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Unveiling Smyd-2's Role in Cytoplasmic Nrf-2 Sequestration and Ferroptosis Induction in Hippocampal Neurons After
Daohang Liu1, Yizhun Zhu1,2
1School of Pharmacy, Shanghai Key Laboratory of Bioactive Small Molecules, Fudan University, Shanghai 201203, China.
Abstract:
SET and MYND Domain-Containing 2 (Smyd-2), a specific protein lysine methyltransferase (PKMT), influences both histones and non-histones. Its role in cerebral ischemia/reperfusion (CIR), particularly in ferroptosis-a regulated form of cell death driven by lipid peroxidation-remains poorly understood. This study identifies the expression of Smyd-2 in the brain and investigates its relationship with neuronal programmed cell death (PCD). We specifically investigated how Smyd-2 regulates ferroptosis in CIR through its interaction with the Nuclear Factor Erythroid-2-related Factor-2 (Nrf-2)/Kelch-like ECH-associated protein (Keap-1) pathway. Smyd-2 knockout protects HT-22 cells from Erastin-induced ferroptosis but not TNF-α + Smac-mimetic-induced apoptosis/necroptosis. This neuroprotective effect of Smyd-2 knockout in HT-22 cells after Oxygen-Glucose Deprivation/Reperfusion (OGD/R) was reversed by Erastin. Smyd-2 knockout in HT-22 cells shows neuroprotection primarily via the Nuclear Factor Erythroid-2-related Factor-2 (Nrf-2)/Kelch-like ECH-associated protein (Keap-1) pathway, despite the concurrent upregulation of Smyd-2 and Nrf-2 observed in both the middle cerebral artery occlusion (MCAO) and OGD/R models. Interestingly, vivo experiments demonstrated that Smyd-2 knockout significantly reduced ferroptosis and lipid peroxidation in hippocampal neurons following CIR. Moreover, the Nrf-2 inhibitor ML-385 abolished the neuroprotective effects of Smyd-2 knockout, confirming the pivotal role of Nrf-2 in ferroptosis regulation. Cycloheximide (CHX) fails to reduce Nrf-2 expression in Smyd-2 knockout HT-22 cells. Smyd-2 knockout suppresses Nrf-2 lysine methylation, thereby promoting the Nrf-2/Keap-1 pathway without affecting the PKC-δ/Nrf-2 pathway. Conversely, Smyd-2 overexpression disrupts Nrf-2 nuclear translocation, exacerbating ferroptosis and oxidative stress, highlighting its dual regulatory role. This study underscores Smyd-2's potential for ischemic stroke treatment by disrupting the Smyd-2/Nrf-2-driven antioxidant capacity, leading to hippocampal neuronal ferroptosis. By clarifying the intricate interplay between ferroptosis and oxidative stress via the Nrf-2/Keap-1 pathway, our findings provide new insights into the molecular mechanisms of CIR and identify Smyd-2 as a promising therapeutic target.
Insights
Smyd-2 protein knockout protects brain cells from ferroptosis, a cell death linked to lipid peroxidation in cerebral ischemia/reperfusion injury. This neuroprotection is mediated by the Nrf-2/Keap-1 pathway, suggesting Smyd-2 as a therapeutic target for stroke.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Cerebral ischemia/reperfusion (CIR) involves complex cell death mechanisms, including ferroptosis, a lipid peroxidation-driven process.
- The role of SET and MYND Domain-Containing 2 (Smyd-2), a protein lysine methyltransferase, in CIR and ferroptosis is not well understood.
- Neuronal programmed cell death (PCD) pathways are critical in the pathophysiology of ischemic stroke.
Purpose of the Study:
- To investigate the expression and function of Smyd-2 in the brain during CIR.
- To elucidate the regulatory mechanism of Smyd-2 in ferroptosis via the Nuclear Factor Erythroid-2-related Factor-2 (Nrf-2)/Kelch-like ECH-associated protein (Keap-1) pathway.
- To evaluate Smyd-2 as a potential therapeutic target for ischemic stroke.
Main Methods:
- Utilized Smyd-2 knockout cell models (HT-22 cells) and in vivo CIR models (middle cerebral artery occlusion - MCAO).
- Induced ferroptosis using Erastin and apoptosis/necroptosis using TNF-α + Smac-mimetic.
- Assessed cell viability, lipid peroxidation, Nrf-2 nuclear translocation, and Nrf-2 lysine methylation.
- Employed Nrf-2 inhibitor (ML-385) and protein synthesis inhibitor (Cycloheximide - CHX).
Main Results:
- Smyd-2 knockout conferred significant neuroprotection against Erastin-induced ferroptosis and CIR-induced ferroptosis in hippocampal neurons.
- Smyd-2 knockout enhanced the Nrf-2/Keap-1 pathway by suppressing Nrf-2 lysine methylation, independent of the PKC-δ/Nrf-2 pathway.
- Smyd-2 overexpression exacerbated ferroptosis and oxidative stress by disrupting Nrf-2 nuclear translocation.
- Nrf-2 inhibition abolished the neuroprotective effects of Smyd-2 knockout, confirming Nrf-2's critical role.
Conclusions:
- Smyd-2 plays a crucial role in regulating ferroptosis in CIR, primarily through its modulation of the Nrf-2/Keap-1 pathway.
- Disruption of the Smyd-2/Nrf-2 interaction compromises antioxidant capacity, leading to ferroptosis in hippocampal neurons.
- Targeting Smyd-2 presents a promising therapeutic strategy for mitigating neuronal damage in ischemic stroke.
