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      C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

      1 , 2 , 3 , 4 , 1 , 2 , 5 , 6 , 7 , 8 , 1 , 2 , 1 , 2 , 1 , 2 , 9 , 1 , 2 , 1 , 2 , 1 , 2 , 10 , 11 , 12 , 5 , 6 , 8 , 1 , 2 , 3 , 13 , 14 , 14 , 10 , 12 , 4 , 15 , 16 , 17 , 11 , 18 , 9 , 5 , 6 , 7 , 8 , 19 , 20 , 1 , 2 , 3 , * , 1 , 2 , 21 , *

      Science Advances

      American Association for the Advancement of Science

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          Abstract

          Arginine-rich dipeptide repeats associated with ALS and FTD inhibit machinery for microtubule-based axonal cargo transport.

          Abstract

          A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we show using patient stem cell–derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, our study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.

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          Most cited references 66

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          Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

          The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.
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            Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ *

            Protein quantification without isotopic labels has been a long-standing interest in the proteomics field. However, accurate and robust proteome-wide quantification with label-free approaches remains a challenge. We developed a new intensity determination and normalization procedure called MaxLFQ that is fully compatible with any peptide or protein separation prior to LC-MS analysis. Protein abundance profiles are assembled using the maximum possible information from MS signals, given that the presence of quantifiable peptides varies from sample to sample. For a benchmark dataset with two proteomes mixed at known ratios, we accurately detected the mixing ratio over the entire protein expression range, with greater precision for abundant proteins. The significance of individual label-free quantifications was obtained via a t test approach. For a second benchmark dataset, we accurately quantify fold changes over several orders of magnitude, a task that is challenging with label-based methods. MaxLFQ is a generic label-free quantification technology that is readily applicable to many biological questions; it is compatible with standard statistical analysis workflows, and it has been validated in many and diverse biological projects. Our algorithms can handle very large experiments of 500+ samples in a manageable computing time. It is implemented in the freely available MaxQuant computational proteomics platform and works completely seamlessly at the click of a button.
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              Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.

              Several families have been reported with autosomal-dominant frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), genetically linked to chromosome 9p21. Here, we report an expansion of a noncoding GGGGCC hexanucleotide repeat in the gene C9ORF72 that is strongly associated with disease in a large FTD/ALS kindred, previously reported to be conclusively linked to chromosome 9p. This same repeat expansion was identified in the majority of our families with a combined FTD/ALS phenotype and TDP-43-based pathology. Analysis of extended clinical series found the C9ORF72 repeat expansion to be the most common genetic abnormality in both familial FTD (11.7%) and familial ALS (23.5%). The repeat expansion leads to the loss of one alternatively spliced C9ORF72 transcript and to formation of nuclear RNA foci, suggesting multiple disease mechanisms. Our findings indicate that repeat expansion in C9ORF72 is a major cause of both FTD and ALS. Copyright © 2011 Elsevier Inc. All rights reserved.
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                Author and article information

                Journal
                Sci Adv
                Sci Adv
                SciAdv
                advances
                Science Advances
                American Association for the Advancement of Science
                2375-2548
                April 2021
                09 April 2021
                : 7
                : 15
                Affiliations
                [1 ]KU Leuven—University of Leuven, Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), Leuven, Belgium.
                [2 ]VIB, Center for Brain & Disease Research, Laboratory of Neurobiology, Leuven, Belgium.
                [3 ]Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, UK.
                [4 ]Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
                [5 ]UK Dementia Research Institute, University of Edinburgh, Edinburgh, UK.
                [6 ]Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK.
                [7 ]The Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, Edinburgh, UK.
                [8 ]The Euan MacDonald Centre, University of Edinburgh, Edinburgh, UK.
                [9 ]KU Leuven—University of Leuven, Department of Development and Regeneration, Stem Cell Institute, Leuven, Belgium.
                [10 ]KU Leuven—University of Leuven, Department of Neurosciences, Laboratory for Molecular Neurobiomarker Research and Leuven Brain Institute (LBI), Leuven, Belgium.
                [11 ]KU Leuven—University of Leuven, Department of Imaging and Pathology, Laboratory for Neuropathology and Leuven Brain Institute (LBI), Leuven, Belgium.
                [12 ]Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium.
                [13 ]Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.
                [14 ]Biogen Idec, Boston, MA, USA.
                [15 ]Hector Institute for Translational Brain Research, Central Institute of Mental Health, University of Heidelberg, Heidelberg, Germany.
                [16 ]Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn, Bonn, Germany.
                [17 ]KU Leuven—University of Leuven, Translational Research Centre for Gastrointestinal Disorders, Leuven, Belgium.
                [18 ]Department of Pathology, University Hospitals Leuven, Leuven, Belgium.
                [19 ]Centre for Brain Development and Repair, inStem, Bangalore, India.
                [20 ]MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
                [21 ]Department of Neurology, University Hospitals Leuven, Leuven, Belgium.
                Author notes
                [* ]Corresponding author. Email: philip.vandamme@ 123456uzleuven.be (P.V.D.); sbullock@ 123456mrc-lmb.cam.ac.uk (S.L.B.)
                [†]

                These authors contributed equally to this work.

                Article
                abg3013
                10.1126/sciadv.abg3013
                8034861
                33837088
                Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

                This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Funding
                Funded by: doi http://dx.doi.org/10.13039/501100000265, Medical Research Council;
                Award ID: MC_U105178790
                Funded by: doi http://dx.doi.org/10.13039/501100000406, Motor Neurone Disease Association;
                Award ID: MR/R001162/1
                Funded by: doi http://dx.doi.org/10.13039/501100003130, Fonds Wetenschappelijk Onderzoek;
                Award ID: G0B2819N
                Funded by: doi http://dx.doi.org/10.13039/501100003130, Fonds Wetenschappelijk Onderzoek;
                Award ID: E0S-D6179-G012718N
                Funded by: doi http://dx.doi.org/10.13039/501100003130, Fonds Wetenschappelijk Onderzoek;
                Award ID: QPG-3A7604
                Funded by: doi http://dx.doi.org/10.13039/501100003130, Fonds Wetenschappelijk Onderzoek;
                Award ID: G.0909.15
                Funded by: doi http://dx.doi.org/10.13039/501100014734, Japan Fisheries Research and Education Agency;
                Award ID: 2017/023
                Funded by: doi http://dx.doi.org/10.13039/501100011878, Vlaamse regering;
                Award ID: 135043
                Funded by: doi http://dx.doi.org/10.13039/501100003130, Fonds Wetenschappelijk Onderzoek;
                Award ID: G0F8516N
                Funded by: doi http://dx.doi.org/10.13039/501100003130, Fonds Wetenschappelijk Onderzoek;
                Award ID: G0B2819N
                Funded by: doi http://dx.doi.org/10.13039/501100003132, Agentschap voor Innovatie door Wetenschap en Technologie;
                Award ID: 150031
                Funded by: doi http://dx.doi.org/10.13039/501100003132, Agentschap voor Innovatie door Wetenschap en Technologie;
                Award ID: 135043
                Funded by: doi http://dx.doi.org/10.13039/501100007155, Medical Research Council Canada;
                Award ID: U105178790
                Funded by: doi http://dx.doi.org/10.13039/501100007155, Medical Research Council Canada;
                Award ID: MR/R001162/1
                Funded by: doi http://dx.doi.org/10.13039/501100011878, Vlaamse regering;
                Award ID: 1165119N
                Funded by: doi http://dx.doi.org/10.13039/501100004727, Vlaams Instituut voor Biotechnologie;
                Funded by: doi http://dx.doi.org/10.13039/501100011620, Center for Life Sciences;
                Funded by: doi http://dx.doi.org/10.13039/501100004497, Onderzoeksraad, KU Leuven;
                Award ID: C1-C14-17-107
                Funded by: doi http://dx.doi.org/10.13039/501100018719, Koç Üniversitesi;
                Award ID: C1-C14-17-107
                Funded by: doi http://dx.doi.org/10.13039/501100016990, Hellenic Society of Cardiology;
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                Mariane Belen

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