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      ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules


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          Mutations in the gene encoding Fused in Sarcoma ( FUS) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predominantly nuclear DNA- and RNA-binding protein that is involved in RNA processing. Large FUS-immunoreactive inclusions fill the perikaryon of surviving motor neurons of ALS patients carrying mutations at post-mortem. This sequestration of FUS is predicted to disrupt RNA processing and initiate neurodegeneration. Here, we demonstrate that C-terminal ALS mutations disrupt the nuclear localizing signal (NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and patient fibroblasts. FUS mislocalization is rescued by the addition of the wild-type FUS NLS to mutant proteins. We also show that oxidative stress recruits mutant FUS to cytoplasmic stress granules where it is able to bind and sequester wild-type FUS. While FUS interacts with itself directly by protein–protein interaction, the recruitment of FUS to stress granules and interaction with PABP are RNA dependent. These findings support a two-hit hypothesis, whereby cytoplasmic mislocalization of FUS protein, followed by cellular stress, contributes to the formation of cytoplasmic aggregates that may sequester FUS, disrupt RNA processing and initiate motor neuron degeneration.

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          A new subtype of frontotemporal lobar degeneration with FUS pathology.

          Frontotemporal dementia (FTD) is a clinical syndrome with a heterogeneous molecular basis. The neuropathology associated with most FTD is characterized by abnormal cellular aggregates of either transactive response DNA-binding protein with Mr 43 kDa (TDP-43) or tau protein. However, we recently described a subgroup of FTD patients, representing around 10%, with an unusual clinical phenotype and pathology characterized by frontotemporal lobar degeneration with neuronal inclusions composed of an unidentified ubiquitinated protein (atypical FTLD-U; aFTLD-U). All cases were sporadic and had early-onset FTD with severe progressive behavioural and personality changes in the absence of aphasia or significant motor features. Mutations in the fused in sarcoma (FUS) gene have recently been identified as a cause of familial amyotrophic lateral sclerosis, with these cases reported to have abnormal cellular accumulations of FUS protein. Because of the recognized clinical, genetic and pathological overlap between FTD and amyotrophic lateral sclerosis, we investigated whether FUS might also be the pathological protein in aFTLD-U. In all our aFTLD-U cases (n = 15), FUS immunohistochemistry labelled all the neuronal inclusions and also demonstrated previously unrecognized glial pathology. Immunoblot analysis of protein extracted from post-mortem aFTLD-U brain tissue demonstrated increased levels of insoluble FUS. No mutations in the FUS gene were identified in any of our patients. These findings suggest that FUS is the pathological protein in a significant subgroup of sporadic FTD and reinforce the concept that FTD and amyotrophic lateral sclerosis are closely related conditions.
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            Rules for nuclear localization sequence recognition by karyopherin beta 2.

            Karyopherinbeta (Kapbeta) proteins bind nuclear localization and export signals (NLSs and NESs) to mediate nucleocytoplasmic trafficking, a process regulated by Ran GTPase through its nucleotide cycle. Diversity and complexity of signals recognized by Kap betas have prevented prediction of new Kap beta substrates. The structure of Kap beta 2 (also known as Transportin) bound to one of its substrates, the NLS of hnRNP A1, that we report here explains the mechanism of substrate displacement by Ran GTPase. Further analyses reveal three rules for NLS recognition by Kap beta 2: NLSs are structurally disordered in free substrates, have overall basic character, and possess a central hydrophobic or basic motif followed by a C-terminal R/H/KX(2-5)PY consensus sequence. We demonstrate the predictive nature of these rules by identifying NLSs in seven previously known Kap beta 2 substrates and uncovering 81 new candidate substrates, confirming five experimentally. These studies define and validate a new NLS that could not be predicted by primary sequence analysis alone.
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              TDP-43 A315T mutation in familial motor neuron disease.

              To identify novel causes of familial neurodegenerative diseases, we extended our previous studies of TAR DNA-binding protein 43 (TDP-43) proteinopathies to investigate TDP-43 as a candidate gene in familial cases of motor neuron disease. Sequencing of the TDP-43 gene led to the identification of a novel missense mutation, Ala-315-Thr, which segregates with all affected members of an autosomal dominant motor neuron disease family. The mutation was not found in 1,505 healthy control subjects. The discovery of a missense mutation in TDP-43 in a family with dominantly inherited motor neuron disease provides evidence of a direct link between altered TDP-43 function and neurodegeneration.

                Author and article information

                Hum Mol Genet
                Hum. Mol. Genet
                Human Molecular Genetics
                Oxford University Press
                1 July 2013
                7 March 2013
                7 March 2013
                : 22
                : 13
                : 2676-2688
                [1 ]Department of Clinical Neuroscience and
                [2 ]Department of Neuroscience, King's College London, Centre for Neurodegeneration Research, Institute of Psychiatry , London SE5 8AF, UK
                [3 ]Faculty of Medical and Health Sciences, Department of Pharmacology and the National Research Centre for Growth and Development, The University of Auckland , Auckland, New Zealand
                [4 ]Department of Biotechnology, Jozef Stefan Institute , Jamova 39, SI-1000 Ljubljana, Slovenia
                Author notes
                [* ]To whom correspondence should be addressed at: Christopher E. Shaw, Department of Clinical Neuroscience, PO 43, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK. Tel: +44 2078485180; Fax: +44 2078485190; Email: christopher.shaw@ 123456kcl.ac.uk

                The authors wish it to be known that these authors contributed equally to the paper.

                © The Author 2013. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permission@ 123456oup.com

                : 7 February 2013
                : 5 March 2013



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