Proteomics Comparison of Cerebrospinal Fluid of Relapsing Remitting and Primary Progressive Multiple Sclerosis

Background

Based on clinical representation of disease symptoms multiple sclerosis (MScl) patients can be divided into two major subtypes; relapsing remitting (RR) MScl (85–90%) and primary progressive (PP) MScl (10–15%). Proteomics analysis of cerebrospinal fluid (CSF) has detected a number of proteins that were elevated in MScl patients. Here we specifically aimed to differentiate between the PP and RR subtypes of MScl by comparing CSF proteins.

Methodology/Principal Findings

CSF samples (n = 31) were handled according to the same protocol for quantitative mass spectrometry measurements we reported previously. In the comparison of PP MScl versus RR MScl we observed a number of differentially abundant proteins, such as protein jagged-1 and vitamin D-binding protein. Protein jagged-1 was over three times less abundant in PP MScl compared to RR MScl. Vitamin D-binding protein was only detected in the RR MScl samples. These two proteins were validated by independent techniques (western blot and ELISA) as differentially abundant in the comparison between both MScl types.

Conclusions/Significance

The main finding of this comparative study is the observation that the proteome profiles of CSF in PP and RR MScl patients overlap to a large extent. Still, a number of differences could be observed. Protein jagged-1 is a ligand for multiple Notch receptors and involved in the mediation of Notch signaling. It is suggested in literature that the Notch pathway is involved in the remyelination of MScl lesions. Aberration of normal homeostasis of Vitamin D, of which approximately 90% is bound to vitamin D-binding protein, has been widely implicated in MScl for some years now. Vitamin D directly and indirectly regulates the differentiation, activation of CD4+ T-lymphocytes and can prevent the development of autoimmune processes, and so it may be involved in neuroprotective elements in MScl.     

Habitat-Specific Effects of Fishing Disturbance on Benthic Species Richness in Marine Soft Sediments

Around the globe, marine soft sediments on continental shelves are affected by bottom trawl fisheries. In this study, we explore the effect of this widespread anthropogenic disturbance on the species richness of a benthic ecosystem, along a gradient of bottom trawling intensities. We use data from 80 annually sampled benthic stations in the Dutch part of the North Sea, over a period of 6 years. Trawl disturbance intensity at each sampled location was reconstructed from satellite tracking of fishing vessels. Using a structural equation model, we studied how trawl disturbance intensity relates to benthic species richness, and how the relationship is mediated by total benthic biomass, primary productivity, water depth, and median sediment grain size. Our results show a negative relationship between trawling intensity and species richness. Richness is also negatively related to sediment grain size and primary productivity, and positively related to biomass. Further analysis of our data shows that the negative effects of trawling on richness are limited to relatively species-rich, deep areas with fine sediments. We find no effect of bottom trawling on species richness in shallow areas with coarse bottoms. These condition-dependent effects of trawling suggest that protection of benthic richness might best be achieved by reducing trawling intensity in a strategically chosen fraction of space.     

Transfer of central nervous system autoantigens and presentation in secondary lymphoid organs

Dendritic cells are thought to regulate tolerance induction vs immunization by transferring Ags and peripheral signals to draining lymph nodes (LN). However, whether myelin Ag transfer and presentation in LN occurs during demyelinating brain disease is unknown. In this study, we demonstrate redistribution of autoantigens from brain lesions to cervical LN in monkey experimental autoimmune encephalomyelitis (EAE) and in multiple sclerosis (MS). Immunohistochemical analysis revealed significantly more cells containing myelin Ags in cervical LN of monkeys with EAE compared with those of healthy control monkeys. Myelin Ags were observed in cells expressing dendritic cell/macrophage-specific markers, MHC class II, and costimulatory molecules. Moreover, these cells were directly juxtaposed to T cells, suggesting that cognate interactions between myelin-containing APC and T cells are taking place in brain-draining LN. Indeed, myelin Ag-reactive T cells were observed in cervical LN from marmosets and rhesus monkeys. Importantly, these findings were paralleled by our findings in human tissue. We observed significantly more myelin Ag-containing cells in LN of individuals with MS compared with those of control individuals. These cells expressed APC markers, as observed in marmosets and rhesus monkeys. These findings suggest that during MS and EAE, modulation of T cell reactivity against brain-derived Ags also takes place in cervical LN and not necessarily inside the brain. A major implication is that novel therapeutic strategies may be targeted to peripheral events, thereby circumventing the blood-brain barrier.     

Perspectives on the use of multiple sclerosis risk genes for prediction

Objective

A recent collaborative genome-wide association study replicated a large number of susceptibility loci and identified novel loci. This increase in known multiple sclerosis (MS) risk genes raises questions about clinical applicability of genotyping. In an empirical set we assessed the predictive power of typing multiple genes. Next, in a modelling study we explored current and potential predictive performance of genetic MS risk models.

Materials and Methods

Genotype data on 6 MS risk genes in 591 MS patients and 600 controls were used to investigate the predictive value of combining risk alleles. Next, the replicated and novel MS risk loci from the recent and largest international genome-wide association study were used to construct genetic risk models simulating a population of 100,000 individuals. Finally, we assessed the required numbers, frequencies, and ORs of risk SNPs for higher discriminative accuracy in the future.

Results

Individuals with 10 to 12 risk alleles had a significantly increased risk compared to individuals with the average population risk for developing MS (OR 2.76 (95% CI 2.02–3.77)). In the simulation study we showed that the area under the receiver operating characteristic curve (AUC) for a risk score based on the 6 SNPs was 0.64. The AUC increases to 0.66 using the well replicated 24 SNPs and to 0.69 when including all replicated and novel SNPs (n = 53) in the risk model. An additional 20 SNPs with allele frequency 0.30 and ORs 1.1 would be needed to increase the AUC to a slightly higher level of 0.70, and at least 50 novel variants with allele frequency 0.30 and ORs 1.4 would be needed to obtain an AUC of 0.85.

Conclusion

Although new MS risk SNPs emerge rapidly, the discriminatory ability in a clinical setting will be limited.     

Profiling and identification of cerebrospinal fluid proteins in a rat EAE model of multiple sclerosis

The experimental autoimmune encephalomyelitis (EAE) model resembles certain aspects of multiple sclerosis (MScl), with common features such as motor dysfunction, axonal degradation, and infiltration of T-cells. We studied the cerebrospinal fluid (CSF) proteome in the EAE rat model to identify proteomic changes relevant for MScl disease pathology. EAE was induced in male Lewis rats by injection of myelin basic protein (MBP) together with complete Freund's adjuvant (CFA). An inflammatory control group was injected with CFA alone, and a nontreated group served as healthy control. CSF was collected at day 10 and 14 after immunization and analyzed by bottom-up proteomics on Orbitrap LC-MS and QTOF LC-MS platforms in two independent laboratories. By combining results, 44 proteins were discovered to be significantly increased in EAE animals compared to both control groups, 25 of which have not been mentioned in relation to the EAE model before. Lysozyme C1, fetuin B, T-kininogen, serum paraoxonase/arylesterase 1, glutathione peroxidase 3, complement C3, and afamin are among the proteins significantly elevated in this rat EAE model. Two proteins, afamin and complement C3, were validated in an independent sample set using quantitative selected reaction monitoring mass spectrometry. The molecular weights of the identified differentially abundant proteins indicated an increased transport across the blood-brain barrier (BBB) at the peak of the disease, caused by an increase in BBB permeability.     

R-Spondin potentiates Wnt/β-catenin signaling through orphan receptors LGR4 and LGR5

The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.     

The rate of false positive sequence matches of peptides profiled by MALDI MS and identified by MS/MS

In most MALDI peptide profiling cases, sequencing is required to identify peptides of interest, preferentially by using different mass spectrometry techniques. Using identical samples, we determined the number of false positive matches in sequence of peptide identification using different mass spectrometers. This paper demonstrates that the reliability of the identification phase greatly benefits from concerted MS-technologies and determines the influence of mass accuracy, signal-to-noise and statistical score on peptide identification.     

Lateral migrations of fishes between floodplain lakes and their drainage channels at the Lower Rhine: diel and seasonal aspects

Although the total migratory activity of fishes from floodplain lakes in the Lower Rhine (Germany) into the connected drainage channels, estimated using electrofishing, fyke nets and visual observations, was low during summer 1997, conspicuous diel migrations of adult bream Abramis brama in June and July were recorded in one of the channels studied. A considerable proportion of the population of the floodplain lake immigrated into the channel at dusk and left it at dawn. During two winters (1994–1995, 1997–1998) the abundance of fishes increased considerably in the channels. The winter migrations, studied in detail in one of the channels in 1997–1998, started in late October, when the water temperature dropped below 10° C. In contrast to the mean of 500 fishes per 100 m channel length between May and October, >5000 fishes were found from November to March, with maximum values of >50 000 fishes per 100 m channel length in February 1998. More than 90% of these shoals were 0+ year fishes, dominated by roach Rutilus rutilus , bream, perch Perca fluviatilis and rudd Scardinius erythrophthalmus . These shoals entered the channel at sunrise and left it around sunset. Generally, the 0+ year fishes in the shoals swam separately from the older fishes, and different reactions towards the attack of predatory fishes were observed. The migrating shoals of juvenile fishes during the winter were interpreted as a strategy to minimize predation risk.