Brain Ependyma Research Articles

P73 C-terminus is dispensable for multiciliogenesis

ABSTRACT The p53 family transcriptional factor p73 plays a pivotal role in development. Ablation of p73 results in severe neurodevelopmental defects, chronic infections, inflammation and infertility. In addition to this, Trp73−\\- mice display severe alteration in the ciliated epithelial lining and the full-length N-terminal isoform TAp73 has been implicated in the control of multiciliogenesis transcriptional program. With our recently generated Trp73Δ13/Δ13 mouse model, we interrogate the physiological role of p73 C-terminal isoforms in vivo. Trp73Δ13/Δ13 mice lack exon 13 in Trp73 gene, producing an ectopic switch from the C-terminal isoforms p73α to p73β. Trp73Δ13/Δ13 mice show a pattern of expression of TAp73 comparable to the wild-type littermates, indicating that the α to β switch does not significantly alter the expression of the gene in this cell type. Moreover, Trp73Δ13/Δ13 do not display any significant alteration in the airway ciliated epithelium, suggesting that in this context p73β can fully substitute the function of the longer isoform p73α. Similarly, Trp73Δ13/Δ13 ciliated epithelium of the brain ependyma also does appear defective. In this district however expression of TAp73 is not detectable, indicating that expression of the gene might be compensated by alternative mechanisms. Overall our work indicates that C-terminus p73 is dispensable for the multiciliogenesis program and suggests a possible tissue-specific effect of p73 alternative splicing.

Tissue-specific roles of p73 in development and homeostasis.

p73 (TP73) belongs to the p53 family of transcription factors. Its gene locus encodes two opposing types of isoforms, the transcriptionally active TAp73 class and the dominant-negative DNp73 class, which both play critical roles in development and homeostasis in an astonishingly diverse array of biological systems within specific tissues. While p73 has functions in cancer, this Review focuses on the non-oncogenic activities of p73. In the central and peripheral nervous system, both isoforms cooperate in complex ways to regulate neural stem cell survival, self-renewal and terminal differentiation. In airways, oviduct and to a lesser extent in brain ependyma, TAp73 is the master transcriptional regulator of multiciliogenesis, enabling fluid and germ cell transport across tissue surfaces. In male and female reproduction, TAp73 regulates gene networks that control cell-cell adhesion programs within germinal epithelium to enable germ cell maturation. Finally, p73 participates in the control of angiogenesis in development and cancer. While many open questions remain, we discuss here key findings that provide insight into the complex functions of this gene at the organismal, cellular and molecular level.

Mutations in the Motile Cilia Gene DNAAF1 Are Associated with Neural Tube Defects in Humans.

Neural tube defects (NTDs) are severe malformations of the central nervous system caused by complex genetic and environmental factors. Among genes involved in NTD, cilia-related genes have been well defined and found to be essential for the completion of neural tube closure (NTC). We have carried out next-generation sequencing on target genes in 373 NTDs and 222 healthy controls, and discovered eight disease-specific rare mutations in cilia-related gene DNAAF1. DNAAF1 plays a central role in cytoplasmic preassembly of distinct dynein-arm complexes, and is expressed in some key tissues involved in neural system development, such as neural tube, floor plate, embryonic node, and brain ependyma epithelial cells in zebrafish and mouse. Therefore, we evaluated the expression and functions of mutations in DNAAF1 in transfected cells to analyze the potential correlation of these mutants to NTDs in humans. One rare frameshift mutation (p.Gln341Argfs*10) resulted in significantly diminished DNAAF1 protein expression, compared to the wild type. Another mutation, p.Lys231Gln, disrupted cytoplasmic preassembly of the dynein-arm complexes in cellular assay. Furthermore, results from NanoString assay on mRNA from NTD samples indicated that DNAAF1 mutants altered the expression level of NTC-related genes. Altogether, these findings suggest that the rare mutations in DNAAF1 may contribute to the susceptibility for NTDs in humans.

Enhanced resistance of CXCR3 deficient mice to ocular HSV-1 infection is due to control of replication in the brain ependyma

CXCR3 deficient (CXCR3−/−) mice are resistant to ocular HSV-1 infection in that less mice develop encephalitis and succumb to infection in comparison to wild type (WT) animals. A region of the brain previously identified to be crucial for development of encephalitis was evaluated in HSV-1-infected CXCR3−/− and WT mice. In this region, known as the ependyma, viral titer, infiltrating leukocyte populations, and key anti-viral cytokine message levels were evaluated. We found that CXCR3−/− mice possessed significantly less HSV-1 and expressed significantly more IFN-β mRNA in the brain ependyma compared to WT animals during the development of encephalitis.

The establishment of rotational polarity in the airway and ependymal cilia: analysis with a novel cilium motility mutant mouse

The airway is covered by multicilia that beat in a metachronous manner toward the mouth to eliminate debris and infectious particles. Coordinated one-directional beating is an essential feature of multicilia in the airway to guarantee proper mucociliary clearance. Defects in ciliary motility lead to primary ciliary dyskinesia (PCD), with major symptoms including bronchitis and other chronic respiratory diseases. Recent work suggested that ciliary motility and planar polarity are required in the process of ciliary alignment that produces coordinated beating. However, the extent to which cilia motility is involved in this process in mammals has not yet been fully clarified. Here, to address the role of ciliary motility in the process of coordinated ciliary alignment, we analyzed Kintoun mice mutants (Ktu(-/-)). Ktu(-/-) exhibited typical phenotypes of PCD with complete loss of ciliary motility in trachea and another ciliated tissue, the brain ependyma. Immunohistochemistry using antibodies against axonemal dynein confirmed the loss of multiple axonemal dynein components in mutant cilia. Observation of cilia orientation based on basal foot directions revealed that ciliary motility was not required in the alignment of airway cilia, whereas a strong requirement was observed in brain ependymal cells. Thus we conclude that the involvement of ciliary motility in the establishment of coordinated ciliary alignment varies among tissues.

Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation

Multiciliate cells function prominently in the respiratory system, brain ependyma, and female reproductive tract to produce vigorous fluid flow along epithelial surfaces. These specialized cells form during development when epithelial progenitors undergo an unusual form of ciliogenesis, in which they assemble and project hundreds of motile cilia. Notch inhibits multiciliate cell formation in diverse epithelia, but how progenitors overcome lateral inhibition and initiate multiciliate cell differentiation is unknown. Here we identify a coiled-coil protein, termed multicilin, which is Notch regulated and highly expressed in developing epithelia where multiciliate cells form. Inhibiting multicilin function specifically blocks multiciliate cell formation in the Xenopus skin and kidney, while ectopic expression induces the differentiation of multiciliate cells in ectopic locations. Multicilin localizes to the nucleus, where it directly activates the expression of genes required for multiciliate cell formation, including FoxJ1 and genes mediating centriole assembly. Multicilin is also necessary and sufficient to promote multiciliate cell differentiation in mouse airway epithelial cultures. These findings suggest that multicilin initiates multiciliate cell differentiation in diverse tissues, by coordinately promoting the transcriptional changes required for motile ciliogenesis and centriole assembly.

T cells can mediate viral clearance from ependyma but not from brain parenchyma in a major histocompatibility class I- and perforin-independent manner

Viral infection of the central nervous system can lead to disability and death. Yet the majority of viral infections with central nervous system involvement resolve with only mild clinical manifestations, if any. This is generally attributed to efficient elimination of the infection from the brain coverings, i.e. the meninges, ependyma and chorioplexus, which are the primary targets of haematogeneous viral spread. How the immune system is able to purge these structures from viral infection with only minimal detrimental effects is still poorly understood. In the present work we studied how an attenuated lymphocytic choriomeningitis virus can be cleared from the central nervous system in the absence of overt disease. We show that elimination of the virus from brain ependyma, but not from brain parenchyma, could be achieved by a T cell-dependent mechanism operating independently of major histocompatibility class I antigens and perforin. Considering that cytotoxic T lymphocyte-mediated cytotoxicity is a leading cause of viral immunopathology and tissue damage, our findings may explain why the most common viral intruders of the central nervous system rarely represent a serious threat to our health.

Cytomegalovirus Ventriculoencephalitis in a Peripheral Blood Stem Cell Transplant Recipient

Cytomegalovirus encephalitis occurs rarely in transplant recipients. We describe a patient with cytomegalovirus ventriculoencephalitis who had a very high CSF viral load but a low peripheral blood viral load. No resistance mutations were present in cerebrospinal fluid viral DNA, whereas DNA from blood showed a resistance mutation in the UL54 gene but not in the UL97 gene. Viral replication was intense in the brain ependyma and periventricular areas without evidence of peripheral cytomegalovirus disease. The data provide evidence for compartmentalization of cytomegalovirus infection. Levels of ganciclovir and foscarnet in the cerebrospinal fluid may be inadequate for treatment, even for some drug-susceptible strains, and, together with periventricular replication, may explain the disparity between cerebrospinal fluid viral load and peripheral blood viral load.

Systemic proliferative changes and clinical signs in cynomolgus monkeys administered a recombinant derivative of human epidermal growth factor.

Epidermal growth factor (EGF) effects have been explored extensively in vivo in rodents, but little is known about trophic responses in nonhuman primates. A previous publication reports the hyperplastic epithelial/parenchymal changes noted in the digestive tract (tongue, esophagus, stomach, intestine, liver, gallbladder, pancreas, and salivary glands) of adult cynomolgus monkeys treated with recombinant human EGF(1-48) (rhEGF(1-48)). This report documents clinical findings and structural effects in the remaining epithelium-containing tissues of these animals. Two monkeys/sex/dose received rhEGF(1-48) by intravenous bolus at 0 (vehicle), 10, 100, 500 (females only), or 1,000 microg/kg/day (males only) daily for up to 2 weeks. Treatment- and dose-related clinical findings included emesis, fecal alterations (soft feces and diarrhea), lacrimation, nasal discharge, hypoactivity, transient hypotension, and salivation after dosing. Male monkeys administered 1,000 microg/kg became moribund after 5 days of treatment and were necropsied. All other monkeys completed the 2-week treatment period. Necropsy findings in nongastrointestinal tissues were: enlarged, pale kidneys at 100 microg/kg and greater; small thymuses seen sporadically at all doses; and enlarged adrenals and small thyroids in males at 1,000 microqg/kg. Respective organ-to-brain weight ratios at 500 and 1,000 microg/kg for kidneys were 1.5- and 2.6-fold greater and for heart were 1.7- and 1.3-fold greater than controls. Microscopically, pronounced dose-related epithelial hypertrophy and hyperplasia were evident in kidney, urinary bladder, skin (epidermis and adnexa), mammary gland, prostate, seminal vesicles, epididymis, uterus, cervix, vagina, thyroid, thymus, tonsillar crypts, cornea, trachea, and pulmonary airways. Epitheliotrophic effects were conspicuous in many tissues at 100 to 1,000 microg/kg. Changes to renal collecting ducts were present at 10 microg/kg, suggesting that kidneys were a relatively sensitive target. Proliferative alterations were not apparent in testes, intraocular structures, brain ependyma and choroid plexus at any dose. Aside from the noted exceptions, rhEGF(1-48) was a pantrophic epithelial mitogen in cynomolgus monkeys when used intravenously at suprapharmacologic doses.

Fibroblast growth factor and hydrocephalus

The Immunohistochemical localization of basic fibroblast growth factor (bFCF) was studied in ventricular ependyma and choroid plexus of aged-matched normotensive and spontaneously hypertensive rats at different ages using a polyclonal antibody against bFCF. The bFCF-like immunoreactivity was observed in brain ependyma and choroid plexus of young and old normotensive rats. However, a progressive loss of immunoreactivity was observed with age in spontaneously hypertensive rats, that was associated with a progressive cerebroventricuiar dilation. These results show a new neuroendocrine anomaly to be added to the many others previously observed in this rat strain, when they develop hydrocephalus as they age. [Neurol Res 2000; 22: 102-104]

The brain ependyma: An epithelial model for the study of the tight junction repression

The brain ependyma: An epithelial model for the study of the tight junction repression

Expression of neuropeptide processing enzymes and neurosecretory proteins in ependyma and choroid plexus epithelium

Recent studies suggest that brain ependyma and choroid plexus produce neuropeptide processing enzymes. To facilitate the understanding of these cells and their ability to produce biologically active peptides, we developed cultures of defined cell type. Ependymal cells were characterized by morphological criteria, and choroid plexus epithelial cell lines were characterized by the presence of the mRNA for IGF-II and transthyretin, a thyroxine binding protein produced in liver and choroid plexus. The ependymal cells and the choroid plexus epithelial cell lines were then examined for the presence of mRNAs for various neuropeptide processing enzymes. Northern blot analysis revealed high levels of furin, car☐ypeptidase E, and peptidyl glycine α-amidating monooxygenase mRNAs, with levels in ependymal cells comparable to those in brain or pituitary. Car☐ypeptidase E activity was detected in medium from cultured ependymal cells; this activity was identified as car☐ypeptidase E based on the acidic pH optimum and sensitivity to various inhibitors. The mRNAs for other neuropeptide processing enzymes, such as prohormone convertases 1 and 2, were not detected on Northern blots of RNA from ependyma or choroid plexus epithelium. Since ependyma and choroid plexus epithelium express a subset of processing enzymes, we suggest that these cells have the capacity to produce biologically active peptides. Initial screening by reverse transcriptase-polymerase chain reaction assays has demonstrated the presence of mRNA for the neurosecretory proteins chromogranin B and secretogranin II in both ependyma and choroid plexus epithelium.

High-level expression of the 32.5-kilodalton calelectrin in ductal epithelia as revealed by immunocytochemistry

Calelectrins are a family of antigenically related Ca 2+-binding proteins that have only recently been described. They have the important property of binding to membranes only in the presence of Ca 2+. We systematically studied the tissue localization of one calelectrin, the 32.5-kilodalton species, in rats using immunocytochemistry. We found that high levels were exclusively present in the epithelial cells of bile and pancreatic ducts, renal collecting ducts, bronchial epithelia, and brain ependyma. In all of these organs, the other cells were not immunoreactive. In addition, strong immunoreactivity was found in the intercalated disks of myocardial cells, and mild immunoreactivity was observed in several endocrine tissues. In contrast, the cellular distribution of the 67-kilodalton calelectrin was more diffuse, involving most parenchymal cells in addition to the already-mentioned cells. Due to the presence of high levels of 32.5-kilodalton calelectrin in some cell types, this protein may be used as a histochemical marker for differentiated ductal epithelial cells, some specialized epithelia, myocardial cells, and Paneth cells.

Coexistence of cytokeratin, vimentin and neurofilament protein in human choroid plexus

The expression of intermediate filament proteins in human brain ependyma and choroid plexus epithelium has been studied by immunohistochemistry using a panel of monoclonal antibodies directed against all five classes of intermediate filaments. Ependymal cells express GFAP and vimentin filaments, whereas plexus epithelium simultaneously contains neurofilaments, cytokeratins and vimentin, a phenomenon not previously observed in normal cells in vivo. By means of specific antibodies we were able to establish that cytokeratins 8 and 18 but not 19 are present in plexus epithelium.

High-level expression of the 32.5-kilodalton calelectrin in ductal epithelia as revealed by immunocytochemistry

Calelectrins are a family of antigenically related Ca 2+-binding proteins that have only recently been described. They have the important property of binding to membranes only in the presence of Ca 2+. We systematically studied the tissue localization of one calelectrin, the 32.5-kilodalton species, in rats using immunocytochemistry. We found that high levels were exclusively present in the epithelial cells of bile and pancreatic ducts, renal collecting ducts, bronchial epithelia, and brain ependyma. In all of these organs, the other cells were not immunoreactive. In addition, strong immunoreactivity was found in the intercalated disks of myocardial cells, and mild immunoreactivity was observed in several endocrine tissues. In contrast, the cellular distribution of the 67-kilodalton calelectrin was more diffuse, involving most parenchymal cells in addition to the already-mentioned cells. Due to the presence of high levels of 32.5-kilodalton calelectrin in some cell types, this protein may be used as a histochemical marker for differentiated ductal epithelial cells, some specialized epithelia, myocardial cells, and Paneth cells.

The mononuclear phagocyte system of the mouse defined by immunohistochemical localisation of antigen F4/80: macrophages associated with epithelia.

The tissue distribution of the murine macrophage-specific antigen F4/80 has been analysed using an immunohistochemical technique. The antigen is observed on all known macrophage populations (including Kupffer cells and bronchoalveolar macrophages) and is absent from any cell types that are definitely not mononuclear phagocytes. Microglial cells from brain express F4/80. F4/80+ macrophages observed associated with epithelia can be divided into two categories, intraepithelial and periepithelial. The former includes epidermal Langerhans cells and cells with similar morphology in other stratified squamous epithelia (cervix, oesophagus), pseudostratified epithelium (trachea), transitional epithelium of urinary bladder, and simple epithelia lining various ducts (salivary gland, common bile duct, tracheobronchial gland). Periepithelial F4/80+ cells, apparently spread immediately below the basal lamina, are associated with simple epithelia throughout the gastrointestinal, respiratory, and male and female reproductive tract as well as the brain ependyma. A major class of periepithelial F4/80+ cells is associated with capillaries throughout the microcirulation. The role of these macrophage populations in control of epithelial function is discussed.

Skin calcium-binding protein: distribution in other tissues.

The distribution of epidermal calcium-binding protein was examined in rat tissues using immunodiffusion and immunofluorescence techniques to investigate its possible physiological role. Epidermal calcium-binding protein was demonstrated in the basal proliferative cell layer of all Malpighian epithelia and related tissues (epidermis, sebaceous glands, cornea, esophagus, and vagina) as well as in ependyma of brain and in the epithelia of the lens. No immunoreactivity for epidermal calcium-binding protein was found in other tissues including dermis, muscle, cartilage, blood vessels, nerve tissue, liver, endocrine glands, urogenital tract, and intestinal and respiratory epithelium. The presence of a protein immunologically indistinguishable from epidermal calcium-binding protein in proliferative cells suggests that it may be involved in the control of calcium-dependent processes perhaps related to mechanical damage and continued proliferation.

Hyaline membrane disease of lungs: Further observations

<h2>Summary</h2> By the use of hemochromogenspectroscopy the substance giving positive benzidine and Nadi reactions in hyaline membrane has been identified as a hemoglobin-like compound. Proof of its iron content is further advanced by tinctorial demonstration of iron in most of the hyaline membrane found in the lungs of an adult case of mitral stenosis. From this it is argued that the development of pulmonary hemosiderosis in rheumatic mitral stenosis is on the basis of repeated episodes of hyaline membrane disease. It is reasoned that this heme in the membrane must come either from lysed extravasated red blood cells or from the mucosal epithelial cells of the respiratory bronchioles and alveolar ducts. Hemochromogen spectroscopy of other tissues has revealed hemoglobin-like compounds in sites other than red blood cells, viz., in the cells of the convoluted tubules of the kidney, ependyma of brain, and especially in the cells covering the chorionic villi of the placenta. It is pointed out that this layer frequently shows development of a condition not unlike hyaline membrane. These findings may mean that hemoglobin-like pigments are distributed throughout the cells of the body in proportion to their metabolic needs and may answer to the description of the "unspecific cell hematin"¹² of Keilin. A corollary of this work is that the Nadi reaction is not specific, at least in histological studies, for the cytochrome C and B compounds or for cytochrome oxidase, but that it is given catalytically by heme compounds in general. Also, diphenylthiocarbazone may be a useful stain for ferrous-porphyrins. Pilocarpine has been shown to enhancethe effect of oxygen in the genesis of hyaline membrane disease in experimental animals. Preliminary evidence is adduced to show that metabolic upset precedes the development of hyaline membranes, and work is now in progress to complete the picture of this metabolic upset.