Transcriptomic profiling reveals sex-specific epigenetic dynamics involving kdm6b and H3K27 methylation in cerebral ischemia-induced neurogenesis and recovery

doi:10.21203/rs.3.rs-4907768/v1

Download PDF

Research Article

Transcriptomic profiling reveals sex-specific epigenetic dynamics involving kdm6b and H3K27 methylation in cerebral ischemia-induced neurogenesis and recovery

https://doi.org/10.21203/rs.3.rs-4907768/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Cerebral ischemic stroke ranks among the leading causes of death and disability worldwide. A significant challenge, beyond the lack of effective therapies, is the frequent oversight of sex as a vital factor in stroke research. This study focuses on elucidating the sex-specific epigenetic mechanisms that contribute to neural damage and recovery in cerebral ischemia.In our previously reported study, we demonstrated that following ischemia-induced cerebral artery occlusion (ICAO), female striatal tissue exhibited an early reinstatement of H3K9me2 marks on the promoters of inflammatory genes compared to male striatal tissue. This restoration led to a reduction in the expression of inflammatory cytokines, ultimately contributing to accelerated recovery in females. Building upon these findings, the current study aimed to investigate the unidentified molecular pathways responsible for the accelerated recovery observed in females. To explore this, we performed illumina-RNA sequencing on striatal tissues 24 hours post-ICAO. Interestingly, our analysis revealed differential regulation of H3K27me2 marks on the promoters of various neurogenic genes at an early stage, which facilitated early neurogenesis in the female striatum. This investigation identifies an epigenetic modulator, kdm6b/jmjd3, targeting H3K27, and delineates its sex-specific role in neural stem cell proliferation. The findings contribute to a comprehensive model linking gender-specific epigenetic regulation, neurogenesis, and post-ICAO recovery. In conclusion, the identified epigenetic modulators and their roles in neurogenesis offer potential targets for refined therapeutic interventions, emphasizing the importance of personalized and sex-specific considerations in stroke studies.

Cerebral ischemia

Sex-difference

Neurogenesis

kdm6b/jmjd3

Illumina-RNA sequencing

Cerebral ischemic stroke is a major neurovascular cause of disability with a high incidence of morbidity in surviving victims, caused by risk factors like, high BMI (Body mass index), smoking, hypertension, life style etc, which are also crucial in other cardiovascular and cerebrovascular diseases. The Global Burden of disease 2019 database, predicted on the basis of age standardized mortality rate and disability adjusted life, that ischemic stroke would be counted as major risk factor of death from 2020 to 2030 [1].Ischemia triggers a cascade that leads to membrane dysfunction, disrupting calcium influx and resulting in calcium-dependent excitotoxicity. This cascade leads to the release of reactive oxygen species and ultimately apoptosis. It is warranted to comprehend fully the molecular etiopathology of ischemic stroke risk, mortality, and functional loss for the development of novel therapies [2]. There remains a substantial requirement for the development of therapeutic medications focussing on neuroprotection in ischemic stroke. These medications should ideally aim at preventing brain injury before and during recanalization, extend the time window for effective intervention, and enhance overall functional recovery [3]. An unfathomable void in stroke research is that, despite gender differences in stroke risk factors, including those associated with atrial fibrillation, metabolic disorders, and age-associated perturbations in sex hormones, studies on stroke have not yet included sex as a significant variable. The outcomes of experimental in vivo and in vitro research reflect the significant influence of sex on clinical ischemic stroke.

Recent studies indicate disparity in ischemic stroke incidence, death and functional outcome in clinical populations [2, 4]. Clinically, there is a disproportionately high and rising female stroke population, which is distinguished from the male-biased usage of study animals. Stroke in women is largely understudied, and it's crucial to include both sexes in preclinical and clinical research that assesses prospective stroke treatments [5]. Therefore, it is essential to investigate and determine if women describe stroke symptoms differently. Over last four decades, in order to identify the mechanisms underlying cerebral ischemia and to develop novel stroke therapies, numerous animal stroke models have ben established. The cerebral vessels which are most often damagedby ischemic stroke in humans include the middle cerebral artery (MCA) and its branches, which account for a significant death rate. Since MCAo model closely simulates stroke condition in humans, most of the cellular and molecular level studies have been done in MCAo model till now [6]. In contrast, investigations using the MCAO model indicate that sensitivity to ischemic stroke varies among males and females. Interestingly, this distinction is linked to the impact of gonadal hormones rather than sex chromosome complement, challenging conventional assumptions and emphasizing the crucial role of biological factors in comprehending sex disparities in susceptibility to ischemic stroke [7].

25% of the total ischemic stroke incidents are caused by occlusion in internal carotid artery, where the patient shows up symptomatic to asymptomatic condition. We successfully established an internal carotid artery occlusion (ICAO) [8, 17] model in both males and females which induces mild to moderate levels of differential neural damage in males and females primarily in the striatal region and partly in the hippocampus and hypothalamic region [9]. Recent molecular studies employing mouse stroke models have revealed that epigenetic changes such as DNA methylation, histone lysine (K) acetylation and deacetylation are involved in ischemia-induced brain damage and recovery [10, 11]. There is growing evidence that histone modifications play crucial role in the pathophysiology of ischemic stroke, however most studies on this subject emphasize the involvement of histone acetylation. There is dearth of investigations on the significance of histone methylation [12]. We have reported previously that females have lower incidence of stroke and are more protected and recover faster from ischemia induced neural damage and a series of motor coordination behavioural paradigms and found that females recovered faster than males.

The assessment of behavioural functional outcome was accomplished through various tests, including the Neurodeficit score (NDS), Rotarod test, and grip strength measurement. Our findings demonstrated that ICAO significantly impaired motor coordination in both the sexes.Females exhibited early recovery from ICAO-induced impairment by day 7 post-ICAO, while males exhibited delayed recovery.Additionally, we examined the role of the histone demethylase kdm4b/jmjd2b and its target, H3K9me2 in the differential regulation of the neuroinflammatory response post-ICAO. Our novel study revealed the crucial role of the epigenetic modification, H3K9me2 and kdm4b/jmjd2b, which use H3K9 as a substrate, in the damage and recovery processes that occurs after stroke and reperfusion. Our molecular analysis suggests that H3K9me2 could be a potential therapeutic target, with sex or gender as an important factor to consider [9]. In the aftermath of ischemia, inflammation exhibits dualistic outcomes contingent upon the severity and temporal progression of the ischemic event. Initial inflammatory responses may exacerbate the injury caused by ischemia, whereas subsequent reactions at later stages can instigate mechanisms for recovery and repair. Our previous observations highlighted the presence of a time-dependent inflammatory response in both male and female subjects following ICAO [9]. Acute neuroinflammation has been shown to enhance neurogenesis and might promoteneuronal survival as well. The long-term survival of these newly formed neurons is a crucial element in post-stroke functional recovery [13].Our previous research also indicated that ICAO led unique vulnerabilities in mitochondria-associated brain regions, and differential regulation of processes such as autophagy and epigenetics, particularly involving H3K9me2 [9].

Expanding on these findings, we posit that the observed boost in neurogenesis in females, contributing to faster recovery, is mediated by specific epigenetic regulators. In order to investigate the differential recovery rates between females and males following ICAO, we employed a high-throughput transcriptomic approach using RNA-seq. Analysis of the data identified a group of genes that were differentially regulated in both sexes, influencing inflammation, neurogenesis, as well as histone lysine methyltransferases and demethylases.Interestingly, we observed early and distinct differential regulation of neurogenic markers in females. These markers were found to be regulated by a specific set of epigenetic regulators such as kdm6b/jmjd3, a demethylase targeting H3K27, along with its methyltransferases like ezh1 and ezh2. This suggests a potential molecular mechanism of epigenetic regulation that differs between females and males in response to ischemic stroke. H3K27-specific demethylase kdm6b/jmjd3 and few histone methylases like,ezh1 and ezh2 as the key stimulators of neurogenesis post-ICAO. kdm6b/jmjd3 is necessary for neural commitment when embryonic stem cells differentiate into NSCs and regulates the dynamics of H3K27me2 to govern key neurogenesis regulators and markers [14]. By studying the sex-specific roles of these epigenetic modulators, we aimed to establish a comprehensive understanding of how neurogenesis is differentially regulated in males and females following ICAO. This novel insight not only links back to our prior observations but suggests targeted therapeutic interventions to enhance post-ICAO recovery by manipulating controlled neurogenesis.

2.1 Animals and Housing

Adult male and female CD1 mice, bred and housed in the CCMB animal facility, were utilized for the entire study. The Institutional Animal Ethics Committee (20/GO/RBi/99/CPCSEA)granted approval for all animal procedures at the Centre for Cellular and Molecular Biology, Hyderabad, India. Adult male and female CD1 mice, aged 7 months, were utilized in the experiments. The mice were acclimated to standard experimental conditions, maintained at 24°C ± 2°C with a 12-hour light/dark cycle, and housed in individually ventilated cage (IVC) system. Food and water were available ad libitum to the animals.Following the habituation period, the animals were divided into sham and ICAO groups (n = 9 for each group), with both males and females included, which subsequently underwent SHAM or ICAO surgery respectively.

2.2 Surgical Procedure

Internal carotid artery occlusion (ICAO) was carried out following established procedures[9, 17].Briefly, adult CD1 mice of both sexes were first anesthetized using an intraperitoneal injection of a ketamine (100 mg/kg) and xylazine (10 mg/kg) mixture.The surgical site was prepared and a midline neck incision was made to expose the left common carotid arteryensuring separation from the vagus nerve. The external carotid artery was identified, separated, and permanently occluded with knots. The internal carotid artery (ICA) was then traced and occluded for 90 minutes near its bifurcation using a cotton thread. After the occlusion period, the cotton thread was removed to allow for reperfusion. The mice were then allowed to recover on a heating pad.Mice in the sham-operated group underwent the same surgical procedure, but the ICA was not occluded, serving as a control group for comparison.

2.3 Behavioral Assays

Behavioral assessments, including neuro-deficit evaluation, rotarod performance, and grip strength measurement (GSM), were conducted at various time points, specifically 1 day and 7 days following the ICAO procedure. To evaluate neuro-deficits, animals were subjected to a series of standardized tests to assess motor and sensory functions. Behavioral tracking and analysis were performed using the video tracking software Ethovision 3.1 (Noldus Information Technology, Leesburg, VA). This software allowed for precise monitoring and analysis of the mice’s movements and performance during these tasks. Data collected from these assessments were analyzed to determine the impact of the ICAO procedure on behavioral and motor functions over time.

2.3.1. Neurobehavioral Assessment

Mice subjected to internal carotid artery occlusion (ICAO) and subsequent reperfusions were evaluated for neurological deficits using the Neurological Deficit Score (NDS) at various time points post-procedure. Assessments were conducted at 1 day and 7 days after ICAO, following a modified version of established methods [9]. Briefly a score of 1 was given for mild symptoms such as eyelid ptosis or localized inflammation, while a score of 1.5 was used when both symptoms were present. A score of 2 represented reduced body bending or axial flexion away from the lesion side when the mouse was lifted by the tail, and a score of 2.5 was assigned for consistent bending or flexion in the same condition. A score of 3 combined the symptoms of scores 1/1.5 and 2/2.5. A score of 4 included the symptoms of score 3, with additional deficits like reduced resistance to lateral pushing, impaired gait towards the paretic side, or diminished gripping ability. A score of 5 indicated symptoms from score 3 plus pronounced circling towards the paretic side.

2.3.2. Grip strength Measurement

The Grip Strength Test (GST) is utilized to evaluate grip strength in rodents. This test was conducted at 1 day and 7 days post-ICAO using a grip strength meter (Ugo Basile, Italy) following established protocols [9]. During the test, the animals were placed in an inclined position on a mesh connected to a transducer. A gentle pulling force was applied to the animal to detach it from the mesh. The force exerted by the animal to resist this pull was measured in newtons (N). Each animal's grip strength was assessed through three separate measurements, and the average of these measurements was calculated to determine the forelimb grip strength of both experimental and sham-operated mice.

2.3.3. Rotarod Performance Test

The Rotarod performance test is a widely used behavioral assessment to evaluate motor coordination in rodents. In this study, the Rotarod apparatus (Ugo Basile, Italy) was employed to assess the motor function of experimental mice. Prior to the induction of internal carotid artery occlusion (ICAO), mice underwent a pre-training phase spanning 3 to 4 consecutive days to familiarize them with the equipment and protocol. During the acclimatization phase, mice were placed on the Rotarod, which was set at a constant speed for 3 to 5 minutes to help them adjust. Following this initial acclimatization, the training phase commenced. Over the course of 3 days, mice were subjected to 2 to 3 trials each day. During these trials, the Rotarod was set to accelerate from 4 to 40 RPM over a duration of 5 minutes. This training aimed to improve the mice's ability to remain on the rotating rod and to gauge their motor coordination and balance. After the ICAO procedure, the mice were tested at various time points, specifically on days 1 and 7 post-ICAO. Each test involved a single trial on the accelerated Rotarod, and the latency to fall was recorded for each animal. This measure provided insights into the motor coordination and balance of the mice following the surgical procedure.

After the completion of behavioral assays, microdissection of striatal regions was performed for further molecular studies, including quantitative polymerase chain reaction (qPCR), Western blot analysis, Illumina RNA sequencing and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). These brain tissues were collected to assess the molecular changes in response to ICAO and reperfusion.

2.4 Tissue collection and fixation

Twenty-four hours after the internal carotid artery occlusion (ICAO), the mice were humanely euthanized using cervical dislocation to prepare for neurobiological studies. Following euthanasia, the animals were dissected to isolate the striatal tissue. For accuracy in molecular investigations, specific regions of interest within the striatum were identified and carefully dissected using a stereomicroscope. The collected tissue samples were quickly frozen in liquid nitrogen and stored at -80°C to preserve them for subsequent analyses, including RNA sequencing, quantitative PCR (qPCR), chromatin immunoprecipitation qPCR (ChIP-qPCR), and Western blotting.For histological examination, the mice were anesthetized and underwent transcardial perfusion with cold phosphate-buffered saline (1×PBS) to clear blood from the brain. This was followed by fixation with 4% paraformaldehyde [8]. The fixed brains were then sectioned into 30 µm thick slices using a Leica cryostat from Germany. These brain slices were further processed according to the specific requirements of the histological analyses to be performed.

2.5 High-throughput RNA-Sequencing using Illumina platform

Striatal tissues were harvested from both male and female subjects, specifically from the ipsilateral cerebral hemisphere of the Internal Carotid Artery Occlusion (ICAO) group and the SHAM group, 1-Day post-procedure (n = 9 per group). The extraction of total RNA from the striatum was accomplished utilizing the mirVana™ RNA Isolation Kit (AM1560). RNA integrity was checked using Agilent 4200 TapeStation systems. Only samples with an RIN > 9.0 were selected. Quantification was performed using Qubit™ RNA High Sensitivity (HS) Assay Kit. RNA libraries were generated using the Illumina® TruSeq® Stranded Total RNA Library Prep workflow with Ribo-Zero Gold. Total RNA sequencing was performed on the IllumiaNovaSeq. An average of 40 million paired end reads were generated for each sample. Quality scores of the reads were checked using fastQC. All reads with an average Phred score of 28 or lesser were removed from the file using fastX trimmer. The paired end reads were aligned to the annotated mouse genome, Mus musculus GRCm39 procured from the NCBI database using GCF_000001635.27 as the annotation file. All reads were aligned using STAR aligner. The SAM files obtained were further quantified using Cuffdiff from the Tuxedo suite for sequencing analysis. Graphs were obtained using the CummeRbund package in R4.0.2This comprehensive process ensures the collection of high-quality RNA and subsequent generation of cDNA libraries for precise and reliable analysis, contributing to a comprehensive understanding of gene expression patterns in response to ischemia and under sham conditions.

2.6 Bioinformatic Analysis

The whole transcriptome RNA sequence data were analyzed using IPA (Ingenuity pathway analysis) software with p value < 0.05 and log₂FC > |1.2|. Using IPA, Disease function analysis, differentially expressed genes (DEGs) and crucial upstream regulators were analyzed post 24 hours of ICAO in both males and females. Followed by, pathway analysis through KEGG: Kyoto Encyclopedia of Genes and Genomes - http://www.genome.jp/kegg/, PANTHER - http://www.pantherdb.org/ and interaction network analysis by STRING - http://string-db.org/.The mutually expressed and exclusively expressed genes were analyzed and listed out using Venny 2.1.0.

2.7 Quantitative Polymerase Chain Reaction

To assess gene expression, total RNA was extracted from the striatum using TRIzol Reagent (Invitrogen, USA) following the manufacturer’s protocol. cDNA was then synthesized using the Thermo Fisher First Strand cDNA Synthesis Kit, adhering to the provided guidelines. Primer sequences used in the study are detailed in Supplementary Table 1. Quantitative real-time PCR (qPCR) was conducted in triplicate using the SYBR Green PCR Master Mix Detection System (Bio-Rad CFX 96). Gene expression analysis focused on genes relevant to focal cerebral ischemia, with RPL32 serving as the housekeeping gene. Data were processed using the ∆∆Ct method for relative quantification [9].

2.8 Western Blotting

Striatal tissue samples were homogenized in 8M Urea buffer to ensure thorough breakdown and maximum protein extraction. The homogenized samples were then subjected to sonication, which further enhanced protein yield by disrupting any remaining cellular structures. For protein separation, 40 µg of each protein sample was loaded into individual lanes of SDS-PAGE gels. Following electrophoresis, the proteins were transferred onto a PVDF membrane according to established protocols[9, 8]. The membrane was probed with a Rabbit Polyclonal H3 di-methyl K27 antibody (16 kDa) and normalized with alpha-tubulin (55 kDa). An anti-rabbit secondary antibody was used for detection.The resulting Western blots were analyzed using ImageJ software, where densitometry was performed to quantify protein band intensity. The intensity values were subsequently depicted as bar graphs for clear visualization and comparison.

2.9 Immunohistochemistry

The immunohistochemical assay was conducted following previously established methods (14). To assess cell proliferation, mice received an intraperitoneal injection of BrdU (50 mg/kg body weight) immediately after ICAO surgery. 24 hours post ICAO, the mice were perfused intracardially with 4% paraformaldehyde (PFA), and their brains were dissected and preserved in 30% sucrose. Coronal brain sections, 30 µm thick, were obtained from the striatum region and serially collected into six wells. From each well, 8 sections were randomly selected and mounted on positively charged glass slides. To evaluate neurogenesis post-ICAO in the striatal tissue, the neural stem cell marker Sox-2 (1:200) and the proliferation marker BrdU (1:200) were used. Following permeabilization, the sections were blocked with a solution containing 5% BSA and 5% normal horse serum (NHS) for 2 hours and then incubated overnight at 4°C with anti-Sox-2 and anti-BrdU antibodies. After washing, the sections were incubated with Cy5 (1:500) and FITC (1:500) secondary antibodies for 1 hour at room temperature. The sections were then mounted with DAPI, and images were captured using an Olympus laser scanning confocal system (FV-10i). Cell counting was performed using ImageJ software [16].

2.10 ChIP-qPCR

A Chromatin Immunoprecipitation (ChIP) assay was conducted following previously established protocols [9]. Briefly, cross-linked chromatin samples were collected from both ICAO and sham groups and sonicated until the chromatin fragments were reduced to a size between 200 and 250 base pairs, as demonstrated in Supplementary Fig. 3. This was accomplished through approximately 75 cycles of sonication, each consisting of 30 seconds on and 30 seconds off.Following sonication, 30 µg of chromatin from each sample was pre-cleared using Sure Beads Protein G Magnetic beads (161–4023, Bio-Rad). The pre-cleared chromatin was then incubated with an anti-rabbit H3K27me2 antibody (ab24684). After incubation, DNA was purified through the phenol-chloroform-isoamyl alcohol method following reverse cross-linking and a series of washes with various salt buffers. Finally, unique primers (listed in Supplementary Table 1) targeting the gene-specific 5′ region upstream of the transcription start site were utilized to assess the enrichment of the H3K27me2 histone mark.

2.11 Statistical analysis

In this study, various experimental techniques were employed with meticulous attention to statistical rigor and data robustness. Western blot and immunohistochemistry analyses were conducted with a sample size of 4 to 5 for each group. For quantitative polymerase chain reaction (qPCR) experiments, a larger sample size of 9 was used. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was performed with a sample size of 4. The data were analyzed using two-way ANOVA to assess statistical significance, with p-values calculated accordingly. Results were consistently presented as mean ± standard error of the mean (SEM), with error bars included where appropriate to indicate variability. Significance levels were set at p < 0.05, denoted as *, p < 0.01 as **, p < 0.001 as ***, and p < 0.0001 as ****. For RNA sequencing (RNA-seq), significance was defined by a fold change </> 1.2 and a p-value < 0.05. This comprehensive approach ensures the reliability and validity of the findings across diverse experimental methodologies.

3.1. Behavioral functional analysis shows faster recovery post-ICAO in females than males

Behavioral functional assessments were conducted using the Neurodeficit score (NDS), Rotarod test and grip strength measurement. The neurological deficit score (NDS, Fig. 1A) was assessed at multiple time points, specifically 1 day (1D) and 7 days (7D) after ICAO, by examining various phenotypic symptoms. Forelimb grip strength (Fig. 1B) was also evaluated at these time points, revealing a significant decrease in both male and female mice at 1D post-ICAO compared to the Sham group. In the Rotarod test (Fig. 2C), ICAO markedly impaired motor coordination in both sexes when compared to the Sham group. Notably, female mice exhibited a longer latency to fall at 7D post-ICAO compared to males. While female mice fully recovered from ICAO-induced motor impairments by 7D, male mice did not achieve complete recovery. Additionally, previous studies have provided detailed behavioral analyses, highlighting accelerated recovery mechanisms in females across various behavioral tests conducted at different time points[9]. Overall, our findings highlight that females recover faster than males from ICAO-induced neural damage.

3.2 Whole transcriptome profiling of the affected striatum revealed sex-specific differentially regulated genes and pathways post-ICAO

To understand the differential molecular mechanism in male and female brain regions affected post-ICAO and underlying early or faster recovery, animals were divided into four groups: Male Sham, Male ICAO, Female Sham, Female ICAO, comprising n = 9 in each group). RNA sequencing was done using Illumina platform, which was followed by the bioinformatic analysis. Validation of identified targets was performed using diverse molecular techniques (Fig. 2). High-throughput RNA sequencing analysis revealed distinct gene expression profiles in the affected striatum of male and female mice post-ICAO, with a total of 4747 genes differentially regulated in males and 4372 genes in females. These differences are graphically depicted through a volcano plot, showcasing genes with fold changes ≥ 1.2 and p-values < 0.05 (Supplementary Fig. 1A, B).

Ingenuity Pathway Analysis (IPA) software was employed for disease function annotation, providing insights into the predicted activation states, activation z-scores, p-values, and molecules associated with each disease function category, and the top 20 differentially regulated signalling pathways were identified (Supplementary Fig. 2). Heatmaps were constructed based on z-scores to visualize the differential regulation of genes in males (Fig. 3A) and females (Fig. 3B) across various disease functions. Among the DEGs, 2087 genes in males and 1956 genes in females exhibited upregulation, while 2660 genes in males and 2416 genes in females showed downregulation (Fig. 3C, D).Notably, the top differentially regulated signalling pathways differed between males and females. Utilizing Venny 2.1.0-BioinfoGP, a comparative analysis revealed that 2081 genes in males and 1725 genes in females were uniquely regulated, while 2595 genes exhibited mutual regulation in both males and females (Fig. 3E). Further, Using IPA analysis, we predicted distinct expression patterns of transcriptional regulators and pathways specific to males and females indicating if they are in activated or inhibited state post-ICAO (Fig. 4).

3.3 Sex specific differential regulation of neurogenesis in the affected striatum post-ICAO influences differential rate of recovery

Following ICAO, we observed a marked disparity in the rate of behavioural recovery with females exhibiting accelerated recovery compared to males. To elucidate the underlying molecular mechanism in this sex-specific differential recovery, the transcriptome data was analysed and intriguingly, we identified a specific subset of neurogenic markers showing significant upregulation in females at this time point compared to males. Given the potential importance of induced neurogenesis in promoting recovery post-ICAO, we proceeded to validate the expression profiles of the key neurogenic genes using quantitative polymerase chain reaction (qPCR). The data suggest significant level of elevation in the expression of neurogenic genes NeuroD1, Sox-2, and DCX in female striatum compared to that in male striatum (Fig. 5: B, C, D).

3.4 Immunohistochemistry analysis revealed sex specific difference in BrdU and sox-2 positive cell count post-ICAO

To further substantiate our understanding of the sex-specific modulation of neurogenesis, we employed supplementary investigative approaches. Specifically, we administered bromodeoxyuridine (BrdU) at a dose of 50mg/kg to both male and female subjects, as illustrated in Fig. 5: A. Following this, we quantified the populations of BrdU- and Sox-2-positive cells within the subventricular zone (SVZ) of the striatum 24 hours post-induced post-ICAO(Fig. 6).Consistent with our expectations, our analysis unveiled a notable augmentation in the counts of both BrdU- and Sox-2-positive cells in females in comparison to males (Fig. 7: A-D). These findings provide robust supplementary evidence reinforcing the heightened neurogenic response observed in females following 24 hours of ICAO in the transcriptomic study. The increased numbers of BrdU- and Sox-2-positive cells in the female cohort underscore the existence of distinct sex-specific differences in the neurogenic reactions elicited by ICAO.

3.5 Interaction network analysis revealed sex-specific epigenetic regulatory mechanism in triggering neurogenesis post-ICAO

Interaction network analysis by STRING (http://string-db.org/) for DEGs from both males and females revealed the interaction of a few inflammatory genes, histone lysine demethylases (KDMs) and methylases (KMTs) with few neurogenic genes and its isoforms (Fig. 8: A, B), and enlisted the differentially regulated genes in both males and females (Fig. 9). The detail analysis showed sex specific differential levels of expression of H3K27me2 specific kdm6b, a hallmark histone lysine demethyalse that controls neurogenesis and neural stem cell migration [14, 15]. Interestingly, we could find that the expression of kdm6b specific to H3K27me2 was significantly upregulated in females when compared with males post 24 hrs of ICAO (Fig. 10A). As expected, the histone lysine methylases specific to H3K27me2 such as ezh2 and ezh1 were significantly downregulated when compared with males (Fig. 10: B, D). Apparently, kdm6a remained unchanged in both males and females 24 hrs post-ICAO (Fig. 10C). Hence, here we confirmed that the differential regulation of neurogenesis in males and females is through H3K27me2 specific kdm6b/jmjd3, ezh2 and ezh1. To confirm the same, protein level expression pattern of H3K27me2 showed significant upregulation in males where females didn’t show any significant changes (Fig. 10: E, F).

3.6 Differential levels of transcriptional activation of sox-2 and dcx in male and female striatum is through altered H3K27me2 occupancy on their promoters

Twenty-four hours after ICAO, male subjects displayed a significant increase in the transcriptionally repressive epigenetic marker H3K27me2 in the striatum. In contrast, females exhibited a notable reduction in H3K27me2 occupancy at the promoters of neurogenic markers. To quantify H3K27 methylation levels at the promoter regions of neurogenic genes such as sox-2 and dcx, we conducted a chromatin immunoprecipitation (ChIP) assay using an H3K27me2-specific antibody (ab 24684) on pooled striatal tissue samples from males and females at 24 hours post-ICAO and sham controls. Interestingly, ChIP-qPCR results indicated a significant decrease in the occupancy of H3K27me2 on the sox-2 and dcx promoters in females, unlike males in the striatum of ICAO group when compared with sham controls. These results confirm the boost in neurogenesis post-ICAO and an accelerated recovery mechanism in females than males (Fig. 11: A, B).

Cerebral ischemic stroke is a pressing global health concern, and our study investigated the molecular mechanisms including the epigenetic ones, underlying the recovery following ICAO in the mouse model, focusing on sex-specific differences. Roughly 80% of strokes arise from ischemia, marked by thromboembolic blockage in a major cerebral artery or its branches. This vascular occlusion triggers a series of events known as the ischemic cascade, involving oxygen and energy deprivation, the generation of reactive oxygen species, glutamate release, intracellular calcium build up, and initiation of inflammatory processes, ultimately leading to irreversible tissue damage or infarction. Within this process, the ischemic penumbra, the region surrounding the infarcted core, holds potential for salvage if timely intervention is applied within a defined therapeutic window [18]. Despite efforts, preclinical neuroprotective agents have had limited success in translation to effective stroke therapies, highlighting the importance of choosing appropriate animal stroke models for successful preclinical research. To investigate the pathophysiology and molecular mechanisms of post-stroke cerebral infarction several models to induce cerebral ischemic stroke have been established utilising rodents [26, 27, 28]. These models offer reproducibility and standardization, allowing precise analysis of stroke pathophysiology and drug effects. Despite the abundance of animal models, including widely used ones like MCAo [8, 9, 17], there remains a gap in the availability of a suitable model for transient ischemic attacks (TIAs) or acute strokes with mild to moderate symptoms. Our model accurately mimics mild to moderate strokes in humans, where symptoms may be symptomatic or asymptomatic, addresses this limitation. Several clinical studies have shown that internal carotid artery occlusion is responsible for a significant proportion of stroke cases, estimated between 13% and 25%. This condition, which causes cerebral ischemia, can be present with or without symptoms [19–23].

Stroke-induced neurogenesis stands out as a promising therapeutic target, providing an opportunity for the brain to undergo rewiring and rejuvenation, thereby facilitating recovery from ischemic damage [24]. Experimental evidence suggests that the damaged adult brain post-stroke, attempts self-repair by generating new neurons, even in regions where neurogenesis doesn't typically occur. The functional outcome of stroke-induced neurogenesis and the integration of new neurons into existing neural circuits are not well-understood. To significantly induce stroke recovery, it is crucial to enhance this self-repair mechanism, focusing on improving the survival and differentiation of the generated neuroblasts [25].From our previous findings, females exhibited a significantly faster recovery post-ICAO compared to males, as evidenced by behavioural tests, including the NDS, grip strength measurement, open field test and the Rotarod test. This finding is consistent with earlier research demonstrating the neuroprotective benefits of female sex hormones, particularly estrogen, and their influence on post-stroke recovery [29]. Epigenetic modifications are pivotal in regulating neuronal gene expression and are implicated in neurodegenerative disorders, including damage induced by ischemia. Replication, transcription, repair, and recombination are basic processes that are regulated by chromatin dynamics, which preserves genomic stability [30].

In our previous investigation; we observed that following ischemia, there was an early restoration of H3K9me2 marks on inflammatory gene promoters in the female striatum compared to the male brain. This rapid restoration was associated with reduced expression of inflammatory cytokines, facilitating a quicker recovery in females. Importantly, inflammatory response initiation in females was evident at 6 hours, characterized by H3K9me2 downregulation, leading to accelerated recovery compared to males. These distinct patterns of H3K9me2 regulation post-ischemia highlight sex-specific differences in the epigenetic response to cerebral ischemia. Our transcriptomic analysis in this study revealed previously unidentified genes and pathways that are differentially regulated between males and females. This approach enabled us to uncover molecular mechanisms involving epigenetic regulation of neurogenesis that contribute to the accelerated recovery observed in females.Specifically, we identified a group of KDMs, KMTs, and miRNAs that were uniquely and reciprocally regulated in males and females. These findings potentially explain the sex-specific variations in the regulation of damage and repair following ICAO (Supplementary Fig. 4).

To understand the molecular basis of these differences, our whole transcriptome profiling led us to reveal hundreds of differentially regulated genes in both sexes post-ICAO. These findings emphasize the complexity of the molecular response to ischemic attack and suggest potential sex-specific therapeutic strategies. Our investigation unveiled a significant upregulation of neurogenic markers in females compared to males, indicating that enhanced neurogenesis contributes to the observed faster recovery in females. Epigenetic factors played a crucial role, with H3K27me2-specific kdm6b/jmjd3 emerging as a key player in controlling neurogenesis, upregulated in females, while methylases specific to H3K27me2 were downregulated. Interestingly, the analysis of transcriptomic dataset obtained from zebrafish brain following an acute hypoxic insult yielded similar result; lysine demethylases of different classes were found dysregulated, including the kdm6ba and kdm6bb. The change in kdm6 regulation in zebrafish model was also associated with altered neurogenesis markers (unpublished results). The altered epigenetic landscape further highlights the influence of epigenetic regulation on recovery.

Further, High-throughput RNA sequencing analysis revealed pronounced sex-specific differences in gene expression profiles following induced cerebral ischemia (ICAO). Specifically, 4747 genes exhibited differential regulation in males, while 4372 genes were identified in females, as visualized in a volcano plot depicting genes with fold changes ≥ 1.2 and p-values < 0.05. Experimental samples, distributed among sham and ICAO groups for both sexes (n = 9/group), underwent categorization for subsequent transcriptome assembly. Ingenuity Pathway Analysis (IPA) facilitated a nuanced exploration of predicted activation states and signalling pathways, thereby elucidating sex-specific molecular responses and signalling pathway disparities in the context of cerebral ischemia. The investigation extended to the realm of neurogenesis, where a substantial sex-specific variation in recovery rates post-ICAO was observed. This prompted an in-depth analysis of high-throughput data, uncovering a subset of neurogenic markers significantly upregulated in females 24 hours post-ICAO compared to males. Validation through quantitative polymerase chain reaction (q-PCR) confirmed the overexpression of neurogenic genes, including neuro d1, sox-2, and dcx, in females. Immunohistochemistry analysis further substantiated these findings, as bromodeoxyuridine (BrdU) administration revealed a significant augmentation in both BrdU and sox-2 positive cell populations within the subventricular zone (SVZ) of the striatum in females compared to males post-ICAO. This supplementary evidence highlights the heightened neurogenic activity in females following cerebral ischemia. In light of this finding, we have uncovered the differences in the regulation and triggers of neurogenesis between males and females in the hippocampal dentate gyrus as it holds yet another neurogenic niche in brain, following ICAO. Interestingly, in females, neurogenesis appears to be triggered at multiple time points (6 hours, 1 day, and 5 days post-ICAO), whereas in males the trigger is only observed at 1-day post-ICAO. This discrepancy in neurogenic response appears to contribute to the accelerated recovery rate observed in females [9].

Interaction pathway analysis using STRING highlighted sex-specific epigenetic regulation in triggering neurogenesis post-ICAO. Histone lysine demethylase kdm6b, a crucial player in controlling neurogenesis, exhibited significant upregulation in female post-ICAO, in contrast to males. This was complemented by the downregulation of histone lysine methylases ezh2 and ezh1 specific to H3K27me2, further emphasizing the role of epigenetic modulation in the observed sex-specific neurogenic responses. The exploration extended to the protein level, confirming a significant upregulation of H3K27me2 in males, while females exhibited no significant changes. Transcriptional activation analysis elucidated a differential regulation in males and females through altered H3K27me2 occupancy on the promoters of neurogenic genes, including sox-2 and dcx. Males displayed an increase in the transcriptionally repressive epigenetic marker, H3K27me2, whereas females exhibited a significant decrease in H3K27me2 occupancy. In summation, these findings highlight the nuanced sex-specific responses in the aftermath of cerebral ischemia, shedding light on potential therapeutic avenues targeting controlled neurogenesis for enhanced post-ICAO recovery.

The outcome of this study suggests that the internal carotid artery occlusion (ICAO)-induced mild to moderate ischemic damage to striatal region that led to distinctneurological deficits in CD1 mice was associated with dysregulation of thousands of genes, as revealed by high throughput RNA sequencing analysis. The differential rate of recovery in male and female animals is due to an alteration in the timing of neurogenesis post-ICAO, and is regulated by a few key epigenetic players such as kdm6b/jmjd3,ehz1 and ezh2. In conclusion, our study provides comprehensive insights into sex-specific differential rate of recovery post-ICAO, paving the way for better treatment strategies in ischemic stroke by targeting neurogenesis. This study contributes valuable knowledge towards a more tailored and effective approach to stroke treatment, recognizing the significance of sex-specific factors in shaping recovery outcomes.

Acknowledgments

The authors extend their sincere gratitude to B. Jyothi Lakshmi for her diligent care and maintenance of the animals throughout the study. They also wish to thank the sequencing facility at the Centre for Cellular and Molecular Biology (CCMB) in Hyderabad for their support. The KIM Department at CSIR-IICT is also acknowledged for providing the institutional publication number IICT/Pubs./2024/237.

CRediT authorship contribution statement

MR was involved in conceptualization, data curation, formal analysis, writing, original draft preparation and graphical editing. AU, SP and PS were involved in data curation, formal analysis, writing, graphical editing and original draft preparation. AK and SC participated in data curation, formal analysis, conceptualisation, funding acquisition, supervision, writing, reviewing and editing.

Funding

This study was originally funded by the Council of Scientific and Industrial Research (CSIR), India, under the network projects [(BSC0103-UNDO) to SC and AK], and later received additional support from the Indian Council of Medical Research (ICMR) grant [5/4-5/3/17/Neuro/2022-NCD-1 to SC]. MR, AU, and SP would like to express their gratitude to CSIR India for providing their doctoral fellowships.

Declaration of Competing Interest

The authors declare that they have no competing interests.

Data availability

Experimental data will be made available on request to corresponding author.Biosample and SRA metadata are available at https://dataview.ncbi.nlm.nih.gov/object/PRJNA1129570?reviewer=cuciaqsrdpnusg62v15haeuq91

Wafa, H. A., Charles, D. A., Wolfe, E., Emmett, G. A., Roth, C. O., Johnson, & Wang, Y. (2020). Burden of stroke in Europe: thirty-year projections of incidence, prevalence, deaths, and disability-adjusted life years. Stroke 51, no. 8 : 2418–2427.
George, P. M., & Gary, K. (2015). Steinberg. Novel stroke therapeutics: unraveling stroke pathophysiology and its impact on clinical treatments. Neuron 87, no. 2 : 297–309.
Rha, J. H. (2007). and Jeffrey L. Saver. The impact of recanalization on ischemic stroke outcome: a meta-analysis. stroke 38, no. 3 : 967–973.
Reeves, M. J., Cheryl, D., Bushnell, G., Howard, J. W., Gargano, P. W., & Duncan (2008). Gwen Lynch, Arya Khatiwoda, and Lynda Lisabeth. Sex differences in stroke: epidemiology, clinical presentation, medical care, and outcomes. The Lancet Neurology, 7(10), 915–926.
Ahnstedt, H., & McCullough, L. D. (2016). Cipolla. The importance of considering sex differences in translational stroke research. Translational stroke research, 7, 261–273.
Fluri, F., Schuhmann, M. K., & Kleinschnitz, C. (2015). Animal models of ischemic stroke and their application in clinical research. Drug design development and therapy : 3445–3454.
Manwani, B., Bentivegna, K., Benashski, S. E., Venna, V. R., Xu, Y., Arnold, A. P., & Louise, D. (2015). McCullough. Sex differences in ischemic stroke sensitivity are influenced by gonadal hormones, not by sex chromosome complement. Journal of Cerebral Blood Flow & Metabolism, 35(2), 221–229.
Chakravarty, S., Jhelum, P., Bhat, U. A., Rajan, W. D., Maitra, S., Pathak, S. S., Patel, A. B., & Arvind Kumar. (2017). Insights into the epigenetic mechanisms involving histone lysine methylation and demethylation in ischemia induced damage and repair has therapeutic implication. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1863(1), 152–164.
Mydhili Radhakrishnan, V., Vijay, B., Supraja Acharya, P., Basuthakur, S., Patel, K., Soren, A., & Kumar (2024). Uncovering Sex-Specific Epigenetic Regulatory Mechanism Involving H3k9me2 in Neural Inflammation, Damage, and Recovery in the Internal Carotid Artery Occlusion Mouse Model. NeuroMolecular Medicine, 26(1), 3.
Schweizer, S., Meisel, A., & Märschenz, S. (2013). Epigenetic mechanisms in cerebral ischemia. Journal of Cerebral Blood Flow & Metabolism, 33(9), 1335–1346.
Uzdensky, A. B., & Demyanenko, S. V. (2019). Epigenetic mechanisms of ischemic stroke. Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology, 13, 289–300.
Ng, G. Y. Q., Lim, Y. A., & Sobey, C. G. (2018). Thameem Dheen, David Yang-Wei Fann, and Thiruma V. Arumugam. Epigenetic regulation of inflammation in stroke. Therapeutic advances in neurological disorders, 11, 1756286418771815.
Rahman, A. A., Amruta, N., Pinteaux, E., & Gregory, J. (2021). Bix. Neurogenesis after stroke: a therapeutic perspective. Translational stroke research, 12(1), 1–14.
Ding, Y., Yao, Y., Gong, X., Zhuo, Q., Chen, J., Tian, M., & Farzaneh, M. (2021). JMJD3: a critical epigenetic regulator in stem cell fate. Cell Communication and Signaling, 19(1), 1–9.
Wijayatunge, R. (2012). The H3K27 Histone Demethylase Kdm6b (Jmjd3) is Induced by Neuronal Activity and Contributes to Neuronal Survival and Differentiation. PhD diss.
Das, T., Soren, K., Yerasi, M., Kumar, A., & Chakravarty, S. (2019). Revealing sex-specific molecular changes in hypoxia-ischemia induced neural damage and subsequent recovery using zebrafish model. Neuroscience Letters, 712, 134492.
Jhelum, P., Radhakrishnan, M., Paul, A. R. S., Dey, S. K., Kamle, A., Kumar, A., Sharma, A., & Chakravarty, S. (2022). Neuroprotective and Proneurogenic Effects of Glucosamine in an Internal Carotid Artery Occlusion Model of Ischemia. NeuroMolecular Medicine : 1–6.
Fluri, F., Schuhmann, M. K., & Kleinschnitz, C. (2015). Animal models of ischemic stroke and their application in clinical research. Drug design development and therapy : 3445–3454.
Flaherty, M. L., Flemming, K. D., McClelland, R., Jorgensen, N. W., & Brown, R. D. Jr. (2004). Population-based study of symptomatic internal carotid artery occlusion: incidence and long-term follow-up. Stroke; a journal of cerebral circulation, 35(8), e349–352.
Fisher, M. (1954). Occlusion of the carotid arteries: further experiences. AMA archives of neurology and psychiatry, 72(2), 187–204.
Mead, G. E., Shingler, H., Farrell, A., O'Neill, P. A., & McCollum, C. N. (1998). Carotid disease in acute stroke. Age and ageing, 27(6), 677–682.
Thanvi, B., & Robinson, T. (2007). Complete occlusion of extracranial internal carotid artery: clinical features, pathophysiology, diagnosis and management. Postgraduate Medical Journal, 83(976), 95–99.
Thiele, B. L., Young, J. V., Chikos, P. M., Hirsch, J. H., & Strandness, D. E. Jr. (1980). Correlation of arteriographic findings and symptoms in cerebrovascular disease. Neurology, 30(10), 1041–1046.
Rahman, A. A., Amruta, N., Pinteaux, E., & Gregory, J. (2021). Bix. Neurogenesis after stroke: a therapeutic perspective. Translational stroke research, 12(1), 1–14.
Lindvall, O., & Kokaia, Z. (2015). Neurogenesis following stroke affecting the adult brain. Cold Spring Harbor perspectives in biology, 7(11), a019034.
Stenzel-Poore, M. P., Stevens, S. L., King, J. S., & Roger, P. (2007). Simon. Preconditioning reprograms the response to ischemic injury and primes the emergence of unique endogenous neuroprotective phenotypes: a speculative synthesis. Stroke 38, no. 2 : 680–685.
McAuley, M. A. (1995). Rodent models of focal ischemia. Cerebrovascular and brain metabolism reviews 7, no. 2 : 153–180. [6] Mhairi, Macrae I. New models of focal cerebral ischaemia. British journal of clinical pharmacology 34, no. 4 (1992): 302–308.
Leng, X., Fang, H., Leung, T. W. H., Mao, C., Miao, Z., Liu, L., Wong, K. S., & David, S. (2016). Liebeskind. Impact of collaterals on the efficacy and safety of endovascular treatment in acute ischaemic stroke: a systematic review and meta-analysis. Journal of Neurology Neurosurgery & Psychiatry, 87(5), 537–544.
Amanollahi, M., Jameie, M., Heidari, A., & Nima Rezaei. (2023). and. The dialogue between neuroinflammation and adult neurogenesis: mechanisms involved and alterations in neurological diseases. Molecular neurobiology 60, no. 2 : 923–959.
Patel, S., Vijay, V., Kumar, A., & Chakravarty, S. (2023). Epigenetics of Altered Circadian and Sleep Cycle Induced Effects on Aging and Longevity. Sleep and Clocks in Aging and Longevity (pp. 363–390). Springer International Publishing.

No competing interests reported.

Download PDF

Editorial decision: Revision requested
19 Sep, 2024
Reviews received at journal
18 Sep, 2024
Reviewers agreed at journal
01 Sep, 2024
Reviews received at journal
27 Aug, 2024
Reviewers agreed at journal
19 Aug, 2024
Reviewers invited by journal
16 Aug, 2024
Editor assigned by journal
15 Aug, 2024
Submission checks completed at journal
15 Aug, 2024
First submitted to journal
13 Aug, 2024

You are reading this latest preprint version

Transcriptomic profiling reveals sex-specific epigenetic dynamics involving kdm6b and H3K27 methylation in cerebral ischemia-induced neurogenesis and recovery

Status:

Version 1

Abstract

Figures

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1 Animals and Housing

2.2 Surgical Procedure

2.3 Behavioral Assays

2.3.1. Neurobehavioral Assessment

2.3.2. Grip strength Measurement

2.3.3. Rotarod Performance Test

2.4 Tissue collection and fixation

2.5 High-throughput RNA-Sequencing using Illumina platform

2.6 Bioinformatic Analysis

2.7 Quantitative Polymerase Chain Reaction

2.8 Western Blotting

2.9 Immunohistochemistry

2.10 ChIP-qPCR

2.11 Statistical analysis

3. RESULTS

3.1. Behavioral functional analysis shows faster recovery post-ICAO in females than males

3.2 Whole transcriptome profiling of the affected striatum revealed sex-specific differentially regulated genes and pathways post-ICAO

3.4 Immunohistochemistry analysis revealed sex specific difference in BrdU and sox-2 positive cell count post-ICAO

3.5 Interaction network analysis revealed sex-specific epigenetic regulatory mechanism in triggering neurogenesis post-ICAO

4. DISCUSSION

5. CONCLUSION

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1