Neuronal microRNA regulation in Experimental Autoimmune Encephalomyelitis

Abstract

Multiple sclerosis (MS) is an autoimmune, neurodegenerative disease but the molecular mechanisms underlying neurodegenerative aspects of the disease are poorly understood. microRNAs (miRNAs) are powerful regulators of gene expression that regulate numerous mRNAs simultaneously and can thus regulate programs of gene expression. Here, we describe miRNA expression in neurons captured from mice subjected to experimental autoimmune encephalomyelitis (EAE), a model of central nervous system (CNS) inflammation. Lumbar motor neurons and retinal neurons were laser captured from EAE mice and miRNA expression was assessed by next-generation sequencing and validated by qPCR. We describe 14 miRNAs that are differentially regulated in both neuronal subtypes and determine putative mRNA targets though in silico analysis. Several upregulated neuronal miRNAs are predicted to target pathways that could mediate repair and regeneration during EAE. This work identifies miRNAs that are affected by inflammation and suggests novel candidates that may be targeted to improve neuroprotection in the context of pathological inflammation.

MicroRNA-223 protects neurons from degeneration in Experimental Autoimmune Encephalomyelitis

“…Cortical neurons from wildtype (WT) C57Bl/6 mice were prepared as previously described (Juzwik et al ., 2018; Pare et al ., 2018). Cortical neurons were seeded in 48-wells at 80 000 cells/well and aged 4 DIV prior to addition of PBMC-CM.…”

“…Total RNA extraction was done using miRNeasy Mini Kit, according to manufacturer’s instruction. miRNA expression was assessed using multiplex qRTPCR with Taqman miRNA Assays (ThermoFisher) and snoRNA202 as the endogenous control, as previously described (Moore et al ., 2013; Juzwik et al ., 2018). Fold change calculations for miRNA expression were performed using the –ΔΔCT method (Livak and Schmittgen, 2001).…”

“…The spinal cord and retina were isolated and frozen at –80°C in Tissue-Tek ® optimal cutting temperature (OCT) compound. Preparation of slides for LCM, LCM procedure, and extraction of RNA from laser-captured material were done as described previously(Juzwik et al ., 2018). For LCM, lumbar motor neurons were isolated from EAE mice at naïve and peak stage; and the retinal ganglion cell (RGC) layer was isolated from EAE mice at pre-symptomatic and peak.…”

“…These include: Diana-microT (Reczko et al ., 2012; Paraskevopoulou et al ., 2013), microRNA.org (Betel et al ., 2008), miRDP (Wong and Wang, 2015), miRTarBase (Chou et al ., 2016), RNA22 (Miranda et al ., 2006), TargetScan (Agarwal et al ., 2015), and TarBase (Vlachos et al ., 2015). Only mRNAs that were identified as putative targets across 4 of the 7 prediction programs were analyzed further, termed ‘filtered targets’(Juzwik et al ., 2018). Pathways regulated by overlapping filtered targets were determined using an overrepresentation test by Protein ANalysis THrough Evolutionary Relationships (PANTHER) classification system (http://www.pantherdb.org/) (Mi et al ., 2013; Mi et al ., 2017).…”

“…Using the EAE model miRNAs have been profiled in purified cell types including immune cell subsets, oligodendrocytes and neurons providing valuable information about molecular substrates that may be targeted to manipulate cell specific biologies (Jr Ode et al ., 2012; Lewkowicz et al ., 2015; Juzwik et al ., 2018). We profiled miRNA expression in lumbar motor neurons and retinal neurons in EAE to identify molecular substrates that may be targeted to promote neuroprotection (Juzwik et al ., 2018). Here, we study two miRNAs, miR-223-3p and miR-27a-3p that are upregulated in motor neurons in mice subjected to EAE.…”