title: Quantification of the Relative Contributions of Loss-of-function and Gain-of-function Mechanisms in TAR DNA-binding Protein 43 (TDP-43) Proteinopathies.
abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, it has been proposed that TDP-43 proteinopathies originate from either a loss-of-function (LOF) mechanism, a gain-of-function (GOF) process, or both. We have addressed this issue by transfecting murine NSC34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs). These two strategies allowed the depletion of nuclear TDP-43 and the accumulation of cytoplasmic TDP-43 aggregates to occur separately and independently. Endogenous and exogenous TDP-43 were monitored and quantified using both immunofluorescence and Western blotting analysis, and nuclear functional TDP-43 was measured by monitoring the sortilin 1 mRNA splicing activity. Various degrees of TDP-43 cytoplasmic accumulation and nuclear TDP-43 depletion were achieved and the resulting cellular viability was evaluated, leading to a quantitative global analysis on the relative effects of LOF and GOF on the overall cytotoxicity. These were found to be ∼55% and 45%, respectively, in both cell lines and using both readouts of cell toxicity, showing that these two mechanisms are likely to contribute apparently equally to the pathologies of ALS and FTLD-U.
authors.affiliation: From the Section of Biochemistry, Department of Experimental and Clinical Biomedical Sciences, University of Florence, V.le GB Morgagni 50, 50134 Florence, Italy and.
authors.forename: Roberta
authors.initials: R
authors.lastname: Cascella
authors.affiliation: From the Section of Biochemistry, Department of Experimental and Clinical Biomedical Sciences, University of Florence, V.le GB Morgagni 50, 50134 Florence, Italy and.
authors.forename: Claudia
authors.initials: C
authors.lastname: Capitini
authors.affiliation: From the Section of Biochemistry, Department of Experimental and Clinical Biomedical Sciences, University of Florence, V.le GB Morgagni 50, 50134 Florence, Italy and.
authors.forename: Giulia
authors.initials: G
authors.lastname: Fani
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW United Kingdom.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.affiliation: From the Section of Biochemistry, Department of Experimental and Clinical Biomedical Sciences, University of Florence, V.le GB Morgagni 50, 50134 Florence, Italy and.
authors.forename: Cristina
authors.initials: C
authors.lastname: Cecchi
authors.affiliation: From the Section of Biochemistry, Department of Experimental and Clinical Biomedical Sciences, University of Florence, V.le GB Morgagni 50, 50134 Florence, Italy and fabrizio.chiti@unifi.it.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Chiti
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D066263:Protein Aggregation, Pathological / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000099183:Sortilin
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D000099183:Sortilin; C000598624:TARDBP protein, human
keywords: Frontotemporal Dementia; Frontotemporal lobar degeneration; TAR DNA-binding protein 43 (TDP-43) (TARDBP); amyloid; amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); motor neuron disease; neurodegeneration; neurodegenerative disease; protein aggregation; protein misfolding