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Differential expression of SAPK isoforms in the rat brain. An in situ hybridisation study in the adult rat brain and during post-natal development
Molecular Brain Research 60 Ž1998. 57–68
Research report
Differential expression of SAPK isoforms in the rat brain. An in situ hybridisation study in the adult rat brain and during post-natal development Lucia Carboni ) , Renzo Carletti, Stefano Tacconi, Corrado Corti, Francesco Ferraguti Department of Pharmacology, Glaxo Wellcome Medicines Research Centre, Verona, Italy Accepted 7 July 1998
Abstract MAPK pathways transduce a broad variety of extracellular signals into cellular responses. Despite their pleiotropic effects and their ubiquitous distribution, surprisingly little is known about their involvement in the communication network of nerve cells. As a first step to elucidate the role of MAPK pathways in neuronal signalling, we studied the distribution of SAPK arJNK2, SAPK brJNK3, and SAPK grJNK1, three isoforms of SAPKrJNK, a stress-activated MAPK subfamily. We compared the mRNA localisation of the three main isoforms in the adult and developing rat brain using in situ hybridisation. In the adult brain, SAPK a and b were widely but heterogeneously distributed, reproducing the pattern of a probe that does not discriminate the isoforms. Differently, high labelling for the SAPK g probe was exclusively localised in the endopiriform nucleus and medial habenula. Intermediate staining was detected in the hippocampus. During post-natal development, SAPK b showed the same localisation as in the adult. Nevertheless, the semi-quantitative analysis of optical densities showed significantly different mRNA levels. In the adult, SAPK g signal was weak, whereas in newborn rats the labelling was intense and widely distributed. SAPK g mRNA levels decreased during development, to reach the low signals detected in the adult. These results suggest that in the central nervous system SAPK-type MAP kinases perform significant physiological functions which are particularly relevant during post-natal development. The distinct distribution patterns of SAPK isoforms in the adult rat brain support the hypothesis that separate functions are performed by the products of the three SAPK genes. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Stress-activated protein kinase; JNK; MAPK; Central nervous system
1. Introduction Mitogen-activated protein kinase ŽMAPK. pathways transduce extracellular stimuli into cellular responses both by direct phosphorylation of target proteins and by transactivation of transcription factors, which consequently modulate gene expression. Typical MAPK cascades are composed of three enzymes, which are sequentially activated by phosphorylation: a MAPK kinase kinase ŽMAPKKK.; a MAPK kinase ŽMAPKK.; and a MAPK ŽFig. 1.. Several parallel pathways have been described in mammalian cells w24x. Stress-activated protein kinasesrc-Jun NH 2-terminal kinases ŽSAPKsrJNKs. belong to a more recently discov) Corresponding author. Molecular Pharmacology, Glaxo Wellcome Medicines Research Centre, via A. Fleming, 4, 37135 Verona, Italy. Fax: q39-45-9218174; E-mail: [email protected]
ered MAPK pathway as compared to extracellular-signal regulated kinases ŽERKs.. SAPKrJNK cascade is stimulated by stressful signals, such as osmotic stress, ultraviolet and ionising radiation, heat shock, and reperfusion after ischemia w22,17x. It is also activated by physiological stimuli, such as hematopoietic cytokines w11x, and G protein-coupled receptors w5x. The activation of SAPKrJNK pathway has been associated with many final cellular responses, some of which generate opposite outcomes. Depending on cell or tissue type and experimental conditions, SAPKrJNK stimulation has been reported to induce as different effects as apoptosis w32x, rescue from apoptosis w21x, proliferation w19x, and differentiation w33x. How such a wide range of specific responses can be obtained by the activation of MAPK is not presently clear. The existence of multiple kinases which belong to the stress-activated cascade may contribute to these diverse
0169-328Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 1 6 6 - 1
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Fig. 1. The MAPK pathway transduces extracellular stimuli through a cascade of activated kinases, ending in phosphorylation of transcription factors and cellular proteins. For the SAPK pathway, many different enzymes exist which can work as activators of MAPKKK; as MAPKKK; as MAPKK, and as MAPK. STE20-HOMs: mammalian kinases homologous to STE20, the activator of MAPKKK in S. cereÕisiae. 3pK: chromosome 3p kinase; ASK: apoptosis signal-regulated kinase; ATF2: activating transcription factor 2; DLK: dual leucine zipper bearing kinase; GCK: germinal centre kinase; GLK: GCK-like kinase; HPK: hematopoietic progenitor kinase; HSF: heat shock factor; JNKK: JNK kinase; KHS: kinase homologous to SPS1rSTE20; MEKK: MAPK Erk kinase kinase; MKK: MAPK kinase; MLK: mixed lineage kinase; MST: mammalian STE20-like protein kinase; MTK: MAP three kinase; MUK: MAPK-upstream kinase; NFAT: nuclear factor of activated T cells; NIK: Nck interacting kinase; PAK: p21-activated protein kinase; PTK: protein tyrosine kinase; SEK: SAPK ERK kinase; SPRK: Src-homology 3 domain-containing proline-rich Kinase; TAK: TGF b-activated kinase; Tpl-2: tumor progression locus 2; ZPK: zipper protein kinase.
physiological responses. Many distinct enzymes have been discovered that diverge in selectivity and tissue distribution, and which act as MAPKKK w8x, or MAPKK w22,28x in the SAPKrJNK pathway ŽFig. 1.. Many distinct isoforms of SAPKrJNK have been reported as well, which exhibit different properties of affinity and substrate specificity w7,13,15,16,20,26x. In the rat, three main SAP kinases, probably arising from three separate genes, have been described. They have been named SAPK a , b and g w6,16x. Each SAPK gene gives rise to two RNA variants of different length, which diverge at the 3X-end. The two mRNA forms are translated into a short Žp46. or long
Žp55. protein. Moreover, SAPK a exhibits an additional site of alternative splicing in protein kinase subdomains IX and X, therefore producing four a isoforms. In human tissue, the three homologous gene products have been called JNK 2, 3 Žor p49 3F12 . and 1, respectively. Shorter and longer mRNA forms were reported for each gene. The corresponding site of alternative splicing that was characterised for SAPK a has been described for both JNK2 and 1, therefore creating ten isoforms in all ŽFig. 1. w7,13,15,20x. Available data show that the central nervous system ŽCNS. is highly enriched in SAPK mRNA and protein, which are widely and heterogeneously distributed in brain
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w3,7,13,18,29x. As compared with other tissues, brain displays a higher baseline activation of SAPKrJNK, activation that can be modulated by environmental stimuli w29x. However, no detailed description of the distribution of the different isoforms has yet been reported. At least certain isoforms appear to mediate specific activities. Indeed, products of the Jnk3 r SAPK b r p49 3F12 gene, which are selectively expressed in brain w20,18x, are reported to mediate glutamate signalling and excitotoxicity in hippocampus w31x. Therefore, a detailed analysis of the relative distribution of individual isoforms in brain was needed to provide a neuroanatomical basis on which to support hypotheses as to their relative function. The aim of this investigation was to study the localisation of the three SAPK gene products both in the adult brain and during post-natal development. To this end, we performed in situ hybridisation with oligonucleotide probes which specifically recognised each isoform.
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mm thick sections; groups of three consecutive slices were cut, to be hybridised to each probe. Sections were thawmounted on poly-L-lysine-coated slides and stored at y808C until the day of the experiment. Groups of three rats at different post-natal days ŽP: 1, 3, 6, 9, 12, 15, 18 and 21. were decapitated after urethane anaesthesia; brains were removed, frozen and cut as previously described. 2.2. Probe choice, synthesis and labelling Forty-five-mer sequences were chosen in the SAPK a , b and g cDNAs. SAPK a probe was complementary to nucleotides 1914–1958 in sequence L27111, SAPK b to 189-233 in sequence L27128, SAPK g to 131-175 in sequence L27129 in GenBank. The specificity of the probes was evaluated with FastA in the Genetic Computer Group ŽGCG, 1991. package. Each sequence was specific for the selected isoform and did not display any significant similarity with the other isoforms or with other sequences in databases. As several splice variants have been identified for SAPK a , b and g, the probes were chosen in a region that do not distinguish between the different splice variants. Oligodeoxynucleotides were synthesised on a Millipore Expedite 8909 DNA synthesiser using a standardcyanoethyl phosphoramidite chemistry. Phosphoramidites were purchased from Millipore, Milan, Italy. Forty-fivemers were subsequently purified by reverse-phase chromatography on Cruachem cartridges ŽGlasgow, UK. following the manufacturer’s instructions. The probes were 3X-end-labelled with w35 SxdATP Žspecific activity 1100 Cirmmol, from New England Nuclear, Frankfurt, Germany. using terminal transferase ŽAmersham Italy, Milan,
2. Materials and methods 2.1. Tissue preparation Adult male Ž300 g. and pregnant female Sprague–Dawley rats were purchased from Charles River, Calco, Italy. The research complied with national legislation and with the company policy on the Care of Use of Animals and with related codes of practice. Adult animals Ž n s 7. were deeply anaesthetised with urethane Ž1.5 grkg, i.p.. and intracardially perfused with ice-cold saline. Brains were immediately removed, frozen in dry-ice cold isopentane and cryostat-cut into coronal, parasagittal or horizontal 14
Table 1 Semi-quantitative analysis of in situ hybridisation signals at different stages of post-natal development A
STR CTX DG CA1 CA3
P1
P3
P6
P9
P12
P15
P18
P21
Adult
113 " 4)) 162 " 6) 236 " 7 240 " 7 284 " 10
89 " 4)) 128 " 5 214 " 8 203 " 10 234 " 10))
65 " 3)) 119 " 5)) 193 " 6 206 " 6 257 " 9))
27 " 2)) 64 " 4)) 108 " 6)) 128 " 7)) 157 " 9))
33 " 4 76 " 6)) 115 " 10)) 130 " 10)) 164 " 10))
36 " 3) 93 " 6)) 137 " 5)) 176 " 8)) 212 " 9))
31 " 4)) 115 " 5)) 160 " 9)) 207 " 9 238 " 11))
52 " 4 160 " 5 217 " 10 256 " 10)) 308 " 11
47 " 2 142 " 4 209 " 6 217 " 7 300 " 7
P1
P3
P6
P9
P12
P15
P18
P21
Adult
37 " 4)) 80 " 5)) 82 " 5)) 92 " 4)) 97 " 5)) 122 " 7))
31 " 3)) 68 " 5)) 84 " 6)) 81 " 5)) 89 " 6)) 114 " 8))
32 " 3)) 74 " 4)) 88 " 5)) 103 " 5)) 126 " 6)) 148 " 11))
14 " 7 34 " 6)) 56 " 6)) 67 " 8)) 73 " 9)) 103 " 19
7"3 36 " 3)) 55 " 5)) 58 " 5)) 66 " 6)) 109 " 20)
10 " 2 34 " 3)) 47 " 5) 50 " 5)) 72 " 6)) 142 " 11))
B SRR CTX DG CA1 CA3 MHB
14 " 1 32 " 2)) 49 " 4) 41 " 2)) 59 " 3)) 78 " 8
9"2 21 " 2 37 " 3 29 " 3 45 " 3 97 " 9
Table shows mean " S.E.M. of relative optical density values measured in film autoradiographs. Panel A displays values measured in slides treated with the SAPK b probe whereas panel B shows values for SAPK g. P: post-natal day; STR: striatum; CTX: cerebral cortex; DG: dentate gyrus; CA1 and CA3: hippocampal fields; MHB: medial habenula. ) p - 0.05 and )) p - 0.01 in Dunnett’s test vs. adult values ŽSNK results are not shown in the table but are discussed in the text..
8"1 15 " 1 28 " 2 19 " 2 35 " 2 74 " 3
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Italy.. Radiolabelled oligonucleotides were separated from unincorporated nucleotides using Nensorb columns ŽNew England Nuclear, Frankfurt, Germany. according to manufacturer’s instructions. Specific activity of the 35 S y labelled probes was routinely between 5 = 10 5 and 8 = 10 5 cpmrml probe. 2.3. In situ hybridisation histochemistry Hybridisations were carried out as previously described w3,4x. Sections were exposed to Amersham 3 H y Hyperfilms and developed after 18–25 days. Afterwards they were dipped in Kodak NTB2 photographic emulsion, exposed for 8 to 12 weeks at y208C, developed for 2 min in Kodak D19 developer, fixed and counterstained with Cresyl violet. In order to assess the amount of non-specific hybridisation, adjacent sections were incubated with 100fold excess unlabelled probe added together with the radioactive oligodeoxynucleotide. 2.4. Analysis of results Microdensitometric analyses of film autoradiographs were performed using a computer imaging device ŽImaging Research, Canada.. The specific optical density of sampled areas was evaluated by subtracting the optical density of the non-specific labelling Žas defined by the mean optical density measured in sections treated with excess of unlabelled probe.. The atlas of Paxinos and Watson w23x was used to identify brain structures. Each experiment contained all experimental groups. Samples belonging to the various experimental groups of each experiment were treated under the very same conditions and hybridisation carried out with the same radiolabelled probe. The statistical analysis of optical density values measured in brain areas at different post-natal days was performed by ANOVA, followed by the Student–Newman–Keuls ŽSNK. test or the Dunnett’s post-hoc comparison. In Table 1 only the results of Dunnett’s test Žvs. adult levels. are shown.
3. Results 3.1. Localisation of SAPK a , b , and g hybridisation signals in adult rat brain In order to analyse the expression pattern of SAPK isoforms in rat brain, three 45-mer probes were chosen.
Fig. 2. In situ hybridisation film autoradiographs of parasagittal sections of adult rat brain. Each section was hybridised with an oligonucleotide probe that specifically recognised SAPK a ŽPanel A. or SAPK b ŽPanel B. or SAPK g ŽPanel C.. The hybridisation signals obtained with the SAPK a and SAPK b probes are widely although heterogeneously distributed in the adult brain and appear to be localised in the very same brain regions. On the other hand, SAPK g specific labelling is restricted to fewer brain areas. Scale bar: 2.7 mm.
Fig. 3. In situ hybridisation film autoradiographs showing a rostrocaudal series of adult rat brain coronal sections hybridised with oligonucleotide probes that recognise SAPK b ŽPanels a, b, c, g, h, i, m, n, o. or SAPK g ŽPanels d, e, f, j, k, l, p, q, r.. The hybridisation signal obtained with SAPK g probe shows high levels only in the endopiriform nucleus ŽEn. and in the medial habenula ŽMHb.. Panels m and p show sections hybridised with a 100-fold excess of unlabelled SAPK b or g oligonucleotide, respectively, used to evaluate non-specific binding ŽNSB.. Scale bar: 1.6 mm.
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Each probe was designed to specifically recognise only one of the three SAPK Ž a , b or g . isoforms w16x, as evaluated with FastA Žsee Section 2.. The probes were unable to discriminate among the splice variants of every isoform, since they were chosen in conserved regions. Consecutive coronal and parasagittal slices were cut from adult rat brains and hybridised to the three labelled probes. Non-specific labelling was measured in slices hybridised to each probe in the presence of 100-fold excess of the corresponding unlabelled oligonucleotide. The hybridisation patterns were compared between each other and with the signal obtained with a probe which does not distinguish among the three isoforms Ž pan-SAPK probe, Ref. w3x.. The hybridisation signals obtained with the probes for SAPK a and b were generally localised in the same brain areas, although the signal intensity was slightly different ŽFig. 2, Panels A, B.. The hybridisation of the probes for SAPK a and b showed a similar pattern to that detected with the pan-SAPK probe w3x. On the other hand, SAPK g labelling appeared markedly different, with only few brain areas intensely labelled ŽFigs. 2 and 3.. High mRNA levels were measured exclusively in the endopiriform nucleus ŽFig. 3, Panels f, j, k, l. and in the medial habenula ŽFig. 3, Panels q, r.. Intermediate to low staining was detected in CA1 and CA3 fields of the pyramidal layer of the hippocampus and in the granule cell layer of the dentate gyrus ŽDG. ŽFig. 3, Panels q, r.. Intermediate to low levels were also observed in the piriform cortex ŽFig. 3, Panels d, e, f, j, k.. SAPK g hybridisation signal appeared weak in the majority of the brain regions analysed, with low levels in the cerebral cortex, diencephalon, basal ganglia, and brainstem ŽFig. 3..
In order to obtain cellular resolution of the hybridisation signal, selected sections were processed for emulsion autoradiography. Given the overlap of hybridisation pattern between SAPK b and SAPK a , and the selective expression of SAPK b in the CNS, all subsequent experiments were processed only with the SAPK b and SAPK g probes. Strong SAPK b labelling was found on the vast majority of neuronal-like cell bodies of the cerebral cortex, hippocampus and diencephalic areas. Grain density over striatal cellular profiles was substantially lower. The labelling pattern of the cerebral cortex followed the cellular architectural with no apparent differences between layers or cortical sub-areas. Although specific labelling appeared to be located mainly over neuronal cells, the presence of mRNA in glial cells cannot be ruled out. The identification of labelled small cell bodies was often difficult in sections processed for in situ hybridisation. Very intense labelling was observed in the principal cells of the pyramidal cell layer of the hippocampal fields CA1 and CA3 and the subiculum, as well as in the granule cells of the dentate gyrus ŽFig. 4.. Marked staining was also detected in numerous interneuron-like cells, in particular in the stratum oriens of CA1 and CA3 fields. A large number of cellular profiles in the polymorphic area of the dentate gyrus were also intensely labelled ŽFig. 4.. In the habenula, both SAPK b and SAPK g hybridisations produced a moderate labelling of all the medium-small sized cells in the medial areas ŽFig. 5.. On the other hand, in the lateral part SAPK g signal appeared consistently lower ŽFig. 5.. Ependimal cells of the third ventricle clearly showed no hybridisation signal for SAPK g, whereas several silver grains could be detected with the SAPK b probe. In the
Fig. 4. Dark-field photomicrograph of hippocampus in a coronal section of adult rat brain hybridised with SAPK b probe. Pyramidal cells in CA1 and CA3 fields and the granule cells in the dentate gyrus ŽDG. show a high hybridisation signal. 3V: third ventricle. Scale bar: 500 mm.
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Fig. 5. Dark-field ŽPanels A, C. or bright-field ŽPanels B, D. photomicrographs of the medial habenula in coronal sections of adult rat brain. Upper panels ŽA and B. show sections hybridised with SAPK b probe; lower panels ŽC and D. show sections hybridised with SAPK g probe. MHb: medial habenula; LHb: lateral habenula. In panels B and D the medial habenula is shown on the left and is delimited by a dotted line. In cells of the medial habenula both SAPK b and g probes produce a moderate signal. In the lateral habenula the hybridisation signal is low for SAPK g, whereas SAPK b maintains an intermediate level. Scale bars: in dark-field panels: 350 mm; in bright-field panels: 70 mm.
endopiriform nucleus of slices processed with the SAPK g probe, many large neuronal cell bodies, identified with Cresyl violet staining, were strongly labelled with silver grains ŽFig. 6.. SAPK b hybridisation was also detected over endopiriform neuronal cells, although apparently with lower number of transcript copies ŽFig. 6.. In the cerebellum, Purkinje cells appeared labelled only with the SAPK b probe ŽFig. 7.. In those slides hybridised with the SAPK g probe occasional silver grains could be detected over Purkinje cells, although with density and distribution resembling that of the background ŽFig. 7.. In the granular layer relatively few SAPK g hybridisation grains were found. However, they showed a higher density with res-
pect to the background, suggesting a very low, yet specific, level of expression. On the other hand, SAPK b labelling in the granular layer was intermediate and diffuse with respect to other brain areas. 3.2. Localisation of SAPK b and g hybridisation signals in post-natal deÕeloping rat brain Consecutive horizontal sections from adult and postnatal day ŽP. 1, 3, 6, 9, 12, 15, 18 and 21 rat brains were hybridised at the same time and under identical conditions with SAPK b or SAPK g probes. Hybridisation patterns
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Fig. 6. Dark-field ŽPanels A, C. or bright-field ŽPanels B, D. photomicrographs of the endopiriform nucleus in coronal sections of adult rat brain. Upper panels ŽA and B. show sections hybridised with a probe that recognises SAPK b; lower panels ŽC and D. show sections hybridised with the SAPK g probe. The probe for SAPK g strongly labels cell bodies in the endopiriform nucleus ŽEn.. SAPK b staining is detected as well, although at lower intensity. CPu: caudate putamen; cc: corpus callosum. Scale bars: in dark-field panels: 350 mm; in bright-field panels: 45 mm.
obtained with the probes at different stages of development were studied. As illustrated in Fig. 8, at the sampled stages of post-natal development, the labelling for SAPK b
showed the same localisation pattern as that observed in the adult Župper panels.. Interestingly, the SAPK g probe showed a high and widespread hybridisation signal which
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Fig. 7. Bright-field photomicrographs of coronal sections of the adult rat cerebellar cortex. Panel A shows a section hybridised with the oligonucleotide probe that recognises SAPK b, whereas the section depicted in panel B was hybridised with the SAPK g probe. Purkinje cells ŽP, pointed by arrows. are labelled only by the probe for SAPK b. In the granule cell layer Žg. SAPK b shows an intermediate level of staining and SAPK g displays a low signal. ml: molecular layer. Scale bar: 50 mm.
decreased substantially during development to reach the highly discrete staining observed in the adult ŽFig. 8, lower panels..
age analysis system. The optical densities were compared in the sampled stages of development in order to evaluate whether mRNA levels were modulated.
3.3. Semi-quantitatiÕe analysis of SAPK b and g labelling during post-natal deÕelopment
3.3.1. SAPK b In the striatum, optical density values were significantly higher at P1 as compared with later days Ž p - 0.01 vs. any other group in the SNK test.. The signal decreased slowly at P3 and P6, maintaining levels that were both different to any other group Ž p - 0.01, SNK test.. Optical density values reached a minimum at P9 and remained essentially unmodified to match those measured in the adult ŽTable 1, Panel A.. SAPK b labelling in the cerebral cortex and the
Hybridisation signals of SAPK b and g were measured in specific brain regions at different stages of post-natal development. Sampled regions were selected according to feasibility of sampling and presence of high hybridisation signal. The microdensitometric analysis of film autoradiograms was performed by means of a computer-based im-
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Fig. 8. In situ hybridisation film autoradiographs of rat brain horizontal sections at different post-natal developmental stages hybridised with oligonucleotide probes which recognise SAPK b Župper panels. or SAPK g Žlower panels.. SAPK b maintains throughout post-natal development the same localisation observed in the adult brain. SAPK g hybridisation signal is high and widely distributed in the rat brain at early post-natal days ŽP.. During development signal intensity decreases substantially in the majority of brain areas remaining relatively high only in the endopiriform nucleus and in the medial habenula ŽMHb.. A: adult. Scale bar: 2.7 mm.
hippocampal fields CA1, CA3, and DG showed a different temporal pattern, with a degree of hybridisation in the early stages of post-natal development comparable to that measured in the adult. Starting from P3, optical density values started to decrease, reaching a minimum at P9 and P12. Signals measured at P9 and P12 were not significantly different, but were different from any other group Ž p - 0.01, SNK test.. At P15, the labelling increased again, reaching adult levels at P21 ŽTable 1, Panel A.. 3.3.2. SAPK g Statistically significant differences in optical density values for SAPK g were observed during development. In the hippocampus, the pyramidal cell layer in CA3 and CA1 displayed high hybridisation signals at P1 and P3 that slightly increased at P6 Ž p - 0.01 vs. any other group, SNK test.. Starting from P9, mRNA levels progressively decreased to reach those observed in the adult ŽTable 1, Panel B.. In caudate-putamen, high mRNA levels were also detected at P1 and remained high until P6. Afterwards, optical density values steeply decreased. At P9, signals appeared as low as those detected in the adult ŽTable 1, Panel B.. The same temporal trend was observed in the cerebral cortex and DG, but optical density values from P9 to P18 were slightly higher than in the adult ŽTable 1, Panel B.. On the other hand, medial habenula displayed a high hybridisation signal from P1 and re-
mained high throughout the post-natal development. A slight reduction was observed at P18 and P21, which exhibited the same levels as in adult ŽTable 1, Panel B.. 4. Discussion We studied by means of in situ hybridisation with specific oligonucleotide probes the localisation of the mRNA transcripts of the three SAPK genes in the adult rat brain. In addition, we investigated the mRNA expression of SAPK b and g during post-natal development. Our results show that SAPK a and b are the main isoforms expressed in the adult brain. The probes that recognise these isoforms marked the same brain regions and reproduced the same hybridisation pattern observed with a probe that does not distinguish among the different SAPK variants. On the other hand, the SAPK g probe revealed a selective localisation in the adult brain, with high levels restricted to the endopiriform nucleus and the medial habenula. In these regions, the presence of the other isoforms could be detected as well. Hybridisation signals for SAPK a , b and g were all present in the hippocampus, with SAPK g showing the lowest signal. We detected low labelling for all of the three isoforms in striatum. Nevertheless, SAPK protein has been detected in this region, and its activity can be stimulated by glutamate w25x.
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In newborn rats, SAPK b appeared expressed in the same brain areas as in adults. A generalised reduction in the levels of expression was found between P9 and P12. Values from P15 to the adult returned gradually to those measured in the early days after birth. On the other hand, SAPK g showed a completely different pattern, with high and diffuse expression in newborn rats. Labelling for this isoform progressively declined during post-natal development, to become restricted to few areas in the adult rat. Interestingly, these findings are consistent with a previous observation that JNK1 mRNA could be detected with Northern blot analysis in human foetal but not adult brain w7x, suggesting a specific role being played by this kinase during development. A function for the stress-activated MAPKs during development is supported by studies in Drosophila. Kinases belonging to the fruitfly homologue of SAPK pathway ŽDJNKrBsk and Hep. exert an essential role in embryonic development w12,27x. This function is likely maintained in mammals, as targeted disruption of the MKK4 gene, a MAPKK for SAPK, causes embryonic death in mice w30x. SAPK brJNK3 protein is highly expressed in mouse embryonic brain w18x, and SAPK arJNK2 is localised in neuroepithelium of chick embryos w14x. In this study, we report a regional regulation of SAPK b and g mRNA levels during different post-natal developmental stages, thus suggesting that this cascade may exert a role in the formation of synaptic circuitry. The mRNA levels of both SAPK b and g varied during the stages of post-natal development. SAP kinase activity is usually reported to be regulated through the control of its phosphorylated state exerted by MAPKK, instead of through the modulation of mRNA transcription or protein translation. Our data suggest that this level of control may be important as well, at least in the CNS and in agreement with Ferrer et al., who report a variation in SAPK immunoreactivity in rat brain after excitotoxic stimuli w9,10x. The different distribution pattern of SAPK isoforms in the adult rat brain suggests the existence of separate, non-redundant, functions being performed by the products of the three SAPK genes. A large body of evidence supports the hypothesis that different isoforms mediate different outputs. Indeed, proteins deriving from each gene Ž Jnk1, 2 or 3 . exhibit diverse selectivity and affinity for the transcription factors whose activity they modulate w1,6,13,15,26x. It has been suggested that, at physiological concentrations in cells, only one of the isoforms is able to phosphorylate a specific transcription factor, therefore inducing a distinct gene expression response w13,15x. Sluss et al. reported that JNK1, but not JNK2, complements a defect in the expression of the HOG MAPK in Saccaromyces cereÕisiae, demonstrating that the isoforms have different properties w26x. Distinct SAPK forms have been suggested to be responsible for the activation of protective or apoptotic responses in small cell lung cancer cells w2x
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and only a p46 SAPK mediates the c-myc-dependent apoptosis in rat fibroblasts w34x. Only one SAPK isoform, which may correspond to SAPK brJNK3, or to a still unknown subtype, has been involved in the induction of staurosporine-mediated differentiation in PC12 cells w33x. Furthermore, the generation of mice with targeted disruption of individual Jnk genes has provided evidence for selectivity of function among isoforms, in particular in the brain. Mice lacking the expression of the Jnk3 gene displayed reduced sensitivity to kainic acid-induced toxicity, which was not shared by the Jnk1Žyry . or Jnk2 Žyry . animals w31x. In conclusion, this study shows that SAPK isoforms are differently localised in specific brain regions. The selective localisation supports the hypothesis that specific functions are performed by each isoform. This study also reports a different modulation of mRNA levels of SAPK isoforms in the developing rat brain. These findings suggest that SAPK isoforms may be differently involved in signal transduction during post-natal development.
Acknowledgements The authors wish to thank Dr. Valerie Matarese and Dr. Francesco Belardetti for critical review of the manuscript and helpful suggestions.
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Research report
Differential expression of SAPK isoforms in the rat brain. An in situ hybridisation study in the adult rat brain and during post-natal development Lucia Carboni ) , Renzo Carletti, Stefano Tacconi, Corrado Corti, Francesco Ferraguti Department of Pharmacology, Glaxo Wellcome Medicines Research Centre, Verona, Italy Accepted 7 July 1998
Abstract MAPK pathways transduce a broad variety of extracellular signals into cellular responses. Despite their pleiotropic effects and their ubiquitous distribution, surprisingly little is known about their involvement in the communication network of nerve cells. As a first step to elucidate the role of MAPK pathways in neuronal signalling, we studied the distribution of SAPK arJNK2, SAPK brJNK3, and SAPK grJNK1, three isoforms of SAPKrJNK, a stress-activated MAPK subfamily. We compared the mRNA localisation of the three main isoforms in the adult and developing rat brain using in situ hybridisation. In the adult brain, SAPK a and b were widely but heterogeneously distributed, reproducing the pattern of a probe that does not discriminate the isoforms. Differently, high labelling for the SAPK g probe was exclusively localised in the endopiriform nucleus and medial habenula. Intermediate staining was detected in the hippocampus. During post-natal development, SAPK b showed the same localisation as in the adult. Nevertheless, the semi-quantitative analysis of optical densities showed significantly different mRNA levels. In the adult, SAPK g signal was weak, whereas in newborn rats the labelling was intense and widely distributed. SAPK g mRNA levels decreased during development, to reach the low signals detected in the adult. These results suggest that in the central nervous system SAPK-type MAP kinases perform significant physiological functions which are particularly relevant during post-natal development. The distinct distribution patterns of SAPK isoforms in the adult rat brain support the hypothesis that separate functions are performed by the products of the three SAPK genes. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Stress-activated protein kinase; JNK; MAPK; Central nervous system
1. Introduction Mitogen-activated protein kinase ŽMAPK. pathways transduce extracellular stimuli into cellular responses both by direct phosphorylation of target proteins and by transactivation of transcription factors, which consequently modulate gene expression. Typical MAPK cascades are composed of three enzymes, which are sequentially activated by phosphorylation: a MAPK kinase kinase ŽMAPKKK.; a MAPK kinase ŽMAPKK.; and a MAPK ŽFig. 1.. Several parallel pathways have been described in mammalian cells w24x. Stress-activated protein kinasesrc-Jun NH 2-terminal kinases ŽSAPKsrJNKs. belong to a more recently discov) Corresponding author. Molecular Pharmacology, Glaxo Wellcome Medicines Research Centre, via A. Fleming, 4, 37135 Verona, Italy. Fax: q39-45-9218174; E-mail: [email protected]
ered MAPK pathway as compared to extracellular-signal regulated kinases ŽERKs.. SAPKrJNK cascade is stimulated by stressful signals, such as osmotic stress, ultraviolet and ionising radiation, heat shock, and reperfusion after ischemia w22,17x. It is also activated by physiological stimuli, such as hematopoietic cytokines w11x, and G protein-coupled receptors w5x. The activation of SAPKrJNK pathway has been associated with many final cellular responses, some of which generate opposite outcomes. Depending on cell or tissue type and experimental conditions, SAPKrJNK stimulation has been reported to induce as different effects as apoptosis w32x, rescue from apoptosis w21x, proliferation w19x, and differentiation w33x. How such a wide range of specific responses can be obtained by the activation of MAPK is not presently clear. The existence of multiple kinases which belong to the stress-activated cascade may contribute to these diverse
0169-328Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 1 6 6 - 1
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Fig. 1. The MAPK pathway transduces extracellular stimuli through a cascade of activated kinases, ending in phosphorylation of transcription factors and cellular proteins. For the SAPK pathway, many different enzymes exist which can work as activators of MAPKKK; as MAPKKK; as MAPKK, and as MAPK. STE20-HOMs: mammalian kinases homologous to STE20, the activator of MAPKKK in S. cereÕisiae. 3pK: chromosome 3p kinase; ASK: apoptosis signal-regulated kinase; ATF2: activating transcription factor 2; DLK: dual leucine zipper bearing kinase; GCK: germinal centre kinase; GLK: GCK-like kinase; HPK: hematopoietic progenitor kinase; HSF: heat shock factor; JNKK: JNK kinase; KHS: kinase homologous to SPS1rSTE20; MEKK: MAPK Erk kinase kinase; MKK: MAPK kinase; MLK: mixed lineage kinase; MST: mammalian STE20-like protein kinase; MTK: MAP three kinase; MUK: MAPK-upstream kinase; NFAT: nuclear factor of activated T cells; NIK: Nck interacting kinase; PAK: p21-activated protein kinase; PTK: protein tyrosine kinase; SEK: SAPK ERK kinase; SPRK: Src-homology 3 domain-containing proline-rich Kinase; TAK: TGF b-activated kinase; Tpl-2: tumor progression locus 2; ZPK: zipper protein kinase.
physiological responses. Many distinct enzymes have been discovered that diverge in selectivity and tissue distribution, and which act as MAPKKK w8x, or MAPKK w22,28x in the SAPKrJNK pathway ŽFig. 1.. Many distinct isoforms of SAPKrJNK have been reported as well, which exhibit different properties of affinity and substrate specificity w7,13,15,16,20,26x. In the rat, three main SAP kinases, probably arising from three separate genes, have been described. They have been named SAPK a , b and g w6,16x. Each SAPK gene gives rise to two RNA variants of different length, which diverge at the 3X-end. The two mRNA forms are translated into a short Žp46. or long
Žp55. protein. Moreover, SAPK a exhibits an additional site of alternative splicing in protein kinase subdomains IX and X, therefore producing four a isoforms. In human tissue, the three homologous gene products have been called JNK 2, 3 Žor p49 3F12 . and 1, respectively. Shorter and longer mRNA forms were reported for each gene. The corresponding site of alternative splicing that was characterised for SAPK a has been described for both JNK2 and 1, therefore creating ten isoforms in all ŽFig. 1. w7,13,15,20x. Available data show that the central nervous system ŽCNS. is highly enriched in SAPK mRNA and protein, which are widely and heterogeneously distributed in brain
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w3,7,13,18,29x. As compared with other tissues, brain displays a higher baseline activation of SAPKrJNK, activation that can be modulated by environmental stimuli w29x. However, no detailed description of the distribution of the different isoforms has yet been reported. At least certain isoforms appear to mediate specific activities. Indeed, products of the Jnk3 r SAPK b r p49 3F12 gene, which are selectively expressed in brain w20,18x, are reported to mediate glutamate signalling and excitotoxicity in hippocampus w31x. Therefore, a detailed analysis of the relative distribution of individual isoforms in brain was needed to provide a neuroanatomical basis on which to support hypotheses as to their relative function. The aim of this investigation was to study the localisation of the three SAPK gene products both in the adult brain and during post-natal development. To this end, we performed in situ hybridisation with oligonucleotide probes which specifically recognised each isoform.
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mm thick sections; groups of three consecutive slices were cut, to be hybridised to each probe. Sections were thawmounted on poly-L-lysine-coated slides and stored at y808C until the day of the experiment. Groups of three rats at different post-natal days ŽP: 1, 3, 6, 9, 12, 15, 18 and 21. were decapitated after urethane anaesthesia; brains were removed, frozen and cut as previously described. 2.2. Probe choice, synthesis and labelling Forty-five-mer sequences were chosen in the SAPK a , b and g cDNAs. SAPK a probe was complementary to nucleotides 1914–1958 in sequence L27111, SAPK b to 189-233 in sequence L27128, SAPK g to 131-175 in sequence L27129 in GenBank. The specificity of the probes was evaluated with FastA in the Genetic Computer Group ŽGCG, 1991. package. Each sequence was specific for the selected isoform and did not display any significant similarity with the other isoforms or with other sequences in databases. As several splice variants have been identified for SAPK a , b and g, the probes were chosen in a region that do not distinguish between the different splice variants. Oligodeoxynucleotides were synthesised on a Millipore Expedite 8909 DNA synthesiser using a standardcyanoethyl phosphoramidite chemistry. Phosphoramidites were purchased from Millipore, Milan, Italy. Forty-fivemers were subsequently purified by reverse-phase chromatography on Cruachem cartridges ŽGlasgow, UK. following the manufacturer’s instructions. The probes were 3X-end-labelled with w35 SxdATP Žspecific activity 1100 Cirmmol, from New England Nuclear, Frankfurt, Germany. using terminal transferase ŽAmersham Italy, Milan,
2. Materials and methods 2.1. Tissue preparation Adult male Ž300 g. and pregnant female Sprague–Dawley rats were purchased from Charles River, Calco, Italy. The research complied with national legislation and with the company policy on the Care of Use of Animals and with related codes of practice. Adult animals Ž n s 7. were deeply anaesthetised with urethane Ž1.5 grkg, i.p.. and intracardially perfused with ice-cold saline. Brains were immediately removed, frozen in dry-ice cold isopentane and cryostat-cut into coronal, parasagittal or horizontal 14
Table 1 Semi-quantitative analysis of in situ hybridisation signals at different stages of post-natal development A
STR CTX DG CA1 CA3
P1
P3
P6
P9
P12
P15
P18
P21
Adult
113 " 4)) 162 " 6) 236 " 7 240 " 7 284 " 10
89 " 4)) 128 " 5 214 " 8 203 " 10 234 " 10))
65 " 3)) 119 " 5)) 193 " 6 206 " 6 257 " 9))
27 " 2)) 64 " 4)) 108 " 6)) 128 " 7)) 157 " 9))
33 " 4 76 " 6)) 115 " 10)) 130 " 10)) 164 " 10))
36 " 3) 93 " 6)) 137 " 5)) 176 " 8)) 212 " 9))
31 " 4)) 115 " 5)) 160 " 9)) 207 " 9 238 " 11))
52 " 4 160 " 5 217 " 10 256 " 10)) 308 " 11
47 " 2 142 " 4 209 " 6 217 " 7 300 " 7
P1
P3
P6
P9
P12
P15
P18
P21
Adult
37 " 4)) 80 " 5)) 82 " 5)) 92 " 4)) 97 " 5)) 122 " 7))
31 " 3)) 68 " 5)) 84 " 6)) 81 " 5)) 89 " 6)) 114 " 8))
32 " 3)) 74 " 4)) 88 " 5)) 103 " 5)) 126 " 6)) 148 " 11))
14 " 7 34 " 6)) 56 " 6)) 67 " 8)) 73 " 9)) 103 " 19
7"3 36 " 3)) 55 " 5)) 58 " 5)) 66 " 6)) 109 " 20)
10 " 2 34 " 3)) 47 " 5) 50 " 5)) 72 " 6)) 142 " 11))
B SRR CTX DG CA1 CA3 MHB
14 " 1 32 " 2)) 49 " 4) 41 " 2)) 59 " 3)) 78 " 8
9"2 21 " 2 37 " 3 29 " 3 45 " 3 97 " 9
Table shows mean " S.E.M. of relative optical density values measured in film autoradiographs. Panel A displays values measured in slides treated with the SAPK b probe whereas panel B shows values for SAPK g. P: post-natal day; STR: striatum; CTX: cerebral cortex; DG: dentate gyrus; CA1 and CA3: hippocampal fields; MHB: medial habenula. ) p - 0.05 and )) p - 0.01 in Dunnett’s test vs. adult values ŽSNK results are not shown in the table but are discussed in the text..
8"1 15 " 1 28 " 2 19 " 2 35 " 2 74 " 3
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Italy.. Radiolabelled oligonucleotides were separated from unincorporated nucleotides using Nensorb columns ŽNew England Nuclear, Frankfurt, Germany. according to manufacturer’s instructions. Specific activity of the 35 S y labelled probes was routinely between 5 = 10 5 and 8 = 10 5 cpmrml probe. 2.3. In situ hybridisation histochemistry Hybridisations were carried out as previously described w3,4x. Sections were exposed to Amersham 3 H y Hyperfilms and developed after 18–25 days. Afterwards they were dipped in Kodak NTB2 photographic emulsion, exposed for 8 to 12 weeks at y208C, developed for 2 min in Kodak D19 developer, fixed and counterstained with Cresyl violet. In order to assess the amount of non-specific hybridisation, adjacent sections were incubated with 100fold excess unlabelled probe added together with the radioactive oligodeoxynucleotide. 2.4. Analysis of results Microdensitometric analyses of film autoradiographs were performed using a computer imaging device ŽImaging Research, Canada.. The specific optical density of sampled areas was evaluated by subtracting the optical density of the non-specific labelling Žas defined by the mean optical density measured in sections treated with excess of unlabelled probe.. The atlas of Paxinos and Watson w23x was used to identify brain structures. Each experiment contained all experimental groups. Samples belonging to the various experimental groups of each experiment were treated under the very same conditions and hybridisation carried out with the same radiolabelled probe. The statistical analysis of optical density values measured in brain areas at different post-natal days was performed by ANOVA, followed by the Student–Newman–Keuls ŽSNK. test or the Dunnett’s post-hoc comparison. In Table 1 only the results of Dunnett’s test Žvs. adult levels. are shown.
3. Results 3.1. Localisation of SAPK a , b , and g hybridisation signals in adult rat brain In order to analyse the expression pattern of SAPK isoforms in rat brain, three 45-mer probes were chosen.
Fig. 2. In situ hybridisation film autoradiographs of parasagittal sections of adult rat brain. Each section was hybridised with an oligonucleotide probe that specifically recognised SAPK a ŽPanel A. or SAPK b ŽPanel B. or SAPK g ŽPanel C.. The hybridisation signals obtained with the SAPK a and SAPK b probes are widely although heterogeneously distributed in the adult brain and appear to be localised in the very same brain regions. On the other hand, SAPK g specific labelling is restricted to fewer brain areas. Scale bar: 2.7 mm.
Fig. 3. In situ hybridisation film autoradiographs showing a rostrocaudal series of adult rat brain coronal sections hybridised with oligonucleotide probes that recognise SAPK b ŽPanels a, b, c, g, h, i, m, n, o. or SAPK g ŽPanels d, e, f, j, k, l, p, q, r.. The hybridisation signal obtained with SAPK g probe shows high levels only in the endopiriform nucleus ŽEn. and in the medial habenula ŽMHb.. Panels m and p show sections hybridised with a 100-fold excess of unlabelled SAPK b or g oligonucleotide, respectively, used to evaluate non-specific binding ŽNSB.. Scale bar: 1.6 mm.
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Each probe was designed to specifically recognise only one of the three SAPK Ž a , b or g . isoforms w16x, as evaluated with FastA Žsee Section 2.. The probes were unable to discriminate among the splice variants of every isoform, since they were chosen in conserved regions. Consecutive coronal and parasagittal slices were cut from adult rat brains and hybridised to the three labelled probes. Non-specific labelling was measured in slices hybridised to each probe in the presence of 100-fold excess of the corresponding unlabelled oligonucleotide. The hybridisation patterns were compared between each other and with the signal obtained with a probe which does not distinguish among the three isoforms Ž pan-SAPK probe, Ref. w3x.. The hybridisation signals obtained with the probes for SAPK a and b were generally localised in the same brain areas, although the signal intensity was slightly different ŽFig. 2, Panels A, B.. The hybridisation of the probes for SAPK a and b showed a similar pattern to that detected with the pan-SAPK probe w3x. On the other hand, SAPK g labelling appeared markedly different, with only few brain areas intensely labelled ŽFigs. 2 and 3.. High mRNA levels were measured exclusively in the endopiriform nucleus ŽFig. 3, Panels f, j, k, l. and in the medial habenula ŽFig. 3, Panels q, r.. Intermediate to low staining was detected in CA1 and CA3 fields of the pyramidal layer of the hippocampus and in the granule cell layer of the dentate gyrus ŽDG. ŽFig. 3, Panels q, r.. Intermediate to low levels were also observed in the piriform cortex ŽFig. 3, Panels d, e, f, j, k.. SAPK g hybridisation signal appeared weak in the majority of the brain regions analysed, with low levels in the cerebral cortex, diencephalon, basal ganglia, and brainstem ŽFig. 3..
In order to obtain cellular resolution of the hybridisation signal, selected sections were processed for emulsion autoradiography. Given the overlap of hybridisation pattern between SAPK b and SAPK a , and the selective expression of SAPK b in the CNS, all subsequent experiments were processed only with the SAPK b and SAPK g probes. Strong SAPK b labelling was found on the vast majority of neuronal-like cell bodies of the cerebral cortex, hippocampus and diencephalic areas. Grain density over striatal cellular profiles was substantially lower. The labelling pattern of the cerebral cortex followed the cellular architectural with no apparent differences between layers or cortical sub-areas. Although specific labelling appeared to be located mainly over neuronal cells, the presence of mRNA in glial cells cannot be ruled out. The identification of labelled small cell bodies was often difficult in sections processed for in situ hybridisation. Very intense labelling was observed in the principal cells of the pyramidal cell layer of the hippocampal fields CA1 and CA3 and the subiculum, as well as in the granule cells of the dentate gyrus ŽFig. 4.. Marked staining was also detected in numerous interneuron-like cells, in particular in the stratum oriens of CA1 and CA3 fields. A large number of cellular profiles in the polymorphic area of the dentate gyrus were also intensely labelled ŽFig. 4.. In the habenula, both SAPK b and SAPK g hybridisations produced a moderate labelling of all the medium-small sized cells in the medial areas ŽFig. 5.. On the other hand, in the lateral part SAPK g signal appeared consistently lower ŽFig. 5.. Ependimal cells of the third ventricle clearly showed no hybridisation signal for SAPK g, whereas several silver grains could be detected with the SAPK b probe. In the
Fig. 4. Dark-field photomicrograph of hippocampus in a coronal section of adult rat brain hybridised with SAPK b probe. Pyramidal cells in CA1 and CA3 fields and the granule cells in the dentate gyrus ŽDG. show a high hybridisation signal. 3V: third ventricle. Scale bar: 500 mm.
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Fig. 5. Dark-field ŽPanels A, C. or bright-field ŽPanels B, D. photomicrographs of the medial habenula in coronal sections of adult rat brain. Upper panels ŽA and B. show sections hybridised with SAPK b probe; lower panels ŽC and D. show sections hybridised with SAPK g probe. MHb: medial habenula; LHb: lateral habenula. In panels B and D the medial habenula is shown on the left and is delimited by a dotted line. In cells of the medial habenula both SAPK b and g probes produce a moderate signal. In the lateral habenula the hybridisation signal is low for SAPK g, whereas SAPK b maintains an intermediate level. Scale bars: in dark-field panels: 350 mm; in bright-field panels: 70 mm.
endopiriform nucleus of slices processed with the SAPK g probe, many large neuronal cell bodies, identified with Cresyl violet staining, were strongly labelled with silver grains ŽFig. 6.. SAPK b hybridisation was also detected over endopiriform neuronal cells, although apparently with lower number of transcript copies ŽFig. 6.. In the cerebellum, Purkinje cells appeared labelled only with the SAPK b probe ŽFig. 7.. In those slides hybridised with the SAPK g probe occasional silver grains could be detected over Purkinje cells, although with density and distribution resembling that of the background ŽFig. 7.. In the granular layer relatively few SAPK g hybridisation grains were found. However, they showed a higher density with res-
pect to the background, suggesting a very low, yet specific, level of expression. On the other hand, SAPK b labelling in the granular layer was intermediate and diffuse with respect to other brain areas. 3.2. Localisation of SAPK b and g hybridisation signals in post-natal deÕeloping rat brain Consecutive horizontal sections from adult and postnatal day ŽP. 1, 3, 6, 9, 12, 15, 18 and 21 rat brains were hybridised at the same time and under identical conditions with SAPK b or SAPK g probes. Hybridisation patterns
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Fig. 6. Dark-field ŽPanels A, C. or bright-field ŽPanels B, D. photomicrographs of the endopiriform nucleus in coronal sections of adult rat brain. Upper panels ŽA and B. show sections hybridised with a probe that recognises SAPK b; lower panels ŽC and D. show sections hybridised with the SAPK g probe. The probe for SAPK g strongly labels cell bodies in the endopiriform nucleus ŽEn.. SAPK b staining is detected as well, although at lower intensity. CPu: caudate putamen; cc: corpus callosum. Scale bars: in dark-field panels: 350 mm; in bright-field panels: 45 mm.
obtained with the probes at different stages of development were studied. As illustrated in Fig. 8, at the sampled stages of post-natal development, the labelling for SAPK b
showed the same localisation pattern as that observed in the adult Župper panels.. Interestingly, the SAPK g probe showed a high and widespread hybridisation signal which
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Fig. 7. Bright-field photomicrographs of coronal sections of the adult rat cerebellar cortex. Panel A shows a section hybridised with the oligonucleotide probe that recognises SAPK b, whereas the section depicted in panel B was hybridised with the SAPK g probe. Purkinje cells ŽP, pointed by arrows. are labelled only by the probe for SAPK b. In the granule cell layer Žg. SAPK b shows an intermediate level of staining and SAPK g displays a low signal. ml: molecular layer. Scale bar: 50 mm.
decreased substantially during development to reach the highly discrete staining observed in the adult ŽFig. 8, lower panels..
age analysis system. The optical densities were compared in the sampled stages of development in order to evaluate whether mRNA levels were modulated.
3.3. Semi-quantitatiÕe analysis of SAPK b and g labelling during post-natal deÕelopment
3.3.1. SAPK b In the striatum, optical density values were significantly higher at P1 as compared with later days Ž p - 0.01 vs. any other group in the SNK test.. The signal decreased slowly at P3 and P6, maintaining levels that were both different to any other group Ž p - 0.01, SNK test.. Optical density values reached a minimum at P9 and remained essentially unmodified to match those measured in the adult ŽTable 1, Panel A.. SAPK b labelling in the cerebral cortex and the
Hybridisation signals of SAPK b and g were measured in specific brain regions at different stages of post-natal development. Sampled regions were selected according to feasibility of sampling and presence of high hybridisation signal. The microdensitometric analysis of film autoradiograms was performed by means of a computer-based im-
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Fig. 8. In situ hybridisation film autoradiographs of rat brain horizontal sections at different post-natal developmental stages hybridised with oligonucleotide probes which recognise SAPK b Župper panels. or SAPK g Žlower panels.. SAPK b maintains throughout post-natal development the same localisation observed in the adult brain. SAPK g hybridisation signal is high and widely distributed in the rat brain at early post-natal days ŽP.. During development signal intensity decreases substantially in the majority of brain areas remaining relatively high only in the endopiriform nucleus and in the medial habenula ŽMHb.. A: adult. Scale bar: 2.7 mm.
hippocampal fields CA1, CA3, and DG showed a different temporal pattern, with a degree of hybridisation in the early stages of post-natal development comparable to that measured in the adult. Starting from P3, optical density values started to decrease, reaching a minimum at P9 and P12. Signals measured at P9 and P12 were not significantly different, but were different from any other group Ž p - 0.01, SNK test.. At P15, the labelling increased again, reaching adult levels at P21 ŽTable 1, Panel A.. 3.3.2. SAPK g Statistically significant differences in optical density values for SAPK g were observed during development. In the hippocampus, the pyramidal cell layer in CA3 and CA1 displayed high hybridisation signals at P1 and P3 that slightly increased at P6 Ž p - 0.01 vs. any other group, SNK test.. Starting from P9, mRNA levels progressively decreased to reach those observed in the adult ŽTable 1, Panel B.. In caudate-putamen, high mRNA levels were also detected at P1 and remained high until P6. Afterwards, optical density values steeply decreased. At P9, signals appeared as low as those detected in the adult ŽTable 1, Panel B.. The same temporal trend was observed in the cerebral cortex and DG, but optical density values from P9 to P18 were slightly higher than in the adult ŽTable 1, Panel B.. On the other hand, medial habenula displayed a high hybridisation signal from P1 and re-
mained high throughout the post-natal development. A slight reduction was observed at P18 and P21, which exhibited the same levels as in adult ŽTable 1, Panel B.. 4. Discussion We studied by means of in situ hybridisation with specific oligonucleotide probes the localisation of the mRNA transcripts of the three SAPK genes in the adult rat brain. In addition, we investigated the mRNA expression of SAPK b and g during post-natal development. Our results show that SAPK a and b are the main isoforms expressed in the adult brain. The probes that recognise these isoforms marked the same brain regions and reproduced the same hybridisation pattern observed with a probe that does not distinguish among the different SAPK variants. On the other hand, the SAPK g probe revealed a selective localisation in the adult brain, with high levels restricted to the endopiriform nucleus and the medial habenula. In these regions, the presence of the other isoforms could be detected as well. Hybridisation signals for SAPK a , b and g were all present in the hippocampus, with SAPK g showing the lowest signal. We detected low labelling for all of the three isoforms in striatum. Nevertheless, SAPK protein has been detected in this region, and its activity can be stimulated by glutamate w25x.
L. Carboni et al.r Molecular Brain Research 60 (1998) 57–68
In newborn rats, SAPK b appeared expressed in the same brain areas as in adults. A generalised reduction in the levels of expression was found between P9 and P12. Values from P15 to the adult returned gradually to those measured in the early days after birth. On the other hand, SAPK g showed a completely different pattern, with high and diffuse expression in newborn rats. Labelling for this isoform progressively declined during post-natal development, to become restricted to few areas in the adult rat. Interestingly, these findings are consistent with a previous observation that JNK1 mRNA could be detected with Northern blot analysis in human foetal but not adult brain w7x, suggesting a specific role being played by this kinase during development. A function for the stress-activated MAPKs during development is supported by studies in Drosophila. Kinases belonging to the fruitfly homologue of SAPK pathway ŽDJNKrBsk and Hep. exert an essential role in embryonic development w12,27x. This function is likely maintained in mammals, as targeted disruption of the MKK4 gene, a MAPKK for SAPK, causes embryonic death in mice w30x. SAPK brJNK3 protein is highly expressed in mouse embryonic brain w18x, and SAPK arJNK2 is localised in neuroepithelium of chick embryos w14x. In this study, we report a regional regulation of SAPK b and g mRNA levels during different post-natal developmental stages, thus suggesting that this cascade may exert a role in the formation of synaptic circuitry. The mRNA levels of both SAPK b and g varied during the stages of post-natal development. SAP kinase activity is usually reported to be regulated through the control of its phosphorylated state exerted by MAPKK, instead of through the modulation of mRNA transcription or protein translation. Our data suggest that this level of control may be important as well, at least in the CNS and in agreement with Ferrer et al., who report a variation in SAPK immunoreactivity in rat brain after excitotoxic stimuli w9,10x. The different distribution pattern of SAPK isoforms in the adult rat brain suggests the existence of separate, non-redundant, functions being performed by the products of the three SAPK genes. A large body of evidence supports the hypothesis that different isoforms mediate different outputs. Indeed, proteins deriving from each gene Ž Jnk1, 2 or 3 . exhibit diverse selectivity and affinity for the transcription factors whose activity they modulate w1,6,13,15,26x. It has been suggested that, at physiological concentrations in cells, only one of the isoforms is able to phosphorylate a specific transcription factor, therefore inducing a distinct gene expression response w13,15x. Sluss et al. reported that JNK1, but not JNK2, complements a defect in the expression of the HOG MAPK in Saccaromyces cereÕisiae, demonstrating that the isoforms have different properties w26x. Distinct SAPK forms have been suggested to be responsible for the activation of protective or apoptotic responses in small cell lung cancer cells w2x
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and only a p46 SAPK mediates the c-myc-dependent apoptosis in rat fibroblasts w34x. Only one SAPK isoform, which may correspond to SAPK brJNK3, or to a still unknown subtype, has been involved in the induction of staurosporine-mediated differentiation in PC12 cells w33x. Furthermore, the generation of mice with targeted disruption of individual Jnk genes has provided evidence for selectivity of function among isoforms, in particular in the brain. Mice lacking the expression of the Jnk3 gene displayed reduced sensitivity to kainic acid-induced toxicity, which was not shared by the Jnk1Žyry . or Jnk2 Žyry . animals w31x. In conclusion, this study shows that SAPK isoforms are differently localised in specific brain regions. The selective localisation supports the hypothesis that specific functions are performed by each isoform. This study also reports a different modulation of mRNA levels of SAPK isoforms in the developing rat brain. These findings suggest that SAPK isoforms may be differently involved in signal transduction during post-natal development.
Acknowledgements The authors wish to thank Dr. Valerie Matarese and Dr. Francesco Belardetti for critical review of the manuscript and helpful suggestions.
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