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Updated: May 1, 2026
Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke
Published on: January 3, 2025
Selective 14-3-3γ induction quenches p-β-catenin Ser37/Bax-enhanced cell death in cerebral cortical neurons during
1Department of Pathophysiology, School of Basic Medicine, Key Laboratory of Neurological Diseases, Ministry of Education, Hubei Provincial Key Laboratory of Neurological Diseases, Huazhong University of Science and Technology, Wuhan, China.
Abstract:
Ischemia-induced cell death is a major cause of disability or death after stroke. Identifying the key intrinsic protective mechanisms induced by ischemia is critical for the development of effective stroke treatment. Here, we reported that 14-3-3γ was a selective ischemia-inducible survival factor in cerebral cortical neurons reducing cell death by downregulating Bax depend direct 14-3-3γ/p-β-catenin Ser37 interactions in the nucleus. 14-3-3γ, but not other 14-3-3 isoforms, was upregulated in primary cerebral cortical neurons upon oxygen-glucose deprivation (OGD) as measured by quantitative PCR, western blot and fluorescent immunostaining. The selective induction of 14-3-3γ in cortical neurons by OGD was verified by the in vivo ischemic stroke model. Knocking down 14-3-3γ alone or inhibiting 14-3-3/client interactions was sufficient to induce cell death in normal cultured neurons and exacerbate OGD-induced neuronal death. Ectopic overexpression of 14-3-3γ significantly reduced OGD-induced cell death in cultured neurons. Co-immunoprecipitation and fluorescence resonance energy transfer demonstrated that endogenous 14-3-3γ bound directly to more p-β-catenin Ser37 but not p-Bad, p-Ask-1, p-p53 and Bax. During OGD, p-β-catenin Ser37 but not p-β-catenin Ser45 was increased prominently, which correlated with Bax elevation in cortical neurons. OGD promoted the entry of 14-3-3γ into the nuclei, in correlation with the increase of nuclear p-β-catenin Ser37 in neurons. Overexpression of 14-3-3γ significantly reduced Bax expression, whereas knockdown of 14-3-3γ increased Bax in cortical neurons. Abolishing β-catenin phosphorylation at Ser37 (S37A) significantly reduced Bax and cell death in neurons upon OGD. Finally, 14-3-3γ overexpression completely suppressed β-catenin-enhanced Bax and cell death in neurons upon OGD. Based on these data, we propose that the 14-3-3γ/p-β-catenin Ser37/Bax axis determines cell survival or death of neurons during ischemia, providing novel therapeutic targets for ischemic stroke as well as other related neurological diseases.
Insights
The protein 14-3-3γ protects brain cells from death after stroke by regulating the 14-3-3γ/p-β-catenin Ser37/Bax pathway. This finding offers new therapeutic targets for ischemic stroke and related neurological diseases.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Ischemia-induced cell death is a primary cause of disability and mortality following stroke.
- Identifying intrinsic protective mechanisms against ischemia is crucial for developing effective stroke treatments.
Purpose of the Study:
- To investigate the role of 14-3-3γ as a selective ischemia-inducible survival factor in cerebral cortical neurons.
- To elucidate the molecular mechanism by which 14-3-3γ protects neurons from ischemic cell death.
Main Methods:
- Quantitative PCR, western blot, and immunofluorescence staining to measure 14-3-3γ expression.
- In vivo ischemic stroke model to verify selective induction of 14-3-3γ.
- Co-immunoprecipitation and fluorescence resonance energy transfer to analyze protein interactions.
- Gene knockdown and overexpression studies to assess the functional role of 14-3-3γ and β-catenin phosphorylation.
Main Results:
- 14-3-3γ, but not other isoforms, was selectively upregulated in cortical neurons during oxygen-glucose deprivation (OGD).
- 14-3-3γ directly interacted with phosphorylated β-catenin (p-β-catenin Ser37) in the nucleus, downregulating Bax expression and reducing neuronal death.
- Overexpression of 14-3-3γ protected neurons from OGD-induced death, while its knockdown exacerbated cell death.
- Inhibition of β-catenin phosphorylation at Ser37 also reduced OGD-induced Bax expression and cell death.
Conclusions:
- The 14-3-3γ/p-β-catenin Ser37/Bax axis is a critical determinant of neuronal survival or death during ischemia.
- Targeting this pathway presents a novel therapeutic strategy for ischemic stroke and other neurological disorders.
