MicroRNAs dysregulated in multiple sclerosis affect the differentiation of CG-4 cells, an oligodendrocyte progenitor cell line.

Introduction: Demyelination is one of the hallmarks of multiple sclerosis (MS). While remyelination occurs during the disease, it is incomplete from the start and strongly decreases with its progression, mainly due to the harm to oligodendrocyte progenitor cells (OPCs), causing irreversible neurological deficits and contributing to neurodegeneration. Therapeutic strategies promoting remyelination are still very preliminary and lacking within the current treatment panel for MS. Methods: In a previous study, we identified 21 microRNAs dysregulated mostly in the CSF of relapsing and/or remitting MS patients. In this study we transfected the mimics/inhibitors of several of these microRNAs separately in an OPC cell line, called CG-4. We aimed (1) to phenotypically characterize their effect on OPC differentiation and (2) to identify corroborating potential mRNA targets via immunocytochemistry, RT-qPCR analysis, RNA sequencing, and Gene Ontology enrichment analysis. Results: We observed that the majority of 13 transfected microRNA mimics decreased the differentiation of CG-4 cells. We demonstrate, by RNA sequencing and independent RT-qPCR analyses, that miR-33-3p, miR-34c-5p, and miR-124-5p arrest OPC differentiation at a late progenitor stage and miR-145-5p at a premyelinating stage as evidenced by the downregulation of premyelinating oligodendrocyte (OL) [Tcf7l2, Cnp (except for miR-145-5p)] and mature OL (Plp1, Mbp, and Mobp) markers, whereas only miR-214-3p promotes OPC differentiation. We further propose a comprehensive exploration of their change in cell fate through Gene Ontology enrichment analysis. We finally confirm by RT-qPCR analyses the downregulation of several predicted mRNA targets for each microRNA that possibly support their effect on OPC differentiation by very distinctive mechanisms, of which some are still unexplored in OPC/OL physiology. Conclusion: miR-33-3p, miR-34c-5p, and miR-124-5p arrest OPC differentiation at a late progenitor stage and miR-145-5p at a premyelinating stage, whereas miR-214-3p promotes the differentiation of CG-4 cells. We propose several potential mRNA targets and hypothetical mechanisms by which each microRNA exerts its effect. We hereby open new perspectives in the research on OPC differentiation and the pathophysiology of demyelination/remyelination, and possibly even in the search for new remyelinating therapeutic strategies in the scope of MS.

Impact of calcitriol and PGD2-G-loaded lipid nanocapsules on oligodendrocyte progenitor cell differentiation and remyelination.

Multiple sclerosis (MS) is a demyelinating and inflammatory disease of the central nervous system (CNS) in need of a curative treatment. MS research has recently focused on the development of pro-remyelinating treatments and neuroprotective therapies. Here, we aimed at favoring remyelination and reducing neuro-inflammation in a cuprizone mouse model of brain demyelination using nanomedicines. We have selected lipid nanocapsules (LNC) coated with the cell-penetrating peptide transactivator of translation (TAT), loaded with either a pro-remyelinating compound, calcitriol (Cal-LNC TAT), or an anti-inflammatory bioactive lipid, prostaglandin D2-glycerol ester (PGD2-G) (PGD2-G-LNC TAT). Following the characterization of these formulations, we showed that Cal-LNC TAT in combination with PGD2-G-LNC TAT increased the mRNA expression of oligodendrocyte differentiation markers both in the CG-4 cell line and in primary mixed glial cell (MGC) cultures. However, while the combination of Cal-LNC TAT and PGD2-G-LNC TAT showed promising results in vitro, no significant impact, in terms of remyelination, astrogliosis, and microgliosis, was observed in vivo in the corpus callosum of cuprizone-treated mice following intranasal administration. Thus, although calcitriol's beneficial effects have been abundantly described in the literature in the context of MS, here, we show that the different doses of calcitriol tested had a negative impact on the mice well-being and showed no beneficial effect in the cuprizone model in terms of remyelination and neuro-inflammation, alone and when combined with PGD2-G-LNC TAT.

MicroRNAs are dysregulated in peripheral blood mononuclear cells in multiple sclerosis and correlate with T cell mediators.

T cell mediators and microRNAs are involved in the pathogenesis of multiple sclerosis (MS), but their interaction largely remains undetermined. We investigated by RT-qPCR the dysregulation of microRNAs in peripheral blood mononuclear cells of MS patients versus healthy controls, according to radiological disease activity or treatment. Several microRNAs correlated positively/negatively with IL21/FOXP3 mRNA expression, but not with serum neurofilament light chain levels. Cytokine expression is conceivably balanced by several regulators, whereas microRNAs possibly target upstream transcription factors rather than directly cytokine mRNAs. Functional studies are needed to investigate their interaction, notably for the predicted targeting of FOXP3 by miR-34c-5p.

Molecular Mechanisms of Immunosenescene and Inflammaging: Relevance to the Immunopathogenesis and Treatment of Multiple Sclerosis.

Aging is characterized, amongst other features, by a complex process of cellular senescence involving both innate and adaptive immunity, called immunosenescence and associated to inflammaging, a low-grade chronic inflammation. Both processes fuel each other and partially explain increasing incidence of cancers, infections, age-related autoimmunity, and vascular disease as well as a reduced response to vaccination. Multiple sclerosis (MS) is a lifelong disease, for which considerable progress in disease-modifying therapies (DMTs) and management has improved long-term survival. However, disability progression, increasing with age and disease duration, remains. Neurologists are now involved in caring for elderly MS patients, with increasing comorbidities. Aging of the immune system therefore has relevant implications for MS pathogenesis, response to DMTs and the risks mediated by these treatments. We propose to review current evidence regarding markers and molecular mechanisms of immunosenescence and their relevance to understanding MS pathogenesis. We will focus on age-related changes in the innate and adaptive immune system in MS and other auto-immune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. The consequences of these immune changes on MS pathology, in interaction with the intrinsic aging process of central nervous system resident cells will be discussed. Finally, the impact of immunosenescence on disease evolution and on the safety and efficacy of current DMTs will be presented.

CSF microRNAs discriminate MS activity and share similarity to other neuroinflammatory disorders.

OBJECTIVE: To perform a comprehensive multicompartment analysis of microRNA (miRNA) expression in multiple sclerosis (MS) linked to disease activity and compared with other neuroinflammatory diseases through a retrospective cross-sectional study. METHODS: One hundred twenty-seven miRNAs were measured by PCR arrays on pooled CSF, serum, and peripheral blood mononuclear cell (PBMC) samples of 10 patients with relapsing MS and 10 controls. Sixty-four miRNAs were then measured by quantitative PCR on individual CSF samples of patients with relapsing or remitting MS and controls (n = 68). Fifty-seven miRNAs were analyzed in the CSF from a second cohort (n = 75), including patients with MS, neuroinfectious, or neuroinflammatory diseases and controls. MiRNAs significantly dysregulated in the CSF were analyzed on individual serum/PBMC samples (n = 59/48) of patients with relapsing or remitting MS and controls. Post hoc analysis consisted of principal component analysis (PCA), gene set, and pathway enrichment analysis. RESULTS: Twenty-one miRNAs were differentially expressed, mainly upregulated in the CSF during MS relapses. Relapsing MS and neuroinfectious/inflammatory diseases exhibited a partially overlapping CSF miRNA expression profile. Besides confirming the association of miR-146a-5p/150-5p/155-5p with MS, 7 miRNAs uncharacterized for MS emerged (miR-15a-3p/124-5p/149-3p/29c-3p/33a-3p/34c-5p/297). PCA showed that distinct miRNA sets segregated MS from controls and relapse from remission. In silico analysis predicted the involvement of these miRNAs in cell cycle, immunoregulation, and neurogenesis, but also revealed that the signaling pathway pattern of remitting MS is more akin to controls rather than patients with relapsing MS. CONCLUSIONS: This study highlights the CSF-predominant dysregulation of miRNAs in MS by identifying a signature of disease activity and intrathecal inflammation among neuroinflammatory disorders.

Hydrocephalus in children under the age of five from diagnosis to short-/medium-/long-term progression: a retrospective review of 142 children.

The aim of this study is to evaluate the clinical history and prognosis of children with early-onset hydrocephalus. The retrospective study's inclusion criteria were hydrocephalus diagnosis before the age of 5 years, independent of aetiology, and birth details, January 1, 2000 to December 31, 2014. Overall, 142 children were entered into the study, divided into 11 aetiological groups: premature-birth post-intraventricular haemorrhage (16%), brain tumours (16%), spina bifida (15%), aqueductal stenosis (8%), post-meningitis (8%), post-haemorrhage (8%), Dandy-Walker malformation (6%), unknown origin (6%), arachnoid cyst (5%), miscellaneous obstruction (4%), and various causes (8%). In total, 23 patients died, primarily from the tumour group. Ventriculostomy, performed 42 times, was successful in 20 patients. Overall, 226 internal shunts were placed in 99 children. Infectious complications affected 19% of children after shunt placement and 51% after mechanical complications. Mean follow-up was 4 years 10 months, with 61% of children progressing fairly well, especially those with aqueductal stenosis, cysts, and unknown or diverse obstructive causes. Post-meningitis hydrocephalus displayed the poorest outcome. Isolated obstructive hydrocephalus exhibited better prognosis, with most obstructive aetiologies effectively treated via ventriculostomy. Children treated by shunt placement were more at risk of complications. Aetiologies with associated abnormalities and neurological sequelae had poorer outcomes.