A pancreatic-like ribonuclease is synthesized in rat brain

Molecular Brain Research, 14 (1992) 1-6 (~) 1992 Elsevier Soence Pubhshers B.V All rights reserved 0169-328X/92/$05.00 BRESM 70415

1

Research Reports

A pancreatic-like ribonuclease is synthesized in rat brain G. Grassi Zucconi a, C. Cosi b, M. Palmieri b, A. Furia c, M . A . Bassetti a and A. Carsana b alnstttute of Cellular Btology, Umverstty of Perugta, Perugta (Italy) and blnstttute of Btologwal Chemistry, University of Verona, Verona (Italy) and CDepartment of Organic and Btologzcal Chemzstry, University of Naples, Naples (Italy) (Accepted 10 December 1991)

Key words Pancreatic RNAase; Rat brim; Gene expression. RNA blot, In situ bybn&zatlon, Immunocytochemistry The &stnbutlon and cell localization of a pancreatic-hke rlbonuclease (RNAase) m the rat brain has been studied by RNA blot analysis and m sltu hybridization using as a probe the cDNA coding for the rat pancreas RNAase, and by lmmunocytochemlstry using an antiserum raised against the rat pancreas RNAase. RNA blot analysis and in situ hybridization experiments have shown that the RNAase mRNA is present in all the cerebral areas investigated and that neurons appeared to be actively expressing RNAase mRNA while ghal cells were devoid of hybridization signals In agreement with these results the ~mmunocytochemical analysis has shown that neurons are specifically lmmunostained These experiments demonstrate that a pancreatlc-hke rlbonuclease is synthesized in the neurons of the rat brain INTRODUCTION Until a few years ago the physiological role of extracellular

pancreatic-like

ribonucleases

(RNAases)

was

t h o u g h t to b e a s s o c i a t e d e x c l u s i v e l y w i t h d i g e s t i v e p r o cesses. T h e p r e s e n c e o f r i b o n u c l e a s e s i d e n t i c a l o r v e r y s i m i l a r to t h o s e p r o d u c e d b y t h e p a n c r e a s in o t h e r m a m m a l i a n o r g a n s o r b i o l o g i c a l fluidsSsuggests t h a t t h e s e e n zymes could, however, have different roles. Moreover, ribonucleases

have

recently

gained

renewed

interest

through the discovery that some extracellular proteins such

as

a n g i o g e n i n 14,

eosinophil-derived

neurotoxin

solution 2% bovine serum albumin, 2% Ficoll, 2% polyvlnyl pyrrolidone)/0.5% SDS/80 ktg/ml denatured salmon sperm DNA/10 ng/ml labelled probe at 42°C. The probes used were the rat pancreas rlbonuclease cDNA 9'I2 and the human actln cDNA labelled with 3Zp using the Multiprime DNA labelling system (Amersham, 1-1 4 109 cpm/pg). After hybn&zatlon filters were washed in 0.1 × SSC/0 1% SDS at 60°C, exposed to Kodak films, and the autoradiograms were scanned with a laser densttometer (LKB) Northern blot analysl~ was carried out using 40 pg of each RNA sample electrophoresed on 1% agarose gel containing 6% formaldehyde and running buffer (20 mM MOPS, 5 mM sodium acetate and 1 mM Na 2 EDTA, pH 7), and transferred to Hybond N membranes. Hybn&zatlon with rat pancreas rlbonuclease cDNA and washings were carried out following the same condmons used for the slot blot analysis

( E D N ) 8, e o s i n o p h i l c a t i o n i c p r o t e i n ( E C P ) 4 a r e h o m o l -

In sttu hybridization

o g o u s to p a n c r e a t i c r i b o n u c l e a s e s a n d t h e i r b i o l o g i c a l

Tissues from 3-month-old animals (3 specimens) were fixed in paraformaldehyde (4% paraformaldehyde in 150 mM phosphate buffer, pH 7 4), embedded in paraffin and cut at a thickness of 5/~m. Sections were mounted onto gelatin/chrome alum-treated slides After deparaffinatlon and hydration slices were incubated with protelnase K (1 ktg/ml in 100 mM Trls-C1/50 mM EDTA, pH 8 0) for 30 rain at 37°C, treated with 0.25% acetic anhydride in 100 mM trlethanolamme-HCl, pH 8 0, for 10 mln at room temperature and dehydrated. Hybn&zatlon to the ass-labelled rat pancreas cDNA 9'12 (MultiprIme DNA labelling system, Amersham, spec radIoact > 5 108 cpm//~g) was carried out at a concentration of 1"107 cpm/ml in a buffer containing 50% formamlde, 2 × SSC, 1 × Denhardt's solution, 10% dextran sulfate, 500 #g/ml denatured salmon sperm DNA and 500/~g/ml tRNA for 16 h at 50°C. Following hybridization, the sections were washed at a moderate stringency in 0.5 × SSC for 15 mln at 52°C, dehydrated and exposed to Hyperfilm-flmax (Amersham) for 3-5 days The sections were then dipped in K5 Ilford nuclear track emulsion for 8-10 days at 4°C, developed in Kodak D 19 and counterstained with hematoxyhn To verify the RNAase mRNA hybridization specificity three different controls were carried out (1) hybridization with unrelated DNA (pBR322 plasmid fragments) under identical conditions used for RNAase cDNA hybridization no localized signal was observed, (2) pretreatment of tissue sections with a solution containing 20/~g/ml

f u n c t i o n s a r e r e l a t e d t o t h e i r r i b o n u c l e o l y t i c activities 1°' 17.19 A c i d a n d a l k a l i n e r i b o n u c l e a s e activities h a v e b e e n d e t e c t e d in m a m m a l i a n

brain both by biochemical and

h i s t o c h e m i c a l m e t h o d s t'3"7aa'1s. I n this s t u d y w e h a v e a d d r e s s e d t w o q u e s t i o n s : (1) is a p a n c r e a t i c - l i k e r i b o n u c l e a s e s y n t h e s i z e d in t h e r a t b r a i n , a n d (2) w h i c h a r e t h e cell t y p e s i n v o l v e d in t h e s y n t h e s i s o f this e n z y m e .

MATERIALS AND METHODS

Slot blot and Northern blot analyses Total RNA was extracted from regional rat brain sections as reported I6. 27 pg, 9 ktg, 3 ktg of each RNA sample were denatured by heating at 65°C for 15 mm in 5% formaldehyde/6 × SSC (1 × SSC" 150 mM NaC1, 15 mM sodium citrate), loaded into the wells of a slot blot apparatus (Schleicher & Schuell) and transferred to Hybond N membranes (Amersham) Filters were hybridized in 50% formamlde/6 x SSC/5 × Denhardt's soluuon (1 x Denhardt's

Correspondence A Carsana, Institute of Biological Chemistry, University of Verona, Strada Le Grazle 8, 37134 Verona, Italy

R N A a s e A and R N A a s e T1 for 30 min at 37°C before hybridization the autoradiographlc signal was dramatically reduced; (3) hybridization of rat pancreas sections with R N A a s e c D N A ' only acmar cells appeared to be labelled, whereas Langerhans islets were devoid of signals The percentage of R N A a s e m R N A - c o n t a i n i n g neurons was calculated using a calibrated eyepiece reticule at 600x The size of the analysed area in different brain regions varied from [) 187 to 0 625 ram-" Only neurons covered with silver grains at a density 10 times over background were considered to be labelled Background levels were determined with sections hybridized with unrelated D N A (pBR322 plasmld fragment) of the same specific activity Neuronal and ghal cells were morphologycally dlstln200 R N A a 8 e

150

m R N A

100

I e

v

50

@ I 8

TH

f3

HIP

Cx

CB TH

81"R

HY

OT

BR

B8

OB

OB

guished on the basis of their perlkaryal profiles and Nlssl staining characteristics ~

lmmunocytochemtstry Shdes with adjacent sections of the same area of tissues prepared for in situ hybridization were premcubated in 0 3% H 2 0 2 in methanol for 20 rain at room temperature to inactivate endogenous peroxldase, treated with 0 05% protease XIV (Sigma) for 15 mln at 37°C, and finally with normal swine serum &luted ten fold in PBS (137 m M NaCI, 2 7 m M NazHPO 4, 1 4 m M KHzPO4) at room temperature for 1-2 h Sections were incubated overnight at 4°C with rabbit antiserum raised against rat pancreas R N A a s e (1 100 in PBS containing 1% BSA) The rat pancreas rlbonuclease was a kind gift of Prof J J Belntema, the University of Groningen The sections were then washed in PBS and were incubated with blOtlnylated anti-rabbit donkey serum ( A m e r s h a m , 1 50) for 1 h at room temperature, processed with Vectastain avldin-biotln peroxldase kit (Vector Labs, Inc ) and reacted with a solution containing 0 05c~ 3,3'-dlammobenzldlne tetrachlorlde ( D A B ) and 0 01% hydrogen peroxide Control sections were processed as described, except that the primary antibody was replaced by normal rabbit serum Moreover, as positive and negative controls, rat pancreas sections were analysed with rat pancreas R N A a s e antibodies only aclnar cells revealed strong positive reactions Ghal cells were distinguished on the basis of their lmmunoreactlvlty with a monoclonal antibody to ghal fibrillary acidic protein (GFAP) (Dakopatts) After the incubation with Vectastaln avldm-blotm peroxidase kit the sections were reacted with a solution containing 0 05% carbazole and 6% N,N-dlmethylformamlde in 20 m M sodium acetate buffer, pH 5 2 Both hghtfield and darkfield microphotographs were taken on a Nikon photomlcroscope.

BS

S Pi 28S-

18S-

C C 'vJ

G r J

Fig 1 A. levels of R N A a s e m R N A m &fferent areas of the rat brain Results were normalized relative to the level of actin m R N A and expressed as a percentage of the R N A a s e m R N A present in the whole brain B Northern blot analysis HIP, hlppocampus, TH, thalamus, OT, olfactory tubercule, HY, hypothalamus, STR, striaturn, CX, cortex, BR, whole brain, BS, bralnstem, CB, cerebellum, O B , olfactory bulb

Fig 2 A u t o r a & o g r a m s of representative coronal sections of the rat brain hybridized to the R N A a s e c D N A CC, corpus callosum, STR, strlatum, PICX, plrlform cortex, D G , dentate gyrus of hlppocampus, CA1 and CA3, regions of the hlppocampus, Gr, cerebellum granules

of total R N A isolated from different cerebral areas to

RESULTS

the rat pancreas R N A a s e c D N A . All the results were normalized relative to the level of actin m R N A and expressed as a percentage of the R N A a s e m R N A present

The level of ribonuclease m R N A in various areas of the rat brain was determined by slot blot hybridization

13

A

f t

C

D

I:

.,;

.4

Fig. 3 Expression of RNAase mRNA in different regions of the rat brain A: darkfield photomicrograph showing cortex (1), corpus callosum (2), alveus (3) and CA/ (4) regions of the hlppocampus. B-F bright-field photomicrographs of cortex (B), corpus callosum (left side m C), cmgulate cortex (right side in C), CA1 region of the hlppocampus (D). tela chonoldea (E) and cerebellum (F) Gr, granules, Pu, Purklnje cells. Bars = 40/~m

ebellum and olfactory bulb showing the highest levels. Northern blot analysis of total R N A isolated from three cerebral areas revealed a single R N A species of about 900 nucleotides hybridizing to the rat pancreaUc R N A a s e c D N A (Fig. 1B) This transcript is similar in length to the R N A a s e m R N A found m rat pancreas ~, although ~ts amount m the brain is much lower than that found m the pancreas ~2. The topological distribution of this R N A species in the rat brain was analysed by in situ hybridization. The specificity of labelling was verified by three i n d e p e n d e n t controls (see Materials and Methods). Fig. 2 shows the autoradiograms of r e p r e s e n t a t w e coronal sections of rat brain. The highest hybridization signal was observed in the dentate gyrus of hippocampus and in the cerebellar cortex, while a relatively low hybridization signal was detected in the cerebral cortex, striatum and thalamlc nuclm. No slgmficant labelling could be detected m the corpus callosum. The localization of m R N A coding for ribonuclease in rat brain was examreed at the cellular level on sections dipped in nuclear track emulsion Hematoxylin staining revealed sdver grams associated specifically with neuronal cells. Neu-

100

80

t.,a 4 o

=°

y

PCX

P i CX

TH

CA 1

CA~

DG

6~

I~

Fig 4. Percentage of RNAase mRNA-posmve neurons (stripped bars) and of RNAase lmmunostalned neurons (black bars) Panetal cortex (PCX), preform cortex (P1CX); thalamus (TH); regions of the hlppocampus' CA1, CA3 and dentate gyrus (DG); cerebellum granules (Gr) and Purklnje cells (Pu)

m the whole brain (Fig. I A ) . Pancreatic R N A a s e m R N A was detected in all the regions investigated, with the cer-

A

C

B

"2

D

Fig 5. Distribution of RNAase-lmmunoreactlve neurons in the rat brain. A cortex, B. corpus callosum (left side) and clngulate cortex (right side), C hlppocampus, DG dentate gyrus, D: cerebellum Bars = 40 um

rons containing RNAase m R N A were found in all the regions examined (Fig. 3) and represent a significant proportion of neuronal population (Fig. 4). No differences were detected between any of the three animals studied. In contrast glial cells were negative following the hybridization with the RNAase specific probe. This was clearly visible in the corpus callosum, where glial cells appeared to be devoid of specific signal (Fig. 3C). The presence of the ribonuclease molecule in the rat brain was determined by the immunocytochemical technique using rabbit antiserum raised against rat pancreas ribonuclease. The immunostaining revealed a specific pattern (Fig. 5) which is similar to that obtained by the in situ hybridization method, although the percentage of RNAase immunopositive neurons is lower than that of the neurons expressing the RNAase m R N A (Fig. 4). Glial cells appeared to be RNAase-immunonegative as demonstrated by immunocytochemical experiments performed using both RNAase-specific antiserum (revealed with DAB) and GFAP-specific antibodies (revealed with carbazole). Sections were processed either with RNAasespecific antiserum first and then with GFAP-specific antibody or with GFAP-specific antibody first and then wtth RNAase-specific antiserum. Identical results were obtained: no overlapping between the two different immunostainings (data not shown). DISCUSSION In the present study we report the distribution and cell localization in the rat brain of a ribonuclease identical or very similar to the enzyme synthesized by the exocrine pancreas. Slot blot and in situ hybridization analyses revealed the presence of RNAase m R N A in all regions of the brain investigated. It is worth mentioning that the apparent discrepancy between the low level of RNAase m R N A determined in the total hippocampus. (Fig. 1) and the high signal observed in the dentate gyrus and the CA1 region of the hippocampus after the in situ hybridization analysis (Fig. 3) can be explained by the higher neuronal cell density of these two regions relative to the other areas of the hippocampus. Neurons appeared to be actively expressing RNAase m R N A , while glial cells were devoid of hybridization signals (Fig. 3). Similar results were obtained by immu-

REFERENCES 1 Alberghlna, M. and Gmffnda Stella, A.M., Age-related changes of ribonuclease activities in various regions of the rat central nervous system, J. Neurochem , 51 (1988) 21-24. 2 Alhnquant, B , Musenger, C. and Schuller, E., Intrathecal orIgin of CSF nbonuclease, Acta Neurol S c a n d , 69 (1984) 1219.

nocytochemical staining using polyclonal antibodies raised against homogeneous rat pancreas RNAase (Fig. 5). The number of RNAase mRNA-positive neurons appears to be higher than RNAase-immunostained neurons (Fig. 4). This may reflect either translational or posttranslational regulation of the ribonuclease molecule or greater sensitivity of the technique of in situ hybridization. The number of ribonuclease-containing neurons could be underestimated because of the failure of the method to detect low levels of antigen. Although the percentages vary, ribonuclease expressing neurons appear to constitute a significant proportion of neuronal population in the rat brain. A secretory ribonuclease has been isolated from bovine brain 7 and its amino acid sequence 2° shows a high degree of identity (78%) to the bovine pancreatic ribonuclease sequence. The bovine brain RNAase is the product of a distinct gene 6 originated by a duplication event occurred rather recently in the ancestor of ruminants after divergence from other artiodactyls s. On the contrary, only one ribonuclease gene of the pancreatic type seems to be present in the rat genome as suggested by Southern blot experiments 15. Therefore, the ribonuclease we investigated in the rat brain should be coded by the same gene as that expressed in the exocrine pancreas. Ribonuclease activities have been detected in the brain of some mammals including man and the rat 1'3"7' 13,18, and in the human cerebrospinal fluid 2. RNAase activity has been found in the cortex, hippocampal formation and ventricles of rat brain sections, whereas only very low levels of activity have been detected in the corpus callosum and internal capsule is. The experiments described herein demonstrate that the RNAase activities detected in the rat brain at neutral or alkaline pH may be, at least in part, due to an extracellular enzyme identical or very similar to the enzyme produced by the pancreas and that neuronal cells are responsible for the synthesis of this pancreatic-like ribonuclease.

Acknowledgements. We are indebted to Prof M. Libonatl for his constant support and encouragement throughout this study and thank Dr. M G. Tovey for critical reading of the manuscript This work was partially supported by EF Ingegnena Genetlca, CNR, and by a grant from the Ministero dell'Universlt~t e della Rlcerca Sclentlfica e Tecnologlca, Italy

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6

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