Journal of Neuroscience Research 29:261-270 (1991)

Rapid Communication Expression of Protein Kinase C Isozymes in Primary Neuronal Cultures of the Rat Cerebellum S. Shimohama, Y. Uehara-Kunugi, K. Terai, T. Taniguchi, J. Kimura, and T. Saitoh Department of Neurology, Faculty of Medicine, Kyoto University (S.S., J . K . ) , and Department of Neurobiology, Kyoto Pharmaceutical University, (Y .U.-K., K.T., T.T.) Japan; Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, California (T.S.)

Protein kinase C (PKC), a family of closely related enzymes, has been implicated in molecular processes involved in differentiation in a variety of cells, including neuronal cells. We studied the presence and distribution of four PKC isozymes immunocytochemically in primary neuronal cultures of the rat cerebellum. We employed four anti-PKC antisera raised against synthetic peptides predicted from the cDNA sequence of the C-terminal portion of four PKC isozymes, a, PI, PII, and y. The majority of neurons were PKC(PI1) immunoreactive both in the early and late (14 days) stage of culture, whereas PKC(a)-, (PI)-, and (y)-immunoreactive neurons were most abundant in the late stage of culture. Immunoreactivity of each PKC was high in the cytoplasm, processes, and growth cones. Prominent nuclear staining was observed with anti-PKC(y) antibody. These results are in contrast with in vivo results where each PKC isozyme is localized in a distinct population of neurons and subcellular compartment, suggesting the presence of regulatory mechanisms for PKC expression and compartmentalization in vivo. Key words: protein kinase C, isozyme, primary neuronal culture, rat cerebellum, development

INTRODUCTION Through cell surface signal transduction mechanisms, protein kinase C (PKC), a phospholipid-dependent kinase, plays a crucial role in the regulation of many cellular functions including secretion, proliferation, and differentiation (Nishizuka, 1986, 1988). PKC is distributed throughout the body and is especially abundant in the brain (Takai et al., 1977), where its role in neurons has been extensively examined (Kikkawa et al., 1986; 0 1991 Wiley-Liss, Inc.

Miller, 1986). Several groups of investigators have demonstrated different forms of the enzyme in the brain (Huang et al., 1987, 1988; Jaken et al., 1987; Shearman et al., 1987; Woodgett and Hunter, 1987). Moreover, the use of molecular cloning techniques has revealed cDNAs for seven PKC transcripts ( O m et a\., 1988), and the biochemical characteristics of four PKC isozymes (a, PI, PII, and y ) have been well studied (Sekiguchi et al., 1987). In the central nervous system (CNS), PKC appears to be involved in several important specialized functions. First, the efficiency of neurotransmission seems to be controlled by the degree of phosphorylation of proteins, which is, at least partly, controlled by PKC. In fact, PKC activation is proposed to be a critical part of the process of long-term potentiation (Routtenberg et al., 1986). Second, PKC is involved in the survival of neurons, and neuronal trophic factors exert many of their functions through PKC activation (Hama et al., 1986; Montz et al., 1985). Thus, PKC seems to be altered in cases of ncuronal degeneration or dysfunction observed due to many different causes such as Alzheimer’s disease, ischemia, and axonal transection (Cole et al., 1988; Kochhar et al., 1989; Masliah et al., 1990; Saitoh et al., 1991; Shimohama et al., 1988). Third, PKC has been implicated in molecular processes involved in differentiation in neurons. During nervous system development, neurons complete their mitotic phase of growth and extend neurites to form axons and dendrites. At the tips of extending axons and dendrites, these developing neurons have

Received February 25, 1991; revised April 10, 1991; accepted April 17, 1991. Address reprint requests to Shun Shimohama, M . D . , Department of Neurology, Faculty of Medicine, Kyoto University, Sakyoko, Kyoto 606 Japan.

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highly motile growth cones which are important not only in neuritic growth but also in synaptogenesis. PKC enzyme activity has recently been demonstrated in growth cones isolated from fetal rat brain (Katz et al., 1985), and extensive binding of 3H-phorbol dibutyrate was detected in elongating cellular processes in fetal rat brain (Murphy et al., 1983). In addition, low concentrations of 12-0tetradecanoyl-phorbol- 13-acetate (TPA) have been shown to enhance neurite outgrowth from sensory ganglia cultures (Hsu et al., 1984). Fourth, the presence of PKC associated with the brain cell nuclei (Nishizuka, 1988; Shimohama et a]., 1990; Wood et al., 1986) suggests a possible role for the enzyme in nuclear events such as the regulation of gene expression. It is probable that this nuclear role of PKC is essential for the above function, maintenance of neurons, and differentiation. To better understand the role of PKC in neuronal growth and in differentiation, we studied immunocytochemically the presence and distribution of PKC isozymes in primary neuronal cultures of the rat cerebellum. We found that PKC isozymes were, to a certain extent, differentially expressed during neuronal development. Notably, there was a very high expression of PKC(PI1) (92% of cells immunopositive) compared to other isozymes on day 1 of culture. PKC(y), the brainspecific form of PKC, was strongly stained in the nuclei relative to other isozymes. Most importantly, the specific and differential distribution of PKC isozymes, which is the characteristic feature in vivo, was not observed in the late stage of neuronal culture, suggesting the existence of regulatory mechanisms that suppress PKC expression and control PKC compartmentalization.

MATERIALS AND METHODS Antibodies The preparation of polyclonal antisera to four PKC isozymes is described in detail elsewhere (Masliah et al., 1990; Shimohama et al., 1990). Briefly, antisera were raised in rabbits against synthetic peptides predicted from the human cDNA sequence of the C-terminal portion of four PKC isozymes, a, PI, PXI, and y. The sequences used were PQFVHPILQSAV (PKC(a), 661672), SYTNPEFVINV (PKC(PI), 661-671), NSEFLKPEVKS (PKC( PII), 663 -673), and SPISPTVPVM (PKC(y), 687-697). The C-terminal cysteine was added to the peptides so that they could be conjugated to proteins by the method of Green et al. (1982). All the antisera gave a titer between 1/2,000 and 1/5,000on dot blots with the individual peptides. Antisera were further affinity purified by affinity chromatography on immobilized antigen columns prepared by coupling free peptide antigens to activated aldehyde-agarose (Actigel A, Sterogene, Arcadia, CA). These antibodies

stained a characteristic cellular population on rat and human brain sections and a Mr 80,000 protein on Western blots of brain homogenates. The staining on brain sections and Western blots was eliminated by the preincubation of antibodies with the free corresponding peptide but not with other peptides demonstrating the specificity of our antisera. Further characterization of these antibodies is found in previous publications (Masliah et al., 1990; Shimohama et al., 1990).

Cell Culture Enriched cultures of rat CNS neurons were prepared from brains aseptically removed from 17-day embryos. Cerebellum was dissected, freed of meninges, and collected in Puck's solution. The tissue was dissected into small pieces with a pair of scissors and enzymatically dissociated by 0.1% trypsin. The cells that remained in suspension after 7 min were collected, pelleted by centrifugation, resuspended in Dulbecco's modified Eagle's minimum essential medium (DMEM)/Ham's F 12 (5050) with 20% horse serum, and filtrated through 45- to 60-pm pore nylon meshes. Cells were plated on poly-L-lysin-coated tissue culture dishes (at a density of 5 X lo4 of trypan blue-excluding cells/cm2) and incubated for 15 min at 30°C with 5% CO,. The medium was changed into DMEM/Ham's F 12 with 15% fetal calf serum and cells were grown at 37°C with 5% CO,. Two days later 20 p M cytosine arabinoside was added to the medium for 24 hr to inhibit the growth of glial cells. The medium was changed every 4 days. Immunocytochemical Procedures Immunocytochemistry was performed according to a modification of the avidin-biotin peroxidase procedure. The cultures were rinsed in phosphate-buffered saline (PBS) and then fixed by adding 4% paraformaldehydei 0.3% glutarldehyde/0.2% picric acid in PBS to the dishes for 30 min on ice according to the method of Kimura et al. (1981). The fixed cultures were postfixed by adding 4% paraformaldehyde/O.2% picric acid overnight at 4"C, soaked in 0.1 M PBS with 15% sucrose for 2 days at 4"C, and then rinsed in PBS followed by rinses in 0.1 M PBS with 0.3% Triton X-100 (PBST). The cultures were incubated for 30 min with 0.01% H,O, in PBST to inhibit the endogenous peroxidase and for I hr with 3% normal goat serum (NGS) in PBST to block the nonspecific binding sites of proteins. The cultures were rinsed in PBST, incubated with one of four different antibodies (anti-PKC(a), -(PI), -(PII), or -(y) for 18 hr at 4°C on a rotator in a humid environment with sodium azide added to prevent bacterial growth, rinsed 3 X 5 min in PBST, and then incubated with biotinylated antirabbit IgG (1:800, Vector Laboratories) in PBST for 1 hr. The cultures were rinsed 3 X 5 min in PBST and

PKC Isozymes in Primary Neuronal Culture

incubated in avidin D peroxidase (1:2,000, Vector Laboratories) in PBST with 1% NGS for 1 hr and then rinsed 3 X 5 min in Tris-buffered saline (TBS), pH 7.4. PKCpositive structures were visualized by incubating the tissue in 0.02% 3-3'-diaminobenzidine tetrachloride (DAB) with 0.0045% H202 and 0.3% Ni ammonium sulfate for 5-10 min. Immunocytochemistry for neurofilament protein and glial fibrillary acidic protein (GFAP) was also performed in the same manner, except using mouse monoclonal anti-200-kDa neurofilament (Labsystem, Helsinki, Finland) and rabbit anti-cow GFAP (Dako Corp., Santa Barbara, CA) as a primary antibody, respectively. Specificity of the immunocytochemical reaction was confirmed by the absence of labeled profiles in tissue sections incubated with either normal goat serum or with antibody preabsorbed with the corresponding synthetic peptides . All experiments were repeated three times. To determine the percentage of cells immunopositive for a given PKC isozyme, 100 neurons in one dish were evaluated. Four determinations were made for each experiment. Because the values for three experiments were comparable, all 12 determinations were averaged for each time point.

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immunoreactive neurons for each antibody indicated that each PKC isozyme is regulated differently during the course of development (Fig. 5 ) . The majority of neurons were PKC(PI1) immunoreactive from the start of the culture whereas other PKC isozyme-immunoreactive neurons were less than 50% at the start of the culture and increased in number after 5 days in culture. The increase in the number of PKC(a)- and PKC(P1)-immunoreactive neurons preceded that of PKC(y). Interestingly, the number of PKC(a)-positive neurons peaked at 5 days in culture and declined thereafter to a 50% steady state level. In earlier stages, cytosolic staining was observed in cultured neurons for each PKC isozyme (Fig. 3a-d), and prominent nuclear staining was observed with anti PKC(y) antibody, whereas little nuclear staining was observed with anti-PKC(a), -(PI), or -(PII) antibodies in any stages (Fig. 6). In newly plated cultures up to a few days, neurons could be seen with extending processes. The growth cones at the tips of these growing processes showed intense staining for each PKC antibody (Fig. 7). In many cultures where fine processes of individual neurons could be seen, immunoreactivity for each PKC was present within axonal varicosities (Fig. 7).

RESULTS Under our culture conditions, the great majority of cells extended neurites and stained with the neuron-selective monoclonal antibody to 200-kDa neurofilament protein (Fig. 1). Astroglia, positive for GFAP, did not proliferate in this medium: counting of anti-GFAPstained cells showed that these cell types represented less than 1% of the total cell population 2 weeks after plating (data not shown). To examine closely the localization of each PKC isozyme within individual differentiating neurons, we studied the distribution of immunoreactivity in rat 17-day embryonal cerebellum neurons grown 1 to 14 days. In all stages examined, PKC immunoreactivity was present, with varying intensity. We divided PKC-immunoreactive neurons into the three classes according to the intensity of the staining: ( + +), intensely stained, (+), moderately stained, and (-) negative (Fig. 2). Figure 3 shows the immunoreactivity for each PKC isozyme on day 1. PKC(PI1)-immunoreactive neurons were most abundant on day 1. We estimated the percentage of the intensely, moderately, and negatively immunoreactive neurons for each antibody from day 1 to day 14 (Fig. 4). The results demonstrate that both number of PKC-immunopositive neurons and the intensity of PKC immunoreactivity in neurons change as a function of time in culture. The results are replotted as the percentage of the total immunoreactive neurons among four PKC isozymes during development. Comparison of the percentage of

DISCUSSION Several reports have previously shown that levels of PKC increase in rat cortex during the first postnatal month as estimated by enzyme activity (Turner et al., 1984) and by an immunoblot analysis (Girard et a]., 1985). An increase in the level of PKC in the cerebellum during the first 3 weeks of postnatal development was also reported (Girard et al., 1988; Huang et al., 1990). Recently, a 20-fold increase in phorbol dibutyrate binding sites was detected during the first week in primary neuronal cultures of the rat embryonal brains with a concomitant increase in Ca2 /phosphatidylserine-dependent protein phosphorylation in soluble neuronal extracts (Burgess et al., 1986). Thus, cultured neurons taken from 16-day rat embryos appeared to be already committed to the increase in phorbol ester binding sites which occurred between days 3 and 8 in culture (Burgess et al., 1986). Our present studies confirmed this result and further showed the differential expression of PKC isozyme during development in culture. Notably, as much as 92% of the neurons on day 1 were PKC(PI1) immunopositive whereas less than 50% were PKC(a), -(PI), or -(y) immunopositive. The high levels of PKC(PI/pII) isozyme in neonatal rat cerebellum have been reported previously using a monoclonal antibody that recognizes both PKC(P1) and -(pH) (Huang et al., 1990). These observations are consistent with the idea that, among the four PKC isozymes, PKC(PI1) might +

Fig. 1. Immunolocalization of 200-kDa neurofilament protein on day S neuronal culture. Bar = SO p m . Fig. 2. Immunolocalization of PKC(-y) on day 5 neuronal culture. moderately stained; -, negative. Bar = 50 p m .

+ + , intensely stained; + ,

Fig. 3. Immunoreactivity for each PKC isozyrne on day I neuronal culture. a, PKC(a); b, PKC(P1); c , PKC(PI1); and d, PKC(y). Bar = 50 pm.

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life of neurons because the remarkable abnormality in PKC(PII), as compared to less remarkable changes in other PKC isozymes, has been observed in Alzheimer's disease, where neuronal death is prominent (Masliah et a]., 1990; Saitoh et al., 1991). We have previously reported the differential expression of PKC isozymes in adult rat cerebellum by 73 using the same antibodies (Shimohama et al., 1990), .-Cm consistent with previous reports from other laboratories i 5 20 (Kitano et al., 1987; Hidaka et a]., 1988; Huang et al., 0 2 4 6 8 10 12 14 1988). In the molecular layer, stellate and basket cells Age of culture (days) were stained with anti-PKC(a). In the Purkinje cell layer Fig. 5. Comparison of the percentage of the immunoreactive Purkinje cells were stained with anti-PKC(PI1) and -(y). neurons among four PKC isozymes during development. Val- In the granule cell layer, both granule and Golgi cells ues are the mean SEM of 12 determinations from three were stained with anti-PKC(P1) and -(PII) but not with experiments. anti-PKC(a) or -(y). Dentate nucleus neurons were positive with anti-PKC( PI) and -(pII) and negative with play an important role, such as growth regulation, during anti-PKC(a) and -(y). Thus, in the adult cerebellum, the early developmental stage. The growth-supporting PKC isozymes are restricted to certain neuronal subfunction of PKC(PI1) might be important throughout the types, in contrast with the current study using cultured 0)

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Fig. 6. PCK(PI1) and -(y) immunoreactivity in the nucleus. Prominent staining was observed in the nucleus with anti-PKC(y) antibody. Bar = 50 pm.

neurons, where the sum of the percentage of intensely immunostained cells for each antibody was far more than loo%, indicating that many neurons contain more than one PKC isozyme. It is tempting to speculate that the intact neuronal connections and activities are required for suppressing the expression of some PKC isozymes or degrading PKC. In this context, it is interesting to note that there is an induction of anti-PKC staining in hippocampus neurons after dissecting axonal input from midsepta1 neurons (Shimohama et al., 1988). As neuronal cells cease proliferation and begin migrating and extending processes, there is a concomitant increase in PKC immunoreactivity (Huang et al., 1990). During this stage of development, the four PKC isozymes, PKC(a), -(PI), -(pII), and -(y), appeared to be localized in extending processes and growth cones, consistent with the findings from the phorbol ester binding studies (Murphy et al., 1983) and with studies utilizing isolated nerve growth cone (Katz et al., 1985). It is also consistent with the report that addition of low concentrations of TPA, an activator of PKC, to serumfree growth medium promotes neuritic outgrowth from explants of chick dorsal root ganglia (Hsu et al., 1984).

It is of interest to note that a major substrate for PKC in brain is GAP-43 (B-50), a growth-associated protein (Aloyo et al., 1983; Coggins and Zwiers, 1989; Dekker et al., 1989; Zwiers et al., 1980). GAP-43 is an acidic 43-kDa protein that is part of a family of proteins whose levels are elevated during periods of axonal growth (Freeman et al., 1985; Meiri et al., 1986). The regulation of Go, a major signal transductor in the growth cone, by GAP-43 (Strittmatter et al., 1990), its increased level in development and regeneration (Masliah et al., 1991 ), and its increased phosphorylation accompanying the induction of long-term potentiation in the rat hippocampal formation (Akers et al., 1986; Routtenberg et al., 1986) are consistent with a major role of GAP-43 and its phosphorylation by PKC in neuritic outgrowth. Although GAP-43 has been reported to be a preferred substrate for PKC(PI/PII) (Sheu et al., 1990), high levels of immunoreactivity of all PKC isozymes were localized in processes and synaptic terminals in the current in vitro study. We have observed prominent nuclear staining with anti-PKC(y) antibody in later stages of cultured neurons and little staining with anti-PKC(a), -(PI), or -(PII),

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Fig. 7. PKC(y) immunolocalization in the growth cones (arrowhead) and the varicosities of extending neurites on day 5 . The growth cones showed staining for all four PKC antibodies as well. Bar = 25 pm

suggesting a role of PKC(y) in nuclear events such as regulation of gene expression as proposed previously (Nishizuka, 1988). The preferential nuclear staining by anti-PKC(y) over anti-PKC(a), -(PI), or -(pH) was also observed in vivo (Shimohama et al., 1990). At the ultrastructural level, PKC(y) has been found in the nucleoplasm in addition to the membrane and cytoplasm of dendrites, axons, and perikarya (Kose et al., 1988). Therefore, it is conceivable that PKC(y) has some nuclear functions in addition to its involvement in the regulation of neurotransmitter release (Taniyama et al., 1990). In conclusion, our present results suggest that PKC might be involved in earlier stages of neuronal differentiation and that several PKC isozymes are expressed with different time-courses in the cytoplasm, nucleus, processes, and growth cones during development in primary neuronal cultures of the rat cerebellum. Using the same fixative, more neurons are stained by each anti-PKC isozyme in culture than in vivo, suggesting the existence of mechanisms which regulate the expression of each PKC isozyme in vivo.

ACKNOWLEDGMENTS We acknowledge the editorial help of Robert Davignon. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (01658514 and 122402 15) from the Ministry of Education, Science and Culture, Japan; by a Grant-in-Aid from the Tokyo Biochemical Research Foundation; by a Grant-in-Aid from the Sasagawa Medical Research Foundation; and by National Institutes of Health grants (AGO8205 and NS28 121).

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Expression of protein kinase C isozymes in primary neuronal cultures of the rat cerebellum.

Protein kinase C (PKC), a family of closely related enzymes, has been implicated in molecular processes involved in differentiation in a variety of ce...
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