WITHDRAWN: CPEB3 can regulate seizure susceptibility by inhibiting the transcriptional activity of STAT3 on NMDARs expression

doi:10.21203/rs.3.rs-4818920/v1

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WITHDRAWN: CPEB3 can regulate seizure susceptibility by inhibiting the transcriptional activity of STAT3 on NMDARs expression

https://doi.org/10.21203/rs.3.rs-4818920/v1

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Background

The pathogenesis of epilepsy is complex, and current antiepileptic drugs do not effectively control the seizures. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) regulates neuronal excitability, but its mechanism of action in epilepsy is not clear. In this paper, we investigated the effect of CPEB3 on seizures and elucidated its underlying molecular mechanism.

Methods

Bioinformatics-based search for genes closely associated with epilepsy. Changes in expression and cellular localization of CPEB3 in epilepsy were verified by Western blotting (WB) and Immunofluorescence staining. Subsequently, The adeno-associated virus was employed to overexpress or Knockdown in mice. Behavioral experiments verified the effect of CPEB3 on epileptic phenotype, and the molecular mechanism of CPEB3 affecting epileptic phenotype was explored by WB, Real-time quantitative polymerase chain reaction (RT-qPCR), RNA immunoprecipitation (RIP), and Chromatin immunoprecipitation (CHIP).

Results

The results were that CPEB3 was downregulated epilepsy in model mice and patients with temporal lobe epilepsy and co-expressed with neurons. Behavioral experiments have shown that CPEB3 negatively regulates seizure susceptibility and excitability. In addition, CPEB3 can also bind to the mRNA of signal transducer and activator of transcription 3 (STAT3) and inhibit its translation, resulting in lower levels of STAT3 and p-STAT3, reduced nuclear translocation of STAT3, and decreased STAT3-mediated transcriptional activity of GluN1, GluN2A, and GluN2B, suppressing the expression of NMDAR subunits and attenuating epilepsy phenotype.

Conclusion

These findings confirm that CPEB3 can alter the excitability and susceptibility of epilepsy by inhibiting the translation of STAT3 and inhibiting its transcription to NMDAR. These results provide new ideas and therapeutic targets to treat epilepsy.

epilepsy

CPEB3

STAT3

transcription factor

NMDARs

Epilepsy is a complex neurological disorder characterized by spontaneous recurrent seizures(Devinsky et al. 2018; Gan et al. 2021). Approximately 70 million people worldwide suffer from epilepsy of varying degrees(Thijs et al. 2019). Patients with epilepsy suffer severe physical and psychological consequences, which further burden families and society financially(Shlobin et al. 2022). Although more than a dozen antiepileptic drugs commonly used in clinics can control the symptoms of epilepsy, 30% of patients still show drug resistance, which is most common in temporal lobe epilepsy (TLE)(Qin et al. 2022). Currently, there is a lack of an effective drug to inhibit the onset or progression of epilepsy. Therefore, exploring new effective therapeutic targets for TLE is urgently needed.

RNA-binding proteins (RBPs) are important proteins that interact with RNA targets and are widely involved in various post-transcriptional regulatory processes, including splicing, localization, degradation, modification, and translation of cellular RNAs, thereby regulating specific gene expression(Liu et al. 2020; Xiao et al. 2024; Zhou et al. 2024). The cytoplasmic polyadenylation element-binding proteins (CPEBs) are a key family of RBPs(Chao et al. 2013; Mendez et al. 2001). Four family members of CPEB have been identified: CPEB1, CPEB2, CPEB3, and CPEB4. CPEB3 is abundantly expressed in the central nervous system(Huang et al. 2006; Morgan et al. 2010; Theis et al. 2003). Studies have shown that CPEB3 regulates neuron-specific selective splicing and neurogenesis gene expression(Qu et al. 2020). In the current study, CPEB3 can mediate the alteration in neuronal excitatory(Hwang et al. 2022). Altered neural excitability is an important factor contributing to seizures(Casillas-Espinosa et al. 2012; Ke et al. 2023). This suggests that CPEB3 may be closely related to epilepsy.

Activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is observed in several central nervous system disorders(such as epilepsy, stroke, Alzheimer's disease, and Parkinson's disease)(Han et al. 2020; He et al. 2020; Hristova et al. 2016; Li et al. 2021; Toral-Rios et al. 2020). Research has demonstrated that the JAK/STAT pathway is involved in epileptic progression and remodeling of synaptic plasticity in hippocampal neurons(Nicolas et al. 2012). Activator of signal transducer and activator of transcription 3 (STAT3) is a key factor in the JAK/STAT pathway. It is also a member of the mammalian signal converter and activator of the transcription family that plays an important role in signal transduction(Kisseleva et al. 2002; Li et al. 2024; Xu et al. 2023). When STAT3 is activated, it can increase phosphorylated STAT3(p-STAT3) expression, inducing STAT3 to enter the nucleus and bind to the promoter of the target gene(Levy et al. 2002; Yu et al. 2014). Recently, it has been reported that the inhibition phosphorylation of STAT3 affects the level of gene transcription of the N-methyl-D-aspartic acid receptors (NMDARs), altering the synaptic plasticity of neurons(Hong et al. 2020).

In this study, we identified a key mechanism by which CPEB3 plays a role in TLE. Interestingly, we used bioinformatics to discover that CPEB3 may be closely linked to epilepsy. Subsequently, we confirmed that CPEB3 was downregulated in epilepsy mice models and patients with TLE. CPEB3 negatively regulated seizure severity and susceptibility in epileptic mice. Further mechanistic studies showed that CPEB3 binds to STAT3 mRNA and inhibits its translation, changing the nuclear translocation of STAT3, reducing STAT3-mediated transcription of GluN1, GluN2A, and GluN2B, down-regulating expression of NMDARs and reducing seizure severity and susceptibility in epileptic mice. These results provide new ideas and therapeutic targets to treat epilepsy.

Human brain tissue

This study was approved by the Ethics Committee of the Second Hospital of Harbin Medical University and was conducted per the Declaration of Helsinki (Approval Number:KY2024-036). In this study, the human brain tissue samples were collected from the Second Hospital of Harbin Medical University, and written informed consent was obtained from the patients. Tissue samples were collected from 12 patients. These tissues were obtained from patients with TLE and patients with traumatic brain injury (TBI). All tissue specimens were stored in liquid nitrogen or 4% paraformaldehyde solution immediately after sampling until use. The clinical characteristics of the patients were shown in Table S1.

Animals

In this study, 6–8 weeks C57BL/6J male mice were purchased from the Animal Laboratory Center of the Second Affiliated Hospital of Harbin Medical University. The mice were randomly assigned to the experimental group. All mice were kept in a constant temperature and humidity environment. All animal experiments were approved by the Ethics Committee Second Affiliated Hospital of Harbin Medical University (Approval Number:KY2018-108).

Bioinformatics analysis

Briefly, bioinformatics analysis includes Gene Expression Omnibus (GEO) dataset collection, extraction of RBPs-related genes, differential expression analysis, screening of Hub genes, and construction of diagnostic model and single-cell analysis. The detailed experimental steps can be seen Supplementary document.

Kainic acid (KA) model

Mice were anesthetized with 4% sodium pentobarbital. Following general anesthesia, mice were positioned in a stereotaxic apparatus and the hippocampal region was injected with 0.6 µL of KA (0.5 µg/µL) (Sigma, USA).The specific coordinates of the bregma origin were: anteroposterior (AP): -2.0 mm; mediolateral (ML): ±1.5 mm; dorsoventral (DV): -2.0 mm(Gao et al. 2023). The amount of saline given to the control mice was the same. The needle was inserted and held in for five minutes before being gradually removed. Seizure scores were evaluated using the Racine scale(Racine 1972). A score of IV and higher was considered successful modeling.

Pentylenetetrazole (PTZ) model

PTZ was injected intraperitoneally into the mice. Seizures were induced from repeated intraperitoneal injections of a subconvulsive dose (35 mg/kg) of PTZ (Sigma, USA) every two days(Yu et al. 2021; Zhang et al. 2022). Each injection was followed by a half-hour of animal observation. Throughout the trial, seizures were tracked and assessed using the Racine scale.

Adeno-associated virus (AAV) injection

Overexpression of CPEB3 virus was purchased from Obio Biotechnology Co.(Shanghai, China). For overexpression of CPEB3 pAAV-CMV-MCS-3xFLAG-WPRE was utilised as a vector. Knockdown CPEB3 virus was purchased from the Shanghai Genechem. (Shanghai, China) For knockdown of CPEB3 was achieved by CPEB3-RNAi piggybacking pAAV-U6 -CAG-WPRE as a vector.

A 1 ul microsyringe was used to inject viruses into the mouse CA1 brain area stereotactically. The specific coordinates of the bregma origin were: AP: -2.0 mm; ML: ±1.5 mm; DV: -1.7 mm. The injections were performed bilaterally at a dose of 0.5 ul per side and a rate of 0.2 ul/min; the needle was kept in place for 5 min after completion of the injections. KA or PTZ-induced epilepsy was performed three weeks after the virus injection.

Western blotting (WB)

Brain tissue samples were collected for WB analyses. Total protein was extracted using RIPA buffer (Beyotime, China), a protease inhibitor (Beyotime, China), and a phosphatase inhibitor (Biosharp, China). The other components follow the manufacturer's instructions (cytoplasmic and nuclear proteins: Thermo Fisher, MA, USA; membrane proteins: Beyotime, China; synaptosome proteins: Invitrogen, CA, USA). Initially, protein samples were separated using sodium dodecyl SDS-polyacrylamide gel and electrotransferred onto a PVDF membrane (Millipore, MA, USA). After that, the membranes were sealed for two hours, and the primary antibody was then incubated at 4°C for the entire night. Finally, the secondary antibody was incubated at room temperature for 2 h, followed by rinsing with TBST, and the blots were visualized using an ECL reagent (Epizyme, China). Specific antibody information is shown in Table S2.

Immunofluorescence staining

After deparaffinization, the brain tissue pieces were submerged in a 1× citrate solution and heated for 10 minutes under high pressure. Triton X-100 (Beyotime, China) permeabilized the cell membranes for 15 min. The goat serum working solution (Boster, China) was blocked at 37°C for 30 min. Then, the primary antibody was incubated overnight at 4°C, and a secondary antibody corresponding to the primary antibody was used. DAPI has stained the nucleus (Beyotime, China) and photographed under a fluorescence microscope (Nikon, Japan). Specific antibody information is shown in Table S2.

Local field potentials (LTP) recording

Briefly, two screws were implanted into the mouse skull as grounding screws, and electrodes (Kedou, China) were subsequently implanted into the CA1 brain region of mice using a stereotactic apparatus(Tang et al. 2023). 2h LFP signals were recorded and data were analyzed using NeuroExplorer (Nex Technologies, MA, USA).

Drug administration

WP1066 (MCE, USA) (a STAT3 inhibitor) was dissolved in dimethyl sulfoxide (DMSO). Knockdown CPEB3 mice were injected intraperitoneally with 40 mg/kg WP1066. Equal volumes of DMSO were administered to control animals. Intraperitoneal injection once daily for 7 days before induction of KA or PTZ.

Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from mouse hippocampal tissues using the TRIzol reagent (Invitrogen, USA). To get cDNA, the manufacturer's (TransGen Biotech, China) instructions were followed. The qPCR procedure was carried out using the PerfectStart® Green qPCR SuperMix kit (TransGen Biotech, China). Primer sequences are shown in the Table S3.

RNA immunoprecipitation (RIP)

An RNA immunoprecipitation kit (Geneseed, China) was used to perform RIP. Briefly, fresh hippocampal tissue from mice overexpressing CPEB3 was lysed. Immunoprecipitation was performed using an anti-FLAG antibody. IgG was used as a control. Subsequently, RT-qPCR was used for quantitative analysis. The primer sequences are shown in Table S2.

Chromatin immunoprecipitation (CHIP)

CHIP was performed according to the instructions of the Sonication CHIP Kit (Abclonal, China). In summary, fresh hippocampal tissues were crosslinked using 1% formaldehyde, followed by nucleus extraction, chromatin sonication and fragmentation, and co-incubation with sonication lysate using an anti-STAT3 antibody. The chromatin was eluted and de-crosslinked. Finally, DNA was purified using a DNA purification kit (Abclonal, China). RT-qPCR was performed to quantitatively analyze the experimental results. The primer sequences are shown in Table S2.

Statistical analysis

The data were analyzed with GraphPad Prism (version 8.0) and given as mean ± standard deviation (SD). Tukey's post-hoc test was used in conjunction with a one-way ANOVA for multiple comparisons. Statistical significance was set at p < 0.05. Non-significant differences were designated as “ns” (p ≥ 0.05), whereas ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 represent significant differences.

Bioinformatics results of RBPs-associated gene expression profiles in TLE patients

We assessed the batch effects before and after merging GSE63808 and GSE29738. After removing the batch effects, the data suggested that the batch effect was better removed from the merged dataset. The dataset was used for subsequent analyses (Fig.S1). Subsequently, we performed a differential expression analysis of the merged and extracted RBPs-associated gene expression profiles. We identified 18 RBPs-associated differentially expressed genes, including 6 upregulated and 12 downregulated genes (Table S4). Notably, the gene of CPEB3 was downregulated in epilepsy patients (Fig. 1A, B). Furthermore, we explored the possible signaling pathways involved in 18 differentially expressed genes using GO and KEGG enrichment analysis (Fig.S2). We used two algorithms to screen for potential Hub genes in the TLE. The 18 RBPs-associated differential genes listed above were reduced using the LASSO regression technique, resulting in the identification of the 11 differentially expressed genes most strongly linked to TLE. (Fig. 1C, D). A subset of 14 features from the 18 RBPs-associated differential genes was identified using the SVM-RFE algorithm (Fig. 1E, F). Finally, 10 overlapping feature genes between the two algorithms were selected as Hub genes (Fig. 1G).

The GSE190452 dataset provided the gene expression patterns of 28 500 cells from four TLE patient samples (Fig.S3A). nCount_RNA was positively correlated with nFeature_ RNA (Fig.S3B). Variance plots showed 25 431 genes in all cells, with red dots representing the top 2 000 highly variable genes (Fig.S3C). Seventy-one marker genes were used to identify different clusters, and the corresponding cell types were identified. Thirty independent clusters and seven cell types were identified (Fig.S3D, Fig. 1H). We conducted cellular localization analysis on the Hub gene and the results showed that CPEB3 is expressed in both excitatory and inhibitory neurons (Fig. 1I, J).

Meanwhile, based on the 10 Hub genes, we used a logistic regression algorithm to establish a diagnostic model for epilepsy classification (Fig.S4). Subsequently, we selected the top three genes in terms of AUC value for validation. q-PCR results showed that only CPEB3 mRNA was reduced in both hippocampus and cortex of epilepsy mice compared with control group (Fig. 1K). In summary, we speculate that downregulation of CPEB3 gene may be associated with epilepsy.

CPEB3 expression and subcellular localization in epileptic brain tissue

We use WB analysis to verify the expression of CPEB3 in epileptic brain tissues. The results demonstrated that the CPEB3 expression was downregulated in the hippocampal tissue of the KA model mice than in the control group since KA-induced sustained epileptic status (SE) at 1, 3, 7, 14, and 30 d, whereas CPEB3 did not show any significant change in the cortex at 1 d after KA injection; however, it was significantly reduced on 3, 7, 14, and 30 d (Fig. 2A, B). Meanwhile, we examined the expression of CPEB3 in PTZ model mice. The results revealed that CPEB3 protein expression consistently decreased in the hippocampus and cortex of PTZ model mice compared to the control group (Fig. 2C, D). Additionally, we investigated CPEB3 protein expression in TLE patients' surgical tissues. We discovered that CPEB3 expression was substantially lower in TLE patients' brain tissues than in TBI patients (Fig. 2E). Although our bioinformatics analysis showed that the CPEB3 gene was localized in neurons, the subcellular localization of CPEB3 remains undetermined. Therefore, we examined the distribution of CPEB3 in epileptic brain tissues using immunofluorescence staining. We found that CPEB3 was primarily co-expressed with neurons in the brain tissues of TLE patients and KA model mice (Fig. 2F, G and S5 ). These experimental results suggest that CPEB3 may be closely related to epilepsy.

CPEB3 modulates seizure susceptibility and severity

To explore whether CPEB3 affects seizures, we injected AAVs into the CA1 region of the mouse hippocampus to overexpress or knockdown CPEB3. WB was used to detect the overexpression or knockdown of CPEB3 efficiency after three weeks (Fig.S6). Next, we administered an intraperitoneal injection of PTZ to induce seizures in the mice and recorded the level of each seizure. The results showed that CPEB3 overexpression significantly decreased the severity of seizures and the number of seizures at level IV/V ( Fig. 3A, B). However, the severity of seizures and the number of seizures at level IV/V increased dramatically after CPEB3 knockdown (Fig. 3C, D). We recorded LFP for spontaneous recurrent seizures (SRSs) in mice, and we found that the amplitude of LFP for SRSs in overexpressing CPEB3 mice was significantly smaller than that of control mice, CPEB3 knockdown mice showed the opposite phenomenon (Fig. 3F, G). We also recorded the latency to the first generalized seizures in mice by behavioral tests. Behavioral observation revealed that overexpression of CPEB3 significantly increased seizure latency (Fig. 3I). Knockdown of CPEB3 had the opposite effect (Fig. 3H). These results suggest that CPEB3 negatively regulates epilepsy susceptibility, and reduces seizure severity.

CPEB3 can alter the nuclear translocation of STAT3 by inhibiting its translation

It has been reported that CPEB3 can affect the activation of the JAK/STAT pathway(Fang et al. 2020). Next, we verified whether this regulatory mechanism exists in epilepsy. We examined changes in STAT3 in total protein lysates from mouse hippocampal tissue to verify whether this mechanism exists in epilepsy. We found that CPEB3 overexpression inhibited STAT3 and p-STAT3 protein expression, while STAT3 and p-STAT3 expression was elevated after CPEB3 knockdown (Fig. 4A, D). STAT3 is a key transcription factor mainly localized in the cytoplasm in the normal state but activated STAT3 changes nuclear translocation. Therefore, we further examined the changes in STAT3 protein expression in the nuclear and cytoplasmic of mouse hippocampal tissues using WB. We found that CPEB3 overexpression decreased STAT3 and p-STAT3 protein expression in the nuclear and cytoplasmic, while CPEB3 knockdown showed the opposite result (Fig. 4B, C, E, F). Immunofluorescence assay showed that nuclear STAT3 level was decreased in overexpression CPEB3 mouse hippocampal and knockdown of CPEB3 induces STAT3 into the nuclear (Fig. 4G, H). This suggests that CPEB3 inhibits the nuclear translocation of STAT3.

We used qPCR to investigate the mRNA levels of STAT3 in hippocampal tissues to investigate further how CPEB3 regulates STAT3 expression. The results showed that CPEB3 did not alter STAT3 mRNA expression (Fig. 4I). Meanwhile, we verified exogenous CEPE3 expression in mouse hippocampal tissues (Fig.S7). RIP experiments showed that exogenous CPEB3 could bind to STAT3 mRNA (Fig. 4J). The above experimental results demonstrated that CPEB3 could bind to the mRNA of STAT3 to inhibit its translation, thereby altering the distribution of STAT3 in the nucleus.

CPEB3 can regulate the expression of NMDAR subunits

NMDARs overexpression causes a neuronal E/I imbalance, leading to epileptic seizures. Previous studies have shown that STAT3 is a transcriptional activator of NMDARs(Wan et al. 2021). We have demonstrated that CPEB3 can regulate the expression of STAT3, but whether CPEB3 can affect the expression of NMDARs needs to be further explored. Therefore, we investigated whether CPEB3 regulates NMDARs expression in epilepsy. To verify this speculation, we examined the mRNA levels of GluN1, GluN2A, and GluN2B in hippocampal tissues. The results showed that the mRNA expression levels of the NMDAR subunit (GluN1, GluN2A, and GluN2B) in the hippocampus were similarly regulated by CPEB3 and were negatively correlated (Fig. 5A, B). We extracted proteins from total lysates of mouse hippocampal tissues and examined NMDAR subunit expression. The results showed that the protein expression of total GluN1, GluN2A, and GluN2B in hippocampal tissues was decreased by CPEB3 overexpression and increased by CPEB3 knockdown (Fig. 5C, F). Subsequently, we extracted proteins from the cell membrane lysates of hippocampal tissues. The results demonstrated that CPEB3 had the same mechanism of negatively regulating the distribution of GluN1, GluN2A, and GluN2B in the cell membranes (Fig. 5D, G). Finally, we examined the protein expression changes of GluN1, GluN2A, and GluN2B in synaptosome lysates of hippocampal tissues. The results showed that only GluN1 is regulated by CPEB3. GluN2A and GluN2B were unaffected by CPEB3 in the synaptosome lysates (Fig. 5E, H). The results indicate that CPEB3 can negatively regulate GluN1, GluN2A, and GluN2B protein expression.

CPEB3 can alter epilepsy susceptibility and severity by inhibiting the expression of NMDARs via STAT3

The above results indicate that CPEB3 regulates STAT3 transcriptional activity. We injected WP1066 (a STAT3 inhibitor) intraperitoneally into knockdown CPEB3 mice and examined the protein and mRNA expression of GluN1, GluN2A, and GluN2B to verify whether CPEB3 inhibits the transcriptional activity of NMDARs via STAT3 in epilepsy. The results showed that the protein and mRNA expression of GluN1, GluN2A, and GluN2B were reduced after inhibiting STAT3 activation (Fig. 6A, B). CHIP experiments showed that STAT3 bound to the transcriptional promoters of GluN1, GluN2A, and GluN2B in mice with CPEB3 knockdown (Fig. 6C-E). Meanwhile, we found that epilepsy phenotype was reduced after STAT3 activation was inhibited (Fig. 6F-I). This suggests that CPEB3 may reduce STAT3-mediated transcriptional activity of NMDARs, inhibit NMDAR subunit expression, and attenuate epilepsy phenotypes.

In this study, we investigated the mechanism of action of CPEB3 using a mice model of epilepsy. We found that CPEB3 was closely linked to epilepsy using bioinformatics analysis. CPEB3 was expressed at low levels in the brain tissues of patients with epilepsy and epileptic mouse models, and CPEB3 co-localized with neurons. CPEB3 overexpression in the hippocampus of mice reduced seizure susceptibility and severity, whereas CPEB3 knockdown increased the seizure susceptibility and severity. Further mechanistic studies revealed that CPEB3 inhibited STAT3 translation and STAT3-mediated transcriptional activity of NMDARs, thereby suppressing NMDAR subunit expression and attenuating epilepsy phenotype. Our findings provide a new theoretical basis for the pathogenesis of epilepsy.

The main pathological mechanisms underlying TLE include neural death, glial cell proliferation, and altered synaptic plasticity. The development of TLE is usually accompanied by cognitive dysfunction and memory impairment. CPEB3 is a key RBP that maintains long-term memory and synaptic plasticity(Qu et al. 2020; Si et al. 2010). However, whether CPEB3 plays a role in epilepsy has not yet been investigated. Our study found that the CPEB3 expression level was reduced in mice models and patients with TLE brain tissues. We also focused on the cellular localization of CPEB3 in brain tissues and found that CPEB3 was co-expressed in neurons. Next, behavioral tests revealed that CPEB3 overexpression attenuated seizures in mice. In contrast, CPEB3 knockdown exacerbated the seizure phenotype. In conclusion, CPEB3 is involved in the pathogenesis of epilepsy; however, the specific molecular mechanisms require further investigation.

The JAK/STAT pathway is involved in the pathogenesis of epilepsy by affecting various biological processes, including, synaptic plasticity, and ferroptosis (Chen et al. 2023; Ouyang et al. 2022; Xu et al. 2024). STAT3 is an important transcription factor, mainly located in the cytoplasm in the normal state, and p-STAT3 expression increases in the activated JAK/STAT pathway. Increased p-STAT3 induces the nuclear translocation of STAT3 and increases its binding to DNA, thereby affecting target gene transcription(Liu et al. 2024). Recently, researchers found that CPEB3 negatively regulates the activation of the JAK/STAT pathway(Zhong et al. 2020). Our study found that CPEB3 inhibited STAT3 and p-STAT3 in total protein extracts, cytoplasmic and nuclear extracts. Moreover, we found that CPEB3 did not alter STAT3 mRNA expression level. Therefore, we speculated that CPEB3 inhibits the translation of STAT3 by binding to STAT3 mRNA. Subsequent RIP experiments confirmed our speculation. This suggests that CPEB3 may alters the epilepsy phenotype by inhibiting the transcriptional activity of STAT3.

NMDARs are glutamate receptors widely distributed in the central nervous system and play an important role in brain development and synaptic plasticity(Goldsmith 2019; Mony et al. 2023; Wang et al. 2021; Zhou et al. 2021). NMDAR dysfunction can cause various central nervous system disorders, including schizophrenia, epilepsy, and Alzheimer's disease. Additional studies have shown that the expression and function of NMDARs are closely related to epilepsy(Sadeghi et al. 2021; Strehlow et al. 2019; Zhang et al. 2024). Excessive activation of NMDARs can cause neuronal depolarization and calcium overload, leading to neuronal death, and blocking NMDAR activation can reduce neuronal death(Ge et al. 2020). GluN1, GluN2A, and GluN2B are the main isoforms of NMDARs and are widely distributed in the cortex and hippocampus(Chen et al. 2021). Mutations in the gene encoding can lead to epilepsy(Sabo et al. 2022). In animal models of epilepsy, the expression of GluN1, GluN2A, and GluN2B subunits is increased(Postnikova et al. 2017). A nonspecific blocker of NMDARs-memantine can reduce the severity and susceptibility of epileptic mice(Gu et al. 2023). In our study, we found that CPEB3 can negatively regulate the mRNA expression levels of GluN1, GluN2A, and GluN2B in the hippocampus. The following results shown that in the hippocampus of epileptic mice, overexpression or knockdown of CPEB3 expression negatively regulated the protein expression of GluN1, GluN2A, and GluN2B in total protein lysates and cell membrane lysates in hippocampal tissues; it did not alter the protein expression of GluN1 and GluN2B in synaptosome lysates. We analyzed that this may be because the expression of GluN1 and GluN2B in hippocampal synapses is regulated by another mechanism.

In this study, we demonstrated that CPEB3 inhibited the activation of STAT3 by negatively regulating the translation of STAT3 and reducing the nuclear translocation of STAT3. This indicated that CPEB3 alters the transcription of STAT3 downstream target genes. Previous studies have disclosed that STAT3 is involved in regulating genes associated with synaptic plasticity(Tipton et al. 2023). Consequently, we speculated that CPEB3 can reduce STAT3-mediated transcription of NMDARs by inhibiting STAT3 translation. Next, we performed rescue experiments using WP1066, a specific inhibitor of STAT3. The results showed that the inhibition of STAT3 activation in mice with CPEB3 knockdown increased the expression of NMDARs and the mRNAs expression levels of GluN1, GluN2A, and GluN2B. CHIP experiments showed that STAT3 directly binds to the transcriptional promoters of GluN1, GluN2A, and GluN2B. Additionally, behavioral experiments demonstrated that WP1066 reduced the severity and susceptibility to epilepsy. These experimental results suggest that CPEB3 can reduce seizure severity and susceptibility in mice by inhibiting the STAT3-mediated transcription of NMDARs.

Our study has several limitations. First, we only confirmed the role of exogenous CPEB3 with STAT3 mRNA due to the lack of specific CPEB3 antibodies that can perform RIP. Second, our results showed that CPEB3 does not regulate GluN1 and GluN2B in hippocampal synaptic lysates, and the specific mechanism underlying this phenomenon must be further explored.

In conclusion, our experimental results suggest that CPEB3 can reduce STAT3-mediated transcription of NMDARs by inhibiting STAT3 translation, which attenuates the epileptic phenotype. These results provide new insights into the search for new therapeutic targets and pathogenesis of epilepsy.

Abbreviation	Full name
CPEB3	Cytoplasmic polyadenylation element-binding protein
STAT3	Signal transducer and activator of transcription 3
p-STAT3	phosphorylated STAT3
TLE	Temporal lobe epilepsy
RBPs	RNA-binding proteins
NMDARs	N-methyl-D-aspartic acid receptors
TBI	Traumatic brain injury
GEO	Gene Expression Omnibus
KA	Kainic acid
PTZ	Pentylenetetrazole
WB	Western blotting
AAV	Adeno-associated virus
LTP	Local field potentials
RT-qPCR	Real-time quantitative polymerase chain reaction
RIP	RNA immunoprecipitation
CHIP	Chromatin immunoprecipitation
SE	Epileptic status
SRSs	Spontaneous recurrent seizures

Ethics approval and consent to participate

All participants provided their informed consent prior to utilizing the study's specimens. This study was approved by the Ethics Committee of the Second Hospital of Harbin Medical University (Approval Number:KY2024-036) and was conducted per the Declaration of Helsinki. All animal experiments were approved by the Ethics Committee Second Affiliated Hospital of Harbin Medical University (Approval Number:KY2018-108).

Consent for publication

Not applicable.

Data availability

All data generated or analyzed during this study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Nos. 81871016), China Postdoctoral Science Foundation (Nos. 2018M640306), Heilongjiang Postdoctoral Grant (Nos. LBH-Q21132 and LBH-Q20045).

Authors' contributions

Conceptualization, methodology, and formal analysis: YZP and HC. Data curation and writing – original draft preparation:WF. Visualization and investigation:LJR, LY and LXA. Supervision and formal analysis:FZJ. Resources and validation: WF and FZJ. Writing – reviewing and editing and project administration: GXY and SJH. All authors read and approved the final manuscript.

Acknowledgements

We extend our appreciation to all the authors for their valuable contributions.

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WITHDRAWN: CPEB3 can regulate seizure susceptibility by inhibiting the transcriptional activity of STAT3 on NMDARs expression

Status:

Version 1

Editorial Note

Abstract

Background

Methods

Results

Conclusion

Figures

BACKGROUND

MATERIALS AND METHODS

Human brain tissue

Animals

Bioinformatics analysis

Kainic acid (KA) model

Pentylenetetrazole (PTZ) model

Adeno-associated virus (AAV) injection

Western blotting (WB)

Immunofluorescence staining

Local field potentials (LTP) recording

Drug administration

Real-time quantitative polymerase chain reaction (RT-qPCR)

RNA immunoprecipitation (RIP)

Chromatin immunoprecipitation (CHIP)

Statistical analysis

RESULTS

Bioinformatics results of RBPs-associated gene expression profiles in TLE patients

CPEB3 expression and subcellular localization in epileptic brain tissue

CPEB3 modulates seizure susceptibility and severity

CPEB3 can alter the nuclear translocation of STAT3 by inhibiting its translation

CPEB3 can regulate the expression of NMDAR subunits

CPEB3 can alter epilepsy susceptibility and severity by inhibiting the expression of NMDARs via STAT3

DISCUSSION

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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