Hypothiocyanous acid oxidizes collapsin response mediator protein 2 in brain pericytes

Abstract

AbstractBackgroundPericytes cover brain endothelium to enable maintenance of the blood‐brain barrier (BBB). Decreasing pericyte numbers are reported in Alzheimer’s disease and could lead to BBB dysfunction, triggering vascular inflammation and the recruitment of immune cells. Neutrophils contain high contents of myeloperoxidase, a heme enzyme that produces several oxidising species, including hypothiocyanous acid (HOSCN). HOSCN specifically targets cysteine residues in proteins and low molecular weight thiols, and is more stable than other oxidants in biological systems. A microtubule stabilising protein, collapsin response mediator protein 2 (CRMP2), is oxidised by hydrogen peroxide (H2O2) in some cell types, purportedly through a peroxiredoxin (Prx) relay mechanism. Here, we have tested whether HOSCN can oxidise CRMP2 and lead to disruption of microtubules (MT) in pericytes.MethodMurine pericytes were harvested and cultured, then treated with sub‐lethal concentrations of HOSCN and H2O2. Western blotting was used to observe CRMP2 and Prx oxidation, along with CRMP2 phosphorylation at sites important for MT binding. Fluorescent staining, live‐cell imaging and in vitro scratch assays were used to view cytoskeleton alterations and pericyte migration. Immunoprecipitation and proximity ligation assays were used to investigate physical associations between CRMP2 and Prxs.ResultTreatment of pericytes with HOSCN led to maximal CRMP2 oxidation after 60 min, whereas H2O2 reaches this in 5 min. Oxidation was closely associated with pericyte shrinkage, less growing microtubules and lower pericyte cell migration. CRMP2 oxidation occurred at the same time as Prx2 oxidation, and there was a physical association between these proteins, but it is not possible to conclude a redox relay occurs.ConclusionWe hypothesized that the longer‐lived oxidant HOSCN could oxidize CRMP2 and impair pericyte function at sub‐lethal doses. Indeed, HOSCN led to less dynamic MTs and subsequent lower migration of pericytes, likely lowering their ability to cover empty area on the inflamed vasculature to protect endothelial function. This effect is reversible upon HOSCN removal, nevertheless HOSCN could be a cause of BBB disruption during neuroinflammation. The mechanisms of CRMP2 oxidation are currently being investigated. Strategies that protect this MT regulatory protein from oxidation could have therapeutic benefit in neurological disease.