Class III beta-tubulin isotype (beta III) in the adrenal medulla: III. Differential expression of neuronal and glial antigens identifies two distinct populations of neuronal and glial-like (sustentacular) cells in the PC12 rat pheochromocytoma cell line maintained in a Gelfoam matrix system.

BACKGROUND: The rat PC12 pheochromocytoma cell line provides an established system for the study of neuronal differentiation. To our knowledge, glial differentiation has not been reported in this cell line. METHODS: We have studied, by immunohistochemistry and immunoblotting, the presence of neuronal cytoskeletal antigens [class III beta-tubulin isotype (beta III), microtubule associated proteins MAP2, MAP1B and tau, and different neurofilament (NF) protein components], and synaptophysin in comparison with the glial fibrillary acidic protein (GFAP) and S-100 protein in the PC12 cell line. In three different experiments, PC12 cells were maintained in a three-dimensional gelatin foam (Gelfoam) matrix system for up to 34 days with and without treatment with 1 mM dibutyryl cyclic (dc)AMP. Immunohistochemistry was performed on explants ranging from 2 to 32 days-in vitro, which were fixed in either Bouin's solution, 70% ethanol, or 10% neutral-buffered formalin and embedded in paraffin. Immunoblotting was performed on Gelfoam explants with a panel of antibodies against all aforementioned neuronal and glial markers. Additional immunoblot experiments using anti-GFAP and anti-beta III monoclonal antibodies in cell suspensions and homogenates from PC12 monolayer cultures were carried out to compare growth conditions in relation to the expression of these proteins. RESULTS: Beta III and MAP2 were demonstrated by immunohistochemistry and immunoblotting of PC12 explants maintained for up to 32 days in Gelfoam matrices with and without treatment with dcAMP. Intense filamentous and granular beta III staining of PC12 cells was observed in dcAMP-treated cultures concomitant with neuronal morphologic alterations (neuritogenesis and ganglionic phenotype). In untreated cultures, beta III staining was present in less differentiated cells, as well in cells undergoing neuritic development. The neuronal phenotype of PC12 cells was confirmed by staining for MAP2, tau, and NF proteins, as well as for synaptophysin. The presence of beta III, MAP2, MAP1B, tau, and NF proteins was confirmed by immunoblotting. Clusters of GFAP-positive and S-100 protein-positive spindle cells, phenotypically distinct from the chromaffin-like or neuronal cells, were demonstrated in Gelfoam explants at 5-30 days in vitro. In 30-day-old cultures treated with dcAMP, there was strong filamentous GFAP and diffuse S-100 protein staining in an increased number of sustentacular-like PC12 cells. GFAP staining was corroborated by immunoblotting of explants maintained under identical conditions in vitro. In contrast, immunoblots performed on homogenates from PC12 suspension and monolayer cultures were GFAP-negative. CONCLUSIONS: Neuronal and glial-like, presumed sustentacular, phenotypes were demonstrated in PC12 cells grown in Gelfoam matrices with and without treatment with dcAMP for up to 34 days. To our knowledge, the occurrence of glial differentiation in the PC12 line is a hitherto unreported finding. Adult rat medullary sustentacular cells are known to express S-100 and GFA proteins (Suzuki and Kachi, Kaibogaku Zasshi-Anat 70(2): 130-139, 1995), and the organ culture system employed in our study may well have favored this direction of differentiation.

High-grade human brain tumors exhibit increased expression of myelin transcription factor 1 (MYT1), a zinc finger DNA-binding protein.

Detection and characterization of distinct central nervous system (CNS) tumor cell types is clinically important since distinct tumor types are associated with different prognoses and treatments. However, there is currently a lack of markers to identify certain glioma types and insufficient understanding as to which cells give rise to different glioma cell types. In the present study, biopsy specimens from human brain tumors were analyzed for expression of Myelin Transcription Factor 1 (MYT1) to explore the extent to which glioma cells reflect characteristic expression of MYT1 in developing glial progenitor cells. Immunostaining with an antibody against MYT1 revealed widespread immunoreactivity that was most prominent in high-grade oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas as well as in a dysembryoplastic neuroepithelial tumor. MYT1 immunoreactivity in tumor regions generally correlated with the prevalence of cells exhibiting nuclear immunolabeling with an antibody against Ki-67, suggesting an association of MYT1 with cell proliferation that was also observed in normal adult human and rat brain in the germinal subependymal zone. The MYT1 immunoreactivity was frequently nuclear, appearing as dotted or punctate, but in some cases it was localized to the cytoplasm. In combination with histopathological studies and analysis of Ki-67 immunoreactivity, examination of MYT1 immunolabeling may provide additional information to aid in the detection and diagnosis of CNS tumors.

Maturation of intracranial immature teratomas. Report of two cases.

Immature teratomas arising within the central neuraxis are rare neoplasms. These tumors contain diverse cell lineages that retain an embryonal character and display phenotypic differentiation attributed to the three classic germ layers. The clinical management of these lesions is unclear, due in part to their low incidence and to an incomplete understanding of their natural history. Although the potential for phenotypic differentiation and cellular maturation within immature teratomas arising in the gonads is well documented, this has not been described in the intracranial tumors. In the present report, the authors describe two cases of intracranial immature teratomas, one involving the pineal region and the other involving the left frontotemporal lobes, which underwent cellular differentiation and maturation. At initial resection, the tumors from both cases were composed predominantly of primitive neuroepithelial tissue that was admixed with immature and differentiating mesenchymal and epithelial structures. No foci of germinoma, endodermal sinus, choriocarcinoma, or embryonal carcinoma tissue were present. Subsequent resections in both cases revealed an absence of immature tissue. The tumor in Case 1 contained only differentiated epithelial and mesenchymal tissue with no neuroepithelial component, whereas the tumor in Case 2 demonstrated abundant mature neuronal and glial tissue. These two cases from different intracranial sites suggest that spontaneous maturation may be a significant aspect of the natural history of intracranial immature teratomas.

Interphotoreceptor retinoid-binding protein (IRBP): expression in the adult and developing Xenopus retina.

Apposition of the neural retina and pigment epithelium is critical to photoreceptor development and function. Interphotoreceptor retinoid-binding protein (IRBP) is a major component of the extracellular matrix separating these epithelia in the African clawed frog Xenopus laevis (Gonzalez-Fernandez et al., [1993], J. Cell Sci. 105:7-21). In the adult retina, IRBP appears to mediate the transport of hydrophobic molecules, particularly retinoids and fatty acids, within the hydrophilic extracellular domain. In this paper, we compare the distribution of IRBP and its mRNA in adult and embryonic Xenopus retina. Xenopus IRBP antisense RNA, labeled with tritium or digoxigenin, was used for in situ hybridizaton studies. For immunohistochemistry, we used an antiserum against Xenopus IRBP expressed in Escherichia coli. In the adult, we found that IRBP is synthesized at similar levels by both rods and cones. The protein is restricted to the interphotoreceptor matrix, with lesser amounts in the pigment epithelial cytoplasm. In the embryo, expression of the mRNA for IRBP is restricted to the central retina, where photoreceptor differentiation has taken place. By contrast, the protein is distributed throughout the embryonic subretinal space. Therefore, the presence of IRBP precedes photoreceptor differentiation. In summary, IRBP is synthesized by both rods and cones and may be internalized by the pigment epithelium. In the embryo, IRBP is synthesized by the central retina and diffuses through the matrix, reaching the undifferentiated peripheral retina. In view of its ligand-binding properties, diffusion of IRBP may provide the peripheral neural retina with a vehicle to transport retinoids and docosahexaenoic acid (molecules critical to normal retinal development) from the pigment epithelium.

Goldfish cones secrete a two-repeat interphotoreceptor retinoid-binding protein.

Vitamin A and fatty acids are critical to photoreceptor structure, function, and development. The transport of these nutrients between the pigment epithelium and neural retina is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP, a 133-kDa (human) glycolipoprotein, is the major protein component of the extracellular matrix separating these two cell layers. In amphibians and mammals, IRBP consists of four homologous repeats of about 300 amino acids which form two retinol and four fatty acid-binding sites. Here we show that IRBP in teleosts is a simpler protein composed of only two repeats. Western blot analysis shows that goldfish IRBP is half the size (70 kDa) of IRBP in higher vertebrates. Metabolic labeling studies employing Brefeldin A taken together with in situ hybridization studies and the presence of a signal peptide show that goldfish IRBP is secreted by the cone photoreceptors. The translated amino acid sequence has a calculated molecular weight of 66.7 kDa. The primary structure consists of only two homologous repeats with a similarity score of 52.5%. The last repeats of human and goldfish IRBPs are 69.1% similar with hydrophobic regions being the most similar. These data suggest that two repeats were lost during the evolution of the ray-finned fish (Actinopterygii), or that the IRBP gene duplicated between the emergence of bony fish (Osteichthyes) and amphibians. Acquisition of a multirepeat structure may reflect evolutionary pressure to efficiently transport higher levels of hydrophobic molecules within a finite space. Quadruplication of an ancestral IRBP gene may have been an important event in the evolution of photo-receptors in higher vertebrates.

Cytoskeletal immunohistochemistry of central neurocytomas.

Central neurocytomas are rare intraventricular tumors. Patients with such tumors have a favorable prognosis after surgical removal. These tumors may be misdiagnosed as neuroblastomas or gliomas, risking the complications of adjuvant therapy. Diagnosis of central neurocytoma requires that the tumor shows the ultrastructural features of mature neuronal differentiation, including the presence of synapses and dense-core and clear vesicles in addition to profiles of neuritic processes with microtubules. The cytoskeletal phenotype of central neurocytomas has not been previously characterized, but it may facilitate their definitive recognition when ultrastructural examination is not possible. Ten central neurocytomas were examined by immunohistochemistry for phosphorylation-dependent/independent neurofilament epitopes, neuron-associated class III beta-tubulin, microtubule-associated proteins (MAP2, tau), and glial fibrillary acidic protein (GFAP). The neuronal nature of all neoplasms was documented by immunoreactivity for synaptophysin in nine tumors and for phosphorylation-independent neurofilament-H/M in the remaining case. Electron microscopy in four cases showed synapses and dense core vesicles. All tumors were immunoreactive for class III beta-tubulin and MAP2, which were seen in cytoskeletal structures by immunoelectron microscopy. Two thirds of the cases were immunohistochemically positive for neurofilament epitopes. None of the tumor cells displayed GFAP immunoreactivity, although reactive astrocytes were present. These data suggest that central neurocytomas may be recognized by synaptophysin immunoreactivity and that the expression of cytoskeletal epitopes indicates that these tumors are well-differentiated neuronal neoplasms.