The Sirtuin 5 Inhibitor MC3482 Ameliorates Microglia-Induced Neuroinflammation following Ischaemic Stroke by Upregulating the Succinylation Level of Annexin-A1

Background

Ischaemic stroke is the most common type of stroke and a leading cause of death and disability worldwide. Its pathogenesis is complex, involving processes such as oxidative stress, neuroinflammation, excitotoxicity, endoplasmic reticulum stress, and energy imbalance, which activate multiple signaling cascades promoting neuronal apoptosis. Current treatments mainly focus on thrombolysis, anti-inflammation, and neuroprotection, but with limited efficacy. Drugs like tissue plasminogen activator (tPA) can dissolve blood clots and restore blood circulation, but have a short therapeutic window and risk of hemorrhagic transformation. Therefore, developing effective anti-ischaemic stroke drugs remains a significant challenge.

Among the mammalian deacetylase family (sirtuins, sirts), Sirtuin 5 (Sirt5) is unique for its strong protein desuccinylase activity. In ischaemic stroke, Sirt5 expression significantly increases in microglia, triggering excessive neuroinflammation and neuronal damage. Thus, interventions targeting Sirt5 may help reduce neuroinflammation and protect against ischaemic brain injury.

Paper Source

This paper was published in the Journal of Neuroimmune Pharmacology in 2024, titled “The Sirtuin 5 Inhibitor MC3482 Ameliorates Microglia-Induced Neuroinflammation following Ischaemic Stroke by Upregulating the Succinylation Level of Annexin-A1”. The main authors include Qian Xia et al., from the Department of Anesthesiology and Pain Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China, and the Department of Neurosurgery, The Third Hospital of Shanxi Medical University (Shanxi Bethune Hospital), China.

Research Process

Experimental Design

The study used the middle cerebral artery occlusion (MCAO) surgery to establish a mouse model of ischaemic stroke. Male C57BL/6JNifdc mice aged 8-12 weeks and weighing 22-25 grams were used, totaling 180 mice, with 13 excluded due to death from anesthesia or surgical failure. Grouping for experimental and control groups was done by random drawing. The study was conducted after approval from the Experimental Animal Ethics Committee of Tongji Hospital.

Drug Treatment and Neurobehavioral Tests

Mice underwent MCAO surgery and received intracerebroventricular injection of MC3482 at doses of 1, 2, or 5 mg/kg 3 hours post-surgery, once daily for 7 days. The control group received an equal volume of saline. Brain slices and triphenyltetrazolium chloride (TTC) staining were performed 48 hours later to assess infarct volume. A series of neurobehavioral tests (e.g., adhesive test, cylinder test, rotarod test, pole test, and foot fault test) were conducted to evaluate long-term neurological function.

Cellular and Molecular Studies

Real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to analyze mRNA and protein levels of pro-inflammatory cytokines and chemokines in brain tissue. Co-immunoprecipitation (Co-IP) and Western blot were used to detect the succinylation level of Annexin-A1 and its interaction with Sirt5.

Main Experimental Results

Protective Effect of MC3482 on Ischaemic Brain Injury

Results showed that MC3482 significantly reduced infarct volume in ischaemic brain injury and improved neurobehavioral scores and long-term neurological function. TTC staining showed that compared to the control group, the MC3482 treatment group had significantly smaller infarct volumes, exhibiting a dose-dependent effect. Water maze and novel object recognition experiments revealed that MC3482 improved spatial learning and memory abilities in mice.

Reduction of Microglia-Induced Neuroinflammation

Experiments showed that MC3482 significantly downregulated mRNA and protein levels of pro-inflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, etc.), inhibiting excessive activation of microglia following ischaemic stroke.

Inhibition of Neuronal Apoptosis

Western blot analysis showed that MC3482 reduced the expression of pro-apoptotic molecules (BAX, Caspase-3, Caspase-9) and increased levels of anti-apoptotic molecules (Bcl-XL). Additionally, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed significantly reduced neuronal apoptosis in the MC3482 treatment group.

Effects on Annexin-A1 Succinylation Level and Subcellular Localization

Co-immunoprecipitation results showed that MC3482 blocked the interaction between Sirt5 and Annexin-A1, significantly increasing the succinylation level of Annexin-A1. Immunofluorescence and subcellular fractionation analysis showed that MC3482 reduced nuclear translocation of Annexin-A1 and increased its recruitment to the membrane and extracellular secretion.

Discussion and Conclusion

This study provides compelling evidence that the Sirt5-specific inhibitor MC3482 alleviates microglia-mediated neuroinflammation induced by ischaemic stroke and improves long-term neurological function by inhibiting Annexin-A1 desuccinylation. MC3482 not only significantly reduced infarct volume but also improved cognitive function and reduced anxiety-like behavior in mice. Mechanistic studies indicated that MC3482 inhibited the interaction between Sirt5 and Annexin-A1, increasing Annexin-A1’s succinylation level and its recruitment to the membrane and extracellular secretion.

Research Highlights

1. Key Finding: MC3482 significantly reduced ischaemic brain injury and improved long-term neurological function by upregulating the succinylation level of Annexin-A1.
2. Problem Solved: This study demonstrated that the Sirt5 inhibitor MC3482 can effectively combat neuroinflammation caused by ischaemic stroke, providing a potential therapeutic strategy for this disease.
3. Methodological Innovation: The study employed a mouse MCAO model and systematically evaluated the mechanism of action of MC3482 in ischaemic stroke.

The conclusion of this study is that MC3482, as a specific inhibitor of Sirt5, may become an effective new drug candidate for treating ischaemic stroke by regulating the succinylation level of Annexin-A1. Future research should further explore its specific mechanisms of action and clinical application prospects.