Hemopexin increases the neurotoxicity of hemoglobin when haptoglobin is absent

Abstract

Hemopexin (Hpx) binds heme with extraordinary affinity, and after haptoglobin may provide a second line of defense against the toxicity of extracellular hemoglobin (Hb). In this series of experiments, the hypothesis that Hpx protects neurons from Hb neurotoxicity was evaluated in murine primary cultures containing neurons and glial cells. Contrary to hypothesis, Hpx increased neuronal loss due to micromolar concentrations of Hb by 4- to 12-fold, as measured by LDH release assay; conversely, the neurotoxicity of hemin was completely prevented. The endogenous fluorescence of Hpx was quenched by Hb, consistent with transfer of Hb-bound heme to Hpx. This was associated with precipitation of globin chains, as detected by immunostaining and fluorescent Hb labeling. A portion of this precipitate attached firmly to cells and could not be removed by multiple washes. Concomitant treatment with haptoglobin (Hp) prevented globin precipitation and most of the increase in neuronal loss. Hpx weakly attenuated the increase in culture non-heme iron produced by Hb treatment, quantified by ferrozine assay. However, Hb-Hpx toxicity was iron-dependent, and was blocked by deferoxamine and ferrostatin-1. Up-regulation of cell ferritin expression, a primary cell defense against Hb toxicity, was not observed on western blots of culture lysates that had been concomitantly treated with Hpx. These results suggest that Hpx destabilizes Hb in the absence of haptoglobin, leading to globin precipitation and exacerbation of iron-dependent oxidative cell injury. Combined therapy with hemopexin plus haptoglobin may be preferable to hemopexin alone after CNS hemorrhage.

Protective effect of vitreous against hemoglobin neurotoxicity

“…Consistent with prior observations [17], treatment with 10 μM Hb for 24 hours resulted in degeneration of most phase-bright cells with the typical appearance of neurons in this model, without injury to the background glial monolayer (Fig. 1 A-D ).…”

“…Multiple prior studies (e.g. [17]) have demonstrated that micromolar Hb concentrations are not toxic to glial cells in this model. After sampling medium (25 μl) for LDH release assay, cultures were washed with MEM10 and incubated with 13 μg/ml propidium iodide (PI) at 37°C for 5 minutes.…”

Deciphering the Iron Side of Stroke: Neurodegeneration at the Crossroads Between Iron Dyshomeostasis, Excitotoxicity, and Ferroptosis

“…In this regard, deletion/knockout of the Hpx aggravates brain injury by ICH (Chen L. et al, 2011; Ma et al, 2016) and, although endogenous levels of Hpx are insufficient to counteract the massive heme overload following ICH, increasing brain Hpx levels has been found to improve the outcome after ICH (Leclerc et al, 2018). Conversely, Hpx has recently been reported to increase the neurotoxicity of Hb in the absence of Hp (Chen-Roetling et al, 2018). Therefore, although both Hp and Hpx might provide a robust line of defense during ICH, additional knowledge on the fine tuning of this system is still required to design new possible neuroprotective treatments for ICH.…”

What Is Next in This “Age” of Heme-Driven Pathology and Protection by Hemopexin? An Update and Links with Iron

“…In vivo studies with mice or rats or in vitro studies with non-neuronal cells have not yet pinpointed globin as a toxic agent. However, a recent in vitro study with mixed mouse neurons and glia in culture incubated with hemoglobin (Hb) and HPX (i.e., in the absence of haptoglobin (Hp) led to white precipitates of globin protein in the medium on the surface of cells and within cells [43]. This was associated with significant levels of neurotoxicity, based on lactate dehydrogenase release from neurons, although not glia, after incubation with Hb (4 µM, i.e., 16 µM heme) and HPX (1mg/mL, i.e.,~17.5 µM).…”