C9ORF72 hexanucleotide repeat exerts toxicity in a stable, inducible motor neuronal cell model, which is rescued by partial depletion of Pten.

Amyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease, characterised by progressive failure of the neuromuscular system. A (G4C2)n repeat expansion in C9ORF72 is the most common genetic cause of ALS and frontotemporal dementia (FTD). To date, the balance of evidence indicates that the (G4C2)n repeat causes toxicity and neurodegeneration via a gain-of-toxic function mechanism; either through direct RNA toxicity or through the production of toxic aggregating dipeptide repeat proteins. Here, we have generated a stable and isogenic motor neuronal NSC34 cell model with inducible expression of a (G4C2)102 repeat, to investigate the gain-of-toxic function mechanisms. The expression of the (G4C2)102 repeat produces RNA foci and also undergoes RAN translation. In addition, the expression of the (G4C2)102 repeat shows cellular toxicity. Through comparison of transcriptomic data from the cellular model with laser-captured spinal motor neurons from C9ORF72-ALS cases, we also demonstrate that the PI3K/Akt cell survival signalling pathway is dysregulated in both systems. Furthermore, partial knockdown of Pten rescues the toxicity observed in the NSC34 (G4C2)102 cellular gain-of-toxic function model of C9ORF72-ALS. Our data indicate that PTEN may provide a potential therapeutic target to ameliorate toxic effects of the (G4C2)n repeat

Co-ordinated Airborne Studies in the Tropics (CAST)

This is the author accepted manuscript. The final version is available from the American Meteorological Society via http://dx.doi.org/10.1175/BAMS-D-14-00290.1The Co-ordinated Airborne Studies in the Tropics (CAST) project is studying the chemical composition of the atmosphere in the Tropical Warm Pool region to improve understanding of trace gas transport in convection. The main field activities of the CAST (Co-ordinated Airborne Studies in the Tropics) campaign took place in the West Pacific in January/February 2014. The field campaign was based in Guam (13.5°N, 144.8°E) using the UK FAAM BAe-146 atmospheric research aircraft and was coordinated with the ATTREX project with the unmanned Global Hawk and the CONTRAST campaign with the Gulfstream V aircraft. Together, the three aircraft were able to make detailed measurements of atmospheric structure and composition from the ocean surface to 20 km. These measurements are providing new information about the processes influencing halogen and ozone levels in the tropical West Pacific as well as the importance of trace gas transport in convection for the upper troposphere and stratosphere. The FAAM aircraft made a total of 25 flights between 1°S-14°N and 130°-155°E. It was used to sample at altitudes below 8 km with much of the time spent in the marine boundary layer. It measured a range of chemical species, and sampled extensively within the region of main inflow into the strong West Pacific convection. The CAST team also made ground-based measurements of a number of species (including daily ozonesondes) at the Atmospheric Radiation Measurement program site on Manus Island, Papua New Guinea (2.1°S, 147.4°E). This article presents an overview of the CAST project focussing on the design and operation of the West Pacific experiment. It additionally discusses some new developments in CAST, including flights of new instruments on the Global Hawk in February/March 2015.CAST is funded by NERC and STFC, with grant NE/ I030054/1 (lead award), NE/J006262/1, NE/J006238/1, NE/J006181/1, NE/J006211/1, NE/J006061/1, NE/J006157/1, NE/J006203/1, NE/J00619X/1, and NE/J006173/1. N. R. P. Harris was supported by a NERC Advanced Research Fellowship (NE/G014655/1). P. I. Palmer acknowledges his Royal Society Wolfson Research Merit Award. The BAe-146-301 Atmospheric Research Aircraft is flown by Directflight Ltd and managed by the Facility for Airborne Atmospheric Measurements, which is a joint entity of the Natural Environment Research Council and the Met Office. The authors thank the staff at FAAM, Directflight and Avalon Aero who worked so hard toward the success of the aircraft deployment in Guam, especially for their untiring efforts when spending an unforeseen 9 days in Chuuk. We thank the local staff at Chuuk and Palau, as well as the authorities in the Federated States of Micronesia for their help in facilitating our research flights. Special thanks go to the personnel associated with the ARM facility at Manus, Papua New Guinea without whose help the ground-based measurements would not have been possible. Thanks to the British Atmospheric Data Centre (BADC) for hosting our data and the NCAS Atmospheric Measurement Facility for providing the radiosonde and ground-based ozone equipment. Chlorophyll-a data used in Figure 1 were extracted using the Giovanni online data system, maintained by the NASA GES DISC. We also acknowledge the MODIS mission scientists and associated NASA personnel for the production of this data set. Finally we thank many individual associated with the ATTREX and CONTRAST campaigns for their help in the logistical planning, and we would like to single out Jim Bresch for his excellent and freely provided meteorological advice

SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits.

Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. Here, we show that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. We further demonstrate that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, we show that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection.MRC, ERC, FP

Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy

GGGGCC repeat expansions of C9ORF72 represent the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We and others have proposed that RNA transcribed from the repeat sequence is toxic via sequestration of RNA-binding factors. Both GGGGCC-repeat (sense) and CCCCGG-repeat (antisense) molecules are detectable by fluorescence in situ hybridisation as RNA foci, but their relative expression pattern within the CNS and contribution to disease has not been determined. Blinded examination of CNS biosamples from ALS patients with a repeat expansion of C9ORF72 showed that antisense foci are present at a significantly higher frequency in cerebellar Purkinje neurons and motor neurons, whereas sense foci are present at a significantly higher frequency in cerebellar granule neurons. Consistent with this, inclusions containing sense or antisense derived dipeptide repeat proteins were present at significantly higher frequency in cerebellar granule neurons or motor neurons, respectively. Immunohistochemistry and UV-crosslinking studies showed that sense and antisense RNA molecules share similar interactions with SRSF2, hnRNP K, hnRNP A1, ALYREF, and hnRNP H/F. Together these data suggest that, although sense and antisense RNA molecules might be expected to be equally toxic via their shared protein binding partners, distinct patterns of expression in various CNS neuronal populations could lead to relative differences in their contribution to the pathogenesis of neuronal injury. Moreover in motor neurons, which are the primary target of pathology in ALS, the presence of antisense foci (χ2, p 2, p = 0.75) correlated with mislocalisation of TDP-43, which is the hallmark of ALS neurodegeneration. This has implications for translational approaches to C9ORF72 disease, and furthermore interacting RNA-processing factors and transcriptional activators responsible for antisense versus sense transcription might represent novel therapeutic targets

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Postauditing in the EIA process

ABSTACT Environmental impact assessment (EIA) is an integral part of the protection of the environment. Although it is a tool which considers the impact on many different elements of the environment, the process also has many deficiencies. Because of its limitations, an audit has been forming since the beginning of EIA. An EIA audit evaluates the performance of an EIA by comparing actual impacts detected after the realization of the project to those that were predicted, those that were listed in the environmental impact statement. This paper will offer an insight into the EIA and the post-auditing process and contains three basic parts of these areas. The first part explores the EIA, its historical evolution and application in the Czech Republic. The second part will discuss the post-audit itself. In this part, the different post-auditing processes, which were carried out in different parts of the world, were chosen. Within the selected post-auditing, the predicted data from the environmental impact statements are statistically compared with resultant data from reality. It is therefore necessary to analyze the percentage given for how many predictions were wrong in a positive way, accurate or wrong in negative way. From different results of post-auditing, we can tell that only a small percentage of the..

MNTs attenuated survival of mouse motor neurons co-cultured with human astrocytes from SOD1 ALS patients.

<p>a) Survival of mouse motor neurons (MNs), expressing GFP under the Hb9 promoter, was significantly decreased in co-cultures with human astrocytes (iAstrocytes) from an ALS patient a SOD1 mutation (Patient 2/SOD1 210; light grey) compared to those from 3 compiled control patients (3 compiled; white) (p<0.0001, unpaired t-test). BNN20, BNN27, and BNN23 were each tested at 1 μM (dark grey), 10 μM (black), and 30 μM (medium grey) in iAstrocyte-mouse MN co-cultures. At 10 μM, BNN20 (p<0.01, Tukey’s test) and BNN27 (p<0.001, Tukey’s test) significantly increased the survival of MNs co-cultured with SOD1 iAstrocytes relative to untreated samples. b) The effects of BNN27 (red) were tested, along with Riluzole (dark grey), on iAstrocytes from 3 control patients (compiled) and 2 individual ALS patients each carrying a distinct SOD1 mutation (Patient 1/SOD1 91 and Patient 2/SOD1 210). Left, BNN27 (10 μM) treatment yielded a significant increase in MN survival in co-cultures with iAstrocytes from Patient 1 (p<0.0001, Tukey’s test) and Patient 2 (p<0.0001, Tukey’s test) relative to untreated co-cultures (light grey). Similarly, Riluzole (10 μM) treatment also significantly increased MN survival in co-cultures with Patient 1 (p<0.0001, Tukey’s test) and Patient 2 (p<0.0001, Tukey’s test) iAstroctyes. Right, representative images of mouse MNs co-cultured with iAstrocytes from control untreated (control), SOD1 210 untreated (untreated), SOD1 210 Riluzole (Riluzole), and SOD1 210 BNN27 (BNN27). Scale bar = 20μM. c) Oxidative stress levels were significantly increased in iAstrocytes from 2 ALS patients containing a SOD1 mutation (Patient 1/SOD1 91 and Patient 2/SOD1 210; light grey) compared to those from 2 control patients (white) (p<0.0001, Tukey’s test). BNN27 treatment (red) produced a significant decrease in oxidative stress levels from SOD1 iAstrocytes relative to untreated cells (p<0.001, Tukey’s test). Scale bar = 10μM. Experiments were performed in triplicate (a, c) and quadruplicate (b). Data are presented as mean ± SEMs. ** p<0.01, ***p<0.001, ****p<0.0001.</p

Effect of BNN27 treatment on stride length and width in male and female mice.

<p>Male Tg vehicle-treated mice exhibited a decrease in stride length (a, left) at p115 (p = 0.0136, Mann-Whitney <i>U</i> test) and stride width (b, left) at p95 (p = 0.0202, Mann-Whitney <i>U</i> test) relative to Wt vehicle-treated mice. Female Tg vehicle-treated mice showed a decrease in stride length (c, left) at p95 (p = 0.0155, Mann-Whitney <i>U</i> test), p115 (p = 0.0024, Mann-Whitney <i>U</i> test), and p135 (p = 0.0010, Mann-Whitney <i>U</i> test) but no change in stride width (d, left) (p>0.05, Mann-Whitney <i>U</i> test) compared to Wt vehicle-treated mice. There was no significant effect of BNN27 treatment (10 mg/kg or 50 mg/kg) on stride length or width in either male or female Tg mice compared to vehicle treatment (p>0.05, Kruskal-Wallis test) (a-d, right). Wt-vehicle (male: n = 10, female: n = 9; grey), Tg-vehicle (male: n = 8, female: n = 11; grey), Tg-BNN27 10 mg/kg (male: n = 9, female: n = 9; black), Tg-BNN27 50 mg/kg (male: n = 9, female: n = 9; red). Data are presented as median ± interquartile ranges. * p<0.05, ** p<0.01, ***p<0.001.</p

Effect of BNN27 treatment on motor neuron survival in male and female mice.

<p>There was no change in motor neuron counts in a) male and b) female mice following BNN27 treatment. Top, representative H&E stained longitudinal sections (10μM) of lumbar spinal cord from Wt vehicle-treated mice and Tg mice treated with vehicle, BNN27 10 mg/kg, or BNN27 50 mg/kg. Arrows indicate motor neurons; scale bar = 150μM. Bottom, quantification of motor neuron counts from Tg mice treated with vehicle (male: n = 7, female: n = 11; grey), BNN27 10 mg/kg (male: n = 8, female: n = 10; black), and BNN27 50 mg/kg (male: n = 9, female: n = 12; red) normalized to Wt vehicle-treated mice (male: n = 12, female: n = 9; white). Tg vehicle-treated mice showed a decrease in the relative motor neuron number compared to Wt vehicle-treated mice in both males (a; p = 0.0095, Mann-Whitney <i>U</i> test) and females (b; p = 0.0042, Mann-Whitney <i>U</i> test). BNN27 treatment (10 mg/kg or 50 mg/kg) had no effect on motor neuron counts in male (a; p>0.05, Kruskal-Wallis test) or female (b; p>0.05, Kruskal-Wallis test) mice. Data are presented as median and 10-90th percentiles. ** p<0.01.</p

Effect of BNN27 treatment on paw grip endurance (PaGE) and rotarod in male and female mice.

<p>Male Tg vehicle-treated mice demonstrated a significant decrease in time spent on the PaGE test (a, left) at p95 (p = 0.0023, Mann-Whitney <i>U</i> test), p115 (p = 0.0003, Mann-Whitney <i>U</i> test), and p135 (p = 0.0385, Mann-Whitney <i>U</i> test) and the rotarod test (b, left) at p55 (p = 0.0089, Mann-Whitney <i>U</i> test), p95 (p = 0.0192, Mann-Whitney <i>U</i> test), and p135 (p = 0.0152, Mann-Whitney <i>U</i> test) relative to Wt vehicle-treated mice. Female Tg vehicle-treated mice demonstrated a decrease in time spent on the PaGE test (c, left) at p95 (p = 0.0128, Mann-Whitney <i>U</i> test), p115 (p<0.0001, Mann-Whitney <i>U</i> test), and p135 (p<0.0001, Mann-Whitney <i>U</i> test) and the rotarod test (d, left) at p55 (p = 0.0379, Mann-Whitney <i>U</i> test), p95 (p = 0.0026, Mann-Whitney <i>U</i> test), and p135 (p = 0.0002, Mann-Whitney <i>U</i> test) relative to Wt vehicle-treated mice. In Tg female mice, BNN27 10 mg/kg treatment significantly increased the time spent on the PaGE (c, right) (p<0.05, Dunn’s multiple comparison test) and rotarod (d, right) (p<0.01, Dunn’s multiple comparison test) tests compared to vehicle treatment at p95. Wt-vehicle (male: n = 10, female: n = 9; grey), Tg-vehicle (male: n = 11, female: n = 11; grey), Tg-BNN27 10 mg/kg (male: n = 9, female: n = 9; black), Tg-BNN27 50 mg/kg (male: n = 9, female: n = 9; red). Data are presented as median ± interquartile ranges. * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001.</p