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

Molecular Brain Research, 22 (1994) 139-143

139

© 1994 Elsevier Science B.V. All rights reserved 0169-328X/94/$07.00

BRESM 70719

Localization of mRNA for protein phosphatase 2A in the brain of adult rats Hiroshi Abe

a,,,

H i r o s h i S h i m a b, M a s a k i S e k i g u c h i a, H u a n g G u o Shinri T a m u r a c, H i s a t a k e K o n d o d

a,

M i n a k o N a g a o b,

Department of Morphology, Division of Human Structure and Function, Tokai University School of Medicine, lsehara, Japan, b Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan, c Department of Biochemistry, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan, a Department of Anatomy, Tohoku University School of Medicine, Sendai, Japan

a

(Accepted 24 August 1993)

Key words: Protein phosphatase 2A; mRNA; Brain; Rat; In situ hybridization

The gene expression for a and /3 isoforms of type 2A protein phosphatase (PP2A-a and -/3) in the adult rat brain was examined by in situ hybridization analysis. No marked difference in the gene expression was discerned between the two isoforms in large portions of brain, except for the thalami in which the expression level for the a isoform was similar to that in the cerebral neocortex whereas that for the/3 was lower than that in the neocortex. The gene expression was observed intensely in the piriform cortex, the cerebellar Purkinje and granule cell layers, and the hippocampal pyramidal and dentate granule cell layers, and the locus ceruleus, whereas the moderate levels of its expression were observed in the olfactory mitral cells and the pontine nuclei. The cerebral neocortex expressed the mRNA moderately to weakly without any laminar patterns, whereas the expression level in the caudate-putamen was very low. This expression pattern is basically similar to that of PP2C reported previously, except for the plexus choroideus and ependyma having no significant expression for PP2A.

INTRODUCTION Reversible addition and removal of phosphate esters on serine and threonine hydroxyls by protein kinases and phosphatases have been regarded as major mechanisms modulating activity of various intracellular processes 9. Four subtypes of the protein-serine/ threonine phosphatases have been identified on the basis of their sensitivity to thermostable proteins, inhibitors-1 and 2, of their preferential dephosphorylation of a or fl subunit of phosphorylase kinase, and of their divalent cation requirements, and they are termed protein phosphatase type 1 (PP-1), PP-2A, PP-2B and PP-2C 5'8'19. All PPs except for PP-2C are composed of catalytic and regulatory subunits and their substrate specificities are suggested to depend on their subunit conformations 5'6'19. However, the detailed physiological roles in vivo of the PPs remain largely to be elucidated. Localization of sites of expression of PPs and their genes in the brain is the first step in understanding their significance in the brain function. In this regard,

previous analyses have revealed the heterogenous expression in the brain of mRNAs for PP-2B (calcineurin) by others 18 and PP-2C by us ~ in the brain. The present study was thus undertaken, as the second of a series of our analyses, to clarify the gene expression of PP-2A in the adult rat brain by in situ hybridization histochemistry. MATERIALS AND METHODS As cDNA probes specific to catalytic subunits of PP2Aa and PP2A/3, PstI-EcoRI fragment, about 400 bp fragment of 3' noncoding region of rat PP2Aa-cDNA and Bst XI-Rsa I fragment, about 400 bp fragment of 3'-non-coding region of rat PP2A/3-cDNA were selected on the basis of the original reports l°'H. The probes were labeled with [35S]dATP by random primed cDNA labeling kit (Boehringer Mannheim, Germany). In situ hybridization histochemistry was performed in essentially the same way as described 2° with minor modifications. Briefly, brains of male albino rats of 150--200 g body weight were removed, frozen on powdered dry ice. Frozen sections, 20--30 p.m thick, were made on a cryostat and mounted on slide glasses. The slides were dipped into 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 15 min and 2 m g / m l of glycine in phosphate buffered saline for 20 min, and subsequently acetylated in 0.25% acetic

* Corresponding author. Department of Morphology, Tokai University School of Medicine, Isehara 259-11, Japan. Fax: (81) 463-94-9052.

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140 anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min. After dehydration in graded series of ethanol, sections were prehybridized for 2 h at room temperature in a prehybridization mixture containing 4×SSC, 50% deionized formamide, 1 ×Denhardt's solution, 2% Sarcosyl, and 250/zg/ml of heat denatured salmon sperm DNA in a (/.l M sodium phosphate buffer (pH 7.2). Hybridization was done at 42°C overnight with a [35S]dATP-labeled probe at a concentration of 5×105 cpm/50 /zl of hybridization mixture, consisting of fresh prehybridization mixture, 10% dextran sulfate and 0.1 M dithiothreitol. The slides were washed 3 times in 0.1 xSSC-0.1% Sarcosyl at 42°C for 40 min and then autoradiographed using NTB2 nuclear track emulsion (Kodak) for 8 weeks. RESULTS L o c a l i z a t i o n of the g e n e expression for P P 2 A s was d e t e c t e d by the a c c u m u l a t i o n o f a u t o r a d i o g r a p h i c silver grains. T h e e x p r e s s i o n signals w e r e o b s e r v e d m o r e o r less in all n e u r o n s in the gray m a t t e r o f the c e n t r a l nervous system, while those in the white m a t t e r , such as c o r p u s c a l l o s u m a n d p y r a m i d a l tract, w e r e very w e a k or negligible (Fig. 1). N o m a r k e d d i f f e r e n c e s in the gene expression w e r e o b s e r v e d b e t w e e n P P 2 A a a n d PP2A/3 in large p o r t i o n s of the b r a i n except for the thalamus. T h e r e f o r e , t h e d e s c r i p t i o n of the g e n e e x p r e s s i o n was c o n f i n e d to P P 2 A a except for the thalamus. In the olfactory bulb, the m R N A was e x p r e s s e d m o d e r a t e l y in the mitral cells a n d weakly in the p e r i g l o m e r u l a r cells, the i n t e r n a l g r a n u l e layer a n d the tufted cells (Fig. 2). A n t e r i o r olfactory nucleus also e x p r e s s e d the g e n e weakly. In the c e r e b r u m , w e a k e x p r e s s i o n was d e t e c t e d in the olfactory tubercle, a m y g d a l o i d nucleus, s e p t u m a n d e n t o r h i n a l cortex, while i n t e n s e expression signals w e r e o b s e r v e d in the p i r i f o r m cortex a n d the s e p t o h i p -

p o c a m p a l nucleus (Fig. 3). T h e signals in the c a u d a t e p u t a m e n was very w e a k o r negligible a n d no b o u n d a r y was actually visible in the d a r k - f i e l d microscopy between the c a u d a t e - p u t a m e n and c o r p u s callosum. M o d e r a t e to w e a k expression was d i s c e r n e d in the d i a g o n a l b a n d a n d the n e o c o r t e x (layers I I - V I ) without l a m i n a r p a t t e r n s . In the h i p p o c a m p u s , i n t e n s e e x p r e s s i o n was dist r i b u t e d in the p y r a m i d a l cell layers of the C A 1 - 3 regions. G r a n u l e cells o f the d e n t a t e gyrus also exp r e s s e d the m R N A intensely (Fig. 4). In the d i e n c e p h a l o n , m o d e r a t e to w e a k expression for P P 2 A a was d i s c e r n e d in the m e d i a l h a b e n u l a r nucleus a n d t h a l a m i c nuclei (Fig. 4), w h e r e a s w e a k expression for PP2A/3 was o b s e r v e d in the t h a l a m i c nuclei. In the thalamus, the g e n e expression level for P P 2 A a was similar to that in the neocortex, w h e r e a s that for PP2A/3 was lower t h a n that in the neocortex. H y p o t h a l a m i c nuclei a n d m a m i l l a r y nuclei e x p r e s s e d the g e n e weakly. In the m i d b r a i n , m o d e r a t e to w e a k expression was d i s c e r n e d in the p a r s c o m p a c t a o f t h e s u b s t a n t i a nigra, the r e d nucleus, the o c u l o m o t o r nucleus, a n d the ventral t e g m e n t a l nucleus. W e a k e x p r e s s i o n was o b s e r v e d t h r o u g h o u t t h e m i d b r a i n including t h e s u p e r i o r a n d inferior colliculi, a n d t h e d o r s a l r a p h e nucleus (Figs. 5 a n d 6). In the p o n s and m e d u l l a o b l o n g a t a , i n t e n s e expression was d i s c e r n e d in t h e locus ceruleus, while the p o n t i n e nuclei e x p r e s s e d the m R N A m o d e r a t e l y (Figs. 5 a n d 6). M o d e r a t e to w e a k expression was d i s c e r n e d in the motor, spinal, m e s e n c e p h a l i c a n d p r i n c i p a l sensory t r i g e m i n a i nuclei, t h e facial nucleus, the c o c h l e a r

Fig. 1. A saginal section of adult rat brain showing expression of the mRNA of phosphatase (PP) 2Aa. Intense expression is found in the cerebellum (CBL), hippocampus (H), and moderate expression is observed in the pontine nucleus (Pn), and the mitral cell layer of the olfactory bulb (M). Cx, cerebral neocortex. Bar = 1 mm. Fig. 2. A coronal section of olfactory bulb. Mitral cells (M) express the mRNA moderately, whereas periglomerular cells in the glomerular layer (G) and granule cells in the internal granule cell layer (I) express the gene weakly. EP, external plexiform layer. Bar = 0.1 mm. Fig. 3. A coronal section through the caudate-putamen (CP). Intense expression of the PP2Ac~ is found in the piriform cortex (Pi) and the septohippocampal nucleus (SH), whereas neocortex (Cx) expresses the gene weakly. The expression level in the caudate-putamen (CP) is very low. CC, corpus callosum. Bar = 1 mm. Fig. 4. A coronal section through the thalamus. The hippocampal pyramidal cell layers (H) and granule cells of the dentate gyrus (D) express the gene intensely. Moderate to weak expression is seen in the thalamic nuclei (T). Note intense expression in the piriform cortex (Pi) and the anterior cortical amygdaloid nucleus (AC). Bar = 1 mm. Fig. 5. A coronal section through the pons. Note moderate expression of the gene in pontine nucleus (Pn), oculomotor nucleus (3) and red nucleus (R). Aq, aqueductus cerebri. CG, central gray. SC, superior colliculus. Bar = 1 mm. Figs. 6 and 7. Coronal sections through cerebellum. Intense expression is seen in the granule cells (G), Purkinje cells (arrows) and the locus ceruleus (LC). Moderate to weak expression is discerned in the cochlear nucleus (Co), the dorsal motor nucleus vagus (10), hypoglossal nucleus (12), inferior olivary nucleus (InO), principal sensory trigeminal nucleus (Pr5) and the external cuneate nucleus (EC). Bars = 1 mm. Fig. 8. A transverse section of spinal cord. Weak expression is seen in the gray matter. AH, anterior horn; C, central canal; PH; posterior horn. Bar = 0.5 ram.

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142 nucleus, the vestibular nucleus, the hypoglossal nucleus, the lateral reticular nucleus, the external cuneate nucleus, the posterodorsal and retieulotegmental nucleus and the inferior olive (Fig. 7). In the cerebellum, Purkinje cells and granule cell layers expressed the m R N A intensely, Deep cerebellar nuclei expressed the gene moderately, while no expression signals were discerned in the molecular layer (Fig. 6). In the spinal cord, weak expression was recognized in the gray matter, while the expression level in the white matter was very weak (Fig. 8). No significant expression was observed in the plexus choroideus of the lateral, third and fourth ventricle, or the ependyma. For control, some brain sections were pretreated with 100 /zg/izl RNase A in 2 × SSC at 37°C for 60 min before hybridization. No significant expression signals above the background levels were detected on the sections of control experiments. DISCUSSION The present study disclosed for the first time widespread expression with differential intensity of m R N A s for PP2A in the adult rat brain, although no marked difference in the expression pattern was disc e r n e d between their two isoforms, PP2A-a and -/3. The high expression was observed in the hippocampal pyramidal cell and dentate granule cell layers, the cerebellar Purkinje cell and granule cell layers. These neuronal populations have also been shown to express highly genes for PP-2B t8 and PP2C 1, and further various subtypes or subunits of cAMP, Ca2+/calmodulin and Ca/phospholipid-dependent protein kinases 2'3'4, suggesting the occurrence of very active phosphorylation/dephosphorylation cycle in these neurons. When compared with our previous finding about PP2C 1, the gene expression for PP2A in the brain shows a localization pattern similar to that of PP2C, except for the plexus choroideus and ependyma. This similarity, together with the absence of marked difference in the expression between a and /3 isoforms of PP2A is in contrast to protein kinases of the s e r i n e / threonine type whose subspecies and subunits show discrete expression patterns 2'3'4 and these features are well compatible with the fact that these PPs have broad and overlapping specificities in vitro 5'6'9. The modulation of Ca-activated potassium channels by PP2A has recently been demonstrated in the rat brain ~5. Thus setting the phosphorylation state of multiple sites in the pottasium channels by PP2A and protein kinases would be crucial to exert diverse func-

tion of given neurons in response to extracetlular stimuli. The gene cloning and subsequent detailed localization of m R N A s for the Ca-activated pottasium channels and comparison with the present finding might help understand more clearly the functional significance of the protein phosphatase. Furthermore, because it has recently been shown that PP1 as well as PP2A and even PP2C are involved in the reversal of the Ca2+-independent activity of Ca2+/catmodulin dependent protein kinase II by autophosphorylation 7't2-14-t6"t7. it is necessary to examine the detailed localization of m R N A for PP1, another major protein phosphatase, which will be published elsewhere by us. The authors wish to thank Mr. H. lwasa and Miss Tomoko lnoue for their technical assistance. This work was supported by grants from the Ministry of Education, Science and Culture of Japan No. 04670032 to H. Abe. Nos. 04770002 and 04404020 to H. Kondo, and also by a grant from Tanaka Orthopedic Clinic Foundation. Hamamatsu. Japan. 1 Abe. H.. Tamura. S. and Kondo. H., Localization of mRNA for protein phosphatase 2C in the brain of adult rats. Mol. Brain Res., 13 (1992) 283-288. 2 Brandt, S.J., Niedel. J.E.. Bell. R.M. and Young, W.S.. III. Distincl patterns of expression of different protein kinase C mRNAs in rat tissues, Cell, 49 (1987) 57-63. 3 Burgin, K.E., Waxham, M.N.. Ridding, S., Westgate, S.A., Mobley, W.C. and Kelly, P.T., In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain, J. Neurosci., 10 (1990) 1788-1798. 4 Cadd, G. and McKnight, G.S.. Distinct patterns of cAMP-dependent protein kinase gene expression in mouse brain, Neuron. 3 (1989) 71-79. 5 Cohen. P.. The structure and regulation of protein phosphatases. Annu. Rev. Biochem., 58 (1989) 453-508. 6 Cohen, P.. Signal integration at the level of protein kinases, protein phosphatases and their substrates, TIBS. 17 (1992) 408413. 7 Fukunaga, K., Kobayashi, T., Tamura, S. and Miyamoto, E., Dephosphorylation of autophosphorylated Ca2+/calmodulindependent protein kinase II by protein phosphatase 2C, J. Biol. Chem., 268 (1993) 133-137. 8 Ingebritsen, T.S. and Cohen, P., The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities, Eur. J. Biochem., 132 (1983) 255-261. 9 Ingebritsen, T.S. and Cohen, P., Protein phosphatases: properties and role in cellular regulation, Science, 221 (t983) 331-338. 10 Kitagawa, Y., Tahira, T., Ikeda, I., Kikuchi, K., Tsuiki, S., SUgimura, T. and Nagao, M., Molecular cloning of cDNA for the catalytic subunit of rat liver type 2A protein phosphatase, and detection of high levels of expression of the gene in normal and cancer cells, Biochem. Biophys. Acta, 951 (1988) 123-129. 11 Kitagawa,Y., Sakai, R., Tahira, T., Tsuda, H., Ito, N., Sugimura, T. and Nagao, M., Molecular cloning of rat phosphoprotein phosphatase 2Aft eDNA and increased expressions of phosphatase 2Aa and 2A/3 in rat liver tumors, Biochem. Biophys. Res. Commun., 157 (1988) 821-827. 12 Lai, Y., Nairn, A.C. and Greengard, P., Autophosphorylation reversibly regulates the Ca2+/catmodulin-dependence of Ca2+/ calmodulin-dependent protein kinase II, Proc. Natl. Acad. Sci. USA, 83 (1986) 4253-4257. 13 Lou, L.L. and Schulman, H., Distinct autophosphorylation sites sequentially produce autonomy and inhibition of the multifunctional caa+/calmodulin-dependent protein kinase, J. Neurosci., 9 (1989) 2020-2032.

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