct roles in PC functions. Methods ANIMAL TREATMENT AND TISSUE PREPARATION
Adult male Wistar rats of 6 weeks old (body weight, 130–150 g) were kept at a constant ambient temperature of 24◦ C under a 12-hour-light/dark cycle with free access to food and water. The rats were anesthetized by the intraperitoneal administration of sodium pentobarbital (50 mg/kg body weight), and were then transcardially perfused with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). The brain was then rapidly dissected out, fixed in 4% PFA in PBS overnight at 4◦ C, and finally in 30% sucrose in PBS overnight at 4◦ C. Frozen brain tissues were cut into 14-µm-thick slices using a cryostat (Leica). Immunohistochemistry and in situ hybrydization experiments were carried out as described below. Experiments on a PLA2 subtype were repeated independently in more than five rats. IMMUNOHISTOCHEMISTRY
For staining cPLA2 α and iPLA2 , cerebellar slices were treated with a blocking solution (5% goat serum, 2% bovine serum
298 albumin (BSA), 0.4% Triton X-100 in PBS) for 30 min at room temperature. The cerebellar slices were then incubated for 2 h at room temperature with an anti-cPLA2 α polyclonal antibody (Santa Cruz, CA, USA) or an anti-iPLA2 polyclonal antibody (Cayman, Ann Arbor, MI, USA). They were then washed in PBS three times, and incubated in 1/1000 diluted Alexa 546 anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) for 1 h. Fluorescence images were acquired using an Olympus FV500 confocal laser scanning microscope. Images were stored using the computer program Fluoview version 4.3 (Olympus) and then arranged and adjusted to match the contrast with the program Adobe Photoshop 6.0 (Adobe Systems Inc.). For labeling sPLA2 IIA and sPLA2 V, slices were treated with the aforementioned blocking solution for 1 h at room temperature, followed by treatment with 1% (w/v) saponin in PBS for 30 min at 4◦ C to permeabilize the membrane. An antisPLA2 IIA or an anti-sPLA2 V polyclonal antibody (Cayman) was diluted with the blocking solution and applied to the slices. The slices were incubated for 2 h at room temperature, washed in PBS three times, and then incubated in 1/1000 diluted Alexa 546 anti-rabbit IgG (Molecular Probes, Eugene, OR, USA). For staining calbindin, an anti-calbindin monoclonal antibody (Sigma, Saint Louis, MS, USA) was diluted (1/1000) with the blocking solution and applied to the specimens. The specimens were incubated for 2 h at room temperature, washed in PBS, and then incubated in 1/1000 diluted Alexa 488 anti-mouse IgG (Molecular Probes).
S H I R A I and I TO In situ HYBRIDIZATION RNA isolation, reverse transcription, and polymerase chain reaction Total RNA was isolated from frozen rat cerebella by the acidphenol-guanidine thiocyanate extraction method (Chomczynski & Sacchi, 1987). Poly (A)+ RNA was purified from total RNA using Oligotex-dT30 (Takara, Shiga, Japan). RNA was converted to cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase (Gibco, Rockville, MD, USA) and random primer (random sequence nonadeoxyribonucleotide mixture) in a 20 µl reaction mixture. Alternatively, RNA was converted to cDNA using Superscript II reverse transcriptase (Stratagene, La Jolla, CA, USA) and oligo (dT) primer in a 20 µl reaction mixture. After the reaction was terminated by heating at 70◦ C, polymerase chain reaction (PCR) was performed using specific primers for each PLA2 subtype listed in Table 1 (for iPLA2 , see also Fig. 4A). For cPLA2 α, cPLA2 β and iPLA2 , PCR was performed in a 10 µl reaction mixture, containing 0.5 µl of reverse transcription (RT) mixture, 0.4 mM sense or antisense primer, 1× ExTaq buffer, 200 µM dNTPs, 0.5 µl of dimethyl sulfoxide (DMSO), and 0.1 µl of ExTaq DNA polymerase (Takara). For sPLA2 IIA and sPLA2 V, PCR was performed in a 10 µl reaction mixture, containing 0.5 µl of RT mixture, 0.4 mM sense primer or antisense primer, 1× Pfu buffer, 200 µM dNTPs, 0.5 µl of DMSO, and 0.1 µl of PfuTurbo DNA polymerase (Stratagene). The PCR protocol for cPLA2 α was as follows: 95◦ C for 5 min, followed by 50 cycles of 95◦ C for 30 sec, 52◦ C for 30 sec, and 72◦ C for 30 sec, and then a final extension at 72◦ C
Table 1. Specific sense and antisense primers for PLA2 s. cPLA2 α sense primer cPLA2 β sense primer iPLA2 sense primer iPLA2 F3 sense primer iPLA2 -exon9F sense primer iPLA2 F4 sense primer sPLA2 IIA sense primer sPLA2 V sense primer cPLA2 α antisense primer cPLA2 β antisense primer iPLA2 antisense primer iPLA2 R3 antisense primer iPLA2 -exon9R antisense primer iPLA2 R4 antisense primer sPLA2 IIA antisense primer sPLA2 V antisense primer
(5 -CGGAATTCATGTCTTTCATAGATCCTTATC-3 ) (5 -GCGGATCCATGGCGGAGGCGGCTTTGGAAG-3 ) (5 -GCGGATCCATGCAGTTCTTTG-3 ) (5 -GCTCTGCAATGCCCGCTGCA-3 ) (5 -CGGAATTCTGATTACCAGGAAGG-3 ) (5 -ATTACCAGGAAGGCGCTCTT-3 ) (5 -GCGAATTCATGAAGGTCCTCCTGTTGCT-3 ) (5 -GCGAATTCATGAAGCGCCTCCTCACGCT-3 ) (5 -CCCTCGAGTGTTCTCTTTTATGTTCTCTTT-3 ) (5 -CCCTCGAGCCAGCCAGAAGTTCACAGCATC-3 ) (5 -CCCTCGAGCCAGAATCTCACT-3 ) (5 -ATGCGTTCGCTTCTCATCCC-3 ) (5 -CATCTCGAGCTAGGTTGTTTAAGC-3 ) (5 -AGCAGCAGCTGGACAAGCTT-3 ) (5 -CCCTCGAGACTGGGCGTCTTCCCTTTGC-3 ) (5 -CCCTCGAGGAGGAAGTTGGGGTAATACT-3 )
Sense and antisense primers for PLA2 subtypes used for PCR. Sense primers for cPLA2 α and cPLA2 β corresponded to about 20 bases upstream of the start codon of the expected open reading frames (ORFs) of cPLA2 α and cPLA2 β cDNAs, respectively. The sense primers for iPLA2 , sPLA2 IIA and sPLA2 V corresponded to the start codon of the expected ORFs of iPLA2 , sPLA2 IIA and sPLA2 V cDNAs, respectively. Antisense primers for cPLA2 α, cPLA2 β and iPLA2 corresponded to about 500 bases downstream of the start codon of cPLA2 α, cPLA2 β and iPLA2 cDNAs, respectively, while antisense primers for sPLA2 IIA and sPLA2 V corresponded to the stop codon of the expected ORFs of sPLA2 IIA and sPLA2 V cDNAs, respectively. The iPLA2 -exon9F sense primer corresponded to the junction between exon 8 and exon 9, and the iPLA2 -exon9R antisense primer corresponded the junction between exon 9 and exon 10. The iPLA2 F3 sense primer, iPLA2 R3 antisense primer, iPLA2 F4 sense primer, and iPLA2 R4 antisense primer are specific to exon 5, exon 10, exon 9, and exon 17 of iPLA2 mRNA, respectively (see Fig. 4A).
Phospholipase A2 subtypes in cerebellum for 5 min. For cPLA2 β: 95◦ C for 5 min, followed by 50 cycles of 95◦ C for 30 sec, 70◦ C for 30 sec, and 72◦ C for 30 sec, and then a final extension at 72◦ C for 5 min. For iPLA2 : 95◦ C for 10 min, followed by 40 cycles of 95◦ C for 1 min, 55◦ C for 1 min, and 72◦ C for 1 min, and then a final extension at 72◦ C for 5 min. For sPLA2 IIA and sPLA2 V: 96◦ C for 5 min, followed by 30 cycles of 96◦ C for 45 sec, 57◦ C for 30 sec, and 72◦ C for 1 min, and then a final extension at 72◦ C for 10 min.
Preparation of digoxigenin (DIG)-labeled RNA probes of PLA2 s The PCR products corresponding to cPLA2 α (500 bp), cPLA2 β (500 bp), iPLA2 (500 bp), iPLA2 -exon 9 (165 bp), sPLA2 IIA (441 bp), or sPLA2 V (414 bp) were separated by 1% lowmelting agarose gel electrophoresis and then purified. Fragments containing EcoRI and XhoI sites (cPLA2 α, iPLA2 -exon 9, sPLA2 IIA, and sPLA2 V) were cloned into the EcoRI-XhoI site of pBlueScript KS+ (Stratagene) and sequenced. Fragments containing BamHI and XhoI sites (cPLA2 β and iPLA2 ) were cloned into the BamHI-XhoI site of pBlueScript KS+ (Stratagene) and sequenced. The antisense and sense RNA probes for a region of the cDNA clone of PLA2 were labeled with DIG-dUTP from linearized templates using a DIG RNA labeling mix (Roche Diagnostics, Mannheim, Germany) and T3 or T7 RNA polymerase (Roche Diagnostics). The synthesis of the RNA probes were confirmed by 1% agarose gel electrophoresis.
Expression We adopted the previously described protocols (Ausubel et al., 1997; Wilkinson, 1992) with some modifications. Frozen sectioned brain slices were thaw-mounted onto gelatincoated slides, and stored at − 80◦ C until in situ hybridization.
299 The slices were postfixed in 4% PFA in PBS for 10 min, followed by acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min, and then hybridized with a DIGlabeled RNA probe (0.5 µg/ml) in a hybridization buffer (50% formamide, 1× Denhart’s solution, 500 µg/ml yeast tRNA, 500 µg/ml salmon sperm DNA) for 12 h at 72◦ C. The slices were washed once in 5× SSC, three times in 0.2× SSC for 30 minutes each at 72◦ C, then washed in 0.2 × SSC and washed further in Tris-buffered saline (TBS) at room temperature. The immunohistochemical detection of DIG-labeled RNA probes was carried out using alkaline phosphatase (AP)-conjugated anti-DIG Fab fragments (Roche Diagnostics). AP activity was visualized with nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as substrates (Roche Diagnostics). Color development was continued for 12 h. Slices were observed under a microscope (BX51; Olympus), and images were acquired using an Olympus DP12 digital camera. Since in situ hybridization using sense RNA probes revealed no signals, Figures 1–5 illustrate only data obtained with antisense but not sense RNA probes.
Results The expression of PLA2 proteins and mRNAs in the adult rat brain was investigated by immunohistochemistry and in situ hybridization. DISTRIBUTION OF C a2+ - DEPENDENT CYTOSOLIC PLA 2 S ( C PLA 2 S )
cPLA2 α Strong to moderate immunoreactivity of cPLA2 α as detected in the cytoplasm of PC somata and dendrites
Fig. 1. Expression of cPLA2 α in rat cerebellum: A: cPLA2 α immunoreactivity visualized using anti-cPLA2 α polyclonal antibody (Santa Cruz) and the fluorescence of Alexa 546. Note that the dendrites and cytoplasm of Purkinje cells are specifically labeled. B: cPLA2 α mRNA visualized by in situ hybridization with DIG-labeled 500 bp antisense RNA probes. Sections were cut sagitally (14 µm thick). Scale bars indicate 100 µm. ML: molecular layer; PC: Purkinje cell; GCL: granule cell layer.
300 (Fig. 1A). Beside PCs, weak immunoreactivity was also observed in molecular and granule cell layers. cPLA2 α immunoreactivity was also observed outside the cerebellum, particularly, in the hippocampus, olfactory bulb, pineal gland, and pituitary gland (Data not shown), as reported by Kishimoto et al. (1999). In situ hybridization revealed a strong expression of cPLA2 α mRNA in the cytoplasm of PCs (Fig. 1B). Granule cells showed only a weak expression (Fig. 1A). cPLA2 β No monoclonal antibodies were available to label cPLA2 β. In situ hybridization revealed a strong to moderate expression of cPLA2 β mRNA in the granule cell layer, a weak expression in the molecular layer, but no expression in the PC layer (Fig. 2A, B). A weak expression was detected in hippocampal CA1-3, and dentate gyrus, and a very weak expression in the olfactory bulb, amygdala, basal forebrain, cerebral cortex, thalamus and midbrain (Data not shown). cPLA2 γ Because rat cPLA2 γ has not been cloned yet, we used data on human cPLA2 γ and a mouse clone (GenBank accession number XM 145370) that shows significant homology with human cPLA2 γ to construct DIGlabeled RNA probes for in situ hybridization. Since human cPLA2 γ was detected by northern blot analysis of human brains (Pickard et al., 1999), rat cPLA2 γ was expected to be expressed in the rat brain. However, no significant expression was observed in the cerebellum by
S H I R A I and I TO in situ hybridization (Data not shown). Alternatively, RT-PCR was performed using a sequence data of rat protein 2 that shows significant homology to human cPLA2 γ (Shinzawa & Tsujimoto, 2003). However, rat protein 2 mRNA was not amplified from mRNA extracted from rat cerebella. Hence, there is no evidence indicating that cPLA2 γ is present in rat cerebella. DISTRIBUTION OF C a2+ - INDEPENDENT CYTOSOLIC PLA 2 ( : PLA 2 )
The strong immunoreactivity of iPLA2 was detected in PCs, particularly in their nuclei (Fig. 3A, compared with PC in Fig. 3B labeled by anti-calbindin antibody: specific to PCs). Stellate cells and granule cells were also labeled. iPLA2 mRNA was strongly expressed in PCs as well as in the granule cell layer, and moderately to weakly in the molecular layer (Fig. 3C). iPLA2 mRNA was ubiquitously expressed in the brain. Strong signals of iPLA2 were detected in the olfactory bulb, hippocampal CA1-3, dentate gyrus, and brain stem. Moderate signals were detected in the cerebral cortex, thalamus, and midbrain, and weak signals in the amygdala and basal forebrain (Data not shown). There are five splice variants of iPLA2 as shown in Figure 4A. While iPLA2 -1 lacks exon 9 (165 bp), the other four subtypes (iPLA2 -2, iPLA2 -3, iPLA2 -ankyrin1, and iPLA2 -ankyrin-2) have it, which converts cytosolic iPLA2 -1 to the other four membrane-bound forms because exon 9 encodes hydrophobic amino acids (Larsson et al., 1998; Larsson Forsell et al., 1999). Since the antigen polypeptide (the amino acid sequence is
Fig. 2. Expression of cPLA2 β in rat cerebellum. A and B: cPLA2 β mRNA visualized by in situ hybridization with DIG-labeled 500 bp antisense RNA probes and presented at increasing magnifications. Note cPLA2 β mRNA expression in the granule cell layer, but not in the PC layer (B). Scale bars indicate 100 µm. PCL, Purkinje cell layer.
Phospholipase A2 subtypes in cerebellum
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Fig. 3. Expression of iPLA2 in rat cerebellum. A: iPLA2 immunoreactivity visualized using anti-iPLA2 polyclonal antibody (Cayman) and the fluorescence of Alexa 546. B: the same slice of the cerebellum as in A, but labeled using anti-calbindin monoclonal antibody (Sigma) that is specific to PCs and Alexa 488. Note that iPLA2 is expressed in both PCs and stellate cells (arrows) in ML. In situ hybridization with DIG-labeled 500 bp antisense RNA probes was used for C. Note that the soluble form of iPLA2 mRNA is expressed in PCs (arrows), granule cells and stellate cells. Scale bars indicate 100 µm.
PRCNQNINLKPPTQPADQLV) of the anti-iPLA2 antibody used in our immunohistochemistry corresponded to the junction between exon 13 and exon 14 of iPLA2 , the iPLA2 protein expressed in the cerebellum should be iPLA2 -1, iPLA2 -2, or iPLA2 -3, but not iPLA2 -ankyrin1 or iPLA2 -ankyrin-2 that has neither exon 13 nor 14 (Fig. 4A). To determine whether PCs express the soluble form or membrane-bound forms of iPLA2 , we performed in situ hybridization experiments and RT-PCR using a primer set (F3/R3) (Table 1) that specifically amplifies the junction containing exon 8 and exon 10. Two fragments (550 bp and 715 bp) were amplified from mRNA extracted from the cerebellum, and both fragments were cloned and sequenced (Data not shown).
The 550 bp fragment lacks exon 9, on the other hand, in the 715 bp fragment, exon 9 was inserted between exon 8 and exon 10 (see Fig. 4A; the blue bar for the 550 bp fragment and the red bar for the 715 fragment). Next, we performed RT-PCR using another primer set (F4/R4) (Table 1) that specifically amplifies a 1.2 kbp fragment of iPLA2 -2 (see Fig. 4A; green bar). The 1.2 kbp fragment was amplified from mRNA extracted from the cerebellum (Data not shown). Furthermore, we performed in situ hybridization using a DIG-labeled RNA probe specific for the exon 9 sequence, and a moderate signal was observed in PCs (Fig. 4B). These results show that both the soluble form (iPLA2 -1) and membrane-bound forms (at least, iPLA2 -2) of iPLA2 are expressed in PCs.
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S H I R A I and I TO
Fig. 4. Expression of iPLA2 membrane-bound form in Purkinje cells. A: A schematic of the exon-intron structure of iPLA2 mRNA (explanation is in the text). Boxes illustrate the exons. A 550 bp fragment specific to iPLA2 -1 (blue bar), or a 715 bp fragment specific to iPLA2 -2, iPLA2 -3, and iPLA2 -ankyrin-1 (red bars) were amplified by RT-PCR using the primer set F3/R3. A 1.2 kbp fragment specific to iPLA2 -2 (green bar) was amplified using another primer set F4/R4. B: expression of membrane-bound form of iPLA2 . mRNA was visualized by in situ hybridization with DIG-labeled 165 bp antisense RNA probes specific for exon 9. Scale bar indicates 100 µm.
Phospholipase A2 subtypes in cerebellum
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Fig. 5. Expression of sPLA2 IIA and sPLA2 V in rat cerebellum. A: immunoreactivity of sPLA2 IIA detected using anti-sPLA2 IIA polyclonal antibody (Cayman) and Alexa 546. B: expression of sPLA2 IIA mRNA visualized by in situ hybridization with DIGlabeled 441 bp antisense RNA probes. C: immunoreactivity detected using anti-sPLA2 V polyclonal antibody (Cayman) and Alexa 546. D: expression of sPLA2 V mRNA visualized by in situ hybridization with DIG-labeled 414 bp antisense RNA probes. The staining pattern of sPLA2 V is typical of the morphology of Bergmann glia cells (C). Scale bars indicate 100 µm.
DISTRIBUTION OF SECRETORY PLA 2 ( S PLA 2 )
sPLA2 IIA sPLA2 IIA immunoreactivity was observed in perinuclear regions of PCs (Fig. 5A), consistent with the reported localization of sPLA2 IIA in the endoplasmic reticulum (ER) in bone marrow-derived mast cells (Bingham et al., 1999) and HEK293 cells (Murakami et al., 1999). sPLA2 IIA immunoreactivity was moderate in granule cells and weak in the molecular layer. The weak diffuse immunoreactivity of sPLA2 IIA seen in the molecular layer could indicate sPLA2 IIA expression in PC dendrites, but other sources such as parallel fibers or Bergmann glia cells cannot be excluded. In situ hybridization revealed a moderate to weak expression of sPLA2 IIA mRNA in the PC layer, particularly in the
PC somata (Fig. 5B). A weak expression was observed in the granule cell layer. Weak signals were also detected in the olfactory bulb and hippocampal CA1-3, while only very weak signals were observed in the amygdala, basal forebrain, cerebral cortex and midbrain (Data not shown). sPLA2 V sPLA2 V immunoreactivity was detected in the molecular and PC layers, with a characteristic staining pattern suggesting sPLA2 V localization in Bergmann glia cells (Fig. 5C). In situ hybridization revealed a weak expression of sPLA2 V mRNA in the Purkinje cell layer, which, however, was not observed in PCs, but presumably in Bergmann glia cells (Fig. 5D).
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Fig. 6. Specific differential expression of PLA2 subtypes in rat cerebellum. In D, small dots in ML indicate weak diffuse expression of sPLA2 IIA.
Weak signals of sPLA2 V were also detected in the olfactory bulb and hippocampal CA1-3, while only very weak signals were observed in the basal forebrain, cerebral cortex and midbrain (Data not shown).
Other sPLA2 s We investigated the expression of the other nine sPLA2 s (sPLA2 IB, sPLA2 IIC, sPLA2 IID, sPLA2 IIE, sPLA2 IIF, sPLA2 III, sPLA2 X, sPLA2 XII-1, and sPLA2 XII-2) by the PCR method using the rat or mouse cDNA library. However, none of these sPLA2 s, except sPLA2 IIC, was amplified from the rat whole-brain cDNA library or mouse whole-brain cDNA library by PCR (Data not shown). sPLA2 IIC mRNA was amplified from the rat brain cDNA library by PCR, however, no significant signals of sPLA2 IIC mRNA were observed in the cerebellum by in situ hybridization (Data not shown). Hence, there is no evidence indicating the presence of these other sPLA2 s in rat cerebella, but, except for sPLA2 IIC, we cannot immediately negate their presence because the negative results might be due to the absence of cDNA or PCR primers might not been effective.
Discussion The present results are summarized in Fig. 6A–E. Of the five subtypes of PLA2 expressed in rat cerebellum (cPLA2 α, cPLA2 β, iPLA2 , sPLA2 IIA and sPLA2 V) three are present in PCs. cPLA2 α is present specifically in PC dendrites and the cytoplasm of PC somata (A). sPLA2 IIA is present in the perinuclear regions of PCs and is possibly very weakly expressed in their dendrites (D), while iPLA2 is expressed preferentially in the nucleus of PCs (C). In contrast, cPLA2 β is strongly expressed in granule cells (B), and sPLA2 V in Bergmann glia cells (E). ROLES OF C PLA 2 α
The preferential localization of cPLA2 α in dendritic synapses of PCs favors the view that these subtypes play roles in LTD induction in parallel fiber-to-PC synapses. cPLA2 α is activated through its phosphorylation by mitogen-associated protein kinase (MAPK) (Lin et al., 1993), which is activated via the Ras-RafMAPK kinase (MEK) cascade. Phosphorylated cPLA2 α increases the production of AA, which in PCs would activate protein kinase C (Nishizuka, 1992), that is the
Phospholipase A2 subtypes in cerebellum α and/or β isoform (Hirono et al., 2001). PKC in turn activates MAPK. In a computer simulation study, Kuroda et al. (2001) assumed that the closed positive feedback loop including cPLA2 α, AA, PKC, and MAPK plays a role in the self-regenerating development of LTD. However, this hypothesis includes still unverified assumptions (Ito, 2002). First, since AA is known to inhibit PLA2 (Bonventre, 1992), the self-regenerating development of LTD is questionable. Second, although exogenous AA counteracts for the effect of PLA2 inhibitors in LTD induction (Linden, 1995), it is unknown whether AA induces LTD. ROLES OF S PLA 2 IIA
The preferential localization of sPLA2 IIA in PCs indicates the possibility that this subtype also plays roles in LTD induction. Mepacrine and manoalide that Linden (1995) used to block LTD are nonspecific inhibitors of PLA2 s. Manoalide sensitively inhibits low-molecularweight secretory subtypes of PLA2 including sPLA2 IIA (Jacobson et al., 1990). This leaves the possibility that sPLA2 IIA plays a role in LTD induction. Mice deficient in both cPLA2 α and sPLA2 IIA exhibit no significant abnormalities in either histological structures of the brain or behavior (Uozumi et al., 1997; Bonventre et al., 1997). This may not negate the possibility that either cPLA2 α or sPLA2 IIA PLA2 s is required for LTD induction, because NOS- or GFAP-deficient mice show few of such phenotypes even when they lack LTD (Shibuki et al., 1996; Lev-Ram et al., 1997). It remains to be confirmed whether LTD is lacking in mice deficient in cPLA2 α or sPLA2 IIA.
305 less, AA produced by these cells would diffuse beyond membranes, possibly reaching PCs. Because Bergmann glia cell processes surround parallel fiber-PC (PF-PC) synapses, it is possible that AA produced via the action of sPLA2 V in Bergmann glia cells plays a role in LTD. The participation of some sPLA2 subtype(s) in signal transduction through a heparan sulfate proteoglycan (HSPG) has been suggested (Murakami et al., 2000). sPLA2 IIA binds to glypican-1, a glycosylphosphatidylinositol (GPI)-anchored HSPG, on the cell surface, through endocytotic pathways, and is transported to nuclear envelopes or ER membranes, from which AA is liberated (Murakami et al., 1998; Murakami et al., 1999). Since both sPLA2 IIA and sPLA2 V are expressed in the cerebellum, and glypican-1 is expressed in PC nuclei (Liang et al., 1997), there is a possibility that secreted sPLA2 proteins can affect PCs, for example, through glypican-1. In summary, not only cPLA2 α, but also at least four other subtypes of PLA2 (cPLA2 β, iPLA2 , sPLA2 IIA, and sPLA2 V) are expressed in the rat cerebellum (Fig. 6). All of these have the potential to be involved in LTD induction. Further pharmacological and genetic analyses are required to determine their different roles in the cerebellum.
Acknowledgments We thank Ms. Hiroko Ono for technical assistance, and Dr. Tsutomu Hashikawa and Dr. Shogo Endo for helpful discussions.
ROLES OF iPLA 2
References
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