Proteasome inhibition results in cytoplasmic TDP-43 accumulation in cultured neurons.
(A) Example of immunohistochemical staining for TDP-43 on human frontotemporal lobar degeneration (FTLD) brain and amyotrophic lateral sclerosis (ALS) spinal cord sections. While TDP-43 is normally in the nucleus (asterisk), it accumulates in cytoplasmic aggregates (arrow), while being depleted from the nucleus in affected dentate gyrus neurons in FTLD-TDP. Similarly, TDP-43 that is normally in the nucleus of motor neurons (asterisk), accumulates (arrowhead) and forms cytoplasmic aggregates (arrow) and is depleted from the nucleus in ALS. (B) Primary neurons were treated with vehicle (DMSO) or different substances and then stained with antibodies to TDP-43 to assess its subcellular distribution, and neurofilament (NF) for cellular integrity. Doses and treatment times are specified in Materials and methods. TDP-43 localizes primarily to the nucleus upon treatment with H2O2 (oxidative stress), N-Methyl-D-aspartic acid (NMDA, excitotoxicity), staurosporine (caspase activation), thapsigargin or tunicamycine (both endoplasmic reticulum (ER) stress). In contrast, proteasome inhibition with lactacystin or MG-132 treatment induces cytoplasmic accumulation of TDP-43 (arrowheads). DAPI stains nuclei. Representative neurons from three independent experiments are shown.