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.

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.

Figures

SB216763 Effectively Inhibits GSK3β in the Brains of WT Mice, but Not in H67D Mice. Effects of SB216763 on β‐Catenin levels in the ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) non‐ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) (independent sample t‐test). Data are expressed as means ± standard deviation; *p < 0.05; p < 0.0001.

SB216763 Does Not Affect Hematoma Size in Either Wild‐Type or H67D Animals. Effects of SB216763 on hematoma volume at 3‐days post‐ICH demonstrate no significant differences. (A) WT‐Vehicle‐Control (n = 4, male = 2, female = 2, mean = 0.088 μm3, STD = 0.006 μm3) (B) WT‐SB216763‐Treated mice (n = 4, male = 3, female = 1, mean = 0.077 μm3, STD = 0.009 μm3) (C) H67D‐Control mice (n = 4, male = 2, female = 2, mean = 0.084 μm3, STD = 0.011 μm3), and (D) H67D‐SB216763‐Treated mice (n = 4, male = 2, female = 2, mean = 0.083 μm3, STD = 0.01 μm3). Data are expressed as means ± standard deviation.

WT, but not H67D Mice, Demonstrate Improved Functional Recovery After SB216763 Treatments. Latency to fall (in seconds) was used to evaluate functional motor recovery and provide a reflection of neurological recovery across different groups (one‐way ANOVA). No differences were found in the first 2 days post‐ICH. WT‐Vehicle‐Control (n = 15, male = 8, female = 7) shows statistically decreased functional motor recovery at 3 days post‐ICH when compared to WT‐SB216763‐Treated (n = 14, male = 10, female = 4), H67D‐Vehicle‐Control (n = 16, male = 8, female = 8), and H67D‐SB216763‐Treated groups (n = 13, male = 7, female = 6). Data are expressed as means ± standard deviation; *p < 0.05; p < 0.01.

WT, but not H67D Mice, Demonstrate Reduced Perihematomal Neurodegeneration After SB216763 Treatments. Fluorojade‐B staining was utilized to determine differences in the number of degenerated neurons in the perihematomal region: (A) WT‐Vehicle‐Control (n = 8, male = 4, female = 4) mice showed significantly increased FJB‐positive cells in the perihematomal region compared to (B) WT‐SB216763‐Treated mice (n = 7, male = 5, female = 2), (C) H67D‐Control mice (n = 8, male = 3, female = 3), and (D) H67D‐SB216763‐Treated mice (n = 6, male = 3, female = 3). Data are expressed as means ± standard deviation; *p < 0.05; p < 0.01.

WT, but not H67D Mice, Demonstrate Increased Levels of Nrf2 in the ICH‐Affected Hemisphere After SB216763 Treatments. Effects of SB216763 on Nrf2 levels in the ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) non‐ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) (independent sample t‐test). Data are expressed as means ± standard deviation; *p < 0.001.

WT, but not H67D Mice, Demonstrate Increased Levels of GPX4 in the ICH‐Affected Hemisphere After SB216763 Treatments. Effects of SB216763 on GPX4 levels in the ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) non‐ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) (independent sample t‐test). Data are expressed as means ± standard deviation; p < 0.0001.

WT and H67D Mice Demonstrate No Significant Increase in FTH1 in ICH‐Affected Hemisphere After SB216763 Treatments. Effects of SB216763 on FTH1 levels in the ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) non‐ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) (independent sample t‐test). Data are expressed as means ± standard deviation.

Full text

Introduction

1

Ferrous iron released from erythrolysis and heme breakdown plays a crucial role in mediating oxidative injury following an intracerebral hemorrhage (ICH). Its redox‐active properties spontaneously catalyze the formation of reactive oxygen species (ROS) through the Fenton reaction, leading to a build‐up of oxidative stress, DNA damage, mitochondrial dysfunction, and ultimately ferroptosis.

Nuclear Factor‐Erythroid 2 Related‐Factor (Nrf2), the master transcription factor for the antioxidant response, is essential in reversing and limiting oxidative stress following an ICH. At baseline, Nrf2 is bound by Kelch‐like ECH‐associated protein 1 (Keap1) in the cytoplasm where, under physiologic conditions, it is readily degraded. Keap1 contains a number of reactive cysteine groups that become modified in response to oxidative stress, allowing for Nrf2 stabilization and translocation to the nucleus (Baird and Dinkova‐Kostova 2011; Suzuki and Yamamoto 2015). Here, Nrf2 binds to regulatory sequences called antioxidant response elements (AREs) to express antioxidant enzymes such as Glutathione Peroxidase‐4 (GPX4) and FTH1, which directly reduces lipid peroxides to lipid alcohols and combats the detrimental effects of oxidative stress (Dodson et al. 2019; Baird and Dinkova‐Kostova 2011; Raghunath et al. 2018). Studies have consistently demonstrated an increase in Nrf2 levels within the perihematomal region, thus demonstrating its crucial role in mediating oxidative stress following an ICH (Chen‐Roetling and Regan 2016; Wang et al. 2007).

Previously, we demonstrated that a common mutation in the Homeostatic Iron Regulatory (HFE) Gene enhances recovery following an ICH in mice (Helmuth, Kumari, Palsa, Neely, et al. 2023). We found that following an ICH, mice harboring the H67D HFE mutation have improved functional motor recovery, decreased neuronal degeneration in the perihematomal region, and reduced markers of ferroptosis. Within the perihematomal tissue, we also discovered an enhanced antioxidant response through an upregulation of Nrf2, GPX4, and FTH1 when compared to wild‐type animals. Normally, HFE negatively regulates transferrin‐bound iron uptake by competitively binding to the transferrin receptor (TfR) at the cell membrane. However, the H67D mutation results in an HFE with decreased affinity to TfR, leading to subclinical brain iron accumulation in humans and mice. It is hypothesized that animals with this mutation demonstrate an adaptive or hormetic response to iron‐related stress as the increased iron acts as a low‐dose stressor that builds tolerance against oxidative stress over time (Marshall Moscon et al. 2024). Notably, the equivalent mutation in humans, H63D HFE, has a worldwide prevalence of approximately 20%, which may impact ICH recovery in the clinical population (Hanson et al. 2001).

Studies in our laboratory using RNAseq and immunoblotting data of brain tissue from H67D animals find that the positive regulator of Nrf2, peroxisome proliferator‐activator receptor‐gamma coactivator‐1alpha (PGC‐1α), is upregulated (Song 2020). PGC‐1α functions to inactivate glycogen synthase kinase‐3 beta (GSK3β) via phosphorylation which in turn stabilizes Nrf2. Normally, GSK3β negatively regulates Nrf2 activation by directly phosphorylating Nrf2 to promote its degradation (Aschner and Culbreth 2018; Rada et al. 2011). Therefore, inactivating GSK3β positively regulates Nrf2 activation by preventing its breakdown and maintaining its nuclear levels once it has been released from Keap1. Mechanistically, this pathway could explain differences in the antioxidant response following ICH in H67D mice that lead to better outcomes following ICH.

Pharmacological manipulation of the Nrf2 pathway in animal models of ICH has been well established (Zhao, Sun, Ting, et al. 2015; Zhao et al. 2007; Yin et al. 2015; Iniaghe et al. 2015; Zhao, Sun, Zhang, et al. 2015; Sukumari‐Ramesh and Alleyne 2016; Imai et al. 2021). However, all of these compounds target the cysteine residues of Keap1 to promote Nrf2 dissociation and have yet to be tried or shown effective in clinical populations despite their impressive effectiveness in animal studies. Given the lack of positive clinical outcomes and continued evidence that the Nrf2 pathway is indeed critical to promoting recovery from ICH, recent studies have focused on novel activation of Nrf2 through GSK3β inhibition (Zhao et al. 2017; Shi et al. 2024).

The neuroprotective landscape produced by the H67D HFE mutation holds immense potential to understand key biological pathways that mitigate ICH damage. Our prior data demonstrate that the mutation results in better outcomes following ICH and lower GSK3β activity at baseline. However, it is unknown if GSK3β is the critical point within this pathway that is responsible for ramping up the antioxidant system to improve ICH outcomes in H67D animals or if manipulation of this pathway results in additional neuroprotection in these animals. Therefore, the objective of the following study is to determine the contributions of GSK3β to the observed genotype‐dependent neuroprotection through pharmacologic manipulation. Overall, this study will unlock vital understandings of the GSK3β/Nrf2 pathway to ICH recovery in a common HFE variant which may impact future therapeutic strategies.

Methods

2

Animal studies were performed following the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Penn State Institutional Animal Care and Use Committee (Protocol: 20190115). The experimenters were blinded to the genotype and treatment condition of each animal during behavioral evaluations and immunohistochemical analysis. They were then unblinded to complete the statistical analysis. Additionally, data were obtained under ARRIVE guidelines.

Drug Administration

2.1

To determine the effects of GSK3β inhibition on ICH outcomes, each animal was injected daily with vehicle or a selective inhibitor of GSK3β (SB 216763, Tocris, Cat# 1616) (10 mg/kg, i.p.) for 14 days prior to ICH and again each day following ICH until euthanasia. This dose was found in previous literature to successfully inhibit GSK3β in the brains of mice (D'Angelo et al. 2016). Each day, SB 216763 was prepared fresh to a concentration of 2 mg/mL containing 5% DMSO in corn‐oil. Vehicle treatments consisted of an equivalent volume containing 5% DMSO in corn‐oil.

Animal ICH Model

2.2

Female and male, 6‐month‐old C57BL/6J × 129 mice carrying either the H67D HFE (n = 29, male = 18, female = 11) or Wild‐Type HFE (n = 29, male = 15, female = 14) gene were used for this study. This sample size was calculated for all experiments with 0.8 power to detect a 0.2 difference at an alpha level of 0.05 based on our initial study (Helmuth, Kumari, Palsa, et al. 2023). The H67D knock‐in was created by ingenious Targeting Laboratory (Ronkonkoma, NY) (Tomatsu et al. 2003). These mice were housed in autoclaved, plastic cages under 12‐h light and dark cycles with free access to standard chow and water at the Penn State College of Medicine Animal Care Facility. Animals were housed with up to three cage mates. One WT‐Vehicle‐Control and two H67D‐SB216763‐Treated animals died following ICH. No other animals were excluded from the study. Additionally, the animals were selected and assigned to an experimental group arbitrarily based on availability. An intracerebral hemorrhage was created using a two‐step autologous blood infusion model according to previously published methods. Here, mice were anesthetized under 1%–2% inhaled isoflurane and maintained in a stereotactic frame. The appropriate depth of anesthesia was assessed via an absent toe‐pinch reflex frequently during the duration of the procedure. The hair on the superior aspect of the animal's skull was removed and the surgical site was prepped using an ethanol swab followed by a betadine scrub. A midline incision was then made to reveal bregma and lambda. A 1 mm burr hole was then drilled 2.0 mm lateral and 0.5 mm anterior to bregma. Next, 30 μL of blood, drawn from the animal's cheek vein, was directly infused through the burr hole at a rate of 1 μL/min into the right caudate using a Hamilton Syringe (depth of 3.5 mm). The syringe dwelled for 20‐min before it was slowly removed to minimize blood regurgitation. The burr hole was then sealed using bone wax and the scalp incision was closed using three interrupted 3–0 vicryl sutures. The animals were then removed from the stereotactic frame and placed in a 37°C incubator to recover. Extended‐release buprenorphine (3.25 mg/kg) was administered subcutaneously to each animal prior to the procedure to reduce pain during the experiment. Each animal was monitored for several hours following surgery and carefully evaluated daily by both research staff and trained veterinarians at the Penn State College of Medicine. No animals demonstrated excessive pain as indicated by hunching, severe lethargy, respiratory distress, or severe dehydration following surgery. Three days following ICH, the mice were anesthetized using an intraperitoneal injection of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). Once fully anesthetized as assessed by an absent toe‐pinch reflex, mice were euthanized following a transcardial perfusion using a lactated ringer solution. The brains of each animal were then removed and maintained for subsequent analysis.

Functional Motor Assessment

2.3

Motor function was assessed using a previously published ramp‐up paradigm on the rotarod (Ruan and Yao 2020). Briefly, each animal was trained 3 days prior to ICH induction. During training, each animal was acclimated on the rod for 30 s without rotation. The rod then rotated at 4 rpm (RPM). Mice were considered trained once they completed 60 s on the rod at this speed. During testing, the rotation of the rod increased from 4 RPM to 20 RPM over 120 s and then remained at 20 RPM for an additional 60 s. Each animal was tested daily following ICH induction until sacrifice.

Fluorojade‐B & H&E Staining

2.4

A subset of animals was sacrificed at 3‐days post ICH to determine differences in degenerated neurons by utilizing Fluorojade‐B Staining (FJB). Brains fixed with paraformaldehyde as described above were frozen in optimal cutting temperature compound and sectioned into 15 μm coronal sections. Three sections within 100 μm of the needle track from each animal were taken, washed with PBS and stained with FJB according to the manufacturer's instructions (VWR, Cat# 76459‐462). The images of each section were captured using a Nikon Fluorescent microscope (Nikon H600L Intensilight C‐HGFIE). Total perihematomal FJB‐positive cells within the striatum were quantified blindly using ImageJ software (RRID: SCR_003070).

Additionally, we have previously validated our procedure for the consistency of hemorrhage size in each genotype (Helmuth, Kumari, Palsa, Neely, et al. 2023). To ensure this, twenty‐micrometer serial, coronal sections of the hematoma were taken and stained with Hematoxylin & Eosin according to the manufacturer's instructions (Abcam, Cat: ab245880). A blinded researcher then calculated the cross‐sectional area of each section using ImageJ software.

Immunoblotting

2.5

Protein levels were detected via immunoblotting as previously described (Palsa et al. 2023). Animals were sacrificed at 3‐days post ICH and perfused with lactated ringers. The brains were rapidly harvested and divided in the sagittal plane into two equal hemispheres and immediately snap frozen in isopentane. Each hemisphere was homogenized individually in NP40 buffer (Thermo, Cat# FNN0021) and protease inhibitor cocktail (Sigma, Cat# P8340), and total protein was quantified using a bicinchonic acid assay (Thermo, Cat# 23225). Then, 25 μg cellular protein was loaded onto a 4% to 20% Criterion TGX Precast Protein Gel (Bio‐Rad). Proteins were transferred onto a PVDF membrane. The membranes were blocked with 5% milk in Tris‐buffered saline with 1% Tween20 and then probed for FTH1 (Cell Signaling Technology, 1:1000, 4393S; RRID: AB_11217441), Nrf2 (Abcam, 1:1000, ab92946, RRID: AB_10561604), GPX4 (Abcam, 1:1000, ab125066, RRID: AB_10973901), β‐Catenin (Abcam, 1:1000, ab223075, RRID: AB_3083545), or β‐actin (Abcam, 1:1000, ab115777, RRID: AB_10899528) which served as a loading control. PVDF membranes were then incubated with the appropriate secondary antibody conjugated to HRP (1:5000, GE Amersham), and bands were visualized using ECL reagents (PerkinElmer) on a ChemiDoc MP Imaging System (Biorad, RRID: SCR_019037). Target proteins were normalized to beta‐actin and expressed as a ratio.

Statistics

2.6

Statistical analysis was conducted using GraphPad Prism 9. Independent sample t‐tests were used to compare treatment differences between groups (Wild‐Type‐Vehicle‐Controls vs. Wild‐Type‐SB216763‐Treated and H67D‐Vehicle‐Controls vs. H67D‐SB216763‐Treated). One‐way ANOVA and Tukey's post hoc test were used to compare the functional motor recovery and neuronal degeneration between groups. The data sets passed Shapiro–Wilk tests for normality. No tests for outliers were conducted. All data are expressed as means and standard deviations while significance was determined by a p‐value < 0.05.

Results

3

SB 216763 Inhibits GSK3β in the Brains of WT Mice, but Not H67D Mice

3.1

GSK3β plays an important role in the Wnt/β‐Catenin signaling pathway. In the absence of Wnt signaling, β‐Catenin is phosphorylated by GSK3β which promotes its destabilization and subsequent degradation. Therefore, pharmacological GSK3β inhibition should increase β‐catenin levels. In order to measure the efficacy of SB 216763 on GSK3β‐Inhibition, β‐Catenin was measured using western blot in both the ipsilateral, ICH‐affected hemisphere and the contralateral, non‐ICH‐affected hemisphere (Figure 1). Independent samples t‐tests yield a significant increase in β‐catenin levels in WT‐SB216763‐Treated animals (mean = 0.946, SD = 0.065, n = 7) compared to WT‐Vehicle‐Controls (Mean = 0.476, SD = 0.065, n = 7) (p < 0.0001); however, there was not a significant difference in β‐catenin levels in H67D‐SB216763‐Treated animals (mean = 1.002, SD = 0.076, n = 7) compared to H67D‐vehicle‐controls (mean = 1.083, SD = 0.094, n = 8) (p = 0.201). Similar to the ipsilateral hemisphere, independent t‐tests demonstrate a significant increase in β‐catenin levels in WT‐SB216763‐Treated animals (mean = 0.915, SD = 0.073, n = 7) compared to WT‐Vehicle‐Controls (mean = 0.603, SD = 0.061, n = 7) (p = 0.0490); however, there was not a significant difference in β‐catenin levels in H67D‐SB216763‐Treated animals (mean = 1.259, SD = 0.230, n = 7) compared to H67D‐vehicle‐controls (mean = 1.128, SD = 0.326, n = 8) (p = 0.638).

*SB216763 Effectively Inhibits GSK3β in the Brains of WT Mice, but Not in H67D Mice. Effects of SB216763 on β‐Catenin levels in the ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) non‐ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) (independent sample t‐test). Data are expressed as means ± standard deviation; *p < 0.05; ***p < 0.0001.

SB216763 Does Not Affect Hematoma Size in Either Wild‐Type or H67D Animals

3.2

To measure the effects of SB216763 on hematoma volume at 3‐days post‐ICH, serial coronal sections of WT‐Vehicle‐Control (n = 4, male = 2, female = 2), WT‐SB216763‐Treated (n = 4, male = 3, female = 1), H67D‐Control (n = 4, male = 2, female = 2), and H67D‐SB216763‐Treated (n = 4, male = 2, female = 2) animals were taken and stained using Hematoxylin and Eosin. WT‐Vehicle‐Control (mean = 0.088 μm^3^, STD = 0.006 μm^3^), WT‐SB216763‐Treated (mean = 0.077 μm^3^, STD = 0.009 μm^3^), H67D‐Control (mean = 0.084 μm^3^, STD = 0.011 μm^3^), and H67D‐SB216763‐Treated (mean = 0.083 μm^3^, STD = 0.01 μm^3^) animals demonstrated no significant difference in lesion volume (Figure 2).

SB216763 Does Not Affect Hematoma Size in Either Wild‐Type or H67D Animals. Effects of SB216763 on hematoma volume at 3‐days post‐ICH demonstrate no significant differences. (A) WT‐Vehicle‐Control (n = 4, male = 2, female = 2, mean = 0.088 μm3, STD = 0.006 μm3) (B) WT‐SB216763‐Treated mice (n = 4, male = 3, female = 1, mean = 0.077 μm3, STD = 0.009 μm3) (C) H67D‐Control mice (n = 4, male = 2, female = 2, mean = 0.084 μm3, STD = 0.011 μm3), and (D) H67D‐SB216763‐Treated mice (n = 4, male = 2, female = 2, mean = 0.083 μm3, STD = 0.01 μm3). Data are expressed as means ± standard deviation.

WT, But Not H67D Mice, Demonstrate Improved Functional Recovery After SB216763 Treatments

3.3

Each animal had its functional motor ability assessed by its latency to fall from a rotarod each day following ICH (Figure 3). Animals that are more functionally impaired will fall from the rod more quickly. There are no statistically significant differences between the groups after the first two days following ICH. However, at Day 3, WT‐Vehicle‐Controls (Mean = 54.93 s, SD = 22.69 s, n = 15) perform significantly worse than WT‐SB216763‐Treated (Mean = 87.36 s, SD = 27.80 s, n = 14, p = 0.011), H67D‐Vehicle‐Controls (Mean = 112.60 s, SD = 45.13 s, n = 15, p = 0.0013), and H67D‐SB216763‐Treated (mean = 102.08 s, SD = 40.71 s, n = 13, p = 0.0079). There was no statistically significant difference in motor function at 3 days post‐ICH in H67D‐Vehicle‐Controls and H67D‐SB216763‐Treated animals (p = 0.915) or between H67D‐Vehicle‐Controls and WT‐SB216763‐Treated animals (p = 0.286).

*WT, but not H67D Mice, Demonstrate Improved Functional Recovery After SB216763 Treatments. Latency to fall (in seconds) was used to evaluate functional motor recovery and provide a reflection of neurological recovery across different groups (one‐way ANOVA). No differences were found in the first 2 days post‐ICH. WT‐Vehicle‐Control (n = 15, male = 8, female = 7) shows statistically decreased functional motor recovery at 3 days post‐ICH when compared to WT‐SB216763‐Treated (n = 14, male = 10, female = 4), H67D‐Vehicle‐Control (n = 16, male = 8, female = 8), and H67D‐SB216763‐Treated groups (n = 13, male = 7, female = 6). Data are expressed as means ± standard deviation; *p < 0.05; *p < 0.01.

WT, But Not H67D Mice, Demonstrate Reduced Perihematomal Neurodegeneration After SB216763 Treatments

3.4

Fluorojade‐B (FJB) is an anionic fluorescein that stains degenerating neurons. Using this dye, the number of degenerating neurons in the perihematomal region was investigated between groups (Figure 4). Two‐way ANOVA results in a significant treatment × genotype interaction [F (1, 23) = 8.86; p = 0.006]. Multiple comparisons determined that WT‐Vehicle‐Controls (Mean = 385.13, SD = 125.38, n = 8) have significantly more degenerating neurons when compared to WT‐SB216763‐Treated (Mean = 181.50, SD = 85.60, p = 0.004, n = 7) and H67D‐Vehicle‐Controls (Mean = 206.28, SD = 125.38, p = 0.017). Additionally, there were no significant differences in FJB staining between H67D‐Vehicle‐Controls and H67D‐SB216763‐Treated animals (p = 0.952) or between WT‐SB216763‐Treated and H67D‐Vehicle‐Controls (p = 0.971).

*WT, but not H67D Mice, Demonstrate Reduced Perihematomal Neurodegeneration After SB216763 Treatments. Fluorojade‐B staining was utilized to determine differences in the number of degenerated neurons in the perihematomal region: (A) WT‐Vehicle‐Control (n = 8, male = 4, female = 4) mice showed significantly increased FJB‐positive cells in the perihematomal region compared to (B) WT‐SB216763‐Treated mice (n = 7, male = 5, female = 2), (C) H67D‐Control mice (n = 8, male = 3, female = 3), and (D) H67D‐SB216763‐Treated mice (n = 6, male = 3, female = 3). Data are expressed as means ± standard deviation; *p < 0.05; *p < 0.01.

WT, But Not H67D Mice, Demonstrate an Enhanced Antioxidant Response After SB216763 Treatments

3.5

To measure the effects of GSK3β‐Inhibition on the antioxidant response, the master transcription factor, Nrf2, was investigated (Figure 5). Independent samples t‐tests demonstrate a significant increase in Nrf2 levels in WT‐SB216763‐Treated animals (Mean = 0.797, SD = 0.232, n = 7) compared to WT‐Vehicle‐Controls (Mean = 0.494, SD = 0.100, n = 7) (p = 0.0005). Additionally, there was no significant difference in Nrf2 levels in H67D‐SB216763‐Treated animals (Mean = 0.823, SD = 0.081, n = 7) compared to H67D‐vehicle‐controls (mean = 1.012, SD = 0.110, n = 8) (p = 0.0724). Within the contralateral, non‐ICH‐affected hemisphere, no significant differences were appreciated.

*WT, but not H67D Mice, Demonstrate Increased Levels of Nrf2 in the ICH‐Affected Hemisphere After SB216763 Treatments. Effects of SB216763 on Nrf2 levels in the ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) non‐ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) (independent sample t‐test). Data are expressed as means ± standard deviation; **p < 0.001.

We also measured GPX4 levels, a transcriptional target of Nrf2 that directly combats the effects of ferroptosis by reversing the effects of lipid peroxidation (Figure 6). Here, GPX4 levels were significantly increased in WT‐SB216763‐Treated animals (Mean = 0.940, SD = 0.141, n = 7) compared to WT‐Vehicle‐Controls (Mean = 0.615, SD = 0.146, n = 7) (p = 0.0003). Additionally, there was no significant difference in GPX4 levels in H67D‐SB216763‐Treated animals (Mean = 1.062, SD = 0.085, n = 7) compared to H67D‐vehicle‐controls (mean = 1.171, SD = 0.124, n = 8) (p = 0.359). Within the contralateral, non‐ICH‐affected hemisphere, no significant differences were appreciated.

*WT, but not H67D Mice, Demonstrate Increased Levels of GPX4 in the ICH‐Affected Hemisphere After SB216763 Treatments. Effects of SB216763 on GPX4 levels in the ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) non‐ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) (independent sample t‐test). Data are expressed as means ± standard deviation; ***p < 0.0001.

Finally, we measured FTH1, an important protein for sequestering and transporting iron that is also under Nrf2 transcriptional activation (Figure 7). Independent samples t‐tests in the stroke‐affected hemisphere demonstrate that FTH1 levels were not significantly increased in WT‐SB216763‐Treated animals (Mean = 0.971, SD = 0.096, n = 7) compared to WT‐Vehicle‐Controls (Mean = 0.892, SD = 0.132, n = 7) (p = 0.656). Further, there was no significant difference in FTH1 levels in H67D‐SB216763‐Treated animals (Mean = 1.11, SD = 0.064, n = 7) compared to H67D‐vehicle‐controls (mean = 1.167, SD = 0.181, n = 8) (p = 0.827). Similar effects were seen within the contralateral, non‐stroke‐affected hemisphere, with no statistically significant differences in FTH1 in this hemisphere.

WT and H67D Mice Demonstrate No Significant Increase in FTH1 in ICH‐Affected Hemisphere After SB216763 Treatments. Effects of SB216763 on FTH1 levels in the ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) non‐ICH‐affected hemisphere (WT‐Vehicle‐Controls n = 7, male = 4, female = 3 compared to WT‐SB216763‐Treated n = 7, male = 5, female = 2, and H67D‐Vehicle‐Controls n = 8, male = 4, female = 4 compared to H67D‐SB216763‐Treated n = 7, male = 4, female = 3) (independent sample t‐test). Data are expressed as means ± standard deviation.

Discussion

4

The aim of our current study sought to determine the contributions of GSK3β to the genotype‐dependent neuroprotection following ICH through pharmacologic manipulation. We determined that the drug SB216763, a selective and ATP‐competitive inhibitor, successfully and significantly reduced GSK3β activity within the brains of WT mice as seen by increases in β‐Catenin levels. Compared to WT‐Vehicle‐Control mice, WT‐SB216763 treated mice demonstrate increased levels of Nrf2 within the stroke‐affected hemisphere as an effect of this GSK3β inhibition. Consequently, this resulted in corresponding increases in GPX4 levels which may contribute to the observed decreased neurodegeneration in the perihematomal area and improved functional recovery of WT‐SB216763 treated mice compared to WT‐Vehicle‐Controls. Still, GSK3β inhibition did not lead to significant increases in FTH1 in WT‐SB216763 treated mice, despite the FTH1 gene being known to contain an ARE. Moreover, the degree of GSK3β inhibition at the study dose in WT animals did not result in the reproduction of the endogenous levels of Nrf2, GPX4, or FTH1 in H67D‐Vehicle‐Control animals at baseline following ICH. Nevertheless, these levels significantly reduced neurodegeneration in the perihematomal area and improved functional outcomes in WT‐SB216763‐treated mice. Overall, these results not only reinforce previous studies that demonstrate the therapeutic potential of GSK3β inhibition in WT animals following ICH but also provide the novel and potentially clinically significant finding that H67D (or the human H63D) is not responsive to this therapeutic approach.

Naturally, GSK3β negatively regulates the antioxidant system through direct phosphorylation and ubiquitin‐mediated destruction of Nrf2. GSK3β additionally indirectly limits the action of Nrf2 through phosphorylation of Fyn, a tyrosine kinase, that promotes nuclear export of Nrf2. Critically, the actions of GSK3β‐mediated regulation of Nrf2 are independent of the canonical Keap1‐mediated pathway. This property is evident within the contralateral, non‐stroke‐affected hemisphere where there were no significant differences in Nrf2 despite treatments with SB216736 and increases in β‐catenin. This is because the lack of oxidative stress and ROS build‐up in this control hemisphere does not allow for Nrf2 dissociation from Keap1, meaning there is limited cytosolic Nrf2 for GSK3β to regulate. Again, the majority of Nrf2 boosting studies for the treatment of ICH have targeted its dissociation from Keap1, thus promoting cytosolic Nrf2 levels. However, the actions of Nrf2 through these means may be limited if other regulators such as GSK3β are actively promoting its breakdown and nuclear export. Regardless, in the setting of ICH, oxidative stress and ROS build‐up should sufficiently release Nrf2 from Keap1. Therefore, actions to maintain the effects of Nrf2 perhaps through the inhibition of GSK3β‐mediated Keap1‐independent pathways may prove more effective clinically than simply increasing cytosolic levels.

Interestingly, H67D mice did not receive any additional neuroprotective benefits from SB216763 treatment as seen by non‐significant differences in β‐catenin levels, functional motor recovery, degenerated neurons in the perihematomal space, or antioxidant proteins. Notably, H67D‐Vehicle‐Control mice already demonstrate decreased GSK3β activity as seen by higher levels of β‐catenin. Previous studies in our laboratory have observed these effects in that H67D primary astrocytes show a 1.4‐fold higher β‐catenin levels and decreased GSK3β activity compared to WT primary astrocytes (Song 2020). Therefore, it is possible that this pathway has already achieved a ceiling effect and no longer responds to increased stimulation due to the mutation alone. Though these pathways are not fully elucidated, there are feedback loops and other regulatory mechanisms that prevent over‐inhibition of GSK3β or over‐activation of Nrf2 signaling.

GSK3β is a primary player in the canonical Wnt signaling pathway that is necessary for several biological processes such as cell proliferation. In the brain, Wnt signaling plays an important role in maintaining neuronal function and mitochondrial health. Briefly, this pathway is initiated when a Wnt ligand binds to a Frizzled (Fz) receptor, which then recruits the scaffold protein disheveled (Dvl). Dvl then goes on to inhibit GSK3β leading to cytosolic β‐Catenin accumulation and changes in gene transcription (Sharma et al. 2024). This inhibition of GSK3β has also been shown to increase Nrf2 levels by preventing GSK3‐dependent phosphorylation and subsequent proteasomal degradation (Rada et al. 2015). There also exist two main non‐canonical Wnt signaling pathways. These pathways, Wnt/PCP and Wnt/Ca^2+^, are independent of β‐Catenin signaling and largely alter cellular migration and calcium signaling. Importantly, studies have found that activation of the non‐canonical Wnt pathways are active following injuries to the central nervous system and can inactivate the canonical pathway (Park et al. 2015; Topol et al. 2003; Ackers and Malgor 2018; Niu et al. 2012). Since SB216763 selectively inhibits GSK3β, a part of the canonical Wnt pathway, it is possible that effectors of the non‐canonical pathway are limiting the action of the drug in H67D animals. However, further experiments are needed to confirm possible differences in non‐canonical signaling in H67D animals. Altogether, these suggest additional therapeutic targets that can be leveraged to understand the genotypic differences in ICH recovery in H67D animals.

Overall, the innate neuroprotection produced by the H67D HFE mutation is due to the chronic exposure of high iron. This concept, known in toxicology as hormesis, suggests that a stressor at a low dose confers long‐term protection against a similar stress at a higher dose (Mattson 2008). In the setting of the H67D HFE mutation, the endogenous, higher levels of intracellular iron increase oxidative stress that ultimately induces an adaptive antioxidant response to limit cellular damage following an ICH. Our data further demonstrate that part of this adaptation includes alterations in the GSK3β pathway which ultimately may confer neuroprotection. This pathway may be manipulated in WT animals to mimic the neuroprotection seen in H67D animals. However, H67D animals receive no additional neuroprotective benefits from pharmacological manipulation of this pathway at the level of GSK3β inhibition.

Although these results are significant, there are some limitations to consider. The first is that our study tested only 10 mg/kg doses of SB216763 that were previously found to be effective and tolerable in prior studies. It is possible that a higher dose may result in a therapeutic effect in H67D animals at the expense of drug toxicity. Further, we did not perform sham surgeries as our initial study demonstrated no differences in outcomes between WT and H67D sham conditions. Finally, our study paradigm followed 14 injections of SB216763 prior to ICH induction. Ultimately, this was done to determine the mechanistic contribution of GSK3β to ICH recovery by ensuring that the target was maximally inhibited prior to ICH. Though this might not seem clinically relevant, some ICH patients experience re‐bleeding following their initial hemorrhage. Therefore, in the future, it may be viable to prophylactically treat some ICH patients with these types of drugs following hospitalization.

Conclusion

5

Our data establishes GSK3β inhibition significantly promotes recovery in WT animals following an ICH through an enhanced antioxidant response, but does not contribute to the genotype‐dependent recovery established in H67D animals. This suggests that the regulation of the antioxidant response, at the level of GSK3β, has reached its biological limit in H67D animals. Therefore, effectors further upstream in the GSK3β pathway may need to be evaluated to reveal how the H67D mutation enhances recovery following ICH. Lastly, given the prevalence of the H63D HFE mutation in the human population, these data strongly argue for genotype stratification in clinical trials or in treatment strategies for ICH as this mutation may alter recovery regardless of therapy. Furthermore, H63D individuals may not respond to GSK3β‐Inhibition studies, of which there are promising preclinical studies which may prove beneficial to those without the mutation.

Author Contributions

Timothy B. Helmuth: conceptualization, investigation, funding acquisition, writing – original draft, methodology, validation, visualization, formal analysis, data curation. Kondaiah Palsa: conceptualization, investigation, validation, writing – review and editing. Aurosman Pappus Sahu: conceptualization, investigation, writing – review and editing. Elizabeth B. Neely: writing – review and editing, investigation, project administration, methodology. Rashmi Kumari: investigation, writing – review and editing. Becky Slagle‐Webb: investigation, writing – review and editing, methodology, project administration. Scott D. Simon: conceptualization, investigation, funding acquisition, methodology, validation, visualization, writing – review and editing, supervision, resources. James R. Connor: software, supervision, resources, writing – review and editing, conceptualization, funding acquisition, methodology, investigation.

Funding

Funding provided by the Penn State Department of Neurosurgery.

Disclosure

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: jnc70393‐sup‐0001‐supinfo1.pdf.