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

Multiple sclerosis (MS) is a disorder of the CNS in which autoreactive immune cells migrate through a damaged blood-brain barrier, resulting in focal demyelinating lesions. Beyond focal lesions, there are also diffuse 'surface-in' gradients of pathology in MS, wherein damage is most severe directly adjacent to CSF-contacting surfaces, such as the subpial and periventricular areas. This observation suggests that toxic factors within MS CSF contribute 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. We first conducted single-cell RNA sequencing of fresh human periventricular brain tissue containing ependymal cells from patients with MS and non-MS disease controls. We subsequently collected CSF from patients with MS and exposed cultured rodent ependymal cells to this CSF to evaluate the 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 an animal model of MS, experimental autoimmune encephalomyelitis (EAE), and designed a novel transgenic animal model to evaluate the cellular and behavioural effect(s) of adult ependymal ciliary disruption. Single-cell RNA sequencing analysis of human ependymal cells in MS demonstrated large-scale 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 demonstrated altered cilia gene and protein expression. We showed that IFNγ, which is elevated in MS CSF, could alter cilia protein expression and motility. Lastly, conditional knockout of Ccdc39 in ependymal cells of adult mice led to transient ventricular enlargement, increased periventricular microglial density and alterations in nesting behaviour. These data suggest that motile cilia in ependymal cells are dysregulated in CNS autoimmunity. More importantly, they suggest that ependymal cilia disruption could play a role in periventricular pathology formation in MS and be associated with behavioural deficits underlying non-motor symptomatology.