Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immunoperoxidase labeling.

Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirmation of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia. Astrocytic processes were stained in white matter, in the granular layet surrounding synaptic glomerular complexes, and in the molecular layer in the form of radially oriented fibers and of sheaths surrounding Purkinje cell dendrites. Astrocytic endfeet impinging on meninges and perivascular membranes were also antigen positive. In astrocytic perikarya and processes, the immunohistochemical reaction product appears both as a diffuse cytoplasmic label and as elongated strands, which by their distribution and frequency could be considered glial filaments.

Aggregations of 35-nanometer particles associated with neuronal cytopathic changes in natural scrapie.

Neuronal vacuolation and intravacuolar budding of vesicles and cytoplasmic processes appear to be the most characteristic cellular lesion in natural scrapie, a chronic degenerative disease of the central nervous system of sheep which is transmissible by injection. Membrane-bound accumulations of the 35-nm particles are fotnd in the cytoplasmic processes that project inside the vacuoles of the neuronal perikaryon. Such particles are present only in a small number of vacuolated neurons.

Astroglial and axonal proteins in isolated brain filaments. I. Isolation of the glial fibrillary acidic protein and of an immunologically active cyanogen bromide peptide from brain filament preparations of bovine white matter.

The glial fibrillary acidic protein and an immunologically active cyanogen bromide peptide were purified by immunoaffinity chromatography from 8 M urea extracts of brain filament preparations isolated from bovine white matter according to Norton's procedure. The protein accounted for approximately 30% of the total protein in this preparation and for the largest fraction in the 50 000 molecular weight range. The fraction not absorbed to the immuno-Sepharose column reacted with neurofilament antisera by double immunodiffusion. On sodium dodecyl sulfate gel electrophoresis the main bands in the non-adsorbed fraction were at 74 000 daltons and above 100 000. Several bands were seen in the 50 000 molecular weight range. It is concluded that glio- and neurofilaments co-purify together in Norton's procedure and that neurofilaments are probably heterogeneous in polypeptide composition.

Glial fibrillary acidic protein from normal and gliosed human brain. Demonstration of multiple related polypeptides.

An improved purification method for the glial fibrillary acidic protein from normal human brain is reported. Preparations of high purity were obtained by substituting DEAE and phosphocellulose chromatography with one step of hydroxylapatite chromatography. The glial fibrillary acidic protein from normal and gliosed brain was separated into 4 bands (components 1-4) ranging in molecular weight from 54 000 plus or minus 1000 to 40500 plus or minus 1000 by sodium dodecylsulfate gel electrophoresis at 7.5% and 12.5% acrylamide concentration. A better separation of the components was obtained on 12.5% acrylamide gels by increasing the time of electrophoresis to 15-17 h. In these conditions each component was split into a doublet. Preparations identical to those previously reported, i.e. 2-band preparations with an average molecular weight of 43 000, were obtained by incubating multiple sclerosis plaques at 24C for 48 h. These 2-band preparations co-migrated with the 2 lower molecular weight components (component 3, 45 000 plus or minus 1000; component 4, 40 500 plus or minus 1000) in 4-band preparations. The components cross-reacted with antisera against different preparations with an immunodiffusion pattern of complete identity and appeared to be chemically related. Most cyanogen bromide peptides were common to 2-band and 4-band preparations. A unique amino-terminal sequence of alanine-glycine-phenyl-alanine was found in all preparations, regardless of the source and of the number of components. The amino acid composition of 2-band and 4-band preparations was similar.

Interaction of a brain extracellular matrix protein with hyaluronic acid.

A glial hyaluronate-binding protein (GHAP) was isolated from bovine spinal cord and partially characterized. Bovine GHAP consisted of three immunologically related polypeptides with molecular masses of 76, 64, and 54 kDa and isoelectric points of 4.1, 4.2, and 4.4, respectively. Peptide mapping and partial amino acid sequencing showed that all three polypeptides derive from the same protein. The protein was localized immunohistochemically with rabbit antisera in the white matter surrounding the myelinated axons. Sugar analyses indicated that the three polypeptides are glycosylated and the sugar residues account for at least 30% of their weight. After enzymatic deglycosylation, the apparent molecular mass of the bovine GHAP was reduced to 43 kDa. The biochemical properties of bovine GHAP were compared to those of human GHAP. Initial peptide mapping indicated similarities between bovine and human GHAP. Partial amino acid sequencing of bovine GHAP showed a striking identity (up to 90%) with human GHAP and with the hyaluronate binding domain of the large human fibroblast proteoglycan, versican. Bovine and human GHAP were demonstrated to bind specifically to hyaluronic acid (HA) with one protein molecule binding to an average 17 disaccharide repeating units. The binding of bovine and human GHAP was inhibited by oligosaccharides of HA and specifically by the octamer. Salt concentrations of up to 1 M NaCl had very little effect on the binding of the GHAP to HA. The GHAP-HA interaction was pH dependent. Dissociation only took place at low pH (less than 3.5). Analysis of several polypeptides derived from GHAP by limited proteolysis allowed us to conclude that one of the tandem repeated sequences is sufficient for HA binding and that the aminoterminal domain (which contains an immunoglobulin-like fold) is not involved in the GHAP-HA-binding event.

Structural properties of the glial fibrillary acidic protein. Evidence for intermolecular disulfide bonds.

Glial fibrillary acidic protein was purified from 4 M urea extracts of bovine brain by DEAE Bio-Gel A chromatography, 30% ammonium sulfate precipitation and hydroxylapatite chromatography. Subunits of about 54 000 daltons are present in solution as polydisperse distributions of polymers largely constrained by the presence of interchain disulfide linkages. Circular dichroism measurements indicate a native conformation containing some alpha-helical structure. The relevance of these findings to the cytoskeletal function of intermediate (80-100 A) filaments is discussed.

Possible involvement of prolactin in endocrine-resistant metastatic prostate cancer.

The hormone resistance of prostate cancer has been proved to depend at least in part on enhanced neuroendocrine activity and the resultant increase in blood concentrations of chromogranin A. Other experimental observations have suggested the involvement of prolactin (PRL), which appears to be a potential growth factor for prostate cancer. Abnormally high levels of PRL have been detected in metastatic prostate cancer, but the clinical significance of this finding has still to be clarified. In an attempt to explain the prognostic significance of serum PRL levels in prostate cancer, in this preliminary study we have analyzed the PRL levels in a group of metastatic prostate cancer patients with hormone-dependent or hormone-resistant cancer. The study included 50 patients with metastatic prostate cancer, 15 of whom had hormone-resistant tumors. The serum levels of PRL were measured by the RIA method. Abnormally high concentrations of PRL were found in 11/50 (22%) patients. Moreover, the percent of patients with cancer-related hyperprolactinemia was significantly higher in the hormone-resistant group than in the hormone-dependent group (8/15 vs 3/35, p < 0.01). This study confirms the possible existence of a hyperprolactinemic state in metastatic prostate cancer, as previously reported by other authors. Moreover, it appears to demonstrate that the occurrence of hyperprolactinemia is more frequent in hormone-resistant neoplasms, suggesting the possible involvement of PRL in hormone independence. Further studies concomitantly evaluating PRL and chromogranin A blood concentrations will be necessary to establish whether the hyperprolactinemia precedes and promotes the onset of hormone resistance in prostate cancer, or whether it is simply a consequence of the hormone independence.

The favorable prognostic significance of surgery-induced hyperprolactinemia in node-positive breast cancer patients: ten-year disease-free survival results.

It has been shown that each manipulation of the mammary region, including breast surgery, may stimulate prolactin secretion. However, it has also been observed that in more than 50% of breast cancer patients surgical removal of the tumor is not followed by enhanced prolactin secretion. This might be indicative of an altered psychoneuroendocrine control of the mammary gland, which could lead to the onset of more biologically aggressive breast cancer. In fact, surgery-induced hyperprolactinemia has been proven to be associated with a better prognosis in terms of survival in node-negative breast cancer patients. The present study was performed to investigate the impact of postoperative hyperprolactinemia on the disease-free survival (DFS) of breast cancer patients with axillary node involvement. The study included 100 consecutive node-positive breast cancer patients who were followed for at least 10 years. Surgery-induced hyperprolactinemia occurred in 45/100 (45%) patients without any significant correlation with the main prognostic variables including number of involved nodes and ER status. The two groups of patients received the same adjuvant therapies. After a median follow-up of 151 months, the recurrence rate in patients with surgery-induced hyperprolactinemia was significantly lower than in patients with no postoperative hyperprolactinemia (23/45 vs 43/55, p<0.01). Moreover, DFS was significantly longer in hyperprolactinemic patients than in patients who had no enhanced secretion of prolactin postoperatively. In agreement with the results described previously in node-negative breast cancer, our study demonstrates the favorable prognostic significance of surgery-induced hyperprolactinemia in terms of DFS duration also in breast cancer patients with axillary node involvement, independent of the other well-known prognostic variables, thereby confirming that the psychoneuroendocrine status of cancer patients may influence the prognosis of their disease.

Vimentin, the 57 000 molecular weight protein of fibroblast filaments, is the major cytoskeletal component in immature glia.

Comparison of cytoskeletal preparations obtained from newborn and adult rat brain showed similar patterns on SDS-PAGE. However, coelectrophoresis of the newborn and adult preparations revealed distinct differences in the mobility of 2 major bands in the molecular weight range of 50--70 000. In adult brain cytoskeletons, the main band in the 50 000 range co-migrated with purified rat GFA protein (apparent molecular weight 53 000). No major band co-migrated with purified rat vimentin (apparent molecular weight 57 000). The reverse was true for newborn brain cytoskeleton. In adult and newborn brain cytoskeleton a major band co-migrated with the 150 000 neurofilament polypeptide isolated from rat spinal cord by immunoaffinity chromatography. Another neurofilament polypeptide (apparent molecular weight 72 000) was prominent in adult but not in newborn brain cytoskeleton. Conversely, newborn brain cytoskeleton comprised a band trailing behind the 72 000 neurofilament polypeptide. This band was not present in adult brain cytoskeleton. The distribution of vimentin in newborn rat brain was studied by immunofluorescence microscopy and compared to the distribution of GFA protein. As previously reported, a relatively limited number of GFA positive cells are present in the brain at this stage compared to later in development. Conversely, the large number of vimentin positive cells in newborn brain was well in keeping with the presence of a prominent vimentin band in cytoskeletal preparations obtained from this tissue. With the exception of meninges and blood vessels, vimentin appeared to be mainly localized in immature glia: periventricular glia; glia in non-myelinated white matter; radial glia in cerebral cortex and basal ganglia; Bergmann glia in cerebellum (Bergmann glia are still GFA negative in newborn rat). The neuroblastic germinal layers in hippocampus and cerebellum did not stain with vimentin antisera.

Laminin in rat sciatic nerve undergoing Wallerian degeneration. Immunofluorescence study with laminin and neurofilament antisera.

Immunofluorescence with laminin antisera revealed a striking change in the localization of this basal membrane glycoprotein in rat sciatic nerve as a result of Wallerian degeneration. The staining was confined to the endoneurium in normal sciatic nerve and during the first days of degeneration. On day 11 endoneurial tubes were no longer identified in the distal stump of crushed nerves or of nerves that had been transected and tightly ligated to prevent regeneration. In both crushed and ligated nerves proliferating Schwann cells forming the cell-bands of Büngner were intensely laminin positive. With double-labeling experiments, laminin and neurofilament antisera revealed similar but not identical staining patterns in crushed nerves, which suggests a close relation between laminin and regenerating axons. Crushed nerves had recovered their normal appearance 18 days after operation while anti-laminin reactivity was decreased in parts of ligated nerves undergoing fibrosis. The localization of laminin in reactive Schwann cells was confirmed by electron microscopy using the indirect immunoperoxidase procedure. Axons did not contain reaction product.

Glial hyaluronate-binding protein in polar spongioblastoma.

Glial hyaluronate-binding (GHA) protein is a 60 kDa glycoprotein isolated from human white matter by affinity chromatography on immobilized hyaluronate. It is localized in white matter astrocytes by immunofluorescence with monoclonal antibodies. Amino acid sequences have not revealed similarities with other proteins except cartilage extracellular matrix proteins, the region of similarity being located within the hyaluronate-binding region. Cryostat sections of 13 intracranial neoplasms removed at surgery were tested for the presence of GHA protein by indirect immunofluorescence with monoclonal antibodies. These included seven astrocytomas, one oligodendroglioma, one medulloblastoma and one spinal cord ependymoma. All tumors were negative with the exception of one astrocytoma in which the GHA protein-positive areas had the typical appearance of polar spongioblastoma, i.e. small cells palisading around blood vessels and very delicate glial fibrillary acidic (GFA) protein-positive fibrils. Conversely, neoplastic as well as reactive GFA protein-positive astrocytes were GHA protein-negative. We suggest that polar spongioblastoma derives from a GHA protein-positive glial precursor and pertinent to this suggestion is the observation that the periventricular germinal layer was found GHA protein-positive in a 22-week human fetus.

The fate of axonal debris in Wallerian degeneration of rat optic and sciatic nerves. Electron microscopy and immunofluorescence studies with neurofilament antisera.

Immunofluorescence demonstrated that axonal debris reacting with neurofilament antisera persist up to 4 months in rat optic nerves undergoing Wallerian degeneration. Antisera used in this study allowed the isolation of the 72,000- and 150,000-dalton neurofilament polypeptides from rat spinal cord by immunoaffinity chromatography. After 2 weeks of degeneration, proteins co-migrating with these neurofilament polypeptides were no longer identifiable in rat optic nerves, which suggests that immunofluorescent structures persisting in the nerves after this period contained neurofilament degradation products of different molecular weight. Additional evidence as to the persistence of axonal debris in degenerated optic nerves was obtained by electron microscopy. Two distinct types of axonal degeneration were observed in rat optic nerves by this method, floccular swelling and increased electron density of the axoplasm. In both types of degeneration, axoplasmic filaments and tubules were not identifiable. Although floccular material disappeared after 2 weeks of degeneration, so that only empty myelin sheaths remained, electron-dense axons persisted longer and were probably phagocytosed together with their myelin sheaths. In sciatic nerves, cross-reaction with neurofilament antisera had almost completely disappeared 10 days after transection. The same was true for nerves which had been tightly ligated to prevent axonal growth and to squeezed nerves which showed vigorous regeneration. A few scattered, brightly immunofluorescent fragments which persisted in nerves up to 2 weeks after transection were exception to these findings.

Preparation of antisera to neurofilament protein from chicken brain and human sciatic nerve.

Antigens isolated by hydroxyapatite chromatography from human sciatic nerve (SN1 protein) and from 8 M urea extracts of chicken brain were selectively localized by immunofluorescence to neurofibrils in rat and chicken CNS. Absorption of the antisera with SN1 protein, chicken antigen or GFA protein abolished the staining. Antisera raised against antigen isolated with the same procedure from buffer extracts of chicken brain stained both neurofibrils and glial fibrils by immunofluorescence. Neurofibrillary staining was selectively abolished by absorption of the antisera with SN1 protein. Antisera prepared against axonal preparations isolated from bovine white matter only stained astroglia and were thus undistinguishable from anti-GFA sera in this respect. The data suggested that the protein subunits of neurofilament and glial filaments, although difficult to separate in brain extracts by standard biochemical procedures and by subcellular fractionation in bovine white matter, still retain immunological specificity. In addition, the immunological cross reactivity between human and chicken antigens suggested that neurofilaments, as other constituents of the cytoskeleton such as microtubules and actin microfilaments, show a high degree of evolutionary stability.

Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies.

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet. Of these nine antibodies, two stained motor neuron perikarya in the spinal cord, while the remaining seven antibodies were axon-specific in this location. Subpopulations of stained and unstained motor neurons were not observed. With all 12 antibodies, the staining pattern in the lumbar dorsal root ganglia and spinal cord remained unchanged following sciatic nerve crush and ligature. The findings suggest that, in the neurofilament, some phosphorylated epitopes are axon specific, while other phosphorylated epitopes are present in both axons and perikarya. Furthermore, they suggest that differences exist between neuronal populations as to the presence of phosphorylated epitopes in perikaryal neurofilaments. It remains to be seen whether phosphorylation events in perikarya and axons have similar or different effects on neurofilament structure and function.

Neurofilament phosphorylation in neuronal perikarya following axotomy: a study of rat spinal cord with ventral and dorsal root transection.

Rat spinal cord was stained by indirect immunofluorescence with 11 neurofilament monoclonal antibodies that recognize phosphorylated epitopes. All monoclonals were axon-specific in this location. The large motoneurons containing bundles of neurofilaments did not stain and the pattern remained unchanged after transection of the sciatic nerve in the thigh. With nine monoclonals, stained motoneurons were observed in the ventral horns 3 days, 5 days, 1 week, and 2 weeks after transection of the ventral roots close to the spinal cord. The abnormal motoneurons were typically scattered among normal (i.e., nonstained) cells. Even in animals showing the most severe reaction, the whole motoneuron population at the site of rhizotomy was not affected, stained and nonstained perikarya often coexisting side by side. Stained motoneurons were no longer observed 3 weeks after ventral root transection. Changes in neuronal immunoreactivity were also observed after dorsal root transection. However, a different population was affected, i.e., middle-sized neurons in dorsal horns and at the base of ventral horns. With two monoclonals (A9 and D21), cell bodies remained negative following all operations. It is concluded that axotomy in proximity of the cell body may induce certain neurofilament phosphorylation events in motor neuron perikarya, whereas other phosphorylation events remain confined to the axons under these experimental conditions. The absence of changes after transection of the sciatic nerve in the thigh suggests that neurofilament phosphorylation is a reaction to cell injury rather than a cellular event related to nerve regeneration.

Neurofilament proteins in fish: a study with monoclonal antibodies reacting with mammalian NF 150K and NF 200K.

Monoclonal antibodies were obtained upon immunization of mice with chicken brain antigen and with the two high molecular weight neurofilament proteins (NF 150K and NF 200K) isolated from bovine spinal cord by anion exchange chromatography. By the immunoblotting procedure, the antibodies selected for this study reacted with bovine NF 150K and NF 200K. By the same procedure the antibodies reacted with sea raven, goldfish, sea bass, shark, and trout spinal cord extracts. In goldfish and sea raven the antibodies stained a single band at approximately 150 kDa and 200 kDa, respectively. Two bands were stained in the shark, sea bass, and trout. In the shark and sea bass these bands were in the molecular weight range of mammalian NF 150K and NF 200K. In the trout the upper band was approximately 150 kDa and the lower band 130 kDa. Our findings suggest an early origin of NF 150K and NF 200K in vertebrate phylogeny as well as considerable divergence in several species.

Immunohistochemical localization of the 150K neurofilament protein in the rat and the rabbit.

Antisera to the 150K-dalton polypeptide of the bovine neurofilament triplet and chicken neurofilament antisera reacting with the 70K protein in isolated bovine brain filaments stained the same structures in rat cerebellum by immunofluorescence and peroxidase-antiperoxidase methods, that is Purkinje cell baskets, thin nerve fibers in the lower half of the molecular layer and myelinated axons. The 150K bovine neurofilament antisera did not stain large motor neurons in the anterior horns of the spinal cord in rat and rabbit, nor aluminum-induced neurofibrillary tangles in the rabbit. All these structures were demonstrated by the chicken neurofilament antisera and by silver neurofibrillary methods. IDPN-induced axonal balloons containing accumulations of neurofilaments were equally well stained by bovine 150K and chicken neurofilament antisera. These data suggest that the 150K polypeptide of the neurofilament triplet is not a subunit of the neurofilament core and probably plays a role in axonal transport.

Glial fibrillary acidic (GFA) protein in vertebrates: immunofluorescence and immunoblotting study with monoclonal and polyclonal antibodies.

We report a comparative immunofluorescence and immunoblotting study of GFA protein, the subunit of glial filaments, in nonmammalian vertebrates. The study was conducted with polyclonal antibodies raised to human and shark antigen and with monoclonal antibodies isolated from mice immunized with chicken and bovine antigen. With the exception of cyclostomes, glial filaments appeared remarkably conserved in vertebrate phylogeny, both with respect to the molecular weight and immunoreactivity of their protein subunit. In most species, the antibodies decorated a single band in brain, spinal cord, and optic nerve extracts by the immunoblotting procedure. This band had the same molecular weight in the different CNS regions. With the exception of the turtle, species differences in the molecular weight of the band were not greater than those observed among mammalian vertebrates (human, bovine, and rat). However, there were some exceptional findings in fish. In goldfish and trout brain and spinal cord extracts, the antibodies decorated with the same intensity two bands. In accordance with previous immunofluorescence findings, goldfish optic nerve extracts were negative by the immunoblotting procedure. In four fishes (sea bass, tautog, trout, and scup), optic nerves reacted with the antibodies. However, the band decorated by the antibodies was higher in molecular weight than that obtained from brain and spinal cord extracts. Glial fibers were demonstrated by immunofluorescence in the brain, spinal cord, optic nerve, and retina of most species studied. In amphibia immunofluorescent structures were comparatively few, probably accounting for the negative results by immunoblotting. A comparative immunohistological study of the cerebellum showed the presence of perpendicular glial fibers in the molecular layer of most species examined. Birds and amphibia were different in this respect. Bergmann glia in chicken were GFA negative. In the frog and the toad, immunofluorescent fibers in the molecular layer of the cerebellum were haphazardly oriented. Ependymal radial glia was GFA-negative in the cerebellum of subavian vertebrates. Antisera raised in rabbit to shark GFA protein reacted with the same bovine GFA fragments recognized by polyclonal and monoclonal antibodies raised to human and bovine antigens, respectively, i.e., 30-kDa N-bromosuccinimide fragment (tryptophan cleavage); 35-kDa 2-nitro-5-thiocyanobenzoic acid fragment (cysteine cleavage); 18-kDa cyanogen bromide fragment (methionine cleavage). Conversely, the chicken GFA monoclonal antibodies selected for this study only reacted with noncleaved protein.

Two spinal cord lesions in a patient with ankylosing spondylitis and cervical spine injury.

A patient with ankylosing spondylitis sustained C3-C4 vertebral subluxation and C4-C5 myelopathy after a hyperextension trauma. Autopsy showed that several segments below the main cervical cord lesion at the fractured site, there was a second spinal cord lesion at the T1 vertebral level with no corresponding local bony or ligamentous damage. The thoracic cord lesion was probably secondary to traction of the upper thoracic cord, where the blood supply is poor, in a narrow and rigid spinal canal at the moment of extreme hyperextension.