Nimodipine confers clinical improvement in two models of experimental autoimmune encephalomyelitis

Multiple sclerosis is characterised by inflammatory neurodegeneration, with axonal injury and neuronal cell death occurring in parallel to demyelination. Regarding the molecular mechanisms responsible for demyelination and axonopathy, energy failure, aberrant expression of ion channels and excitotoxicity have been suggested to lead to Ca²⁺ overload and subsequent activation of calcium‐dependent damage pathways. Thus, the inhibition of Ca²⁺ influx by pharmacological modulation of Ca²⁺ channels may represent a novel neuroprotective strategy in the treatment of secondary axonopathy. We therefore investigated the effects of the L‐type voltage‐gated calcium channel blocker nimodipine in two different models of mouse experimental autoimmune encephalomyelitis (EAE ), an established experimental paradigm for multiple sclerosis. We show that preventive application of nimodipine (10 mg/kg per day) starting on the day of induction had ameliorating effects on EAE in SJL /J mice immunised with encephalitic myelin peptide PLP _139–151, specifically in late‐stage disease. Furthermore, supporting these data, administration of nimodipine to MOG _35–55‐immunised C57BL /6 mice starting at the peak of pre‐established disease, also led to a significant decrease in disease score, indicating a protective effect on secondary CNS damage. Histological analysis confirmed that nimodipine attenuated demyelination, axonal loss and pathological axonal β‐amyloid precursor protein accumulation in the cerebellum and spinal cord in the chronic phase of disease. Of note, we observed no effects of nimodipine on the peripheral immune response in EAE mice with regard to distribution, antigen‐specific proliferation or activation patterns of lymphocytes. Taken together, our data suggest a CNS ‐specific effect of L‐type voltage‐gated calcium channel blockade to inflammation‐induced neurodegeneration.

     

Phylogeography of the southern skua complex-rapid colonization of the southern hemisphere during a glacial period and reticulate evolution

Whilst we have now a good understanding how past glaciation influenced species at the northern hemisphere, our knowledge of patterns and modes of speciation is far more limited for the southern hemisphere. We provide mtDNA based data on the phylogeography of a circumpolar distributed southern hemisphere seabird group-the southern skua complex (Catharacta spp.). Diversification of southern skuas dates between 210,000 yBP and 150,000 yBP and coincides with a glacial spanning 230,000-140,000 yBP. Skuas most likely first inhabited the Antarctic continent, in the course of global cooling and increasing glaciation spread to the sub-antarctic islands and Tristan da Cunha and finally colonized Patagonia and the Falkland Islands at the glacial maximum. Despite significant differences between taxa most populations still exchange genes with neighboring populations of other taxa and speciation is incomplete.     

Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A

Successful mitosis requires the right protein be degraded at the right time. Central to this is the spindle checkpoint that prevents the destruction of securin and cyclin B1 when there are improperly attached chromosomes. The principal target of the checkpoint is Cdc20, which activates the anaphase-promoting complex/cyclosome (APC/C). A Drosophila Cdc20/fizzy mutant arrests in mitosis with high levels of cyclins A and B, but paradoxically the spindle checkpoint does not stabilize cyclin A. Here, we investigated this paradox and found that Cdc20 is rate limiting for cyclin A destruction. Indeed, Cdc20 binds efficiently to cyclin A before and in mitosis, and this complex has little associated Mad2. Furthermore, the cyclin A complex must bind to a Cks protein to be degraded independently of the checkpoint. Thus, we identify a crucial role for the Cks proteins in mitosis and one mechanism by which the APC/C can target substrates independently of the spindle checkpoint.     

Isoaspartate-containing amyloid precursor protein-derived peptides alter efficacy and specificity of potential beta-secretases

Neuritic plaques of Alzheimer patients are composed of multiple protein components. Among them, the amyloid beta-peptides (Abeta) 1-40/42 and further N- and C-terminally modified fragments of Abeta are highly abundant. Most prominent are the isoaspartate (isoAsp)-Abeta peptides and pyroglutamyl (pGlu)-Abeta. While pGlu-Abeta can only be formed from an N-terminal glutamate by glutaminyl cyclase, spontaneous isoAsp-isomerization cannot occur at an N-terminal aspartate of peptides. This means that isoAsp-Abeta formation must precede proteolysis of the amyloid precursor protein (APP). Abeta generation from APP by beta- and gamma-secretases initiates the amyloid peptide aggregation and deposition process. Two aspartate proteases have been identified as secretases: BACE-1 (beta-site amyloid precursor protein cleaving enzyme) and the intramembrane gamma-secretase multiprotein complex. However, recent evidence supports more than one beta-secretase initiating this cascade. Formation of Abeta1-40/42 was predominantly studied by expression of mutated human APP sequences in cell culture and transgenic animals, generating Abeta fragments that did not contain such multiple posttranslational modifications as in Alzheimer's disease. This prompted us to investigate the catalytic turnover of Asp- or isoAsp-containing APP-derived peptide sequences by BACE-1 and cathepsin B, another potential beta-secretase. While cathepsin B is more effective than BACE-1 in processing the Asp-containing peptide derivatives, only cathepsin B can cleave the isoAsp-containing peptides, which occurs with high catalytic efficiency.     

Alternative pathways for production of beta-amyloid peptides of Alzheimer's disease

This highlight article describes three Alzheimer's disease (AD) studies presented at the 5th General Meeting of the International Proteolysis Society that address enzymatic mechanisms for producing neurotoxic beta-amyloid (Abeta) peptides. One group described the poor kinetics of BACE 1 for cleaving the wild-type (WT) beta-secretase site of APP found in most AD patients. They showed that cathepsin D displays BACE 1-like specificity and cathepsin D is 280-fold more abundant in human brain than BACE 1. Nevertheless, as BACE 1 and cathepsin D show poor activity towards the WT beta-secretase site, they suggested continuing the search for additional beta-secretase(s). The second group reported cathepsin B as an alternative beta-secretase possessing excellent kinetic efficiency and specificity for the WT beta-secretase site. Significantly, inhibitors of cathepsin B improved memory, with reduced amyloid plaques and decreased Abeta(40/42) in brains of AD animal models expressing amyloid precursor protein containing the WT beta-secretase site. The third group addressed isoaspartate and pyroglutamate (pGlu) posttranslational modifications of Abeta. Results showed that cathepsin B, but not BACE 1, efficiently cleaves the WT beta-secretase isoaspartate site. Furthermore, cyclization of N-terminal Glu by glutaminyl cyclase generates highly amyloidogenic pGluAbeta(3-40/42). These presentations suggest cathepsin B and glutaminyl cyclase as potential new AD therapeutic targets.     

EST and Mitochondrial DNA Sequences Support a Distinct Pacific Form of Salmon Louse, <Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis>

Nuclear deoxyribonucleic acid sequences from approximately 15,000 salmon louse expressed sequence tags (ESTs), the complete mitochondrial genome (16,148bp) of salmon louse, and 16S ribosomal ribonucleic acid (rRNA) and cytochrome oxidase subunit I (COI) genes from 68 salmon lice collected from Japan, Alaska, and western Canada support a Pacific lineage of Lepeophtheirus salmonis that is distinct from that occurring in the Atlantic Ocean. On average, nuclear genes are 3.2% different, the complete mitochondrial genome is 7.1% different, and 16S rRNA and COI genes are 4.2% and 6.1% different, respectively. Reduced genetic diversity within the Pacific form of L. salmonis is consistent with an introduction into the Pacific from the Atlantic Ocean. The level of divergence is consistent with the hypothesis that the Pacific form of L. salmonis coevolved with Pacific salmon (Onchorhynchus spp.) and the Atlantic form coevolved with Atlantic salmonids (Salmo spp.) independently for the last 2.5–11 million years. The level of genetic divergence coincides with the opportunity for migration of fish between the Atlantic and Pacific Ocean basins via the Arctic Ocean with the opening of the Bering Strait, approximately 5 million years ago. The genetic differences may help explain apparent differences in pathogenicity and environmental sensitivity documented for the Atlantic and Pacific forms of L. salmonis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Anaesthesia Monitoring by Recurrence Quantification Analysis of EEG Data

Appropriate monitoring of the depth of anaesthesia is crucial to prevent deleterious effects of insufficient anaesthesia on surgical patients. Since cardiovascular parameters and motor response testing may fail to display awareness during surgery, attempts are made to utilise alterations in brain activity as reliable markers of the anaesthetic state. Here we present a novel, promising approach for anaesthesia monitoring, basing on recurrence quantification analysis (RQA) of EEG recordings. This nonlinear time series analysis technique separates consciousness from unconsciousness during both remifentanil/sevoflurane and remifentanil/propofol anaesthesia with an overall prediction probability of more than 85%, when applied to spontaneous one-channel EEG activity in surgical patients.     

Distribution of ergot alkaloids and ricinoleic acid in different milling fractions

The sclerotia of the fungus Claviceps sp. are still a challenge for the milling industry. Ergot sclerotia are a constant contamination of the rye crop and have to be removed by modern milling technologies. Changing sizes and coloration of the sclerotia make it difficult to separate them from the grain. Ergot sclerotia are a problem when cleaning is insufficient and non-separated specimens or sclerotia fragments get into the milling stream and thus ergot alkaloids are distributed into the different cereal fractions. In model milling experiments, the residues of ergot in rye flour and the distribution of ergot into different milling fractions were investigated. Rye grains were mixed with whole ergot sclerotia and in another experiment with ergot powder and cleaned afterwards before milling. The ergot alkaloids ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, ergocristineand their related isomeric forms (-inine-forms), and additionally ricinoleic acid as a characteristic component of ergot, were quantified in the different milling fractions. From the first experiment, it can be shown that after harvesting even simple contact of sclerotia with bulk grains during ordinary handling or movement of bulk grain in the granary is sufficient to contaminate all the healthy or sound rye grains with ergot alkaloids. Thereby, the amount of ergot residue correlates with the amount of peripheral layers of rye grains in the flour. In an additional experiment without sclerotia specimens, bulk rye grains were loaded with powder of sclerotia. After subsequent cleaning, aconcentration of ergot alkaloids was detected, which was tenfold higher than the ergot alkaloidconcentration of the experiment with intact ergot sclerotia.     

Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.