Localization of mRNA for protein phosphatase 2C in the brain of adult rats

Molecular Brain Research, 13 (1992) 283-288 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$05.00

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Localization of m R N A for protein phosphatase 2C in the brain of adult rats H i r o s h i A b e a, Shinri T a m u r a b a n d H i s a t a k e K o n d o a aDepartment of Anatomy, School of Medicine and bDepartment of Biochemistry, Research Institute of Tuberculosis and Cancer, Tohoku University, Sendai (Japan) (Accepted 19 November 1991)

Key words: Protein phosphatase 2C; mRNA; Central nervous system; Rat; In situ hybridization

q~pe 2C protein phosphatase (PP2C) is one of four major serine-threonine specific phosphoprotein phosphatases which modulate various intraceilular activities. By in situ hybridization analysis of the adult rat, expression signals of mRNA for PP2C were observed most highly in the granule cells and Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and granule cells of the dentate gyrus, and plexus choroideus of the lateral ventricle, whereas moderate levels of its expression were observed in the medial habenula, piriform cortex and the pineal body. Several discrete nuclei of the brainstem including pars compacta of the substantia nigra, the pontine nuclei, and the locus ceruleus expressed the mRNA moderately. Weak expression of PP2C mRNA was observed in mitral and internal granule cells of the olfactory bulb, spinal cord gray matter, the cerebral neocortex, thalamic and hypothalamic nuclei, Only faint expression was detected in the caudate putamen. These patterns of expression are different from that of calcineurin/PP2B reported by other immunohistochemical studies and it is suggested that various neuronal proteins are differentially dephosphorylated by the different types of PP. INTRODUCTION It is well known that reversible addition or removal of p h o s p h a t e ester on serine and threonine hydroxyls by protein kinases and phosphatases m o d u l a t e s the activity of m a n y intracellular substrate proteins which contribute to such characteristic functions of neurons as neurotransmitter biosynthesis and release, axoplasmic intergrity and transport, ion channel conductance, and neuronal plasticity 1'8. F o u r subtypes of the protein-serine/threonine phosphatases have been indentified on the basis of their sensitivity to t h e r m o s t a b l e proteins, inhibitors-1 and -2, and of divalent cation requirements, and they are t e r m e d protein p h o s p h a t a s e type 1 (PP1), P P 2 A , PP2B and PP2C 6'7. A m o n g them, PP2C is m o n o m e r i c and requires Mg 2+ for activity, and it has a widespread tissue distribution with some enrichment in the brain, whereas PP2B is C a / C a l m o d u l i n - d e p e n d e n t and often t e r m e d calcineurin because of highest concentrations in the brain 6'1t. Molecular cloning studies have revealed that there is some h o m o l o g y in amino acid sequences a m o n g PP1, 2 A and 2B, while no h o m o l o g y is present b e t w e e n PP2C and the r e m a i n d e r 1°. This suggests that PP2C has a distinct function among PPs. Localization of the sites of expression of PPs and their genes in the brain and o t h e r tissues is a first step in un-

derstanding their functional significance. In this regard, previous immunohistochemical studies have revealed the heterogenous distribution of calcineurin in the brain 5. H o w e v e r , no information is available concerning the localization of the o t h e r PPs and of m R N A s for all the four PPs in any tissues. Therefore, in the present study we p e r f o r m e d the in situ hybridization analysis of m R N A for PP2C in the whole brain of adult rats in detail. MATERIALS AND METHODS As a cDNA probe specific to PP2C, Rsa I fragment, about 500 bp fragment of 3'-non-coding region of rat PP2C-cDNA was selected on the basis of the original report 1° and the appropriate size for the in situ hybridization analysis. The probes were labeled with [35S]dCTP or [32p]dATP by a random-primed DNA labeling kit (Boehringer Mannheim, FRG). Male albino adult rats of 150-200 g body weight were sacrificed by decapitation under ether anesthesia. Brains were removed, frozen on dry ice and stored at -80°C. Frozen sections, 20-30/~m thick, were made on a cryostat and mounted on gelatin-coated slide glasses. The slides were dipped into 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 20 min and 2 mg/ml of glycine in phosphate-buffered saline for 20 min, and subsequently acetylated in 0.25% acetate in 0.1 M Tris-HCl (pH 8.0). Hybridization was done at 37°C overnight with a [35S]dCTP-labeled probe at a concentration of 5 × 105cpm/50 pl of hybridization mixture consisting of 4× SSC, 50% formamide, 1× Denhardt's solution, 10% dextran sulfate, 0.1 M sodium phosphate buffer (pH 7.2), 2% sarcosyl, 0.1 M dithiothreitol and 250 /~g/ml of heat-denatured salmon sperm DNA. The slides were washed 3 times in 0.1 x SSC-

Correspondence: H. Abe, Department of Anatomy, Tohoku University School of Medicine, Seiryo-machi 2-1, Sendai, Japan. Fax: (81) 22272-7273.

284 0.1% sarcosyl at 42°C for 40 min and then autoradiographed using NTB2 nuclear track emulsion (Kodak) for 8 weeks. Northern blot analyses were performed to determine the sizes of the mRNA species detected. Poly(A) RNA of the brain was isolated from adult rat by homogenization in 5 M guanidinium thiocyanate followed by direct precipitation of RNA from the guanidinium solution with 4 M lithium chloride and oligo(dT) cellulose chromatography. The poly(A) RNA (2 ktg) was electrophoresed on a horizontal 1.5% agarose/2.2 M formaldehyde gel and transferred onto a nitrocellulose filter membrane. Blots were prehybridized at 37°C for 2 h in buffer containing 50% formamide, 5× SSC (0.15 M NaC1 and 0.015 M sodium citrate), 1 × Denhardt's solution, 50 mM sodium phosphate buffer (pH 6,5) and 250 ktg/ml of heat-denatured salmon sperm DNA. After hybridization in the same buffer containing [32p]dATP-labeled probe, the filter was washed three times in 2× SSC-0.1% SDS (sodium dodecyl sulfate) at room temperature, and then in 0.1x SSC-0.1% SDS at 42°C for 30 min, and autoradiographed for two days. Similar to previous studies 1°, the PP2C probe detected a single 2.4 kb band (Fig. 1).

RESULTS The expression signals for PP2C m R N A were represented by accumulation of autoradiographic silver grains and they were more or less observed in somata of virtually all neurons examined, whereas glial cells in the white matter did not show any significant amounts of the expression signals. In the olfactory bulb, the expression of PP2C m R N A was detected weakly in the mitral and internal granule cells in forms of discrete grain accumulations, while the silver grains faintly and diffusely deposited in the periglomerular zones, representing the periglomerular cells (Figs. 2 and 3). In the forebrain, weak expression of the m R N A was

detected in the septohippocampus and diagonal band of Broca, and in the neocortex, layers I I - V I , neurons of the thalamus, zona incerta, hypothalamic nuclei and mammillary nuclei. Cells in the piriform cortex, the amygdala and olfactory tuberculum expressed it weakly to moderately, whereas the habenular nuclei and the pineal body expressed the m R N A moderately. However, only faint levels of expression were seen in the caudateputamen. Intense expression levels of the m R N A were detected in the pyramidel cell layers of the hippocampus and the granule cell layer of the dentate gyrus without distinct regional differences in its intensity. In addition, moderate expression of the m R N A was detected in the plexus choroideus of the lateral and third ventricles (Figs. 4 and 5). In the midbrain and hindbrain, moderate expression levels of PP2C m R N A were noted in the pontine nuclei, the locus ceruleus, the mesencephalic trigeminal nuclei and the pars compacta of substantia nigra, the red nuclei, the oculomotor nuclei, the facial nuclei and the lateral reticular nuclei, whereas most other nuclei including the raphe expressed relatively low levels of the m R N A (Figs. 6-8 and 10). In the cerebellum, the granule cell layer expressed PP2C m R N A intensely and a distinct expression of the m R N A was also identified in the Purkinje cells (Fig. 9). The level of the expression in these two cell populations was highest in the central nervous system. No expression of the m R N A was seen in the molecular layer (Fig. 9). In the spinal cord, cells in both ventral and dorsal horns expressed the m R N A weakly in forms of discrete grain aggregations (Fig. 11). The localization of PP2C m R N A and the relative intensity of the expression levels in the brain is summarized in Table I. DISCUSSION

28s~

q

JI

18SD"

1 Fig. 1. Northern blot analysis of expression of phosphatase 2C mRNA in the brain of adult rat. The position of the 28S and 18S ribosomal subunits are indicated.

The present study disclosed for the first time regional variation in the intensity of expression of PP2C m R N A in the brain. When compared with the localization of immunoreactivity for calcineurin/PP2B in the brain reported by G o t o et al. 5, and assuming that the expression level of m R N A for calcineurin is comparable to that of the enzyme itself, some differences and similarities in the expression intensity are pointed out in several brain loci between these two phosphates: the caudate-putamen expresses high levels of calcineurin, but low levels of PP2C. In the cerebellum, some Purkinje cells and granule cells express calcineurin, while all Purkinje and granule cells express PP2C intensely. The plexuses choroideus express PP2C moderately but they exhibit no expression of calcineurin. On the other hand, both PP2C and calcineurin

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Figs. 2 and 3. Coronal sections of olfactory bulb in dark field (Fig. 2) and bright field (Fig. 3). Mitral cells in the mitral cell layer (M) and granule cells in internal granule cell layer (I) express the mRNA weakly, whereas periglomerular cells in the glomerular layer (G) express the gene faintly. EP, external plexiform layer. Bars = 0.5 mm. Fig. 4. A coronal section through the caudate-putamen. Moderate expression is found in the piriform cortex (P), whereas neocortox (Cx) and septohippocampus (SH) express the gene weakly. Note faint and diffuse labeling in the caudate-putamen (CP). CC, corpus callosum. Bar = 1 mm. Fig. 5. A coronal section through thalamus. The hippocampal pyramidal cell layers (CAI-3) and granule cell layer of the dentate gyrus (D) express the gene intensely. Moderate expression is seen in the medial habenula (Hb) and the plexus choroideus of the third and the fourth ventricles (arrowheads). Weak expression is seen in the thalamic (T) and hypothalamic (HT) nuclei. Bar = 1 mm.

287 TABLE I Comparison of expression levels of protein phosphatase 2C m R N A among neurons and plexus choroideus in the central nervous system of the rat

Nomenclature for the brains is drawn from the atlases of Paxinos and Watson9. Relative expression intensities are graded: + + + , intense; ++, moderate; +, weak; - / + , faint or partial; - , absent. Olfactory bulb perigiomerular cell mitral cells tufted cells internal granule cells Cerebrum olfactory tubercle piriform cortex amygdaloid nucleus hippocampus CA1 CA2 CA3 dentate gyrus caudate-putamen accumbens nucleus globus pallidus septum (medial, lateral) septohippocampal nucleus diagonal band neocortex Diencephalon hypothalamus preoptic area paraventricular nucleus supraoptic nucleus mammillary nucleus habenula medial lateral pineal body zona incerta thalamic nuclei Mesencephalon substantia nigra red nucleus oculomotor trochlear nucleus central gray

-/+ + + + ++ + +++ +++ +++ +++ -/+ + + -/+ + + +

+ + + + ++ + ++ + + ++ ++ ++ + +

are expressed intensely in the hippocampal formation and weakly to moderately in the neocor tex of cerebrum. These patterns of expression suggest that various neuronal proteins are differentially dephosphorylated in different loci by PP2C and calcineurin. F u r t h e r m o r e , the importance of protein phosphatases in the phosphorylation/dephosphorylation cycle leads us to compare the expression patterns of m R N A s for PP2C and serine-threonine protein kinases in the brain. There are a large n u m b e r of serine-threonine protein kinases, most of which have several subtypes. A t present our information on the localization of gene expression is confined to a limited n u m b e r of kinases such as cAMP-de-

superior colliculus inferior colliculus mesencephalic trigeminal nucleus locus ceruleus dorsal raphe nucleus Pons and mudulla oblongata pontine nucleus pontine reticular nucleus motor trigeminal nucleus spinal trigeminal nucleus facial nucleus cochlear nucleus vestibular nucleus medial spinal lateral dorsal motor nucleus vagus hypoglossal nucleus lateral reticular nucleus gracil nucleus cuneate nucleus external cuneate nucleus inferior olivary nucleus superior olivary nucleus raphe magnus nucleus

+ + ++ ++ + ++ + + + ++ + + + + + + ++ + + + + + +

Cerebellum Purkinje cells granule cells molecular layer deep cerebellar nuclei

+++ +++ +

Spinal cord ventral horn dorsal horn lateral horn

+ + +

Plexus choroideus lateral ventricle third ventricle fourth ventricle

++ ++ +

nucleus

p e n d e n t protein kinases (A-kinase), Cae+-phospholipid d e p e n d e n t protein kinases (C-kinases), and type II Ca 2+c a l m o d u l i n - d e p e n d e n t protein kinases (CaM kinases 11)24,12. Similarities and differences in the gene expression levels between these kinases and PP2C are noted in several brain loci. For instance, in the cerebellar granule layer and the hippocampal formations, m R N A s for A-kinases R I a and Rift, C-kinases III, and CaM kinase Ilfl are expressed at high levels which were well compatible with the intense expression of PP2C m R N A . This indicates the occurrence of very active phosphorylation/dephospholylation cycle in these brain regions. O n the other hand, a considerable n u m b e r of the kinase tran-

288 scripts are expressed highly in the c a u d a t e - p u t a m e n , in which the expression of P P 2 C is at low levels, a n d n o n e of the p r o t e i n kinases have b e e n d e t e c t e d highly in the plexus c h o r o i d e u s in which PP2C is c o n c e n t r a t e d at the t r a n s c r i p t i o n level. T h e identification of n e u r o n a l sub-

icance of PP2C, in relation to the p h o s p h o r y a t i o n / d e p h o s p h o r y l a t i o n cycle.

strate proteins specific for P P 2 C a n d m o r e detailed localization of o t h e r p r o t e i n kinase genes in b r a i n s are crucial to u n d e r s t a n d m o r e clearly the f u n c t i o n a l signif-

Acknowledgements. This work was supported by grants from Asaoka Eye Hospital Foundation, Hamamatsu, Japan and from JCR Pharmaceuticals Co. Ltd., Ashiya, Japan. The authors wish to thank Mr. H. Iwasa for his photographic assistance.

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