Bra#l Research, 587 (I 992) 123-129 ~ 1992 Elsevier Science Publishers B.V. All rights reserved (1006-8993/92/$05,00
123
BRES 17946
Immunohistochemical distribution of protein kinase C isozymes is differentially altered in ischemic gerbil hippocampus Masayuki Y o k o t a a, J o h n W. P e t e r s o n ~, Marios K a o u t z a n i s " and Nell W. Kowall
b
" Laboratoo,.h~r Cerehrocasculm" Biophysics, Neurosurge~' Sen'ice, and h Eaperimental Neuropathology Laboratol3.', Neurolo~.' Sere'ice. Massachusetts General Hospital, Harcard Medical Sclu~d, Boston, MA 02114 (USA) (Accepted 10 March 1992)
Key words: Protein kinase C, Isozyme; Global ischemia: Gerbil; Immunohistochemistry
We used monoclonal antibodies to examine the immunohistochcmical distribution of the three major Ca~+-dependent protein kinase C (PKC) is~xymes (L il, and lID in ischemic gerbil hippocampus. Groups of four anim:ds were sacrificed at 15 min, 4 h, 1 day, 2 days, 3 days, .and 7 days after a 10-min episode of global forebrain isehemia, In control animals, PKC-I immunoreactivity was greater in CAI neurons th:m in CA3-4. Terminal-like staining was not evident. PKC-II immunoreactivity was observed in :dl CA fields :rod in the outer molecular layer of the dentate gyrus. PKC-Ill staining was present in the CA fields, the inner molecular layer of the dentate gyrus and the subiculum. Dentate granule cells and mossy fibers were not stained with any of the PKC antibodies. Fifteen minutes and 4 h after ischemia, PCK-i, -It and -Ill immunoreactivity were all increased in CAI neurons and PKC-Ill immunoreactivity alone was visualized in gr:mule cells and mossy fibers. Staining patterns returned to baseline one day after ischemia. PKC-II and -Ill terrain:d-like staining were preserved in the stratum lacunosum-molcculare for 3 days .'rod 2 days alter ischemia respectively and then disappeared, The altered palterns of PKC st:fining in the hippoeampus may reflect activation and/or down-retlulation of PKC isozymes. Ca" "-dependent PKC isozymes may, therefore, potentially play a role in the pathogenesis of delayed ischemi¢ neuronal death,
INTRODUCTION Transient global forehrain ischemia in the gerbil induces a stereotyped pattern of selective degeneration affecting hippocampal neurons in CAI and CA4 with relative sparing of CA3 neurons and dentate granule cells ~.%a~.Delayed degeneration affects CAI neurons which are normal for 2 days after the insult, The molecular mechanisms responsible for differential neuronal vulnerability to ischemic injury are incompletely understood but pharmacological studies showing that glutamate receptor antagonists block neuronal degeneration suggest that glutamate-mediated excitotoxicity is involved .a.s..,...4. Glutamate receptor subtypes are linked to both ion channels and G proteins that activate phospholipase C 3.. Activation of Ca '~+- and diacylglycerol-dependent signalling pathways by glutamate may, therefore, contribute to the development of ischemic neuronal injury 4,7.22,32,33
The term protein kinasc C (PKC) describes an important family of seri,lc/threoninc kinascs that require phospholipid and are activated by diacylglyccrol :s. To date, nine subspecies of PK(." have been identified by molecular cloning -'. Initially, four e-DNA clones were identified (c¢, ~!, /311, 'y) in bovine, rat and huma,1 brains. Activities of these subspecies arc Ca:+-dcpend • ent. Recently, five more eDNA clones have bccn described; (/~, e, 'r/, ~ and L). Activities of these subspecics arc Ca~+-independent and their cnzymatological properties have not been clarified. The PKC species designated a, ~ and ~, correspond to PKC-III, -11 and -! respectively as identified by hydroxyapatite column chromatography. Activated PKC is translocatcd from the cytoplasm to cellular membranes. PKC phosphorylates a wide variety of substrates including cytoskeletal proteins and substrate-specific kinases. The various isoforms of PKC have distinctive substrate specificities, arc differentially activated by second messengers, and
Correspondence: N.W. Kowall, Neurology Service. Massachusetts General Hospital, Boston MA 2114, USA. Fax: (I) (617) 726-2353.
124 have specific cellular and intracellular distribution patterns. T h e cascade o f cellular phosphorylation initiated by activation of P K C has been implicated in many intracellular processes including cellular differentiation, smooth muscle cell contraction, long-term potentiation, immediate early gene expression and prog r a m m e d cell death o r apoptosis . . ,s . . .,, . Activation o f CaZ+-dependent P K C isozymes may, therefore, contribute to the differential sensitivity o f n e u r o n s to ischemia and their specific patterns o f cellular response. Several authors showed that the activity o f P K C is transiently increased and then decreased (down-regulated) immediately after global cerebral ischemia in rat ha~'~'j'zT'34. Weiloch and colleagues showed that PKC-I and -ll but not P K C - i l I are transiently redistributed to cellular m e m b r a n e s and down-regulated in the rat striatum after transient ischemia .~4. Cerebellar granule cell cultures pretreated with phorbol esters to deplete PKC are protected from ischemic injury s. In vivo studies on experimental animals pretreated with P K C inhibitors also suggest that modulation o f P K C activity may alter the extent of ischemic n e u r o n a l injury and death l l.zo.zl In order to clarify the potential role o f C a ' + - d e p e n d e n t PKC isozymcs in the pathogenesis o f ischemic neuronal injury in the hippocampus, we examined their immunocytochemical distribution using specific m o n o clonal antibodies at several time points after the induction of transient global ischcmia in the gerbil.
PKC, I, -Ii and -!11 (MBL, Nagoya. Japan) diluted to yield a final concentration of l0 gg/ml IgG. The characteristics of the monoclonal antibodies have been established by Hagiwara et al. ~. After rinsing, sections were incubated in goat antimous¢ lgG (Dako) diluted 1:50 for I h followed by further washing and incubation in mouse peroxidase antiperoxidase complex (Dako) diluted 1:250 for ! h at room temperature. After rinsing, immunolabel was visualized with 0.015% diaminobenzidine tetrahydrochloride (Sigma) and 0.003% H20, in 50 mM Tris-HCl buffer (pH 7.6). Immunohistochemical controls for the specificity included substitute of PBS and normal mouse serum (Dako) for primary antibodies and preabsorplion of the primary antibodies with excess of PKC antigen (20 /zg/ml). RESULTS C o m p l e t e ischemia of the forebrain for 10 rain produced •consistent neuronal d e g e n e r a t i o n in the hipp o c a m p a l region in all animals surviving at 3 a n d 7 days after the ischemic insult (Fig. 1). All the CA1 pyramidal n e u r o n s were affected bilaterally, and n e u rons in C A 3 and granule cells in the d e n t a t e gyrus were morphologically intact. in s h a m - t r e a t e d control animals, PKC-I antibody stained C A I neuronal perikarya heavily and C A 3 - 4 n e u r o n s moderately (Fig. 2At. Terminal.like staining was not seen and astrocytes were not immunoreactiv¢.
"5
M A T E R I A L S AND METHODS
Thirty adult I'~male mongolian ~crhils weighing 51)-7i) g (Tumb, lehrouk Farms, W~:st Brookficld, MAt housed under diurnal lighting
conditions and allowed food and water ad libitum were anesthetized durin8 the ischcmia and reperfusion period by ethyl ether and room air, Body tcmperatur~ was maintained at 3fl-3"/°Cwith a homeother. mic heating blanket (Harvard Apparatus, South Natick, MAt during anesthesia. Follnwit~g a midline cervical incision, both common carotid arteries (CCAs) were dissected in 24 gerbils and occluded with small aneu~smal clips. After I(I rain occlusion, the clips were removed to allow recirculation, in 4 sham,operated animals, CCAs were dissected but not occluded, Four of each group of animals were sacrificed at times 15 min, 4 h, I day, 2 days, 3 days and 7 days after recirculalion. Sham animals were sacrified at 3 h after operation, At each time point, the animals were euthanized with an overdose of sodium pentoharbital (60 mg/kg i,p,) and then perfused at 40C through ascending aorta with 50 ml of (I,9~ saline followed by 4% parah~rmaldehyd¢ in 0.1 M phosphat~ buffer (PB, pH "14~ The pcrfuscd brains were removed and fixed with the same fixative for 24 h. Then the brain was immersed in 0,Ill M phosphate-buffered saline (PBS) containing 1(I-2()r~ sucrose flw 24 h. The brains were c~)ro. nally sectioned at 50 gm on a freezing micmtome. Sections of the hippocampus and ~erebellum were processed hr PKC immunohisto. chemistry and/or stained fi)r Cresyl violet {CVI to assess tissue architecture, The sections were rinsed fi)r 15 min at r~vom temr~rature with PBS followed by {).3C; tI,O z in PBS for 5 rain and incubated for 20 rain with 3~ normal goat serum (Dako) in PBS. The sections were then incubated at 4°C for 2 h with monoclonal antibodies against
Fig. I. Normal (At and ischemic gerbil hippoeampus (B) 7 days after I(I rain of transient forebrain ischemia stained with Cresyl violet. Isehemia produces degeneration of CAt pyramidal neurons while CA3 neurons and dentate granule cells are preserved. Bar = 400 gm.
125
PKC-II and -III antibody stained CAI neurons lightly and CA3-4 neurons moderately (Fig. 2B,C). Moderately intense, diffuse, terminal-like PKC-ll staining was seen throughout the CA fields with the notable exception of the mossy fiber terminations in CA4 and stratum lucidum of CA3 (Fig. 2B). PKC-lll terminal-like staining was seen throughout the CA fields but was most intense in the stratum oriens of CA1. The subiculure was most heavily stained with PKC-III antibody and lightly stained with PKC-i antibody. PKC-II im-
?
Fig. 2. PKC iso~mes showdistinct distribution patterns in the gerbil hi00ocampus. Prominent PKC-I immunoreactivity(IR) was present in neuronal perikarya in CAl. CA3-4 neurons were more lightly stained (A). Moderate PKC-II IR was observed in CAI-4 neurons and in the outer molecular layer of the dentate gyrus (B). PKC-III IR was present in CA1-3 neurons and in the inner molecular layer of the dentate gyrus (C). The subiculum was stained heavily with PKC-I1 antibodyand lightlywith PKC-Iantibody. Bar -- 400/~m.
munoreactivity was not present. Granule cells of the dentate gyrus and mossy fibers were not normally stained with any of the PKC antibodies. A band of PKC-Iil immunoreactivity delineated the inner molecular layer of the dentate gyrus. A complementary staining pattern was seen with PKC-II antibody which labeled the outer molecular layer. PKC-II immunoreactivity was most intense in the middle third of the dentate gyrus molecular layer (Fig. 2B). In the cerebellum of sham-treated animals, consistent with previous reports 12, PKC-I and -III staining was found in Purkinje cells, and PKC-II and -III immunoreactivity was found in the granule cell layer. These patterns of immunoreactivity in the cerebellum were not affected by the induction of forebrain ischemia. These distinct patterns of PKC immunoreactivity were not observed with PBS, normal goat serum or monoclonal antibody preabsorbed with PKC antigen. PKC-I, -ll, and -lit immunoreactivities were markedly increased in the dendrites and perikarya of CA1 neurons from 15 rain to 4 h after ischemia and recovered to control levels 24 h after ischemia (Fig. 3). Surviving neurons continued to contain PKC immunoreactivity 3 days after ischemia. By day 7, there was extensive degeneration of neurons in CA1 and widespread loss of PKC immunoreactivity. Granule cells in the dentate gyrus and mossy fibers were transiently PKC-lll immunoreactive from 15 min to 4 h after ischemia (Fig. 4). PKC-II and -Ill immunoreactivities in the stratum lacunosum-moleculare (SLM) were preserved until 3 days and 2 days after ischemia respectively and then disappeared (Fig. 5). On the other hand, PKC.I! and -I!1 immunoreactivities in the CAI stratum oricns and radiatum were preserved throughout the study (Fig, 5).
DISCUSSION The detailed regional distribution of PKC isozymes in the gerbil hippocampus has not been previously reported. In sham animals, each PKC isozymc showed a distinctive distribution pattern in the hippoeampus (Fig. 2). Patterns of PKC immunoreactivity may be highly variable among different species. For example neuronal perikarya in CAI of the gerbil were most intensely labeled with PKC-I antibody, whereas canine CA1 neurons are prominently PKC-ill-immunoreactive (unpublished data). Although previous studies have not addressed the immunohistochemical localization of PKC after ccrcbral ischemia, several groups have reported that PKC activity in rat brain is initially increased and subse-
126 quently decreased after global ischemia ,.17.~...7. WieIoch and co-workers 34 demonstrated that PKC isozymes were activated and redistributed from cytosolic fraction to membrane fraction in rat striatum following forebrain transient ischemia by immunoblotting method. Crumrine and co-workers " suggested that the decline of PKC activity resulted from activation of endogenous PKC inhibitors. in the present study, we found that immunoreactivity for all PKC isozymes in the cell body and dendrites of CAI neurons, and PKC-III immunoreactivity in the dentate granule cells were enhanced 15 rain and 4 h
after an ischemic insult (Figs. 3 and 4), This may reflect activation and redistribution of PKC isozymes to cellular membranes as reported by Wieioch and coworkers 34. Activation and membrane binding of PKC may lead to enhanced dendritic and perikaryal staining. The rapid appearance of PKC-II! immunoreactivity in dentate granule cells and mossy fibers 15 rain after the ischemie insult suggests that rapid post-translational changes related to the activation of PKC-Ill produce increased immunoreactivity, Conformational changes affecting PKC-III may increase the affinity of the monoclonal antibody or expose an antigenic site in
¢ 3
, d d i ~% ',, ' ;
X,/i ~|tl
Fig, 3, PKC-I (A-D), -II (E-H) and -I11 ( i - L ) immunoreactive CAI neurons sequentially examined at selected time points after the induction of IO rain ~f fl)rehrain ischemia (sham, 15 rain, 4 h and I day after ischemia, in that order), All PKC iso~mes showed enhanced immunoreactivities (IR) in dendrites of CAI neuron,~ 15 rain (B, F, J) and 4 h (C, G, K) after ischemia, which returned to control levels I day after ischemia (D, H, L), Bar = 50 p.m,
127 the PKC-III molecule. In situ hybridization studies have not been performed to confirm that dentate granule cells synthesize PKC-Ill. The selective activation of PKC-III is consistent with Kruger et al.'s is suggestion that PKC isozymes are differentially regulated. We found that PKC-II and -Ill terminal-like immunoreactivity was differentially depleted in the SLM of CAI, 7 and 3 days after ischemia respectively (Fig. 5). This delayed depletion may be due to late degeneration of axonal terminals projecting to this region. Extrinsic projections to the SLM of CA1 arise from the entorhinal cortex and midline thalamic nuclei ~. Intrinsic neurons in stratum oriens of CA1 also project locally to the SLM. The entorhinal cortex and midline thalamic nuclei are not damaged by transient forebrain
C
ischcmia (data not shown), so local loss of PKC immunoreactivity is most likely a consequence of local neuronal degeneration in CAl. PKC isozymes are differentially sensitive to degradation by calpain, a calcium-activated neutral protease L~.,,. This differential degradation might explain the early disappearance of PKC-lll and the later disappearance of PKC-I! immunoreactivity. PKC-II and -lIl immunoreactivities were preserved in CAI stratum oriens and radiatum throughout the study. This observation might reflect that inhibitory interneurons in CA1 stratum oriens and most axons in CA1 stratum radiatum are resistent to transient global ischemia as Johansen described ~4 The potential role of PKC activation in the pathogenesis of ischemic neuronal injury has been examined
k,
!,,?
Fig. 4. Normal dentate granule cells and mossy fibers do not show PKC-111 immunoreactivity (IR) (A), but 15 min (B) and 4 h (C) after ischemia. PKC-Ill IR was present in both granule cells and mossy fibers which returned to control levels I day (D) after ischemia. High power photomicrograph shows normal dentate gyrus (E) and the same region 15 min after the induction of ischemia (F). Bar = 400 ~m (A-D) and 50 /~m (E and F).
128 using activators and inhibitors of PKC ~al,.,0.,i. Hara and coworkers tt reported that topical administration of staurosporine, a relatively specific and potent PKC inhibitor, prevented postischemic neuronal damage in the CA! field of rat and gerbil, whereas inhibitors of cyclic AMP-dependent, cyclic GMP-dependent, and calmodulin-dependent kinases had no neuroprotective effect, Magal and co-workers 2o found that pretreatment of gravid rats with gangliosides that prevent activation and down-regulation of PKC increase the tolerance of rat fetuses to deprivation of maternal blood supply. Favaron and co-workers x showed that downregulation of PKC produced by pretreatment with phorbol esters protects cerebellar granule cells from ischemic injury in vitro. In contrast, Madden and coworkers 20 found that staurosporine reduced the tolerance of rabbit spinal cord to ischemia, while 1,2oleoylacetylglycerol, an activator of PKC, was neuroprotective. These inconsistent results may be a consequence of the differences in test systems, methods and species examined.
Activation of PKC isozymes by ischemic injury could play a direct role in the pathogenesis of neuronal degeneration. Activation of PKC has been implicated in the process of programmed cell death termed apoptosis m.,6. Ojeda et al. 26 demonstrated that steroid-induced apoptosis of thymocytes is mediated by PKC activation. Glucocorticoids have also been shown to potentiate ischemic neuronal injury ~.29. The specific substrates phosphorylated by PKC that mediate the subsequent cellular responses to ischemia are not known. Activation of substrate specific kinases or immediate early gene transcription by PKC could potentially be important 2s. Definition of the specific properties that distinguish the Ca2+-dcpendent isozymes of PKC, including substrate specificity and differential regulation, may provide further insight into the pathogenesis of ischemic neuronal death. Acknowledgements. We thank Mr. Larry Cherkas for photographic expertise.This workwas supportedby grants fromNHLB!to J.W.P. (HL44554.01) and from NIH to N.W.K. (AG05134, NA25588, NS1()828).
.H Fig. 5. PKC-II (A-D) and -lll(E-l-I)immunoreactivity(IR) in stratum lacunosum-moleculare (SLM) of CAt showing sequentialalterationsof stainingat differenttime points(sham, 2 days,3 days and 7 days afterischemia,in that order),PKC-II IR in the 5 L M disappeared by 7 days after ischemia (Ck while PKC-Ill IR disapl~:aredby 3 days after ischemia (G). PKC-II and -III IR were preserved in stratum oriens and radiatum throughout the study.Arrow show.~boundary betwcen 5 L M and the molecular layerof dentate gyrus. Bar = 200 p.m.
129 REFERENCES 1 Araki, S., Simada, Y., Kaji, K. and Hayashi, H., Role of protein kinase C in the inhibition by fibroblast growth factor of apoptosis in serum-depleted endothelial cells, Biochem. Bit~hys. Reg. Cornman., 172 (1990) 1081-1085. 2 Bell, R,M. and Burns, D J., Lipid activation of protein kinase C, 1. Biol. Chem., 266 (1991)4661-4664. 3 Benveniste, H., Drejer, J., Schousboe, A. and Diemer. N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral mierodialysis, ./. Neorochem., 43 (1984) 1369-1374. 4 Benveniste, H. and Diemer, N.H., Early postisehemic 45Ca accumulation in rat dentate hilus, J. Cereb. Blood Flow Metabol., 8 (1988) 713-719. 5 Benveniste, H., Jorgensen, M.B., Sandberg, M., Christensen, T., Hagberg, H, and Diemer, N.H., Ischemie damage in hippocampal CAI is dependent on glutamate release and intact innetvation from CA3, J. Cereh. Blood Flow Metabol., 9 (1989) 628-639. 6 Crumrine, R,C,, Dubyak, G, and LaManna, J,C, Decreased protein kinase C activity during cerebral ischemia and after reperfusion in the adult rat, J. Neurochem., 55 (1990) 2001-2007. 7 Dubinsky, J,M. and Rothman, S.M., lntracellular calcium concentrations during "chemical hypoxia" and excitotoxic neuronal injury, J. NeuroscL, I1 (1991)2545-2551. 8 Favaron, M., Manev, H., Siman, R,, Bertolino, M., Szekely, A.M., DeErausquin, G., Guidotti, A. and Costa, E,, Down-regulation of protein kinase C protects ¢ercbellar granule neurons in primary culture from glutamate-induced neuronal death, Proc, Natl. Acad. Sci. USA, 87 (1990) t983-1987,
9 Hagiwara, M,, Hachiya, T., Watanabe, M., Usuada, N,, iida. F., Tamai, K. and Hidaka, H., Assessment of protein kinase C isozymes by enzyme immunoassay and overexpression of type II in thyroid adenocarcinoma, Cancer Res., 50 (1990) 5515-5519, 10 Hagberg, H,, Lehmann, A., Sandberg, M., Nystrom, B,, Jacobson, I. and Hamberger, A,, lschemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments, J. Cereb. BIotM Flow Mt,tahol,, 5 0985) 413-419. I1 Iolara,H., Onndera, H., Yoshidoml, M., Matsuda, Y. and Kogure, K,, Stautosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal damage in the gerbil and rat, J. Celvh. BIo¢~I Flow Metabol,, 10 (1')90) 646-653. 12 Huan8, F.L., Yoshidu, Y., Nukubayashi, H., Young, W,S. and Huang, K.-P,, Immunocytochemical localization of protein kinuse C i~zymes in rat brain, J. Neun~scl., 8 (1988) 4734-4744. 13 Huang, F,L., Yoshida, Y,, Cunha,Mclo, J.R., Beaven, M.A. and Huan8, K.-P,, Differential down-regulation of protein kinase C i~zymes, J, Biol. Chem,, 264 (1989) 4238-4243, 14 Johunsen, F.F,, Jorgensen, M,B,, Ekstrom yon Lubitz, D.KJ, and Diemer, N,H., Selective dendrite damage in hippocampal CAI stratum radiatum with unchanged axon ultrastructure and glutamate uptake after transient cerebral ischemia in the rat, Brain Res., 291 (1984) 373-377. 15 Kirino, T,, Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57-69. 16 Kishimoto, A., Mikawa, K,, Yasuda, I., Tanaka, S., Tominaga, M., Kuroda, T. and Nishizuka, Y., Limited proteolysis of protein kinase C subspecies by calcium.dependent neutral protease (¢alpain), £ Biol. Chem., 264 (1980)4088-4092. 17 Koehhar, A., Saitoh, T. and Zivin, J., Reduced protein kinase C activity in ischemic spinal cord, J. Neurochem., 53 (1989) 946-952.
18 Kruger, E.K., Sossin, W.S., Sacktor, T.C., Bergold, P.J., Beu~hausen, S, and Schwartz. J.H., Cloning and characterization of Ca2+-dependent and Ca2÷-independent PKCs expressed in Aplysia sensory cells, J. Neurosci., I I (1991) 2302-2313. 19 Louis, J.-C., Magal, E., Brixi, A., Steinberg, R., Yavin, E. and Vincendon, G., Reduction of protein kinase C activity in the adult rat brain following transient forebrain ischemia, Brain Res., 541 O991) 171-174. 20 Madden, K.P., Clark, W.M., Kochhar, A. and Zivin, J.A., Effect of protein kinase C modulation on outcome of experimental CNS ischemia, Brain Rex., 547 (1991) 193-198. 21 Magal, E., Louis, J.-C., Aguilera, J. and Yavin, E., Gangliosides prevent ischemia-induced down-regulation of protein kinase C in fetal rat brain, J. Neurochem., 55 (1990) 2126-2131. 22 Martins, E., lnamura, K,, Themner, K., Malmqvist, K.G. and Siesjo, B.K., Accumulation of calcium and loss of potassium in the hippocampus following transient cerebral ischemia: a proton microprobe study, J. Cereb. Blood Flow Metabol., 8 (1988) 531538. 23 Masters, J.N., Finch, CE. and Sapolsky, R.M., Glucocorticoid endangerment of hippocampal neurons does not involve deoxyribonucleic acid cleavage, Endocrinology, 124 (1989) 3{)83-3088. 24 Monaghan, D.T, and Cotman, C.W,, Distribution of N-methylD-aspartate-sensitiveL-['~H]glutamate-bindingsitesin rat brain, J. Nearosci., 5 (1985) 2909-2919. 25 Nishizuka, Y., The molecular heterogeneity of protein kinase C and its implications for cellular regulation, Nature, 334 {1988) 661-665. 26 Ojeda, F,, Guarda, M,, Maldonado, C. and Folch, H., Protein kinase C involvement in thymocyte apoptosis induced by hydrocortisone, Cell. Immunol,, 125 (1990) 535-539. 27 Onodera, H., Araki, T. and Kogure, K., Protein kinase C activity in the rat hippocampus after forebrain ischemia: autoradiographic analysis by [3H]phorbol 12,13.dibutyrate, Brain Res.. 481 (1989) 1-7. 28 Rosene, D.L. and Van Hoesen, G.W, The hippocampal formation of the primate brain, A review of some comparative aspects of cytoarchiteeture and connections, in E.G. Jones and A. Peters (Eds.), Cerebral Cortex, Vol. 6, Plenum, New York, 1987, pp. 345-456. 29 Sapols~, R.M. and Pulsinelli, W.A,, Glucocorticoids p0tcnliate ischemic injury to neurons: therapeutic implications, Sciem'e, 229 (1985} 1397-14{}0. 30 Scholz, W.K. and Palfrey, H.C., Glutamate.stimulated protein phosphorylailon in cultured hippncampal pyramidal neurons, ). Ncurosci,, 11 (1991) 2422-2432, 31 Shmidt.Kastner, R. and Freund, T,F,, Selective vulnerability of the hippocampus in brain ischemia, Nt,uroscle.ce, 40 (1991)599636, 32 Tsuda, T., Kogure, K., Ishii, K, and Orihar~,, H,, Postischemic changes of calcium and endogenous antagonist in the rat hippocampus studied by proton-induced X-ray emission analysis, Brain Res., 484 (1989) 228-233. 33 Vaccarino, F.M., Liljequist, S, and Tallman, J.F., Modulation of protein kinase C translocation by excitatory and inhibitory amino acids in primary cultures of neurons, J, Neumchem,, 57 (1991) 391-396, 34 Wieloch, T., Cardell, M,, Bingren, H,, Zivin, J. and Saitoh, T,, Changes in the activity of protein kinase C and the differential subcellular redistribution of its isozymes in the rat striatum during and following transient forebrain ischemia, J. N,,urochem., 56 (1991) 1227-1235.
123
BRES 17946
Immunohistochemical distribution of protein kinase C isozymes is differentially altered in ischemic gerbil hippocampus Masayuki Y o k o t a a, J o h n W. P e t e r s o n ~, Marios K a o u t z a n i s " and Nell W. Kowall
b
" Laboratoo,.h~r Cerehrocasculm" Biophysics, Neurosurge~' Sen'ice, and h Eaperimental Neuropathology Laboratol3.', Neurolo~.' Sere'ice. Massachusetts General Hospital, Harcard Medical Sclu~d, Boston, MA 02114 (USA) (Accepted 10 March 1992)
Key words: Protein kinase C, Isozyme; Global ischemia: Gerbil; Immunohistochemistry
We used monoclonal antibodies to examine the immunohistochcmical distribution of the three major Ca~+-dependent protein kinase C (PKC) is~xymes (L il, and lID in ischemic gerbil hippocampus. Groups of four anim:ds were sacrificed at 15 min, 4 h, 1 day, 2 days, 3 days, .and 7 days after a 10-min episode of global forebrain isehemia, In control animals, PKC-I immunoreactivity was greater in CAI neurons th:m in CA3-4. Terminal-like staining was not evident. PKC-II immunoreactivity was observed in :dl CA fields :rod in the outer molecular layer of the dentate gyrus. PKC-Ill staining was present in the CA fields, the inner molecular layer of the dentate gyrus and the subiculum. Dentate granule cells and mossy fibers were not stained with any of the PKC antibodies. Fifteen minutes and 4 h after ischemia, PCK-i, -It and -Ill immunoreactivity were all increased in CAI neurons and PKC-Ill immunoreactivity alone was visualized in gr:mule cells and mossy fibers. Staining patterns returned to baseline one day after ischemia. PKC-II and -Ill terrain:d-like staining were preserved in the stratum lacunosum-molcculare for 3 days .'rod 2 days alter ischemia respectively and then disappeared, The altered palterns of PKC st:fining in the hippoeampus may reflect activation and/or down-retlulation of PKC isozymes. Ca" "-dependent PKC isozymes may, therefore, potentially play a role in the pathogenesis of delayed ischemi¢ neuronal death,
INTRODUCTION Transient global forehrain ischemia in the gerbil induces a stereotyped pattern of selective degeneration affecting hippocampal neurons in CAI and CA4 with relative sparing of CA3 neurons and dentate granule cells ~.%a~.Delayed degeneration affects CAI neurons which are normal for 2 days after the insult, The molecular mechanisms responsible for differential neuronal vulnerability to ischemic injury are incompletely understood but pharmacological studies showing that glutamate receptor antagonists block neuronal degeneration suggest that glutamate-mediated excitotoxicity is involved .a.s..,...4. Glutamate receptor subtypes are linked to both ion channels and G proteins that activate phospholipase C 3.. Activation of Ca '~+- and diacylglycerol-dependent signalling pathways by glutamate may, therefore, contribute to the development of ischemic neuronal injury 4,7.22,32,33
The term protein kinasc C (PKC) describes an important family of seri,lc/threoninc kinascs that require phospholipid and are activated by diacylglyccrol :s. To date, nine subspecies of PK(." have been identified by molecular cloning -'. Initially, four e-DNA clones were identified (c¢, ~!, /311, 'y) in bovine, rat and huma,1 brains. Activities of these subspecies arc Ca:+-dcpend • ent. Recently, five more eDNA clones have bccn described; (/~, e, 'r/, ~ and L). Activities of these subspecics arc Ca~+-independent and their cnzymatological properties have not been clarified. The PKC species designated a, ~ and ~, correspond to PKC-III, -11 and -! respectively as identified by hydroxyapatite column chromatography. Activated PKC is translocatcd from the cytoplasm to cellular membranes. PKC phosphorylates a wide variety of substrates including cytoskeletal proteins and substrate-specific kinases. The various isoforms of PKC have distinctive substrate specificities, arc differentially activated by second messengers, and
Correspondence: N.W. Kowall, Neurology Service. Massachusetts General Hospital, Boston MA 2114, USA. Fax: (I) (617) 726-2353.
124 have specific cellular and intracellular distribution patterns. T h e cascade o f cellular phosphorylation initiated by activation of P K C has been implicated in many intracellular processes including cellular differentiation, smooth muscle cell contraction, long-term potentiation, immediate early gene expression and prog r a m m e d cell death o r apoptosis . . ,s . . .,, . Activation o f CaZ+-dependent P K C isozymes may, therefore, contribute to the differential sensitivity o f n e u r o n s to ischemia and their specific patterns o f cellular response. Several authors showed that the activity o f P K C is transiently increased and then decreased (down-regulated) immediately after global cerebral ischemia in rat ha~'~'j'zT'34. Weiloch and colleagues showed that PKC-I and -ll but not P K C - i l I are transiently redistributed to cellular m e m b r a n e s and down-regulated in the rat striatum after transient ischemia .~4. Cerebellar granule cell cultures pretreated with phorbol esters to deplete PKC are protected from ischemic injury s. In vivo studies on experimental animals pretreated with P K C inhibitors also suggest that modulation o f P K C activity may alter the extent of ischemic n e u r o n a l injury and death l l.zo.zl In order to clarify the potential role o f C a ' + - d e p e n d e n t PKC isozymcs in the pathogenesis o f ischemic neuronal injury in the hippocampus, we examined their immunocytochemical distribution using specific m o n o clonal antibodies at several time points after the induction of transient global ischcmia in the gerbil.
PKC, I, -Ii and -!11 (MBL, Nagoya. Japan) diluted to yield a final concentration of l0 gg/ml IgG. The characteristics of the monoclonal antibodies have been established by Hagiwara et al. ~. After rinsing, sections were incubated in goat antimous¢ lgG (Dako) diluted 1:50 for I h followed by further washing and incubation in mouse peroxidase antiperoxidase complex (Dako) diluted 1:250 for ! h at room temperature. After rinsing, immunolabel was visualized with 0.015% diaminobenzidine tetrahydrochloride (Sigma) and 0.003% H20, in 50 mM Tris-HCl buffer (pH 7.6). Immunohistochemical controls for the specificity included substitute of PBS and normal mouse serum (Dako) for primary antibodies and preabsorplion of the primary antibodies with excess of PKC antigen (20 /zg/ml). RESULTS C o m p l e t e ischemia of the forebrain for 10 rain produced •consistent neuronal d e g e n e r a t i o n in the hipp o c a m p a l region in all animals surviving at 3 a n d 7 days after the ischemic insult (Fig. 1). All the CA1 pyramidal n e u r o n s were affected bilaterally, and n e u rons in C A 3 and granule cells in the d e n t a t e gyrus were morphologically intact. in s h a m - t r e a t e d control animals, PKC-I antibody stained C A I neuronal perikarya heavily and C A 3 - 4 n e u r o n s moderately (Fig. 2At. Terminal.like staining was not seen and astrocytes were not immunoreactiv¢.
"5
M A T E R I A L S AND METHODS
Thirty adult I'~male mongolian ~crhils weighing 51)-7i) g (Tumb, lehrouk Farms, W~:st Brookficld, MAt housed under diurnal lighting
conditions and allowed food and water ad libitum were anesthetized durin8 the ischcmia and reperfusion period by ethyl ether and room air, Body tcmperatur~ was maintained at 3fl-3"/°Cwith a homeother. mic heating blanket (Harvard Apparatus, South Natick, MAt during anesthesia. Follnwit~g a midline cervical incision, both common carotid arteries (CCAs) were dissected in 24 gerbils and occluded with small aneu~smal clips. After I(I rain occlusion, the clips were removed to allow recirculation, in 4 sham,operated animals, CCAs were dissected but not occluded, Four of each group of animals were sacrificed at times 15 min, 4 h, I day, 2 days, 3 days and 7 days after recirculalion. Sham animals were sacrified at 3 h after operation, At each time point, the animals were euthanized with an overdose of sodium pentoharbital (60 mg/kg i,p,) and then perfused at 40C through ascending aorta with 50 ml of (I,9~ saline followed by 4% parah~rmaldehyd¢ in 0.1 M phosphat~ buffer (PB, pH "14~ The pcrfuscd brains were removed and fixed with the same fixative for 24 h. Then the brain was immersed in 0,Ill M phosphate-buffered saline (PBS) containing 1(I-2()r~ sucrose flw 24 h. The brains were c~)ro. nally sectioned at 50 gm on a freezing micmtome. Sections of the hippocampus and ~erebellum were processed hr PKC immunohisto. chemistry and/or stained fi)r Cresyl violet {CVI to assess tissue architecture, The sections were rinsed fi)r 15 min at r~vom temr~rature with PBS followed by {).3C; tI,O z in PBS for 5 rain and incubated for 20 rain with 3~ normal goat serum (Dako) in PBS. The sections were then incubated at 4°C for 2 h with monoclonal antibodies against
Fig. I. Normal (At and ischemic gerbil hippoeampus (B) 7 days after I(I rain of transient forebrain ischemia stained with Cresyl violet. Isehemia produces degeneration of CAt pyramidal neurons while CA3 neurons and dentate granule cells are preserved. Bar = 400 gm.
125
PKC-II and -III antibody stained CAI neurons lightly and CA3-4 neurons moderately (Fig. 2B,C). Moderately intense, diffuse, terminal-like PKC-ll staining was seen throughout the CA fields with the notable exception of the mossy fiber terminations in CA4 and stratum lucidum of CA3 (Fig. 2B). PKC-lll terminal-like staining was seen throughout the CA fields but was most intense in the stratum oriens of CA1. The subiculure was most heavily stained with PKC-III antibody and lightly stained with PKC-i antibody. PKC-II im-
?
Fig. 2. PKC iso~mes showdistinct distribution patterns in the gerbil hi00ocampus. Prominent PKC-I immunoreactivity(IR) was present in neuronal perikarya in CAl. CA3-4 neurons were more lightly stained (A). Moderate PKC-II IR was observed in CAI-4 neurons and in the outer molecular layer of the dentate gyrus (B). PKC-III IR was present in CA1-3 neurons and in the inner molecular layer of the dentate gyrus (C). The subiculum was stained heavily with PKC-I1 antibodyand lightlywith PKC-Iantibody. Bar -- 400/~m.
munoreactivity was not present. Granule cells of the dentate gyrus and mossy fibers were not normally stained with any of the PKC antibodies. A band of PKC-Iil immunoreactivity delineated the inner molecular layer of the dentate gyrus. A complementary staining pattern was seen with PKC-II antibody which labeled the outer molecular layer. PKC-II immunoreactivity was most intense in the middle third of the dentate gyrus molecular layer (Fig. 2B). In the cerebellum of sham-treated animals, consistent with previous reports 12, PKC-I and -III staining was found in Purkinje cells, and PKC-II and -III immunoreactivity was found in the granule cell layer. These patterns of immunoreactivity in the cerebellum were not affected by the induction of forebrain ischemia. These distinct patterns of PKC immunoreactivity were not observed with PBS, normal goat serum or monoclonal antibody preabsorbed with PKC antigen. PKC-I, -ll, and -lit immunoreactivities were markedly increased in the dendrites and perikarya of CA1 neurons from 15 rain to 4 h after ischemia and recovered to control levels 24 h after ischemia (Fig. 3). Surviving neurons continued to contain PKC immunoreactivity 3 days after ischemia. By day 7, there was extensive degeneration of neurons in CA1 and widespread loss of PKC immunoreactivity. Granule cells in the dentate gyrus and mossy fibers were transiently PKC-lll immunoreactive from 15 min to 4 h after ischemia (Fig. 4). PKC-II and -Ill immunoreactivities in the stratum lacunosum-moleculare (SLM) were preserved until 3 days and 2 days after ischemia respectively and then disappeared (Fig. 5). On the other hand, PKC.I! and -I!1 immunoreactivities in the CAI stratum oricns and radiatum were preserved throughout the study (Fig, 5).
DISCUSSION The detailed regional distribution of PKC isozymes in the gerbil hippocampus has not been previously reported. In sham animals, each PKC isozymc showed a distinctive distribution pattern in the hippoeampus (Fig. 2). Patterns of PKC immunoreactivity may be highly variable among different species. For example neuronal perikarya in CAI of the gerbil were most intensely labeled with PKC-I antibody, whereas canine CA1 neurons are prominently PKC-ill-immunoreactive (unpublished data). Although previous studies have not addressed the immunohistochemical localization of PKC after ccrcbral ischemia, several groups have reported that PKC activity in rat brain is initially increased and subse-
126 quently decreased after global ischemia ,.17.~...7. WieIoch and co-workers 34 demonstrated that PKC isozymes were activated and redistributed from cytosolic fraction to membrane fraction in rat striatum following forebrain transient ischemia by immunoblotting method. Crumrine and co-workers " suggested that the decline of PKC activity resulted from activation of endogenous PKC inhibitors. in the present study, we found that immunoreactivity for all PKC isozymes in the cell body and dendrites of CAI neurons, and PKC-III immunoreactivity in the dentate granule cells were enhanced 15 rain and 4 h
after an ischemic insult (Figs. 3 and 4), This may reflect activation and redistribution of PKC isozymes to cellular membranes as reported by Wieioch and coworkers 34. Activation and membrane binding of PKC may lead to enhanced dendritic and perikaryal staining. The rapid appearance of PKC-II! immunoreactivity in dentate granule cells and mossy fibers 15 rain after the ischemie insult suggests that rapid post-translational changes related to the activation of PKC-Ill produce increased immunoreactivity, Conformational changes affecting PKC-III may increase the affinity of the monoclonal antibody or expose an antigenic site in
¢ 3
, d d i ~% ',, ' ;
X,/i ~|tl
Fig, 3, PKC-I (A-D), -II (E-H) and -I11 ( i - L ) immunoreactive CAI neurons sequentially examined at selected time points after the induction of IO rain ~f fl)rehrain ischemia (sham, 15 rain, 4 h and I day after ischemia, in that order), All PKC iso~mes showed enhanced immunoreactivities (IR) in dendrites of CAI neuron,~ 15 rain (B, F, J) and 4 h (C, G, K) after ischemia, which returned to control levels I day after ischemia (D, H, L), Bar = 50 p.m,
127 the PKC-III molecule. In situ hybridization studies have not been performed to confirm that dentate granule cells synthesize PKC-Ill. The selective activation of PKC-III is consistent with Kruger et al.'s is suggestion that PKC isozymes are differentially regulated. We found that PKC-II and -Ill terminal-like immunoreactivity was differentially depleted in the SLM of CAI, 7 and 3 days after ischemia respectively (Fig. 5). This delayed depletion may be due to late degeneration of axonal terminals projecting to this region. Extrinsic projections to the SLM of CA1 arise from the entorhinal cortex and midline thalamic nuclei ~. Intrinsic neurons in stratum oriens of CA1 also project locally to the SLM. The entorhinal cortex and midline thalamic nuclei are not damaged by transient forebrain
C
ischcmia (data not shown), so local loss of PKC immunoreactivity is most likely a consequence of local neuronal degeneration in CAl. PKC isozymes are differentially sensitive to degradation by calpain, a calcium-activated neutral protease L~.,,. This differential degradation might explain the early disappearance of PKC-lll and the later disappearance of PKC-I! immunoreactivity. PKC-II and -lIl immunoreactivities were preserved in CAI stratum oriens and radiatum throughout the study. This observation might reflect that inhibitory interneurons in CA1 stratum oriens and most axons in CA1 stratum radiatum are resistent to transient global ischemia as Johansen described ~4 The potential role of PKC activation in the pathogenesis of ischemic neuronal injury has been examined
k,
!,,?
Fig. 4. Normal dentate granule cells and mossy fibers do not show PKC-111 immunoreactivity (IR) (A), but 15 min (B) and 4 h (C) after ischemia. PKC-Ill IR was present in both granule cells and mossy fibers which returned to control levels I day (D) after ischemia. High power photomicrograph shows normal dentate gyrus (E) and the same region 15 min after the induction of ischemia (F). Bar = 400 ~m (A-D) and 50 /~m (E and F).
128 using activators and inhibitors of PKC ~al,.,0.,i. Hara and coworkers tt reported that topical administration of staurosporine, a relatively specific and potent PKC inhibitor, prevented postischemic neuronal damage in the CA! field of rat and gerbil, whereas inhibitors of cyclic AMP-dependent, cyclic GMP-dependent, and calmodulin-dependent kinases had no neuroprotective effect, Magal and co-workers 2o found that pretreatment of gravid rats with gangliosides that prevent activation and down-regulation of PKC increase the tolerance of rat fetuses to deprivation of maternal blood supply. Favaron and co-workers x showed that downregulation of PKC produced by pretreatment with phorbol esters protects cerebellar granule cells from ischemic injury in vitro. In contrast, Madden and coworkers 20 found that staurosporine reduced the tolerance of rabbit spinal cord to ischemia, while 1,2oleoylacetylglycerol, an activator of PKC, was neuroprotective. These inconsistent results may be a consequence of the differences in test systems, methods and species examined.
Activation of PKC isozymes by ischemic injury could play a direct role in the pathogenesis of neuronal degeneration. Activation of PKC has been implicated in the process of programmed cell death termed apoptosis m.,6. Ojeda et al. 26 demonstrated that steroid-induced apoptosis of thymocytes is mediated by PKC activation. Glucocorticoids have also been shown to potentiate ischemic neuronal injury ~.29. The specific substrates phosphorylated by PKC that mediate the subsequent cellular responses to ischemia are not known. Activation of substrate specific kinases or immediate early gene transcription by PKC could potentially be important 2s. Definition of the specific properties that distinguish the Ca2+-dcpendent isozymes of PKC, including substrate specificity and differential regulation, may provide further insight into the pathogenesis of ischemic neuronal death. Acknowledgements. We thank Mr. Larry Cherkas for photographic expertise.This workwas supportedby grants fromNHLB!to J.W.P. (HL44554.01) and from NIH to N.W.K. (AG05134, NA25588, NS1()828).
.H Fig. 5. PKC-II (A-D) and -lll(E-l-I)immunoreactivity(IR) in stratum lacunosum-moleculare (SLM) of CAt showing sequentialalterationsof stainingat differenttime points(sham, 2 days,3 days and 7 days afterischemia,in that order),PKC-II IR in the 5 L M disappeared by 7 days after ischemia (Ck while PKC-Ill IR disapl~:aredby 3 days after ischemia (G). PKC-II and -III IR were preserved in stratum oriens and radiatum throughout the study.Arrow show.~boundary betwcen 5 L M and the molecular layerof dentate gyrus. Bar = 200 p.m.
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