CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases

CRISPR/Cas9 screen in human iPSC‐derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity

Introduction The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. Methods We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. Results Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. Discussion We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS

Screening for Toxic Amyloid in Yeast Exemplifies the Role of Alternative Pathway Responsible for Cytotoxicity

The relationship between amyloid and toxic species is a central problem since the discovery of amyloid structures in different diseases. Despite intensive efforts in the field, the deleterious species remains unknown at the molecular level. This may reflect the lack of any structure-toxicity study based on a genetic approach. Here we show that a structure-toxicity study without any biochemical prerequisite can be successfully achieved in yeast. A PCR mutagenesis of the amyloid domain of HET-s leads to the identification of a mutant that might impair cellular viability. Cellular and biochemical analyses demonstrate that this toxic mutant forms GFP-amyloid aggregates that differ from the wild-type aggregates in their shape, size and molecular organization. The chaperone Hsp104 that helps to disassemble protein aggregates is strictly required for the cellular toxicity. Our structure-toxicity study suggests that the smallest aggregates are the most toxic, and opens a new way to analyze the relationship between structure and toxicity of amyloid species

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

Ecological performance underlying ecosystem multifunctionality is promoted by organic farming and hedgerows at the local scale but not at the landscape scale

International audienceThe relative contributions of agricultural intensity and semi-natural habitats to the multifunctionality and sustainability of ecosystems at different spatial scales remain largely under-investigated. In this study, we assessed the multifunctionality of 40 winter cereal fields and 40 hedgerows based on ecological, agronomic and socio-economic performance using data from field surveys and interviews with farmers. We specifically focused on the effects of local farming systems (organic vs. conventional) and management (cereals intercropped with legumes vs. monocrops), the effects of landscape heterogeneity related to hedgerow density, and the spatial extent of semi-natural habitat and organic farming. Multifunctionality indices associated with increased values of proxies for biodiversity conservation and pest control functions were higher for hedgerows than crop fields. Agroecosystem multifunctionality was similar between organic and conventional farming as a consequence of antagonistic responses of individual function proxies. While organic farming promoted the ecological performance of crops, conventional farming resulted in higher agronomic performance (i.e. yield). Interestingly, lower yields of organic crops were not systematically associated with reductions in socio-economic performance in terms of farmer income and labour. At the landscape scale, hedgerow density and the extent of semi-natural habitats and organic farming had little influence on agroecosystem multifunctionality or individual function proxies. Synthesis and applications. Our results confirm the high value of hedgerows and organic farming at the local scale for the ecological performance of ecosystems. Our study suggests that, among existing agri-environment schemes in Europe, hedgerow planting and organic farming are appropriate tools to optimise the ecological performance of ecosystems at the local scale even if antagonistic effects with agronomic performance should not be neglected

Targeted Exon Capture and Sequencing in Sporadic Amyotrophic Lateral Sclerosis

<div><p>Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in progressive degeneration of motor neurons, ultimately leading to paralysis and death. Approximately 10% of ALS cases are familial, with the remaining 90% of cases being sporadic. Genetic studies in familial cases of ALS have been extremely informative in determining the causative mutations behind ALS, especially as the same mutations identified in familial ALS can also cause sporadic disease. However, the cause of ALS in approximately 30% of familial cases and in the majority of sporadic cases remains unknown. Sporadic ALS cases represent an underutilized resource for genetic information about ALS; therefore, we undertook a targeted sequencing approach of 169 known and candidate ALS disease genes in 242 sporadic ALS cases and 129 matched controls to try to identify novel variants linked to ALS. We found a significant enrichment in novel and rare variants in cases versus controls, indicating that we are likely identifying disease associated mutations. This study highlights the utility of next generation sequencing techniques combined with functional studies and rare variant analysis tools to provide insight into the genetic etiology of a heterogeneous sporadic disease.</p></div

Comparison with Irish population.

<p>Genes are sorted into two categories, depending on whether they where implicated in the Mendelian form of the disease (equivalent to the “known ALS” category in this paper) or are low penetrance/tentative ALS genes (“associated”), as used in the original publication <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004704#pgen.1004704-Kenna2" target="_blank">[35]</a>.</p><p>Comparison with Irish population.</p

Sequenced genes and hits category repartitions.

<p>All genes fall into the four following categories: known ALS disease genes (Known ALS), genes potentially associated with ALS (Associated), candidate genes from a previously published analysis of ALS trios (Trios) and genes containing RNA Recognition Motifs (RRM) bearing high prion scores or are very toxic when expressed in yeast. A) Pie chart showing the four categories of genes sequenced. B) Bar graph showing the number of rare or novel variants found in cases in controls for the genes in which the most variants were found. The number of variants found in controls was adjusted for a control cohort of the same size than the ALS cohort. C) Localization of novel variants (in red) identified in this study for some of the top hits. Position of some selected variants already linked with ALS, or other diseases (Floating-Harbor syndrome, SRCAP), are indicated in orange (or in orange heat map, SOD1).</p

Predicted effects of new TAF15 variant on aggregation and conservation.

<p>A) ZipperDB prediction of the increase of the TAF15 p.R150K variant fibrilization propensity versus the wild type. B) TAF15 p.R150 residue is highly conserved within mammals.</p

ALS variants prevalence among different genetic backgrounds.

<p>Prevalence of ANG, FUS, OPTN and SOD1 variants in ALS between our American cohort and Irish, Italian and Korean populations <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004704#pgen.1004704-Kenna2" target="_blank">[35]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004704#pgen.1004704-Chi2" target="_blank">[38]</a>.</p><p>ALS variants prevalence among different genetic backgrounds.</p