Unbiased metabolome screen leads to personalized medicine strategy for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a rapidly progressive neurodegenerative disease that affects 1/350 individuals in the United Kingdom. The cause of amyotrophic lateral sclerosis is unknown in the majority of cases. Two-sample Mendelian randomization enables causal inference between an exposure, such as the serum concentration of a specific metabolite, and disease risk. We obtained genome-wide association study summary statistics for serum concentrations of 566 metabolites which were population matched with a genome-wide association study of amyotrophic lateral sclerosis. For each metabolite, we performed Mendelian randomization using an inverse variance weighted estimate for significance testing. After stringent Bonferroni multiple testing correction, our unbiased screen revealed three metabolites that were significantly linked to the risk of amyotrophic lateral sclerosis: Estrone-3-sulphate and bradykinin were protective, which is consistent with literature describing a male preponderance of amyotrophic lateral sclerosis and a preventive effect of angiotensin-converting enzyme inhibitors which inhibit the breakdown of bradykinin. Serum isoleucine was positively associated with amyotrophic lateral sclerosis risk. All three metabolites were supported by robust Mendelian randomization measures and sensitivity analyses; estrone-3-sulphate and isoleucine were confirmed in a validation amyotrophic lateral sclerosis genome-wide association study. Estrone-3-sulphate is metabolized to the more active estradiol by the enzyme 17β-hydroxysteroid dehydrogenase 1; further, Mendelian randomization demonstrated a protective effect of estradiol and rare variant analysis showed that missense variants within HSD17B1, the gene encoding 17β-hydroxysteroid dehydrogenase 1, modify risk for amyotrophic lateral sclerosis. Finally, in a zebrafish model of C9ORF72-amyotrophic lateral sclerosis, we present evidence that estradiol is neuroprotective. Isoleucine is metabolized via methylmalonyl-CoA mutase encoded by the gene MMUT in a reaction that consumes vitamin B12. Multivariable Mendelian randomization revealed that the toxic effect of isoleucine is dependent on the depletion of vitamin B12; consistent with this, rare variants which reduce the function of MMUT are protective against amyotrophic lateral sclerosis. We propose that amyotrophic lateral sclerosis patients and family members with high serum isoleucine levels should be offered supplementation with vitamin B12

Means simplifying data entry in large ERPbased on SAP framework

The thesis deals with the problem how are the various information system user interfaces suitable for the various groups of users of the IS. The author examines the impact of using suitable or unsuitable user interface on data entry process effectiveness in the scale of large IS. The thesis studies the user demands on IS and its interfaces and how well the interfaces solve these users demands. Author of the thesis suggests the measurement of the relationship quality between the users and the interface and using the exact method of search for the deficiencies in this relationship. The eforms technology is used to demonstrate that the new-fashioned and innovative user interfaces exists and can solve some users demands in the appropriate manner

A new zebrafish model produced by TILLING of SOD1-related amyotrophic lateral sclerosis replicates key features of the disease and represents a tool for in vivo therapeutic screening

Mutations in the superoxide dismutase gene (SOD1) are one cause of familial amyotrophic lateral sclerosis [ALS; also known as motor neuron disease (MND)] in humans. ALS is a relentlessly progressive neurodegenerative disease and, to date, there are no neuroprotective therapies with significant impact on the disease course. Current transgenic murine models of the disease, which overexpress mutant SOD1, have so far been ineffective in the identification of new therapies beneficial in the human disease. Because the human and the zebrafish (Danio rerio) SOD1 protein share 76% identity, TILLING (‘targeting induced local lesions in genomes’) was carried out in collaboration with the Sanger Institute in order to identify mutations in the zebrafish sod1 gene. A T70I mutant zebrafish line was characterised using oxidative stress assays, neuromuscular junction (NMJ) analysis and motor function studies. The T70I sod1 zebrafish model offers the advantage over current murine models of expressing the mutant Sod1 protein at a physiological level, as occurs in humans with ALS. The T70I sod1 zebrafish demonstrates key features of ALS: an early NMJ phenotype, susceptibility to oxidative stress and an adult-onset motor neuron disease phenotype. We have demonstrated that the susceptibility of T70I sod1 embryos to oxidative stress can be used in a drug screening assay, to identify compounds that merit further investigation as potential therapies for ALS

Characterisation of the phenotype of adult MFN2^L285X mutant zebrafish.

<p>(A) Survival curve showing loss of viability of MFN2^L285X/L285X zebrafish from 170 days onwards (p<0.0001, Log-rank (Mantel-Cox) test). (B) Bar graph of body weight for all three groups of siblings at 60–70 days old (p<0.0001, Kruskal-Wallis with Dunn’s multiple comparisons test). (C) Bar graph of the nose to tail length of MFN2^L285X mutant and sibling zebrafish at 60–70 days old (p<0.001, Kruskal-Wallis with Dunn’s multiple comparisons test).</p

MFN2L285X/L285X zebrafish have a progressive swimming defect.

<p>(A) Kaplan-Meier plot of swimming endurance in 100 and 200 day-old zebrafish of each genotype. MFN2+/+ and MFN2^L285X/+ zebrafish swam for 30 minutes at both 100 and 200d old. For clarity only the 200d data is plotted, although the lines are superimposed. (MFN2^L285X/L285X: p<0.001 at both ages, Log-rank (Mantel-Cox) test). (B) Frequency distribution plot of swimming angle (measured from horizontal) of adult wild type zebrafish. Representative frames captured from digital video recordings of adult zebrafish and cartoons are shown. The upper panel shows a wild type sibling, and the lower panel shows a MFN2^L285X/L285X zebrafish showing abnormal posture, with the body angled at 30<b>°</b> from horizontal. (C) Bar graph showing the percentage of time spent with normal swimming posture during 1 minute of observation in 90 day-old zebrafish (p<0.0001, 2-sided T test Mann-Whitney). (D) Bar graph showing the mean duration of each bout of abnormal swimming in 90 day-old old zebrafish (p<0.0001, 2-sided T test Mann-Whitney). (E, F) Graphs showing the decline in normal swimming posture (E) and the increasing duration of each bout of abnormal swimming (F), between 105 and 155 days old.</p

Analysis of axonal transport of mitochondria.

<p>(A) Representative kymographs showing defective axonal transport of mitochondria along axons of cultured zebrafish neurons from MFN2^L285X/L285X embryos compared with MFN2^+/+ and MFN2^L285X/+ neurons. (B) Mitochondria in cultured MFN2^L285X/L285X neurons are significantly less motile; anterograde displacement of mitochondria is not significantly altered, but retrograde displacement is significantly reduced (p<0.01, one-way ANOVA). (C) The velocity of retrograde but not anterograde mitochondrial transport is significantly reduced in cultured MFN2^L285X/L285X neurons (p<0.001, Kruskal-Wallis with Dunn’s multiple comparisons test). (D) Representative images of axons from neurons of each genotype. The cell body is to the left, and distal axon is to the right hand side (scale bar  = 10 <b>µ</b>m) (E) Comparison of mitochondrial density in proximal and distal axonal segments, there is a significant reduction in mitochondrial density in the proximal but not distal segment of MFN2^L285X/L285X neurons (p<0.05, 2-way ANOVA with Bonferroni’s multiple comparisons test). (F) There is a significant increase in the inter-mitochondrial distance in the proximal axon of MFN2^L285X/L285X neurons (p<0.01, 2-way ANOVA with Bonferroni’s multiple comparisons test).</p

Alterations of NMJ pathology in MFN2^L285X larvae (A–D) and adults (E–H).

<p>(A) Representative images of dual immunofluorescence staining of whole mount 15d larvae of each genotype for <b>α</b>-Bungarotoxin (green) and SV2 (red). (B) ICQ analysis of 3 larvae per group reveals no alteration of SV2/<b>α</b>-Bungarotoxin co-localisation in MFN2^L285X/+ or MFN2^L285X/L285X larvae. Compared to siblings the (C) pre-synaptic area, and (D) post-synaptic area are not significantly different in MFN2^L285X/+ or MFN2^L285X/L285X larvae. (E) Representative images of a-Bungarotoxin (green) and SV2 (red) in 200 day-old zebrafish. (F) ICQ is significantly reduced in MFN2^L285X/L285X at this stage, and there are significant reductions in the pre- and post-synaptic area (G and H). Scale bars  = 10 <b>µ</b>m.</p

A genetic model of amyotrophic lateral sclerosis in zebrafish displays phenotypic hallmarks of motoneuron disease

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that, for ∼80% of patients, is fatal within five years of diagnosis. To better understand ALS, animal models have been essential; however, only rodent models of ALS exhibit the major hallmarks of the disease. Here, we report the generation of transgenic zebrafish overexpressing mutant Sod1. The construct used to generate these lines contained the zebrafish sod1 gene and ∼16 kb of flanking sequences. We generated lines expressing the G93R mutation, as well as lines expressing wild-type Sod1. Focusing on two G93R lines, we found that they displayed the major phenotypes of ALS. Changes at the neuromuscular junction were observed at larval and adult stages. In adulthood the G93R mutants exhibited decreased endurance in a swim tunnel test. An analysis of muscle revealed normal muscle force, however, at the end stage the fish exhibited motoneuron loss, muscle atrophy, paralysis and premature death. These phenotypes were more severe in lines expressing higher levels of mutant Sod1 and were absent in lines overexpressing wild-type Sod1. Thus, we have generated a vertebrate model of ALS to complement existing mammal models