Cerebrospinal fluid-driven ependymal motile cilia defects are implicated in multiple sclerosis pathophysiology

Abstract

BackgroundMultiple sclerosis is a neurodegenerative autoimmune disorder of the central nervous system (CNS) in which autoreactive immune cells migrate through a damaged blood brain barrier, resulting in focal demyelinating lesions of both the white and grey matter. Of increasing interest is the repeated observation that beyond focal lesions, there are also diffuse, surface-in gradients of pathology in MS, wherein damage is most severe directly adjacent to cerebrospinal fluid (CSF)-contacting surfaces, such as the subpial and periventricular areas. This observation suggests that toxic factors within MS CSF may be contributing to the emergence and/or evolution of surface-in gradients. Directly separating the CSF from the periventricular parenchyma are ependymal cells - a glial epithelium that are equipped with tufts of motile cilia which are critical for circulating CSF solutes and regulating local fluid flow. While damage to ependymal cilia has the potential to drastically modify CSF homeostasis and thus contribute to the damage of CSF exposed regions, these motile cellular structures have yet to be investigated in the context of MS. MethodsWe first conducted single cell RNA sequencing of fresh human periventricular brain tissue containing ependymal cells from MS patients and non-MS disease controls. We subsequently collected CSF from MS patients and exposed cultured rodent ependymal cells to this CSF in order to evaluate impact on ependymal ciliary function. To complement our direct evaluation of cilia in the context of MS, we also confirmed whether cilia were altered in a classic animal model of MS, experimental autoimmune encephalomyelitis (EAE), and also designed a novel transgenic animal model to evaluate the cellular and behavioural effect(s) of adult ependymal ciliary disruption. ResultsSingle cell RNA sequencing analysis of human ependymal cells in MS demonstrated largescale dysregulation of ciliary genes and in situ stains of MS brain tissue confirmed a loss of ependymal cilia. Exposure of ependymal cells to MS CSF led to transcriptional modification of ciliary gene and protein expression and reduced ciliary beating frequency. Likewise, analysis of ependymal cells in EAE also demonstrated altered cilia gene and protein expression. Conditional knockout in adult mice, of the critical cilia-associated gene Ccdc39 in ependymal cells led to transient ventricular enlargement, increased periventricular microglial density, and alterations in nesting behaviour. ConclusionThese data suggest that motile cilia in ependymal cells are dysregulated in CNS autoimmunity. More importantly, however, they provide evidence to suggest that ependymal cilia disruption could play an active role in the development of periventricular pathology in MS and can lead to behavioural deficits that may underlie aspects non-motor MS symptomatology.