Abstract
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/β-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.
Highlights
Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord
Postnatal day 1 (P1) and adult ventricular wall samples were positive for IIIG9 by RT-PCR analysis, and three principal bands (42, 51 and 53 kDa) were detected by Western blot (Fig. 1a, b; Supplementary Figs. 1, 2) in a similar way to protein extract isolated from trachea (Supplementary Fig. 4)
The ventricular zone en face wholemount technique is an adequate approach to study the polarity of protein distribution in different cells[25]; we analyzed adult ependymal cells (Fig. 1c–l) using anti-acetylated α-tubulin, anti-β-catenin or anti-cadherin antibodies with this approach (Fig. 1d)
Summary
Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord. Adult ependymal cells coexist primarily as E1 multiciliate cells (approximately 50 cilia per cell) and E2 biciliated cells (less than 5% total), lining the walls of the lateral ventricles, the third dorsal ventricle, and the fourth ventricle to create a cerebrospinal fluid (CSF) flow constant[1] Both cell types constitute highly polarized cells generated from radial glial cells[2,3,4], that protect the brain parenchyma from direct contact with CSF, among other functions[5,6]. The stability of this epithelium is maintained by the presence of functional adherens junctions[6,7] Loss of these junction complexes induces the detachment and death of ependymal cells, generating pathological consequences and changing normal ventricular CSF flow, resulting in ventricular dilation (ventriculomegaly) followed by hydrocephalus and brain tissue edema[8,9,10,11]. IIIG9 has been detected in other regions of the cell, suggesting that it may have diverse intracellular functions
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