minocycline and lactacystin

Neuroinflammation plays a role in the pathomechanism of many neurodegenerative diseases, including Parkinson disease (PD). Proteasome inhibition has also been known to be involved in the pathology of PD. Recent studies have reported that microglial activation and dopaminergic cell death were observed in in vivo lactacystin-induced models of PD. In the present study, we investigated whether proteasome inhibition had a direct effect on the inflammatory reaction. Lactacystin treatment increased the amount of nitric oxide and tumor necrosis factor alpha (TNF-alpha) in culture media containing murine microglia (BV-2). Neuronal cell death was more pronounced when the culture media containing BV-2 cells (BV-2 conditioned media; BV-2 CM) were harvested and treated with human dopaminergic neurons (SH-SY5Y) than when treated with lactacystin alone. Apoptosis was markedly increased by treatment with BV-2 CM, which could be mitigated by pretreatment with minocycline and N(omega)-nitro-l-arginine methyl ester (L-NAME). These results suggest that proteasome inhibition can directly trigger neuroinflammation, which leads to neuronal death.

Topics: Acetylcysteine; Animals; Anti-Bacterial Agents; Apoptosis; Cell Line; Cell Survival; Culture Media, Conditioned; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; I-kappa B Proteins; Inflammation; Macrophage Activation; Mice; Microglia; Minocycline; Neurons; NF-KappaB Inhibitor alpha; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type I; Proteasome Endopeptidase Complex

High molecular weight detergent-insoluble complexes of superoxide dismutase 1 (SOD1) enzyme are a biochemical abnormality associated with mutant SOD1-linked familial amyotrophic lateral sclerosis (FALS). In the present study, SOD1 protein from spinal cords of transgenic FALS mice was fractionated according to solubility in saline, zwitterionic, non-ionic or anionic detergents. Both endogenous mouse SOD1 and mutant human SOD1 were least soluble in SDS, followed by NP-40 and CHAPS, with an eight-fold greater detergent resistance of mutant protein overall. Importantly, high molecular weight mutant SOD1 complexes were isolated with SDS-extraction only. To reproduce SOD1 aggregate pathology in vitro, primary fibroblasts were isolated and cultured from neonatal transgenic FALS mice. Fibroblasts expressed abundant mutant SOD1 without spontaneous aggregation over time with passage. Proteasomal inhibition of cultures using lactacystin induced dose-dependent aggregation and increased the SDS-insoluble fraction of mutant SOD1, but not endogenous SOD1. In contrast, paraquat-mediated superoxide stress in fibroblasts promoted aggregation of endogenous SOD1, but not mutant SOD1. Treatment of cultures with peroxynitrite or the copper chelator diethyldithiocarbamate (DDC) alone did not modulate aggregation. However, DDC inhibited lactacystin-induced mutant SOD1 aggregation in transgenic fibroblasts, while exogenous copper slightly augmented aggregation. These data suggest that SOD1 aggregates may derive from proteasomal or oxidation-mediated oligomerisation pathways from mutant and endogenous subunits respectively. Furthermore, these pathways may be affected by copper availability. We propose that non-neural cultures such as these transgenic fibroblasts with inducible SOD1 aggregation may be useful for rapid screening of compounds with anti-aggregation potential in FALS.

Topics: Acetylcysteine; Amyotrophic Lateral Sclerosis; Animals; Animals, Newborn; Blotting, Western; Cell Survival; Chelating Agents; Chlorides; Copper; Detergents; Disease Models, Animal; Ditiocarb; Fibroblasts; Humans; Immunohistochemistry; Mice; Mice, Transgenic; Minocycline; Mutation; Oxidative Stress; Paraquat; Peroxynitrous Acid; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Solubility; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Zinc Compounds