IIIG9 inhibition in adult ependymal cells changes adherens junctions structure and induces cellular detachment.

Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord. In cancer, the loss of ependymal cell polarity promotes the formation of different types of tumors, such as supratentorial anaplastic ependymomas, which are highly aggressive in children. IIIG9 (PPP1R32) is a protein restricted to adult ependymal cells located in cilia and in the apical cytoplasm and has unknown function. In this work, we studied the expression and localization of IIIG9 in the adherens junctions (cadherin/ß-catenin-positive junctions) of adult brain ependymal cells using confocal and transmission electron microscopy. Through in vivo loss-of-function studies, ependymal denudation (single-dose injection experiments of inhibitory adenovirus) was observed, inducing the formation of ependymal cells with a "balloon-like" morphology. These cells had reduced cadherin expression (and/or delocalization) and cleavage of the cell death marker caspase-3, with "cilia rigidity" morphology (probably vibrational beating activity) and ventriculomegaly occurring prior to these events. Finally, after performing continuous infusions of adenovirus for 14 days, we observed total cell denudation and reactive parenchymal astrogliosis. Our data confirmed that IIIG9 is essential for the maintenance of adherens junctions of polarized ependymal cells. Eventually, altered levels of this protein in ependymal cell differentiation may increase ventricular pathologies, such as hydrocephalus or neoplastic transformation.

Disruption of CDH2/N-cadherin-based adherens junctions leads to apoptosis of ependymal cells and denudation of brain ventricular walls.

Disruption/denudation of the ependymal lining has been associated with the pathogenesis of various human CNS disorders, including hydrocephalus, spina bifida aperta, and periventricular heterotopia. It has been traditionally considered that ependymal denudation is a consequence of mechanical forces such as ventricular enlargement. New evidence indicates that ependymal disruption can precede ventricular dilation, but the cellular and molecular mechanisms involved in the onset of ependymal denudation are unknown. Here, we present a novel model to study ependymal cell pathophysiology and demonstrate that selective disruption of N-cadherin-based adherens junctions is sufficient to provoke progressive ependymal denudation. Blocking N-cadherin function using specific peptides that interfere with the histidine-alanine-valine extracellular homophilic interaction domain caused early pathologic changes characterized by disruption of zonula adherens and abnormal intracellular accumulation of N-cadherin. These changes then triggered massive apoptosis of ependymal cells and denudation of brain ventricular walls. Because no typical extrinsic mechanical factors such as elevated pressure or stretching forces are involved in this model, the critical role of N-cadherin-based adherens junctions in ependymal survival/physiology is highlighted. Furthermore, the results suggest that abnormal adherens junctions between ependymal cells should be considered as key components of the pathogenesis of CNS disorders associated with ependymal denudation.

New ependymal cells are born postnatally in two discrete regions of the mouse brain and support ventricular enlargement in hydrocephalus.

A heterogeneous population of ependymal cells lines the brain ventricles. The evidence about the origin and birth dates of these cell populations is scarce. Furthermore, the possibility that mature ependymal cells are born (ependymogenesis) or self-renewed (ependymal proliferation) postnatally is controversial. The present study was designed to investigate both phenomena in wild-type (wt) and hydrocephalic α-SNAP mutant (hyh) mice at different postnatal stages. In wt mice, proliferating cells in the ventricular zone (VZ) were only found in two distinct regions: the dorsal walls of the third ventricle and Sylvian aqueduct (SA). Most proliferating cells were monociliated and nestin+, likely corresponding to radial glial cells. Postnatal cumulative BrdU-labeling showed that most daughter cells remained in the VZ of both regions and they lost nestin-immunoreactivity. Furthermore, some labeled cells became multiciliated and GLUT-1+, indicating they were ependymal cells born postnatally. Postnatal pulse BrdU-labeling and Ki-67 immunostaining further demonstrated the presence of cycling multiciliated ependymal cells. In hydrocephalic mutants, the dorsal walls of the third ventricle and SA expanded enormously and showed neither ependymal disruption nor ventriculostomies. This phenomenon was sustained by an increased ependymogenesis. Consequently, in addition to the physical and geometrical mechanisms traditionally explaining ventricular enlargement in fetal-onset hydrocephalus, we propose that postnatal ependymogenesis could also play a role. Furthermore, as generation of new ependymal cells during postnatal stages was observed in distinct regions of the ventricular walls, such as the roof of the third ventricle, it may be a key mechanism involved in the development of human type 1 interhemispheric cysts.

Impact of very old age on the expression of cervical spinal cord cell markers in rats.

Aging is a process associated with both anatomical changes and loss of expression of some cell markers. Intermediate filaments are known to impart mechanical stability to cells and tissues. Some of them are present in different cell populations of the central nervous system. In order to explore the impact of extreme age we immunohistochemically characterized the changes in intermediate filaments and other cellular markers present in cells populating the gray matter cervical spinal cord of very old rats (28 months) taking young (5 months) counterparts as a reference. The spinal cord weight of the senile animals (12.6+/-1.1 g) was significantly higher (P<0.001) than that of the young animals (8.4+/-1.1 g). Spinal cord length also increased significantly (P<0.05) with age (7.9+/-0.3 cm vs. 8.28+/-0.1 cm for young and senile, respectively). An increase in both neurofilament staining area and density was observed in senile rats in comparison to young animals. A significant (P<0.05) age-related increment in the mean area of the cervical segments was observed. Vimentin expression in the ependymal zone decreased in area and intensity during aging. Our data show that there are some significant changes in the morphological and histochemical patterns of the cervical spinal cord in senile rats. However, they do not necessarily represent a pathologic situation and may rather reflect plastic reorganization.

Sodium vitamin C cotransporter SVCT2 is expressed in hypothalamic glial cells.

Kinetic analysis of vitamin C uptake demonstrated that different specialized cells take up ascorbic acid through sodium-vitamin C cotransporters. Recently, two different isoforms of sodium-vitamin C cotransporters (SVCT1/SLC23A1 and SVCT2/SLC23A2) have been cloned. SVCT2 was detected mainly in choroidal plexus cells and neurons; however, there is no evidence of SVCT2 expression in glial and endothelial cells of the brain. Certain brain locations, including the hippocampus and hypothalamus, consistently show higher ascorbic acid values compared with other structures within the central nervous system. However, molecular and kinetic analysis addressing the expression of SVCT transporters in cells isolated from these specific areas of the brain had not been done. The hypothalamic glial cells, or tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid with different neurons of the region. Our hypothesis postulates that SVCT2 is expressed selectively in tanycytes, where it is involved in the uptake of the reduced form of vitamin C (ascorbic acid), thereby concentrating this vitamin in the hypothalamic area. In situ hybridization and optic and ultrastructural immunocytochemistry showed that the transporter SVCT2 is highly expressed in the apical membranes of mouse hypothalamic tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of the SVCT2 isoform in these cells. The results demonstrate that tanycytes express a high-affinity transporter for vitamin C. Thus, the vitamin C uptake mechanisms present in the hypothalamic glial cells may perform a neuroprotective role concentrating vitamin C in this specific area of the brain.

Cellular mechanisms involved in the stenosis and obliteration of the cerebral aqueduct of hyh mutant mice developing congenital hydrocephalus.

Two phases may be recognized in the development of congenital hydrocephalus in the hyh mutant mouse. During embryonic life the detachment of the ventral ependyma is followed by a moderate hydrocephalus. During the first postnatal week the cerebral aqueduct becomes obliterated and a severe hydrocephalus develops. The aim of the present investigation was to elucidate the cellular phenomena occurring at the site of aqueduct obliteration and the probable participation of the subcommissural organ in this process. Electron microscopy, immunocytochemistry, and lectin histochemistry were used to investigate the aqueduct of normal and hydrocephalic hyh mice from embryonic day 14 (E-14) to postnatal day 7 (PN-7). In the normal hyh mouse, the aqueduct is an irregularly shaped cavity with 3 distinct regions (rostral, middle, and caudal) lined by various types of ependyma. In the hydrocephalic mouse, these 3 regions behave differently; the rostral end becomes stenosed, the middle third dilates, and the caudal end obliterates. The findings indicate that the following sequence of events lead to hydrocephalus: 1) denudation of the ventral ependyma (embryonic life); 2) denudation of dorsal ependyma and failure of the subcommissural organ to form Reissner fiber (first postnatal week); 3) obliteration of distal end of aqueduct; and 4) severe hydrocephalus. No evidence was obtained that NCAM is involved in the detachment of ependymal cells. The process of ependymal denudation would involve alterations of the surface sialoglycoproteins of the ependymal cells and the interaction of the latter with macrophages.

The pineal recess of the opossum: a scanning and transmission electron microscope study.

Scanning and transmission electron microscope studies were performed in the pineal recess region of eighteen adult Didelphis albiventris opossums. With the scanning electron microscope, three concentric regions could be observed in the pineal recess: a central zone lined by cells free of cilia or microvilli, or by a meshwork of interdigitating cell processes; an intermediate zone lined by microvillous cells; and a peripheral zone, lined by ciliated cells. The transmission electron microscope revealed that the central zone is lined mainly by pinealocytes. The lining cells are frequently overlaid by a meshwork of pinealocytic and glial processes. A "subsurface labyrinth" (a meshwork of cellular processes underneath the lining cells) is constantly seen in the central zone. The intermediate zone is lined by elongated microvillous ependymal cells, and the peripheral zone by typical ependyma. The large liquor-contacting area in the pineal recess region, as well as the peculiar organization of its surface, suggest a complex interrelationship between the liquor and the pineal gland of the opossum.

The pineal recess of the opossum: a scanning and transmission electron microcospe study

Scanning and transmission electron microscope studies were performed in the pineal recess region of eighteen adult Didelphis albiventris opossums. With the scanning electron microscope, three concentric regions could be observed in the pineal recess: a central zone lined by cells free of cilia or microvilli, or by a meshwork of interdigitating cell processes; an intermediate zone lined by microvillous cells; and a peripheral zone, lined by ciliated cells. The transmission electron microscope revealed that the central zone is lined mainly by pinealocytes. The lining cells are frequently overlaid by a meshwork of pinealocytic and gliak oricesses. A <> (a meshwork of cellular processes underneath the lining cells) is contantly seen in the central zone. The intermediate zone is lined by elongated microvillous ependymal cells, and the peripheral zone by typical ependyma. The large liquor-contacting area in the pineal recess region, as well as the peculiar organization of its surface, suggest a complex interrelationship between the liquor and the pineal gland of the opossum (AU)

The pineal recess of the opossum: a scanning and transmission electron microcospe study

Scanning and transmission electron microscope studies were performed in the pineal recess region of eighteen adult Didelphis albiventris opossums. With the scanning electron microscope, three concentric regions could be observed in the pineal recess: a central zone lined by cells free of cilia or microvilli, or by a meshwork of interdigitating cell processes; an intermediate zone lined by microvillous cells; and a peripheral zone, lined by ciliated cells. The transmission electron microscope revealed that the central zone is lined mainly by pinealocytes. The lining cells are frequently overlaid by a meshwork of pinealocytic and gliak oricesses. A <> (a meshwork of cellular processes underneath the lining cells) is contantly seen in the central zone. The intermediate zone is lined by elongated microvillous ependymal cells, and the peripheral zone by typical ependyma. The large liquor-contacting area in the pineal recess region, as well as the peculiar organization of its surface, suggest a complex interrelationship between the liquor and the pineal gland of the opossum

Correlative scanning and transmission electron microscopic study on the ependymal surface of Cebus apella.

Topographical variations of the ependymal surface of the whole brain ventricular system of Cebus apella, a wild Brazilian species, were studied at the scanning electron microscope after CO2 critical point drying and carbon-gold coating. Certain selected areas were also studied at the transmission electron microscope, following standard technical procedures. We observed distinct patterns in the distribution of cilia, microvilli, other cell projections, and the so called supraependymal structures. Based on the morphological findings, functional considerations are made.

Correlative scanning and transmission electron microscopic study on the ependymal surface of cebus apella

Variaçöes topográficas da superfície ependimária de todo o sistema ventricular encefálico do Cebus apella, uma espécie silvestre brasileira, foram estudadas mediante microscopia eletrônica de varredura, após secagem pelo método do ponto crítico do CO2 e metalizaçäo pelo ouro-carbono. Determinadas áreas foram também estudadas no microscópio eletrônico de transmissäo, após os procedimentos técnicos usuais. Observamos distintos padröes na distribuiçäo de cílios, microvilosidades, e outras projeçöes celulares, além das assim chamadas estruturas supraependimárias. Säo feitas consideraçöes morfofuncionais, baseadas nos achados morfológicos

Correlative scanning and transmission electron microscopic study on the ependymal surface of Bradypus tridactylus.

Topographical variations of the ependymal surface of the whole brain ventricular system of Bradypus tridactylus were studied at the scanning electron microscope after CO2 critical point drying and carbon-gold coating. Certain selected areas were also studied at the transmission electron microscope, following standard technical procedures. We observed distinct patterns in the distribution of cilia, microvilli and supraependymal structures (nerve-like and fibrous astrocyte fibers), and also a small number of blebs. It the lateral transition zone between the floor and roof of the lateral ventricle were found interconnected stellate bulgings measuring an average 98 x 190 microns, constituted by a complex meshwork of processes from fibrous astrocytes, without ependymal lining and with few cell bodies. In the junction between these formations and the underlying neuropil, ciliated ependymal-cell-like glial cells were found to delimit complex labyrinthic spaces. Based on the morphological findings, morpho-functional considerations are made.

Ependymal astrocytoma: subependyoma or subependymal glomerate astrocytoma.

The histological study of four cases and one by electron microscopy of subependyomomas allowed us to obtain morphostructural characteristics of ependymocytes and astrocytes. Comparing these findings to those of the present day literature, we propose to name these tumours ependymal astrocytomas.

Ultrastuctural study on the relations among nerve elements and ependymal cells of the "Bradypus tridactylus".

On hand of the electonmicroscope, the authors study 24 parts of the encephalic ventricles in samples of Bradypus tridactylus, relating the nervous structures to the ependymal lining, chiefly emphasizing the presence of intraventricular unmyelinated fibres. In various of these regions, typical nerve fibres and other intraventricular cell projections, that cannot be taken as nerve structures, because of the continuity they show along with ependymal cells, were observed. Finally they make an analysis of some functions related to arrangements of this nature.