Rbm8a regulates neurogenesis and reduces Alzheimer’s disease-associated pathology in the dentate gyrus of 5×FAD mice

Chenlu Zhu ,Xiao Ren ,Chen Liu ,Yawei Liu ,Yonggang Wang

Abstract Alzheimer’s disease is a prevalent and debilitating neurodegenerative condition that profoundly affects a patient’s daily functioning with progressive cognitive decline,which can be partly attributed to impaired hippocampal neurogenesis.Neurogenesis in the hippocampal dentate gyrus is likely to persist throughout life but declines with aging,especially in Alzheimer’s disease.Recent evidence indicated that RNA-binding protein 8A (Rbm8a) promotes the proliferation of neural progenitor cells,with lower expression levels observed in Alzheimer’s disease patients compared with healthy people.This study investigated the hypothesis that Rbm8a overexpression may enhance neurogenesis by promoting the proliferation of neural progenitor cells to improve memory impairment in Alzheimer’s disease.Therefore,Rbm8a overexpression was induced in the dentate gyrus of 5×FAD mice to validate this hypothesis.Elevated Rbm8a levels in the dentate gyrus triggered neurogenesis and abated pathological phenotypes (such as plaque formation,gliosis reaction,and dystrophic neurites),leading to ameliorated memory performance in 5×FAD mice.RNA sequencing data further substantiated these findings,showing the enrichment of differentially expressed genes involved in biological processes including neurogenesis,cell proliferation,and amyloid protein formation.In conclusion,overexpressing Rbm8a in the dentate gyrus of 5×FAD mouse brains improved cognitive function by ameliorating amyloid-beta-associated pathological phenotypes and enhancing neurogenesis.

Key Words: Adora2a;Alzheimer’s disease;astrocyte;cAMP signaling pathway;dentate gyrus;dystrophic neurites;microglia;neurogenesis;plaque;Rbm8a

Introduction

Alzheimer’s disease (AD) is a prevalent neurodegenerative disorder that typically manifests with memory loss and impaired learning ability (Eimer and Vassar,2013).The pathological phenotypes in AD are multifaceted and include extracellular senile plaque formation,intracellular tau neurofibrillary tangles,and irreversible functional impairment in neurons (Edwards,2019;Hondius et al.,2021;Roda et al.,2022;Gonçalves et al.,2023).There is no effective curative treatment for AD.Recent evidence suggests that neural stem cells in the dentate gyrus (DG) can promote neurogenesis throughout the lifespan (Abbott and Nigussie,2020).Nevertheless,neurogenesis sharply declines with aging.Therefore,exploring methods to enhance neurogenesis in the DG shows potential for improving cognitive impairment and mitigating AD-related pathological features.

Located on the chromosomal region 1q21.1,RNA-binding protein 8A (Rbm8a;also known as Y14) is a ribonucleoprotein that contains a conserved RNAbinding motif necessary for maintaining normal mRNA splicing and stability (Chuang et al.,2013,2019).Rbm8aparticipates in the regulation of selfrenewal properties associated with cortical neural progenitor cells and retinal progenitor cells (Zou et al.,2015;Zhang et al.,2017).However,aberrant expression ofRbm8acan lead to various diseases in both central and peripheral organs.For example,impaired expression ofRbm8aresults in cell cycle deficiency,apoptosis,and developmental disorders such as thrombocytopenia-absent radius syndrome and microcephaly,which can ultimately lead to early postnatal lethality (Ishigaki et al.,2013;Al-Qattan,2016;Monteiro et al.,2022).Interestingly,increased expression ofRbm8ahas been observed in tumor-related diseases such as stomach cancer,liver cancer,and glioblastoma,and plays a role in promoting tumor cell proliferation and migration (Liang et al.,2017;Lv and Cheng,2020;Lin et al.,2021).In contrast,expression levels ofRbm8awere lower in the brains of AD patients compared with healthy individuals.This induced the downregulation of autophagyrelated genes such as focal adhesion kinase family interacting protein of 200 kD (Fip200),Beclin 1,and nuclear receptor binding factor 2 (Nrbf2),thereby increasing the risk of amyloid protein accumulation in AD (Zou et al.,2019).However,the function and regulatory mechanism ofRbm8ain AD remain elusive,specifically regarding its effect on neurogenesis,plaque formation,and gliosis reaction.Hence,further investigation is needed to determine whether restoringRbm8aexpression can improve cognitive function in AD.

As an AD disease model,5×FAD mice exhibit AD-related pathological phenotypes such as amyloid deposition,plaque formation,gliosis,and neuronal loss,with the mice demonstrating relatively early and aggressive cognitive dysfunction.5×FAD mice develop amyloid deposition at 1.5 months,followed by decreased neurogenesis at 2 months,and cognitive impairment at 4 months (Oakley et al.,2006;Moon et al.,2014;Forner et al.,2021).Thus,it is interesting to explore whether overexpression ofRbm8ain the DG of 5×FAD mice could increase neurogenesis,reduce amyloid-beta (Aβ)-related pathology,and improve cognitive function,as well as to further investigate the underlying molecular mechanisms ofRbm8ain the progression of AD.

Methods

Animals

Male and female 5×FAD mice (20-30 g,aged 2-3 months,Stock No.VSM40051,RRID: MGI: 3693208) were obtained from Beijing View Solid Biotechnology (Beijing,China,license No.SCXK (Jing) 2021-0010).All the mice used for experiments were naïve.No drug tests were performed.All mice had free access to food and water.All mice were housed at 22 ± 1°C with a relative humidity of 60%.A controlled cylinder was used to maintain a 12-hour day/night cycle.There were two to four mice in each cage.All transgenic 5×FAD animals (n=60) were confirmed by polymerase chain reaction (PCR) before experiments.5×FAD mice express human amyloid precursor protein (APP) and presenilin-1 (PSEN1) transgenes with five AD-linked mutations: the Swedish (K670N/M671L),Florida (I716V),and London (V717I) mutations in APP,and the M146L and L286V mutations inPSEN1(Bundy et al.,2019).All mice were specific-pathogen-free (SPF) grade and had a C57BL/6 genetic background of at least six generations.To investigate age-dependent changes in RBM8A expression levels,wild-type (WT) mice (n=15,20-30 g,aged 2-12 months) from the same litter as 5×FAD mice were selected from January,February,June,and December.WT mice were housed in the same environment as 5×FAD mice.All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai Tech University (approval No.20230322001,approval date: March 22,2023) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8thed.,National Research Council,2011).This study was reported in accordance with ARRIVE 2.0 guidelines (Animal Research: Reporting ofIn VivoExperiments;Percie du Sert et al.,2020).To examine the effect of RBM8A on plaque accumulation in AD,both male and female 5×FAD mice were used in one study.A subsequent set of investigations focused solely on female 5×FAD mice owing to their greater level of plaque burden when compared with male 5×FAD mice.

Design and construction of functional AAV vectors

In this study,overexpression ofRbm8awas achieved via virus replication by producing recombinant adeno-associated viruses (AAV) particles through an AAV Helper-Free System,which was generated by Taitool Bioscience (Shanghai,China).The first step involved cloning theRbm8agene into the pAOV.SYN.GFP vector provided by Taitool Bioscience.To expressRbm8a,the gene was introduced into the pAOV.SYN.GFP vector before transfecting AAV-293 cells (a subclonal cell line derived from modified HEK293 to increase AAV production capacity,provided by Taitool Bioscience) with a recombinant expression plasmid alongside helper and pAAV-RC plasmids.The AAV typically remains active for an extended period,but mainly in metabolically active cells such as AAV-293 cells.Green fluorescent protein (GFP) fluorescence was detected after 24 hours of virus infection.Within 2 to 3 days of transfection,packaged cells were successfully assembled with the recombinant AAV.The pAOV.SYN.GFP vector contains a cytomegalovirus (CMV) promoter and other components required for high gene expression levels of foreign genes cloned into a multiple cloning site (MCS).The vector also contains AAV reverse terminal repeats (ITRs) that guide virus replication and packaging.pAOV.SYN.GFP has a 3xFLAG-tag at the C-end of the MCS.Furthermore,the pAAVRC plasmid containsRep/Capgenes that encode replication proteins of AAV and viral capsid proteins.The pAAV-RC uses two different promoters to control the expression ofRep/Capand ensure optimal expression levels and proportions of each gene product.The helper plasmid contains a collection of adenovirus genes (VA,E2A,and E4) required for AAV-293 cells to produce high-titer viruses.The adenovirus proteins,E1A and E1B,are essential for AAV production and were stably expressed in AAV-293 cells.

Experimental design and stereotaxic injection

First,the expression levels ofRbm8awere measured in the hippocampal DG region of WT mice at 1,2,6,and 12 months of age.Rbm8aexpression levels of WT and 5×FAD mice were obtained at 2 months of age.DecliningRbm8aexpression patterns correlated with aging,and its function in AD was explored by forming two groups.The pAAV2-CMV-bGI-Rbm8a-3xFlag-2A-GFPSV40PA overexpression virus and pAAV2-CMV-bGI-GFP-WPRE-PA control virus were separately injected into the bilateral DG region (2.0 mm from bregma,1.5 mm from the midline,2.0 mm vertical from the cortical surface;Paxinos and Franklin,2013).The virus was administered at a dosage of 600 nL per DG with a titer of 1.92×1013transducing units (TU)/mL.This was followed by behavioral tests and molecular experiments such as RNA-seq,western blotting test,quantitative PCR test,and enzyme-linked immunosorbent assay (ELISA).Finally,immunofluorescence assays were conducted at 14 days after virus injection for neural progenitor cell analysis and 2 months after virus injection for Aβ-related pathology analysis.

Behavioral tests

Cognitive,depressive,and anxiety-like behaviors in 4-month-old 5×FAD mice were assessed.Behavioral experiments were conducted in a controlled environment,which was cleaned preemptively with 75% ethanol and by removing any feces.Different experiments were conducted at least 24 hours apart.The behavioral experiments were carried out in a calm and neutralsmelling environment equipped with fluorescent lighting.A video camera was mounted above the testing apparatus to ensure optimal brightness and capture animal behavior.

As previously described (Kraeuter et al.,2019),the Y-maze was made of white,opaque polyvinyl plastic and consisted of three equidistant arms (each measuring 21 cm length,7 cm width,and 15.5 cm height),arranged at 120-degree angles to one another.Each of the three arms in the Y-maze was labeled A,B,or C,with A serving as the entry point for mice to begin their exploration for 8 minutes.The percentage of spontaneous alterations and number of arm entries were quantified to evaluate spatial working memory.The former was calculated using the following formula: Percentage of spontaneous alterations=(number of alternations)/(the total number of arm entries -2)×100 (Kang et al.,2018).

Considering the innate tendency of mice to explore novel objects,the novel object recognition test (NORT) is a memory test designed to measure hippocampal-dependent spatial working memory (Lueptow,2017).The testing device consists of a rectangular plastic box (50 cm length,35 cm width,15 cm height) as our testing device.Trials involved a combination of training and testing phases lasting five minutes each.The experiment began with the introduction of two identical spherical objects presented to the mouse,which the mouse was allowed to freely explore for five minutes.Two hours later,one of the spheres was replaced with a cube of equal size,and the mouse was allowed to resume exploration for an additional 5 minutes.The exploration process inside the box was monitored by an overhead camera,recording the mouse as it interacted with both familiar and novel objects.The time spent interacting with these objects was subsequently analyzed to calculate the discrimination index (DI),a widely acknowledged standard for the assessment of cognitive function.Exploration was defined as any form of interaction,including touching and sniffing,occurring within a 2 cm vicinity of the object’s location inside the testing box.Finally,the time spent exploring the novel object was divided by the total duration of exploration to calculate the DI.

The contextual fear conditioning task (CFC) is used to evaluate learning and memory abilities,particularly contextual memory,which is dependent on the hippocampus (Danielson et al.,2016).The test was performed in cylindrical chambers (18 cm diameter of the bottom,30 cm high),as reported previously (Kimura and Ohno,2009).The mice were exposed to an unfamiliar environment for 5 minutes per day for 2 consecutive days.Subsequently,the mice were introduced to a conditioning chamber where they were exposed to a tone accompanied by a foot shock administered three times daily over the next 3 days.The 70-decibel tone was emitted alongside a 0.5-milliampere,2-second foot shock,with a rest period of 20 seconds between stimuli.The CFC task was assessed 4 days later using the TSE system (TSE Systems China,Thuringia,Germany),which automatically recorded their freezing behavior as a measure of memory function.Freezing behavior is a stress response in rodents (like mice) when threatened or harmed.It is specific,showing a resting state dominated by respiratory activity and little else (Curzon et al.,2009).

A previous study reported depression as a concomitant symptom in AD (Chan et al.,2020).Therefore,the tail suspension test (TST) and forced swimming test (FST) were performed to assess the depression status of 5×FAD mice.The FST used cylindrical tanks made of transparent Plexiglas (30 cm height,20 cm diameter).Within the cylindrical tanks,the water level was 15 cm deep to ensure the mouse could not touch the bottom of the tank.For the TST,tail suspension boxes (55 cm height,60 cm width,and 11.5 cm depth) were manufactured out of wood.The mice were suspended with tape within the three-walled rectangular compartment (55 cm height,15 cm width,11.5 cm depth) without touching any of the surroundings.The above two tests were performed for 6 minutes and the immobility time was calculated.

The open field test was conducted to assess anxiety-like behavior,as previously described with minor modifications (Seibenhener and Wooten,2015).The open field test involved placing a 5×FAD mouse within the central region (22.5 cm width,22.5 cm long) of a larger apparatus (45 cm length,45 cm width,50 cm height),preceded by a 2-minute habituation period.The mouse was then allowed to explore freely for 10 minutes.Exploration time spent within the confined central region serves as an indicator of anxiety-like behavior and was recorded by video in the TSE system (TSE Systems China) for subsequent analysis.

RNA-seq data processing

To investigate the role ofRbm8ain AD,RNA-seq was used to analyze DG regions extracted from 4-month-old 5×FAD female mice (both 5×FAD-Ctrl and 5×FAD-Rbm8agroups,n=3 mice/group).RNA-seq was performed by the Beijing Genomics Institute (BGI,Wuhan,China).Only samples with an RNA Integrity Number (RIN) >2 were selected for extraction to ensure RNA integrity.Subsequently,six samples were sequenced using the Dense Bead-Based Sequencing platform (BGI),resulting in an average yield of 1.19 G bases per sample.The average mapping ratio to the reference genome was 96.62%,while the average mapping ratio to genes was 75.85%.Subsequently,RNA was further purified using the RNeasy micro kit (QIAGEN,Dusseldorf,Germany,Cat# 74004).Pearson correlation coefficients were used to assess gene expression correlations across sample pairs.Furthermore,Hierarchical Indexing for Spliced Alignment of Transcripts (HISAT,a R package to align the raw file,https://bioconductor.org/packages/release/bioc/html/Rhisat2.html) was used to align clean reads to the reference genome.Thereafter,differentially expressed genes (DEGs) in samples from the 5×FAD-Ctrl and 5×FAD-Rbm8agroups were detected using DEseq2 in R (University of Auckland,New Zealand,https://bioconductor.org/packages/release/bioc/html/DESeq2.html) (Love et al.,2014).DEGs were defined by aP-value ≤ 0.05 and |Log2 fold change| ≥ 1.DEGs data are listed inAdditional Table 1.These DEGs were then presented in a heatmap using TBtools software (https://github.com/CJ-Chen/TBtools) (Chen et al.,2020).Significantly enriched Gene Ontology (GO) terms were identified by aq-value ≤ 0.05.Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed by Cluster Profile in R and a Venn diagram was generated online (online.visual-paradigm.com).

Immunofluorescence staining

The 5×FAD male and female mice (n=40/group) were anesthetized by intraperitoneal injection with 3% 2,2,2-tribromoethanol (Bide Pharmatech,Shanghai,China,Cat# BD63827) and perfused with 20 mL 1× phosphatebuffered saline (PBS,pH=7.4),followed by 20 mL 4% paraformaldehyde (PFA) in 1× PBS.Following extraction,brain tissues were fixed overnight with 4% paraformaldehyde (PFA).Using a vibratome (Leica,Wetzlar,Germany,Cat# VT1000S),40-µm-thick coronal slices were obtained from fixed tissues.The tissue sections were washed in PBS three times for 5 minutes each before staining.Subsequently,primary antibodies were diluted in a buffer consisting of 0.3% Triton X-100 and 1% bovine serum albumin (BSA) in 1× PBS and incubated overnight with free-floating tissue sections at 4°C.The following day,the brain slices were washed three times with 1× PBS for 5 minutes each.Next,the tissue sections were incubated at room temperature with secondary antibodies diluted in buffer (consisting of 0.3% Triton X-100 and 1% BSA in 1× PBS) for 2 hours.Throughout this procedure,all experimental measures were conducted within a dark environment.Finally,the nuclei were stained with 4,6-diamino-2-phenyl indole (DAPI) (Yeasen Biotech Co.,Ltd.,Shanghai,China,Cat# 40728ES03) for 20 minutes.Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-neuron specific nuclear protein (NeuN,1:500,Abcam,Cambridge,UK,Cat# ab177487,RRID: AB_2532109),rabbit anti-glial fibrillary acidic protein (GFAP,1:500,Abcam,Cat# ab7260,RRID: AB_305808),goat anti-ionized calcium-binding adapter molecule 1 (IBA1,1:500,Abcam,Cat# ab5076),rabbit anti-autophagy related protein 9A (ATG9A,1:400,Abcam,Cat# ab108338,RRID: AB_10863880),rabbit anti-light chain-3B (LC3B,1:500,Abcam,Cat# ab192890,RRID: AB_2827794),rabbit anti-doublecortin (DCX,1:500,Abcam,Cat# ab18723,RRID: AB_732011),mouse anti-5-bromo-2-deoxyuridine (BrdU,1:500,BD Biosciences,Franklin Lakes,NJ,USA,Cat# 347580),goat anti-SRY-Box transcription factor 2 (SOX2,1:200,R&D Systems,Emeryville,CA,USA,Cat# AF2018,RRID: AB_355110),and mouse anti-beta amyloid (Aβ) 1-16 (6E10,1:500,Bio Legend,San Diego,CA,USA,Cat# 803004,RRID: AB_2715854).Secondary antibodies were: goat anti-mouse Alexa Fluor 647 (1:1000,Jackson Immuno-Research Labs,West Grove City,PA,USA,Cat# 115-605-003,RRID: AB_2338902),goat anti-rabbit Alexa Fluor 555 (1:1000,Thermo Fisher Scientific,Shanghai,China,Cat# A-21428,RRID: AB_2535849),donkey anti-goat Alexa Fluor 555 (1:1000,Thermo Fisher Scientific,Cat# A-21432,RRID: AB_2535853),and donkey anti-mouse Alexa Fluor 647 (1:1000,Thermo Fisher Scientific,Cat# A-31571,RRID: AB_162542).

Image analysis

To ensure unbiased analysis,a Zeiss LSM980 inverted microscope (Carl Zeiss,Oberkochen,Germany) was used to randomly capture confocal images with fixed gains and exposure times for each fluorescent channel.Immunofluorescent images of slides stained for plaques,microglia,astrocytes,neurons,radial glial stem cells,newborn neurons,and dystrophic neurites (DNs) were obtained at 10× and 63× magnification.

Moreover,10× magnification confocal images were processed by ImageJ software 1.8.0 (National Institutes of Health,Bethesda,MD,USA) to accurately assess the total number of plaques (mm2) and mean plaque area (µm2) within the DG.A threshold level mask was established to ensure uniformity across all analyses.The average plaque area (µm2) was then determined by dividing the total plaque coverage area (mm2) in the DG by the total number of plaques in the DG.

The number of Aβ-associated DNs expressing GFP+ATG9A+or GFP+LC3B+in the DG were manually evaluated from confocal images at 10× magnification.To determine the number of DNs in both groups,and assess the role ofRbm8ain the establishment of DNs,the percentages of Aβ-associated 6E10+GFP+ATG9A+/6E10+GFP+or 6E10+GFP+LC3B+/6E10+GFP+cells were examined.The Aβ-associated area was defined in the same manner as prior publications: a 50-µm-diameter region surrounding the plaque core (Ren et al.,2022).

Plaque-associated astrocytes or microglia were defined as those surrounding plaques within a 50 µm diameter.The average number of these cells per plaque was obtained using ImageJ-win64 software version 1.8.0 (National Institutes of Health) from 10× confocal images.The total number of microglia or astrocytes (mm2) surrounding each plaque were calculated and then divided by the total number of plaques associated with these cells.

Each brain section was selected for a similar position of DG region.Three to five brain slices per mouse were used for immunostaining quantification.

ELISA assay

The 5×FAD mouse hippocampus was homogenized on ice with a protease inhibitor cocktail (Apex Bio,Houston,TX,USA,Cat# K1007).Frozen tissues were stored at -80°C.Tissue was retrieved and placed into a 2 mL centrifuge tube with PBS buffer,then centrifuged at 12,000×g,4°C for 30 minutes until no visible pieces were seen.The supernatant was collected for soluble Aβ40and Aβ42ELISA analysis,which was conducted using a fluorescent-based kit including a Human beta Amyloid 40 kit (ExCell Bio,Shanghai,China,Cat# EH039-48) and Human beta Amyloid 42 kit (ExCell Bio,Cat# EH039-48),according to the manufacturer’s protocol.

Quantitative reverse transcription-PCR

The cDNA was extracted from the DG of 5×FAD mouse brain tissue with TRIzol (Invitrogen,Carlsbad,CA,USA,Cat# 15596018) according to the manufacturer’s protocol,and synthesized using a cDNA Synthesis Kit (Transgene Biotech,Beijing,China,Cat# AT311-03).Quantitative reverse transcription-PCR (qRT-PCR) was performed with 2×SYBR Green qPCR Master Mix (Bimake,Houston,Texas,USA,Cat# B21202) using a Quant Studio 7 Flex reverse transcription-PCR system (Thermo Fisher Scientific,Cat# 4485700).The reaction conditions were as follows: 20 seconds at 95°C for predenaturation,followed by 40 cycles of 15 seconds at 95°C for denaturation,30 seconds at 60°C for annealing,and 30 seconds at 72°C for extension.Primer sequences are shown inAdditional Table 2.The relative expression ratio of target gene mRNA was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression using the 2-ΔΔCTmethod.

Western blots

Following deep anesthetization of WT and 5×FAD mice using 2% sodium pentobarbital at a dose of 400 mg per body weight,tissues were extracted for western blotting.Supernatants were collected and protein concentration determined with a BCA Protein Assay Kit (Beyotime Biotechnology,Shanghai,China,Cat# P0010) using the bicinchoninic acid (BCA) method (Walker,1994).The samples were loaded onto polyacrylamide gels and electrophoresed.Then,these protein samples were transferred to polyvinylidene fluoride membranes,followed by blocking of the membranes in 5% non-fat milk for 2 hours at room temperature.After that,the membranes were incubated with the following primary antibodies overnight (prepared in antibody solution,5% BSA,1× Tris-buffered saline [TBS] with 0.1% Tween 20): rabbit anti-RNA binding motif 8A (RBM8A,1:5000,ProSci,San Diego,CA,USA,Cat# 29-348,RRID: AB_10910328),rabbit anti-amyloid precursor protein (APP,1:5000,Abcam,Cat# ab180140,RRID:AB_2895244),mice anti-3xFlag (1:5000,Abcam,Cat# ab124462,RRID:AB_11000959),and mouse antibeta actin (β-Actin,1:5000,Multi Sciences,Zhejiang,China,Cat# 70-ab008-100,RRID:AB_2750915).The membranes were washed in TBS-Tween and incubated for 1 hour at room temperature with the following secondary antibodies: goat anti-rabbit IgG (H+L;1:20000,Bio World,Dublin,OH,USA,Cat# BS13278,RRID: AB_2773728) and goat anti-mouse IgG (H+L;1:5000,Jackson Immuno-Research Labs,Cat# 112-035-003).After washing the membranes in TBS-Tween,ECL Chemiluminescent Substrate Reagent (EpiZyme,Shanghai,China,Cat# SQ201) was used to visualize immunoreactive bands with an Amersham Imager 600 Chemiluminescent Imaging System (GE Healthcare,Beijing,China).Finally,the band densities were evaluated with ImageJ Software version 1.8.0.Protein band densities were normalized to β-actin as an internal reference.

BrdU administration

To examine the proliferation of neural progenitor cells,5×FAD mice were given daily intraperitoneal injections of BrdU (Sigma-Aldrich,St.Louis,MO,USA,Cat# 59-14-3) for 3 consecutive days,beginning 11 days after virus injection into the DG region.The BrdU dose was 50 mg/kg in sterile PBS.The mice were euthanized 24 hours after their final BrdU injection.Brain slices were then collected and pretreated prior to immunofluorescence staining.Brain slices were washed three times with 1× PBS for 5 minutes and treated with pre-cooled 1 M hydrogen chloride at 4°C for 10 minutes.Subsequently,the slices were incubated with 2 M hydrogen chloride at room temperature for an additional 10 minutes,followed by further 20-minute incubation with 2 M hydrogen chloride preheated to 37°C.Finally,the slices were incubated with 0.1 M sodium borate solution at room temperature for 12 minutes.

Statistical analysis

No statistical methods were used to predetermine sample sizes;however,our sample sizes are similar to those reported in a previous publication (Liu et al.,2023).Blinded assessment was performed in both biochemical and histological analyses to minimize potential bias.All the samples were coded and read by a reviewer who had no knowledge of the experimental groups during analysis.All data in the present study are presented as mean ± standard deviation (SD).An unpaired Student’st-test was used to evaluate differences between two groups.For multiple group comparisons,the Tukey correction after one-way analysis of variance (ANOVA) was used to assess significant significance.All statistical analyses were performed on GraphPad Prism 9.0.0 software (GraphPad,San Diego,CA,USA,www.graphpad.com).Data indicated statistical significance when theP-value was <0.05.

Results

Overexpression of Rbm8a in the DG brain region of 5×FAD mice

qRT-PCR and western blotting were performed to investigate age-relatedRbm8achanges in 1,2,6,and 12-month WT mice,revealing a marked decrease inRbm8aexpression with aging (P<0.01 orP<0.05;Additional Figure 1A-C).Additionally,potential changes inRbm8aexpression were explored in 5×FAD mice,showing statistically significant decreases compared with WT mice (P<0.05;Additional Figure 1DandE).Moreover,5×FAD mice had significantly higher levels of APP compared with WT mice (P<0.05;Additional Figure 1DandE),which confirmed the validity of the 5×FAD mice as an AD mouse model.Based on these observations,overexpression ofRbm8amight represent a viable therapeutic strategy for addressing the cognitive decline in AD.Here,we generated aRbm8aoverexpression virus and its control virus (Additional Figure 2A).Immunofluorescence staining ofRbm8aconfirmedRbm8aoverexpression mediated by AAV infection,as indicated by co-immunostaining ofRbm8a(red) and AAV-GFP (green) (Additional Figure 2B).We quantified 3×FLAG-tag and RBM8A protein expression in the DG of 5×FAD-Ctrl and 5×FAD-Rbm8amice.5×FAD-Rbm8amice showed significantly elevated expression of 3xFLAG-tag and RBM8A compared with the 5×FAD-Ctrl group (P<0.01;Additional Figure 2C-E).To confirm the specific cell types targeted by AAV,an experiment was designed (Figure 1AandB) to label common cell types within the DG region (including astrocytes,microglia,and neurons) using immunofluorescence staining.The results revealed that AAV primarily infected mature neurons,constituting about 47.5% to 51.7% of infected cells (Figure 1C-F),with no observed infection in astrocytes or microglia.Additionally,other infected cells were speculated to be neural cells such as stem cells,neural progenitor cells,and immature neurons.The number of mature neurons infected by AAV in the DG was quantified,indicating a statistically significant increase in the 5×FADRbm8agroup compared with the control group (P<0.01;Figure 1G).These results strongly supported the notion thatRbm8aplays an integral role in the production of neurons.

Figure 1｜Neurons but not glial cells were infected by the AAV2-GFP virus in the DG region of the hippocampus.

Figure 2｜Reduced soluble Aβ and plaque formation in the DG of 5×FAD mice with Rbm8a overexpression.

Reduced soluble Aβ and plaque formation in the DG of 5×FAD mice with

Rbm8a overexpression

Extensive studies have shown that the accumulation of amyloid protein in the brain is a pathological hallmark of the onset and progression of AD (Abdul-Hay et al.,2012).This study’s primary goal was to determine if augmenting the expression of RBM8A can mitigate amyloid protein deposition and plaque formation in AD.Therefore,immunofluorescence staining was performed to analyze brain slices with Aβ-specific antibodies (Figure 2A),revealing a significant decrease in plaque coverage area (P<0.001 orP<0.05;Figure 2B) and an overall reduction in the number of plaques present (P<0.001 orP<0.01;Figure 2C) among both male and female 5×FAD mice followingRbm8aoverexpression in the DG.Interestingly,no changes were noted in terms of individual plaque size within the 5×FAD-Rbm8aexperimental group compared with the 5×FAD-Ctrl group (P>0.05;Figure 2D).Additionally,our results revealed that females in both the 5×FAD-Rbm8aand 5×FAD-Ctrl groups had a greater plaque-covered area and number of plaques within the DG region compared with males (P<0.01 orP<0.001;Figure 2A-D).ELISA testing was used to analyze the levels of soluble human Aβ40and Aβ42within the DG region of female 5×FAD mouse brain.The findings revealed a significant reduction in soluble Aβ production in 5×FAD-Rbm8amice compared with 5×FAD-Ctrl mice (P<0.01;Figure 2EandF).Hence,elevating RBM8A levels could potentially be an effective approach to reduce amyloid buildup in individuals with AD.

Reduced DNs surrounding plaques in the DG of 5×FAD mice following overexpression of Rbm8a

In the DG region of 5×FAD mice,a disrupted GFP signal was observed near plaques that appeared to be DNs,characterized by an misshapen cell morphology and higher GFP signal intensity.This indicates more GFP+cells might be impaired in 5×FAD-Ctrl mice,with reduced cell loss in 5×FAD-Rbm8amice.The disrupted GFP signal was confirmed by both anti-ATG9A (Figure 3A) and anti-LC3B (Figure 3D) markers.As opposed to ATG9A (a marker for early-onset DNs),LC3B is a marker for late-onset DNs,which are distributed relatively further from the core of amyloid plaques,as previously reported (Sharoar et al.,2019).Compared with the 5×FAD-Ctrl group,mice in the 5×FAD-Rbm8agroup exhibited a significant decrease in the number of Aβassociated DNs,including ATG9A+DNs (P<0.01;Figure 3B) and LC3B+DNs (P<0.05;Figure 3E).Furthermore,the ratio of 6E10+GFP+ATG9A+/GFP+6E10+(P<0.05;Figure 3C) or 6E10+GFP+LC3B+/GFP+6E10+(P<0.05;Figure 3F) was significantly reduced in the 5×FAD-Rbm8agroup relative to the 5×FADCtrl group.These findings indicate thatRbm8acan significantly inhibit the formation of DNs,offering a promising treatment option to reduce neuronal dysfunction and loss in AD.

Figure 3｜Reduced dystrophic neurites surrounding plaques following overexpression of Rbm8a in the DG of 5×FAD mice.

Rbm8a overexpression leads to a reduction in the number of microglia and astrocytes in the DG region of 5×FAD mice

Accumulating evidence suggests that glial cells can engulf and aggregate plaques,thereby reducing local plaque-associated toxicity (Sosna et al.,2018).Therefore,the effect ofRbm8aon gliosis and the phagocytic activity of surrounding glial cells towards amyloid plaques were explored.Immunofluorescence staining was used to detect microglia distribution and gliosis reaction (Figure 4A),revealing a decreased area occupied by IBA1+microglia (P<0.01;Figure 4B) in the DG region of the 5×FAD-Rbm8agroup compared with the 5×FAD-Ctrl group,while no significant difference in the average number of microglia surrounding each plaque was observed (P>0.05;Figure 4C).Furthermore,the astrocyte-occupied area in the 5×FAD-Rbm8agroup was reduced (P<0.05;Figure 4DandE),but no significant change was found in the average number of astrocytes surrounding each plaque (P>0.05;Figure 4F),compared with the 5×FAD-Ctrl group.Overall,these results suggested thatRbm8asuppressed the gliosis response of glial cells without affecting their phagocytic function towards amyloid plaques.

Figure 4｜ Reduced gliosis response of astrocytes and microglia in the DG of 5×FAD mice following Rbm8a overexpression.

Rbm8a overexpression in the DG improves cognitive impairment but not depression and anxiety-like behavior in 5×FAD mice

The reduction in plaque formation and gliosis reaction due toRbm8aoverexpression in the DG indicates thatRbm8acould potentially alleviate memory impairment in AD.To confirm this hypothesis,an open field test was performed to eliminate any confounding variables related to motor function on behavior tests (Figure 5A).The findings demonstrated no significant changes in behavior metrics such as distance traveled during movement (P>0.05;Figure 5B) and central activity duration (P>0.05;Figure 5C) compared between 5×FAD-Ctrl and 5×FAD-Rbm8agroup mice.Subsequently,a series of behavioral experiments was performed to verify the effect ofRbm8aon memory in AD,including the Y-maze,NORT,and CFC tests.The Y-maze test was conducted to evaluate spatial working memory (Figure 5D).A significant increase in the percentage of spontaneous alteration was observed in 5×FADRbm8agroup mice compared with 5×FAD-Ctrl group mice (P<0.0001;Figure 5E),but no significant difference was found in the number of arm entries (P>0.05;Figure 5F).In the NORT (Figure 5G),the DI showed a substantial increase in the 5×FAD-Rbm8agroup compared with 5×FAD-Ctrl mice (P<0.0001;Figure 5H).Finally,the CFC test (Figure 5I) found an increase in freezing behavior in 5×FAD-Rbm8amice compared with 5×FAD-Ctrl mice (P<0.05;Figure 5J).In addition,to investigate whetherRbm8aplayed a role in depressive-like behavior in AD,several depression-associated tests were conducted including the TST (Figure 5K) and FST (Figure 5L).However,no significant differences were observed in immobility time (Figure 5KandL).Our results suggested thatRbm8aoverexpression substantially ameliorated memory dysfunction in AD without demonstrating any concurrent effect on mood-related symptoms such as anxiety or depression.

Figure 5｜ Cognition but not depression and anxiety-like behavior were improved by Rbm8a overexpression in the DG of 5×FAD mice.

Rbm8a overexpression increases immature neurons in the DG of 5×FAD miceIn addition to plaque formation in the DG,neuron loss also contributes to memory impairment in AD.However,Rbm8ahas been shown to play a critical role in neural progenitor cell proliferation (Al Mamun et al.,2022).Therefore,whetherRbm8aoverexpression in the DG can lead to an increase in neuronal number is of great interest.This study found that the number of immature neurons (DCX+) and the percentage of DCX+GFP+cells relative to GFP+cells in the DG were significantly increased in 5×FAD-Rbm8a mice compared with 5×FAD-Ctrl mice (P<0.01;Figure 6A-C).Moreover,the relative expression levels of DCX mRNA showed a significant increase in 5×FAD-Rbm8amice compared with the 5×FAD-Ctrl group mice (P<0.01;Figure 6D).This indicates thatRbm8acan stimulate an increase in the number of immature neurons in AD.

Figure 6｜Rbm8a overexpression increases the number of immature neurons in the DG of 5×FAD mouse brain.

Increased neurogenesis by Rbm8a overexpression in the DG of 5×FAD mouse brain

Immature neurons develop from stem cells,and an increased number indicates neurogenesis in the DG.An experiment was conducted (Figure 7A) to determine the neurogenesis stage in whichRbm8ais involved (the process of neurogenesis is described by a schematic diagram [Figure 7B]).Anti-GFAPtargeted radial glial stem cells were not infected by the virus (Figure 7CandD),which indicated thatRbm8aoverexpression did not affect this stage in neurogenesis.In other neurogenesis stages,a significant increase in the number of SOX2+cells (P<0.05;Figure 7EandF) and DCX+cells (P<0.05;Figure 7HandI) were observed,as well as an increase in the percentage of SOX2+GFP+/GFP+cells (P<0.01;Figure 7G) and percentage of DCX+GFP+/GFP+cells (P<0.001;Figure 7J).These results suggested that the increase in immature neurons was due to increased neural progenitor cells induced byRbm8aoverexpression in the DG.

Figure 7｜Rbm8a overexpression promotes neurogenesis in the DG of 5xFAD mouse brain.

Increased proliferation of neural progenitor cells induced by Rbm8a overexpression in the DG of 5×FAD mouse brain

During neurogenesis,neural stem cells and progenitor cells proliferate and differentiate into newborn neurons (Lindsey et al.,2018).The upregulation ofRbm8ain the DG was hypothesized to induce self-proliferation of neural progenitor cells,resulting in increased cell numbers.To confirm this hypothesis,the experiment illustrated in the schematic diagram was performed (Figure 8A).Immunofluorescence staining using anti-BrdU and anti-SOX2 (Figure 8B) revealed a significant rise in the quantity of SOX2+BrdU+cells (P<0.01;Figure 8C),as well as an increase in the proportion of SOX2+BrdU+/SOX2+cells (P<0.01;Figure 8D) and BrdU+GFP+SOX2+cells (P<0.001;Figure 8E).In addition,a significantly greater percentage of GFP+SOX2+BrdU+/SOX2+GFP+cells was observed in the DG region of the 5×FAD-Rbm8agroup mice (P<0.0001;Figure 8F).These data indicated thatRbm8apromotes the proliferation of neural progenitor cells,which may further lead to an increase in immature neuronal cell density in the DG region.

Figure 8｜Increased proliferation of neural progenitor cells induced by Rbm8a overexpression in the DG.

Decreased expression of Adora2a and increased neurogenesis pathway by overexpression of Rbm8a

RNA sequencing was performed to explore howRbm8apromotes neurogenesis,reduces amyloid protein formation,and enhances cognitive function in AD.DEGs were identified by volcano plot,revealing both upregulated and downregulated expression (Figure 9AandB).Subsequently,the five most significantly upregulated genes were identified,namelyRbm8a,Rxfp1,Pou4f1,Otop2,andBace2,while the five most downregulated genes wereTac1,Adora2a,Rgs9,Rarb,andSerpina9(Figure 9C).According to the results of the GO-based analysis,the DEGs were found to be mainly enriched in processes related to amyloid protein formation,neurogenesis,and proliferation (Figure 9D).Based on the quantification of enrichment of different gene sets in various diseases,our results showed that neurodegenerative disorders are the second most gene-enriched disorder (Figure 9E).A Venn diagram was used to compare DEGs from neuronal cells,neurodegenerative disease,and cell proliferation datasets,identifying the adenosine A2a receptor gene (Adora2a) as the hub gene (Figure 9F).The enriched pathways withAdora2ain AD included pathways related to alcoholism,neuroactive ligand-receptor interaction pathway,and cyclic AMP (cAMP) signaling pathway (Figure 9G).Further experiments revealed that the relative mRNA expression levels ofRbm8awere significantly increased (P<0.001;Figure 9H),whileAdora2aexhibited a significant decrease (P<0.01;Figure 9I) in the 5×FAD-Rbm8a group mice compared with 5×FAD-Ctrl group mice.These findings suggested that overexpression ofRbm8ain the DG of 5×FAD mice promoted the proliferation of neural progenitor cells,leading to increased densities of newborn and mature neurons (Figure 10A).In addition,Rbm8a overexpression reduced AD-associated pathological phenotypes,such as plaque formation and gliosis response,and improved cognitive function in AD (Figure 10B).

Figure 9｜Decreased expression of Adora2a and increased neurogenesis pathway by Rbm8a overexpression in the DG.

Figure 10｜Working Model: Rbm8a overexpression promotes neurogenesis,alleviates pathological phenotypes,and improves cognitive function in 5×FAD mice.

Discussion

Our research shows thatRbm8aoverexpression decreased Aβ deposition,plaque formation,gliosis reaction,and reduced DNs but increased neurogenesis within the DG by enhancing neural progenitor cell proliferation,which ultimately mitigated cognitive behavior impairments in 5×FAD mice.Collectively,these observations indicate that expression levels ofRbm8aare critical for AD-associated pathology,which might be a potential target for delaying AD progression.

The DG of the hippocampus receives input from the entorhinal cortex and transmits information to other hippocampal subfields,like CA1 and CA3,serving as a critical component for preserving accurate memory and learning capabilities.Previous research has shown that neurogenesis continues to occur in the DG of the brain throughout lifespan,but decreases significantly during the aging process,particularly in persons diagnosed with AD (Babcock et al.,2021).Neurogenesis can be influenced by physical exercise and environmental enrichment,which show benefit for neurogenerative disease (Trigiani and Hamel,2017;Yu et al.,2021).Here,it was interesting for us to explore the function ofRbm8ain neurogenesis in an AD mouse model and its potential molecular mechanism in regulating Aβ-associated pathologies.

Rbm8ais an essential member of the nonsense-mediated decay pathway,playing a critical role in maintaining mRNA integrity and proper splicing.Additionally,earlier studies have demonstrated the ability ofRbm8ato promote proliferation in both neural progenitor cells and retinal progenitor cells,which indicated the potential ofRbm8ain promoting neurogenesis (Zou et al.,2015;Zhang et al.,2017).Our results demonstrated that the expression levels ofRbm8adeclined with aging,which was similar to alterations in neurogenesis.RestoringRbm8aexpression via AAV-Rbm8ainjection into bilateral DG enhanced spatial memory in 4-month-old 5×FAD mice.The memory improvement may be linked to increased neurogenesis induced byRbm8aoverexpression in the DG of the 5×FAD mice.Based on these findings,restoringRbm8aexpression levels was hypothesized to enhance neurogenesis in the DG.

FollowingRbm8aoverexpression in 5×FAD mice,intraperitoneal BrdU injections were administered for 3 consecutive days to label cells undergoing mitosis in the DG.Our findings indicated increased proliferation among neural progenitor cells,as well as higher numbers of immature and mature neurons.These results suggested that restoringRbm8aexpression enhanced neurogenesis in the DG of 5×FAD mouse brains.As reported previously,impaired neurogenesis is an early event that occurs before plaque formation (Scopa et al.,2020).Hence,the regulation of neurogenesis in the early stage may be a promising treatment strategy for AD.Hence,it is crucial to identify the specific regulatory mechanisms ofRbm8afor improving memory performance in AD.

To further investigate the specific role ofRbm8ain regulating neurogenesis in the DG,RNA sequencing was performed on tissue collected from 5×FAD mice followingRbm8aoverexpression.After observing numerous DEGs related to biological processes such as neurogenesis and cell proliferation,along with additional DEGs associated with neurodegenerative diseases and neural cells,our investigation identifiedAdora2aas the only hub gene.The expression ofAdora2awas downregulated in 5×FAD group mice withRbm8aoverexpression.Hence,we postulated that the enhanced neurogenesis induced byRbm8aoverexpression in the DG may be mediated byAdora2a,but this requires further confirmation in subsequent studies.Nonetheless,previous studies support a relationship betweenAdora2aand neurogenesis and memory function in AD.A previous study found that inhibition of the ADORA2A receptor (A2AR),encoded byAdora2a,can enhance spatial memory and hippocampal plasticity through adult neurogenesis (Laurent et al.,2016).Based on gene expression data from postmortem human brain,A2AR was found to be highly expressed in the hippocampus (including the DG and CA1 and CA2 regions),which was related to neurogenesis.Contrary to the inhibition of A2AR,overactivation of A2AR contributed to synaptic dysfunction and memory impairment in early AD,which was caused by increased synaptic release of ATP and a higher density and activity of ecto-5’-nucleotidase (CD73)-mediated formation of adenosine,thereby selectively activating A2AR (Goncalves et al.,2019).Our own research also provided evidence for a possible correlation betweenAdora2aand neurogenesis or neuron function.We found thatAdora2awas enriched in several signaling pathways related to neurogenesis and neuronal activity,such as the neuroactive ligand-receptor interaction pathway and cAMP signaling pathway.Previous studies have reported that the cAMP signaling pathway is involved in the modulation of neurogenesis,neuronal survival,and axon growth within the hippocampus.Therefore,we hypothesized thatAdora2aregulates neurogenesis via the cAMP signaling pathway (Sabbir and Fernyhough,2018;Zhang et al.,2018).Hence,it will be of great of interest to examine whether the enhanced neurogenesis induced by Rbm8a overexpression is mediated byAdora2ain future studies.

In AD,neuronal function is impaired or can even be lost owing to the buildup of amyloid plaques,which destroy the synaptic structures responsible for transmitting neuronal signals.This results in severe impairments to learning and memory capabilities.Based on our research,we found thatRbm8aoverexpression induced neurogenesis in 4-month-old AD mice.However,further experiments are needed to determine ifRbm8aimproves synaptic connections and function by enhancing neurogenesis.Nevertheless,our results demonstrate that environmental perturbation factors of the synapse can be inhibited byRbm8aoverexpression.For instance,the aggregation of amyloid into plaques tends to decrease,as do DNs and the gliosis response,potentially leading to a reduction in inflammatory factors that worsen synaptic function.These changes not only protect existing neurons and synapses but may also contribute to neurogenesis.Therefore,it would be worthwhile to investigate how Rbm8a regulates the environmental perturbation factors affecting neurogenesis.

As a toxic substance of neurogenesis,Aβ is produced by the cleavage of APP by multiple enzymes within neurons,ultimately forming oligomers that facilitate plaque formation and contribute to impaired neurogenesis in AD (Oyama et al.,1993;Wirths,2017).According to our results,Rbm8aoverexpression participated in reducing amyloid protein buildup and plaque formation.The RNA sequencing data indicated that such alleviation may be linked specifically to a decrease inAdora2agene expression levels induced byRbm8aoverexpression in DG.As reported previously,the elevated levels of Aβ and amyloid plaques increasedAdora2aexpression levels in the hippocampus and neocortex of aging mice (Orr et al.,2018).Caffeine,a nonselective blocker ofAdora2a,can improve cognitive function and reduce brain amyloid plaque production in animal models of amyloid pathology (Arendash et al.,2006;Dall’Igna et al.,2007).Hence,Rbm8awas speculated to reduce Aβ plaque formation by inhibitingAdora2aexpression.But further verification is required to confirm this theory.To summarize,the current study implies that Rbm8a mitigates plaque load by reducing its formation.

Sanchez-Mico et al.(2021) have established a causal relationship between DNs and the accumulation of plaques in AD.Specifically,their findings suggest that DNs may accelerate plaque growth.This discovery has also been corroborated by Sadleir et al.,(2016).The presence of DNs and plaque-induced swollen neurites increased neuronal susceptibility to damage and degeneration in the DG region in AD.This study revealed thatRbm8aupregulation led to a reduction in both early-and late-stage DNs,effectively shielding neurons from the detrimental effects of plaque-induced impairment.Furthermore,there is a well-established connection between plaque-associated neuroinflammation and AD pathology.A previous study found that the presence of plaques can result in the recruitment and activation of astrocytes and microglia (Delizannis et al.,2021).This can enhance plaque clearance through phagocytosis,but the secretion of pro-inflammatory molecules such as TNF-α,IL-6,IL-1β,and COX-2 exacerbate the neuroinflammatory response,accelerating AD pathology.Our data reveal thatRbm8aupregulation led to a reduction in the buildup of microglia and astrocytes within the DG area,indicating a decrease in astrogliosis and microgliosis.Rbm8aindirectly protects neurons from the detrimental effects of secreted inflammatory cytokines by reducing the recruitment of glial cells.

Our animal experiments lend support to the notion that enforcingRbm8aexpression can amplify neurogenesis,dampen amyloidosis,and optimize cognition;however,certain constraints should be acknowledged.First,while our research concentrated exclusively on the effect ofRbm8aon neurogenesis,we did not explore synaptic connectivity or neuronal structural plasticity.As a result,we plan to perform patch clamp tests in the future to unravel the precise mechanisms underlying the effect ofRbm8aon synaptic function.Second,by introducing AAV2-Rbm8a,we concentrated exclusively on a specific brain area;therefore,our findings may not mirror the global effects ofRbm8aacross diverse regions with neural stem cells.Finally,although our study focused on early-stage 5×FAD mice,howRbm8afunctions in late-stage AD where stem cell numbers are depleted remains an open question.

In summary,our present study indicates that overexpression ofRbm8aenhances neurogenesis,mitigates amyloid protein deposition and plaque formation,and enhances cognitive function in 5×FAD mice.Based on our experimental results,we hypothesize that downregulation of theAdora2agene mediates the neurogenesis and alleviates plaque formation induced byRbm8aoverexpression in the DG.Although this investigation provides insightful evidence,more comprehensive studies are necessary to validate our conclusions.

Acknowledgments:We thank Xiaoming Li,Rui Wang,and Ziwei Yang from the Molecular Imaging Core Facility (MICF) and Ying Xiong from the Molecular Cellular Core Facility at the School of Life Science and Technology,ShanghaiTech University,for providing technical support.

Author contributions:YW,CZ and XR designed and performed experiments and data analyses.CZ and CL contributed to sample preparation.XR,YW,and YL supervised the experiments and helped data analyses.CZ and XR wrote the manuscript.All authors have read and approved the manuscript.

Conflicts of interest:The authors declare no competing or financial interests.

Data availability statement:All relevant data are within the paper and its Additional files.

Open access statement:This is an open access journal,and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License,which allows others to remix,tweak,and build upon the work non-commercially,as long as appropriate credit is given and the new creations are licensed under the identical terms.

Additional files:

Additional Table 1:The primer sequences for qRT-PCR.

Additional Table 2:The differentially expressed genes list obtained by RNAseq in the dentate gyrus of 5×FAD-Rbm8a and 5×FAD-Ctrl groups.

Additional Figure 1:The expression of Rbm8a declines with aging in WT mice and is lower in 5×FAD mice compared with the WT mice.

Additional Figure 2:Construction and validation of AAV2 stenotype virus with Rbm8a overexpression.