The Effect of Methyltransferase NSUN2 in Stroke

Stroke is one of the most important diseases that seriously threaten the health and public health of elderly patients.NSUN2 refers to the predominant methyltransferase for RNA m5C methylation, contributing to increased RNA stability, translocation and translation, and playing an important role in the physiopathology. However, there is insignicant progress on the biological functions and mechanisms of NSUN2 in cerebral ischemia-reperfusion injury. Here, C57BL/6 mice were employed to establish a middle cerebral artery ischemia-reperfusion injury model (MCAO) and found to signicantly increase in NSUN2 protein and mRNA expression levels by Western blotting and qRT-PCR. Subsequently, NSUN2 knockout mice were exploited to build the MCAO model. This study reported that knockout of NSUN2 signicantly aggravated brain infarct size and behavioral scores, while reducing 7-day postoperative survival and increasing neuronal apoptosis and injury in MCAO mice. According to the investigation of Western blotting results, decreased PI3K/AKT, ICAM-1 and Bcl-2 protein expressions and increased apoptosis-related protein (Caspase-3/Bax) were found. Overall, this study suggested that NSUN2 may affect cerebral ischemia-reperfusion injury via PI3K/AKT signaling channel and ICAM-1 protein regulation of apoptosis.


Introduction
Stroke is one of the most important diseases that seriously threaten the health and public health of elderly patients. It has a high morbidity, disability and fatality rate. About 75% of survivors lose the ability to live and work to varying degrees (Jiang et al.,2018). And society brings a heavy burden. Therefore, effective prevention and treatment of stroke has become one of the major issues that need to be resolved urgently in the national major chronic disease and public health health strategy.RNA methylation is a critical epigenetic modi cation in post-transcriptional modi cation of RNA, i.e., the frontier and hot spot of research over the past few years. Limited studies have shown that RNA m5C methylation is widespread in cells and exerts vital effects on various physiological and pathological processes, and that the mentioned modi cations are critical to mRNA stability, processing, regulation of stem cell function, stress response and genetic messaging (Oerum et  In addition, NSUN2 is highly expressed in mouse embryogenesis and increases in the brain, and mice knocked out of NSUN2 show more pronounced microcephaly and mental retardation as impacted by over cell apoptosis within the prenatal brain and other disorders attributed to impaired neuronal differentiation and development (Blanco et al.,2014b).Thus far, the mechanism of the mentioned defects is impaired translation as impacted by increased tRNA fragmentation after NSUN2 deletion (Flores et al.,2017). For this reason, NSUN2 is hypothesized to be also critical to stroke.The present study directly tested this hypothesis.
This study more speci cally investigated whether NSUN2 participates to stroke through mediating the phosphatidylinositol 3-kinase/serine/threonine kinase (PI3K/AKT) signaling channel and intercellular adhesion molecule-1 (ICAM-1) onto endothelial cell to determine the mechanism of action of NSUN2 in stroke. Activation of the PI3K/AKT signaling channel, which is critical to the regulation of various cellular functions, inhibits apoptosis in post-ischemic neurons and astrocytes (Zhou et al.,2020). Apoptosis is a programmed cell apoptosis pattern, which is one of the typical pathological features in ischemic stroke and is regulated by a combination of genes, such as Bcl-2 family, Caspase family, NF-κB family (Li et al.,2021;Capece et al.2020).And ICAM-1 is critical to regulating angiogenesis after cerebral ischemiareperfusion injury (Luo et al.,2016).
Nevertheless, little progress has been made on the biological functions and mechanisms of NSUN2 in stroke. Therefore, the effect of NSUN2 on stroke and the effects on PI3K/AKT signaling channel, ICAM-1 and apoptosis were explored here.

Animals
The Animal Experimentation Ethics Committee of Wenzhou Medical University approved the experiment protocol employed here. The present study was conducted based on the Wenzhou Medical University Animal Experimentation Guidelines. Male C57BL/6(20~25g) provided by Beijing Vitonlivar Laboratory Animal Technology Co.NSUN2 knockout mice were generated by mating conditional knockout mice nsun2Flox/Flox with estrogen-induced ER-Cre instrumental mice, identi ed by PCR and used for ear tagging.The mice are all male.Mice were housed in 2-5/cages and placed in a controlled environment (12h light/dark cycle; 21±2°C; 60-70% humidity) for 1 week prior to surgery. Animals have free access to standard laboratory food and water. Based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No.8023,revised 1978), all animal tests were carried out.
Discarded mice were executed using a carbon dioxide execution chamber.

Models and drugs
Anesthesia for each mouse was achieved by intraperitoneally injecting sodium pentobarbital (50 mg/kg, Sigma, USA). Subsequently, the mice under the anesthesia received the ventilation via tracheal intubations for assisting respiration, and the rectal temperature was kept at 37 degrees C with the use of a heating pad. Under the surgical microscope, the internal carotid artery (ICA), external carotid artery and right carotid artery received the careful separation when the of the neck skin was incised at the median position. A silicone-coated 4-0 mono lament nylon lament received the insertion to the right internal carotid artery and the advancement to the middle cerebral artery beginning, which is occluded. This study employed the lament for holding the lament at the appropriate position. At the end of surgery, the neck wound was closed and the animals were enabled to be recovered based on anaesthetization. The vessels received the removal approximately 10 mm after ligation under iso urane anesthesia for reperfusion 2 h after ligation. The success of occlusion was judged by the presence of hemiparesis and elevated body temperature to (37.8-38.8°C) prior to reperfusion. In sham-operated animals, the middle cerebral artery was not embolized but the same procedure was performed.

Survival rate
After establishing the MCAO model, the number of surviving and dead mice was recorded for 7 days.

Neurological evaluation
Mice were scored neurobehaviorally 24 h after MCAO, and an author blind to the experiment group assessed neurological de cits based on a modi ed neurobehavioral scoring system. The neurobehavioral score criteria were: 0, no de cit; 1, no voluntary activity with impaired consciousness; 2, falling to the right; 3, circling to the right; 4, failure to extend the right forelimb. After MCAO, the subjects achieving a neurobehavioral score of 0 should be excluded.

Measurement of infarct size
After 24h of reperfusion, the brain tissue was euthanized with an excess of chloral hydrate, quickly removed, and 2 mm-thick coronal sections (anterior to posterior) were generated and subsequently received the staining by using 2% 2,3,5-triphenyltetrazolium (TTC) solution under the temperature 37°C for 20 min. Subsequently, they received the xing process by using 4% paraformaldehyde for 24h. The images received the photographing, scanning, and investigation based on Image-Pro Plus 6.0 software system. Infarct volume had the expression of infarcted tissue percentage to overall brain tissues.

Western blot
At 24h when the reperfusion was completed, this study carried out euthanasia based on the overdose of chloral hydrate, and the infarct-side hippocampus received the rapid separation. The operating process was conducted on a cold surface, and cell metabolism was stopped with liquid nitrogen. Tissue homogenates received the treatment by using 100 RIPA lysis buffer (Beyotime, China), 10 phosphatase inhibitors (Roche, Germany), and 1 PMSF (Beyotime, China). The tissue extract received the thirty minute centrifugating process based on 12,000g at 4℃. This study carried out western blotting by complying with the general procedure. Next, the protein received the transfer in gel into polyvinylidene uoride membrane (Million, MA, the United States of America) in terms of Western blotting investigation. With the nishing of closure, this study carried out the incubating process for the sample with the use of primary antibody based on 4℃ through the complete night. The major antibodies: rabbit anti-NSUN2(1:1000, ab259941,abcam), rabbit anti-PI3K(1:1000, AF5112,a nity), rabbit anti-AKT(1:1000, 4685,CST), rabbit anti-P-AKT(1:1000,4060,CST), rabbit anti-ICAM-1(1:1000,ab179707,abcam), rabbit anti-GAPDH antibody (1:5000, AP0063, Bioworld, USA). When the incubation was based on the application of applicable horseradish peroxidase-conjugation secondary antibody, this study carried out the detection for antigenantibody reacting process by drawing upon Quantity One investigation software (BioRad, San Francisco, CA, USA) as well as optimized chemiluminescence mechanism.

qRT-PCR investigation
Brain tissue was extracted 24 h after MCAO, the extraction of overall RNA was achieved by adopting Trizol, speci c primers were designed, and the produced PCR reaction solution was arranged onto a Realtime PCR instrument for PCR ampli cation reaction. The reaction conditions are.

Immuno uorescence staining
The collection of brain was achieved 24h with the nishing of MCAO, the arrangement within 4% paraformaldehyde (4℃) for 4-6h, the movement into 15% sucrose solution (4℃) in the base and subsequently into 30% sucrose solution (4℃) till sinking. Brains received the embedment process to embedment agent at -20℃ and sectioned with a freezer (section thickness: 8µm).The sections received the freezing process and then were closed for 1 h with a blocking solution prepared from donkey serum, BSA and Triton. After anti-NSUN2 primary antibody, incubate overnight at 4℃. After PBST elution, add uorescent secondary antibody, incubate and elute, and add DAPI dye to restrain. Leica orthomosaic uorescence microscopy for observation. The primary antibody below was employed: rabbit anti-NSUN2(1:200,ab259941,abcam).

HE staining
This study adopted Hematoxylin and eosin (HE) staining for detecting pathological variations within the ischemic penumbra twenty-four h with the nishing of reperfusion. In brief, the mice received the anesthetizing process, and the brain received the xing through transcardial perfusion by employing 4% paraformaldehyde and 0.9% cold heparinized saline, the para n-embeding process, and sectioning process under 4 µm thickness in terms of HE staining, and Nissl staining. Subsequently, the section received the staining by employing HE, and HE-positive cell number within ischemic penumbra received the counting within ve distinctive aspects in terms of the respective section within a blinded process under a light microscope (BX51; Olympus, Tokyo, Japan).

Nissl staining
This study employed Nissl staining for observing neuronal morphologic variations within the ischemic penumbra 24h after reperfusion. The experiment procedure was rigorously used following the producer's Nissl staining tool manual (#G1432, Solarbio). An author kept from the treatment group determined the overall number of Nissl-positive neurons within the penumbra in 5 distinctive view eld in terms of the respective section under a light microscopy (BX51; Olympus, Tokyo, Japan).

Tunel staining
The mouse brain tissue was para n-embedded, 4µm slices were xed with paraformaldehyde, cleaned two times with PBS repeatedly, and stained with Tunel by complying with the Roche Tunel Fluorescent Staining Kit, and restrained with DAPI.A leica ortho-uorescence microscope was applied for observation. The nuclei were green for positive cells.

Statistical investigation
Data have the expression of mean±standard deviations, except for infarct size and survival, denoted as percentages. Statistical investigation was conducted with GraphPad Prism 8.0 (GraphPad, San Diego, CA). The Kolmogorov-Smirnov test was performed to perform the normal distribution test. One-way ANOVA (One-way ANOVA) was applied for the infarct area determination, Western blotting assay, with Bonferroni correction followed in terms of post hoc t-test. Wilcoxon signed rank sum test (Wilcox test) was employed for comparing neurological de cit score. Non-linear regression (nonlinear regression) was adopted to compare survival rates. This study gave statistical importance as P<0.05. Fig.1 Western blotting and qRT-PCR analyses showed that NSUN2 protein (Fig.1A) and mRNA (Fig.1B) expression noticeably increased in the ischemic half dark zone region of the brain at 24 h after ischemiareperfusion injury in the model group of mice compared with the sham-operated group. To gain insights into the variations in NSUN2 expression in mouse brain ischemia-reperfusion injury, we used immuno uorescence labeling of NSUN2 and found that the number of NSUN2 expression and uorescence intensity increased noticeably and localized in the nucleus (Fig.1C). Fig.2 To further clarify the effect of NSUN2 within cerebral ischemia-reperfusion injury of mice, we used Nsun2-KO mice to establish the MCAO model. In contrast to the MCAO group, neurological function scores increased within the MCAO-KO group, thereby demonstrating a further decrease in neuromotor function ( Fig.2A). In addition, the 7-day postoperative survival rate of mice in the MCAO-KO group signi cantly decreased (Fig.2B). TTC staining for measuring brain injury within three groups (Fig.2C). Fig.  2C illustrates that the MCAO-KO group had 44.20 ± 1.028% infarct volume. HE, Nissl and Tunel staining for assessing survival and neuronal damage within ischemic semidark zone after MCAO injury ( Fig.2D/E). According to Fig. 2D and 2E, cells positive after HE staining (HE+ ) achieved intact nucleolus, compact structure, and clear outline. Cells positive after Nissl staining (Nissl+ ) achieved intact neuron as well as ush cell body, whereas neuron with injury achieved shrunken cell body followed by pyknotic nuclei and Shrunken. Furthermore, according to Tunel staining, in the MCAO-KO group, the percentage of TUNEL-positive cell within the cerebral cortex further increased.

NSUN2 knockdown exacerbates ischemia-reperfusion injury in mouse brain
3.3 NUSN2 knockdown inhibits the expression of PI3K/AKT signaling channel and ICAM-1 in the ischemic half dark zone of mouse brain Figure 3 Next, we examined the effects of NUSN2 knockdown on PI3 K/AKT signaling channel and ICAM-1 expression in mice with cerebral ischemia-reperfusion injury. Western blotting results showed that the PI3K/P-AKT and ICAM-1 protein contents were signi cantly decreased in the MCAO-KO group compared with the MCAO group at 24h after cerebral ischemia-reperfusion injury in the ischemic half dark zone region of the cerebral cortex, indicating that PI3K/AKT signaling channel activity as well as ICAM-1 protein expression were signi cantly inhibited after NUSN2 knockdown (Fig. 3A).
3.4 NUSN2 knockdown promotes apoptosis in the ischemic hemispheric zone of mouse brain Figure 4 Tunel staining results (Fig. 2E) showed that apoptosis was increased. We then measured apoptosis-related proteins(clea-caspase3/caspase3/bcl-2/bax) in the ischemic hemispheric zone of the cerebral cortex 24 h after MCAO mapping by Western blotting. The results of this study showed that the ratio of apoptotic proteins clea-caspase3/caspase3 and bcl-2/bax noticeably increased in the MCAO-KO group compared with the MCAO group, indicating an increase in apoptosis in the ischemic semidark zone of the cerebral cortex 24h after MCAO modeling (Fig. 4A).

Discussion
This study initially investigated the effect of NSUN2 in cerebral ischemia-reperfusion injury. We used a mouse MCAO/R model to analyze the expression pattern and function of NSUN2, as well as the effects and potential mechanisms of cerebral ischemia-reperfusion injury by knocking down NSUN2.
NSUN2 refers to a member of the NOL1/ NOP2/SUN domain-containing protein family, primarily localized in the nucleus. NSun2 was reported to be able to modify some non-coding small RNAs besides tRNA.
NSun2 forms covalent intermediates with the cytosine of the target RNA through the cysteine in the molecule in order to activate the spent methylated pyrimidine loop, allowing SAM to make nucleophilic attack to pyrimidine C5, thereby forming m5C modi cation (Khoddami and Cairns,2013).Neurological phenotypes are often the primary manifestation of mutations affecting the tRNA regulome. The brain's translational machinery bears promise for psychiatric disease treatment(Aguilar-Valles et al.,2021;Fine et al.,2019).NSUN2 is a vital epitranscriptome regulator in mature neurons, and its expression and activity in mature neurons is critical for complex behaviors including synaptic transmission and emotional memory. De ciency of neuronal NSUN2 can cause impaired neurotransmitter transduction and synaptic signaling causing emotional behavioral variations (Blaze et al.,2021).NSUN2 expression is signi cantly reduced in equine tissues of Alzheimer's patients, thereby decreasing tRNA methylation and promoting the development of Alzheimer's disease (Wu et al.,2021). As revealed from the results here, NSUN2 knockdown signi cantly up-regulated neurobehavioral scores, brain infarct size and neuronal death, while decreasing survival in a mouse MCAO/R model. For this reason, this study hypothesized that NSUN2 is critical to cerebral ischemia-reperfusion injury in mice.
Next, the potential mechanisms of NSUN2 in cerebral ischemia-reperfusion injury was further explored. In this study, PI3K/P-AKT, ICAM-1 and bcl-2 levels were suggested to decrease signi cantly as well as clea-caspase3, and bax levels increased noticeably in the brain tissue of NSUN2 knockout mice model by WB investigation. The PI3K/AKT signaling channel is critical to regulating various different cellular functions (e.g., metabolism, growth, proliferation, survival, transcription and protein synthesis) (Manning and Toker,2017). After the occurrence of cerebral ischemia, the body can release some substances that can activate tyrosine kinase receptors (e.g., nerve cell growth factor and integrins) which can activate PI3K/AKT signal channel after phosphorylation of PI3K and AKT, and the effector molecules are then capable of exerting anti-apoptotic effects via apoptotic proteins (e.g., BCL-2 family proteins and Caspase family proteins) and apoptotic genes; also It can regulate mitochondria, endoplasmic reticulum, etc., thus exerting anti-apoptotic effects ( to-matrix contact and binding, thereby participating in cell signaling and activation, with low expression at rest and increased expression in response to stimuli (e.g., in ammatory mediators and oxidative stress) (Lawson and Wolf,2009). Moreover, it has been demonstrated that ICAM-1 is signi cantly correlated with cerebral infarction (Deddens et al.,2017). The study indicated that m5C methyltransferase NSun2 up-regulates the expression of cell adhesion molecule ICAM-1 at the translational level through mRNA methylation, thereby promoting vascular repair through adhesion of leukocytes to vascular endothelial cells. As revealed from the results here, NSUN2 knockdown signi cantly inhibits the expression of PI3K/p-Akt/ICAM-1 protein in the mouse MCAO/R model.
The shortcoming of this study is that the mechanism of the effect of NSUN2 on PI3K/p-Akt/ICAM-1 protein was not further explored.
In brief, the results of this study demonstrated that NSUN2 is critical to stroke in mice, and NSUN2 knockdown signi cantly increases stroke injury and decreases survival in mice, probably attributed to increased apoptosis attributed to NSUN2 knockdown, thereby inhibiting PI3K/Akt signaling channel and ICAM-1 expression.This study will expand the new mechanism of stroke, provide a scienti c theoretical basis for nding new therapeutic targets for stroke injury, and play an important role in reducing diseases in elderly patients and reducing public health pressure.

Declarations Data Availability Statements
All data generated or analysed during this study are included in this published article(and its supplementary information les).

Ethics approval
The study was approved by the Animal Experimentation Ethics Committee of Wenzhou Medical University (approval number wydw2021-0120).

Consent to participate
All participants provided their written informed consent to participate in this study. Content and procedures of written information consent were inspected thoroughly within the ethics approval procedure.

Consent for Publication
Not applicable Availability of data and materials

Supplementary Files
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