Cytoplasmic Accumulation and Aggregation of TDP-43 upon Proteasome Inhibition in Cultured Neurons
Figure 6
TDP-43 reduction increased vulnerability of neurons.
(A) Immunocytochemistry of nuclear TDP-43 in neurons expressing control (siR co) or two different siRNAs against TDP-43 (siR #543 and siR #560). SiR #543 and more so siR #560 reduce TDP-43 levels. Propidium iodine (PI) uptake indicates increased cell death in siR #560 neurons. DAPI counterstains nuclei. (B) Quantification of TDP-43 fluorescence intensity reveals a 42±5.0% and 88±2.2% reduction of TDP-43 levels with siR #543 and siR #560, respectively, compared to control (siR co) neurons (Student's t test P<0.05; *** indicates P<0.0001). Levels of TDP-43 are similar in siR co and wild-type (WT) neurons (n.s., not significant). (C) Western blotting confirms reduction of TDP-43 levels with siR #543 and siR #560, compared to siR co and wild-type (WT) neurons. Actin serves as loading control. (D) Numbers of PI positive neurons upon treatment with vehicle (DMSO) or MG-132 for 16 hours. Numbers of PI positive cells are similar in DMSO- and MG-132-treated siR co neurons, as well as in DMSO-treated siR #543 cells (Student's t test P<0.05; n.s., not significant). MG-132 treatment significantly increases numbers of PI positive siR #543 neurons (P<0.0001). High numbers of PI positive siR #560 neurons (79±1.5%) are also increased by MG-132, with virtually all cells being PI positive (98±1.4%; *** indicates P<0.0001). (E) MG-132 induces cytoplasmic accumulation (arrowheads) of TDP-43 in both siR co and siR #543 neurons. Note the faint staining in siR #543 neurons due to reduced TDP-43 levels.