Oxygen Radicals Elicit Paralysis and Collapse of Spinal Cord Neuron Growth Cones upon Exposure to Proinflammatory Cytokines
Figure 4
NADPH oxidase mediates ROS formation in growth cones exposed to TNFα. (a) Exposure of freshly isolated growth cone particles (GCPs) to TNFα activates MAP kinase. GCPs were plated on laminin in Krebs buffer (2 h), washed, and incubated with the MAP kinase inhibitors PD98059 (25 μM, 20 min) or PBS (equivalent volume) prior to an addition of 100 ng/mL TNFα. Attached GCPs were lysed and equal amounts of total protein were subjected to SDS gel electrophoresis (10%) followed by western blotting and detection (chemiluminescence) of MAP kinase (top panel) and phospho-MAP kinase immunoreactivity (bottom panel). Actin served as a loading control (middle panel). Exposure of GCPs to TNFα stimulated MAP kinase activity (increased phospho-MAP kinase), which was negated by PD98059. Also, TNFα treatment enhanced expression of MAP kinase (increases in total MAP kinase), a response apparently diminished by a presence of PD98059. Confocal images acquired (63x, oil) of cultured chick forebrain neurons (laminin, 24 h) exposed to 100 ng/mL of TNFα (TNFα) revealed phospho-JNK immunoreactivity (white signal) as a discrete, punctate pattern in cell bodies and neuronal process in contrast to the homogenous appearance in neuronal soma under control conditions (PBS, equivalent volume). Cultures were fixed with 4% paraformaldehyde and immunostained against phospho-JNK. (b) Neuronal growth cones contain a functional NADPH oxidase activity. Lysates obtained from SC neuron cultures revealed immunoreactivity against the large membrane subunit NOX2 and the cytosolic subunits and . Incubation of chick (E7) forebrain neurons with the NOX activator PMA (400 ng/mL) increased plasma membrane association of the cytosolic subunit () compared to controls (Con) indicative for the formation of the NOX multiprotein complex. Native gel electrophoresis, combined with in-gel NBT staining, of freshly isolated growth cone particles (GCPs) obtained from E10–12 chick forebrain demonstrated one NADPH oxidoreductases activity () colocalizing with NOX2 immunoreactivity. (c) GCPs were loaded with 10 μM DCF (30 min) in the presence of 2 μM DPI (NOX inhibitor), 500 μM NAC (antioxidant), or PBS (mock, equivalent volume) prior to addition of TNFα (45 min). DCF fluorescence was determined in GCP lysates, adjusted to total soluble protein, and normalized to control (Con, open bar). TNFα stimulated a significant, dose-dependent ROS formation in GCPs (T100 = 100 ng/mL, = 200 ng/mL, ), which was abolished by DPI or NAC. Peroxide (H2O2) served as a positive control. (d) Plasma membrane association of normalized to control conditions was quantified (western blotting) using plasma membrane-enriched fractions. Exposure of GCPs to 200 ng/mL TNFα significantly increased the relative plasma membrane association of (TNF, ) compared to control (Con) indicating the formation of a functional NOX complex. Preincubation of GCPs with methyl-β-cyclodextrin (0.1%) negated plasma membrane translocation of in the presence of TNFα (mCdx-TNF). All data represent SEM from at least two experiments (triplicate conditions each) with .
