thapsigargin and epoxomicin

TDP-43 has been implicated in the pathogenesis of amyotrophic lateral sclerosis and other neurodegenerative diseases. Here we demonstrate, using neuronal and spinal cord organotypic culture models, that chronic excitotoxicity, oxidative stress, proteasome dysfunction and endoplasmic reticulum stress mechanistically induce mislocalization, phosphorylation and aggregation of TDP-43. This is compatible with a lack of function of this protein in the nucleus, specially in motor neurons. The relationship between cell stress and pathological changes of TDP-43 also includes a dysfunction in the survival pathway mediated by mitogen-activated protein kinase/extracellular signal-regulated kinases (ERK1/2). Thus, under stress conditions, neurons and other spinal cord cells showed cytosolic aggregates containing ERK1/2. Moreover, aggregates of abnormal phosphorylated ERK1/2 were also found in the spinal cord in amyotrophic lateral sclerosis (ALS), specifically in motor neurons with abnormal immunoreactive aggregates of phosphorylated TDP-43. These results demonstrate that cellular stressors are key factors in neurodegeneration associated with TDP-43 and disclose the identity of ERK1/2 as novel players in the pathogenesis of ALS.

Topics: Aged; Amyotrophic Lateral Sclerosis; Animals; Animals, Newborn; Case-Control Studies; Cell Line, Transformed; DNA-Binding Proteins; Enzyme Inhibitors; Female; Gene Expression Regulation; Humans; Hydrogen Peroxide; Male; Middle Aged; Mitogen-Activated Protein Kinases; Motor Neurons; Neurons; Oligopeptides; Organ Culture Techniques; Oxidants; Oxidative Stress; Rats; Signal Transduction; Spinal Cord; Thapsigargin; Transfection

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells and basophils results in FcepsilonRI beta and gamma subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FcepsilonRI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FcepsilonRI beta and gamma chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FcepsilonRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FcepsilonRI and activated Syk to the proteasome for degradation.

Topics: Animals; Enzyme Precursors; Intracellular Signaling Peptides and Proteins; Kinetics; Leukemia, Basophilic, Acute; Ligands; Oligopeptides; Phosphorylation; Protein Subunits; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Rats; Receptors, IgE; Syk Kinase; Thapsigargin; Tumor Cells, Cultured; Ubiquitin; Ubiquitin-Protein Ligases; Vaccinia virus