Brigitte Katrin Paap

MicroRNAs in multiple sclerosis and experimental autoimmune encephalomyelitis

MicroRNA (miRNA) are small non-coding RNA molecules about 21-25 nucleotides long. They control gene regulation by translational repression and cleavage. Several studies have shown that many miRNA are associated with the etiology of different diseases. Recent developments in diverse miRNA profiling platforms like microarray and quantitative real-time PCR may enable the identification of specific miRNA as novel diagnostic and predictive markers for various diseases. MiRNAs could even be used as therapeutic drug targets. Multiple sclerosis (MS) is a chronic autoimmune disease affecting the central nervous system. Dysregulated immune system processes result in demyelination of neurons and consequently, electrical impulses that travel along the nerves are disrupted resulting in the impairment of organs. In the past three years, there has been an increased interest in establishing miRNA-based biomarkers for MS. So far, there are six studies on miRNA expression in MS patients in which first miRNAs were discovered as potential disease markers. For instance, one study showed that blood levels of miR-145 can discriminate MS patients from healthy controls, and another showed that active lesions in the brain are characterized by a strong up-regulation of miR-155. Studies on experimental autoimmune encephalomyelitis (EAE), the animal model of MS, further support the significance of miRNA as e.g. mice with miR-155 deletion are highly resistant to EAE. Such investigations help to understand the molecular processes involved in the disease. The identification of miRNA markers that are associated with type of MS, individual disease activity or clinical progression under treatment may open new avenues for early diagnosis and optimized therapy of MS.

Elevated type I interferon-like activity in a subset of multiple sclerosis patients: molecular basis and clinical relevance

BackgroundA subset of patients with multiple sclerosis (MS) shows an increased endogenous IFN-like activity before initiation of IFN-beta treatment. The molecular basis of this phenomenon and its relevance to predict individual therapy outcomes are not yet fully understood. We studied the expression patterns of these patients, the prognostic value of an elevated IFN-like activity, and the gene regulatory effects of exogenously administered IFN-beta.MethodsMicroarray gene expression profiling was performed for 61 MS patients using peripheral blood mononuclear cells obtained before and after 1 month of IFN-beta therapy. Expression levels of genes involved in pathways either inducing or being activated by IFN-beta were compared between patients with high (MX1high cohort) and low (MX1low cohort) endogenous IFN-like activity. Patients were followed for 5 years and relapses as well as progression on the expanded disability status scale (EDSS) were documented.ResultsBefore the start of therapy, 11 patients presented elevated mRNA levels of IFN-stimulated genes indicative of a relatively high endogenous IFN-like activity (MX1high). In these patients, pathogen receptors (for example, TLR7, RIG-I and IFIH1) and transcription factors were also expressed more strongly, which could be attributed to an overactivity of IFN-stimulated gene factor 3 (ISGF3, a complex formed by STAT1, STAT2 and IFN regulatory factor 9). After 1 month of IFN-beta therapy, the expression of many pathway genes was significantly induced in MX1low patients, but remained unaltered in MX1high patients. During follow-up, relapse rate and changes in EDSS were comparable between both patient groups, with differences seen between different types of IFN-beta drug application.ConclusionsTherapeutic IFN-beta induces the transcription of several genes involved in IFN-related pathways. In a subgroup of MS patients, the expression of these genes is already increased before therapy initiation, possibly driven by an overexpression of ISGF3. Patients with high and low endogenous IFN-like activity showed similar clinical long-term courses of disease. Different results were obtained for different IFN-beta drug preparations, and this merits further investigation.

Integration of MicroRNA databases to study MicroRNAs associated with multiple sclerosis.

MicroRNAs (miRNAs) are small non-coding RNAs which regulate many genes post-transcriptionally. In various contexts of medical science, miRNAs gained increasing attention over the last few years. Analyzing the functions, interactions and cellular effects of miRNAs is a very complex and challenging task. Many miRNA databases with diverse data contents have been developed. Here, we demonstrate how to integrate their information in a reasonable way on a set of miRNAs that were found to be dysregulated in the blood of patients with multiple sclerosis (MS). Using the miR2Disease database, we retrieved 16 miRNAs associated with MS according to four different studies. We studied the predicted and experimentally validated target genes of these miRNAs, their expression profiles in different blood cell types and brain tissues, the pathways and biological processes affected by these miRNAs as well as their regulation by transcription factors. Only miRNA–mRNA interactions that were predicted by at least seven different prediction algorithms were considered. This resulted in a network of 1,498 target genes. In this network, the MS-associated miRNAs hsa-miR-20a-5p and hsa-miR-20b-5p occurred as central hubs regulating about 500 genes each. Strikingly, many of the putative target genes play a role in T cell activation and signaling, and many have transcription factor activity. The latter suggests that miRNAs often act as regulators of regulators with many secondary effects on gene expression. Our present work provides a guideline on how information of different databases can be integrated in the analysis of miRNAs. Future investigations of miRNAs shall help to better understand the mechanisms underlying different diseases and their treatments.

Reassessment of Blood Gene Expression Markers for the Prognosis of Relapsing-Remitting Multiple Sclerosis

Despite considerable advances in the treatment of multiple sclerosis, current drugs are only partially effective. Most patients show reduced disease activity with therapy, but still experience relapses, increasing disability, and new brain lesions. Since there are no reliable clinical or biological markers of disease progression, long-term prognosis is difficult to predict for individual patients. We identified 18 studies that suggested genes expressed in blood as predictive biomarkers. We validated the prognostic value of those genes with three different microarray data sets comprising 148 patients in total. Using these data, we tested whether the genes were significantly differentially expressed between patients with good and poor courses of the disease. Poor progression was defined by relapses and/or increase of disability during a two-year follow-up, independent of the administered therapy. Of 110 genes that have been proposed as predictive biomarkers, most could not be confirmed in our analysis. However, the G protein-coupled membrane receptor GPR3 was expressed at significantly lower levels in patients with poor disease progression in all data sets. GPR3 has therefore a high potential to be a biomarker for predicting future disease activity. In addition, we examined the IL17 cytokines and receptors in more detail and propose IL17RC as a new, promising, transcript-based biomarker candidate. Further studies are needed to better understand the roles of these receptors in multiple sclerosis and its treatment and to clarify the utility of GPR3 and IL17RC expression levels in the blood as markers of long-term prognosis.

Multiple sclerosis: modulation of toll-like receptor (TLR) expression by interferon-β includes upregulation of TLR7 in plasmacytoid dendritic cells.

Interferon-β is an established treatment for patients with multiple sclerosis (MS) but its mechanisms of action are not well understood. Viral infections are a known trigger of MS relapses. Toll-like receptors (TLRs) are key components of the innate immune system, which sense conserved structures of viruses and other pathogens. Effects of interferon-β on mRNA levels of all known human TLRs (TLR1-10) and the TLR adaptor molecule MyD88 were analyzed in peripheral blood mononuclear cells (PBMCs) of healthy donors by quantitative real-time PCR and by transcriptome analysis in PBMCs of 25 interferon-β-treated patients with relapsing-remitting MS. Regulation of TLR protein expression by interferon-β was investigated by flow cytometry of leukocyte subsets of healthy subjects and of untreated, interferon-β-, or glatiramer acetate-treated patients with MS. Interferon-β specifically upregulated mRNA expression of TLR3, TLR7, and MyD88 and downregulated TLR9 mRNA in PBMCs of healthy donors as well as in PBMCs of patients with MS. Plasmacytoid dendritic cells (pDCs) were identified as the major cell type responding to interferon-β with increased expression of TLR7 and MyD88 protein. In line with this, expression of TLR7 protein was increased in pDCs of interferon-β-treated, but not untreated or glatiramer acetate-treated patients with MS. Interferon-β-induced upregulation of TLR7 in pDCs is of functional relevance since pre-treatment of PBMCs with interferon-β resulted in a strongly increased production of interferon-α upon stimulation with the TLR7 agonist loxoribine. Flow cytometry confirmed pDCs as the cellular source of interferon-α production induced by activation of TLR7. Thus, upregulation of TLR7 in pDCs and a consequently increased activation of pDCs by TLR7 ligands represents a novel immunoregulatory mechanism of interferon-β. We hypothesize that this mechanism could contribute to a reduction of virus-triggered relapses in patients with MS.

Glatiramer acetate treatment effects on gene expression in monocytes of multiple sclerosis patients

BackgroundGlatiramer acetate (GA) is a mixture of synthetic peptides used in the treatment of patients with relapsing-remitting multiple sclerosis (RRMS). The aim of this study was to investigate the effects of GA therapy on the gene expression of monocytes.MethodsMonocytes were isolated from the peripheral blood of eight RRMS patients. The blood was obtained longitudinally before the start of GA therapy as well as after one day, one week, one month and two months. Gene expression was measured at the mRNA level by microarrays.ResultsMore than 400 genes were identified as up-regulated or down-regulated in the course of therapy, and we analyzed their biological functions and regulatory interactions. Many of those genes are known to regulate lymphocyte activation and proliferation, but only a subset of genes was repeatedly differentially expressed at different time points during treatment.ConclusionsOverall, the observed gene regulatory effects of GA on monocytes were modest and not stable over time. However, our study revealed several genes that are worthy of investigation in future studies on the molecular mechanisms of GA therapy.

Molecular Biomarkers in Multiple Sclerosis

Multiple Sclerosis (MS) is a chronic immune-modulated disorder of the central nervous system (CNS) affecting mainly young adults. Due to the complexity and heterogenic etiology of this disease diagnosis, treatment, and estimations concerning the future course of the disease for the individual patient are challenging. To encounter the variability in phenotype, disease progression and response to treatments, various new drugs are in development to complement existing treatment options. Since years intensive efforts are directed to identify biomarkers that are associated with various aspects of MS on different levels of the organizational hierarchy of the human body (e.g. DNA, RNA, proteins, cells). We researched the last ten years of literature to identify those proposed candidates that had been repeatedly published as being associated with MS etiology, clinical manifestation, disease course, and treatment response. Here, we present a categorized overview over molecular biomarkers in MS. However, despite of the large sum of studies and the long list of candidate markers, today only very few biomarkers are of clinical value. This is mostly due to lack of comparability and statistical power in most studies. However, there are recent advances in the field of applicable molecular biomarkers in MS: For example measurement of anti-AQP4 levels allows differentiation between neuromyelitis optica (NMO) and MS.

An Inventory of Short Term and Long Term Changes in Gene Expression Under Interferon β Treatment of Relapsing Remitting MS Patients

Multiple sclerosis (MS) is a chronic immune modulated disease of the central nervous system (CNS), characterized by inflammation, demyelination and axonal injury. Currently relapsing remitting type of MS patients are most commonly treated with immune- modulators like interferon β (IFN β) or glatiramer acetate (GA). However, while the majority of patients respond well to therapy others do not. Gene expression profiles in blood samples taken over the course of IFN β treatment can document changes in the biology of the patients in response to the drug and disease activity. During the last decade quite a few of such studies profiling expression in response to IFN β treatment had been done. Here, we combine the results of these studies to outline common differential expression patterns in peripheral blood mononuclear cells (PBMCs) over the course of time under IFN β treatment. We set these profiles into the picture of current knowledge about IFN β pathway, MS immunology and IFN β mechanisms of action. IFN β modulates hundreds of genes. In most of the studies the prominent ones like MX 1, OAS 1, and CXCL 10 had been found to be influenced by IFN β drug treatment. We show examples of short term and long term induced expressional changes, up and down regulated genes (STAT1 and IL8), and explain how under drug treatment feedback loops of type I IFN (IFN α and IFN β) regulated genes may be modulated and changes in expression patterns may result in cytological changes.

Clinical and genetic characteristics of sporadic adult-onset degenerative ataxia

Objective: To define the clinical phenotype and natural history of sporadic adult-onset degenerative ataxia and to identify putative disease-causing mutations. Methods: The primary measure of disease severity was the Scale for the Assessment and Rating of Ataxia (SARA). DNA samples were screened for mutations using a high-coverage ataxia-specific gene panel in combination with next-generation sequencing. Results: The analysis was performed on 249 participants. Among them, 83 met diagnostic criteria of clinically probable multiple system atrophy cerebellar type (MSA-C) at baseline and another 12 during follow-up. Positive MSA-C criteria (4.94 ± 0.74, p < 0.0001) and disease duration (0.22 ± 0.06 per additional year, p = 0.0007) were associated with a higher SARA score. Forty-eight participants who did not fulfill MSA-C criteria and had a disease duration of >10 years were designated sporadic adult-onset ataxia of unknown etiology/non-MSA (SAOA/non-MSA). Compared with MSA-C, SAOA/non-MSA patients had lower SARA scores (13.6 ± 6.0 vs 16.0 ± 5.8, p = 0.0200) and a slower annual SARA increase (1.1 ± 2.3 vs 3.3 ± 3.2, p = 0.0013). In 11 of 194 tested participants (6%), a definitive or probable genetic diagnosis was made. Conclusions: Our study provides quantitative data on the clinical phenotype and progression of sporadic ataxia with adult onset. Screening for causative mutations with a gene panel approach yielded a genetic diagnosis in 6% of the cohort. ClinicalTrials.gov registration: NCT02701036.