Ions, energy and axonal injury: towards a molecular neurology of multiple sclerosis

It is now clear that effective treatments for nervous system disorders such as multiple sclerosis (MS) represent achievable objectives. Molecular mechanisms of axonal degeneration - a major pathological substrate for disability in MS - have been identified, pointing to the possibility of neuroprotection. Although previous studies were mainly carried out in laboratory models, recent analyses of human MS tissue have identified molecular targets that are related to mitochondrial function and specific isoforms of ion channels as contributors to axonal degeneration in MS. Taken together, the observations in model systems and in human tissue converge on the identification of a group of molecules that are related to ion fluxes and energetics as significant actors in the axonal-injury cascade, and suggest a set of molecular targets that might be useful in the development of new therapies.     

Treatment of experimental allergic encephalomyelitis with myelin basic protein: Which route is best?

When myelin basic protein (BP) has been used for the treatment of multiple sclerosis (MS), it has been injected intramuscularly (IM) or subcutaneously (SC). Experimental allergic encephalomyelitis (EAE) is widely used as a model for MS, and the use of BP for MS is based on its efficacy in EAE. The present work shows that BP is more effective in EAE when administered by intravenous (IV) route than by IM or SC routes. These observations may be pertinent to therapeutic trials in MS.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry

We describe an improved assay for platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) using HPLC-tandem mass spectrometry (LC-MS/MS). The present method can readily detect as little as 1 pg (1.9 fmol) of PAF, a significant improvement over previously described LC-MS/MS methods, and gives a linear response up to 1,000 pg of PAF. Our method also overcomes the artifacts from isobaric lipids that have limited the usefulness of certain existing LC-MS/MS assays for PAF. In the course of these studies, we detected three novel lipid species in human neutrophils. One of the novel lipids appears to be a new molecular species of PAF, and the other two have chromatographic and mass spectrometric properties consistent with stearoyl-formyl-glycerophosphocholine and oleoyl-formyl-glycerophosphocholine. These observations identify previously unknown potential interferences in the measurement of PAF by LC-MS/MS. Moreover, our data suggest that the previously described palmitoyl-formyl-glycerophosphocholine is not unique but rather is a member of a new and poorly understood family of formylated lipids.     

Morphomechanics: goals, basic experiments and models

Morphomechanics is a branch of developmental biology, studying the generation, space-time patterns and morphogenetic role of mechanical stresses (MS) which reside in embryonic tissues. All the morphogenetically active embryonic tissues studied in this respect have been shown to bear substantial mechanical stresses of tension or pressure. MS are indispensable for organized cell movements, expression of a number of developmentally important genes and the very viability of cells. Even a temporary relaxation of MS leads to an increase in the morphological variability and asymmetry of embryonic rudiments. Moreover, MS may be among the decisive links of morphogenetic feedback required for driving forth embryonic development and providing its regular space-time patterns. We hypothesize that one such feedback is based upon the tendency of cells and tissues to hyperrestore (restore with an overshoot) their MS values after any deviations, either artificial or produced by neighboring morphogenetically active tissues. This idea is supported by a number of observations and experiments performed on the tissue and individual cell levels. We describe also the models demonstrating that a number of biologically realistic stationary shapes and propagating waves can be generated by varying the parameters of the hyperrestoration feedback loop. Morphomechanics is an important and rapidly developing branch of developmental and cell biology, being complementary to other approaches.     

Castration attenuates myelin repair following lysolecithin induced demyelination in rat optic chiasm: an evaluation using visual evoked potential, marker genes expression and myelin staining

Multiple sclerosis (MS) is a demyelinating disease that affects the central nervous system. MS is the most common neurological disorder in young adults with a greater incidence among females. Male gonadal hormones have a protective effect on neural system development and myelin maturation. In this study, we investigate the effect of castration on lysolecithin-induced demyelination and remyelination processes using visual evoked potentials, in addition to measuring the expressions of Olig2, MBP, Nogo-A and GFAP mRNAs as oligodendrocyte or astrocyte markers; and histological assessments by myelin-specific staining. We observed more expanded demyelination with delayed repair process in castrated rats. Expression levels of the aforementioned marker genes confirmed histological and electrophysiological observations. Our results showed a pivotal role for endogenous male hormones in the context of demyelinating insults. It may also account for the different prognosis of MS between male and female genders and provide new insights for therapeutic treatments.     

Strategies for the identification of loci responsible for the pathogenesis of multiple sclerosis

Multiple sclerosis (MS) is a chronic, debilitating disease, which manifests itself by de-myelination of the central nervous system (CNS). MS is predominantly found in Caucasians of European decent and is more prominent in females than males. MS is one of the most prevalent causes of disability of young adults in the world. The exact cause of MS is not known, however genetic susceptibility to MS is linked to the major histocompability complex (MHC). Self reactive CD4+ T cells, specific for CNS antigens, such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and proteolipid protein (PLP), are detectable in MS patients along with pathogenic autoantibodies specific to these CNS antigens produced by B cells. These observations suggest that MS is an autoimmune disease. Epidemiology of MS along with the analysis of sibling pairs and twins suggest that the multiple genetic factors and their interaction with environment contribute to disease susceptibility. Recent developments and advancements in genetic analysis may aid in accurate determination of genetic risk factors for the development of MS. We review these developments, advances in technology and discuss recent results in this article. These authors contributed equally to this paper     

The Evolutionary Reduction of Microsporangia in Microseris (Asteraceae): Transition Genotypes and Phenotypes

Abstract: The loss of the two inner (adaxial) microsporangia (MS) on the anthers is a shared, derived character for three species of the genus Microseris (Asteraceae). In a hybrid between M. douglasii (4 MS) and M. bigelovii (2 MS), one major gene and four modifier loci are responsible for the difference in MS number. The homozygous recessive (2 MS) genotype of the major gene is necessary but not sufficient for the reduction. In addition, at least five M. bigelovii (2 MS) alleles of the three major modifiers are needed for a stable 2‐MS phenotype in all florets of a plant. One, two or three M. bigelovii alleles of the modifiers cause the random reduction or loss of some of the adaxial MS. When the major gene and two modifiers specify 2 MS and only one modifier is homozygous for the M. douglasii (4 MS) alleles, sister plants can have any phenotype from pure 2 MS to pure 4 MS. Here, we examine the phenotypic expression of these genotypes raised under the normal winter annual conditions and under long‐day conditions. In all cases, the phenotypes vary among sister plants, but the range of variation (most notably under long‐day conditions) depends on the specific modifier gene contributing the M. douglasii alleles. The phenotypic variance in one of the genotypes was decreased by a factor of ten in the depauperate heads produced in the long‐day experiment. This effect is mediated by a dependence of the MS phenotype on the position of the floret relative to the edge of the flowering head (capitulum) and directly by the size of the capitulum. Genotypes specifying phenotypes with more or less precisely two or four MS in all florets show hardly any dependence on environmental or developmental factors. The significance of these observations lies in the non‐linear, “canalized” relationship between phenotypic expression and gene dosage, which shows how a qualitative morphological change dependent on a single major gene mutation can pass through a potentially maladaptive intermediate stage.     

The genetic epidemiology of multiple sclerosis

Epidemiological studies have implicated an interplay between genetic and environmental factors in the aetiology of multiple sclerosis (MS). There is a familial recurrence rate of approximately 15%. Meta-analysis of the recurrence risk shows that the rate is highest overall for siblings, then parents and children, with lower rates in second- and third-degree relatives. Recurrence is highest for monozygotic twins. Conversely, the frequency in adoptees is similar to the population lifetime risk. The age-adjusted risk for half siblings is also less than for full siblings. Recurrence is higher in the children of conjugal pairs with MS than the offspring of single affected. These classical genetic observations suggest that MS is a complex trait in which susceptibility is determined by several genes acting independently or epistatically. Comparisons between co-affected sibling pairs provide no evidence for correlation with age or year at onset and mode of presentation or disability. Thus far, the identification of susceptibility genes has proved elusive but genetic strategies are now in place which should illuminate the problem. The main dividend will be an improved understanding of the pathogenesis. To date, population studies have demonstrated an association between the class II major histocompatibility complex (MHC) alleles DR15 and DQ6 and their corresponding genotypes. An association with DR4, with or without the primary DR15 link, is seen in some Mediterranean populations. Candidate gene approaches have otherwise proved unrewarding. Four groups of investigators have undertaken a systematic search of the genome. In common with most other complex traits, no major susceptibility gene has been identified but regions of interest have been provisionally identified. These genetic analyses are predicated on the assumption that MS is one disease. Genotypic and phenotypic analyses are beginning to question this assumption. A major part of future studies in the genetics of MS will be to resolve the question of disease heterogeneity.     

Outlier Detection using Projection Quantile Regression for Mass Spectrometry Data with Low Replication

ABSTRACT: BACKGROUND: Mass spectrometry (MS) data are often generated from various biological or chemical experiments and there may exist outlying observations, which are extreme due to technical reasons. The determination of outlying observations is important in the analysis of replicated MS data because elaborate pre-processing is essential for successful analysis with reliable results and manual outlier detection as one of pre-processing steps is time-consuming. The heterogeneity of variability and low replication are often obstacles to successful analysis, including outlier detection. Existing approaches, which assume constant variability, can generate many false positives (outliers) and/or false negatives non-outliers). Thus, a more powerful and accurate approach is needed to account for the heterogeneity of variability and low replication. FINDINGS: We proposed an outlier detection algorithm using projection and quantile regression in MS data from multiple experiments. The performance of the algorithm and program was demonstrated by using both simulated and real-life data. The projection approach with linear, nonlinear, or nonparametric quantile regression was appropriate in heterogeneous high-throughput data with low replication. CONCLUSION: Various quantile regression approaches combined with projection were proposed for detecting outliers. The choice among linear, nonlinear, and nonparametric regressions is dependent on the degree of heterogeneity of the data. The proposed approach was illustrated with MS data with two or more replicates.     

Using stem cells in multiple sclerosis therapies

Stem cell transplantation approaches offer for the first time the opportunity to design therapeutic approaches for multiple sclerosis (MS) with curative intent. Here we discuss key observations and questions emerging from clinical trials of hematopoietic stem cell transplantation for MS and from studies of myelin/neural repair in experimental models of demyelinating disorders.     

Behavior of melon-headed whales, Peponocephala electra, near oceanic islands

Southall et al . (2006) concluded that a near mass stranding (MS) of melon-headed whales (MHWs), Peponocephala electra , in Hanalei Bay, Kauai, Hawaii, on 3–4 July 2004, was likely related to the operation of mid-frequency sonars (MFS). However, subsequent authors argued that the nearly simultaneous entry of MHWs into Sasanhaya Bay, Rota (∼5,740 km away) made this conclusion untenable. They suggested that both sightings, and other MSs of MHWs, could be related to lunar cycles. To resolve this question, we reviewed information on the biology and behavior of MHWs and compared the two sightings to observations of MHWs around Palmyra Atoll and Nuku Hiva, French Polynesia. We also tested for a relationship between observations and MSs of MHWs with lunar cycles. MHWs near many oceanic islands rest nearshore during the day and feed offshore in deeper water at night. The MHWs at Rota exhibited normal diurnal resting behavior as seen at Palmyra and Nuku Hiva, while those at Kauai showed milling behavior typically seen prior to MS events. Thus, these events were not similar. Neither observations nor MSs of MHWs were related to lunar cycles. Our review of MHW behavior strengthens the case that MFS use played a major role in the near MS in Hanalei Bay.     

Pregnenolone sulfate in the brain: a controversial neurosteroid

Pregnenolone sulfate (PREGS) has been shown, either at high nanomolar or at micromolar concentrations, to increase neuronal activity by inhibiting GABAergic and by stimulating glutamatergic neurotransmission. PREGS is also a potent modulator of sigma type 1 (sigma1) receptors. It has been proposed that these actions of PREGS underlie its neuropharmacological effects, and in particular its influence on memory processes. On the other hand, the PREGS-mediated increase in neuronal excitability may become dangerous under particular conditions, for example in the case of excitotoxic stress or convulsions. However, the physiopathological significance of these observations has recently been put into question by the failure to detect significant levels of PREGS within the brain and plasma of rats and mice, either by direct analytical methods based on liquid chromatography/mass spectrometry (LC/MS) or enzyme linked immunosorbent assay (ELISA) with specific antibodies against PREGS, or by indirect gas chromatography/mass spectrometry (GC/MS) analysis with improved sample workup. These recent results have not come to the attention of a large number of neurobiologists interested in steroid sulfates. However, although available direct analytical methods have failed to detect levels of PREGS above 0.1-0.3 ng/g in brain tissue, it may be premature to completely exclude the local formation of biologically active PREGS within specific and limited compartments of the nervous system. In contrast to the situation in rodents, significant levels of sulfated 3beta-hydroxysteroids have been measured in human plasma and brain. Previous indirect measures of steroid sulfates by radioimmunoassays (RIA) or GC/MS had detected elevated levels of PREGS in rodent brain. The discrepancies between the results of different assay procedures have revealed the danger of indirect analysis of steroid sulfates. Indeed, PREGS must be solvolyzed/hydrolyzed prior to RIA or GC/MS analysis, and it is the released, unconjugated PREG which is then quantified. Extreme caution needs to be exercised during the preparation of samples for RIA or GC/MS analysis, because the fraction presumed to contain only steroid sulfates can be contaminated by nonpolar components from which PREG is generated by the solvolysis/hydrolysis/derivatization reactions.     

Altered expression patterns of complement factor H and miR-146a genes in acute-chronic phases in experimental autoimmune encephalomyelitis mouse

Considerable advances have been made in identification of the involvement of immune modulators in diseases. There is growing evidence on the role of complement pathway in pathogenesis and course of multiple sclerosis (MS). Moreover, it has been recognized that microRNAs (miRNAs) play an essential role in modulation and development of immune response in the central nervous system. We aimed to investigate the expression profile of complement factor H (CFH) and miR-146a genes in experimental autoimmune encephalomyelitis (EAE) mouse model of MS to detect the possible roles of CFH and miR-146a as biomarkers of MS disease stats. Expression of CFH and miR-146a genes in liver and brain tissues of EAE mice was measured in acute and chronic phases of disease compared to matched controls using real-time polymerase chain reaction. In the liver, increased expression of CFH gene was observed in the chronic phase compared to the acute phase. However, no significant difference was observed between acute and chronic phase mice with normal mice, while miR-146a expression was significantly decreased in livers of EAE mice in chronic group compared to acute and control groups. The expression of CFH gene in brain had a significant decrease in acute and chronic phases compared to healthy mice. Taken together, these observations indicate probable implication of complement system and miR-146a in course of immune-related diseases and reveal more facts about the pathogenesis of MS. However, further work is needed to determine protein levels of CFH and other possible targets of miR-146a in serum and cerebrospinal fluid of MS patients.     

Composition of the mitochondrial electron transport chain in acanthamoeba castellanii: structural and evolutionary insights

The mitochondrion, derived in evolution from an α-proteobacterial progenitor, plays a key metabolic role in eukaryotes. Mitochondria house the electron transport chain (ETC) that couples oxidation of organic substrates and electron transfer to proton pumping and synthesis of ATP. The ETC comprises several multiprotein enzyme complexes, all of which have counterparts in bacteria. However, mitochondrial ETC assemblies from animals, plants and fungi are generally more complex than their bacterial counterparts, with a number of 'supernumerary' subunits appearing early in eukaryotic evolution. Little is known, however, about the ETC of unicellular eukaryotes (protists), which are key to understanding the evolution of mitochondria and the ETC. We present an analysis of the ETC proteome from Acanthamoeba castellanii, an ecologically, medically and evolutionarily important member of Amoebozoa (sister to Opisthokonta). Data obtained from tandem mass spectrometric (MS/MS) analyses of purified mitochondria as well as ETC complexes isolated via blue native polyacrylamide gel electrophoresis are combined with the results of bioinformatic queries of sequence databases. Our bioinformatic analyses have identified most of the ETC subunits found in other eukaryotes, confirming and extending previous observations. The assignment of proteins as ETC subunits by MS/MS provides important insights into the primary structures of ETC proteins and makes possible, through the use of sensitive profile-based similarity searches, the identification of novel constituents of the ETC along with the annotation of highly divergent but phylogenetically conserved ETC subunits.     

The fate of b-ions in the two worlds of collision-induced dissociation

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss.     

The Effect of Peptide Identification Search Algorithms on MS2-Based Label-Free Protein Quantification

Abstract Several approaches exist for the quantification of proteins in complex samples processed by liquid chromatography-mass spectrometry followed by fragmentation analysis (MS2). One of these approaches is label-free MS2-based quantification, which takes advantage of the information computed from MS2 spectrum observations to estimate the abundance of a protein in a sample. As a first step in this approach, fragmentation spectra are typically matched to the peptides that generated them by a search algorithm. Because different search algorithms identify overlapping but non-identical sets of peptides, here we investigate whether these differences in peptide identification have an impact on the quantification of the proteins in the sample. We therefore evaluated the effect of using different search algorithms by examining the reproducibility of protein quantification in technical repeat measurements of the same sample. From our results, it is clear that a search engine effect does exist for MS2-based label-free protein quantification methods. As a general conclusion, it is recommended to address the overall possibility of search engine-induced bias in the protein quantification results of label-free MS2-based methods by performing the analysis with two or more distinct search engines.     

Suppressor T cells are activated in vivo in patients with multiple sclerosis coinciding with remission from acute attack

By a variety of assays, investigators (1-4) from several laboratories previously observed changes in numbers of thymus-derived suppressor lymphocytes (Ts) in patients with acute active multiple sclerosis (MS). Changes in numbers of Ts in the peripheral blood compartment of such patients closely follow disease activity. Extending these observations we now report Ts are activated in the peripheral blood of certain patients with MS. This activation occurred in vivo in the majority of patients with active disease, but only during remission after an acute episode of MS. In contrast, activated Ts were found infrequently in patients with chronic progressive or stable MS, with neurologic diseases other than MS, or healthy individuals. The suppressor activity found in peripheral blood T cells during the acute remitting stage of disease was associated with elevated numbers of lymphocytes bearing Fc receptors for IgG and/or OKT8 markers.     

A global analysis of peptide fragmentation variability

Understanding the fragmentation process in MS/MS experiments is vital when trying to validate the results of such experiments, and one way of improving our understanding is to analyze existing data. We here present our findings from an analysis of a large and diverse data set of MS/MS-based peptide identifications, in which each peptide has been identified from multiple spectra, recorded on two commonly used types of electrospray instruments. By analyzing these data we were able to study fragmentation variability on three levels: (i) variation in detection rates and intensities for fragment ions from the same peptide sequence measured multiple times on a single instrument; (ii) consistency of rank-based fragmentation patterns; and (iii) a set of general observations on fragment ion occurrence in MS/MS experiments, regardless of sequence. Our results confirm that substantial variation can be found at all levels, even when high-quality identifications are used and the experimental conditions as well as the peptide sequences are kept constant. Finally, we discuss the observed variability in light of ongoing efforts to create spectral libraries and predictive software for target selection in targeted proteomics.     

The use of 2D-electrophoresis and de novo sequencing to characterize inter- and intra-cultivar protein polymorphisms in an allopolyploid crop

Polyploidy and allopolyploidy have played an important role in the evolution of many plants and crops. Several techniques exist to characterize allopolyploid varieties. Analyzing the consequences of genomic reorganization at the gDNA level is a prerequisite but a better insight into the consequences for the phenotype is also primordial. As such, protein polymorphism analysis is important in understanding plant and crop biodiversity and is a driving force behind crop improvement. Our strategy to analyze protein isoforms and to detect possible gene silencing or deletion in bananas was based on protein analysis. Bananas are a good representative of a complex allopolyploid and important crop. We combined two-dimensional electrophoresis (2DE) and 2D DIGE with de novo MS/MS sequence determination to characterize a range of triploid varieties. Via Principal Component Analysis (PCA) and hierarchical clustering we were able to blindly classify the different varieties according to their presumed genome constitution. We report for the first time the application of an automated approach for the derivatization of peptides for facilitated MS/MS de novo sequence determination. We conclude that the proteome does not always correspond to the presumed genome formulae and that proteomics is a powerful tool to characterize varieties. The observations at the protein level provide good indications for a more complex genome structure and genomic rearrangement in some banana varieties.