Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche.

Prdm16 is required for the maintenance of neural stem cells in the postnatal forebrain and their differentiation into ependymal cells.

We and others showed previously that PR domain-containing 16 (Prdm16) is a transcriptional regulator required for stem cell function in multiple fetal and neonatal tissues, including the nervous system. However, Prdm16 germline knockout mice died neonatally, preventing us from testing whether Prdm16 is also required for adult stem cell function. Here we demonstrate that Prdm16 is required for neural stem cell maintenance and neurogenesis in the adult lateral ventricle subventricular zone and dentate gyrus. We also discovered that Prdm16 is required for the formation of ciliated ependymal cells in the lateral ventricle. Conditional Prdm16 deletion during fetal development using Nestin-Cre prevented the formation of ependymal cells, disrupting cerebrospinal fluid flow and causing hydrocephalus. Postnatal Prdm16 deletion using Nestin-CreERT2 did not cause hydrocephalus or prevent the formation of ciliated ependymal cells but caused defects in their differentiation. Prdm16 was required in neural stem/progenitor cells for the expression of Foxj1, a transcription factor that promotes ependymal cell differentiation. These studies show that Prdm16 is required for adult neural stem cell maintenance and neurogenesis as well as the formation of ependymal cells.

Ependymal cells undergo astrocyte-like reactivity in response to neuroinflammation.

Ependymal cells form a specialized brain-cerebrospinal fluid (CSF) interface and regulate local CSF microcirculation. It is becoming increasingly recognized that ependymal cells assume a reactive state in response to aging and disease, including conditions involving hypoxia, hydrocephalus, neurodegeneration, and neuroinflammation. Yet what transcriptional signatures govern these reactive states and whether this reactivity shares any similarities with classical descriptions of glial reactivity (i.e., in astrocytes) remain largely unexplored. Using single-cell transcriptomics, we interrogated this phenomenon by directly comparing the reactive ependymal cell transcriptome to the reactive astrocyte transcriptome using a well-established model of autoimmune-mediated neuroinflammation (MOG35-55 EAE). In doing so, we unveiled core glial reactivity-associated genes that defined the reactive ependymal cell and astrocyte response to MOG35-55 EAE. Interestingly, known reactive astrocyte genes from other CNS injury/disease contexts were also up-regulated by MOG35-55 EAE ependymal cells, suggesting that this state may be conserved in response to a variety of pathologies. We were also able to recapitulate features of the reactive ependymal cell state acutely using a classic neuroinflammatory cocktail (IFNγ/LPS) both in vitro and in vivo. Taken together, by comparing reactive ependymal cells and astrocytes, we identified a conserved signature underlying glial reactivity that was present in several neuroinflammatory contexts. Future work will explore the mechanisms driving ependymal reactivity and assess downstream functional consequences.

Ependymal cells: roles in central nervous system infections and therapeutic application.

Ependymal cells are arranged along the inner surfaces of the ventricles and the central canal of the spinal cord, providing anatomical, physiological and immunological barriers that maintain cerebrospinal fluid (CSF) homeostasis. Based on this, studies have found that alterations in gene expression, cell junctions, cytokine secretion and metabolic disturbances can lead to dysfunction of ependymal cells, thereby participating in the onset and progression of central nervous system (CNS) infections. Additionally, ependymal cells can exhibit proliferative and regenerative potential as well as secretory functions during CNS injury, contributing to neuroprotection and post-injury recovery. Currently, studies on ependymal cell primarily focus on the basic investigations of their morphology, function and gene expression; however, there is a notable lack of clinical translational studies examining the molecular mechanisms by which ependymal cells are involved in disease onset and progression. This limits our understanding of ependymal cells in CNS infections and the development of therapeutic applications. Therefore, this review will discuss the molecular mechanism underlying the involvement of ependymal cells in CNS infections, and explore their potential for application in clinical treatment modalities.

Loss of RNA-Binding Protein HuR Leads to Defective Ependymal Cells and Hydrocephalus.

Multiciliated ependymal cells line the ventricle wall and generate CSF flow through ciliary beating. Defects in ependymal cells cause hydrocephalus; however, there are still significant gaps in our understanding the molecular, cellular and developmental mechanisms involved in the pathogenesis of hydrocephalus. Here, we demonstrate that specific deletion of RNA-binding protein (RBP) Hu antigen R (HuR) in the mouse brain results in hydrocephalus and causes postnatal death. HuR deficiency leads to impaired ependymal cell development with defective motile ciliogenesis in both female and male mice. Transcriptome-wide analysis reveals that HuR binds to mRNA transcripts related to ciliogenesis, including cilia and flagella associated protein 52 (Cfap52), the effector gene of Foxj-1 and Rfx transcriptional factors. HuR deficiency accelerates the degradation of Cfap52 mRNA, while overexpression of Cfap52 is able to promote the development of HuR-deficient ependymal cells. Taken together, our results unravel the important role of HuR in posttranscriptional regulation of ependymal cell development by stabilizing Cfap52 mRNA.SIGNIFICANCE STATEMENT This study identifies Hu antigen R (HuR) as a genetic factor involved in the pathogenesis of hydrocephalus. Mechanistically, HuR regulates ependymal cell differentiation and ciliogenesis through stabilizing Cfap52 mRNA, the effector gene of Foxj-1 and Rfx transcriptional factors.

The role of lipids in ependymal development and the modulation of adult neural stem cell function during aging and disease.

Within the adult mammalian central nervous system, the ventricular-subventricular zone (V-SVZ) lining the lateral ventricles houses neural stem cells (NSCs) that continue to produce neurons throughout life. Developmentally, the V-SVZ neurogenic niche arises during corticogenesis following the terminal differentiation of telencephalic radial glial cells (RGCs) into either adult neural stem cells (aNSCs) or ependymal cells. In mice, these two cellular populations form rosettes during the late embryonic and early postnatal period, with ependymal cells surrounding aNSCs. These aNSCs and ependymal cells serve a number of key purposes, including the generation of neurons throughout life (aNSCs), and acting as a barrier between the CSF and the parenchyma and promoting CSF bulk flow (ependymal cells). Interestingly, the development of this neurogenic niche, as well as its ongoing function, has been shown to be reliant on different aspects of lipid biology. In this review we discuss the developmental origins of the rodent V-SVZ neurogenic niche, and highlight research which has implicated a role for lipids in the physiology of this part of the brain. We also discuss the role of lipids in the maintenance of the V-SVZ niche, and discuss new research which has suggested that alterations to lipid biology could contribute to ependymal cell dysfunction in aging and disease.

Transforming Growth Factor-ß1 and Bone Morphogenetic Protein-2 Inhibit Differentiation into Mature Ependymal Multiciliated Cells.

Ependymal cilia play pivotal roles in cerebrospinal fluid flow. In the primary culture system, undifferentiated glial cells differentiate well into ependymal multiciliated cells (MCCs) in the absence of fetal bovine serum (FBS). However, the substances included in FBS which inhibit this differentiation process have not been clarified yet. Here, we constructed the polarized primary culture system of ependymal cells using a permeable filter in which they retained ciliary movement. We found that transforming growth factor-ß1 (TGF-ß1) as well as Bone morphogenetic protein (BMP)-2 inhibited the differentiation with ciliary movement. The inhibition on the differentiation by FBS was recovered by the TGF-ß1 and BMP-2 inhibitors in combination.

Correlation of hydromyelia with subarachnoid hemorrhage-related hydrocephalus: an experimental study.

Although the central canal is an integral component of the cerebral ventricular system, central canal dilation has not been examined adequately during the progression of subarachnoid hemorrhage-related hydrocephalus (SAH-H). Central canal dilation-associated ependymal cell desquamation or subependymal membrane rupture has been rarely reported. Herein, we try to describe possible mechanisms of central canal dilation "Hydromyelia," developing after SAH. A total of 25 New Zealand hybrid female rabbits were recruited. Five served as controls, and five received sham operations. In the remaining animals (n = 15), 0.5 mL/kg of autologous blood was injected into the cisterna magna twice on 0 and 2nd days. Five of these animals died within a few days. A total of 10 survivor animals decapitated 3 weeks later, and the brains and cervical spinal cords were histologically examined. Central canal volumes, ependymal cell numbers on the canal surfaces, and the Evans' indices of the ventricles were compared. On histological examination, central canal occlusion with desquamated ependymal cells and basement membrane rupture were evident. The mean Evans' index of the brain ventricles was 0.31, the mean central canal volume was 1.054 mm3, and the normal ependymal cell density was 4.210/mm2 in control animals; the respective values were 0.34, 1.287 mm3, and 3.602/mm2 for sham-operated animals, and 0.41, 1.776 mm3, and 2.923/mm2 in the study group. The differences were statistically significant (p < 0.05). Hydromyelia, an ignored complication of SAH-H, features ependymal cell desquamation, subependymal basement membrane destruction, blood cell accumulation on the subependymal cell basement membrane, and increased CSF pressure. Hydromyelia may be a significant complication following SAH.

A comparative study of the turnover of multiciliated cells in the mouse trachea, oviduct, and brain.

BACKGROUND: In mammals, multiciliated cells (MCCs) line the lumen of the trachea, oviduct, and brain ventricles, where they drive fluid flow across the epithelium. Each MCC population experiences vastly different local environments that may dictate differences in their lifetime and turnover rates. However, with the exception of MCCs in the trachea, the turnover rates of these multiciliated epithelial populations at extended time scales are not well described. RESULTS: Here, using genetic lineage-labeling techniques we provide a direct comparison of turnover rates of MCCs in these three different tissues. CONCLUSION: We find that oviduct turnover is similar to that in the airway (~6 months), while multiciliated ependymal cells turnover more slowly.

Fractone Bulbs Derive from Ependymal Cells and Their Laminin Composition Influence the Stem Cell Niche in the Subventricular Zone.

Fractones are extracellular matrix structures in the neural stem cell niche of the subventricular zone (SVZ), where they appear as round deposits named bulbs or thin branching lines called stems. Their cellular origin and what determines their localization at this site is poorly studied, and it remains unclear whether they influence neural stem and progenitor cell formation, proliferation, and/or maintenance. To address these questions, we analyzed whole-mount preparations of the lateral ventricle of male and female mice by confocal microscopy using different extracellular matrix and cell markers. We found that bulbs are rarely connected to stems and that they contain laminin α5 and α2 chains, respectively. Fractone bulbs were profusely distributed throughout the SVZ and appeared associated with the center of pinwheels, a critical site for adult neurogenesis. We demonstrate that bulbs appear at the apical membrane of ependymal cells at the end of the first week after birth. The use of transgenic mice lacking laminin α5 gene expression (Lama5) in endothelium and in FoxJ1-expressing ependymal cells revealed ependymal cells as the source of laminin α5-containing fractone bulbs. Deletion of laminin α5 from ependymal cells correlated with a 60% increase in cell proliferation, as determined by phospho-histone H3 staining, and with a selective reduction in the number of slow-dividing cells. These results indicate that fractones are a key component of the SVZ and suggest that laminin α5 modulates the physiology of the neural stem cell niche.SIGNIFICANCE STATEMENT Our work unveils key aspects of fractones, extracellular matrix structures that are present in the SVZ that still lack a comprehensive characterization. We show that fractones extensively interact with neural stem cells, whereas some of them are located precisely at pinwheel centers, which are hotspots for adult neurogenesis. Our results also demonstrate that fractones increase in size during aging and that their interactions with neural stem and progenitor cells become more complex in old mice. Last, we show that fractone bulbs are produced by ependymal cells and that their laminin content regulates neural stem cells.

Mcidas and GemC1 are key regulators for the generation of multiciliated ependymal cells in the adult neurogenic niche.

Multiciliated cells are abundant in the epithelial surface of different tissues, including cells lining the walls of the lateral ventricles in the brain and the airway epithelium. Their main role is to control fluid flow and defects in their differentiation are implicated in many human disorders, such as hydrocephalus, accompanied by defects in adult neurogenesis and mucociliary disorder in the airway system. Here we show that Mcidas, which is mutated in human mucociliary clearance disorder, and GemC1 (Gmnc or Lynkeas), previously implicated in cell cycle progression, are key regulators of multiciliated ependymal cell generation in the mouse brain. Overexpression and knockdown experiments show that Mcidas and GemC1 are sufficient and necessary for cell fate commitment and differentiation of radial glial cells to multiciliated ependymal cells. Furthermore, we show that GemC1 and Mcidas operate in hierarchical order, upstream of Foxj1 and c-Myb transcription factors, which are known regulators of ependymal cell generation, and that Notch signaling inhibits GemC1 and Mcidas function. Our results suggest that Mcidas and GemC1 are key players in the generation of multiciliated ependymal cells of the adult neurogenic niche.

SNX27 Deletion Causes Hydrocephalus by Impairing Ependymal Cell Differentiation and Ciliogenesis.

Hydrocephalus is a brain disorder derived from CSF accumulation due to defects in CSF clearance. Although dysfunctional apical cilia in the ependymal cell layer are causal to the onset of hydrocephalus, mechanisms underlying proper ependymal cell differentiation are largely unclear. SNX27 is a trafficking component required for normal brain function and was shown previously to suppress γ-secretase-dependent amyloid precursor protein and Notch cleavage. However, it was unclear how SNX27-dependent γ-secretase inhibition could contribute to brain development and pathophysiology. Here, we describe and characterize an Snx27-deleted mouse model for the ependymal layer defects of deciliation and hydrocephalus. SNX27 deficiency results in reductions in ependymal cells and cilia density, as well as severe postnatal hydrocephalus. Inhibition of Notch intracellular domain signaling with γ-secretase inhibitors reversed ependymal cells/cilia loss and dilation of lateral ventricles in Snx27-deficient mice, giving strong indication that Snx27 deletion triggers defects in ependymal layer formation and ciliogenesis through Notch hyperactivation. Together, these results suggest that SNX27 is essential for ependymal cell differentiation and ciliogenesis, and its deletion can promote hydrocephalus pathogenesis. SIGNIFICANCE STATEMENT: Down's syndrome (DS) in humans and mouse models has been shown previously to confer a high risk for the development of pathological hydrocephalus. Because we have previously described SNX27 as a component that is consistently downregulated in DS, we present here a robust Snx27-deleted mouse model that produces hydrocephalus and associated ciliary defects with complete penetrance. In addition, we find that γ-secretase/Notch modulation may be a candidate drug target in SNX27-associated hydrocephalus such as that observed in DS. Based on these findings, we anticipate that future study will determine whether modulation of a SNX27/Notch/γ-secretase pathway can also be of therapeutic interest to congenital hydrocephalus.

Mechanosensory Genes Pkd1 and Pkd2 Contribute to the Planar Polarization of Brain Ventricular Epithelium.

Directional beating of ependymal (E) cells' cilia in the walls of the ventricles in the brain is essential for proper CSF flow. E cells display two forms of planar cell polarity (PCP): rotational polarity of individual cilium and translational polarity (asymmetric positioning of cilia in the apical area). The orientation of individual E cells varies according to their location in the ventricular wall (location-specific PCP). It has been hypothesized that hydrodynamic forces on the apical surface of radial glia cells (RGCs), the embryonic precursors of E cells, could guide location-specific PCP in the ventricular epithelium. However, the detection mechanisms for these hydrodynamic forces have not been identified. Here, we show that the mechanosensory proteins polycystic kidney disease 1 (Pkd1) and Pkd2 are present in primary cilia of RGCs. Ablation of Pkd1 or Pkd2 in Nestin-Cre;Pkd1(flox/flox) or Nestin-Cre;Pkd2(flox/flox) mice, affected PCP development in RGCs and E cells. Early shear forces on the ventricular epithelium may activate Pkd1 and Pkd2 in primary cilia of RGCs to properly polarize RGCs and E cells. Consistently, Pkd1, Pkd2, or primary cilia on RGCs were required for the proper asymmetric localization of the PCP protein Vangl2 in E cells' apical area. Analyses of single- and double-heterozygous mutants for Pkd1 and/or Vangl2 suggest that these genes function in the same pathway to establish E cells' PCP. We conclude that Pkd1 and Pkd2 mechanosensory proteins contribute to the development of brain PCP and prevention of hydrocephalus. SIGNIFICANCE STATEMENT: This study identifies key molecules in the development of planar cell polarity (PCP) in the brain and prevention of hydrocephalus. Multiciliated ependymal (E) cells within the brain ventricular epithelium generate CSF flow through ciliary beating. E cells display location-specific PCP in the orientation and asymmetric positioning of their cilia. Defects in this PCP can result in hydrocephalus. Hydrodynamic forces on radial glial cells (RGCs), the embryonic progenitors of E cells, have been suggested to guide PCP. We show that the mechanosensory proteins Pkd1 and Pkd2 localize to primary cilia in RGCs, and their ablation disrupts the development of PCP in E cells. Early shear forces on RGCs may activate Pkd1 and Pkd2 in RGCs' primary cilia to properly orient E cells. This study identifies key molecules in the development of brain PCP and prevention of hydrocephalus.

Immunoreactivity of glucose transporter 8 is localized in the epithelial cells of the choroid plexus and in ependymal cells.

High fructose intake is known to be associated with increased plasma triglyceride concentration, impaired glucose tolerance, insulin resistance, and high blood pressure. In addition, excess fructose intake is also thought to be a risk factor for dementia. Previous immunohistochemical studies have shown the presence of glucose transporter 5 (GLUT5), a major transporter of fructose, in the epithelial cells of the choroid plexus and ependymal cells in the brains of humans, rats, and mice, while GLUT2, a minor transporter of fructose, was localized in the ependymal cells of rat brain. In this study, immunoreactivity for the fructose transporter GLUT8 was observed in the cytoplasm of the epithelial cells in the choroid plexus and in the ependymal cells of the brains of humans and mice. These structures were not immunoreactive for GLUT7, GLUT11, and GLUT12. Our findings support the hypothesis of the transport of intravascular fructose through the epithelial cells of the choroid plexus and the ependymal cells.

A Novel Monoclonal Antibody Against Neuroepithelial and Ependymal Cells and Characteristics of Its Positive Cells in Neurospheres.

There are still few useful cell membrane surface antigens suitable for identification and isolation of neural stem cells (NSCs). We generated a novel monoclonal antibody (mAb), designated as mAb against immature neural cell antigens (INCA mAb), which reacted with the areas around a lateral ventricle of a fetal cerebrum. INCA mAb specifically reacted with neuroepithelial cells in fetal cerebrums and ependymal cells in adult cerebrums. The recognition molecules were O-linked 40 and 42 kDa glycoproteins on the cell membrane surface (gp40 INCA and gp42 INCA). Based on expression pattern analysis of the recognition molecules in developing cerebrums, it was concluded that gp42 INCA was a stage-specific antigen expressed on undifferentiated neuroepithelial cells, while gp40 INCA was a cell lineage-specific antigen expressed at the stages of differentiation from neuroepithelial cells to ependymal cells. A flow cytometric analysis showed that fetal and young adult neurospheres were divided into INCA mAb(-) CD133 polyclonal antibody (pAb)(-), INCA mAb(+) CD133 pAb(-), and INCA mAb(+) CD133 pAb(+) cell populations based on the reactivity against INCA mAb and CD133 pAb. The proportion of cells having the neurosphere formation capability in the INCA mAb(+) CD133 pAb(+) cell population was significantly larger than that of undivided neurospheres. Neurospheres formed by clonal expansion of INCA mAb(+) CD133 pAb(+) cells gave rise to neurons and glial cells. INCA mAb will be a useful immunological probe in the study of NSCs.

Immunohistochemical analysis of transporters related to clearance of amyloid-ß peptides through blood-cerebrospinal fluid barrier in human brain.

A large number of previous reports have focused on the transport of amyloid-ß peptides through cerebral endothelial cells via the blood-brain barrier, while fewer reports have mentioned the transport through the choroid plexus epithelium via the blood-cerebrospinal fluid barrier. Concrete roles of these two pathways remain to be clarified. In this study, we immunohistochemically examined the expression of transporters/receptors that are supposed to be related to the clearance of amyloid-ß peptides in the choroid plexus epithelium, the ventricular ependymal cells and the brain microvessels, using seven autopsied human brains. In the choroid plexus epithelium, immunoreactivity for low-density lipoprotein receptor (LDLR), LDLR-related protein 1 (LRP1), LRP2, formylpeptide receptor-like 1 (FPRL1), ATP-binding cassette (ABC) transporter-A1 (ABCA1), ABCC1 and ABCG4 was seen in 7 of 7 brains, while that for ABCB1, ABCG2, RAGE and CD36 was seen in 0-2 brains. In the ventricular ependymal cells, immunoreactivity for CD36, LDLR, LRP1, LRP2, FPRL1, ABCA1, ABCC1 and ABCG4 was seen in 6-7 brains, while that for ABCB1, ABCG2 and RAGE was seen in 0-1 brain. Immunoreactivity for insulin-degrading enzyme (IDE) was seen in three and four brains in the choroid plexus epithelium and the ventricular ependymal cells, respectively. In addition, immunoreactivity for LDLR, ABCB1 and ABCG2 was seen in over 40 % of the microvessels (all seven brains), and that for FPRL1, ABCA1, ABCC1 and RAGE was seen in over 5 % of the microvessels (4-6 brains), while that for CD36, IDE, LRP1, LRP2 and ABCG4 was seen in less than 5 % of the microvessels (0-2 brains). These findings may suggest that these multiple transporters/receptors and IDE expressed on the choroid plexus epithelium, ventricular ependymal cells and brain microvessels complementarily or cooperatively contribute to the clearance of amyloid-ß peptides from the brain.

Motor protein Kif6 regulates cilia motility and polarity in brain ependymal cells.

Motile cilia on ependymal cells that line brain ventricular walls beat in concert to generate a flow of laminar cerebrospinal fluid (CSF). Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how kinesin motors contribute to cilia motility. Here, we show that Kif6 is a slow processive motor (12.2±2.0 nm/s) on microtubules in vitro and localizes to both the apical cytoplasm and the axoneme in ependymal cells, although it does not display processive movement in vivo. Using a mouse mutant that models a human Kif6 mutation in a proband displaying macrocephaly, hypotonia and seizures, we found that loss of Kif6 function causes decreased ependymal cilia motility and, subsequently, decreases fluid flow on the surface of brain ventricular walls. Disruption of Kif6 also disrupts orientation of cilia, formation of robust apical actin networks and stabilization of basal bodies at the apical surface. This suggests a role for the Kif6 motor protein in the maintenance of ciliary homeostasis within ependymal cells.

Deup1 Expression Interferes with Multiciliated Differentiation.

A recent study revealed that the loss of Deup1 expression does not affect either centriole amplification or multicilia formation. Therefore, the deuterosome per se is not a platform for amplification of centrioles. In this study, we examine whether gain-of-function of Deup1 affects the development of multiciliated ependymal cells. Our time-lapse study reveals that deuterosomes with an average diameter of 300 nm have two different fates during ependymal differentiation. In the first instance, deuterosomes are scattered and gradually disappear as cells become multiciliated. In the second instance, deuterosomes self-organize into a larger aggregate, called a deuterosome cluster (DC). Unlike scattered deuterosomes, DCs possess centriole components primarily within their large structure. A characteristic of DC-containing cells is that they tend to become primary ciliated rather than multiciliated. Our in utero electroporation study shows that DCs in ependymal tissue are mostly observed at early postnatal stages, but are scarce at late postnatal stages, suggesting the presence of DC antagonists within the differentiating cells. Importantly, from our bead flow assay, ectopic expression of Deup1 significantly impairs cerebrospinal fluid flow. Furthermore, we show that expression of mouse Deup1 in Xenopus embryos has an inhibitory effect on differentiation of multiciliated cells in the epidermis. Taken together, we conclude that the DC formation of Deup1 in multiciliated cells inhibits production of multiple centrioles.

ANKS1A-Deficiency Aberrantly Increases the Entry of the Protein Transport Machinery into the Ependymal Cilia.

In this study, we examine whether a change in the protein levels for FOP in Ankyrin repeat and SAM domain-containing protein 1A (ANKS1A)-deficient ependymal cells affects the intraflagellar transport (IFT) protein transport system in the multicilia. Three distinct abnormalities are observed in the multicilia of ANKS1A-deficient ependymal cells. First, there were a greater number of IFT88-positive trains along the cilia from ANKS1A deficiency. The results are similar to each isolated cilium as well. Second, each isolated cilium contains a significant increase in the number of extracellular vesicles (ECVs) due to the lack of ANKS1A. Third, Van Gogh-like 2 (Vangl2), a ciliary membrane protein, is abundantly detected along the cilia and in the ECVs attached to them for ANKS1A-deficient cells. We also use primary ependymal culture systems to obtain the ECVs released from the multicilia. Consequently, we find that ECVs from ANKS1A-deficient cells contain more IFT machinery and Vangl2. These results indicate that ANKS1A deficiency increases the entry of the protein transport machinery into the multicilia and as a result of these abnormal protein transports, excessive ECVs form along the cilia. We conclude that ependymal cells make use of the ECV-based disposal system in order to eliminate excessively transported proteins from basal bodies.

Pathogenic role of autoantibodies at the ependyma in autoimmune disorders of the central nervous system.

Ependymal cells make up the epithelial monolayer that lines the brain ventricles and the spinal cord central canal that are filled with cerebrospinal fluid. The ependyma has several functions, including regulating solute exchange between the cerebrospinal fluid and parenchyma, controlling microcirculation of cerebrospinal fluid via coordinated ciliary beating, and acting as a partial barrier. Dysregulation of these functions can lead to waste clearance impairment, cerebrospinal fluid accumulation, hydrocephalus, and more. A role for ependymal cells in a variety of neurological disorders has been proposed, including in neuromyelitis optica and multiple sclerosis, two autoimmune demyelinating diseases of the central nervous system, where periventricular damage is common. What is not known is the mechanisms behind how ependymal cells become dysregulated in these diseases. In neuromyelitis optica, it is well established that autoantibodies directed against Aquaporin-4 are drivers of disease, and it has been shown recently that these autoantibodies can drive ependymal cell dysregulation. We propose a similar mechanism is at play in multiple sclerosis, where autoantibodies targeting a glial cell protein called GlialCAM on ependymal cells are contributing to disease. GlialCAM shares high molecular similarities with the Epstein-Barr virus (EBV) protein EBNA1. EBV has recently been shown to be necessary for multiple sclerosis initiation, yet how EBV mediates pathogenesis, especially in the periventricular area, remains elusive. In this perspective article, we discuss how ependymal cells could be targeted by antibody-related autoimmune mechanisms in autoimmune demyelinating diseases and how this is implicated in ventricular/periventricular pathology.