Posttranskriptionale Regulation von Myelin-mRNAs

Die Myelinisierung neuronaler Axone ermöglicht eine schnelle und energieeffiziente Weiterleitung von Informationen im Nervensystem. Durch lokale Synthese von Myelinproteinen kann die Myelinschicht, zeitlich und räumlich reguliert, gebildet werden. Dieser Prozess ist abhängig von verschiedensten axonalen Eigenschaften und muss damit lokal reguliert werden. Die Myelinisierung im zentralen sowie im peripheren Nervensystem hängt unter anderem stark von kleinen regulatorischen RNA Molekülen ab. In Oligodendrozyten wird das Myelin Basische Protein (MBP) von der sncRNA715 translational reguliert, indem diese direkt innerhalb der 3’UTR der Mbp mRNA bindet und damit die Proteinsynthese verhindert. Mbp mRNA wird in hnRNP A2‐enthaltenen RNA Granula in die Zellperipherie transportiert, wo in Antwort auf axonale Signale die membranständige Tyrosin‐ Kinase Fyn aktiviert wird, welche Granula‐Komponenten wie hnRNP A2 und F phosphoryliert wodurch die lokale Translation initiiert wird. Während des Transports wird die mRNA durch die Bindung der sncRNA715 translational reprimiert. SncRNAs bilden zusammen mit Argonaut‐Proteinen den microRNA induced silencing complex (miRISC), welcher die translationale Inhibition oder den Abbau von mRNAs vermittelt. In der vorliegenden Arbeit sollte zum einen die Regulation der sncRNA715‐abhängigen translationalen Repression der Mbp mRNA in oligodendroglialen Zellen genauer untersucht werden und im zweiten Teil wurde die Rolle der sncRNA715 in den myelinbildenden Zellen des peripheren Nervensystems, den Schwann Zellen, analysiert. Es konnte in oligodendroglialen Zellen die mRNA‐Expression der vier, in Säugern bekannten Argonaut‐Proteinen nachgewiesen werden. Außerdem konnten die beiden Proteine Ago1 und Ago2 in vitro sowie in vivo detektiert werden. Ago2 interagiert mit hnRNP A2, Mbp mRNA und sncRNA715, womit es als neue Komponente des Mbp mRNA Transportgranulas identifiziert werden konnte. Des Weiteren colokalisiert Ago2 mit der Fyn‐Kinase und alle vier Argonaut‐Proteine werden Fyn‐abhängig Tyrosin‐phosphoryliert. Die Fyn‐abhängige Phosphorylierung der Granula‐Komponenten in Antwort auf axo‐glialen Kontakt führt zum Zerfall des RNA‐Granulas und zur gesteigerten MBP Proteinsynthese. Dies wird möglicherweise durch Abstoßungskräfte der negativ geladenen phosphorylierten Proteine vermittelt, wodurch diese sich voneinander und von der mRNA entfernen. Durch die Ablösung des miRISCs von der Mbp mRNA wird die Translation möglicherweise reaktiviert und die Myelinisierung kann starten. Mit der Identifizierung von Ago2 als neuer Mbp mRNA Transportgranula‐Komponente konnte ein weiterer Einblick in die Regulation der lokalen Translation von MBP gewährt werden. Das Verständnis dieses Prozesses ist entscheidend für die Entwicklung neuer Therapien von demyelinisierenden Erkrankungen, da neue Faktoren als eventuelle Ziele für pharmakologische Manipulationen identifiziert und möglichweise neue Therapiemöglichkeiten entstehen könnten. Im zweiten Teil der Arbeit wurde die translationale Regulation von Mbp mRNA in Schwann Zellen untersucht. Auch Schwann Zell‐Mbp wird als mRNA translational inaktiviert zur axo‐glialen Kontaktstelle transportiert, wo vermutlich auch lokale Translation in Antwort auf spezifische Signale stattfindet. Allerdings bleiben die genauen Mechanismen der mRNA‐Lokalisation und damit verbundenen translationalen Repression bislang ungeklärt. Es konnte hier gezeigt werden, dass auch in Schwann Zellen die sncRNA715 exprimiert wird und die Translation von Mbp reguliert. Überexpression der synthetischen sncRNA715 führt zu einer signifikanten Reduktion der MBP Proteinmengen in differenzierten primären Schwann Zellen. Damit kann vermutet werden, dass die Regulation der lokalen MBP Proteinsynthese in Schwann Zellen der in Oligodendrozyten ähnelt

Cathepsin G is differentially expressed in primary human antigen-presenting cells

Cathepsins are required for the processing of antigens in order to make them suitable for loading on major histocompatibility complex (MHC) class II molecules, for subsequent presentation to CD4(+) T cells. It was shown that antigen processing in monocyte-derived dendritic cells (DC), a commonly used DC model, is different from that of primary human DC. Here, we report that the two subsets of human myeloid DC (mDC) and plasmacytoid DC (pDC) differ in their cathepsin distribution. The serine protease cathepsin G (CatG) was detected in mDC1, mDC2, pDC, cortical thymic epithelial cells (cTEC) and high levels of CatG were determined in pDC. To address the role of CatG in the processing and presentation of a Multiple Sclerosis-associated autoantigen myelin basic protein (MBP), we used a non-CatG expressing fibroblast cell line and fibroblasts, which were preloaded with purified CatG. We find that preloading fibroblasts with CatG results in a decrease of MBP84-98-specific T cell proliferation, when compared to control cells. Our data suggest a different processing signature in primary human antigen-presenting cells and CatG may be of functional importance.

Quality control for retinal OCT in multiple sclerosis: validation of the OSCAR-IB criteria.

BACKGROUND Retinal optical coherence tomography (OCT) permits quantification of retinal layer atrophy relevant to assessment of neurodegeneration in multiple sclerosis (MS). Measurement artefacts may limit the use of OCT to MS research. OBJECTIVE An expert task force convened with the aim to provide guidance on the use of validated quality control (QC) criteria for the use of OCT in MS research and clinical trials. METHODS A prospective multi-centre (n = 13) study. Peripapillary ring scan QC rating of an OCT training set (n = 50) was followed by a test set (n = 50). Inter-rater agreement was calculated using kappa statistics. Results were discussed at a round table after the assessment had taken place. RESULTS The inter-rater QC agreement was substantial (kappa = 0.7). Disagreement was found highest for judging signal strength (kappa = 0.40). Future steps to resolve these issues were discussed. CONCLUSION Substantial agreement for QC assessment was achieved with aid of the OSCAR-IB criteria. The task force has developed a website for free online training and QC certification. The criteria may prove useful for future research and trials in MS using OCT as a secondary outcome measure in a multi-centre setting.

A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139–151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139–151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178–191) and myelin oligodendrocyte protein (MOG) residues 92–106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139–151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139–151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.

Poliovirus chimeras replicating under the translational control of genetic elements of hepatitis C virus reveal unusual properties of the internal ribosomal entry site of hepatitis C virus.

Chimeric genomes of poliovirus (PV) have been constructed in which the cognate internal ribosomal entry site (IRES) element was replaced by genetic elements of hepatitis C virus (HCV). Replacement of PV IRES with nt 9-332 of the genotype Ib HCV genome, a sequence comprising all but the first eight residues of the 5' nontranslated region (5'NTR) of HCV, resulted in a lethal phenotype. Addition of 366 nt of the HCV core-encoding sequence downstream of the HCV 5'NTR yielded a viable PV/HCV chimera, which expressed a stable, small-plaque phenotype. This chimeric genome encoded a truncated HCV core protein that was fused to the N terminus of the PV polyprotein via an engineered cleavage site for PV proteinase 3CPpro. Manipulation of the HCV core-encoding sequence of this viable chimera by deletion and frameshift yielded results suggesting that the 5'-proximal sequences of the HCV open reading frame were essential for viability of the chimera and that the N-terminal basic region of the HCV core protein is required for efficient replication of the chimeric virus. These data suggest that the bona fide HCV IRES includes genetic information mapping to the 5'NTR and sequences of the HCV open reading frame. PV chimeras replicating under translational control of genetic elements of HCV can serve to study HCV IRES function in vivo and to search for anti-HCV chemotherapeutic agents.

Insulin-like growth factor I treatment reduces demyelination and up-regulates gene expression of myelin-related proteins in experimental autoimmune encephalomyelitis.

To compare effects of insulin-like growth factor I (IGF-I) and placebo treatment on lesions that resemble those seen during active demyelination in multiple sclerosis, we induced experimental autoimmune encephalomyelitis in Lewis rats with an emulsion containing guinea pig spinal cord and Freund's adjuvant. On day 12-13, pairs of rats with the same degree of weakness were given either IGF-I or placebo intravenously twice daily for 8 days. After 8 days of placebo or IGF-I (200 micrograms/day or 1 mg/day) treatment, the spinal cord lesions were studied by in situ hybridization and with immunocytochemical and morphological methods. IGF-I produced significant reductions in numbers and areas of demyelinating lesions. These lesions contained axons surrounded by regenerating myelin segments instead of demyelinated axons seen in the placebo-treated rats. Relative mRNA levels for myelin basic protein, proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase in lesions of IGF-I-treated rats were significantly higher than they were in placebo-treated rats. PLP mRNA-containing oligodendroglia also were more numerous and relative PLP mRNA levels per oligodendrocyte were higher in lesions of IGF-I-treated rats. Finally, a significantly higher proportion of proliferating cells were oligodendroglia-like cells in lesions of IGF-I-treated rats. We think that IGF-I effects on oligodendrocytes, myelin protein synthesis, and myelin regeneration reduced lesion severity and promoted clinical recovery in this experimental autoimmune encephalomyelitis model. These IGF-I actions may also benefit patients with multiple sclerosis.

Prevention of autoimmune demyelination in non-human primates by a cAMP-specific phosphodiesterase inhibitor.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that serves as a model for the human disease multiple sclerosis. We evaluated rolipram, a type IV phosphodiesterase inhibitor, for its efficacy in preventing EAE in the common marmoset Callithrix jacchus. In a blinded experimental design, clinical signs of EAE developed within 17 days of immunization with human white matter in two placebo-treated animals but in none of three monkeys that received rolipram (10 mg/kg s.c. every other day) beginning 1 week after immunization. In controls, signs of EAE were associated with development of cerebrospinal fluid pleocytosis and cerebral MRI abnormalities. In the treatment group, there was sustained protection from clinical EAE, transient cerebrospinal fluid pleocytosis in only one of three animals, no MRI abnormality, and marked reduction in histopathologic findings. Rolipram-treated and control animals equally developed circulating antibodies to myelin basic protein. Thus, inhibition of type IV phosphodiesterase, initiated after sensitization to central nervous system antigens, protected against autoimmune demyelinating disease.

Analysis of Plasminogen Genetic Variants in Multiple Sclerosis Patients.

Multiple sclerosis (MS) is a prevalent neurological disease of complex etiology. Here, we describe the characterization of a multi-incident MS family that nominated a rare missense variant (p.G420D) in plasminogen (PLG) as a putative genetic risk factor for MS. Genotyping of PLG p.G420D (rs139071351) in 2160 MS patients, and 886 controls from Canada, identified 10 additional probands, two sporadic patients and one control with the variant. Segregation in families harboring the rs139071351 variant, identified p.G420D in 26 out of 30 family members diagnosed with MS, 14 unaffected parents, and 12 out of 30 family members not diagnosed with disease. Despite considerably reduced penetrance, linkage analysis supports cosegregation of PLG p.G420D and disease. Genotyping of PLG p.G420D in 14446 patients, and 8797 controls from Canada, France, Spain, Germany, Belgium, and Austria failed to identify significant association with disease (P = 0.117), despite an overall higher prevalence in patients (OR = 1.32; 95% CI = 0.93-1.87). To assess whether additional rare variants have an effect on MS risk, we sequenced PLG in 293 probands, and genotyped all rare variants in cases and controls. This analysis identified nine rare missense variants, and although three of them were exclusively observed in MS patients, segregation does not support pathogenicity. PLG is a plausible biological candidate for MS owing to its involvement in immune system response, blood-brain barrier permeability, and myelin degradation. Moreover, components of its activation cascade have been shown to present increased activity or expression in MS patients compared to controls; further studies are needed to clarify whether PLG is involved in MS susceptibility.

Prediction of psychological adjustment to multiple sclerosis

This study examined the utility of self-efficacy as a predictor of social activity and mood control in multiple sclerosis (MS). Seventy-one subjects with MS were recruited from people attending an MS centre or from a mailing list and were examined on two occasions that were two months apart. Clinic patients were more disabled than patients who completed assessments by post, but they were of higher socioeconomic status and were less dysphoric. We attempted to predict self-reported performance of mood control and social activity at two months, from self-efficacy or performance on these tasks at pretest. Demographic variables, disorder status, disability, self-esteem and depression were also allowed to compete for entry into multiple regressions. Substantial stability in mood, performance and disability was observed over the two months. In both mood control and social activity, past performance was the strongest predictor of later performance, but self-efficacy also contributed significantly to the prediction. The disability level entered a prediction of socila activity, but no other variables predicted either type of performance. A secondary analysis predicting self-esteem at two months also included self-efficacy for social activity, illustrating the contribution of perceived capability to later assessments of self-worth. The study provided support for self-efficacy as a predictor of later behavioural outcomes and self-esteem in multiple sclerosis.

Circulating microRNAs involved in multiple sclerosis

Multiple sclerosis (MS) is an immune-mediated, demyelinating and neurodegenerative disease of the central nervous system. After traumatic brain injury, it is the leading cause of neurology disability in young adults. Considerable advances have been made in identifying genes involved in MS but the genetic and phenotypic complexity associated with this disease significantly hinders any progress. A novel class of small RNA molecules, microRNAs (miRNAs) has acquired much attention because they regulate the expression of up to 30% of protein-coding genes and may play a pivotal role in the development of many, if not all, complex diseases. Seven published studies investigated miRNAs from peripheral blood mononuclear cells, CD4+, CD8+ T cell, B lymphocytes, peripheral blood leukocytes, whole blood and brain astrocytes with MS risk. The absence of MS studies investigating plasma miRNA prompted the current investigation of identifying a circulating miRNA signature in MS. We conducted a microarray analysis of over 900 known miRNA transcripts from plasma samples collected from four MS individuals and four sex-aged and ethnicity matched healthy controls. We identified six plasma miRNA (miR-614, miR-572, miR-648, miR-1826, miR-422a and miR-22) that were significantly up-regulated and one plasma miRNA (miR-1979) that was significantly down-regulated in MS individuals. Both miR-422a and miR-22 have previously been implicated in MS. The present study is the first to show a circulating miRNA signature involved in MS that could serve as a potential prognostic and diagnostic biomarker for MS.

Modeling the cumulative genetic risk for multiple sclerosis from genome-wide association data

Background: Multiple sclerosis (MS) is the most common cause of chronic neurologic disability beginning in early to middle adult life. Results from recent genome-wide association studies (GWAS) have substantially lengthened the list of disease loci and provide convincing evidence supporting a multifactorial and polygenic model of inheritance. Nevertheless, the knowledge of MS genetics remains incomplete, with many risk alleles still to be revealed. Methods: We used a discovery GWAS dataset (8,844 samples, 2,124 cases and 6,720 controls) and a multi-step logistic regression protocol to identify novel genetic associations. The emerging genetic profile included 350 independent markers and was used to calculate and estimate the cumulative genetic risk in an independent validation dataset (3,606 samples). Analysis of covariance (ANCOVA) was implemented to compare clinical characteristics of individuals with various degrees of genetic risk. Gene ontology and pathway enrichment analysis was done using the DAVID functional annotation tool, the GO Tree Machine, and the Pathway-Express profiling tool. Results: In the discovery dataset, the median cumulative genetic risk (P-Hat) was 0.903 and 0.007 in the case and control groups, respectively, together with 79.9% classification sensitivity and 95.8% specificity. The identified profile shows a significant enrichment of genes involved in the immune response, cell adhesion, cell communication/ signaling, nervous system development, and neuronal signaling, including ionotropic glutamate receptors, which have been implicated in the pathological mechanism driving neurodegeneration. In the validation dataset, the median cumulative genetic risk was 0.59 and 0.32 in the case and control groups, respectively, with classification sensitivity 62.3% and specificity 75.9%. No differences in disease progression or T2-lesion volumes were observed among four levels of predicted genetic risk groups (high, medium, low, misclassified). On the other hand, a significant difference (F = 2.75, P = 0.04) was detected for age of disease onset between the affected misclassified as controls (mean = 36 years) and the other three groups (high, 33.5 years; medium, 33.4 years; low, 33.1 years). Conclusions: The results are consistent with the polygenic model of inheritance. The cumulative genetic risk established using currently available genome-wide association data provides important insights into disease heterogeneity and completeness of current knowledge in MS genetics.

Results of a phase IIa clinical trial of an anti-inflammatory molecule, chaperonin 10, in multiple sclerosis

Background Chaperonin 10 (Cpn10) is a mitochondrial molecule involved in protein folding. The aim of this study was to determine the safety profile of Cpn10 in patients with multiple sclerosis (MS). Methods A total of 50 patients with relapse-remitting or secondary progressive MS were intravenously administered 5 mg or 10 mg of Cpn10 weekly for 12 weeks in a double-blind, randomized, placebo controlled, phase II trial. Clinical reviews, including Expanded Disability Status Scale and magnetic resonance imaging (MRI) with Gadolinium, were undertaken every 4 weeks. Stimulation of patient peripheral blood mononuclear cells with lipopolysaccharide ex vivo was used to measure the in vivo activity of Cpn10. Results No significant differences in the frequency of adverse events were seen between treatment and placebo arms. Leukocytes from both groups of Cpn10-treated patients produced significantly lower levels of critical proinflammatory cytokines. A trend toward improvement in new Gadolinium enhancing lesions on MRI was observed, but this difference was not statistically significant. No differences in clinical outcome measures were seen. Conclusions Cpn10 is safe and well tolerated when administered to patients with MS for 3 months, however, a further extended phase II study primarily focused on efficacy is warranted.

Study of leukemia inhibitory factor polymorphism within an Australian multiple sclerosis population

OBJECTIVE: To examine a polymorphism within the 3' untranslated region of the leukemia inhibitory factor gene for an association with multiple sclerosis within an Australian case-control population. METHODS: A test group of 121 unrelated multiple sclerosis patients, of Caucasian origin, and 121 controls, matched for ethnicity, sex and age (+/-5 years) were included in the study. The LIF 3' UTR StuI polymorphism was genotyped by restriction fragment length polymorphism analysis. Statistical analysis of genotype and allele frequencies included Hardy-Weinberg law and conventional contingency table analysis incorporating the standard chi-squared test for independence. RESULTS: Allelic and genotype frequencies did not demonstrate a significant association between the case and control groups for the tested LIF 3' UTR StuI polymorphism. CONCLUSION: The results indicate that the LIF 3' UTR StuI polymorphism is not associated with multiple sclerosis, however we cannot exclude the hypothesis that other polymorphic alleles of LIF could be implicated in MS susceptibility.