- Email: [email protected]
Stress activated protein kinases, a novel family of mitogen-activated protein kinases, are heterogeneously expressed in the adult rat brain and differentially distributed from extracellular-signal-regulated protein kinases
Neuroscience Vol. 69, No. 4, pp. 1103-I 110, 1995
Elsevier Science Ltd Copyright 0 1995 IBRO
Pergamon
0306-4522(95)00284-7
Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
STRESS ACTIVATED PROTEIN KINASES, A NOVEL FAMILY OF MITOGEN-ACTIVATED PROTEIN KINASES, ARE HETEROGENEOUSLY EXPRESSED IN THE ADULT RAT BRAIN AND DIFFERENTIALLY DISTRIBUTED FROM EXTRACELLULAR-SIGNAL-REGULATED PROTEIN KINASES R. CARLETTI, Department
S. TACCONI,
of Pharmacology,
Glaxo
E. BETTINI
Research
Laboratories,
and
F. FERRAGUTI*
Via Fleming
4, 37100 Verona,
Italy
Abstract-Mitogen-activated protein kinases are important mediators of signal transduction from the cell surface to the nucleus and their activation has been implicated in a wide array of physiological processes. The extracellular-signal-regulated kinases are the archetypal and best studied members of the mitogen activated protein kinases. Recently, additional subgroups of mitogen activated protein kinases have been identified which exhibit distinct regulatory elements, substrate specificity and respond to diverse extracellular stimuli. Among these newly identified protein kinases are the rat stress-activated protein kinases. Despite a rapidly expanding literature on the biochemical properties of stress-activated protein kinases no anatomical data are yet available. In the present study, we have investigated the regional distribution of messenger RNA transcripts for stress-activated protein kinases in the adult rat central nervous system and compared this distribution to that observed for extracellular-signal-regulated kinases. Intense labelling for stress-activated protein kinases could be detected in discrete brain areas with high levels in hippocampus, neocortex and some nuclei of the brain stem. The apparent hybridization signal appeared to be selectively neuronal. Stress-activated protein kinases and extracellular-signal-regulated kinases hybridization patterns appeared generally dissimilar although a certain degree of co-expression in some brain areas, such as the hippocampal formation, could he observed.
These results reveal an extreme complexity in the mitogen-activated protein kinase signalling pathway and suggest the existence of parallel mitogen-activated protein kinase cascades that can be activated independently or in some cases simultaneously, by extracellular stimuli. Key words: SAPK,
ERK,
MAPK,
in situ hybridization,
The mitogen-activated protein kinase (MAPK) cascade is a major signalling system by which cells transduce extracellular stimuli into intracellular responses.6 MAPKs comprise a family of serine/threonine protein kinases which share the unique feature of being activated by phosphorylation on threonine and tyrosine residues by an upstream dualspecificity kinase.2 Until recently, the extracellularsignal-regulated kinases (ERKs) 1 and 2 were the only cloned and relatively well characterized MAPKs. Extracellular-signal-regulated kinases are expressed ubiquitously in vertebrate tissues, nevertheless they appear to be particularly enriched in the developing and adult CNS.3,4 Independent research efforts have recently led to the identification of a new subfamily of MAPKs: the rat stress-activated protein
*To whom correspondence
should be addressed.
Abbreviufions: AD, Alzheimer’s disease; ATFZ, activating transcription factor 2; ERK, extracellular-signal-regulated protein kinase; JNK, c-Jun NH,-terminal kinase; MAPK, mitogen-activated protein kinase; SAPK, stressactivated protein kinase; SEKl, SAPK/ERK kinase; SSC, saline sodium citrate.
central
nervous
system.
kinases (SAPKS)‘~ and the human homologues c-Jun NH,-terminal kinases (JNKs).~ These kinases show 4&45% identity to ERKs, sharing the requirement of both tyrosine and threonine phosphorylation for activation and proline-directed substrate specificity.16 SAPKs/JNKs are activated as a result of the cellular response to intra- and extracellular stress such as heat shock, ultraviolet radiation, protein synthesis inhibitors, inflammatory cytokines and sphingomyelinase all of which only poorly activate ERKs.jrJ6 Three closely related (88-90% identity) classes of SAPKs have been identified from a rat brain cDNA library and designated SAPK-c(, -B and -y.16 The only information available about their anatomical localization is a general description that they are ubiquitously expressed, at low levels, throughout the body. I6 Notably, the upstream regulatory kinase SAPK/ERK kinase 1 (SEKl) is also ubiquitously expressed, as demonstrated by northern blot analysis, although at very high levels in brain and muscle.20 In the present study, the regional distribution of mRNA transcripts for SAPKs in the adult rat CNS was investigated by means of in situ 1103
R. Carletti ez al.
1104
hybridization with 3SS-labelled oligonucleotide probes and compared with that of the well-characterized MAPKs, ERKl and ERK2. SAPK probes were selected for recognizing at once all three classes.
EXPERIMENTAL
PROCEDURES
Materials Adult SpragucDawley rats (> 200 g) were obtained from Charles River, Calco, Italy. [3sS]dATP, specific activity 1100 Ci/mmol was from New England Nuclear, Frankfurt, Germany. Phosphoramidites for oligonucleotide synthesis were purchased from Millipore, Milano, Italy. In situ hybridization histochemistry Animals were deeply anaesthetized with urethane (1.5 g/kg, i.p.) and intracardially perfused with ice cold saline. Brains were immediately removed, frozen in dry-ice cold isopentane and kept at - 80°C until sectioned (14 pmthick-sections) for in situ hybridization studies. Antisense oligonucleotide probes (45mers) were synthesized on a Millipore Expedite 8909 DNA synthesizer complementary to mRNAs of ERKs4 and SAPKs.i6 The ERKs antisense oligonucleotide probes were complementary to nucleotides 141&1454 of ERKl (Gene Bank accession number M61177) and to nucleotides 1201-1245 of ERK2 (Gene Bank accession number M64300). The SAPK probe was complementary to nucleotides 592636 of the mRNA for SAPKa (Gene Bank accession number L27111) a region with 87-98% identity to the other classes of SAPKs. Thus, the probe recognized all the known classes of SAPKs. The percentage homology between oligonucleotide probes and full length sequences was calculated using the GCG (Genetics Computer Group, 1991) package. Oligonucleotides were 3’end-labelled with [35S]dATP. Radiolabelled oligonucleotides were separated from unincorporated nucleotides using Nensorb columns (New England Nuclear) according to manufacturer instructions. Specific activity of the ?Slabelled probes was routinely between 5 x 10’ and 8 x 10’c.p.m./nl probe. Hybridizations were performed essentially as described by Wisden et al.*’Following hybridization, sections were washed twice for 5min at room temperature with 2 x saline sodium citrate (SSC), further washed 4 times for 15 min at 55°C with 0.5 x SSC, rinsed briefly at room temperature in 1 x SSC, 70% ethanol and 95% ethanol and air-dried prior to apposing to Amersham ‘H-Ultrofilms. After appropriate exposure, films were developed and sections were dipped in NTB2 photographic emulsion (Kodak). Sections were exposed -for four- to five weeks at - 20°C. develoued for 2 min in Kodak D19 develooer and counterstained ‘with Cresyl Violet. Adjacent sections were hybridized with excess of unlabelled probe to assess the specificity of hybridization. An identical labelling pattern was observed with different non-overlapping probes. RESULTS
Film or emulsion autoradiographic evaluation of in situ hybridization revealed a strikingly heterogeneous distribution of mRNA for SAPKs throughout the brain. The hybridization was seen to be specific since non-significant hybridization was observed in parallel
experiments using the same labelled probes in the presence of unlabelled probe. The film autoradiography showed strong signal intensity in the hippocampal formation, the cerebral cortex, the granular layer of the cerebellar cortex and the facial nucleus.
Moderate levels of hybridization signal were also observed in diencephalic areas, in septal and basal forebrain regions, in part of the amygdaloid complex and in some nuclei of the brain stem. Results from representative brain sections are illustrated in Figs 1A and 2. The autoradiographs were semiquantitatively evaluated by means of computer-assisted image analysis and results are summarized in Table 1. Specific optical density values were expressed as a percentage of the maximum value observed (hippocampal CA3 pyramidal cell layer). Specific labelling appeared to be located over nerve cell bodies in all brain areas analysed, whereas no apparent labelling was found over glial cells and capillaries (Fig. 3). In neocortical regions, many neuronal cell bodies, of both pyramidal and non-pyramidal neurons were weakly- or moderately-labelled, particularly in layers IV and V (Figs 2 and 3A, 3B). In the limbic cortical regions, the hippocampal formation contained highly-labelled cell bodies of pyramidal neurons. Within the hippocampus, an uneven localization of the message was also found. The CAl/CA2 and dentate gyrus fields displayed lower levels of hybridization signals than that in the CA3 field, although they contained high levels of SAPK-mRNA (Figs 4A and B). Labelling in the subcortical regions of the forebrain was generally weak to moderate. Semiquantitative analysis of autoradiographs showed a high level of relative optical density in the medial habenular nucleus which after emulsion dipping appeared to consist of moderately-labelled neurons. This discrepancy is due to the high density of small neurons contained in this brain area. The majority of neurons in thalamic nuclei were weakly- to moderately-labelled for SAPK-mRNA. Of the forebrain regions, the basal ganglia demonstrated the lowest relative optical density. Striatum, globus pallidus and ventral pallidum contained only scattered, weaklylabelled neurons (Figs 2A and 3C). In the substantia nigra, the pars compacta contained few moderatelylabelled cell bodies, while no hybridization signal was observed in the pars reticulata. Film autoradiographs showed high hybridization signal in the granular layer of the cerebellar cortex (Fig. 2G and H). In emulsion dipped brain sections, however, individual granule neurons were only moderately-labelled (Fig. SA and C). This is due, as mentioned above, to the high cell density of this cerebellar cell layer. Moreover, in the cerebellar cortex Purkinje neurons were also found to be moderately-labelled (Fig. 5A). mRNA for SAPKs in brain stem regions appeared to be discretely localized in relatively few nuclei with the highest level of hybridization signal present in the facial nucleus (Figs 2G and SD). The spinal trigeminal nucleus displayed a low relative optical density due to dispersed distribution of neuronal cell bodies which in emulsion dipped sections appeared generally strongly labelled (Fig. 5E). ERKs mRNA levels in adult rat brain were also investigated and compared to those observed for
SAPKs in adult rat central nervous system
Fig. 1. Distribution of mRNAs for SAPKs, ERKl and ERK2 in adult rat brain. Darkfield photogr ot autoradiograms illustrating the distribution of SAPK-mRNA (‘panel A), ERKI-mRNA (panel B) and ERK2-mRNA (panel C) in parasagittal brain sections. Cb, cerebellar cortex; CPU, caudate-putamen; Cx, cerebral cortex; DG, dentate gyrus; Hi, hippocampus; Th, thalamus; 7, facial nucleus. Scale bar = 1.6 mm.
SAPKs.
ERKl
mRNA
transcripts
were
confined
primarily to dentate gyrus granule cells (Fig. 1B) although hybridization signal was also detected in other areas such as the locus coeruleus and the hypothalamus. ERK2, however, was more widely distributed throughout the rat brain (Fig. 1C) a NSC 69,4--E
finding
which
is in agreement
with
previous
re-
ports. 15z2* Earlier in situ hybridization and immunohistochemical studies reported that ERKs are also localized in reactive astrocytes and in oligodendrocytes, and that ERKs-immunoreactivity in neurons was restricted to cell bodies and dendrites.“~‘5~22
Fig. 2. 1106
SAPKs
in adult rat central nervous system
1107
Table 1. Distribution of stress-activated protein kinase mRNA in the adult rat CNS Relative Optical Density
Regions of CNS Neocortex Frontal cortex Occipital cortex Parietal cortex Temporal cortex Limbic cortex Cingulate cortex Dentate gyrus, granule cells Entorhinal cortex Piriform cortex Retrosplenial cortex Subiculum Hippocampus CAl, pyramidal layer CA3, pyramidal layer Amygdala Basolateral amygdaloid nucleus Basomedial amygdaloid n., anterior p. Central nucleus Lateral amygdaloid nucleus Thalamus Anterodorsal nucleus Lateral geniculate nucleus Medial geniculate nucleus Mediodorsal nucleus Parafascicular nucleus Posterior nuclear group Reticular nucleus Ventromedial nucleus Ventroposterior nuclear group Subthalamus Subthalamic nucleus Zona Incerta Medial habenular nucleus Lateral habenular nucleus Hypothalamus Arcuate nucleus Medial mammillary n., medial part Medial mammillary n., posterior part Premammillary n., dorsal part
++ ++ ++ ++ ++ +++ ++ +++ ++ ++ +++ ++++ ++ ++ + ++ + ++ ++ ++ + ++ + ++ ++ ++ ++ + +++ + ++ ++ + ++
Regions of CNS Posterior hypothalamic area Supramammillary nucleus Tuberomammillary nucleus Ventromedial nucleus Basal Ganglia Accumbcns n. Caudate-putamen Globus pallidus Substantia nigra, pars compacta Substantia nigra, pars reticulata Ventral tegmental area Septal and Basal Forebrain Regions Bed nucleus of the stria terminalis Lateral septal n., dorsal part Lateral septal n., intermediate part Lateral septal n., ventral part Medial preoptic area Medial septal nucleus Nuclei of the diagonal band Cerebellum Cortex, granular layer Cortex, molecular layer Interposed nucleus Medial nucleus Brain Stem Cuneate nucleus Facial nucleus Hypoglossal nucleus Inferior olive Interpeduncular nucleus Lateral reticular nucleus Medial accessory oculomotor nucleus Nucleus of the solitary tract Oculomotor nucleus Parvocellular reticular nucleus Periaqueductal gray Pontine nuclei Red nucleus Spinal trigeminal nucleus
Relative Optical Density
+ ++ ++ ++ ---+ ---+ ---+ + _ + ++ + ++ ---+ ++ ++ ++ ++-+++ _ ++ ++ + +++ ++ + + ++ ++ + + ++ + ++-+++ ++ +
The microdensitometric analysis of SAPK-mRNA labelled autoradiographs was performed by means of an automatic image analyser (Imaging Research, Canada) according to Ferraguti er al.” The optical density of the specific labelling present in sampled areas was evaluated after subtraction of the optical density of non-specific labelling (as defined by the mean optical density measured in sections treated with excess of unlabelled probe). Relative optical densities of sampled areas were expressed as percent of the maximum value observed and grouped into five categories: + + + +, very high (lo&76%); + + +, high (75-51%); + +, moderate (S&26%); +, low (2551%); -, background level. The brain regions were demarcated according to Paxinos and Watson.‘* DISCUSSION
The present study has shown that SAPKs, a recently identified MAPK subfamily, have different regional distribution in the brain as compared to the archetypal MAPKs ERKl and ERK2. Intense labelling for SAPKs was detected in discrete brain areas, such as the hippocampus, neocortex and some nuclei of the brain stem. Marked differences in the degree of
specific labelling were also observed at the various rostrocaudal levels analysed. No confirmatory evidence of expression of SAPKs in glial cells was obtained. The existence of SAPKs in glial cells, however, cannot be excluded entirely, since it was often difficult to identify the nature of small cell bodies which were only poorly labelled in sections processed for in situ hybridization.
Fig. 2. Autoradiograms of rostra1 (A), median (B, C, E, F) and caudal (G, H) coronal brain sections illustrating the lacalization of the hybridization signal generated by an antisense 35S-labelled oligonucleotide probe for SAPKs. In situ hybridization was performed as described in experimental procedures. Sections incubated with excess of unlabelled probe (D) were used as controls. Arc, arcuate nucleus; cc, corpus callosum; CPU, caudate-putamen, Cx, cerebral cortex; gr, cerebellar granule cell layer; Pir, piriform cortex; 7, facial nucleus. Scale bar = 2.5 mm.
1108
R. Carletti et al.
Fig. 3. Photomicrographs of emulsion-dipped sections counterstained with Cresyl Violet showing the cellular localization of the mRNA for SAPKs in adult rat brain. Low (A) and high (B) power photomicrographs of the cerebral cortex. In A, arrowheads denote intensely-labelled pyramidal neurons whereas in B they indicate small- and medium-sized unlabelled cells. Panel C, low power photomicrograph showing a striatal field in which arrowheads mark labelled neurons poorly expressing SAPKs-mRNA. Scale bars A, C = 60 pm; B = 25 pm.
Although, SAPK transcripts showed a distinct pattern of expression from ERKl and ERK2 in many brain areas, a conspicuous degree of co-expression could be observed. ERKl, ERK2 and SAPK mRNAs were all detected in granule cells of the dentate gyrus, and ERK2 and SAPKs were co-localized in pyramidal neurons of the CAl, CA2 and CA3 fields of the Ammons horn.** Furthermore, it can be
postulated that certain neuronal populations also co-express ERKs and SAPKs such as pyramidal neurons of the neocortex, the piriform cortex and the cerebellar granule and Purkinje cells. A clear mismatch between ERKs and SAPKs was observed in basal ganglia which express high levels of ERK2 while the SAPK-mRNA content is very low.
Fig. 4. Photomicrograph of a coronal section showing the distribution of SAPK transcripts in the hippocampal formaTion, panel A. High magnification photomicrograph of the cellular distribution of SAPK-mRNA in the CA3 hippocampal field, panel B. DG, dentate gyrus; MHb, medial habenular nucleus. Scale bars A = 900 pm; B = 25 pm.
SAPKs in adult rat central nervous system
Fig. 5. Cellular distribution of SAPK-mRNA in cerebellar cortex, hippocampus and brain stem. Panel A, high power photomicrograph of the cerebellar cortex, arrowheads show individual Purkinje cells moderately expressing SAPKs. Panel B, localization of SAPKs-mRNA containing neurons in the CA1 hippocampal field. Arrowheads indicate intensely labelled non-pyramidal neurons. Panels C, low power bright-field photomicrograph of granule neurons of the cerebellar cortex. Panel D, the large neurons of the facial nucleus appear densely covered with silver grains indicating a high level of SAPKs-mRNA expression. Panel E, highly-labelled neurons in the spinal trigeminal nucleus are indicated by arrowheads. Scale bars A=25pm; B, C, D, E=60pm. In neural tissues a variety of stimuli e.g. nerve growth factor: phorbol esters and activation of neurotransmitter receptors,‘,” lead to activation of the ERK cascade which do not, or in some cases only poorly, activate SAPKs. I6 These results taken together suggest that SAPK activation reflects an independent and parallel pathway with respect to ERKs in the response of neural cells to extracellular signals. The physiological functions of MAPKs have been investigated primarily in the context of the regulation of the cell cycle in somatic and germ cells. In somatic cells, MAPKs promote the GO-G1 transition.4 Recent reports strongly implicate MAPKs in the phosphorylation of cytoskeletal proteins and thus in the regulation of the cytoskeleton, in particular in microin the regulation tubular dynamics. 8,‘3 Perturbations of the phosphorylated state of cytoskeletal proteins may be involved in pathological processes of human brain such as Alzheimer’s disease (AD). The micro-
tubule-associated protein tau is phosphorylated by MAPKs9 and abnormally phosphorylated tau has been found within neurofibrillary tangles in brains of AD patients. ‘* Recently, a novel human kinase, p493F’2 kinase, has been identified which co-localizes with the ALZ-50 antigen,” a marker for early neurofibrillary degeneration in AD.24 This kinase displays a 98% amino acid identity with the rat SAPKB representing probably the human homologue. SAPKs also phosphorylate the transcription factors c-Jun and activating transcription factor 2 (ATF2)7,‘4~‘6,20thus leading to increased transcriptional activity. Since this class of kinases is highly activated by cellular stresses, they may induce an early genetic response to neuronal injuries such as chronic or acute neurodegenerative events, possibly by enhancing trans activating activity of c-Jun and ATF2. I9 To date the physiological significance of the SAPK cascade is still obscure, however in view of the present
R. Carletti et al.
1110 evidence of a region-specific
distribution
of SAPKs in
the brain an important role for these enzymes in brain
signal transduction
can be envisaged.
Acknowledgements-The authors wish to thank Dr Bernd Bunnemann, Dr Philip Gerrard and Dr Fabio Benfenati for critical review of the manuscript.
REFERENCES
1. Bading H. and Greenberg M. E. (1991) Stimulation of protein tyrosine phosphorylation by NMDA receptor activation. Science 253, 912-914. 2. Blumer K. J. and Johnson G. L. (1994) Diversity in function and regulation of MAP kinase pathway. Trends biochem. Sci. 19, 236240. 3. Boulton T. G., Yancopoulos G. D., Gregory J. S., Slaughter C., Moomaw C., Hsu J. and Cobb M. H. (1990) An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science 249, 6467. 4. Boulton T. G., Nye S. H., Robbins D. J., Ip N. Y., Radziejewska E., Morgenbesser S. D., DePinho R. A., Panayotatos N., Cobb M. H. and Yancopoulos G. D. (1991) ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65, 663-675. 5. Cano E. and Mahadevan L. C. (1995) Parallel signal processing among mammalian MAPKs. Trends biochem. Sci. XI, 117-122. 6. Davies R. (1993) The mitogen-activated protein kinase signal transduction pathway. J. biol. Chem. 268, 14553-14556. 7. Derijard B., Hibi M., Wu I-H., Barret T., Su B., Deng T., Karin M. and Davies R. (1994) JNKl: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-jun activation domain. Cell 76, 1025-1037. 8. Drechsel D. N., Hyman A. A., Cobb M. H. and Kirschner M. W. (1992) Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. Molec. Biol. Cell 3, 1141-l 154. 9. Drewes G., Lichtenberg-Kraag B., Doring F., Mandelkow E. M., Biemat J., Goris J., Doree M. and Mandelkow E. (1992) Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state. Eur. molec. Biol. Org. .Z. 11, 2131-2138. 10. Ferraguti F., Zoli M., Aronsson M., Agnati L. F., Goldstein M., Filer D. and Fuxe K. (1990) Distribution of glutamic acid decarboxylase messenger RNA-containing nerve cell populations of the male rat brain. J. them. Neuroanat. 3, 377-396.
11. Fiore R. S., Bayer V. E.. Pelech S. L.. Posada J., Cooper J. A. and Baraban J. M. (1993) n42 mitoaen-activated kinase in brain! prominent localization in neuronal cell bodies, and dendrites. hi,uro&ience 55,463472. 12 Goedert A. M., Spillantini M. G., Jakes R., Rutherford D. and Crowther R. A. (1989) Multiple isoforms of microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer’s disease.
nrotein * human Neuron
3, 519-526.
13. Gotoh Y., Nishida E., Matsuda S., Shiina N., Kosako H., Shiokawa K., Akiyama T., Otha K. and Sakai H. (1991) In oitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase. Nature 349, 251-254. 14. Gupta S., Campbell D., Derijard B. and Davies R. (1995) Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267, 389-393. 15. Hyman B. T., Elvhage T. E. and Reiter J. (1994) Extracellular signal regulated kinases. Localization of protein and mRNA in the human hippocampal formation in Alzheimer’s disease. Am. .Z. Path 144, 565-572. 16. Kyriakis J. M., Banerjee P., Nikolakaki E., Dai T., Rubie E. A., Ahmad M. F., Avruch J. and Woodgett J. R. (1994) The stress-activated protein kinase subfamily of cdun kinases. Nature 369, 156-160. 17. Mohit A. A.. Martin J. H. and Miller C. A. (1995) ~493F12 Kinase: a novel kinase exoressed in a subset of neurons in the human nervous system. Neuron 14, 67178. ’_ 18. Paxinos G. and Watson C. (1986) The Rat Brain in Stereofaxic Coordinates. Academic Press, Orlando, U.S.A. 19. Pombo C. M., Bonventre J. V., Avruch J., Woodgett J. R., Kyriakis J. M. and Force. T. (1994) The stress-activated protein kinases are major cJun amino-terminal kinases activated by ischemia and reperfusion. J. biol. Chem. 269, 2654626551.
20. Sanchez I., Hughes R. T., Mayer B. J., Yee K., Woodgett J. R., Avruch J., Kyriakis J. M. and Zon L. I. (1994) Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor cJun. Nature 372, 794-798. 21. Stratton K. R., Worley P. F., Huganir R. L., and Baraban J. M. (1989) Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices. Proc. natn. Acad. Sci. U.S.A. 86,2498-2501. 22. Thomas K. L. and Hunt S. P. (1993) The regional distribution of extracellularly regulated kinase-1 and -2 messenger RNA in the adult rat central nervous system. Neuroscience 56, 741-757. 23. Wisden W., Morris B. J. and Hunt S. P. (1991) In situ hybridization with synthetic DNA probes. In Molecular Neurobiology: A Practical Approach (eds Chad J. and Wheal H.), pp. 205225. IRL Press at Oxford University Press, U.K. 24. Wolozin B. L., Pruchnicki A., Dickson D. W. and Davies P. (1986) A neuronal antigen in the brains of Alzheimer patients. Science 232, 648-650. (Accepted 5 July 1995)
Elsevier Science Ltd Copyright 0 1995 IBRO
Pergamon
0306-4522(95)00284-7
Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
STRESS ACTIVATED PROTEIN KINASES, A NOVEL FAMILY OF MITOGEN-ACTIVATED PROTEIN KINASES, ARE HETEROGENEOUSLY EXPRESSED IN THE ADULT RAT BRAIN AND DIFFERENTIALLY DISTRIBUTED FROM EXTRACELLULAR-SIGNAL-REGULATED PROTEIN KINASES R. CARLETTI, Department
S. TACCONI,
of Pharmacology,
Glaxo
E. BETTINI
Research
Laboratories,
and
F. FERRAGUTI*
Via Fleming
4, 37100 Verona,
Italy
Abstract-Mitogen-activated protein kinases are important mediators of signal transduction from the cell surface to the nucleus and their activation has been implicated in a wide array of physiological processes. The extracellular-signal-regulated kinases are the archetypal and best studied members of the mitogen activated protein kinases. Recently, additional subgroups of mitogen activated protein kinases have been identified which exhibit distinct regulatory elements, substrate specificity and respond to diverse extracellular stimuli. Among these newly identified protein kinases are the rat stress-activated protein kinases. Despite a rapidly expanding literature on the biochemical properties of stress-activated protein kinases no anatomical data are yet available. In the present study, we have investigated the regional distribution of messenger RNA transcripts for stress-activated protein kinases in the adult rat central nervous system and compared this distribution to that observed for extracellular-signal-regulated kinases. Intense labelling for stress-activated protein kinases could be detected in discrete brain areas with high levels in hippocampus, neocortex and some nuclei of the brain stem. The apparent hybridization signal appeared to be selectively neuronal. Stress-activated protein kinases and extracellular-signal-regulated kinases hybridization patterns appeared generally dissimilar although a certain degree of co-expression in some brain areas, such as the hippocampal formation, could he observed.
These results reveal an extreme complexity in the mitogen-activated protein kinase signalling pathway and suggest the existence of parallel mitogen-activated protein kinase cascades that can be activated independently or in some cases simultaneously, by extracellular stimuli. Key words: SAPK,
ERK,
MAPK,
in situ hybridization,
The mitogen-activated protein kinase (MAPK) cascade is a major signalling system by which cells transduce extracellular stimuli into intracellular responses.6 MAPKs comprise a family of serine/threonine protein kinases which share the unique feature of being activated by phosphorylation on threonine and tyrosine residues by an upstream dualspecificity kinase.2 Until recently, the extracellularsignal-regulated kinases (ERKs) 1 and 2 were the only cloned and relatively well characterized MAPKs. Extracellular-signal-regulated kinases are expressed ubiquitously in vertebrate tissues, nevertheless they appear to be particularly enriched in the developing and adult CNS.3,4 Independent research efforts have recently led to the identification of a new subfamily of MAPKs: the rat stress-activated protein
*To whom correspondence
should be addressed.
Abbreviufions: AD, Alzheimer’s disease; ATFZ, activating transcription factor 2; ERK, extracellular-signal-regulated protein kinase; JNK, c-Jun NH,-terminal kinase; MAPK, mitogen-activated protein kinase; SAPK, stressactivated protein kinase; SEKl, SAPK/ERK kinase; SSC, saline sodium citrate.
central
nervous
system.
kinases (SAPKS)‘~ and the human homologues c-Jun NH,-terminal kinases (JNKs).~ These kinases show 4&45% identity to ERKs, sharing the requirement of both tyrosine and threonine phosphorylation for activation and proline-directed substrate specificity.16 SAPKs/JNKs are activated as a result of the cellular response to intra- and extracellular stress such as heat shock, ultraviolet radiation, protein synthesis inhibitors, inflammatory cytokines and sphingomyelinase all of which only poorly activate ERKs.jrJ6 Three closely related (88-90% identity) classes of SAPKs have been identified from a rat brain cDNA library and designated SAPK-c(, -B and -y.16 The only information available about their anatomical localization is a general description that they are ubiquitously expressed, at low levels, throughout the body. I6 Notably, the upstream regulatory kinase SAPK/ERK kinase 1 (SEKl) is also ubiquitously expressed, as demonstrated by northern blot analysis, although at very high levels in brain and muscle.20 In the present study, the regional distribution of mRNA transcripts for SAPKs in the adult rat CNS was investigated by means of in situ 1103
R. Carletti ez al.
1104
hybridization with 3SS-labelled oligonucleotide probes and compared with that of the well-characterized MAPKs, ERKl and ERK2. SAPK probes were selected for recognizing at once all three classes.
EXPERIMENTAL
PROCEDURES
Materials Adult SpragucDawley rats (> 200 g) were obtained from Charles River, Calco, Italy. [3sS]dATP, specific activity 1100 Ci/mmol was from New England Nuclear, Frankfurt, Germany. Phosphoramidites for oligonucleotide synthesis were purchased from Millipore, Milano, Italy. In situ hybridization histochemistry Animals were deeply anaesthetized with urethane (1.5 g/kg, i.p.) and intracardially perfused with ice cold saline. Brains were immediately removed, frozen in dry-ice cold isopentane and kept at - 80°C until sectioned (14 pmthick-sections) for in situ hybridization studies. Antisense oligonucleotide probes (45mers) were synthesized on a Millipore Expedite 8909 DNA synthesizer complementary to mRNAs of ERKs4 and SAPKs.i6 The ERKs antisense oligonucleotide probes were complementary to nucleotides 141&1454 of ERKl (Gene Bank accession number M61177) and to nucleotides 1201-1245 of ERK2 (Gene Bank accession number M64300). The SAPK probe was complementary to nucleotides 592636 of the mRNA for SAPKa (Gene Bank accession number L27111) a region with 87-98% identity to the other classes of SAPKs. Thus, the probe recognized all the known classes of SAPKs. The percentage homology between oligonucleotide probes and full length sequences was calculated using the GCG (Genetics Computer Group, 1991) package. Oligonucleotides were 3’end-labelled with [35S]dATP. Radiolabelled oligonucleotides were separated from unincorporated nucleotides using Nensorb columns (New England Nuclear) according to manufacturer instructions. Specific activity of the ?Slabelled probes was routinely between 5 x 10’ and 8 x 10’c.p.m./nl probe. Hybridizations were performed essentially as described by Wisden et al.*’Following hybridization, sections were washed twice for 5min at room temperature with 2 x saline sodium citrate (SSC), further washed 4 times for 15 min at 55°C with 0.5 x SSC, rinsed briefly at room temperature in 1 x SSC, 70% ethanol and 95% ethanol and air-dried prior to apposing to Amersham ‘H-Ultrofilms. After appropriate exposure, films were developed and sections were dipped in NTB2 photographic emulsion (Kodak). Sections were exposed -for four- to five weeks at - 20°C. develoued for 2 min in Kodak D19 develooer and counterstained ‘with Cresyl Violet. Adjacent sections were hybridized with excess of unlabelled probe to assess the specificity of hybridization. An identical labelling pattern was observed with different non-overlapping probes. RESULTS
Film or emulsion autoradiographic evaluation of in situ hybridization revealed a strikingly heterogeneous distribution of mRNA for SAPKs throughout the brain. The hybridization was seen to be specific since non-significant hybridization was observed in parallel
experiments using the same labelled probes in the presence of unlabelled probe. The film autoradiography showed strong signal intensity in the hippocampal formation, the cerebral cortex, the granular layer of the cerebellar cortex and the facial nucleus.
Moderate levels of hybridization signal were also observed in diencephalic areas, in septal and basal forebrain regions, in part of the amygdaloid complex and in some nuclei of the brain stem. Results from representative brain sections are illustrated in Figs 1A and 2. The autoradiographs were semiquantitatively evaluated by means of computer-assisted image analysis and results are summarized in Table 1. Specific optical density values were expressed as a percentage of the maximum value observed (hippocampal CA3 pyramidal cell layer). Specific labelling appeared to be located over nerve cell bodies in all brain areas analysed, whereas no apparent labelling was found over glial cells and capillaries (Fig. 3). In neocortical regions, many neuronal cell bodies, of both pyramidal and non-pyramidal neurons were weakly- or moderately-labelled, particularly in layers IV and V (Figs 2 and 3A, 3B). In the limbic cortical regions, the hippocampal formation contained highly-labelled cell bodies of pyramidal neurons. Within the hippocampus, an uneven localization of the message was also found. The CAl/CA2 and dentate gyrus fields displayed lower levels of hybridization signals than that in the CA3 field, although they contained high levels of SAPK-mRNA (Figs 4A and B). Labelling in the subcortical regions of the forebrain was generally weak to moderate. Semiquantitative analysis of autoradiographs showed a high level of relative optical density in the medial habenular nucleus which after emulsion dipping appeared to consist of moderately-labelled neurons. This discrepancy is due to the high density of small neurons contained in this brain area. The majority of neurons in thalamic nuclei were weakly- to moderately-labelled for SAPK-mRNA. Of the forebrain regions, the basal ganglia demonstrated the lowest relative optical density. Striatum, globus pallidus and ventral pallidum contained only scattered, weaklylabelled neurons (Figs 2A and 3C). In the substantia nigra, the pars compacta contained few moderatelylabelled cell bodies, while no hybridization signal was observed in the pars reticulata. Film autoradiographs showed high hybridization signal in the granular layer of the cerebellar cortex (Fig. 2G and H). In emulsion dipped brain sections, however, individual granule neurons were only moderately-labelled (Fig. SA and C). This is due, as mentioned above, to the high cell density of this cerebellar cell layer. Moreover, in the cerebellar cortex Purkinje neurons were also found to be moderately-labelled (Fig. 5A). mRNA for SAPKs in brain stem regions appeared to be discretely localized in relatively few nuclei with the highest level of hybridization signal present in the facial nucleus (Figs 2G and SD). The spinal trigeminal nucleus displayed a low relative optical density due to dispersed distribution of neuronal cell bodies which in emulsion dipped sections appeared generally strongly labelled (Fig. 5E). ERKs mRNA levels in adult rat brain were also investigated and compared to those observed for
SAPKs in adult rat central nervous system
Fig. 1. Distribution of mRNAs for SAPKs, ERKl and ERK2 in adult rat brain. Darkfield photogr ot autoradiograms illustrating the distribution of SAPK-mRNA (‘panel A), ERKI-mRNA (panel B) and ERK2-mRNA (panel C) in parasagittal brain sections. Cb, cerebellar cortex; CPU, caudate-putamen; Cx, cerebral cortex; DG, dentate gyrus; Hi, hippocampus; Th, thalamus; 7, facial nucleus. Scale bar = 1.6 mm.
SAPKs.
ERKl
mRNA
transcripts
were
confined
primarily to dentate gyrus granule cells (Fig. 1B) although hybridization signal was also detected in other areas such as the locus coeruleus and the hypothalamus. ERK2, however, was more widely distributed throughout the rat brain (Fig. 1C) a NSC 69,4--E
finding
which
is in agreement
with
previous
re-
ports. 15z2* Earlier in situ hybridization and immunohistochemical studies reported that ERKs are also localized in reactive astrocytes and in oligodendrocytes, and that ERKs-immunoreactivity in neurons was restricted to cell bodies and dendrites.“~‘5~22
Fig. 2. 1106
SAPKs
in adult rat central nervous system
1107
Table 1. Distribution of stress-activated protein kinase mRNA in the adult rat CNS Relative Optical Density
Regions of CNS Neocortex Frontal cortex Occipital cortex Parietal cortex Temporal cortex Limbic cortex Cingulate cortex Dentate gyrus, granule cells Entorhinal cortex Piriform cortex Retrosplenial cortex Subiculum Hippocampus CAl, pyramidal layer CA3, pyramidal layer Amygdala Basolateral amygdaloid nucleus Basomedial amygdaloid n., anterior p. Central nucleus Lateral amygdaloid nucleus Thalamus Anterodorsal nucleus Lateral geniculate nucleus Medial geniculate nucleus Mediodorsal nucleus Parafascicular nucleus Posterior nuclear group Reticular nucleus Ventromedial nucleus Ventroposterior nuclear group Subthalamus Subthalamic nucleus Zona Incerta Medial habenular nucleus Lateral habenular nucleus Hypothalamus Arcuate nucleus Medial mammillary n., medial part Medial mammillary n., posterior part Premammillary n., dorsal part
++ ++ ++ ++ ++ +++ ++ +++ ++ ++ +++ ++++ ++ ++ + ++ + ++ ++ ++ + ++ + ++ ++ ++ ++ + +++ + ++ ++ + ++
Regions of CNS Posterior hypothalamic area Supramammillary nucleus Tuberomammillary nucleus Ventromedial nucleus Basal Ganglia Accumbcns n. Caudate-putamen Globus pallidus Substantia nigra, pars compacta Substantia nigra, pars reticulata Ventral tegmental area Septal and Basal Forebrain Regions Bed nucleus of the stria terminalis Lateral septal n., dorsal part Lateral septal n., intermediate part Lateral septal n., ventral part Medial preoptic area Medial septal nucleus Nuclei of the diagonal band Cerebellum Cortex, granular layer Cortex, molecular layer Interposed nucleus Medial nucleus Brain Stem Cuneate nucleus Facial nucleus Hypoglossal nucleus Inferior olive Interpeduncular nucleus Lateral reticular nucleus Medial accessory oculomotor nucleus Nucleus of the solitary tract Oculomotor nucleus Parvocellular reticular nucleus Periaqueductal gray Pontine nuclei Red nucleus Spinal trigeminal nucleus
Relative Optical Density
+ ++ ++ ++ ---+ ---+ ---+ + _ + ++ + ++ ---+ ++ ++ ++ ++-+++ _ ++ ++ + +++ ++ + + ++ ++ + + ++ + ++-+++ ++ +
The microdensitometric analysis of SAPK-mRNA labelled autoradiographs was performed by means of an automatic image analyser (Imaging Research, Canada) according to Ferraguti er al.” The optical density of the specific labelling present in sampled areas was evaluated after subtraction of the optical density of non-specific labelling (as defined by the mean optical density measured in sections treated with excess of unlabelled probe). Relative optical densities of sampled areas were expressed as percent of the maximum value observed and grouped into five categories: + + + +, very high (lo&76%); + + +, high (75-51%); + +, moderate (S&26%); +, low (2551%); -, background level. The brain regions were demarcated according to Paxinos and Watson.‘* DISCUSSION
The present study has shown that SAPKs, a recently identified MAPK subfamily, have different regional distribution in the brain as compared to the archetypal MAPKs ERKl and ERK2. Intense labelling for SAPKs was detected in discrete brain areas, such as the hippocampus, neocortex and some nuclei of the brain stem. Marked differences in the degree of
specific labelling were also observed at the various rostrocaudal levels analysed. No confirmatory evidence of expression of SAPKs in glial cells was obtained. The existence of SAPKs in glial cells, however, cannot be excluded entirely, since it was often difficult to identify the nature of small cell bodies which were only poorly labelled in sections processed for in situ hybridization.
Fig. 2. Autoradiograms of rostra1 (A), median (B, C, E, F) and caudal (G, H) coronal brain sections illustrating the lacalization of the hybridization signal generated by an antisense 35S-labelled oligonucleotide probe for SAPKs. In situ hybridization was performed as described in experimental procedures. Sections incubated with excess of unlabelled probe (D) were used as controls. Arc, arcuate nucleus; cc, corpus callosum; CPU, caudate-putamen, Cx, cerebral cortex; gr, cerebellar granule cell layer; Pir, piriform cortex; 7, facial nucleus. Scale bar = 2.5 mm.
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R. Carletti et al.
Fig. 3. Photomicrographs of emulsion-dipped sections counterstained with Cresyl Violet showing the cellular localization of the mRNA for SAPKs in adult rat brain. Low (A) and high (B) power photomicrographs of the cerebral cortex. In A, arrowheads denote intensely-labelled pyramidal neurons whereas in B they indicate small- and medium-sized unlabelled cells. Panel C, low power photomicrograph showing a striatal field in which arrowheads mark labelled neurons poorly expressing SAPKs-mRNA. Scale bars A, C = 60 pm; B = 25 pm.
Although, SAPK transcripts showed a distinct pattern of expression from ERKl and ERK2 in many brain areas, a conspicuous degree of co-expression could be observed. ERKl, ERK2 and SAPK mRNAs were all detected in granule cells of the dentate gyrus, and ERK2 and SAPKs were co-localized in pyramidal neurons of the CAl, CA2 and CA3 fields of the Ammons horn.** Furthermore, it can be
postulated that certain neuronal populations also co-express ERKs and SAPKs such as pyramidal neurons of the neocortex, the piriform cortex and the cerebellar granule and Purkinje cells. A clear mismatch between ERKs and SAPKs was observed in basal ganglia which express high levels of ERK2 while the SAPK-mRNA content is very low.
Fig. 4. Photomicrograph of a coronal section showing the distribution of SAPK transcripts in the hippocampal formaTion, panel A. High magnification photomicrograph of the cellular distribution of SAPK-mRNA in the CA3 hippocampal field, panel B. DG, dentate gyrus; MHb, medial habenular nucleus. Scale bars A = 900 pm; B = 25 pm.
SAPKs in adult rat central nervous system
Fig. 5. Cellular distribution of SAPK-mRNA in cerebellar cortex, hippocampus and brain stem. Panel A, high power photomicrograph of the cerebellar cortex, arrowheads show individual Purkinje cells moderately expressing SAPKs. Panel B, localization of SAPKs-mRNA containing neurons in the CA1 hippocampal field. Arrowheads indicate intensely labelled non-pyramidal neurons. Panels C, low power bright-field photomicrograph of granule neurons of the cerebellar cortex. Panel D, the large neurons of the facial nucleus appear densely covered with silver grains indicating a high level of SAPKs-mRNA expression. Panel E, highly-labelled neurons in the spinal trigeminal nucleus are indicated by arrowheads. Scale bars A=25pm; B, C, D, E=60pm. In neural tissues a variety of stimuli e.g. nerve growth factor: phorbol esters and activation of neurotransmitter receptors,‘,” lead to activation of the ERK cascade which do not, or in some cases only poorly, activate SAPKs. I6 These results taken together suggest that SAPK activation reflects an independent and parallel pathway with respect to ERKs in the response of neural cells to extracellular signals. The physiological functions of MAPKs have been investigated primarily in the context of the regulation of the cell cycle in somatic and germ cells. In somatic cells, MAPKs promote the GO-G1 transition.4 Recent reports strongly implicate MAPKs in the phosphorylation of cytoskeletal proteins and thus in the regulation of the cytoskeleton, in particular in microin the regulation tubular dynamics. 8,‘3 Perturbations of the phosphorylated state of cytoskeletal proteins may be involved in pathological processes of human brain such as Alzheimer’s disease (AD). The micro-
tubule-associated protein tau is phosphorylated by MAPKs9 and abnormally phosphorylated tau has been found within neurofibrillary tangles in brains of AD patients. ‘* Recently, a novel human kinase, p493F’2 kinase, has been identified which co-localizes with the ALZ-50 antigen,” a marker for early neurofibrillary degeneration in AD.24 This kinase displays a 98% amino acid identity with the rat SAPKB representing probably the human homologue. SAPKs also phosphorylate the transcription factors c-Jun and activating transcription factor 2 (ATF2)7,‘4~‘6,20thus leading to increased transcriptional activity. Since this class of kinases is highly activated by cellular stresses, they may induce an early genetic response to neuronal injuries such as chronic or acute neurodegenerative events, possibly by enhancing trans activating activity of c-Jun and ATF2. I9 To date the physiological significance of the SAPK cascade is still obscure, however in view of the present
R. Carletti et al.
1110 evidence of a region-specific
distribution
of SAPKs in
the brain an important role for these enzymes in brain
signal transduction
can be envisaged.
Acknowledgements-The authors wish to thank Dr Bernd Bunnemann, Dr Philip Gerrard and Dr Fabio Benfenati for critical review of the manuscript.
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