# Neuroprotection From Intracerebral Hemorrhage Following Pharmacological Inhibition of GSK3β Depends on HFE Gene Status

## TL;DR

A brain bleed causes iron-induced damage, but mice with a specific HFE mutation show natural protection, and this protection does not improve further with a drug that inhibits GSK3β.

## Contribution

The study reveals that GSK3β inhibition benefits only non-mutant mice, not those with the H67D HFE mutation, highlighting the importance of HFE genotype in ICH treatment.

## Key findings

- WT mice showed improved recovery and reduced brain damage after GSK3β inhibition.
- H67D mice did not benefit from GSK3β inhibition, suggesting a biological limit to their antioxidant response.
- HFE genotype may influence the effectiveness of GSK3β-targeted therapies for ICH.

## Abstract

Iron release from hemoglobin breakdown following an intracerebral hemorrhage (ICH) is a key mediator in stroke‐induced cytotoxicity. We have previously demonstrated that mice carrying the H67D mutation in the homeostatic iron regulatory gene (HFE) experience marked neuroprotection following ICH. This improvement is likely due to an endogenous upregulation in the Nrf2 antioxidant system. Prior studies in H67D mice discovered decreased activity in GSK3β, a kinase that functions to break down Nrf2. Interestingly, pharmacological inhibition of GSK3β has been shown to vastly improve outcomes in ICH animal models. However, it remains unclear whether this pathway is responsible for the enhanced antioxidant response in H67D animals. In this study, H67D and WT mice received daily injections of intraperitoneal SB216763, a selective inhibitor of GSK3β, 14 days prior to ICH. The functional motor recovery of each animal was assessed by rotarod and neurodegeneration was measured using Fluorojade‐B. Immunoblotting assessed the antioxidant response and GSK3β activity through Nrf2, GPX4, FTH1, and β‐Catenin. At 3 days post‐ICH, SB216763‐treated WT mice display enhanced functional recovery, decreased degenerated neurons, and increased brain levels of Nrf2 and GPX4 compared to WT‐Vehicle‐Controls. Further, SB216763 treatment in H67D mice did not result in any significant changes in measured outcomes compared to H67D‐Vehicle‐Controls. In conclusion, WT mice benefit from GSK3β inhibition following ICH whereas H67D animals do not. This suggests that the regulation of the antioxidant response may have reached its biological limit in H67D animals. Importantly, these data suggest that clinical trials aimed towards improving ICH outcomes, especially through GSK3β inhibition, must take into account HFE genotype as this mutation, present in nearly 20% of individuals worldwide, may alter ICH recovery regardless of therapy.

Following a brain bleed, blood breakdown leads to the release of iron, which injures the brain through oxidative stress. We have previously shown that mice carrying a common mutation in an iron‐regulatory gene (H67D HFE) have an innate protection from this damage due to an enhanced response to the antioxidant system following a brain bleed. Interestingly, this may be due in part to decreased levels of a protein called GSK3β in these animals. This specific protein normally inhibits the antioxidant response. In this study, we demonstrate that mice without the H67D mutation have better recovery following brain bleeds when treated with a drug that inhibits GSK3β. However, mice with the H67D HFE mutation do not demonstrate any additional benefits. Clinically, this may mean that treatments that inhibit GSK3β might help some patients after a brain bleed, but this benefit could depend on the patient's HFE genotype.