Dimethyl fumarate confers neuroprotection by casein kinase 2 phosphorylation of Nrf2 in murine intracerebral hemorrhage

Highlights

• Treatment with dimethyl fumarate after intracerebral hemorrhage in mice:

• Activated casein kinase 2 and Nrf2 signaling pathway

• Ameliorated inflammation

• Improved neurological function and protected blood–brain barrier integrity

Abstract

Background and purpose

Edema formation, inflammation and increased blood–brain barrier permeability contribute to poor outcomes after intracerebral hemorrhage (ICH). This study examined the therapeutic effect of dimethyl fumarate (DMF), a fumaric acid ester that activates nuclear factor erythroid-2 related factor 2 (Nrf2) and Nrf2 heterodimerization effector protein musculo-aponeurotic fibrosarcoma-G (MAFG) in a murine ICH model.

Methods

Male CD-1 mice (n = 176) were subjected to intrastriatal infusion of bacterial collagenase (n = 126), autologous blood (n = 18) or sham surgery (n = 32). Four (4) animals not subjected to ICH (naive) were also included in the study. After ICH, animals either received vehicle, dimethyl fumarate (10 mg or 100 mg/kg) or casein kinase 2 inhibitor (E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid (TBCA). Thirty-two mice also received scrambled siRNA or MAFG siRNA 24 h before ICH. Brain water content and neurological function were evaluated.

Results

Dimethyl fumarate reduced Evans blue dye extravasation, decreased brain water content, and improved neurological deficits at 24 and 72 h after ICH. Casein kinase 2 inhibitor TBCA and MAFG siRNA prevented the effect of dimethyl fumarate on brain edema and neurological function. After ICH, ICAM-1 levels increased and casein kinase 2 levels decreased. Dimethyl fumarate reduced ICAM-1 but enhanced casein kinase 2 levels. Again, casein kinase 2 inhibitor TBCA and MAFG siRNA abolished the effect of dimethyl fumarate on ICAM-1 and casein kinase 2. Dimethyl fumarate preserved pNrf2 and MAFG expression in the nuclear lysate after ICH and the effect of dimethyl fumarate was abolished by casein kinase 2 inhibitor TBCA and MAFG siRNA. Dimethyl fumarate reduced microglia activation in peri-hematoma areas after ICH. The protective effect of dimethyl fumarate on brain edema and neurological function was also observed in a blood injection mouse model.

Conclusion

Dimethyl fumarate ameliorated inflammation, reduced blood-brain barrier permeability, and improved neurological outcomes by casein kinase 2 and Nrf2 signaling pathways after experimental ICH in mice.

Introduction

Intracerebral hemorrhage (ICH) which accounts for about 15–20% of all deaths from stroke is the rupturing of small blood vessels in the brain parenchyma. Currently, no effective treatment options are available for this fatal stroke subtype, and, even if patients survive the initial injury, a series of secondary events may lead to cerebral edema, progression of neurobehavioral deficits, and possible death (Chen et al., 2014, Ikram et al., 2012, Keep et al., 2012, Pandey and Xi, 2014, Chen et al., 2014).

Evidence from clinical and animal studies suggest that inflammation and oxidative stress which occur after hematoma formation are involved in ICH-induced secondary brain injury and neurological dysfunction (Aronowski and Hall, 2005, Chen et al., 2015). Vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), adhesion molecules expressed in the endothelium that are important in inflammation after injury, are increased upon activation of the nuclear factor-κB (NF-κB)-mediated tumor necrosis factor α (TNFα) signaling pathway. TNFα increases early-onset endothelial adhesion by protein kinase C-dependent up-regulation of ICAM-1 expression, which can worsen outcomes following ICH (Javaid et al., 2003, Wang and Doré, 2007).

Nuclear factor erythroid-2 related factor 2 (Nrf2), a major phase II gene regulator, is a broadly expressed transcription factor that binds to the antioxidant response element (ARE) consensus and regulates expression of phase II detoxifying enzymes (Itoh et al., 1997). In addition to protecting against oxidative and electrophilic stress, recent studies demonstrated that Nrf2 responds to pro-inflammatory stimuli and rescues cells/tissues from inflammatory injuries (Chen et al., 2006, Zhao and Aronowski, 2013). As a transcription factor, it is essential for Nrf2 to translocate into the nucleus in order to stimulate the up-regulation of cytoprotective genes (Zhang et al., 2013). Translocation of Nrf2 from the cytoplasm to the nucleus and export of Nrf2 from the nucleus is regulated by nuclear localization signals, nuclear export sequences and phosphorylation (Jain et al., 2005). Nrf2 is the substrate of several protein kinases, including protein kinase C (Numazawa et al., 2003), phosphatidylinositol 3-kinase (Lee et al., 2001), glycogen synthase kinase-3 (Rada et al., 2012), casein kinase 2 (Apopa et al., 2008), and Fyn (Jain and Jaiswal, 2006). Casein kinase 2, an ubiquitous eukaryotic kinase composed of catalytic (α or α1) and regulatory (β) subunits was elucidated as a major kinase for phosphorylating Nrf2 in in-vitro studies using neuroblastoma cells and human keratinocyte cell lines (Apopa et al., 2008, Pi et al., 2007).

Dimethyl fumarate (DMF), a fumaric acid ester that is effective in the treatment of relapsing/remitting multiple sclerosis, promotes Nrf2 activation and stabilization through direct modification of Keap1 at cysteine residue 151 (Kappos et al., 2008, Linker et al., 2011). Stabilization and phosphorylation of Nrf2 facilitate its nuclear import, forming heterodimers with MAFG, subsequently up-regulating cytoprotective genes and inhibiting NF-κB nuclear translocation, thus decreasing expression of NF-κB-dependent genes, including inflammatory cytokines, chemokines, and adhesion molecules (Jain et al., 2005, Stoof et al., 2001). Although dimethyl fumarate stabilizes Nrf2, the role casein kinase 2 plays in phosphorylating Nrf2 and p-Nrf2 conferred neuroprotection after ICH has not been documented. In the present study, we aimed to test 2 hypotheses, (i) administration of dimethyl fumarate will reduce brain edema and neurological dysfunction in mice after ICH and (ii) casein kinase 2 phosphorylation of Nrf2 will promote Nrf2 nuclear translocation and antioxidant response element activation as well as ameliorate inflammation and blood–brain barrier permeability after ICH. A schema of the study design is presented in Appendix 1.