c-neu oncoprotein in developing rostral cerebral cortex: relationship to epidermal growth factor...

THE JOURNAL OF COMPARATIVE NEUROLOGY 372:189-203 (1996)

c-neu Oncoprotein in Developing Rostra1 Cerebral Cortex: Relationship to Epidermal Growth Factor Receptor

PETER E. KUHN AND MICHAEL W. MILLER Program in Cell and Developmental Biology, Rutgers University, Piscataway, New Jersey

08854-1059 (P.E.K., M.W.M.); Research Service, Veterans Affairs Medical Center, Iowa City, Iowa 52246-2208 (M.W.M.); Departments of Psychiatry and Pharmacology,

University of Iowa College of Medicine, Iowa City, Iowa 52242-1000 (M.W.M.)

ABSTRACT The c-neu oncoprotein, p185"-""", is a transmembrane tyrosine kinase that shares

structural similarities with the receptor for epidermal growth factor (EGFr). We used immunoblots, immunoprecipitation, and immunohistochemistry 1) to test the hypothesis that p185"-"'" and EGFr are coordinately expressed in central nervous system tissue and 2) to assess the spatiotemporal expression of both the c-neu oncoprotein and EGFr in the rostra1 cerebral cortex.

In nondenaturing gels, anti-c-neu antibody identified high molecular weight proteins (about 300-400 kDa) that were reduced by EDTA to a molecular weight of 180-200 kDa. Sodium dodecylsulfate polyacrylamide gel electrophoresis broke down this protein into an array of smaller peptides, which were expressed prenatally, transiently during the first three postnatal weeks, or in the adult. Perinatally, c-neu immunoreactivity was evident in subplate neurons, ascending processes of neurons in the cortical plate, and ventricular zone cells. During the second postnatal week, cells throughout cortex expressed somatodendritic immunostaining, but, in the adult, c-neu immunoreactivity was expressed only by pyramidal neurons in layer V and by glia in the white matter and ependyma.

EGFr-positive proteins behaved in the nondenaturing gels as did c-neu-positive oncoproteins, suggesting that both proteins naturally formed dimers. This contention was supported by the EGFr-or c-neu immunolabeling of tissue that was previously immunoprecipitated with anti-c-neu or anti-EGFr, respectively. The pattern of EGFr immunolabeling in the developing and mature cortex was virtually identical to that described for c-neu immunoreactivity.

Cortical neurons express the c-neu oncoprotein and EGFr, probably as heterodimers. The specific immunolabeling of layer V neurons in the adult cortex with anti-c-neu and anti-EGFr suggests that the p185"-""" ligand and EGF regulate the activity of corticofugal systems. The expression of different c-neu- and EGFr-positive peptides is developmentally defined and may be related to specific ontogenetic events.

Indexing terms: cell proliferation, corticofugal projections, neuronal migration, neuronal death,

D 1996 Wiley-Liss, Inc.

subplate

c-neu is a protooncogene that is up-regulated in actively proliferating cancer cells, such as breast tumors (e.g., Berger et al., 1988; De Potter et al., 1989a; Goldman et al., 1990; Stancovski et al., 1991; Bacus et al., 1992). The wild-type gene is expressed in the mouse fibroblast line DHFRIG8, and mutated forms are expressed by rat (B104) and mouse (B104-1-1) fibroblast lines (Schechter et al., 1984, 1985; Stern et al., 1986).

Among the proteins translated by c-neu is a 185 kDa protein, ~ 1 8 5 ' - " ~ ~ . This protein is a cell-surface receptor with extracellular, transmembranous, and intracellular

domains. The extracellular domain of p185"-""" contains six glycosylation sites (Bargmann et al., 1986); glycosylation changes the apparent molecular weight from 160 to 185 kDa (Huang et al., 1990). The ligand-binding region is also part of the extracellular domain. The transmembranous domain is responsible for the formation of dimers and for the development of malignancy (Cao et al., 1992, 1993;

Accepted February 28,1996. Address reprint requests to Michael W. Miller, Department of Psychiatry-

M.E.B., University of Iowa College of Medicine, Iowa City, IA 52242-1000.

o 1996 WILEY-LISS, INC.

190 P.E. KUHN AND M.W. MILLER

Lehtola et al., 1992; Lofts et al., 1993). In fact, the isolation of c-neu was possible, because a point mutation in the transmembranous portion induces carcinogenic growth and, coincidentally, the formation of a more stable dimer (Weiner et al., 1989; Huang et al., 1990). The intracellular domain contains the kinase domain and three tyrosines that are involved in autophosphorylation (Bargmann et al., 1986). The phosphorylated tyrosines are necessary for lowering the sensitivity of the receptor to the ligand and for the down-regulation of p185"-"'".

The oncoprotein has many structural similarities to the receptor for epidermal growth factor (EGFr). EGFr, as it is expressed in human carcinoma cells (A431), is a transmembrane protein, and its size changes from 140 to 175 kDa with the addition of carbohydrate moieties (Bargmann et al., 1986; Yamamoto et al., 1986). Like p185cneu, the extracellular domain of EGFr has six glycosylation sites and is rich in cysteine residues. The intracellular segment of EGFr contains a tyrosine kinase region, which has an 85% homology with ~185"-~"" (Yamamoto et al., 1986). Note that ~ 1 8 5 ~ - " ' ~ and EGFr are coexpressed by dorsal root ganglion neurons (Kokai et al., 1987; Werner et al., 1988).

The method of action of p185"-"'" is similar to EGFr. ~ 1 8 5 " - ' ~ ~ " phosphorylates key signal transduction peptides (Bjorge et al., 1990; Peles et al., 1992a, 1993); however, the role of this phosphorylation during development remains unresolved (Fazioli et al., 1991). Furthermore, the two receptors can be highly interactive. Both ~185"-"~" and EGFr have three tyrosines that can autophosphorylate, and the two proteins can phosphorylate one another (Cochet et al., 1988; Qian et al., 1992). In the presence of EGF, EGFr phosphorylates p185"-"'" in NR6 Swiss mouse 3T3 fibroblasts (Connelly and Stern, 1990). Both proteins can form homodimers or heterodimers in A431, B104-1-1, and DHFR/G8 cells as well as the human breast carcinoma line SKBR-3 (Cochet et al., 1988; Weiner et al., 1989; Goldman et al., 1990). The heterodimer exhibits higher kinase activity than the homo- or monodimer.

Despite the similarities between ~ 1 8 5 ~ - ~ ~ " and EGFr, there are notable differences between the receptors. EGFr has a molecular weight of 175 kDa, whereas ~ 1 8 5 " - " ~ ~ has a molecular weight of 185 kDa. Although the amino acid sequences indicate that the receptors are related, they are not identical. For example, the extracellular domain of ~185~-" '" shares only a 43% identity with the similar region of EGFr, and the sequence of 60 amino acids adjacent to the kinase region shows only a 55% identity with EGFr (Barg- mann et al., 1986). Moreover, in humans, e-neu and the gene for EGFr are located on different chromosomes (Schechter et al., 19851, and the two receptors respond maximally to different ligands (Yarden and Weinberg, 1989; Davis et al., 1991; Dobashi et al., 1991; Yarden and Peles, 1991; Huang and Huang, 1992; Peles et al., 199213; Wen et al., 1992).

~185"-" '~ plays a role in sundry developmental events, including cell proliferation, migration, and differentiation. In proliferating populations, p185"-"'" initiates the cell cycle or maintains cells within the a cycling population. Neu- activating factor and neu differentiation factor stimulate DNA synthesis in cultured breast cells expressing oncogenic or protooncogenic forms of c-neu (Dobashi et al., 1991; Marte et al., 1994), and ~185'-"~" ligands foster the proliferation of Schwann cells (Levi et al., 1995).

~185"-"~" is involved in cell adhesion, hence, it may play a critical role during cell migration. ~ 1 8 5 ~ - " ~ ~ is localized in

microvilli and motile cell membrane protrusions of human breast carcinoma cells (De Potter and Quatacker, 1993; De Corte et al., 1994). A factor released by COLO-16 cells initiates ~ 1 8 5 " - " ~ ~ phosphorylation and, subsequently, the migration of these cells. Anti-c-neu antibodies block motility in the breast cell line SK-BR3. Furthermore, the extracellular domain of p185'-"'" in adenocarcinoma cell lines binds directly to the cell adhesion molecules cadherin and catenin (Kanai et al., 1995).

Cell differentiation is initiated by p185"-"'"; that is, p185C-"eu may push a cell out of the cell cycle and initiate differentiation. Although the p185"-"'" ligand activates the mitotic division of several murine epithelial and human breast lines (see above), in human breast lines, ~ 1 8 5 " ~ ~ ~ ligands can arrest cells in the G1 phase of the cell cycle and induce cell differentiation (Lupu et al., 1992; Peles et al., 199213; Wen et al., 1992; Marte et al., 1994). PC12 pheochro- mocytoma cells engineered to express ~185""'" can develop neurites while retaining their ability to continue proliferate (Gamett and Cerione, 19941, and p185"--""" ligands can stimulate the acetylcholine receptor transcription in cultured muscle cells (Falls et al., 1993; Jo et al., 1995).

EGFr mediates developmental processes similar to those described for p185"--"'". For example, EGF is a glial mitogen (Libermann et al., 1984; Wong et al., 1987) that affects neuronal survival and promotes neurite outgrowth (Morri- son et al., 1987, 1988; Kinoshita et al., 1990; Knusel et al., 1990; Kornblum et al., 1990). It is interesting to note that EGFr is expressed by most neurons in primary cultures of cerebral cortex (Morrison et al., 1987; Kinoshita et al., 1990).

We hypothesize that ~185"-"~" expression in vivo is associated with the development of central nervous system (CNS) neurons, specifically, neocortical neurons. Based on its pivotal role in the development of various nonneural cell lines, p185"-"'" may play a role in the proliferation, migration, and/or differentiation of cortical neurons. Inasmuch as ~185~-" '" and EGFr have similar structures, we compared the spatiotemporal expression of the c-neu oncoprotein and EGFr in the rat cerebral cortex of maturing rats.

MATERIALS AND METHODS Animals

Pregnant Long-Evans rats were obtained from Harlan- Sprague-Dawley (Altamount, NY). Animals were maintained in a humidity- and temperature-controlled facility where the lightidark cycles were 12/12 hours, and they were fed chow and water ad libitum. Rats were mated at the end of the light cycle, and the first day the dams had a vaginal plug was designated G1. Pups were born on G22, also referred to as postnatal day (PI 0. Fetuses and pups were harvested on G16 and G19 and on PO, P3, P6, P9, P12, P15, P21, and P90.

Immunochemical studies Tissue preparation. For each time point, four animals

were used. These animals were drawn from two litters per prenatal time point and three or four litters for the postnatal ages. Animals were anesthetized by intraperito- neal injection of a cocktail of 60 mg/kg ketamine and 7.5 mgikg xylazine and were killed by decapitation. To collect fetuses, the pregnant dams were anesthetized, and the fetuses were removed by Cesarean section.

c-neu IN DEVELOPING CEREBRAL CORTEX 191

The brains were taken from their crania, and the rostra1 one-third of each forebrain was dissected. The olfactory lobes, tissue ventral to the rhinal fissure, and caudate nucleus were removed. Thus, the entire cerebral wall, from the ventricular zone to the pial surface, was used in the final preparation. The block of tissue was quickly homogenized in cold modified RIPA buffer containing protease and phosphatase inhibitors and detergent to prevent protein degradation (Yang et al., 1989; Jalla et d., 1992). This buffer contained 1.0 mM dithiothreitol, 192 mM glycine, 50 pM leupeptin, 1.0% Nonidet P-40, 10 pg phenylmethylsul- fonylfluoride per ml buffer, 150 mM sodium chloride, 1.0% sodium deoxycholate, 0.10% sodium dodecylsulfate (SDS), and 50 FM sodium orthovanadate in 10 mM sodium phosphate buffer, pH 7.2. The DNA concentration of the homogenate was determined by quantifying the absorbance at 260 nm (Sambrook et al., 19891, and the total protein concentration was determined by using a kit obtained from BioRad (Hercules, CAI. Triplicate aliquots of the homogenate from each animal were used in the immunoblot analyses.

Tissues from four different animals (no more than two per litter) were used for the analysis. Samples containing 10 pg DNA equivalents were applied to a polyacrylamide gel (PAG) containing 7.5% SDS (Laemmli, 1970). The samples were electrophoresed, and the protein bands were transferred onto nitrocellulose (30 V, overnight at 4°C) by using the method of Towbin and colleagues (1979). Triplicates from each animal were run.

The immunochemical reactions were carried out by using standard protocols. Nonspecific immunoreactivity was blocked by immersing the blot in 5.0% nonfat dry milk for 30 hours. The blots were incubated overnight at 4°C either with a monoclonal antibody directed against p185"-"'" (Ab-3; Oncogene, Uniondale, NY) or with a polyclonal anti-EGFr antibody (Ab-4; Oncogene). The anti-c-neu and anti-EGFr were each diluted to 150 in 0.10 M phosphate-buffered saline (PBS). There appears to be no cross-reactivity between the antibodies (see, e.g., Lundy et al., 1991; Umekita et al., 1992). Not only do many breast carcinoma cells express immunoreactivity to only one antibody, but the pattern of immunohistochemical staining is substantially different with anti-c-neu and anti-EGFr; c-mu-positive elements are membrane bound, whereas EGFr immunostaining is mostly cytoplasmic.

Immunoreactive bands were detected by an anti-mouse or an anti-rabbit secondary antibody cross linked to alka- line phosphatase (Vector, Burlingame, CA). The sizes of the various immunoreactive bands were determined by compar- ing their weights with those of standard proteins covering the range from 21 to 116 kDa (BioRad) or from 97 to 450 kDa (Sigma, St. Louis, MO).

A series of nondenaturing PAG that did not contain SDS was produced. By omitting the SDS, the native size and conformation of the proteins was retained. Homogenized tissue from 6- and 12-day-old pups and from adults was applied to PAG. Immunoblots were produced by using the electrophoresis, blotting, and immunostaining procedures described above. Divalent cation(s) may have cross linked the c-neu and EGFr peptides to form oligomeric complexes that did not enter the 7.5% SDS-PAG. Therefore, some native gels were prepared with 50 mM EDTA to determine whether the immunoreactive proteins were expressed as dimers.

Preparation of blots.

Zmmunoprecipitation studies. Tissue from 6-, 12-, and 90-day-old rats were used in the immunoprecipitation studies. After spinning the crude homogenate at 3,000 rpm to remove particulate matter, the supernatant (which included the crude protein) was incubated overnight at 4°C with either anti-c-neu or anti-EGFr diluted to 1:20 in 0.10 M phosphate buffer. The bound antigen was precipitated by agarose that was cross-linked to a secondary antibody, and the eluent was processed according to the SDS-PAG electrophoresis and immunoblotting procedures described above.

The amount of a protein on the immunoblots was analyzed with the aid of microdensitometry software in the Bioquant Image Analysis System (R and M Biometrics, Nashville, TN). With this system, the relative density and the size of a band were determined. The product of the density and size of the band was taken as an estimate of the relative amount of a protein in the blot. The means of the triplicates per animal per time point were calculated and the grand means (2S.D.) for all of the age-matched subjects were determined. The statistical significance of the changes in levels of a protein was assessed by using an analysis of variance, and changes over time were examined with a Newman-Keuls test.

Analysis.

Morphological studies Preparation of tissue sections. Fetuses (G16 and G19),

pups (PO, P6, and P12), and adults (P90) were killed. Animals or pregnant dams were anesthetized by intraperi- toneal injection of ketamine/xylazine and transcardially perfused with Bouin's fixative for 30 minutes. After being removed from the skull, the brains were postfixed in fresh fixative for 1-2 hours. Each brain was cryoprotected by immersing it in 10% sucrose in phosphate buffer for 4-8 hours and then in 30% buffered sucrose for up to 2 days (until the brain sank). The brain was frozen and cut into a series of 20 pm coronal sections with a cryostat.

Two series of alter- nate sections were composed: One was immunolabeled for p185"-""", and the other was immunolabeled for EGFr. After blocking nonspecific binding with 5.0% goat serum in 0.4% Triton X-100, sections were incubated in either a 1:25 or a 1:20 dilution of anti-c-neu or anti-EGFr, respectively, in PBS. Immunopositive cells were detected by conjugating an avidin-biotin-horseradish peroxidase complex (Vector) to the primary antibody (Hsu et al., 1981). The peroxidase was reacted with 1.0% hydrogen peroxide in the presence of the chromogen (1.0%) diaminobenzidine.

The sections were examined microscopically to determine spatiotemporal changes in the distribution of immunoreactive elements. The pattern of immunostaining was compared to the changes in the amounts of the c-neu-positive oncoprotein in the irnmunoblots in order to assign an ontogenetic role to the receptors.

Immunohistochemical procedures.

Analysis.

RESULTS c-neu

Immunochemical studies. A relatively large c-neu- positive protein was evident in the nondenaturing gels without EDTA. c-neu-positive proteins ran as a smear, indicating that the antibody recognized a heterogenous population of peptides. Although the resolution of these gels was poor, the molecular weights of the immunoreactive proteins appeared to be 300-400 kDa. Following treatment with EDTA, the anti-c-neu antibody labeled a more homoge-


Fig 1 Developmental expression of c-neu-immunopositive peptides Immunoblots of tissue (25 ~ 1 ) from rats of different ages [gestational day (G) 16 to adult, Ad1 were prepared Various proteins were identified, some immunopositive peptides were evldent in the adult, whereas others were transiently expressed during early or late development

neous population with two bands on the PAG. The main band had a molecular weight of 180-190 kDa, and the minor band was about 80 kDa. We concluded that the main band on the PAG with EDTA was the p185'-""" monomer described in breast carcinomas and that the smear of proteins detected on the nondenaturing gels without EDTA represented a series of homodimers and heterodimers formed by the oncoprotein or assoeations with other proteins.

SDS broke down the homogenized tissue from mature cortex, so that a family of c-neu-positive peptides was identified (Fig. 1). Although the 185 kDa peptide was expressed weakly at best in the SDS-PAG, numerous other c-neu-positive peptides were detected. These included a peptide triplet that had a molecular weights of 34, 40, and 46 kDa and a 60 and 115 kDa peptide. The expression of these peptides during cortical development was biphasic (Fig. 2). An initial burst of expression occurred prenatally. There was a significantly (P < 0.05) higher expression of the 34,46,60, and 115 kDa peptides on G16 or G19 relative to the amount detected on PO. The levels of expression increased steadily and significantly (P < 0.05) between the day of birth and P12. They were maintained at peak levels until at least P21 and then dropped 20-50% to adult levels.

Various c-neu-positive peptides were transiently expressed only during cortical development, i.e., they did not appear in the adult (Fig. 1). These peptides were grouped by the timing of their expression; they were expressed either prenatally (and/or postnatally) or primarily postnatally.

6 z n

? zn i.

z 3 0 2 6 w I-

W oi

1 4

34kDa "1 40kDa 46 kDa

1 7 60kDa T ~ /rT!

5 1

1 185kDa /r/f

0 1

"3 f

I I 1 1 ,'+

GI5 P3 P I 3 P23 Adult

Fig. 2. Temporal expression of c-neu-positive peptides identified in the adult. The anti-c-neu oncoprotein antibody identified five peptides in the adult (34, 40, 46, 60, 115, and 185 kDa peptides). The relative expression of these peptides was quantified microdensitometrically and plotted as a function of time. After being expressed transiently in the fetus, the expression of each peptide increased over the first three postnatal weeks before dropping to adult levels. Each symbol represents the mean amount of a particular protein at a particular time (n = 4). The T-bars indicate the standard deviations.


2 w i ' L fi

T 50kDa

Y GI5 P3 P I 3 P23 Adult

AGE Fig. 3. c-neu-Positive peptides expressed prenatally. Two peptides

(50 and 76) were evident in the fetus. The expression of the 50 kDa protein was confined solely to the prenatal period, the 76 kDa protein was elaborated during the week before and the week after birth. Notations as in Figure 2.

a 30 a 35kDa

300 kDa

G I 5 P3 P I 3 P23 Adult

AGE Fig. 4. c-neu-Positive peptides transiently expressed during the

second postnatal week. The major protein recognized by the anti-c-neu oncoprotein antibody had a molecular weight of 300 kDa. The expression of this oncoprotein occurred mostly during the second postnatal week. A 35 kDa oncoprotein was expressed postnatally as well. Neither of these proteins was produced in the adult. Notations as in Figure 2.

Two peptides were expressed prenatally (Fig. 3) . A 76 kDa protein was evident on G16 and G19; that is, only on these 2 days was the amount of the 76 kDa protein significantly (P < 0.05) greater than it was postnatally. A 50 kDa protein was expressed pre- and postnatally. Be- tween G16 and P6, a relatively constant amount of the 50 kDa protein was expressed. After P6, however, the amount of protein detected was significantly (P < 0.05) lower, and this reduced expression was maintained during the second and succeeding postnatal weeks into adulthood.

Two peptides (300 and 35 kDa) were expressed transiently in the preweanling (Fig. 4). Peak expression for both proteins was attained on P6 and P9. Initially, the 300 kDa protein was expressed prenatally and at least until P21. On

the other hand, the expression of the 35 kDa protein was more restricted: it occurred only between PO and P12.

Based on the size and the shape of the labeled cell bodies, the vast majority of c-neu-immunolabeled cells in the adult cortex were identified as neurons. Most labeled somata were in layer V, principally in layer Vb (Figs. 5,6) . All of the labeled cells in layer V appeared to be pyramidal neurons. They had conspicuously long apical dendrites that extended well into supragranular cortex and a spray of dendrites emanating from the base of the cell body. Although an axonal hillock arising from the base of the cell body could be discerned, the axon did not appear to be immunolabeled. A small number of immunolabeled neuronal somata, including those of local circuit (e.g., stellate and bitufted) neurons, were scattered through layers II/III, IV, and VI. Although cortex was largely devoid of labeled glia, glia in the white matter and ependyma in the ventricular zone did express c-neu immunoreactivity (Fig. 7).

In contrast to the adult, the staining in the immature cortex was quite widespread (Fig. 5). Two overlapping patterns of immunolabeling were identified; each pattern followed a different spatiotemporal sequence. One pattern was evident in the superficial cortical plate between G16 and P6. The first evidence of c-neu immunoreactivity was as radially oriented ribbons in the fetal cortical plate. Examina- tion with phase-interference microscopy showed that these ribbons appeared to be in the apical processes of the bipolar (migrating) neurons. By P6, these ribbons were evident only in the most superficial portion of the cortical plate (the anlage of layer II/III), and, by P9, they were absent.

The second pattern of oncoprotein immunostaining was a generalized somatic staining. The appearance of cells expressing this staining followed an inside-to-outside pattern. That is, the first evidence of somatic immunolabeling occurred in the subplate (on G16), and, over time, cell bodies in succeedingly superficial segments of the cortical plate expressed immunoreactivity. The cytoplasmic localization of the immunoreaction product changed dramatically over a 3 week period. A typical example of the changes in this immunostaining pattern was in subplate neurons (Fig. 8). In the fetal subplate, the immunoreactive elements were perikaryal caps: These foci were generally associated with the sites of origin for dendrites. The amount of cytoplasmic immunostaining generalized during the first postnatal week, and, by P6, the entire perikarya and proximal dendrites were positively stained. At this time, the c-neu-labeled elements were vesicular organelles. During the second postnatal week, the immunostaining became more diffuse and somewhat more restricted, so that, by P12, the staining was confined largely to cell bodies, and dendritic staining was rare. Immunostaining in layer VIb of the adult (the descendant of the subplate) was virtually absent.

A similar progression in the expression of c-neu immunoreactivity was evident in layer Vb neurons (Fig. 6). The only difference between the developmental sequences of staining in layer V and in the subplate was the heterogeneity of the immunostaining in layer V on P12. Whereas, in the subplate, the intensity of the immunolabeling all neurons was similar, some cell bodies of layer V neurons were more intensely stained than others. Presumably, these intensely labeled neurons were the neurons that retained immunoreactivity in the adult.

Anatomical studies.


Cells in the immature ventricular zone expressed c-neu- immunoreactivity (Fig. 7). Such immunostaining was evident as early as G16. This pattern persisted as ependymal cells lining the lateral ventricles of the adult were immunolabeled.

EGFr In the nondenaturing gels of

cortex from 6-, 12-, and 90-day-old rats, anti-EGFr immunolabeled proteins with a broad range of molecular weights between 200 and 400 kDa. Following treatment with EDTA, the molecular weights of the immunoreactive peptides were substantially reduced: The peptides were about 180-200 kDa. Thus, EGFr, like ~ 1 8 5 ~ - " ~ ~ , apparently formed dimers that were broken down to monomers by the EDTA treatment.

Treatment with SDS reduced the EGFr to a series of peptides with molecular weights from 35 to 80 kDa (Fig. 9). The 50 and 80 kDa peptides were expressed in the adult (Fig. 10). Both were expressed transiently in the fetus and then were expressed in increasing amounts over the first 2 postnatal weeks. The expression of the 50 and 80 kDa peptides subsequently declined to adult levels. This temporal pattern was similar to that of the c-neu-positive peptides expressed in the adult (e.g., the 34,40, 46, 60, and 115 kDa peptides). The expression of the 34 kDa peptide contrasted with that of the other EGFr-positive peptides. The 35 kDa EGFr-positive peptide was undetected prenatally and in the adult; its expression was limited to P3 to P12.

Various immunoprecipitation experiments were per- formed to assess the relationship between EGFr and the ~ 1 8 5 " - " ~ ~ . After precipitating homogenized cortex with anti- EGFr, most of the c-neu-positive peptides remained unprec- ipitated in the supernatant; however, a number of c-neu- positive peptides (e.g., with molecular weights of 60, 115, and 300 kDa) were precipitated (Fig. 11). In the converse experiment, the 34, 50, and 80 kDa EGFr-positive peptides were largely immunoprecipitated.

Anatomical studies. Although the background staining was greater with the anti-EGFr antibody than it was with the anti-c-neu antibody, the pattern of immunostaining with both antibodies was remarkably similar. Immunostain- ing was evident throughout adult cortex, but the most intensely labeled cells were layer V pyramidal neurons (Fig. 12). Glia in the white matter and ependyma were also immunolabeled (Fig. 13).

The developmental expression of EGFr immunoreactivity was also quite similar to c-neu immunolabeling. In cortex, EGFr expression appeared during the first week and waned in the second. The intermediate zone was largely

Immunochemical studies.

Fig. 5. Spatiotemporal changes in the distribution of c-neu immunoreactivity in developing rat cortex. The distribution of immunoreactive neurons in the immature cortex was quite broad. In the fetus, immunoreactive elements were distributed in the subplate (SP) and in the cortical plate (CP). During the first 2 postnatal weeks, the immunostaining in the subplate (and its descendant layer VIb) increased, but, by adulthood, it was virtually absent. The density of immunopositive elements was high in the cortical plate and in the most recently segregated cortical laminae (e.g., layer V on postnatal day (PI 0 and layer IIiIII on P6). Over time, the expression of c-neu immunoreactivity in layers 11-VIa became more evenly distributed. In the adult (Ad), the oncoprotein was elaborated primarily by pyramidal neurons with cell bodies in layer V and a small number of neurons scattered through the other layers. MZ, marginal zone. Scale bars = 100 pm.

devoid of labeling prenatally and during the second postnatal week. On the other hand, EGFr immunostaining was present in many ventricular cells (Fig. 13).

DISCUSSION The anti-c-neu oncoprotein antibody recognizes a family

of peptides in SDS-treated immunoblots. These peptides are principally expressed in the adult (34, 40, 46, and 115 kDa), prenatally (50 and 76 kDa), or during early postnatal life (35 and 300 kDa). In the nondenaturing gels, the immunoreactive band has a consistent size, regardless of the age of the rat. Therefore, we conclude that it is not the oncoprotein that changes during development. Rather, all of the c-neu-positive peptides are fragments of the p185C-"eu: Their common feature is that they all include epitope- containing parts of the intracellular domain.

The developmentally regulated array of c-neu-positive peptides likely is produced by posttranslational changes. Other studies on the c-neu oncoprotein support this conjec- ture. For example, in confluent cultures of murine fibroblasts transformed by wild type c-neu, ligand-receptor complexes produce an 80-100 kDa peptide (Yarden and Weinberg, 1989). In addition, a 116 kDa peptide can be identified when substances that interfere with ligand- receptor interactions, e.g., suramin, are added to the cul- ture. In general, receptor down-regulation follows one of two pathways (Lund et al., 1990; Brown and Greene, 1991). One pathway involves the incorporation of phosphorylated c-neu into coated pits during the internalization of the ligand- receptor complex (Gilboa et al., 1995). This pathway is blocked by suramin.

We presume that the labeling in the immunohistochemical preparations (as in the nondenaturing PAG) represents the intact oncoprotein. Thus, based on the limited patterns of expression of the oncoprotein fragments and on the changes in the spatiotemporal distribution of immunohisto- chemically labeled elements, we can ascribe probable ontogenetic roles to the c-neu oncoproteins or to unidentified catabolic enzymes.

Oncoprotein expression in the adult In the adult, primarily layer Vb neurons are immunoreac-

tive. Since only five peptides (34, 40, 46, 115, and 185 kDa) are evident in the adult, it appears that mature layer V neurons possess an enzyme that is unexpressed during most of early development. Layer Vb contains neurons that have specific projections to the brainstem and spinal cord (see, e.g., Hicks and D'Amato, 1977; Wise and Jones, 1977; Miller, 1987; Schofield et al., 1987; Hallman et al., 1988). Although a double-labeling study is needed to determine whether corticofugal projection neurons do express the oncoprotein, it is appealing to speculate that the discreet- ness of the immunostaining indicates that the oncoprotein plays a role in the physiological regulation of corticofugal information.

The spatial distribution of c-neu-positive peptides in the adult is identical to the distribution of neurons that express receptor proteins for cortical neurotrophins (Pitts and Miller, 1995). Antibodies against the high-affinity receptor (trlz) or the low-affinity receptor (p75) label the cell bodies of pyramidal neurons in layer Vb. In fact, double-immunoflu- orescence studies show that many (at least 60%) c-neu- positive layer Vb neurons colocalize an isoform of trlz or p75 (Pitts et al., 1994, 1995). The parallel between the c-neu

Fig. 6. c-neu immunoreactivity in layer V. In neonates, c-neu- positive ribbons (arrows) were evident in the leading (apical) processes of neurons in layer V. The cell bodies of these neurons (asterisks) were unlabeled. On P6 and P12, the cell bodies as well as the apical dendrites

of many layer V neurons were immunolabeled. In the adult, many secondary and tertiary dendritic arbors were labeled. Scale bars = 20 pn.


oncoprotein and neurotrophin receptors is supported fur- ther by their distribution and colocalization in the basal forebrain neurons. Antibodies directed against either the c-neu oncoprotein or the neurotrophin receptors label a continuous sheet of cell bodies extending from hippocampal pyramidal cell layer through layer Vb of neocortex to the basal forebrain. The coexpression of the oncoprotein receptors may be redundant with the neurotrophin receptors, which have a well-described function in cortex; however, such a conclusion may be the facile product of our ignorance of the oncoprotein's actions. Since the oncoprotein can relate to growth factor receptors, such as EGFr (see below), we believe that the oncoprotein plays an independent or augmentary function in the transduction of growth factor signals.

Perinatal expression The expression of the 50 kDa peptide is steady between

G16 and P6 and crashes by P9. This expression is coinci- dent with the immunohistochemical labeling in the superficial cortical plate (i.e., the perinuclear puncta and the ribbons in the leading processes). The timing and the unique distribution of staining suggest that the expression of the 50 kDa protein is associated with neuronal migration. The migration of neurons to frontal cortex begins by G16 and is complete by the beginning of the second postnatal week (Berry and Rogers, 1965; Hicks and D'Amato, 1968; Miller, 1993, in preparation). Because the immunostained cells are in the cortical plate and appear to be cells nearing or at the end of their migration, the enzyme cleaving off the 50 kDa protein may be important in the process that tells migrating neurons when and where to stop their migration.

Posttranslational modification of p185e-neu may permit it to play a role in neuronal migration. Part of the intracellular domain of ~185"-" '~ binds to cell adhesion molecules (Kanai et al., 1995), and chemotaxis can be induced in human SK-BR3 cells in the presence of ~ 1 8 5 ~ - " ~ " ligands (De Potter and Quatacker, 1993; De Corte et al., 1994). We propose that unphosphorylated p185c-"eu mediates the adherence of young c-neu-expressing neurons to radial glial fibers. After binding the ligand, the p185e-"eu autophosphory- lates, and, in this phosphorylated state, the adhesion between the cells is broken, and cell movement begins (Ben-Levy et al., 1994). According to this model, directed migration is promoted by a repeated binding-and-release sequence of the p185c-neu-cadherin complex, i.e., a ratchet- ing process. Blockage of the binding of ~185"-" '~ with its ligand would retain the p185c-"eu-cadherin complex, which, in turn, would increase neuronal-glial adherence and cause abnormalities in neuronal migration. Such an increase in adherence may underlie the defects in neuronal migration associated with the murine mutant reeler (Hoffarth et al., 1995) and with early exposure to ethanol (Miller, 1988a, 1993).

Fig. 7. c-neu immunoreactivity in the ventricular zone and white matter. A: Immunoreactivity was evident in the ventricular zone (vz) on G16. Most of this staining appeared to be anchored at the surface of the ventricle (v). Occasional fibrous projections extended into the subventricular zone (sz). B: c-neu immunostaining in the ventricular zone persisted into adulthood. C: Occasional labeled glia (arrows) were evident in the white matter of the adult. Cau, caudate. Scale bars = 20 pm.

Fig. 8. c-neu immunoreactivity in the subplate (layer VIb). The c-neu oncoprotein was transiently expressed in the subplate. In the fetus and neonate, only primary dendrites (arrows) were immunolabeled. Cell bodies (asterisks) were unlabeled. On P6, immunolabeling

extended into the cell bodies of subplate neurons. By P12, the dendritic label had largely disappeared, and only the somatic labeling remained. In the adult, c-neu immunoreactivity was virtually absent in layer VIb. Scale bars = 20 km.

c-neu IN DEVELOPING CEREBRAL CORTEX

G16 G19 PO P3

116

80

P6 P9 P12 P15 P21 Ad

49

32

Fig. 9. Developmental expression of epidermal growth factor receptor (EGFr) immunoreactivity. EGFr-positive peptides were expressed throughout cortical development. Three sets of proteins were evident. The expression of a peptide triplet at about 50 kDa increased as the rats aged. A 35 kDa peptide and an 80 kDa peptide were transiently expressed postnatally.

a n 15 z

W

I- 1

. 35kDa '1

1

GI5 P3 P I 3 P23 Adult

AGE Fig. 10. Temporal expression of EGFr-positive proteins. The changes

in relative expression of three peptides (35,50, and 80 kDa) with age are plotted. The 50 and 80 kDa peptides had similar profiles of temporal expression. They were maximally expressed on G19 and during the early postnatal period. The 35 kDa peptide was evident only during the first and second postnatal weeks, reaching maximal expression on P9.

The 76 kDa protein is expressed exclusively in the fetus. c-neu immunoreactivity is evident in the fetal ventricular zone, which is the site of neuronal and glial generation (for reviews, see Miller, 198813, 1992; Bayer and Altman, 1991).

199

The transient expression of the 76 kDa peptide may be associated with the proliferation of neuronal precursors. After all, the ventricular zone in the adult is c-neu positive, and the neocortical ventricular zone in the adult rat only generates glia, but the 76 kDa peptide is not expressed postnatally. It is well documented that the c-neu oncoprotein plays a role in the proliferation of mammary carcinomas (see, e.g., Mori et al., 1987; Berger et al., 1988; De Potter et al., 198910). Alternatively, the oncoprotein may be involved in the exiting of the cycling neuronal precursors and the initiation of neuronal migration.

Interestingly, the peptides expressed in the adult are transiently expressed prenatally as well. This is indicative of a dual role for the oncoprotein. That is, that the c-neu oncoprotein [or the enzyme(s) that effect the posttranslational changes] is important at two pivotal developmental time points: during cell proliferation and at the time when survival of the neuron is in the balance. Evidence indicates that neurotrophins, such as nerve growth factor, also play a dual role during neuronal ontogeny (Ucker, 1991; Colombel et al., 1992; Loughlin and Fallon, 1993; Freeman et al., 1994).

Later postnatal expression During the second postnatal week, the expression of the

35 kDa protein peaked. The timing for the expression of the 35 kDa oncoprotein parallels that of the 34 kDa EGFr- positive peptide and the 56 kDa peptide recognized by ALZ-50 (Miller et al., 1994). This coincidence of temporal


Pel let Supernatant

Std P6 P12 Ad P6 P12 Ad

205

116

80

4 9

4 0

205

116

80

49

Fig. 11, Immunoprecipitation studies. A Following immunoprecipitation of cortical tissue with anti-EGFr, the anti-c-neu antibody identified a number of peptides in the precipitate from 35 to 185 kDa, most of which were also in the supernatant. The 60, 115, and 360 kDa peptides were found nearly completely in the pellet. B: In the reverse experiment, the 35,50, and 80 kDa EGFr-positive peptides were largely precipitated by the anti-c-neu antibody.

expression is intriguing, because ALZ-50 apparently identi- fies a protein that is up-regulated during the early stages of neuronal death (&-Ghoul and Miller, 1989; Valverde et al., 1990; Miller et al., 1991; Naegele et al., 1991). It is unclear whether the 35 kDa peptide is related to the ALZ-50- positive antigen or whether the 35 kDa peptide is a cleavage product of an enzyme related to the degenerative process.

There is reason to believe that the oncoprotein is related to neural degeneration/survival. Our data show that the number of c-neu-positive neurons in the ventral, but not the dorsal, principal sensory nucleus of the trigeminal nerve (PSN) increase shortly (2 hours and 2 days) after transecting the infraorbital nerve in a neonate (Kuhn and Miller, 1995). Because deafferentation leads to neuronal death and increases ALZ-50 immunoreactivity in the ventral PSN (Miller et al., 1991), these data suggest that c-neu may be involved in neuronal death. Furthermore, Cohen et al. (1992) have shown that c-neu translation and transcription is increased in Schwann cells in the sciatic nerves of

This Oncogene up-regu1a- tion Occurs in the absence of a change in trk expression. Thus, it is not a generalized up-regulation of tyrosine

Fig. 12. EGFr immunoreactivity in the mature cortex. A: Most EGFr-positive cell bodies were distributed in layer V. B: EGFr-labeled cells appeared to be pyramidal neurons, because they had large cell bodies, each of which gave rise to a urominent ascending urocess. Scale

rats that were

kinase transcription. bars = 100 km.


The temporal expression of the 300 kDa c-neu-positive protein contrasts to that of the 35 kDa peptide. Although the peak expression of both peptides occurs during the

second postnatal week, the 300 kDa protein is initially evident prenatally, and its expression continues well into the third postnatal week. This time profile is most consistent with a role in neuronal differentiation, possibly synap- togenesis.

Relationship of the c-neu oncoprotein and EGFr

p185"-"'" interacts with other proteins. This statement is supported by findings of a 300 kDa c-neu-positive peptide (i.e., one larger than the ~185~-""") in SDS-PAG immunoblots and the change in the molecular weight of the c-neu oncoprotein changes in nondenaturing gels following the addition of EDTA. Our data show that the c-neu oncoprotein associates with EGFr throughout development. The patterns of ~185~-" '" and EGFr labeling (in both immunohistochemical and immunoblot preparations) are remarkably parallel throughout development. Because the available data show that the two antibodies do not cross react, we infer from the immunoprecipitation data that the oncoprotein and EGFr form a heterodimer. Both antibodies are directed against the intracellular segments of the receptors. Hence, it appears that the receptors are conjugated by the intracellular domains of the two receptors.

The oncoprotein and EGFr form heterodimers in nonneural tissues. When the receptors are activated, p185"-""" or EGFr can form 360-400 kDa homodimers in primary breast carcinoma cultures and mouse fibroblasts transformed with rat c-neu protooncogenes that are resist to 2-mercaptoethanol (Weiner et al., 1989; Qian et al., 1992). Furthermore, p185c-fleu and EGFr can form heterodimers in cultured murine fibroblasts (Qian et al., 1992). The interaction of p185"-"'" with other cellular components is important for the activation of c-neu by its ligand (Connelly and Stern, 1990; Qian et al., 1992; Peles et al., 1993). Likewise, EGF can phosphorylate p185c-neu if the cell coexpresses EGFr (Connelly and Stern, 1990).

In summary, although ~185"-"~" is expressed widely among cortical cells during development, in the adult, only a discrete population of cortical neurons and glia are c-neu positive. The oncoprotein appears 1) to form heterodimers with EGFr and 2) to participate in various stages of neuronal ontogeny. The roles proposed for p185"-""" during neuronal development are consistent with data on nonneural tissues. Nevertheless, it must be emphasized that some of the developmental changes that we describe for p185"-""" may not involve for ~185'-"~" per se, but, rather, may involve enzyme(s) that act on p185"-"'". In this sense, ~ 1 8 5 ~ - " ~ " may provide a valuable vehicle to understand some of the mechanisms underlying fundamental developmental events.

ACKNOWLEDGMENTS This research has been supported by the Department of

Veterans Affairs and the National Institutes of Health (AA 06916, AA 07568, and DE 07734).

Fig. 13. EGFr immunoreactivity in the ventricular zone and white matter. A On G16, many of the cells in the fetal ventricular zone (vz) expressed EGFr immunoreactivity. B: Descendants of the ventricular cells, ependymal cells in the adult, were intensely EGFr positive. C: Glia (arrows) in the white matter of the mature rat were also EGFr positive. v, Ventricle; sz, subventricular zone; Cau, caudate nucleus. Scale bars = 20 pm.


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