E. M. Rodríguez

Ependymal denudation and alterations of the subventricular zone occur in human fetuses with a moderate communicating hydrocephalus

In mutant rodents, ependymal denudation occurs early in fetal life, preceding the onset of a communicating hydrocephalus, and is a key event in the etiology of this disease. The present investigation was designed to obtain evidence whether or not ependymal denudation occurs in 16- to 40-week-old human fetuses developing a communicating hydrocephalus (n = 8) as compared to fetuses of similar ages with no neuropathologic alterations (n = 15). Sections through the walls of the cerebral aqueduct and lateral ventricles were processed for lectin binding and immunocytochemistry using antibodies against ependyma, astroglia, neuroblasts, and macrophages markers. Anti-caveolin was used as a functional marker of the fetal ependyma. The structural and functional molecular markers are differentially expressed throughout the differentiation of the human fetal ependyma. Denudation of the ependyma of the aqueduct and lateral ventricles occurred in all fetuses developing a communicating hydrocephalus, including the youngest ones studied. The denuded surface area increased in parallel with the fetus age. The possibility is advanced that in many or most cases of human fetal hydrocephalus there is a common defect at the ependymal cell lineage leading to ependymal detachment. Evidence was obtained that in hydrocephalic human fetuses a process to repair the denuded areas takes place during the fetal life. In hydrocephalic fetuses, detachment of the ependyma of the lateral ventricles resulted in the (i) loss of the germinal ependymal zone, (ii) disorganization of the subventricular zone and, (iii) abnormal migration of neuroblasts into the ventricular cavity. Thus, detachment of the ependymal layer in hydrocephalic fetuses would not only be associated with the pathogenesis of hydrocephalus but also to abnormal neurogenesis.

Spontaneous congenital hydrocephalus in the mutant mouse hyh. Changes in the ventricular system and the subcommissural organ

The subcommissural organ is an ependymal gland located at the entrance of the cerebral aqueduct. It secretes glycoproteins into the cerebrospinal fluid, where they aggregate to form Reissners fiber. This fiber grows along the aqueduct, fourth ventricle, and central canal. There is evidence that the subcommissural organ is involved in the pathogenesis of congenital hydrocephalus. This organ was investigated in the mutant mouse hyh developing a congenital hydrocephalus. The central nervous system of normal and hydrocephalic hyh mice, 1 to 40 days old, was investigated using antibodies recognizing the subcommissural organ secretory glycoproteins, and by transmission and scanning electron microscopy. At birth, the affected mice displayed open communications between all ventricles, absence of a central canal in the spinal cord, ependymal denudation of the ventricles, stenosis of the rostral end of the aqueduct, and hydrocephalus of the lateral and third ventricles and of the caudal end of the aqueduct. Around the 5th postnatal day, the communication between the caudal aqueduct and fourth ventricle sealed, and hydrocephalus became severe. It is postulated that the hyh mice carry a genetic defect affecting the ependymal cell lineage. The subcommissural organ showed signs of increased secretory activity; it released to the stenosed aqueduct a material that aggregated, but it did not form a Reissners fiber. A large area of the third ventricular wall differentiated into a secretory ependyma synthesizing a material similar to that secreted by the subcommissural organ. It is concluded that the subcommissural organ changes during hydrocephalus; whether these changes preceed hydrocephalus needs to be investigated.

Secretory glycoproteins of the rat subcommissural organ are N-linked complex-type glycoproteins. Demonstration by combined use of lectins and specific glycosidases, and by the administration of Tunicamycin.

SummaryTwo experimental protocols were used to investigate the secretory glycoproteins of the subcommissural organ (SCO).nProtocol I: Lectins, specific exoglycosidases and immunocytochemistry were sequentially applied to the same section or to adjacent semithin sections of the rat SCO fixed in Bouins fluid and embedded in methacrylate. Lectins used: concanavalin A (con A), wheat germ agglutinin, Limulus polyphemus agglutinin, Ricinus communis agglutinin and Arachis hypogeae agglutinin. Glycosidases used: neuroaminidase, β-galactosidase, α-mannosidase, α-glucosidase and β-N-acetyl-glucosaminidase. For immunocytochemistry an antiserum against bovine Reissners fiber (AFRU) was used. Lectins and glycosidases were used in sequences that allowed the cleaved sugar residue to be identified as well as that appearing exposed as a terminal residue. This approach led to the following conclusions: (1) the terminal sugar chain of the secreted glycoproteins has the sequence sialic acid-galactose-glucosamine-; (2) the con A-binding material present in the rough endoplasmic reticulum corresponds to mannose; (3) the apical secretory granules and Reissners fibers displayed a strong con A affinity after removing sialic acid, thus indicating the presence of internal mannosyl residues in the secreted material; (4) after removing most of the sugar moieties the secretory material continued to be strongly immunoreactive with AFRU. Protocol II: Rats were injected into the lateral ventricle with Tunicamycin and killed 12, 24, 50 and 60 h after the injection. The SCO of rats from the last two groups showed a complete absence of con A binding sites. The results from the two experiments confirm that the secretory glycoproteins of the rat SCO are N-linked complex-type glycoproteins with the conformation previously suggested (Rodríguez et al. 1986).

Immunocytochemical study of the subcommissural organ of rats with induced postnatal hydrocephalus

SummaryThe subcommissural organ (SCO)-Reissners fiber (RF) complex of rats suffering from postnatal hydrocephalus was investigated immunocytochemically (peroxidase-antiperoxidase technique) by use of an antiserum against bovine RF. Hydrocephalus was induced by injecting kaolin into the cisterna magna or by intracerebral infection with Borna disease virus. The kaolin-injected, hydrocephalie male rats were divided into two groups: (1) possessing an open communication between the fourth ventricle and the central canal of the spinal cord; (2) enduring an obliteration of this communication. In the latter group of rats the dilation of the ventricular cavities was far greater than in the former group. The Borna disease virus-infected female rats developed a severe hydrocephalus although in these animals all ventricular cavities and the central canal were in fully open communication. All rats belonging to the above-mentioned three groups displayed essentially the same alterations of their SCO-RF complex: (i) A reduction in the size of SCO and in the height of the ependymal secretory cells. (ii) A progressive disappearance of the immunoreactive hypendymal cells. (iii) The amount of AFRU-immunoreactive secretory material located in the rough endoplasmic reticulum was reduced. (iv) In contrast, the amount, location and immunoreactivity of the apical secretory granules did not undergo variations in comparison to sham-operated rats. (v) In the area of the SCO the layer of pre-RF material was thin or missing and a RF was not formed, and thus the central canal was also free of such secretory products. (vi) Clusters of AFRU-immunoreactive material were found attached to the wall of the Sylvian aqueduct. It is concluded that in the presented types of hydrocephalus: (i) the secretory material stored in the SCO is partially depleted, thus indicating a probably increased turnover of this material; (ii) the SCO continues to secrete into the ventricle; and (iii) unknown factors prevent the assembly of the released secretory material into the characteristic thread-like structure of the RF.

Astrocytes acquire morphological and functional characteristics of ependymal cells following disruption of ependyma in hydrocephalus

Hydrocephalic hyh mutant mice undergo a programmed loss of the neuroepithelium/ependyma followed by a reaction of periventricular astrocytes, which form a new cell layer covering the denuded ventricular surface. We present a comparative morphological and functional study of the newly formed layer of astrocytes and the multiciliated ependyma of hyh mice. Transmission electron microscopy, immunocytochemistry for junction proteins (N-cadherin, connexin 43) and proteins involved in permeability (aquaporin 4) and endocytosis (caveolin-1, EEA1) were used. Horseradish peroxidase (HRP) and lanthanum nitrate were used to trace the intracellular and paracellular transport routes. The astrocyte layer shares several cytological features with the normal multiciliated ependyma, such as numerous microvilli projected into the ventricle, extensive cell–cell interdigitations and connexin 43-based gap junctions, suggesting that these astrocytes are coupled to play an unknown function as a cell layer. The ependyma and the astrocyte layers also share transport properties: (1) high expression of aquaporin 4, caveolin-1 and the endosome marker EEA1; (2) internalization into endocytic vesicles and early endosomes of HRP injected into the ventricle; (3) and a similar paracellular route of molecules moving between CSF, the subependymal neuropile and the pericapillary space, as shown by lanthanum nitrate and HRP. A parallel analysis performed in human hydrocephalic foetuses indicated that a similar phenomenon would occur in humans. We suggest that in foetal-onset hydrocephalus, the astrocyte assembly at the denuded ventricular walls functions as a CSF–brain barrier involved in water and solute transport, thus contributing to re-establish lost functions at the brain parenchyma–CSF interphase.

Hydrocephalus induced by immunological blockage of the subcommissural organ-Reissner's fiber (RF) complex by maternal transfer of anti-RF antibodies

Abstract. Stenosis of the cerebral aqueduct seems to be a key event for the development of congenital hydrocephalus. The causes of such a stenosis are not well known. Overholser et al. in 1954 (Anat Rec 120:917–933) proposed the hypothesis that a dysfunction of the subcommissural organ (SCO) leads to aqueductal stenosis and congenital hydrocephalus. The SCO is a brain gland, located at the entrance of the cerebral aqueduct, that secretes glycoproteins into the cerebrospinal fluid that, upon release, assemble into a fibrous structure known as Reissners fiber (RF). By the permanent addition of new molecules to its rostral end, RF grows and extends along the aqueduct, fourth ventricle, and central canal of the spinal cord. The immunological blockage of the SCO-RF complex has been used to test Overholsers hypothesis. The following was the sequence of events occurring in pregnant rats that had been immunized with RF glycoproteins: the mother produced anti-RF antibodies and transferred them to the fetus through the placenta and to the pup through the milk, and the antibodies reached the brain of the fetus and pup and blocked the SCO-RF complex. This resulted in a permanent absence of RF that was followed by stenosis of the cerebral aqueduct, and then by the appearance of hydrocephalus. The latter was patent until the end of the 6-month observation period. The chronic hydrocephalic state appeared, in turn, to induce new alterations of the SCO. It is concluded that a selective immunological knock out of the SCO-RF complex leads to hydrocephalus.

PARTIAL SEQUENCING OF REISSNER'S FIBER GLYCOPROTEIN I (RF-GLY I)

Abstractu2002The bulk of the secretion of the subcommissural organ is formed by glycoproteins that appear to be derived from two precursor forms of 540 and 320 kDa. Upon release into the ventricle, these glycoproteins aggregate to form Reissner’s fiber. We report the isolation of three cDNA clones from a cDNA library prepared from bovine subcommissural organ RNA, by using an anti-Reissner’s fiber serum for immunoscreening. Inserts of 0.7, 1.2, and 2.5 kb were amplified by the polymerase chain reaction, subcloned into pUC18 vector, and sequenced. Although restriction mapping of the three inserts initially suggested that all of them were derived from the same mRNA, sequence analysis showed that a short non-homologous region was present in the 0.7-kb insert when compared with the 1.2-kb and 2.5-kb inserts, suggesting that they corresponded to two different, although highly homologous, mRNAs. Northern analyses showed a single mRNA species of approximately 9.5 kb present in the subcommissural organ and missing in the choroid plexus, brain cortex, and liver. In situ hybridization confirmed that the expression of the RNA was restricted to cells of the bovine subcommissural organ. Polyclonal antibodies raised against a synthetic peptide, whose amino-acid sequence was deduced from the 2.5-kb cDNA, reacted specifically with the bovine and rat subcommissural organ-Reissner’s fiber complex. In immunoblots of bovine subcommissural organ, this antibody revealed the precursor 540-kDa form and its putative processed form of 450 kDa. It is concluded that the cloned cDNA encodes for the major constitutive glycoprotein of Reissner’s fiber, here designated as RF-Gly I. The sequenced region of RF-Gly I displays a high degree of homology with some regions of the von Willebrand factor and certain mucins; it also displays two motifs homologous with repeats present in proteins of the spondin family and other proteins. A core sequence of the RF-Gly I repeats suggests that this molecule displays protein-binding properties.

Synthesis of transthyretin by the ependymal cells of the subcommissural organ

Transthyretin (TTR) is a protein involved in the transport of thyroid hormones in blood and cerebrospinal fluid (CSF). The only known source of brain-produced TTR is the choroid plexus. In the present investigation, we have identified the subcommissural organ (SCO) as a new source of brain TTR. The SCO is an ependymal gland that secretes glycoproteins into the CSF, where they aggregate to form Reissner’s fibre (RF). Evidence exists that the SCO also secretes proteins that remain soluble in the CSF. To investigate the CSF-soluble compounds secreted by the SCO further, antibodies were raised against polypeptides partially purified from fetal bovine CSF. One of these antibodies (against a 14-kDa compound) reacted with secretory granules in cells of fetal and adult bovine SCO, organ-cultured bovine SCO and the choroid plexus of several mammalian species but not with RF. Western blot analyses with this antibody revealed two polypeptides of 14xa0kDa and 40xa0kDa in the bovine SCO, in the conditioned medium of SCO explants, and in fetal and adult bovine CSF. Since the monomeric and tetrameric forms of TTR migrate as bands of 14xa0kDa and 40xa0kDa by SDS-polyacrylamide gel electrophoresis, a commercial preparation of human TTR was run, with both bands being reactive with this antibody. Bovine SCO was also shown to synthesise mRNA encoding TTR under in vivo and in vitro conditions. We conclude that the SCO synthesises TTR and secretes it into the CSF. Colocalisation studies demonstrated that the SCO possessed two populations of secretory cells, one secreting both RF glycoproteins and TTR and the other secreting only the former. TTR was also detected in the SCO of bovine embryos suggesting that this ependymal gland is an important source of TTR during brain development.

Immunochemical analysis of the subcommissural organ-Reissner's fiber complex using antibodies against alkylated and deglycosylated glycoproteins of the bovine Reissner's fiber.

Abstract.Reissner’s fiber (RF) has been isolated, solubilized, and used to raise polycloncal antibodies. The present investigation has been designed: (1) to obtain antibodies against RF-glycoproteins in their native form (anti-RF-BI), after irreversible denaturation by alkylation (anti-RF-A), and after alkylation and deglycosylation by using endoglycosidase F (anti-RF-DE); (2) to use these antisera for a comparative immunocytochemical study of the subcommissural organ (SCO)-RF complex; (3) to establish the molecular mass of the deglycosylated RF-glycoproteins. Anti-RF-BI reacts with the SCO of all the species investigated. Anti-RF-A and anti-RF-DE only react with bovine SCO and RF. The three antisera stain the same bands in immunoblots of extracts of bovine SCO and RF, but anti-RF-A and anti-RF-DE reveal additional bands. The epitope common to all species reacting with anti-RF-BI is thus probably conformational in nature and associated with the integrity of the disulfide bonds. The lack of antibodies against conserved sequential epitopes in anti-RF-A does not support previous assumptions on the conserved nature of the SCO secretion. After deglycosylation by using endoglycosidase F, the RF-glycoproteins present a reduction in their molecular mass ranging between 10% and 25%. The three larger compounds (450, 300, and 230 kDa) lose their affinity for Limax flavus agglutinin (affinity=∧sialic acid), whereas the 190-kDa compound (170 kDa after deglycosylation) keeps its affinity for this lectin suggesting that it has N-linked and O-linked carbohydrate moieties, the three larger proteins probably having only N-linked carbohydrates.

Light and electron microscopical demonstration of concanavalin A and wheat-germ agglutinin binding sites by use of antibodies against the lectin or its label (peroxidase)

SummaryThree straining protocols for the ultrastructural visualization of concanavalin A (ConA) and wheat germ agglutinin (WGA) binding sites were applied to samples of nervous tissue embedded in Lowicryl K4M. The hypothalamo-neurohypophysial neurosecretory system was chosen for this investigation because it has two major neuronal populations, one secreting vasopressin, whose precursor is glycosylated, and the other secreting oxytocin whose precursor form is not glycosylated.The series of incubations of the tissue sections for the three protocols were: Protocol 1: i) non labeled ConA or WGA; ii) ConA or WGA antibody; iii) protein A-gold; Protocol 2: i) pre-prepared WGA-anti-WGA complex; ii) protein A-gold; Protocol 3: i) peroxidase-labeled ConA or WGA; ii) anti-peroxidase; iii) protein A-gold.The three methods allowed to detect fine differences in the distribution of sugar residues. This, in turn, made it possible to distinguish vasopressin granules containing precursor forms from those containing processed precursor.At the light microscopic level the three methods were successfully applied to paraffin and 1-μm methacrylate sections by using a second antibody, PAP complex and the diaminobenzidine reaction.