10.6084/m9.figshare.5123491.v1 Nakamura M. Nakamura M. Kaneko S. Kaneko S. Ito H. Ito H. Jiang S. Jiang S. Fujita K. Fujita K. Wate R. Wate R. Nakano S. Nakano S. Fujisawa J. Fujisawa J. Kusaka H. Kusaka H. Supplementary Material for: Activation of Transforming Growth Factor-β/Smad Signaling Reduces Aggregate Formation of Mislocalized TAR DNA-Binding Protein-43 Karger Publishers 2012 Amyotrophic lateral sclerosis Aggregation Smad TAR DNA-binding protein-43 Transforming growth factor-β 2012-07-10 00:00:00 Dataset https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Activation_of_Transforming_Growth_Factor-_Smad_Signaling_Reduces_Aggregate_Formation_of_Mislocalized_TAR_DNA-Binding_Protein-43/5123491 <b><i>Background:</i></b> TAR DNA-binding protein of 43 kDa (TDP-43) is naturally located in the nucleus and has been identified as the major component of cytoplasmic ubiquitinated inclusions in patients with amyotrophic lateral sclerosis (ALS). We have reported that TDP-43 and phosphorylated Smad2 (pSmad2), an intracellular mediator protein of transforming growth factor-β (TGFβ) signaling, are co-localized within cytoplasmic inclusions in the anterior horn cells of sporadic ALS patients. <b><i>Objective:</i></b> To investigate the possible pathophysiological linkage between pathologic cytoplasmic inclusions containing TDP-43 and TGFβ/Smad signaling. <b><i>Methods:</i></b> We replicated cytoplasmic aggregates of TDP-43 in HEK293T cells by transfecting the cells with a nuclear localization signal deletion mutant of TDP-43 and inhibiting proteasome activity, and assessed the effect of TGFβ/Smad signaling on the cytoplasmic aggregate formation. <b><i>Results:</i></b> The aggregates contained ubiquitinated, phosphorylated, and fragmented TDP-43, consistent with the essential features of the human pathology. Moreover, the aggregates were co-localized with pSmad2 under continuous TGFβ stimulation. Overexpression of Smad2 reduced the amount of cytoplasmic aggregates in HEK293T cells, and TGFβ stimulation augmented this reduction effect in a dose-dependent manner. <b><i>Conclusion:</i></b> Activation of the TGFβ/Smad signaling system is protective against aggregate formation of cytoplasmically mislocalized TDP-43 and may be a potential therapeutic approach to delay progression of ALS.