Journal of Neuroscience Research 29:379-389 (1991)
Distribution of Protein Kinase C Isozymes in Rat Optic Nerves S. Komoly, Y. Liu, H. deF. Webster, and K.-F. J. Chan Laboratory of Experimental Neuropathology , National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland
’
Light (LM) and electron (EM) microscopic immuno- ubiquitous in eukaryotes. In mammals, this enzyme is cytochemical methods were used to study the distri- found in high concentrations in the central nervous sysbution of protein kinase C (PKC) isozymes in adult tem. Protein kinase C-mediated protein phosphorylation rat optic nerves. In cryostat and vibratome sections has been implicated in the regulation of a number of examined by LM, type I1 (p) isozyme was localized biological events (for review, see Nishizuka, 1986; Kikalmost exclusively in the axons. In the EM, immu- kawa and Nishizuka, 1986; Kaczmarek, 1987; Huang, noreaction products were found to associate with mi- 1989). In the nervous system, protein kinase C may play crotubules and neurofilaments. The inner surface of important roles in neurotransmitter release, neuritogeneaxonal membranes were occasionally stained. Analy- sis, and memory and learning (Kaczmarek, 1987; Bursis of PKC isozyme composition of the optic nerves by goyne, 1989; Chiarugi et al., 1989). Seven isoforms of using immunoblot techniques revealed that type I1 protein kinase C (a,PI, PII, y, 6, E, 5 ) have thus far (p) isozyme accounted for approximately 80% of the been identified by genetic approaches (Coussens et al., total immunoreactivity. By contrast, type I11 (a) 1986; Nishizuka, 1988; Kikkawa et al., 1989). These isozyme, which accounted for the remaining 20% of isoforms are encoded by different genes with the excepPKC, was found mainly in the astrocytes. Astrocytic tions of PI and PII, which are derived from the same processes next to blood vessels and between myeli- mRNA transcript by alternative splicing (Nishizuka, nated axons were stained. In the EM, immunoreac- 1988). Protein kinase C purified from the brain contains tion products were found in the cytoplasm and along three major isozymes termed type I, type 11, and type 111. astroglial filaments. Segments of plasma membranes These subspecies are encoded by cDNA corresponding also were stained; but nuclei were unstained. Adult to y,PI + PII, and a;respectively. Together, they conglial cells were not stained by an antibody to type I1 stitute over 90% of the total protein kinase C activity in (p) isozyme except for the occurrence of a few punc- the brain (Huang and Huang, 1986). tate cytoplasmic densities in occasional astrocytes. The exact physiological significance of the various Very faint or no immunostaining was observed in sec- PKC isozymes is still unclear. Biochemical studies retions treated with a monoclonal antibody to type I ( y ) vealed that different isozymes have slightly different isozyme. Immunoblot analyses also did not reveal this substrate specificities and requirements of activators subspecies. The absence of type I (y)isozyme in optic (Kikkawa et al., 1989). Several PKC isozymes also were nerves is not due to a down-regulation of the enzyme found to have different subcellular distributions and exduring development. In developing (5 and 11 day) pression during development of the brain. Theoretically, rats, immunoreactivity of protein kinase C was very each isozyme may have distinct biological functions. faint or absent. After 15 days, reaction products of This may be due to the presence of specific protein or both type I11 (a)and type I1 (p) isozymes were found enzyme substrates in different subcellular locations. Althroughout the nerve. These findings suggest that type I1 (p) isozyme may be involved in axonal trans- Received September 28, 1990; revised December 13, 1990; accepted port whereas type I11 (a)isozyme may play a role in December 13, 1990. some astrocyte functions in mature optic nerves. Key words: protein kinase C, isozymes, optic nerve, axons, astrocytes, glial filaments, microtubules INTRODUCTION Protein kinase C (PKC) is a calcium-activated and phospholipid-dependent serinefthreonine protein kinase Published 1991 by Wiley-Liss, Inc.
Address reprint requests to Dr. S . Komoly, National Institutes of Health, Building 36, Room 4A-29, Bethesda, MD 20892.
Dr. K.-F.J. Chan is now at the Department of Biochemistry, University of Hong Kong, Hong Kong. Abbreviations used: PKC, protein kinase C; PBS, phosphate-buffered saline; HEPES N-2-hydroxyethylpiperazine-N’-ethanesulfonicacid; EGTA, [ethylenebis(oxyethylenenitrilo)tetraacetic acid; SDS, sodium dodecyl sulfate.
380
Komoly et al.
ternatively, the presence of a particular PKC isozyme in specific cell types may come as a result of unique cellular regulation of the allosteric activators and inhibitors. So far, most of the brain regions including cerebral cortex, striatum, thalamus, spinal cord, hippocampus, septum, and mesencephalon were found to contain two or more isozymes (Mochly-Rosen et al., 1987; Huang et al., 1989; Saito et al., 1988; Yoshida et al., 1988). Expression of a single subspecies was observed only in a few cell types, e.g., Purkinje cells and olfactory bulb mitral cells (Hidaka et al., 1988; Young, 1988). In this report, we show that the distribution of PKC isozymes in adult rat optic nerves also is cell-specific. Type I1 (6) isozyme was found predominantly in axons whereas type I11 ( a ) isozyme was localized mainly in astrocytes. No significant immunoreactivity was observed for type I (y) isozyme. These results suggest that the optic nerve may be a good anatomical system for further studies of the functions and regulations, as well as the mechanisms of gene expression, of protein kinase C isozymes.
containing 4% paraformaldehyde, 15% saturated picric acid, and 0.1 M PBS. The optic nerves were dissected out and postfixed overnight in the same fixative. Cryoprotection of the samples was carried out in a solution containing 0.9% (w/v) sodium chloride, 20% (w/v) sucrose, and 3% (v/v) polyethylene glycol. After 24 hr, the optic nerves were embedded in Tissue-Tek and frozen in dry ice-cold isopenthane. Serial sections of 6-12 pm were cut in a cryostat (2800 Frigocut, Reichert-Jung) and mounted on gelatin-covered slides. The samples were kept at 4°C until used. The sections were washed in PBS containing 0.5% of Triton X-100 for 3 hr to remove the cryoprotection reagents and to permeabilize the cell membranes. Nonspecific protein binding sites were blocked by incubating the specimen with horse serum. Immunoreaction with different monoclonal antibodies to protein kinase C was carried out at 4°C for 16-24 hr. After washing, the specimens were incubated with biotinylated anti-mouse TgG (1:300 dilution) for 60 min at room temperature. Color reaction was developed by using ABC Elite horseradish peroxidase kit with 0.05% DAB and 0.005% H,O, in PBS. Two sets of control experiments were performed. These included ( 1 ) substitution of monoclonal anti-PKC antibodies with normal mouse IgG; and (2) preincubation of the primary antibodies with purified protein kinase C before immunostaining.
EXPERIMENTAL PROCEDURES Materials Monoclonal antibodies to protein kinase C isozymes type I (y), type I1 (p), and type 111 ( a )were obtained from Seikagaku Kogyo Co., Ltd. The antibodies were reconstituted to a concentration of 0.1 mgiml and stored in aliquots at -20°C. A 1:lOO dilution of these antibodies was used in both immunostaining and Electron Microscopic Immunocytochemistry Four adult (180 g) Wistar rats were perfused as immunoblotting experiments. Affinity purified antimouse biotinylated horse IgG, normal horse serum, nor- described above except that the fixative also contained mal mouse IgG, and the standard ABC Elite horseradish 0.5% glutaraldehyde. The optic nerves were removed peroxidase kit were purchased from Vector Laboratories, with the brain; they were postfixed overnight, and transInc. The sera were diluted in 0.1 M phosphate-buffered ferred into PBS. Sections of 30 pm were cut with a BSK saline (PBS) containing 1% bovine serum albumin and Slicer. Vibratome sections were immunostained as de0.1 % sodium azide. The embedding medium for frozen scribed for LM but without prior detergent treatment. tissue specimens, Tissue-Tek, was from Miles Labora- After color development with DAB, the sections were tories. Nitrocellulose membranes were from Schleicher postfixed with 0.5-1 % osmium tetroxide in the presence and Schuell, Inc. Casein was from Sigma Chemical Co. or absence of 0.6% potassium fenicyanide and 5% su'251-labeledprotein G was a product of Amersham Corp. crose for 60 min and embedded flat in epon. Thin sections Paraformaldehyde, glutaraldehyde, uranyl acetate 3,3- were cut with a Reichert-Jung ultramicrotome and diaminobenzidine tetrahydrochloride (DAB), and Poly/ mounted on Formvar-coated single slot grids. Every secBed 812 were purchased from Polysciences, Inc. Protein ond one of the serial sections was stained with uranyl kinase C was purified to apparent homogeneity from the acetate for 12 min and then with lead citrate for 30 sec in particulate fractions of rat brains essentially according to a LKB 2168 ultrastainer. The remaining sections were not the previously described procedures (Huang et al., stained with heavy metals. The specimens were examined in a Philips EM 410 electron microscope at 80 kV. 1986). Light Microscopic Immunocytochemistry Adult and developing ( 5 , 1 1, 15, 18, and 27 days) Wistar rats were anesthetized and perfused through the heart with physiological saline followed by a fixative
Immunoblotting of Protein Kinase C Isozymes Five adult Wistar rats were anesthetized and killed by decapitation. The optic nerves were removed from the brain and immediately homogenized in 20 mM HEPES,
Protein Kinase C Isozyrnes in Optic Nerve
pH 7.4, 0.5mM EGTA, 2 mM EDTA, 20 mM NaCl, and 0.5 mM dithiothreitol (buffer A). All procedures were carried out at 0-4°C. The homogenates were centrifuged at 15,OOOg for 30 min. The resulting supernatant fluids were saved as soluble fractions. The pellets were resuspended in buffer A and saved as particulate fractions. The proteins (30 pg) were separated by using SDSpolyacrylamide gel (10%) electrophoresis and electroblotted onto nitrocellulose membranes. The blotting buffer contained 30 mM Tris-HC1, 200 mM glycine, pH 8.3, and 20% (v/v) methanol. After washing the membranes with 50 mM Tris-HC1, pH 7.4, and 150 mM NaCl (TBS) containing 0.05% Tween-20, nonspecific binding sites were blocked by overnight incubation with 2% (wl v) casein. Immunoreactions of the monoclonal antibodies (1 pglml) with PKC isozymes types I (y), I1 (p), and I11 ( a )were carried out at 25°C for 2 hr. Reaction products were detected by incubating the membranes with I25 I-labeled protein G (containing 1 X lo7 cpm) in TBS for 30 min at 25°C and subsequent autoradiography. Immunoreactivity was quantitated by both densitometric scanning of the corresponding PKC bands with a Shimadzu CS-9000 flying-spot densitometer and gamma counting of the excised PKC-containing nitrocellulose membranes using a LKB-Wallac 1272 Clini-Gamma counter.
RESULTS Distribution of PKC Isozyrnes in Adult Rat Optic Nerves To better understand the function and regulation of different protein kinase C isozymes in the brain, we searched for a well-defined anatomical system in which separate PKC subspecies are localized in different cell types. The optic nerves of adult rats were found to possess these characteristics. Light microscopic immunocytochemical studies of the cryostat sections revealed an almost exclusive localization of PKC type 111 ( a ) isozyme in the astrocytes (Fig. 1A). Immunoreaction products of a monoclonal antibody to this isozyme were found both in the cytoplasm and in the astrocytic processes around blood vessels and between meylinated axons (Fig. IA,B). The nuclei were unstained. By contrast, immunoreactivity of PKC type I1 (p) isozyme was confined mainly to axons (Fig. lC,D). Most, if not all, of the axons were immunoreactive. Adult glial cells were not stained by the monoclonal antibody to this type I1 (p) subspecies except for the occurrence of a few punctate cytoplasmic densities in occasional astrocytes. Very faint or no immunoreactivity was found in cryostat sections treated with an antibody to PKC type I (y) isozyme (Fig. lE,F).The
381
immunostaining was similar to those obtained by using a biotinylated mouse IgG or after preincubation of the monoclonal antibodies with purified rat brain protein kinase C. Immunostaining of the three PKC isozymes in vibratome sections was similar to those observed in frozen sections. However, no immunoreactivity was found in paraffin-embedded sections. Prior delipidation of vibratome and frozen sections with ethanol also abolished the immunolabeling of protein kinase C by monoclonal antibodies (results not shown). In adult rat retina, immunostaining of type I1 (p) isozyme was localized mainly in ganglion cells whereas type I11 ( a )subspecies was found in Muller cells (results not shown). There were no immunoreaction products to type I (y) isozyme. These findings are in agreement with the immunoblotting data (Yosida et al., 1988) and support our observations on the distribution of protein kinase C in the optic nerves.
Immunoblot Analysis of Protein Kinase C Isozymes The composition of PKC isozymes in adult rat optic nerves were quantitated by using immunoblot analyses. The results are summarized in Table I. Over 80% of the total PKC immunoreactivity was due to type I1 (p) isozyme. Subcellular distribution studies revealed that approximately two-thirds of this PKC subspecies were in the soluble (cytosolic) fractions. The immunoreaction of the monoclonal antibody with type I1 (p) isozyme was specific. Only one radioactive band with an apparent M , of 80,000 was observed in the soluble and the particulate fractions of the optic nerves (Fig. 2). These findings suggest that protein kinase C, in particular, the type I1 (p) isozyme, may play an important role in axonal functions. Type 111 ( a )isozyme accounts for the remaining 20% of PKC immunoreactivity in the optic nerves (Table I). Nearly 70% of this isozyme was found in the cytosolic fractions. By contrast, no significant immunoreactivity of type I (y) isozyme was observed. The absence of immunoreaction of the monoclonal antibody to type I (y) subspecies was not due to its ineffectiveness. This antibody could recognize type I (y) isozyme in purified protein kinase C preparations (data not shown). Electron Microscopic Localization of PKC Isozymes To learn more about the putative functions of protein kinase C isozymes in optic nerves, the subcellular localization of both type I1 (p) and type 111 (a)subspecies were investigated by using electron microscopic immunocytochemical methods. As shown in Figure 3 , immunoreaction products of type I1 (p) isozyme were associated with microtubules in axons. The punctate staining in the cytoplasm might be due to the labeling of neurofilaments (Fig. 3A-C). Occasionally, immunoreaction prod-
Fig. 1. Light microscopic immunocytochemical localization of PKC isozymes in rat optic nerves. A,B: Irnrnunostaining of astrocytes with type 111 (a)monoclonal antibody. C,D: Irnrnunostaining of axons with type I1 (p) monoclonal antibody. E,F: Immunostaining of optic nerve with type I (y) monoclonal antibody. Serial cryostat sections are 6 p,m in thickness. Arrowheads denote the same blood vessels. Magnifications: A, C, E, X 120; B, D, F, ~ 2 4 0 .
Protein Kinase C Isozymes in Optic Nerve TABLE I. Distribution of Protein Kinase C Isozymes in Adult Rat Optic Nerves* Protein kinase C isozymes (%)
I (Y) Total Subcellular Soluble Particulate
11 (PI
I11 (ff)
-
80.8
?
3.3
19.2 ? 3.5
-
67.2 32.8
? ?
7.1 7.2
69.2 30.8
-
& ?
5.8 6.4
*The composition of protein kinase C isozymes in adult rat optic nerves was determined by immunoblot assays as described under Experimental Procedures. lmmunoreactivity of monoclonal antibodies to PKC isozymes type I (y), type I1 (p), and type 111 (a)was determined by using '251-1abeled protein G overlay and quantitated by densitometric scanning of the corresponding radioactive PKC bands.
383
products were observed during the first two postnatal weeks (results not shown). Gradually, dense immunostaining was found similar to the patterns shown in Figure 1C and D. There was no positive staining with the monoclonal antibody to PKC type I (y) isozyme, regardless of age (data not shown). This observation suggests that the failure to detect type I (y) isozyme in adult rat optic nerves is not due to a down-regulation of this subspecies during early development.
DISCUSSION
Protein kinase C has been implicated in the regulation of a number of neural functions (Nishizuka, 1986; Coussen et al., 1986; Kaczmarek, 1987). This protein ucts were found to associate with the internal surface of phosphorylation system also may play important roles in the axolemma and on the outer membranes of the mito- several pathological conditions such as ischemic brain chondria. No diffuse staining was observed in the axo- injury (Louis et al., 1989; Olah et al., 1990) and brain plasm. It should be noted that the intensity of the immu- edema formation (Joo et al., 1989). The exact functional nostaining could vary from axon to axon, and sometimes roles of different protein kinase C isozymes in these and other processes still remain unclear. One problem is that even within the same axon. Immunoreaction products of PKC type 111 (a) many cell types in many regions of the brain express isozyme were localized almost exclusively in astrocytes more than one subspecies. Our findings of an almost (Fig. 4). The astrocytes were identified by their ultra- exclusive distribution of type I1 (p) isozyme in the axons structural features (Peters et al., 1991) as well as by their and type 111 (a)isozyme in the astrocytes of adult rat immunoreactivity with an antibody to glial fibrillary optic nerves (Fig. 1) may provide a unique opportunity acidic protein (GFAP) (results not shown). Within the for further investigation of the function and regulation, astrocytes, diffuse reaction products were observed in and perhaps the mechanisms of gene expression, of these the cytoplasm (Fig. 4A). Glial fibers were stained (Figs. two isozymes. 4B ,C). Mitochondria were sometimes labeled. Strong In the EM, immunoreaction products of type 11 (p) immunoreactivity also was found to associate with cell isozyme were found to associate with microtubules and membranes (Figs. 4C ,D), particularly at contact regions neurofilaments (Fig. 3 ) . This specific localization sugbetween perivascular astroglial processes (Figs. 4D,E). gests that type I1 (p) isozyme may play a role in reguThe nuclei were unstained. A few endothelial cells in the lating cytoskeletal structures and axonal transport prooptic nerve were occasionally found to contain PKC type cesses in axons. Indeed, microtubule-associated proteins 111 ( a ) isozyme immunoreactivity. In addition, faint re- such as MAP-2 and tau, and neurofilament proteins are action products were observed at tight junctions (results putative substrates for protein kinase C (Kaczmarek, not shown). 1987; Nixon et al., 1987; Sihag et al., 1988; Pestronk et No positive immunostaining of PKC type I (y) al., 1990). These proteins are phosphorylated in vivo. isozyme was seen in the EM. Immunostaining of axons with monoclonal antibodies to pII and y subspecies also have been reported in the cerExpression of PKC Isozymes During Development ebellum (Kose et al., 1988; Kikkawa et al., 1989; Saito of the Optic Nerves et al., 1989). The functional significance of the other The distribution of the various PKC isozymes in cytosolic type I1 (p) isozyme in axons (Fig. 2 and Table the optic nerves of developing rats ( 5 , 1 1, 15, 18, and 27 I) is less clear. In some cells, PI1 subspecies may play a days) was investigated by using LM immunocytochem- role in protein processing (Kikkawa et al., 1989). istry. From 5 to 15 days, reaction products of type I11 ( a ) Protein kinase C type I11 ( a ) isozyme is more subspecies were very faint or absent (Fig. 5A-C). After widely distributed in the astrocytes of optic nerves. Im15 days, immunopositive astrocytic-like cells were ob- munoreactivity was observed in the cytoplasm as well as served (Fig. 5D-F). Both the intensity and the number of in association with glial filaments and cell membranes stained cells were found to increase with age. The de- (Figs. 1 and 4). Furthermore, most, and perhaps all, of velopmental expression of PKC type I1 (p) isozyme in the astrocytes in the optic nerve were immunostained. axons of rat optic nerves was similar to the type I11 (a) These results are in agreement with the previous localsubspecies. Only weak and diffuse immunoreaction ization of PKC type I11 ( a )isozyme in cultured astroglial
384
Komoly et al.
CONTROL TYPE II
S
c3 I
0 F
x
P
' s
P '
's
P'
98 67 43
31
IMMUNOBLOT Fig. 2. Immunoblotting of PKC type I1 (p) isozyme in rat optic nerves. Proteins (30 pg) in both soluble (S) and particulate (P) fractions of adult rat optic nerves were separated by using SDS-polyacrylamide gel (10%) electrophoresis. The Coomassie blue protein staining patterns are shown in the left
panel. Duplicate gels were electroblotted onto nitrocellulose membranes. Immunoreactions of a mouse IgG (control) and a monoclonal antibody to type I1 (p) isozyme were determined by '251-labeled protein G overlay and subsequent autoradiography (right panel).
cell lines (Shimosawa et a]., 1990). Thus, type 111 (a) isozyme may have some important regulatory roles in cellular functions that are common to different subpopulations of astrocytes. In cultured astrocytes, treatment with phorbol esters can lead to morphological transformation concomitant with an increase in intermediate filament protein phosphorylation and a translocation of protein kinase C from the cytosolic to the particulate fraction (Pollenz and McCarthy, 1985; Mobley et al., 1986; Neary et al., 1986, 1988; Harrison and Mobley, 1989; Bhat, 1989). No immunoreaction products of PKC type 111 (a) isozyme were found during early development (5 and 11 days) of the optic nerves (Fig. 5). After 15 days, there was a gradual increase in both the number and the intensity of positive astrocytic-like cells. These results support the earlier findings that fully mature glial cells were observed in electron micrographs only after 14 days of development (Skoff et al., 1976). The increase in protein kinase C during development is not unique to the optic nerves. Other biochemical, immunological, and gene expression studies also revealed a 10- to 30-fold increase in PKC during maturation of the brain (Girard et al., 1986, 1988; Stichel 1988; Noguchi et at., 1988; Yoshida et al., 1988; Sposi et al., 1989).
Protein kinase C type I (y) isozyme has been shown to exist solely in the brain and spinal cord (Kikkawa, 1989). But no immunostaining of this subspecies was observed in the optic nerves. The absence of immunoreaction products is not due to a malfunctioning of the monoclonal antibody, because positive immunostaining has been observed in the cerebellum (results not shown). In addition, the antibody can recognize type I isozyme in purified protein kinase C preparations. It remains to be established whether the absence of type I (y) isozyme in optic nerves is a result of transcriptional or translational control. Protein kinase C immunoreactivity also was not found in oligodendrocytes and meylin sheaths. These results are in agreement with several other immunocytochemical and in situ hybridization studies (MochlyRosen et al., 1987; Hidaka et al., 1988; Huang et al., 1988, 1989; Kose et al., 1988; Saito et al., 1988, 1989; Young, 1988, 1989). Yet, protein kinase C has been shown to modulate both oligodendrocyte differentiation and myelin basic protein phosphorylation in vitro (Vartanian et al., 1986; Chan, 1987, 1989; Ritchie et al., 1987; Yong et al., 1988). The failure to detect protein kinase C in optic nerve oligodendrocytes or myelin sheaths by immunocytochemical methods may be due to a different
Protein Kinase C Isozymes in Optic Nerve
385
Fig. 3. Electron microscopic localization of PKC type I1 (p) isozyme in axons. A: Axons stained heavily (ax) and less heavily (a) with a monoclonal antibody to PKC type I1 (p) isozyme. Arrowheads indicate microtubules. The specimen was counterstained with uranyl acetate and lead citrate. X 60,000. B: Immunostained axon without heavy metal treat-
ment. Arrowhead points to an immunostained microtubule. X60,OOO. C: Longitudinal section of an axon showing clustering of immunostaining (arrow). Oblique sections of two unstained axons are represented by a. Contrast was enhanced by treatment with 0.6% potassium ferricyanide and 1 % osmium tetroxide. X 20,000.
orientation or conformation of the enzyme in CNS tissue such that the epitope(s) are not accessible. Protein kinase C type I1 (p) isozyme could be detected in purified rat and guinea pig brain myelin preparations by using immunoblotting techniques (K.-F.J. Chan and Y. Liu, unpublished results). Of course, it is also possible that the major protein kinase C isozyme in both oligodendrocytes and myelin sheaths may correspond to a subspecies other than types I, 11, and 111. The differential distribution of protein kinase C isozymes in optic nerves suggests that each isozyme may
have different functional roles in processing and modulating physiological cellular responses of the visual system. The subspecies in different cell types also may respond to different regulatory mechanisms. For example, purified type 111 (a)isozyme is responsive to Ca2+ and arachidonic acids (Kikkawa, 1989). It is therefore possible that the astrocytes in optic nerves may have a signal transduction pathway that involves the release of these activators. Further investigation of the optic nerves, a less complicated anatomical system than the whole CNS, may provide more information on both gene expres-
Fig. 4. Electron microscopic localization of PKC type 111 (a) isozyme in astrocytes. A: Immunostaining of an astrocytic process (As) is adjacent to an endothelial cell (En) of a blood vessel. x 52,000. B: Immunostaining of filamentous structures in an astrocyte (As). Adjacent pericyte processes (P), an endothelial cell (En), and an astrocytic process (asterisk) were unstained. x 41,000. C: Immunostained (arrowheads) and un-
stained (open arrows) plasma membranes of two adjacent astrocytic processes. Filamentous immunoreaction products are in the cytoplasm. En = endothelial cell. X 31,000. D: Clustering of reaction products at the cytoplasmic sides of two adjacent plasma membranes (arrows). En = endothelial cell. X 41,000. E: Higher magnification ( X 82,000) of D.
Fig. 5 . PKC type I11 (a)immunoreactivity during development of rat optic nerves. Light microscopic immunocytochemical localization of PKC type 111(a)isozyme in the optic nerves of developing (A: 5 days; B: 11 days; C: 15 days; D: 27 days) and adult (E,F) rats. A stained mature astrocyte is shown by an arrow. Magnifications: A-E, X 120; F, X 240.
388
Komoly et al.
sion and activation of different protein kinase C isozymes.
ACKNOWLEDGMENTS We wish to thank Mrs. Yoong Chang for her excellent technical assistance in the EM studies.
REFERENCES Bhat NR (1989): Role of protein kinase C in glial cell proliferation. J Neurosci Res 22:20-27. Burgoyne RD (1989): A role of membrane-inserted protein kinase C in cellular memory? Trends Biol Sci 14:87-88. Chan, K-FJ (1987) Ganglioside-modulated protein phosphorylation in myelin. J Biol Chem 262:2415-2422. Chan, K-FJ (1989) Effects of gangliosides on protein phosphorylation in rat brain myelin. Neurosci Res Commun 5:95-104. Chiarugi VP, Ruggiero M, Corradetti R (1989): Oncogenes, protein kinase C, neuronal differentiation and memory. Neurochem Int 1411-9. Coussens L, Parker PJ, Rhee L, Yang-Feng TL, Chen E, Waterfield D, Francke U, Ullrich A (1986): Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science 233:859-866. Girard PR, Mazzei GJ, Kuo JF (1986): Immunological quantitation of phospholipidiCa’ -dependent protein kinase and its fragments. J Biol Chem 261:370-375. Girard PR, Wood JG, Freshci JE, Kuo JF (1988): Immunocytochemical localization of protein kinase C in developing brain tissue and in primary neuronal cultures. Dev Biol 126:98-107. Harrison BC, Mobley PL (1989): Protein phosphorylation in astrocytes mediated by protein kinase C: Comparison with phosphorylation by cyclic AMP-dependent protein kinase. J Neurochem 53: 1245-1 25 I . Hidaka H, Tanaka T, Onada K, Hagiwara M, Watanabe M, Ohta H, Ito Y , Tsurudome M, Yoshida T (1988): Cell type specific expression of protein kinase C isozymes in the rabbit cerebellum. J Biol Chem 263:4523-4526. Huang, K-P (1989): The mechanism of protein kinase C activation. Trends Neurosci 12:424-432. Huang K-P, Huang FL ( 1986): lmmunochemical characterization of rat brain protein kinase C. J Biol Chem 261:14781-14787. Huang K-P, Chan K-FJ, Singh TJ, Nakabayashi H , Huang FL (1986): Autophosphorylation of rat brain Ca2+-activatedand phospholipid dependent protein kinase. J Biol Chem 261:1213412140. Huang K-P, Huang FL, Nakabayashi H, Yoshida Y (1989): Expression and function of protein kinase C isozymes. Acta Endocrinol 121 :307-3 16. Joo F, Tosaki A, Olah 2, Koltai M (1989): Inhibition by H-7 of protein kinase C prevents formation of brain edema in SpragueDawley CFY rats. Brain Res 490:141-143. Kaczmarek, L (1987) The role of protei kinase C: in the regulation of ion channels and neurotransmitter release. Trends Neurosci 10: 30-34. Kikkawa U , Nishizuka Y (1986): Protein kinase C. The Enzymes 17:167-189. Kikkawa U, Kishimoto A, Nishizuka Y (1989): The protein kinase C family: Heterogeneity and its implications. Annu Rev Biochem 58131-44. Kose A, Saito N , Ito H, Hiroshi I, Kikkawa U, Nishizuka Y, Tanaka
C (1988): Electron microscopic localization of type I protein kinase C in rat Purkinje cells. J Neuroscience 8:4262-4268. Louis JC, Magal E, Yavin E (1988): Protein kinase C alterations in the fetal rat brain after global ischemia. J Biol Chem 36:1928219285. Mobley PL, Scott SL, CNZ EG (1986): Protein kinase C in astrocytes: A determinant of cell morphology. Brain Res 398:366-369. Mochly-Rosen D, Basbaum AI, Koshlan DE (1987): Distinct cellular and regional localization of immunoreactive protein kinase C in rat brain. Proc Natl Acad Sci USA 84:4660-4664. Neary JT, Norenberg LOB, Norenberg MD (1986): Calciumactivated, phospholipid-dependent protein kinase and protein substrates in primary cultures of astrocytes. Brain Res 385: 420-424. Neary JT, Norenberg LOB, Norenberg MD (1988): Protein kinase C in primary astrocyte cultures: cytoplasmic localization and translocation by phorbol ester. J Neurochem 50:1179-1184. Nishizuka Y (1986): Studies and perspectives of protein kinase C. Science 233:305-3 12. Nishizuka Y (1988): The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature (London) 334:661-664. Nixon RA, Lewis SE, Martotta CA (1987): Posttranslational modification of neurofilament proteins by phosphate during axoplasmic transport in retinal ganglion cell neurons. J Neurosci 7: 1145-1 158. Noguchi A, DeGuire J , Zanaboni P (1988): Protein kinase C in the developing rat liver, heart and brain. Dev Pharmaol Ther 11: 31-43. Olah 2 , Ikeda J , Anderson WB, Joo F (1990): Altered protein kinase C activity in different subfields of hippocampus following cerebral ischemia. Neurochem Res 15:515-518. Pestronk A, Watson DF, Yuan CM (1990): Neurofilament phosphorylation in peripheral nerve: Changes with axonal length and growth state. J Neurochem 54:977-982. Peters A, Palay SL, Webster HdeF (1991): “The Fine Structure of the Nervous System. ” New York: Oxford University Press. Pollenz R, McCarthy KD (1985): Regulation of intermediate filament protein phosphorykation in astroglia. Trans Amer Soc Neurochem 16:188. Ritchie T, Cole R, Kim H-M, de Vellis J , Noble EP (1987): Inositol phospholipid hydrolysis in cultured astrocytes and oligodendrocytes. Life Sci 41:31-39. Saito N, Kikkawa U , Nishizuka Y , Yanaka C (1988): Distribution of protein kinase C-like immunoreactive neurons in rat brain. J Neurosci 8:369-382. Saito N , Kose A, Ito H, Hosoda K, Mori M, Hirata M, Ogita K, Kikkawa U, Ono Y, Igarashi K , Nishizuka Y, Tanaka C ( I 989): lmmunocytochemical localization of PI1 subspecies of protein kinase C in rat brain. Proc Natl Acad Sci USA 86: 3409-341 3. Shimosawa S, Hachiya T, Hagiwara M, Usuda N, Sugita K, Hidaka H (1990): Type-specific expression of protein kinase C isozymes in CNS tumor cells. Neurosci Lett 108:11-16. Sihag RK, Jeng AY, Nixon RA (1988): Phosphorylation of neurofilament proteins by protein kinase C. FEBS Lett 233:181-185. Skoff RO, Price LD, Stocks A (1976) Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. J Comp Neurol 169:3 13-334. Sposi NM, Bottero L, Cossu G, Russo G , Testa U, Peschle C (1989): Expression of protein kinase C genes during otogenic development of the central nervous system. Mol Cell Biol 9:22842288. Stichel CC (1988): Ontogenetie changes in the level and subcellular
Protein Kinase C Isozymes in Optic Nerve distribution of protein kinase C in cat visual cortex. Int J Dev Neurosci 6:341-349. Vartanian T, Szuchet S , Dawson G, Campagnoni AT (1986): Oligodendrocyte adhesion activates protein kinase C-mediated phosphorylation of myelin basic protein. Science 234: 1395-1398. Yong VW, Sekiguchi S , Kim MW, Kim SU (1988): Phorbol ester enhances morphological differentiation of oligodendrocytes in culture. J Neurosci Res 19:187-194. Yoshida Y, Huang FL, Nakabayashi H, Huang K-P (1988): Tissue
389
distribution and developmental expression of protein kinase C isozymes. J Biol Chem 263:9868-9873. Young 111 SW (1988): Expression of three (and a putative four) protein kinase C genes in brains of rat and rabbit. J Chem Neuroanat 1:177-1 94. Young 111 SW (1989): Levels of transcripts encoding a member of the protein kinase C family in the paraventricular and supraoptic nuclei are increased by hyperosmolarity . J Neuroendocrinol 1:79-82.
Distribution of Protein Kinase C Isozymes in Rat Optic Nerves S. Komoly, Y. Liu, H. deF. Webster, and K.-F. J. Chan Laboratory of Experimental Neuropathology , National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland
’
Light (LM) and electron (EM) microscopic immuno- ubiquitous in eukaryotes. In mammals, this enzyme is cytochemical methods were used to study the distri- found in high concentrations in the central nervous sysbution of protein kinase C (PKC) isozymes in adult tem. Protein kinase C-mediated protein phosphorylation rat optic nerves. In cryostat and vibratome sections has been implicated in the regulation of a number of examined by LM, type I1 (p) isozyme was localized biological events (for review, see Nishizuka, 1986; Kikalmost exclusively in the axons. In the EM, immu- kawa and Nishizuka, 1986; Kaczmarek, 1987; Huang, noreaction products were found to associate with mi- 1989). In the nervous system, protein kinase C may play crotubules and neurofilaments. The inner surface of important roles in neurotransmitter release, neuritogeneaxonal membranes were occasionally stained. Analy- sis, and memory and learning (Kaczmarek, 1987; Bursis of PKC isozyme composition of the optic nerves by goyne, 1989; Chiarugi et al., 1989). Seven isoforms of using immunoblot techniques revealed that type I1 protein kinase C (a,PI, PII, y, 6, E, 5 ) have thus far (p) isozyme accounted for approximately 80% of the been identified by genetic approaches (Coussens et al., total immunoreactivity. By contrast, type I11 (a) 1986; Nishizuka, 1988; Kikkawa et al., 1989). These isozyme, which accounted for the remaining 20% of isoforms are encoded by different genes with the excepPKC, was found mainly in the astrocytes. Astrocytic tions of PI and PII, which are derived from the same processes next to blood vessels and between myeli- mRNA transcript by alternative splicing (Nishizuka, nated axons were stained. In the EM, immunoreac- 1988). Protein kinase C purified from the brain contains tion products were found in the cytoplasm and along three major isozymes termed type I, type 11, and type 111. astroglial filaments. Segments of plasma membranes These subspecies are encoded by cDNA corresponding also were stained; but nuclei were unstained. Adult to y,PI + PII, and a;respectively. Together, they conglial cells were not stained by an antibody to type I1 stitute over 90% of the total protein kinase C activity in (p) isozyme except for the occurrence of a few punc- the brain (Huang and Huang, 1986). tate cytoplasmic densities in occasional astrocytes. The exact physiological significance of the various Very faint or no immunostaining was observed in sec- PKC isozymes is still unclear. Biochemical studies retions treated with a monoclonal antibody to type I ( y ) vealed that different isozymes have slightly different isozyme. Immunoblot analyses also did not reveal this substrate specificities and requirements of activators subspecies. The absence of type I (y)isozyme in optic (Kikkawa et al., 1989). Several PKC isozymes also were nerves is not due to a down-regulation of the enzyme found to have different subcellular distributions and exduring development. In developing (5 and 11 day) pression during development of the brain. Theoretically, rats, immunoreactivity of protein kinase C was very each isozyme may have distinct biological functions. faint or absent. After 15 days, reaction products of This may be due to the presence of specific protein or both type I11 (a)and type I1 (p) isozymes were found enzyme substrates in different subcellular locations. Althroughout the nerve. These findings suggest that type I1 (p) isozyme may be involved in axonal trans- Received September 28, 1990; revised December 13, 1990; accepted port whereas type I11 (a)isozyme may play a role in December 13, 1990. some astrocyte functions in mature optic nerves. Key words: protein kinase C, isozymes, optic nerve, axons, astrocytes, glial filaments, microtubules INTRODUCTION Protein kinase C (PKC) is a calcium-activated and phospholipid-dependent serinefthreonine protein kinase Published 1991 by Wiley-Liss, Inc.
Address reprint requests to Dr. S . Komoly, National Institutes of Health, Building 36, Room 4A-29, Bethesda, MD 20892.
Dr. K.-F.J. Chan is now at the Department of Biochemistry, University of Hong Kong, Hong Kong. Abbreviations used: PKC, protein kinase C; PBS, phosphate-buffered saline; HEPES N-2-hydroxyethylpiperazine-N’-ethanesulfonicacid; EGTA, [ethylenebis(oxyethylenenitrilo)tetraacetic acid; SDS, sodium dodecyl sulfate.
380
Komoly et al.
ternatively, the presence of a particular PKC isozyme in specific cell types may come as a result of unique cellular regulation of the allosteric activators and inhibitors. So far, most of the brain regions including cerebral cortex, striatum, thalamus, spinal cord, hippocampus, septum, and mesencephalon were found to contain two or more isozymes (Mochly-Rosen et al., 1987; Huang et al., 1989; Saito et al., 1988; Yoshida et al., 1988). Expression of a single subspecies was observed only in a few cell types, e.g., Purkinje cells and olfactory bulb mitral cells (Hidaka et al., 1988; Young, 1988). In this report, we show that the distribution of PKC isozymes in adult rat optic nerves also is cell-specific. Type I1 (6) isozyme was found predominantly in axons whereas type I11 ( a ) isozyme was localized mainly in astrocytes. No significant immunoreactivity was observed for type I (y) isozyme. These results suggest that the optic nerve may be a good anatomical system for further studies of the functions and regulations, as well as the mechanisms of gene expression, of protein kinase C isozymes.
containing 4% paraformaldehyde, 15% saturated picric acid, and 0.1 M PBS. The optic nerves were dissected out and postfixed overnight in the same fixative. Cryoprotection of the samples was carried out in a solution containing 0.9% (w/v) sodium chloride, 20% (w/v) sucrose, and 3% (v/v) polyethylene glycol. After 24 hr, the optic nerves were embedded in Tissue-Tek and frozen in dry ice-cold isopenthane. Serial sections of 6-12 pm were cut in a cryostat (2800 Frigocut, Reichert-Jung) and mounted on gelatin-covered slides. The samples were kept at 4°C until used. The sections were washed in PBS containing 0.5% of Triton X-100 for 3 hr to remove the cryoprotection reagents and to permeabilize the cell membranes. Nonspecific protein binding sites were blocked by incubating the specimen with horse serum. Immunoreaction with different monoclonal antibodies to protein kinase C was carried out at 4°C for 16-24 hr. After washing, the specimens were incubated with biotinylated anti-mouse TgG (1:300 dilution) for 60 min at room temperature. Color reaction was developed by using ABC Elite horseradish peroxidase kit with 0.05% DAB and 0.005% H,O, in PBS. Two sets of control experiments were performed. These included ( 1 ) substitution of monoclonal anti-PKC antibodies with normal mouse IgG; and (2) preincubation of the primary antibodies with purified protein kinase C before immunostaining.
EXPERIMENTAL PROCEDURES Materials Monoclonal antibodies to protein kinase C isozymes type I (y), type I1 (p), and type 111 ( a )were obtained from Seikagaku Kogyo Co., Ltd. The antibodies were reconstituted to a concentration of 0.1 mgiml and stored in aliquots at -20°C. A 1:lOO dilution of these antibodies was used in both immunostaining and Electron Microscopic Immunocytochemistry Four adult (180 g) Wistar rats were perfused as immunoblotting experiments. Affinity purified antimouse biotinylated horse IgG, normal horse serum, nor- described above except that the fixative also contained mal mouse IgG, and the standard ABC Elite horseradish 0.5% glutaraldehyde. The optic nerves were removed peroxidase kit were purchased from Vector Laboratories, with the brain; they were postfixed overnight, and transInc. The sera were diluted in 0.1 M phosphate-buffered ferred into PBS. Sections of 30 pm were cut with a BSK saline (PBS) containing 1% bovine serum albumin and Slicer. Vibratome sections were immunostained as de0.1 % sodium azide. The embedding medium for frozen scribed for LM but without prior detergent treatment. tissue specimens, Tissue-Tek, was from Miles Labora- After color development with DAB, the sections were tories. Nitrocellulose membranes were from Schleicher postfixed with 0.5-1 % osmium tetroxide in the presence and Schuell, Inc. Casein was from Sigma Chemical Co. or absence of 0.6% potassium fenicyanide and 5% su'251-labeledprotein G was a product of Amersham Corp. crose for 60 min and embedded flat in epon. Thin sections Paraformaldehyde, glutaraldehyde, uranyl acetate 3,3- were cut with a Reichert-Jung ultramicrotome and diaminobenzidine tetrahydrochloride (DAB), and Poly/ mounted on Formvar-coated single slot grids. Every secBed 812 were purchased from Polysciences, Inc. Protein ond one of the serial sections was stained with uranyl kinase C was purified to apparent homogeneity from the acetate for 12 min and then with lead citrate for 30 sec in particulate fractions of rat brains essentially according to a LKB 2168 ultrastainer. The remaining sections were not the previously described procedures (Huang et al., stained with heavy metals. The specimens were examined in a Philips EM 410 electron microscope at 80 kV. 1986). Light Microscopic Immunocytochemistry Adult and developing ( 5 , 1 1, 15, 18, and 27 days) Wistar rats were anesthetized and perfused through the heart with physiological saline followed by a fixative
Immunoblotting of Protein Kinase C Isozymes Five adult Wistar rats were anesthetized and killed by decapitation. The optic nerves were removed from the brain and immediately homogenized in 20 mM HEPES,
Protein Kinase C Isozyrnes in Optic Nerve
pH 7.4, 0.5mM EGTA, 2 mM EDTA, 20 mM NaCl, and 0.5 mM dithiothreitol (buffer A). All procedures were carried out at 0-4°C. The homogenates were centrifuged at 15,OOOg for 30 min. The resulting supernatant fluids were saved as soluble fractions. The pellets were resuspended in buffer A and saved as particulate fractions. The proteins (30 pg) were separated by using SDSpolyacrylamide gel (10%) electrophoresis and electroblotted onto nitrocellulose membranes. The blotting buffer contained 30 mM Tris-HC1, 200 mM glycine, pH 8.3, and 20% (v/v) methanol. After washing the membranes with 50 mM Tris-HC1, pH 7.4, and 150 mM NaCl (TBS) containing 0.05% Tween-20, nonspecific binding sites were blocked by overnight incubation with 2% (wl v) casein. Immunoreactions of the monoclonal antibodies (1 pglml) with PKC isozymes types I (y), I1 (p), and I11 ( a )were carried out at 25°C for 2 hr. Reaction products were detected by incubating the membranes with I25 I-labeled protein G (containing 1 X lo7 cpm) in TBS for 30 min at 25°C and subsequent autoradiography. Immunoreactivity was quantitated by both densitometric scanning of the corresponding PKC bands with a Shimadzu CS-9000 flying-spot densitometer and gamma counting of the excised PKC-containing nitrocellulose membranes using a LKB-Wallac 1272 Clini-Gamma counter.
RESULTS Distribution of PKC Isozyrnes in Adult Rat Optic Nerves To better understand the function and regulation of different protein kinase C isozymes in the brain, we searched for a well-defined anatomical system in which separate PKC subspecies are localized in different cell types. The optic nerves of adult rats were found to possess these characteristics. Light microscopic immunocytochemical studies of the cryostat sections revealed an almost exclusive localization of PKC type 111 ( a ) isozyme in the astrocytes (Fig. 1A). Immunoreaction products of a monoclonal antibody to this isozyme were found both in the cytoplasm and in the astrocytic processes around blood vessels and between meylinated axons (Fig. IA,B). The nuclei were unstained. By contrast, immunoreactivity of PKC type I1 (p) isozyme was confined mainly to axons (Fig. lC,D). Most, if not all, of the axons were immunoreactive. Adult glial cells were not stained by the monoclonal antibody to this type I1 (p) subspecies except for the occurrence of a few punctate cytoplasmic densities in occasional astrocytes. Very faint or no immunoreactivity was found in cryostat sections treated with an antibody to PKC type I (y) isozyme (Fig. lE,F).The
381
immunostaining was similar to those obtained by using a biotinylated mouse IgG or after preincubation of the monoclonal antibodies with purified rat brain protein kinase C. Immunostaining of the three PKC isozymes in vibratome sections was similar to those observed in frozen sections. However, no immunoreactivity was found in paraffin-embedded sections. Prior delipidation of vibratome and frozen sections with ethanol also abolished the immunolabeling of protein kinase C by monoclonal antibodies (results not shown). In adult rat retina, immunostaining of type I1 (p) isozyme was localized mainly in ganglion cells whereas type I11 ( a )subspecies was found in Muller cells (results not shown). There were no immunoreaction products to type I (y) isozyme. These findings are in agreement with the immunoblotting data (Yosida et al., 1988) and support our observations on the distribution of protein kinase C in the optic nerves.
Immunoblot Analysis of Protein Kinase C Isozymes The composition of PKC isozymes in adult rat optic nerves were quantitated by using immunoblot analyses. The results are summarized in Table I. Over 80% of the total PKC immunoreactivity was due to type I1 (p) isozyme. Subcellular distribution studies revealed that approximately two-thirds of this PKC subspecies were in the soluble (cytosolic) fractions. The immunoreaction of the monoclonal antibody with type I1 (p) isozyme was specific. Only one radioactive band with an apparent M , of 80,000 was observed in the soluble and the particulate fractions of the optic nerves (Fig. 2). These findings suggest that protein kinase C, in particular, the type I1 (p) isozyme, may play an important role in axonal functions. Type 111 ( a )isozyme accounts for the remaining 20% of PKC immunoreactivity in the optic nerves (Table I). Nearly 70% of this isozyme was found in the cytosolic fractions. By contrast, no significant immunoreactivity of type I (y) isozyme was observed. The absence of immunoreaction of the monoclonal antibody to type I (y) subspecies was not due to its ineffectiveness. This antibody could recognize type I (y) isozyme in purified protein kinase C preparations (data not shown). Electron Microscopic Localization of PKC Isozymes To learn more about the putative functions of protein kinase C isozymes in optic nerves, the subcellular localization of both type I1 (p) and type 111 (a)subspecies were investigated by using electron microscopic immunocytochemical methods. As shown in Figure 3 , immunoreaction products of type I1 (p) isozyme were associated with microtubules in axons. The punctate staining in the cytoplasm might be due to the labeling of neurofilaments (Fig. 3A-C). Occasionally, immunoreaction prod-
Fig. 1. Light microscopic immunocytochemical localization of PKC isozymes in rat optic nerves. A,B: Irnrnunostaining of astrocytes with type 111 (a)monoclonal antibody. C,D: Irnrnunostaining of axons with type I1 (p) monoclonal antibody. E,F: Immunostaining of optic nerve with type I (y) monoclonal antibody. Serial cryostat sections are 6 p,m in thickness. Arrowheads denote the same blood vessels. Magnifications: A, C, E, X 120; B, D, F, ~ 2 4 0 .
Protein Kinase C Isozymes in Optic Nerve TABLE I. Distribution of Protein Kinase C Isozymes in Adult Rat Optic Nerves* Protein kinase C isozymes (%)
I (Y) Total Subcellular Soluble Particulate
11 (PI
I11 (ff)
-
80.8
?
3.3
19.2 ? 3.5
-
67.2 32.8
? ?
7.1 7.2
69.2 30.8
-
& ?
5.8 6.4
*The composition of protein kinase C isozymes in adult rat optic nerves was determined by immunoblot assays as described under Experimental Procedures. lmmunoreactivity of monoclonal antibodies to PKC isozymes type I (y), type I1 (p), and type 111 (a)was determined by using '251-1abeled protein G overlay and quantitated by densitometric scanning of the corresponding radioactive PKC bands.
383
products were observed during the first two postnatal weeks (results not shown). Gradually, dense immunostaining was found similar to the patterns shown in Figure 1C and D. There was no positive staining with the monoclonal antibody to PKC type I (y) isozyme, regardless of age (data not shown). This observation suggests that the failure to detect type I (y) isozyme in adult rat optic nerves is not due to a down-regulation of this subspecies during early development.
DISCUSSION
Protein kinase C has been implicated in the regulation of a number of neural functions (Nishizuka, 1986; Coussen et al., 1986; Kaczmarek, 1987). This protein ucts were found to associate with the internal surface of phosphorylation system also may play important roles in the axolemma and on the outer membranes of the mito- several pathological conditions such as ischemic brain chondria. No diffuse staining was observed in the axo- injury (Louis et al., 1989; Olah et al., 1990) and brain plasm. It should be noted that the intensity of the immu- edema formation (Joo et al., 1989). The exact functional nostaining could vary from axon to axon, and sometimes roles of different protein kinase C isozymes in these and other processes still remain unclear. One problem is that even within the same axon. Immunoreaction products of PKC type 111 (a) many cell types in many regions of the brain express isozyme were localized almost exclusively in astrocytes more than one subspecies. Our findings of an almost (Fig. 4). The astrocytes were identified by their ultra- exclusive distribution of type I1 (p) isozyme in the axons structural features (Peters et al., 1991) as well as by their and type 111 (a)isozyme in the astrocytes of adult rat immunoreactivity with an antibody to glial fibrillary optic nerves (Fig. 1) may provide a unique opportunity acidic protein (GFAP) (results not shown). Within the for further investigation of the function and regulation, astrocytes, diffuse reaction products were observed in and perhaps the mechanisms of gene expression, of these the cytoplasm (Fig. 4A). Glial fibers were stained (Figs. two isozymes. 4B ,C). Mitochondria were sometimes labeled. Strong In the EM, immunoreaction products of type 11 (p) immunoreactivity also was found to associate with cell isozyme were found to associate with microtubules and membranes (Figs. 4C ,D), particularly at contact regions neurofilaments (Fig. 3 ) . This specific localization sugbetween perivascular astroglial processes (Figs. 4D,E). gests that type I1 (p) isozyme may play a role in reguThe nuclei were unstained. A few endothelial cells in the lating cytoskeletal structures and axonal transport prooptic nerve were occasionally found to contain PKC type cesses in axons. Indeed, microtubule-associated proteins 111 ( a ) isozyme immunoreactivity. In addition, faint re- such as MAP-2 and tau, and neurofilament proteins are action products were observed at tight junctions (results putative substrates for protein kinase C (Kaczmarek, not shown). 1987; Nixon et al., 1987; Sihag et al., 1988; Pestronk et No positive immunostaining of PKC type I (y) al., 1990). These proteins are phosphorylated in vivo. isozyme was seen in the EM. Immunostaining of axons with monoclonal antibodies to pII and y subspecies also have been reported in the cerExpression of PKC Isozymes During Development ebellum (Kose et al., 1988; Kikkawa et al., 1989; Saito of the Optic Nerves et al., 1989). The functional significance of the other The distribution of the various PKC isozymes in cytosolic type I1 (p) isozyme in axons (Fig. 2 and Table the optic nerves of developing rats ( 5 , 1 1, 15, 18, and 27 I) is less clear. In some cells, PI1 subspecies may play a days) was investigated by using LM immunocytochem- role in protein processing (Kikkawa et al., 1989). istry. From 5 to 15 days, reaction products of type I11 ( a ) Protein kinase C type I11 ( a ) isozyme is more subspecies were very faint or absent (Fig. 5A-C). After widely distributed in the astrocytes of optic nerves. Im15 days, immunopositive astrocytic-like cells were ob- munoreactivity was observed in the cytoplasm as well as served (Fig. 5D-F). Both the intensity and the number of in association with glial filaments and cell membranes stained cells were found to increase with age. The de- (Figs. 1 and 4). Furthermore, most, and perhaps all, of velopmental expression of PKC type I1 (p) isozyme in the astrocytes in the optic nerve were immunostained. axons of rat optic nerves was similar to the type I11 (a) These results are in agreement with the previous localsubspecies. Only weak and diffuse immunoreaction ization of PKC type I11 ( a )isozyme in cultured astroglial
384
Komoly et al.
CONTROL TYPE II
S
c3 I
0 F
x
P
' s
P '
's
P'
98 67 43
31
IMMUNOBLOT Fig. 2. Immunoblotting of PKC type I1 (p) isozyme in rat optic nerves. Proteins (30 pg) in both soluble (S) and particulate (P) fractions of adult rat optic nerves were separated by using SDS-polyacrylamide gel (10%) electrophoresis. The Coomassie blue protein staining patterns are shown in the left
panel. Duplicate gels were electroblotted onto nitrocellulose membranes. Immunoreactions of a mouse IgG (control) and a monoclonal antibody to type I1 (p) isozyme were determined by '251-labeled protein G overlay and subsequent autoradiography (right panel).
cell lines (Shimosawa et a]., 1990). Thus, type 111 (a) isozyme may have some important regulatory roles in cellular functions that are common to different subpopulations of astrocytes. In cultured astrocytes, treatment with phorbol esters can lead to morphological transformation concomitant with an increase in intermediate filament protein phosphorylation and a translocation of protein kinase C from the cytosolic to the particulate fraction (Pollenz and McCarthy, 1985; Mobley et al., 1986; Neary et al., 1986, 1988; Harrison and Mobley, 1989; Bhat, 1989). No immunoreaction products of PKC type 111 (a) isozyme were found during early development (5 and 11 days) of the optic nerves (Fig. 5). After 15 days, there was a gradual increase in both the number and the intensity of positive astrocytic-like cells. These results support the earlier findings that fully mature glial cells were observed in electron micrographs only after 14 days of development (Skoff et al., 1976). The increase in protein kinase C during development is not unique to the optic nerves. Other biochemical, immunological, and gene expression studies also revealed a 10- to 30-fold increase in PKC during maturation of the brain (Girard et al., 1986, 1988; Stichel 1988; Noguchi et at., 1988; Yoshida et al., 1988; Sposi et al., 1989).
Protein kinase C type I (y) isozyme has been shown to exist solely in the brain and spinal cord (Kikkawa, 1989). But no immunostaining of this subspecies was observed in the optic nerves. The absence of immunoreaction products is not due to a malfunctioning of the monoclonal antibody, because positive immunostaining has been observed in the cerebellum (results not shown). In addition, the antibody can recognize type I isozyme in purified protein kinase C preparations. It remains to be established whether the absence of type I (y) isozyme in optic nerves is a result of transcriptional or translational control. Protein kinase C immunoreactivity also was not found in oligodendrocytes and meylin sheaths. These results are in agreement with several other immunocytochemical and in situ hybridization studies (MochlyRosen et al., 1987; Hidaka et al., 1988; Huang et al., 1988, 1989; Kose et al., 1988; Saito et al., 1988, 1989; Young, 1988, 1989). Yet, protein kinase C has been shown to modulate both oligodendrocyte differentiation and myelin basic protein phosphorylation in vitro (Vartanian et al., 1986; Chan, 1987, 1989; Ritchie et al., 1987; Yong et al., 1988). The failure to detect protein kinase C in optic nerve oligodendrocytes or myelin sheaths by immunocytochemical methods may be due to a different
Protein Kinase C Isozymes in Optic Nerve
385
Fig. 3. Electron microscopic localization of PKC type I1 (p) isozyme in axons. A: Axons stained heavily (ax) and less heavily (a) with a monoclonal antibody to PKC type I1 (p) isozyme. Arrowheads indicate microtubules. The specimen was counterstained with uranyl acetate and lead citrate. X 60,000. B: Immunostained axon without heavy metal treat-
ment. Arrowhead points to an immunostained microtubule. X60,OOO. C: Longitudinal section of an axon showing clustering of immunostaining (arrow). Oblique sections of two unstained axons are represented by a. Contrast was enhanced by treatment with 0.6% potassium ferricyanide and 1 % osmium tetroxide. X 20,000.
orientation or conformation of the enzyme in CNS tissue such that the epitope(s) are not accessible. Protein kinase C type I1 (p) isozyme could be detected in purified rat and guinea pig brain myelin preparations by using immunoblotting techniques (K.-F.J. Chan and Y. Liu, unpublished results). Of course, it is also possible that the major protein kinase C isozyme in both oligodendrocytes and myelin sheaths may correspond to a subspecies other than types I, 11, and 111. The differential distribution of protein kinase C isozymes in optic nerves suggests that each isozyme may
have different functional roles in processing and modulating physiological cellular responses of the visual system. The subspecies in different cell types also may respond to different regulatory mechanisms. For example, purified type 111 (a)isozyme is responsive to Ca2+ and arachidonic acids (Kikkawa, 1989). It is therefore possible that the astrocytes in optic nerves may have a signal transduction pathway that involves the release of these activators. Further investigation of the optic nerves, a less complicated anatomical system than the whole CNS, may provide more information on both gene expres-
Fig. 4. Electron microscopic localization of PKC type 111 (a) isozyme in astrocytes. A: Immunostaining of an astrocytic process (As) is adjacent to an endothelial cell (En) of a blood vessel. x 52,000. B: Immunostaining of filamentous structures in an astrocyte (As). Adjacent pericyte processes (P), an endothelial cell (En), and an astrocytic process (asterisk) were unstained. x 41,000. C: Immunostained (arrowheads) and un-
stained (open arrows) plasma membranes of two adjacent astrocytic processes. Filamentous immunoreaction products are in the cytoplasm. En = endothelial cell. X 31,000. D: Clustering of reaction products at the cytoplasmic sides of two adjacent plasma membranes (arrows). En = endothelial cell. X 41,000. E: Higher magnification ( X 82,000) of D.
Fig. 5 . PKC type I11 (a)immunoreactivity during development of rat optic nerves. Light microscopic immunocytochemical localization of PKC type 111(a)isozyme in the optic nerves of developing (A: 5 days; B: 11 days; C: 15 days; D: 27 days) and adult (E,F) rats. A stained mature astrocyte is shown by an arrow. Magnifications: A-E, X 120; F, X 240.
388
Komoly et al.
sion and activation of different protein kinase C isozymes.
ACKNOWLEDGMENTS We wish to thank Mrs. Yoong Chang for her excellent technical assistance in the EM studies.
REFERENCES Bhat NR (1989): Role of protein kinase C in glial cell proliferation. J Neurosci Res 22:20-27. Burgoyne RD (1989): A role of membrane-inserted protein kinase C in cellular memory? Trends Biol Sci 14:87-88. Chan, K-FJ (1987) Ganglioside-modulated protein phosphorylation in myelin. J Biol Chem 262:2415-2422. Chan, K-FJ (1989) Effects of gangliosides on protein phosphorylation in rat brain myelin. Neurosci Res Commun 5:95-104. Chiarugi VP, Ruggiero M, Corradetti R (1989): Oncogenes, protein kinase C, neuronal differentiation and memory. Neurochem Int 1411-9. Coussens L, Parker PJ, Rhee L, Yang-Feng TL, Chen E, Waterfield D, Francke U, Ullrich A (1986): Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science 233:859-866. Girard PR, Mazzei GJ, Kuo JF (1986): Immunological quantitation of phospholipidiCa’ -dependent protein kinase and its fragments. J Biol Chem 261:370-375. Girard PR, Wood JG, Freshci JE, Kuo JF (1988): Immunocytochemical localization of protein kinase C in developing brain tissue and in primary neuronal cultures. Dev Biol 126:98-107. Harrison BC, Mobley PL (1989): Protein phosphorylation in astrocytes mediated by protein kinase C: Comparison with phosphorylation by cyclic AMP-dependent protein kinase. J Neurochem 53: 1245-1 25 I . Hidaka H, Tanaka T, Onada K, Hagiwara M, Watanabe M, Ohta H, Ito Y , Tsurudome M, Yoshida T (1988): Cell type specific expression of protein kinase C isozymes in the rabbit cerebellum. J Biol Chem 263:4523-4526. Huang, K-P (1989): The mechanism of protein kinase C activation. Trends Neurosci 12:424-432. Huang K-P, Huang FL ( 1986): lmmunochemical characterization of rat brain protein kinase C. J Biol Chem 261:14781-14787. Huang K-P, Chan K-FJ, Singh TJ, Nakabayashi H , Huang FL (1986): Autophosphorylation of rat brain Ca2+-activatedand phospholipid dependent protein kinase. J Biol Chem 261:1213412140. Huang K-P, Huang FL, Nakabayashi H, Yoshida Y (1989): Expression and function of protein kinase C isozymes. Acta Endocrinol 121 :307-3 16. Joo F, Tosaki A, Olah 2, Koltai M (1989): Inhibition by H-7 of protein kinase C prevents formation of brain edema in SpragueDawley CFY rats. Brain Res 490:141-143. Kaczmarek, L (1987) The role of protei kinase C: in the regulation of ion channels and neurotransmitter release. Trends Neurosci 10: 30-34. Kikkawa U , Nishizuka Y (1986): Protein kinase C. The Enzymes 17:167-189. Kikkawa U, Kishimoto A, Nishizuka Y (1989): The protein kinase C family: Heterogeneity and its implications. Annu Rev Biochem 58131-44. Kose A, Saito N , Ito H, Hiroshi I, Kikkawa U, Nishizuka Y, Tanaka
C (1988): Electron microscopic localization of type I protein kinase C in rat Purkinje cells. J Neuroscience 8:4262-4268. Louis JC, Magal E, Yavin E (1988): Protein kinase C alterations in the fetal rat brain after global ischemia. J Biol Chem 36:1928219285. Mobley PL, Scott SL, CNZ EG (1986): Protein kinase C in astrocytes: A determinant of cell morphology. Brain Res 398:366-369. Mochly-Rosen D, Basbaum AI, Koshlan DE (1987): Distinct cellular and regional localization of immunoreactive protein kinase C in rat brain. Proc Natl Acad Sci USA 84:4660-4664. Neary JT, Norenberg LOB, Norenberg MD (1986): Calciumactivated, phospholipid-dependent protein kinase and protein substrates in primary cultures of astrocytes. Brain Res 385: 420-424. Neary JT, Norenberg LOB, Norenberg MD (1988): Protein kinase C in primary astrocyte cultures: cytoplasmic localization and translocation by phorbol ester. J Neurochem 50:1179-1184. Nishizuka Y (1986): Studies and perspectives of protein kinase C. Science 233:305-3 12. Nishizuka Y (1988): The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature (London) 334:661-664. Nixon RA, Lewis SE, Martotta CA (1987): Posttranslational modification of neurofilament proteins by phosphate during axoplasmic transport in retinal ganglion cell neurons. J Neurosci 7: 1145-1 158. Noguchi A, DeGuire J , Zanaboni P (1988): Protein kinase C in the developing rat liver, heart and brain. Dev Pharmaol Ther 11: 31-43. Olah 2 , Ikeda J , Anderson WB, Joo F (1990): Altered protein kinase C activity in different subfields of hippocampus following cerebral ischemia. Neurochem Res 15:515-518. Pestronk A, Watson DF, Yuan CM (1990): Neurofilament phosphorylation in peripheral nerve: Changes with axonal length and growth state. J Neurochem 54:977-982. Peters A, Palay SL, Webster HdeF (1991): “The Fine Structure of the Nervous System. ” New York: Oxford University Press. Pollenz R, McCarthy KD (1985): Regulation of intermediate filament protein phosphorykation in astroglia. Trans Amer Soc Neurochem 16:188. Ritchie T, Cole R, Kim H-M, de Vellis J , Noble EP (1987): Inositol phospholipid hydrolysis in cultured astrocytes and oligodendrocytes. Life Sci 41:31-39. Saito N, Kikkawa U , Nishizuka Y , Yanaka C (1988): Distribution of protein kinase C-like immunoreactive neurons in rat brain. J Neurosci 8:369-382. Saito N , Kose A, Ito H, Hosoda K, Mori M, Hirata M, Ogita K, Kikkawa U, Ono Y, Igarashi K , Nishizuka Y, Tanaka C ( I 989): lmmunocytochemical localization of PI1 subspecies of protein kinase C in rat brain. Proc Natl Acad Sci USA 86: 3409-341 3. Shimosawa S, Hachiya T, Hagiwara M, Usuda N, Sugita K, Hidaka H (1990): Type-specific expression of protein kinase C isozymes in CNS tumor cells. Neurosci Lett 108:11-16. Sihag RK, Jeng AY, Nixon RA (1988): Phosphorylation of neurofilament proteins by protein kinase C. FEBS Lett 233:181-185. Skoff RO, Price LD, Stocks A (1976) Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. J Comp Neurol 169:3 13-334. Sposi NM, Bottero L, Cossu G, Russo G , Testa U, Peschle C (1989): Expression of protein kinase C genes during otogenic development of the central nervous system. Mol Cell Biol 9:22842288. Stichel CC (1988): Ontogenetie changes in the level and subcellular
Protein Kinase C Isozymes in Optic Nerve distribution of protein kinase C in cat visual cortex. Int J Dev Neurosci 6:341-349. Vartanian T, Szuchet S , Dawson G, Campagnoni AT (1986): Oligodendrocyte adhesion activates protein kinase C-mediated phosphorylation of myelin basic protein. Science 234: 1395-1398. Yong VW, Sekiguchi S , Kim MW, Kim SU (1988): Phorbol ester enhances morphological differentiation of oligodendrocytes in culture. J Neurosci Res 19:187-194. Yoshida Y, Huang FL, Nakabayashi H, Huang K-P (1988): Tissue
389
distribution and developmental expression of protein kinase C isozymes. J Biol Chem 263:9868-9873. Young 111 SW (1988): Expression of three (and a putative four) protein kinase C genes in brains of rat and rabbit. J Chem Neuroanat 1:177-1 94. Young 111 SW (1989): Levels of transcripts encoding a member of the protein kinase C family in the paraventricular and supraoptic nuclei are increased by hyperosmolarity . J Neuroendocrinol 1:79-82.
