title: TARDBP pathogenic mutations increase cytoplasmic translocation of TDP-43 and cause reduction of endoplasmic reticulum Ca²⁺ signaling in motor neurons.
abstract: The transactive response DNA binding protein (TDP-43) is a major component of the characteristic neuronal cytoplasmic inclusions seen in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Furthermore, pathogenic mutations in the gene encoding TDP-43, TARDBP, are found in sporadic and familial ALS cases. To study the molecular mechanisms of cellular toxicity due to TDP-43 mutations we generated a novel in vitro cellular model using a fluorescently tagged human genomic TARDBP locus carrying one of two ALS-associated mutations, A382T or M337V, which were used to generate site-specific bacterial artificial chromosome (BAC) human stable cell lines and BAC transgenic mice. In cell lines and primary motor neurons in culture, TDP-M337V mislocalized to the cytoplasm more frequently than wild-type TDP (wt-TDP) and TDP-A382T, an effect potentiated by oxidative stress. Expression of mutant TDP-M337V correlated with increased apoptosis detected by cleaved caspase-3 staining. Cells expressing mislocalized TDP-M337V spontaneously developed cytoplasmic aggregates, while for TDP-A382T aggregates were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin. Lowering Ca(2+) concentration in the ER of wt-TDP cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca(2+) dysregulation as a potential mediator of pathology through alterations in Bcl-2 protein levels. Ca(2+) signaling from the ER was impaired in immortalized cells and primary neurons carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca(2+) stores content and delayed Ca(2+) release compared with cells carrying wt-TDP. The deficits in Ca(2+) release in human cells correlated with the upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored the amplitude of Ca(2+) oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 and Bcl-2 mediated ER Ca(2+) signaling dysregulation.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK; Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: ruxandra.mutihac@ndcn.ox.ac.uk.
authors.forename: R
authors.initials: R
authors.lastname: Mutihac
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK. Electronic address: javier.alegre@uclh.nhs.uk.
authors.forename: J
authors.initials: J
authors.lastname: Alegre-Abarrategui
authors.affiliation: Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: david.gordon@ndcn.ox.ac.uk.
authors.forename: D
authors.initials: D
authors.lastname: Gordon
authors.affiliation: Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: lucy.farrimond@googlemail.uk.
authors.forename: L
authors.initials: L
authors.lastname: Farrimond
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK; Oxford Stem Cell Institute, University of Oxford, Oxford OX1 3QX, UK. Electronic address: michiko.yamasakimann@dpag.ox.ac.uk.
authors.forename: M
authors.initials: M
authors.lastname: Yamasaki-Mann
authors.affiliation: Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, Oxford OX3 9DU, UK. Electronic address: kevin.talbot@ndcn.ox.ac.uk.
authors.forename: K
authors.initials: K
authors.lastname: Talbot
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK. Electronic address: richard.wade-martins@dpag.ox.ac.uk.
authors.forename: R
authors.initials: R
authors.lastname: Wade-Martins
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial; D003593:Cytoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008822:Mice, Transgenic; D009046:Motor Neurons / Q000378:metabolism*; D020125:Mutation, Missense; D018384:Oxidative Stress / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D013116:Spinal Cord / Q000378:metabolism
chemical_list: C000595707:BCL2 protein, human; D004268:DNA-Binding Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C000598624:TARDBP protein, human; C052617:Bcl2 protein, mouse; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D002118:Calcium
keywords: Bcl-2; Ca(2+) dysregulation; Motor neurons; TDP-43