A method for reducing brain damage after stroke using a deacetylase activator

Abstract

The application discloses a kind of by application specific deacetylase activator Targeted elimination mitochondrial apoptosis-inducing factor (AIF) lysine (K295) position 295 Acetylation modification, effectively inhibit ischemic stress under AIF from mitochondria to the abnormal translocation of nucleus, block its mediated chromosomal DNA fragmentation process, and then significantly reduce the level of neuronal programmed death, thereby reduce the brain injury after stroke.In cell and mouse stroke model, after the specific elimination of AIF K295 acetylation modification, the survival rate of neurons is increased by 4 times (p<0.01), and the cerebral infarction volume is reduced by 40% (p<0.01).The application provides a new drug action target and treatment strategy for the development of neuroprotective therapy after stroke.

Description

Technical Field

[0001] This invention relates to the field of stroke treatment and detection. Specifically, it relates to a method for reducing nerve cell death and minimizing brain damage during stroke treatment. Additionally, it relates to the detection and application of lysine acetylation at position 295 of the AIF (a marker for stroke detection). Background Technology

[0002] Stroke, the second leading cause of death worldwide and the leading cause of disability in adults, presents a persistent core challenge in neuroscience, with research into its pathological mechanisms and innovation in treatment methods. According to the World Health Organization, approximately 15 million new stroke cases occur globally each year, with ischemic stroke accounting for as much as 87%. Although reperfusion therapies such as intravenous thrombolysis and endovascular thrombectomy can effectively restore blood flow, their clinical application is limited by a strict time window (within 4.5 hours of onset) and cannot prevent the cascade of neuronal damage triggered by reperfusion. Studies have shown that approximately 65% ​​of patients experience poor prognosis due to missing the treatment window or secondary neuronal death, highlighting the urgent need to develop novel neuroprotective strategies.

[0003] In recent years, programmed necrosis has been identified as a key mechanism of neuronal damage after stroke. Mitochondrial apoptosis-inducing factor (AIF / AIFM1), as the executive protein in this process, is released from mitochondria and translocates to the nucleus under ischemic stress, triggering irreversible cell death by inducing large fragmentation (~50kb) degradation of chromosomal DNA. However, existing research largely focuses on subcellular localization changes of AIF, and the molecular switches regulating its function remain unclear, particularly the impact of post-translational modifications on AIF nuclear translocation and damaging activity. This has resulted in intervention strategies targeting the AIF pathway remaining largely theoretical, with a lack of discoverable drug targets.

[0004] Our research revealed that lysine 295 (K295) of the AIF (acid-dependent fibroblast) undergoes significant acetylation modification after treatment with the programmed cell death inducer MNNG. Biochemical and cellular experiments demonstrated that this acetylation modification is a key molecular switch regulating its nuclear translocation and DNA cleavage activity. In vitro hypoxic-glucose cell experiments (simulating stroke) and animal models of stroke confirmed that AIF K295 acetylation levels were closely related to neuronal mortality in hypoxic-glucose and mouse stroke models. When K295 was mutated to arginine (R), which cannot undergo acetylation modification, through point mutation and complementation experiments, neuronal death and chromosomal DNA degradation were significantly reduced. This discovery overcomes the limitations of traditional theories regarding AIF functional regulation and provides a novel entry point for developing targeted intervention strategies. Further research showed that activation of the deacetylase SIRT1-mediated AIF K295 deacetylation significantly inhibited its nuclear translocation efficiency and its degradation of chromosomal DNA, reducing infarct volume by 40% in animal models. However, there are no reports of the development of diagnostic reagents or specific modulators based on acetylation modification of this site of AIF in the existing technology, and related treatment methods are still lacking. Summary of the Invention

[0005] This invention targets the molecular regulatory mechanism of programmed neuronal death after stroke. For the first time, it proposes to eliminate the acetylation modification of lysine at position 295 (K295) of apoptosis-inducing factor (AIF) by activating SIRT1, a specific deacetylase that removes this modification, thereby inhibiting hypoxic-ischemic programmed cell death and reducing brain injury after stroke. Simultaneously, a site-specific antibody was prepared as a marker for detecting programmed neuronal death and brain injury after stroke.

[0006] To achieve the above objectives, the present invention is implemented through the following solution:

[0007] Previous mechanistic studies confirmed that lysine residues in the AIF protein undergo high acetylation modification in in vitro programmed necrosis induction agents, hypoxia-glucose deficiency, and mouse stroke models. Lysine residue 295 of the AIF protein was screened as the main site for acetylation modification under the aforementioned stress conditions. Antibodies targeting the specific acetylation site at AIF 295 were prepared. The deacetylase SIRT1, which binds to AIF, was identified, and SIRT1 activators were used to inhibit the acetylation modification of lysine residue 295 of the AIF protein under stress conditions. In a mouse stroke model, this activator significantly reduced brain lesion volume. Attached Figure Description

[0008] Figure 1 Patent principle diagram: By activating deacetylase to eliminate the acetylation of lysine at position 295 of AIF, brain damage after stroke can be reduced.

[0009] Figure 2 Identification and verification of the acetylation sites of AIF. Figure 2 a: In 10cm 293T cells, AIF with the Flag tag was transiently overexpressed. After 24 hours of transfection, the cells were treated with OGD (glucose-oxygen deprivation, simulating stroke) and harvested 12 hours later. The samples were eluted with Flag-M2agarose IP, run on a gel, and after gel cutting, sent to mass spectrometry for further detection of AIF modification sites. The figure shows the acetylation sites of AIF identified by mass spectrometry. Figure 2 b: Wild-type AIF was transiently overexpressed in 293T cells. Twenty-four hours post-transfection, cells were treated with OGD for 12 hours, harvested, and lysed using Flag-M2 agarose IP. Simultaneously, His-tagged AIF was expressed and purified using *E. coli*. The cells were then incubated with a purified specific antibody targeting the acetylation modification of lysine at position 295 of AIF (AIF-K295). ac The results showed that the antibody could only recognize wild-type AIF and not the purified, unmodified AIF protein from E. coli, indicating that the antibody has high specificity.

[0010] Figure 3 AIF-K295 acetylation modification is significantly increased during programmed cell death. Figure 3 a: Cells were transiently overexpressing Flag-tagged AIF in 10cm293T cells. Twenty-four hours after transfection, cells were treated with OGD (glucose-oxygen deprivation, simulating stroke), and harvested at different time points. Flag-M2 agarose IP was used. AIF-K295 was also used. ac Antibody detection was used to assess the acetylation level of lysine at position 295 of AIF in the sample. Figure 3 b: A schematic diagram of a mouse middle cerebral artery embolization surgery simulating unilateral stroke, in which a suture is used to enter the internal carotid artery through the common carotid artery to cause middle cerebral artery embolization; Figure 3 c: The left side of the image shows the un-embolized cerebral hemisphere, and the right side shows the embolized cerebral hemisphere. Brain tissue samples within the yellow box were subjected to immunofluorescence and TUNEL assays. The results showed that embolization causes brain tissue damage and nerve cell death. Within the embolized brain region, large amounts of AIF were released from mitochondria and entered the cell nucleus, co-localizing with the TUNEL cell death signal. Figure 3 d: Mouse brains from the sham-operated group (control group) and the middle cerebral artery embolization group were collected, homogenized, lysed, and inoculated with AIF antibody via AIF-K295. ac Antibody detection was used to assess the acetylation status of AIF in the damaged brain region. The results showed that the AIF at position 295 underwent extensive acetylation modification in the brain region caused by suture occlusion.

[0011] Figure 4 In programmed cell death, the acetylation of AIF-K295 determines cell fate.

[0012] Figure 4 a: A stable AIF knockdown cell line and a stable AIF replenishment cell line were established in the SK-N-SH neural cell line, including wild-type and AIF acetylation-modified mutant cell lines. AIF expression was detected using Western blotting. Figure 4 b: Cell viability was assessed using WST-1. The stable cell lines were first treated with OGD (glucose-oxygen deprivation, simulating stroke). After one day, WST-1 was added and the cells were cultured for another 2 hours. The WST-1 readings were then measured using OD450, and cell viability was calculated based on the readings.

[0013] Figure 5 SIRT1 activators reduce AIF-K295 acetylation modification, increase cell survival, and reduce brain damage after stroke.

[0014] Figure 5 a: In a mouse model of stroke, the use of SIRT1 activator SRT1720 can significantly reduce the infarct volume caused by cerebral ischemia and reduce brain injury. Figure 5 b: Obtain brain tissue from mice after modeling, homogenize it, and use AIF antibody IP, followed by AIF-K295. ac The acetylation level of AIF in brain tissue was detected, and the results showed that SRT1720 can significantly reduce the acetylation level of AIF at position 295 after cerebral ischemia. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in this technical field. Of course, the instruments and materials used in the embodiments are not limited to the examples listed herein, but are based on their ability to solve the technical problems of the present invention and achieve the corresponding technical effects. In addition, the molecular biology methods not described in detail in the embodiments are all conventional methods in the art, and specific operations can be found in molecular biology guides or product instructions.

[0016] Example 1

[0017] Identification and verification of acetylation sites in AIF.

[0018] Figure 2 a. Mass spectrometry results showed that lysine 295 of AIF was highly acetylated during OGD (glucose-oxygen deprivation, mimicking stroke) induced programmed cell death. Figure 2b. Preparation of acetylation modification targeting lysine at position 295 of AIF (AIF-K295) ac Rabbit polyclonal antibodies were synthesized and purified, and the specificity was verified using the purified antibodies. AIF protein was purified from eukaryotic 293T cells and *E. coli*, respectively. AIF-K295 ac It can only recognize AIF expressed in eukaryotes, but not AIF purified and unmodified from prokaryotes. This result indicates that AIF-K295... ac The antibody exhibits high specificity. The results are as follows: Figure 2 As shown, mass spectrometry identified the acetylation site of AIF as K295, and the programmed cell death factor OGD can induce acetylation modification at the AIF-K295 site.

[0019] Example 2

[0020] AIF-K295 acetylation modification is significantly increased during stroke-induced programmed cell death.

[0021] Figure 3 a: AIF cells transiently overexpressing the Flag tag were transfected into 10cm 293T cells. Twenty-four hours after transfection, the cells were treated with OGD (oxygen-deficiency hypoxia, an in vitro cell model simulating cerebral ischemia). Cells were harvested at different time points and processed using Flag-M2 agarose IP. AIF-K295 was used. ac Antibody detection of the acetylation level of lysine 295 in AIF samples showed that OGD significantly induced acetylation of lysine 295 in AIF (AIF-K295). ac ); Figure 3 b: A schematic diagram of a mouse middle cerebral artery embolization surgery simulating unilateral stroke. A suture occluder is used to enter the internal carotid artery through the common carotid artery to cause middle cerebral artery embolization, simulating stroke. Figure 3 c: The left side of the image shows the un-embolized cerebral hemisphere, and the right side shows the embolized cerebral hemisphere. Brain tissue samples within the yellow box were subjected to immunofluorescence and TUNEL assays. The results showed that embolization causes brain tissue damage and nerve cell death. Within the embolized brain region, large amounts of AIF were released from mitochondria and entered the cell nucleus, co-localizing with the TUNEL cell death signal. Figure 3 d: Mouse brains were collected from the control group (sham surgery group) and the middle cerebral artery embolization group, respectively. The brains were homogenized and lysed, and then subjected to AIF antibody in vitro (IP) and analyzed using AIF-K295. ac Antibody detection was used to assess the acetylation status of AIF in the damaged brain region. The results showed that the AIF at position 295 underwent extensive acetylation modification in the brain region caused by suture occlusion.

[0022] Example 3

[0023] AIF-K295 acRegulates AIF-mediated programmed cell death.

[0024] Figure 4 a. Construction of stable cell lines with AIF knockdown and reversion mutations: shN (negative control short hairpin RNA), shR (shAIF), shR+AIF, shR+AIF-K295R (mutant that cannot undergo acetylation modification), and shR+AIF-K295Q (mimicking the continuous acetylation modification mutant). The above stable cell lines were cultured under OGD (glucose-oxygen deprivation) for 1 day, and cell viability was assessed using the WST-1 method. Results are as follows... Figure 4 As shown in b, acetylation at position 295 of AIF positively regulates programmed cell death. When acetylation at this site is prevented (AIF-K295R), OGD-induced programmed cell death is significantly inhibited. This result demonstrates that acetylation at position 295 of AIF regulates AIF-mediated programmed cell death.

[0025] Example 4

[0026] Inhibiting the acetylation modification at position 295 of AIF can reduce brain damage after stroke.

[0027] Figure 5 a. The use of SRT1720, a specific activator of the deacetylase SIRT1, can significantly reduce the infarct volume, i.e., brain injury, in a mouse model of stroke. Figure 5 b. Further experiments showed that SRT1720 significantly reduced the acetylation level of lysine at position 295 of AIF in a mouse model of stroke. These results are consistent with those from the cell model, indicating that activation of SIRT1 can reduce brain injury by inhibiting AIF-K295 acetylation.

[0028] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of AIF modified by acetylation of lysine at position 295 as a target in the development of drugs for the treatment of ischemic stroke.

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