Molecular cloning and regional distribution of rat brain cyclophilin

Molecular Brain Research, 9 (1991) 239-244 Elsevier

239

BRESM 70264

Molecular cloning and regional distribution of rat brain cyclophilin Rajnikant P. Lad 1, Mark A. Smith 2 and D a n a C. Hilt 3 1Laboratory of Biochemical Genetics and 2Clinical Neuroendocrinology Branch, National Institutes of Health, Bethesda, MD 20892 (U.S.A.) and 3Departments of Neurology, Biochemistry, and the Medical Biotechnology Center, University of Maryland School of Medicine, Veterans Administration Hospital, Baltimore, MD 21201 (U.S.A) (Accepted 18 September 1990)

Key words: Cyclophilin; Cyclosporine A; In situ hybridization

Cyclosporine A (CsA) is a potent immunosuppressive drug that has widespread clinical uses in organ transplantation and the treatment of autoimmune disorders. However, the drug's clinical applications are on an empiric basis with a poor understanding of the basic mechanism(s) of action. CsA may exert some of its effects by binding to a cellular receptor protein - - the cyclosporine receptor (also called cyclophilin). Cyclophilin (CyP) is an ubiquitous, soluble, cytoplasmic 17 kDa protein which has recently been shown to be a peptide-prolyl isomerase. CsA specifically binds to this protein and inhibits its isomerase activity. A rat cyclophilin cDNA clone was isolated from a rat brain lambda gtll cDNA library. Northern blot analysis shows a single 1 kb messenger RNA in rat brain. In order to determine the regional distribution of the Cyp mRNA in situ hybridization was performed. The Cyp mRNA appeared to be expressed throughout the brain but there were particularly high levels in the cerebral cortex and hippocampus compared to the relatively low levels in white matter areas and tracts. At the cellular level, the Cyp mRNA is expressed at much higher levels in neurons than in glia. The high levels of Cyp in cortical (neuronal) areas may, in part, explain the global encephalopathic symptoms clinically observed in CsA neurotoxicity.

INTRODUCTION

isomerase activity and that C s A inhibits the isomerase activity of the protein s'21.

Cyclosporin A ( C s A ) is a cyclic u n d e c a p e p t i d e produced as a secondary m e t a b o l i t e by fungi imperfecti which has immunosuppressive and antiparasitic p h a r m a cologic activities2. C s A is a p o t e n t immunosuppressive drug which has revolutionized organ transplantation since, unlike o t h e r immunosuppressive drugs, it lacks significant myelotoxicity. C s A acts on the immune response p a t h w a y at a n u m b e r of points, all of which serve to suppress the i m m u n e response by aborting the activation of resting lymphocytes and inhibiting the production of interleukin-2 (IL-2) by helper T-cells 6'14. H o w e v e r , it does not interfere with expression of IL-2 receptors and so cannot affect T-cell proliferation 14. C s A also inhibits the production of g a m m a interferon TM. A l t h o u g h these effects of C s A have been d e m o n s t r a t e d on isolated i m m u n e cells in vitro, much of the clinical use of C s A remains empiric. Cyclophilin (Cyp) is a cytoplasmic protein that specifically binds C s A with high affinity 9. It is found in almost every cell in the body 12. The protein has been purified and the a m i n o acid sequence obtained for both the human and bovine species 11 which show m a r k e d conservation of their p r i m a r y amino acid sequence. Recently, it has been d e m o n s t r a t e d that Cyp has peptide prolyl

C s A has significant clinical side effects including nephrotoxicity and hepatotoxicity. Significant neurologic side effects are being r e p o r t e d with increasing frequency and include t r e m o r , ataxia, seizures, quadriparesis and coma 1'5. Recent studies in rats have d e m o n s t r a t e d the induction of epileptiform discharges d o c u m e n t e d by e l e c t r o e n c e p h a l o g r a p h y with both low and high doses of C s A 17. These manifestations suggest that the drug has global effects on cortical neuronal functioning. We report here the molecular cloning of a rat Cyp c D N A and regional distribution in rat brain of Cyp m R N A by in situ hybridization histochemistry. MATERIALS AND METHODS

Reagents Restriction enzymes and other DNA modifying enzymes were purchased from Bethesda Research Labs. (BRL), New England Biolabs (NEB), Boehringer Mannheim (BM), United States Biochemicals (USB) and International Biotechnologies Inc. (IBI). The following reagents and kits were purchased from the suppliers indicated, Random primer kit (BM), Sequenase Kit (USB), GeneScreen membrane (New England Nuclear (NEN), molecular weight markers (BRL). Radioisotopes were purchased from NEN. All the reagents were of ultrapure molecular biology grade. All reagents and enzymes were used according to the manufacturer's instructions. The rat brain (Sprague-Dawley) lambda gtll cDNA

Correspondence: D.C. Hilt, Department of Neurology, University of Maryland School of Medicine, 22 South Greene St., MD 21201, U.S.A. 0169-328X/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

240 0

50

I

i

i

i

i

I

100 i

i

i

i

150

I

i

i

i

i

I

I

MVNP T VFFD ITADGEP LGRVCFELFADKVPKTAENFRAL$ T GEKGFGYKGS SFHR I I P G F M C Q G G D F T R B N G T G G K S I YGEKFEDZNF I L KflTGPG I LSMAN AGPNTNCSQFF i C TAKTEWLDGK flVVFGKVKgGM$ I V EM4ERFGSRNGKT SKK IT I S D CGQ L

""-""-,'~'L_"-'"-"

"

"-'~,.d-V-,--'"

v,,..,

-v \_,~,

3

Fig. 1. A Kyte-Dolittle hydrophobicity plot and amino acid sequence of the deduced rat cyclophilin protein sequence. Areas of relative hydrophobicity are underlined. library was purchased from Clonetech and used according to manufacturer's instructions.

cDNA library screening and characterization The Cyp cDNA was isolated during screening of the rat brain cDNA library with otigonucleotides directed to an unrelated mRNA species (calbindin) and was present in a cyp:calbindin chimeric cDNA. A virtually full length Cyp cDNA was present in a chimeric cDNA composed of the Cyp cDNA portion and Calbindin cDNA. The Cyp cDNA was subc|oned as a 712 bp EcoRI fragment into either pUC13 (Pharmacia) or ml3mpl8 and sequenced by the dideoxy chain termination technique 19 using [35S]dATP and gradient or wedge gels. Either Klenow or the modified T7 DNA polymerase (USB) was used for the sequencing reactions.

Northern blot analysis Total rat brain RNA (Sprague-Dawley) was prepared 8 and fractionated (10/tg) by etectrophoresis through a 1% formaldehyde denaturing gel x6. The RNA was electroblotted onto a Gene Screen Plus membrane according to manufacturers instructions (NEN) and baked at 80 °C in vacuo for 2 h. The 712 bp Cyp cDNA insert was isolated by electroelution and ethanol precipitation 16. It was labeled to high specific activity (>108 dpm/ug) with [32p]dCTP (spec. act. 3000 Ci/mmol (NEN)) by random, primer extension 7. The blot was prehybridized with 5 × SSC (1 × SSC is 0.15 M sodium chloride-0.015 M sodium citrate), 50% formamide, 100 Mg/ml of salmon sperm DNA, for 12 h at 42 °C. It was then hybridized with the same solution containing approximately 1,000,000 cpm/ml of the labeled probe for 24 h at 42 °C. The blot was washed with a final stringency of 0.2 × SSC, 0.1% SDS at 60 °C for 1 h. The washed blot was exposed to Kodak XAR film overnight with Cronex Lightning Plus intensifying screens. The apparent size of the Cyp mRNA was calculated using RNA size markers (BRL) run in parallel.

brains were removed and frozen by immersion in isopentane on dry ice. Frozen brain sections (15 #m) were cut using a cryostat and thaw-mounted onto gelatin-coated slides. Sections were fixed in 4% formaldehyde, treated with acetic anhydride, dehydrated and defatted. A synthetic oligonucleotide (48 mer), corresponding to nucleotides 172-220 of the rat Cyp sequence was labelled using [35S]dATP with terminal deoxytransferase. Approximately 500,000 cpm of probe was applied to each section and hybridization performed overnight in a humidity chamber at 37 °C. The sections were washed sequentially in 50% formamide/2 x SSC at 40 °C and 1 x SSC at room temperature, and then apposed to Kodak Ortho M film for 5 days. Autoradiographic images were digitized with a video camera and Macintosh II computer-based image analysis system using the IMAGE program developed by Wayne Rasband (Research Services Branch, NIMH). To determine the anatomical localization of probe at the cellular level, sections were dipped in NTB-2 nuclear emulsion (Kodak), exposed for 7 days, developed (D19, Kodak) for 2 min at 16 °C, and counterstained with Cresyl violet. RESULTS

Isolation and nucleotide sequence analysis o f a rat brain Cyp c D N A A 712 bp C y p c D N A was i s o l a t e d d u r i n g s c r e e n i n g of a rat brain library for an u n r e l a t e d c D N A (calbindin). A chimeric cDNA

containing

nearly

a full l e n g t h C y p

c D N A in a d d i t i o n to t h e calbindin c D N A was i s o l a t e d by p l a q u e purification with the c a l b i n d i n d i r e c t e d o l i g o n u cleotide. T h e p r e s e n c e of a n e a r full l e n g t h C y p c D N A ligated to a calbindin c D N A p r e s u m a b l y reflects t h e high

In situ hybridization In situ hybridization was performed as described 23. Briefly, rat

level of the C y p m R N A in rat brain: a c o n c l u s i o n f u r t h e r s u p p o r t e d by s u b s e q u e n t N o r t h e r n b l o t t i n g analysis. T h e Cyp p o r t i o n was s u b c l o n e d as an E c o R I f r a g m e n t into

Kb

pUC

13 and

designated

pCYP7a.

The

cDNA

was

s e q u e n c e d in b o t h strands using e i t h e r restriction frag-

-'--

2.37

m e n t s s u b c l o n e d into the M 1 3 m p l 8 m u l t i c l o n i n g site o r plasmid s e q u e n c i n g . O u r c D N A s e q u e n c e differs f r o m a

1.35

I

recently r e p o r t e d rat brain C y p c D N A 4 s e q u e n c e by only 3 nucleotides. Two of t h e s e c h a n g e s o c c u r in the 3" u n t r a n s l a t e d region. T h e third is a C to T c h a n g e at the third position of the p r o l i n e c o d o n which d o e s n o t c h a n g e

0.33

the e n c o d e d a m i n o acid. T h e p r e d i c t e d a m i n o acid s e q u e n c e and t h e K y t e D o o l i t t l e h y d r o p h o b i c i t y / h y d r o p h i l i c i t y p l o d 5 of the rat brain Cyp is s h o w n in Fig. 1. T h e b u l k o f the p r o t e i n is

Fig. 2. Northern blot analysis of cylclophilin mRNA in rat brain. Ten /~g of total rat brain mRNA was hybridized with a labelled pCyp.7a insert as described in Materials and Methods. The RNA molecular weight markers are indicated on the right of the photograph.

either h y d r o p h i l i c or has n e i t h e r a h y d r o p h o b i c n o r h y d r o p h i l i c character. This is c o m p a t i b l e with the cytoplasmic l o c a t i o n of Cyp. T h e r e are, h o w e v e r , several

241

Fig. 3. Computer generated images of in situ hybridization autoradiographs showing distribution of cyclophilin mRNA in (a) cerebral cortex (Cx) and the caudate putamen (CPu), (b) arcuate (Arc) and ventral medial hypothalamic nucleus (VMH), (c) hippocampus including the CA3 region (Hip) and piriform cortex (PiC), (d) ventral hippocampus and substantia nigra (Sn), (e) cerebellar cortex (Cer Cx), inferior coUiculus (IC), locus ceruleus (LC), motor trigeminal nucleus (Mo5), and raphe pontis nucleus (RPn), and (f) cerebellar cortex and facial nucleus (FNu).

regions of relative hydrophobicity, notably at amino acid residues 20-26, 58-62, 97-101, 112-116. CsA is a very hydrophobic molecule, thus suggesting that one of more of the hydrophobic regions of Cyp may function as a binding site for CsA. Alternatively the relatively short hydrophobic regions of Cyp may have to adopt a co-operative tertiary structure to form the CsA binding domain.

Northern blot analysis The Cyp c D N A probe hybridizes to an approximately 1,000 bp m R N A species in rat brain (Fig. 2). The Cyp m R N A appears to be an abundant species as this blot contains only 10/~g of total R N A and was only exposed for 24 h. There is no evidence of more than one Cyp m R N A transcript in rat brain although one cannot exclude the possibility completely since the hybridization

242 signal is a b r o a d band. Previous investigators have noted multiple genes of pseudogenes by Southern blotting 1°. Additionally, two isoforms of the Cyp protein were previously r e p o r t e d 12. W h e t h e r or not these forms represent different gene transcripts or alternatively spliced gene products is not known. R N a s e protection experiments and/or characterization of multiple Cyp c D N A s will be necessary to clarify this point.

In situ hybridization studies

Fig. 4. In situ hybridization of emulsion coated slide showing prominent silver grains over thalamic neurons and fewer grains in the interneuronal areas and over a nearby glial cell (indicated by arrow). 100x oil immersion. Bar ~- 6/~m.

In o r d e r to d e t e r m i n e the regional distribution of Cyp in rat brain and whether there was cell type-specific expression, in situ hybridization studies were performed. A s shown in Fig. 3 the Cyp m R N A is widely distributed throughout the rat brain. The relative degree of hybridization was roughly p r o p o r t i o n a l to the neuronal density, so areas such as the h i p p o c a m p u s and piriform cortex were p r o m i n e n t in the a u t o r a d i o g r a p h s (Fig. 3C,D). The Cyp m R N A was p r o m i n e n t in the m o t o r nuclei of the

Fig. 5. Dark-field photomicrographs of emulsion coated slides showing in situ hybridization of Cyp mRNA. A section through the left dorsal hippocampus showing layers CA1, CA4 and the dentate gyrus is shown in Fig. 5A. The bar represents approximately 75/~m. In B, the arrow points to the Purkinje cell layer of the cerebellar cell layer which appears to have slightly more grains/neuron than in the adjacent granular layer on the right, despite the densely packed neurons in the granular layer. Bar ~ 30 ltm.

243 brainstem. It also was abundant in catecholaminergic cell bodies such as those in the locus coeruleus and substantia nigra which synthesize norepinephrine and dopamine respectively (Fig. 3D,E). Cyp m R N A was found in the choroid plexus and in the anterior pituitary, although to a lesser degree than in neurons. Cyp m R N A is a marker of neuronal density as shown in the hippocampus (Fig. 5A). However, some neurons may express somewhat higher levels of Cyp m R N A as for instance in the Purkinje cells of the cerebellar cortex (Fig. 5B). At a cellular level, Cyp m R N A was abundant in all neurons but perhaps less so in glial cells (Fig. 4). DISCUSSION CsA has become the most important immunosuppressive drug in clinical use today. Its mechanism of immune suppression in vivo remains unknown. CsA inhibits T-cell activation, proliferation and synthesis of IL-26A4A8. Biochemically the largest amount of intracellular CsA binding acitivity has been attributed to Cyp 9. This cytoplasmic 17 kDa protein exists in at least two isomeric forms in humans 12 and is present in virtually all eukaryotic cells at levels approaching 0.5-1.0% of total cellular protein 11A2. Previous Southern blotting experiments 4'1° and our own data (unpublished observations, R. Lad and D. Hilt) suggest that there are multiple genes or pseudogenes for Cyp in the rat and human genomes. To date, however, there is no evidence that there is more than one Cyp transcript in a given species. Our Northern blotting data do not suggest that there is more than one Cyp transcript expression in the rat brain, but the hybridization signal is broad and multiple m R N A species cannot be excluded. Results from in situ hybridization indicated that cyclophilin m R N A was expressed throughout the brain roughly in proportion to the neuronal cell density with a few exceptions such as the apparent increased grain density over the large Purkinje cells in the cerebellum. Although it was not possible to determine in a quantitative manner the amount of Cyp m R N A in glia, the number of grains over the white matter tracts containing oligodendrocytes was above background indicating that these glial cells expressed Cyp m R N A at least to a small degree. Moreover, although astrocytes which outnumber neurons ten to one were not counterstained by the Cresyl violet and therefore were not visualized, a minority of silver grains was seen in the interneuronal spaces. These regions which presumably contain astrocytes have a much REFERENCES 1 Atkinson, K., Biggs, J., Darveniza, P., Boland, J., Concannon,

lower grain density (Fig. 5). Thus both oligodendrocytes and astrocytes probably express Cyp m R N A but perhaps to a lesser degree than do neurons. The therapeutic use of CsA is associated with multisystern complications including neurologic side effects. These include global neurological manifestations such as encephalopathy and seizures l's. This first r6~port of in situ localization of Cyp in the brain demonstrates that the Cyp m R N A is present in high concentrations in cortical regions and may suggest why the drug produces such complications. Cellular expression of Cyp in the brain appears to be predominantly neuronal and the relatively high levels of Cyp expression by neurons suggests that the generation of the functional neuronal phenotype may require higher levels of Cyp than do glial cell types. Neuronal toxicity manifesting as encephalopathy and seizure indicates that a functional Cyp molecule is necessary for neuronal activity. Recently it has been demonstrated that Cyp is a peptidyl-prolyl isomerase (PPI) catalysing the cis-trans isomerization of peptide bonds at the proline residue. This reaction is the rate limiting step for the refolding of some native proteins from the denatured state. Thus, it appears that Cyp is an enzyme that is required to fold proteins into functional conformations after synthesis. The ubiquity of Cyp observed in our experiment suggests that its protein isomerase activity is necessary in virtually all cells. The PPI activity of Cyp is completely inhibited in the presence of CsA at a concentration that is close to the dissociation constant of CsA from Cyp. Thus, it appears that some biological effects of CsA at a cellular level may be mediated by inhibition of the PPI activity of Cyp. However, this is by no means the complete explanation of CsA activity because firstly, it does not explain the T-cell-specific actions of CsA. Secondly, in lower eukaryotes the presence of CsA retards growth and, in fact, can kill the organism 22. There may be an intracellular component which interacts with the CsA-Cyp complex in order to provide tissue-specific function. With the availability of mutants in Drosophila 2° and yeast 22 it should be possible to work out the network of interaction of CsA with different cellular proteins. Acknowledgements. We are grateful to Dr Michael Brownstein for providing the resources of his laboratory for this project. We also wish to thank Harish Dave for helpful discussion and David Trisler for critically evaluating the manuscript. R.P.L. is the recipient of a fellowship from the Fogarty International Center. D.C.H. is supported by grants from the Veterans Administration and the NIH.

A. and Dodds, A., Cyclosporine associated central nervous system toxicity after allogenic bone marrow transplantation, Transplantation, 38 (1984) 34-37.

244 2 Borel, J.F., Fuerer, C., Gubler, H.U. and Stahelin, H., Biological effects of Cyclosporin A, Agents Actions, 6 (1976) 468-474. 3 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of biologically active RNA from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299. 4 Danielson, P.E., Forss-Petter, S., Brow, M.A., Calavetta, L., Douglass, J., Milner, R.J. and Sutcliffe, J.G., plB15: a cDNA clone of rat mRNA encoding cyclophilin, DNA, 7 (1988) 261-267. 5 Deierhoi, M.H., Kalayoglu, M., Sollinger, H.W. and Belzer, EO., Cyciosporine neurotoxicity in liver transplant recipients: report of three cases, Transplant Proc., XX (1988) 116-118. 6 Elliot, J.F., Lin, Y., Mizel, S.B., Bleackley, R.C., Harnish, D.G. and Paetkau, V., Induction of interleukin 2 mRNA inhibited by Cyclosporin A, Science, 226 (1984) 1439-1441. 7 Feinberg, A.P. and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 137 (1984) 266-267. 8 Fisher, G., Wittman-Liebold, B., Lang, K., Kiefhaber, T. and Schmid, EX., Cyclophilin and peptidyl-propyl cis-trans isomerase are probably identical proteins, Nature, 337 (1989) 476-478. 9 Handschumacher, R.E., Harding, M.W., Rice, J., Drugge, R.J. and Speicher, D.W., Cyclophilin: a specific cytosolic binding protein for cyclosporin A, Science, 226 (1984) 544-547. 10 Haendler, B., Hofer-Warbinek, R. and Hofer, E., Complementary DNA for human T-cell cyclophilin, EMBO J., 6 (1987) 947-950. 11 Harding, M.W., Handschumacher, R.E., Speicher, D.W., Isolation and amino acid sequence of Cyclophilin, J. Biol. Chem., 261 (1986) 8547-8555. 12 Koletsky, A.J., Harding, M.W. and Handschumacher, R.E., Cyclophilin: distribution and variant properties in normal and neoplastic tissues, J. lmmunol., 137 (1986) 1054-1059. 13 Kozak, M., The scanning model for translation: an update, J. Cell Biol., 108 (1989) 229-241.

14 Krone, M., Leonard, W.J., Depper, J.M., Arya, S.K., WongStaal, F., Gallo, R.C., Waldman, T.A. and Greene, W.C., Cyclosporin A inhibits T-cell growth factor gene expression at the level of mRNA transcription, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 5214-5218. 15 Kyte, J. and Doolittle, R.A., A simple method for displaying the hydropathic character of a protein, J. Mol. Biol., 157 (1982) 105-132. 16 Maniatis, T., Fritsch, E.F. and Sambrook, J., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY, 1982. 17 Racusen, L.C., Famiglio, L.M., Fivush, B.A., Olton, D.A. and Solez, K., Neurologic abnormalities and mortality in rats treated with cyclosporin A, Transplan Proc., XX Suppl 3 (1988) 934-936. 18 Reem, G.H., Cook, L.A. and Vilcek, J., Gamma interferon synthesis by human thymocytes and T lymphocytes inhibited by cyclosporin A, Science, 221 (1983) 63-65. 19 Sanger, F., Nicklens, S. and Coulson, A.R., DNA sequencing with chain terminating inhibitors, Proc. Natl. Acad. Sci. U.S.A, 74 (1977) 5463-5467. 20 Shieh, B.H., Stamnes, M.A., Seavello, S., Harris, G.L. and Zuker, C.S., The nin A gene required for visual transduction in Drosophila encodes a homologue of cyclosporin A-binding protein, Nature, 338 (1989) 67-70. 21 Takahashi, N., Hayano, T. and Suzuki, M., Peptidyl-prolpl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin, Nature, 337 (1989) 473-475. 22 Tropschug, M., Barthelmess, I.B. and Neuport, W., Sensitivity to Cyclosporin A is mediated by Cyclophilin in Neurospora crassa and Saccharomyces cerevisiae, Nature, 342 (1989) 953955. 23 Young, W.S., Methods in Enzymology, Hormone Action, Part K. Neuroendocrine Peptides, Vol. 168, Academic Press, New York, 1989, pp. 702-710.