Extracellular aggregated Cu/Zn superoxide dismutase activates microglia to give a cytotoxic phenotype

Abstract

A large body of literature suggests that amyotrophic lateral sclerosis (ALS) pathology is intimately linked with neuroinflammation, specifically activation and recruitment of microglia and astrocytes. The actual cause of gliosis is unclear. Extracellular Cu/Zn superoxide dismutase (SOD1) has recently been shown to activate microglia in a CD14 dependant mechanism providing one potential pathway by which glial cells become activated. As protein inclusions are thought to be an important part of ALS pathology and are associated with all forms of ALS, we sought to determine if aggregated SOD1 would activate microglia. Recombinant SOD1 was aggregated and this, or monomeric forms of SOD1 were then added to EOC.13 microglial cells or primary microglial cells in culture. Although monomeric mutant SOD1 has been shown to promote microglial activation in the past, we found that aggregated SOD1 was able to much more efficiently activate microglia in culture when compared with the unaggregated form of mutant SOD1. In addition to CD14 dependant pathways, aggregated SOD1 also bound to the surface of glial cells and was internalized in a lipid raft and scavenger receptor dependent manner. We have for the first time shown that aggregated mutant SOD1 potently activates microglia. These results suggest that there may be a potential link between protein aggregation and microglial activation in ALS.

Extracellular wildtype and mutant SOD1 induces ER–Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

“…expression and purification of recombinant SOD1 human SOD1 Wt and SOD1 G93a were expressed and purified from E. coli as previously described [28,48]. Briefly, E. coli cells were cultured at 23 °C with 3 mM CuSO 4 and 30 µM ZnSO 4 for metal loading.…”

“…Briefly, E. coli cells were cultured at 23 °C with 3 mM CuSO 4 and 30 µM ZnSO 4 for metal loading. SOD1 Wt and SOD1 G93a proteins were then purified from bacterial lysates, by heat denaturation, size exclusion chromatography (Superdex 75; Ge healthcare), and anion-exchange chromatography using a Q-Sepharose anion exchange column (Ge healthcare), and eluted with a salt gradient of 0-125 mM NaCl, ph 7.5 [28].…”

“…Wildtype human SOD1 (SOD1 Wt ) and aLS-linked human mutant SOD1 G93a proteins were expressed in bacteria and purified to homogeneity as previously described [28]. Both proteins were metallated to the extent that they were enzymically active [28].…”

“…For this purpose a human neuroblastoma cell line (Sh-SY5Y) was used due to the previously reported species specificity in seeding SOD1 Wt aggregation [17]. aggregated recombinant SOD1 Wt and mutant SOD1 G93a proteins were prepared by treatment with 5 mM eDta and 50 mM Dtt to remove metal ions and reduce the native Cys57-Cys146 SOD1 disulfide bond, respectively, as described previously [28,[32][33][34]. the aggregated proteins (aggSOD1 Wt , aggSOD1 G93a ) and native SOD1 Wt and SOD1 G93a were then labeled with alexa Fluor 488 dye.…”

“…In vitro SOD1 aggregation was performed as previously described [28]. Purified SOD1 Wt and G93a SOD1 proteins (1 mg/mL) dissolved in PBS were incubated with 50 mM dithiothreitol (Dtt) and 5 mM ethylenediaminetetraacetic acid (eDta) at 37 °C for 72 h whilst shaking and vortexed briefly to obtain a homogenous solution.…”