Pathogenic variants in autism gene KATNAL2 cause hydrocephalus and disrupt neuronal connectivity by impairing ciliary microtubule dynamics.

Enlargement of the cerebrospinal fluid (CSF)-filled brain ventricles (cerebral ventriculomegaly), the cardinal feature of congenital hydrocephalus (CH), is increasingly recognized among patients with autism spectrum disorders (ASD). KATNAL2, a member of Katanin family microtubule-severing ATPases, is a known ASD risk gene, but its roles in human brain development remain unclear. Here, we show that nonsense truncation of Katnal2 (Katnal2Δ17) in mice results in classic ciliopathy phenotypes, including impaired spermatogenesis and cerebral ventriculomegaly. In both humans and mice, KATNAL2 is highly expressed in ciliated radial glia of the fetal ventricular-subventricular zone as well as in their postnatal ependymal and neuronal progeny. The ventriculomegaly observed in Katnal2Δ17 mice is associated with disrupted primary cilia and ependymal planar cell polarity that results in impaired cilia-generated CSF flow. Further, prefrontal pyramidal neurons in ventriculomegalic Katnal2Δ17 mice exhibit decreased excitatory drive and reduced high-frequency firing. Consistent with these findings in mice, we identified rare, damaging heterozygous germline variants in KATNAL2 in five unrelated patients with neurosurgically treated CH and comorbid ASD or other neurodevelopmental disorders. Mice engineered with the orthologous ASD-associated KATNAL2 F244L missense variant recapitulated the ventriculomegaly found in human patients. Together, these data suggest KATNAL2 pathogenic variants alter intraventricular CSF homeostasis and parenchymal neuronal connectivity by disrupting microtubule dynamics in fetal radial glia and their postnatal ependymal and neuronal descendants. The results identify a molecular mechanism underlying the development of ventriculomegaly in a genetic subset of patients with ASD and may explain persistence of neurodevelopmental phenotypes in some patients with CH despite neurosurgical CSF shunting.

Ependymal polarity defects coupled with disorganized ciliary beating drive abnormal cerebrospinal fluid flow and spine curvature in zebrafish.

Idiopathic scoliosis (IS) is the most common spinal deformity diagnosed in childhood or early adolescence, while the underlying pathogenesis of this serious condition remains largely unknown. Here, we report zebrafish ccdc57 mutants exhibiting scoliosis during late development, similar to that observed in human adolescent idiopathic scoliosis (AIS). Zebrafish ccdc57 mutants developed hydrocephalus due to cerebrospinal fluid (CSF) flow defects caused by uncoordinated cilia beating in ependymal cells. Mechanistically, Ccdc57 localizes to ciliary basal bodies and controls the planar polarity of ependymal cells through regulating the organization of microtubule networks and proper positioning of basal bodies. Interestingly, ependymal cell polarity defects were first observed in ccdc57 mutants at approximately 17 days postfertilization, the same time when scoliosis became apparent and prior to multiciliated ependymal cell maturation. We further showed that mutant spinal cord exhibited altered expression pattern of the Urotensin neuropeptides, in consistent with the curvature of the spine. Strikingly, human IS patients also displayed abnormal Urotensin signaling in paraspinal muscles. Altogether, our data suggest that ependymal polarity defects are one of the earliest sign of scoliosis in zebrafish and disclose the essential and conserved roles of Urotensin signaling during scoliosis progression.

Design of a Stem Cell-Based Therapy for Ependymal Repair in Hydrocephalus Associated With Germinal Matrix Hemorrhages.

BACKGROUND: In preterm birth germinal matrix hemorrhages (GMHs) and the consequent posthemorrhagic hydrocephalus (PHH), the neuroepithelium/ependyma development is disrupted. This work is aimed to explore the possibilities of ependymal repair in GMH/PHH using a combination of neural stem cells, ependymal progenitors (EpPs), and mesenchymal stem cells. METHODS: GMH/PHH was induced in 4-day-old mice using collagenase, blood, or blood serum injections. PHH severity was characterized 2 weeks later using magnetic resonance, immunofluorescence, and protein expression quantification with mass spectrometry. Ependymal restoration and wall regeneration after stem cell treatments were tested in vivo and in an ex vivo experimental approach using ventricular walls from mice developing moderate and severe GMH/PHH. The effect of the GMH environment on EpP differentiation was tested in vitro. Two-tailed Student t or Wilcoxon-Mann-Whitney U test was used to find differences between the treated and nontreated groups. ANOVA and Kruskal-Wallis tests were used to compare >2 groups with post hoc Tukey and Dunn multiple comparison tests, respectively. RESULTS: PHH severity was correlated with the extension of GMH and ependymal disruption (means, 88.22% severe versus 19.4% moderate). GMH/PHH hindered the survival rates of the transplanted neural stem cells/EpPs. New multiciliated ependymal cells could be generated from transplanted neural stem cells and more efficiently from EpPs (15% mean increase). Blood and TNFα (tumor necrosis factor alpha) negatively affected ciliogenesis in cells committed to ependyma differentiation (expressing Foxj1 [forkhead box J1] transcription factor). Pretreatment with mesenchymal stem cells improved the survival rates of EpPs and ependymal differentiation while reducing the edematous (means, 18% to 0.5% decrease in severe edema) and inflammatory conditions in the explants. The effectiveness of this therapeutical strategy was corroborated in vivo (means, 29% to 0% in severe edema). CONCLUSIONS: In GMH/PHH, the ependyma can be restored and edema decreased from either neural stem cell or EpP transplantation in vitro and in vivo. Mesenchymal stem cell pretreatment improved the success of the ependymal restoration.

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.

Multiciliated ependymal cells: an update on biology and pathology in the adult brain.

Mature multiciliated ependymal cells line the cerebral ventricles where they form a partial barrier between the cerebrospinal fluid (CSF) and brain parenchyma and regulate local CSF microcirculation through coordinated ciliary beating. Although the ependyma is a highly specialized brain interface with barrier, trophic, and perhaps even regenerative capacity, it remains a misfit in the canon of glial neurobiology. We provide an update to seminal reviews in the field by conducting a scoping review of the post-2010 mature multiciliated ependymal cell literature. We delineate how recent findings have either called into question or substantiated classical views of the ependymal cell. Beyond this synthesis, we document the basic methodologies and study characteristics used to describe multiciliated ependymal cells since 1980. Our review serves as a comprehensive resource for future investigations of mature multiciliated ependymal cells.

Cytological features of ependymal and choroid plexus tumours.

Ependymal and choroid plexus tumours arise in anatomically related regions. Their intraoperative differential diagnosis is large and depends on factors such as age, tumour site and clinical presentation. Squash cytology can provide valuable information in this context. Cytological features of conventional ependymomas, subependymomas and myxopapillary ependymomas as well as choroid plexus tumours are reviewed and illustrated. Differential diagnostic considerations integrating morphological and clinical information are discussed.

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.

Rethinking the cilia hypothesis of hydrocephalus.

Dysfunction of motile cilia in ependymal cells has been proposed to be a pathogenic cause of cerebrospinal fluid (CSF) overaccumulation leading to ventricular expansion in hydrocephalus, primarily based on observations of enlarged ventricles in mouse models of primary ciliary dyskinesia. Here, we review human and animal evidence that warrants a rethinking of the cilia hypothesis in hydrocephalus. First, we discuss neuroembryology and physiology data that do not support a role for ependymal cilia as the primary propeller of CSF movement across the ventricles in the human brain, particularly during in utero development prior to the functional maturation of ependymal cilia. Second, we highlight that in contrast to mouse models, motile ciliopathies infrequently cause hydrocephalus in humans. Instead, gene mutations affecting motile cilia function impact not only ependymal cilia but also motile cilia found in other organ systems outside of the brain, causing a clinical syndrome of recurrent respiratory infections and situs inversus, symptoms that do not typically accompany most cases of human hydrocephalus. Finally, we postulate that certain cases of hydrocephalus associated with ciliary gene mutations may arise not necessarily just from loss of cilia-generated CSF flow but also from altered neurodevelopment, given the potential functions of ciliary genes in signaling and neural stem cell fate beyond generating fluid flow. Further investigations are needed to clarify the link between motile cilia, CSF physiology, and brain development, the understanding of which has implications for the care of patients with hydrocephalus and other related neurodevelopmental disorders.

De Novo Mutations in FOXJ1 Result in a Motile Ciliopathy with Hydrocephalus and Randomization of Left/Right Body Asymmetry.

Hydrocephalus is one of the most prevalent form of developmental central nervous system (CNS) malformations. Cerebrospinal fluid (CSF) flow depends on both heartbeat and body movement. Furthermore, it has been shown that CSF flow within and across brain ventricles depends on cilia motility of the ependymal cells lining the brain ventricles, which play a crucial role to maintain patency of the narrow sites of CSF passage during brain formation in mice. Using whole-exome and whole-genome sequencing, we identified an autosomal-dominant cause of a distinct motile ciliopathy related to defective ciliogenesis of the ependymal cilia in six individuals. Heterozygous de novo mutations in FOXJ1, which encodes a well-known member of the forkhead transcription factors important for ciliogenesis of motile cilia, cause a motile ciliopathy that is characterized by hydrocephalus internus, chronic destructive airway disease, and randomization of left/right body asymmetry. Mutant respiratory epithelial cells are unable to generate a fluid flow and exhibit a reduced number of cilia per cell, as documented by high-speed video microscopy (HVMA), transmission electron microscopy (TEM), and immunofluorescence analysis (IF). TEM and IF demonstrate mislocalized basal bodies. In line with this finding, the focal adhesion protein PTK2 displays aberrant localization in the cytoplasm of the mutant respiratory epithelial cells.

Wnt produced by stretched roof-plate cells is required for the promotion of cell proliferation around the central canal of the spinal cord.

Cell morphology changes dynamically during embryogenesis, and these changes create new interactions with surrounding cells, some of which are presumably mediated by intercellular signaling. However, the effects of morphological changes on intercellular signaling remain to be fully elucidated. In this study, we examined the effect of morphological changes in Wnt-producing cells on intercellular signaling in the spinal cord. After mid-gestation, roof-plate cells stretched along the dorsoventral axis in the mouse spinal cord, resulting in new contact at their tips with the ependymal cells that surround the central canal. Wnt1 and Wnt3a were produced by the stretched roof-plate cells and delivered to the cell process tip. Whereas Wnt signaling was activated in developing ependymal cells, Wnt activation in dorsal ependymal cells, which were close to the stretched roof plate, was significantly suppressed in embryos with roof plate-specific conditional knockout of Wls, which encodes a factor that is essential for Wnt secretion. Furthermore, proliferation of these cells was impaired in Wls conditional knockout mice during development and after induced spinal cord injury in adults. Therefore, morphological changes in Wnt-producing cells appear to generate new Wnt signal targets.

An eye on the future for defeating hydrocephalus, ciliary dyskinesia-related hydrocephalus: review article.

Congenital hydrocephalus affects approximately one in 1000 newborn children and is fatal in approximately 50% of untreated cases. The currently known management protocols usually necessitate multiple interventions and long-term use of healthcare resources due to a relatively high incidence of complications, and many of them mostly provide a treatment of the effect rather than the cause of cerebrospinal fluid flow reduction or outflow obstruction. Future studies discussing etiology specific hydrocephalus alternative treatments are needed. We systematically reviewed the available literature on the effect of ciliary abnormality on congenital hydrocephalus pathogenesis, to open a discussion on the feasibility of factoring ciliary abnormality in future research on hydrocephalus treatment modalities. Although there are different forms of ciliopathies, we focused in this review on primary ciliary dyskinesia. There is growing evidence of association of other ciliary syndromes and hydrocephalus, such as the reduced generation of multiple motile cilia, which is distinct from primary ciliary dyskinesia. Data for this review were identified by searching PubMed using the search terms 'hydrocephalus,' 'Kartagener syndrome,' 'primary ciliary dyskinesia,' and 'immotile cilia syndrome.' Only articles published in English and reporting human patients were included. Seven studies met our inclusion criteria, reporting 12 cases of hydrocephalus associated with primary ciliary dyskinesia. The patients had variable clinical presentations, genetic backgrounds, and ciliary defects. The ependymal water propelling cilia differ in structure and function from the mucus propelling cilia, and there is a possibility of isolated non-syndromic ependymal ciliopathy causing only hydrocephalus with growing evidence in the literature for the association ependymal ciliary abnormality and hydrocephalus. Abdominal and thoracic situs in children with hydrocephalus can be evaluated, and secondary damage of ependymal cilia causing hydrocephalus in cases with generalized ciliary abnormality can be considered.

A mutation in Ccdc39 causes neonatal hydrocephalus with abnormal motile cilia development in mice.

Pediatric hydrocephalus is characterized by an abnormal accumulation of cerebrospinal fluid (CSF) and is one of the most common congenital brain abnormalities. However, little is known about the molecular and cellular mechanisms regulating CSF flow in the developing brain. Through whole-genome sequencing analysis, we report that a homozygous splice site mutation in coiled-coil domain containing 39 (Ccdc39) is responsible for early postnatal hydrocephalus in the progressive hydrocephalus (prh) mouse mutant. Ccdc39 is selectively expressed in embryonic choroid plexus and ependymal cells on the medial wall of the forebrain ventricle, and the protein is localized to the axoneme of motile cilia. The Ccdc39prh/prh ependymal cells develop shorter cilia with disorganized microtubules lacking the axonemal inner arm dynein. Using high-speed video microscopy, we show that an orchestrated ependymal ciliary beating pattern controls unidirectional CSF flow on the ventricular surface, which generates bulk CSF flow in the developing brain. Collectively, our data provide the first evidence for involvement of Ccdc39 in hydrocephalus and suggest that the proper development of medial wall ependymal cilia is crucial for normal mouse brain development.

The ependymal cell cytoskeleton in the normal and injured spinal cord of mice.

The cytoskeleton of ependymal cells is fundamental to organize and maintain the normal architecture of the central canal (CC). However, little is known about the plasticity of cytoskeletal components after spinal cord injury. Here, we focus on the structural organization of the cytoskeleton of ependymal cells in the normal and injured spinal cord of mice (both females and males) using immunohistochemical and electron microscopy techniques. We found that in uninjured animals, the actin cytoskeleton (as revealed by phalloidin staining) was arranged following the typical pattern of polarized epithelial cells with conspicuous actin pools located in the apical domain of ependymal cells. Transmission electron microscopy images showed microvilli tufts, long cilia, and characteristic intercellular membrane specializations. After spinal cord injury, F-actin rearrangements paralleled by fine structural modifications of the apical domain of ependymal cells were observed. These changes involved disruptions of the apical actin pools as well as fine structural modifications of the microvilli tufts. When comparing the control and injured spinal cords, we also found modifications in the expression of vimentin and glial fibrillary acidic protein (GFAP). After injury, vimentin expression disappeared from the most apical domains of ependymal cells but the number of GFAP-expressing cells within the CC increased. As in other polarized epithelia, the plastic changes in the cytoskeleton may be critically involved in the reaction of ependymal cells following a traumatic injury of the spinal cord.

Prx2 (Peroxiredoxin 2) as a Cause of Hydrocephalus After Intraventricular Hemorrhage.

Background and Purpose- Our recent study demonstrated that release of Prx2 (peroxiredoxin 2) from red blood cells (RBCs) is involved in the inflammatory response and brain injury after intracerebral hemorrhage. The current study investigated the role of extracellular Prx2 in hydrocephalus development after experimental intraventricular hemorrhage. Methods- There were 4 parts in this study. First, Sprague-Dawley rats received an intraventricular injection of lysed RBC or saline and were euthanized at 1 hour for Prx2 measurements. Second, rats received an intraventricular injection of Prx2, deactivated Prx2, or saline. Third, lysed RBC was coinjected with conoidin A, a Prx2 inhibitor, or vehicle. Fourth, rats received Prx2 injection and were treated with minocycline or saline (i.p.). The effects of Prx2 and the inhibitors were examined using magnetic resonance imaging assessing ventriculomegaly, histology assessing ventricular wall damage, and immunohistochemistry to assess inflammation, particularly at the choroid plexus. Results- Intraventricular injection of lysed RBC resulted in increased brain Prx2 and hydrocephalus. Intraventricular injection of Prx2 alone caused hydrocephalus, ventricular wall damage, activation of choroid plexus epiplexus cells (macrophages), and an accumulation of neutrophils. Conoidin A attenuated lysed RBC-induced injury. Systemic minocycline treatment reduced the epiplexus cell activation and hydrocephalus induced by Prx2. Conclusions- Prx2 contributed to the intraventricular hemorrhage-induced hydrocephalus, probably by inducing inflammatory responses in choroid plexus and ventricular wall damage.

Cerebral cysts of ependymal or ventricular origin in a juvenile rhesus macaque (Macaca mulatta) with neurologic signs.

Naturally occurring neurologic disease in non-human primates may be attributable to a wide-range of causes, including infectious agents, congenital or acquired malformations, degenerative diseases, and, rarely, neoplasia. We report a case of ataxia and paresis in a juvenile rhesus macaque with ependymal-lined cerebral cysts.

Cells in the adult human spinal cord ependymal region do not proliferate after injury.

In vertebrates that regenerate the injured spinal cord, cells at the ependymal region proliferate and coordinate the formation of bridges between the lesion stumps. In mammals, these cells also proliferate profusely around the central canal after spinal cord injury, although their actual contribution to repair is controversial. In humans, however, the central canal disappears from early childhood in the majority of individuals, being replaced by astrocyte gliosis, ependymocyte clusters, and perivascular pseudo-rosettes. In this human ependymal remnant, cells do not proliferate under normal conditions, but it is not known if they do after a lesion. Here, we studied the human ependymal remnant after traumatic spinal cord injury using samples from 21 individuals with survival times ranging from days to months post-injury. With three different monoclonal antibodies raised against two different proliferation markers (Ki67 and MCM2), we found that the ependymal remnant in adult humans does not proliferate after injury at any time or distance from the lesion. Our results seriously challenge the view of the spinal cord ependymal region as a neurogenic niche in adult humans and suggest that it would not be involved in cell replacement after a lesion. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cystic dilation of a ventriculus terminalis. Case report and review of the literature.

The ventriculus terminalis (VT) is a small ependyma-lined cavity within the conus medullaris that is in direct continuity with the central canal of the spinal cord. Cystic dilatation of the ventriculus terminalis on its own is an extremely rare pathological event in adults whose pathogenesis is uncertain. VT has been described in children as a normal developmental phenomenon. These lesions are often diagnosed incidentally during imaging and are in most cases asymptomatic, especially in children. Symptomatic dilatation of VT in adults is a rare condition with 61cases being reported to date. Symptomatic dilatation of VT in children has not been reported till now. We present a 5 year-old-boy with a sphincteric and walking disorder. The patient was assessed by clinical, electrophysiological and urodynamic investigations as well as magnetic resonance imaging (MRI) of the lumbar-sacral segment with and without gadolinium enhancement. Lumbar-sacral MRI demonstrated the presence of a cystic lesion containing cerebrospinal fluid (CSF), which did not enhance after gadolinium, compatible with the diagnosis of the ventriculus terminalis dilation.The patient underwent laminectomy and the cyst wall was fenestrated with a midline myelotomy. In 6-month of follow-up, urinary problems and gait disturbance improved.

An ex vivo spinal cord injury model to study ependymal cells in adult mouse tissue.

Traumatic spinal cord injury is characterized by an initial cell loss that is followed by a concerted cellular response in an attempt to restore the damaged tissue. Nevertheless, little is known about the signaling mechanisms governing the cellular response to injury. Here, we have established an adult ex vivo system that exhibits multiple hallmarks of spinal cord injury and allows the study of complex processes that are difficult to address using animal models. We have characterized the ependymal cell response to injury in this model system and found that ependymal cells can become activated, proliferate, migrate out of the central canal lining and differentiate in a manner resembling the in vivo situation. Moreover, we show that these cells respond to external adenosine triphosphate and exhibit spontaneous Ca2+ activity, processes that may play a significant role in the regulation of their response to spinal cord injury. This model provides an attractive tool to deepen our understanding of the ependymal cell response after spinal cord injury, which may contribute to the development of new treatment options for spinal cord injury.