Activation of Transforming Growth Factor-beta/Smad Signaling Reduces Aggregate Formation of Mislocalized TAR DNA Binding Protein-43 (P03.182)

Abstract

Objective: To investigate the possible pathophysiological linkage between TAR DNA binding protein-43 (TDP-43) positive inclusions and transforming growth factor (TGF)-beta/Smad signaling system. Background We have previously reported that phosphorylated Smad2/3 (pSmad2/3) proteins, the major intracellular mediators of TGF-beta signaling, were abnormally accumulated in TDP-43 positive inclusions of anterior horn cells (AHCs) in ALS patients. Moreover, the nuclear pSmad2/3 immunosignal was decreased in inclusion-bearing AHCs in comparison with those without inclusions. These findings suggested that TGF-beta/Smad signaling system and its neuroprotective effect might be disturbed in ALS. Design/Methods: We constructed a plasmid of the nuclear localization signal deletion mutant of TDP-43, and transfected it into HEK293T cells. TDP-43 positive inclusions were formd in cytoplasm of the HEK293T cell under proteasome inhibition. Plasmids of Smad2 protein and constitutive active or dominant negative forms of TGF type1 receptor were also transfected into the HEK293T cells. The direct effects of TGF-beta/Smad signaling on the aggregation formation were analyzed by immunocytochemistry and western blotting. Results: The aggregates formed in cytoplasm of the HEK293T cells contained ubiquitinated, phosphorylated, and fragmented TDP-43, consistent with the essential features of the human pathology. Moreover, the aggregates were colocalized with phosphorylated Smad2 under continuous TGF-beta stimulation. Overexpression of Smad2 reduced the amount of cytoplasmic aggregates in HEK293T cells, and TGF-beta stimulation augmented this reduction effect in a dose-dependent manner. Conclusions: Our data indicate that activation of TGF-beta/Smad signaling system is protective against aggregate formation of cytoplasmically mislocalized TDP-43 and may be a potential therapeutic approach to delay the progression of ALS. Disclosure: Dr. Nakamura has nothing to disclose. Dr. Kaneko has nothing to disclose. Dr. Ito has nothing to disclose. Dr. Fujisawa has nothing to disclose. Dr. Kusaka has nothing to disclose.