Retained introns in phototransduction genes of 5xFAD mouse retina suggest vision impairment as an early diagnostic marker for Alzheimer’s disease

Retained introns in phototransduction genes of 5xFAD mouse retina suggest vision impairment as an early diagnostic marker for Alzheimer’s disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Retained introns in phototransduction genes of 5xFAD mouse retina suggest vision impairment as an early diagnostic marker for Alzheimer’s disease Hyun-Min Lee, Jinho Kim, Ji-Young Kim, Mi-Jin An, Geun-Seup Shin, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6392160/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Alzheimer’s disease (AD) is an age-related neurodegenerative disorder characterized by neuronal and synaptic loss in the brain, which leads to cognitive impairment and dementia. Therefore, early diagnosis by employing various biomarkers is crucial for preventing and treating AD. Although retinal pathology is an emerging biomarker associated with AD, detailed molecular mechanisms of retinal impairments remain unclear. Herein, we identified genome-wide dysfunction of alternative splicing in the early stage of 5xFAD transgenic mouse retina by performing RNA sequencing analysis. Notably, retained intron, highly enriched in phototransduction and retinal genes of 1.5-month-old 5xFAD mouse retina, was significantly associated with retinal physiological impairment of rod photoreceptors, as evidenced by electroretinogram (ERG) analysis. These results indicate that the abnormal scotopic ERG associated with global splicing impairment may be valuable as an early detection biomarker for AD. Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Molecular biology/Transcriptomics Alzheimer's disease (AD) Retina Alternative splicing Retained intron 5xFAD Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Alzheimer’s disease (AD) is a complex neurodegenerative disorder that leads to a gradual decline in cognitive functions, such as memory and behavior 1 , 2 . Although aging is the most notable risk factor for AD, the rate and extent of cognitive decline vary between individuals, influenced by environmental factors, personal experiences, and genetic predispositions 3 . Epigenetic changes and abnormal RNA splicing events at various AD susceptibility loci may contribute to the onset of AD in healthy brains 4 , 5 . With advances in transcriptomic and epigenomic technologies, potential neurodegenerative alterations in the AD brain can be detected based on evidence such as downregulation of neuronal functions and upregulation of the innate immune response 6 – 8 . Employing these approaches could aid in comprehensively clarifying alterations in gene expression regulation in the AD brain, revealing pathophysiology and potential therapeutic options 9 . Identifying molecular alterations as a biomarker in the brain of a living individual prior to AD development remains challenging. Nonetheless, ongoing efforts to develop non-invasive methods for early AD diagnosis without causing damage to the brain tissue are hindered by considerable hurdles. Alternative splicing (AS) is a regulatory mechanism that can markedly enhance proteome diversity by generating multiple mRNA transcripts from a single gene 10 . Disruptions in splicing patterns are frequently observed during normal aging and may be linked to susceptibility to various diseases, including cancers, cardiovascular disease, and several neuropathies 11 , 12 . Retained intron (RI) is a type of AS that is garnering interest in human health and disease investigations. RI occurs when an intron is inappropriately removed from the pre-mRNA transcript, resulting in the retention of an intron sequence in the mature mRNA. RI in the brain affects gene expression and protein production and has been implicated in the development of age-related diseases such as Parkinson’s disease and AD 13 , 14 . Amyloid beta (Aβ) deposition is not limited to the brain and can occur in various tissues and organs, including the bone, heart, and retina, contributing to a range of diseases and conditions beyond AD 15 – 17 . However, the correlation between splicing patterns and physiological functions in Aβ-deposited tissues remains poorly understood. The retina is an extension of the central nervous system, and its structure and functions share several similarities with those of the brain 18 . Owing to the homology between the retina and the brain, AD researchers have focused on the retina as a “window into the brain” 18 , 19 . Some classic ocular diseases exhibit neurodegenerative mechanisms similar to those in central nervous system disorders 20 . For instance, structural degeneration in the retinal nerve fiber layer at an early stage of AD 21 , 22 , visual dysfunction in AD 23 , 24 , Aβ deposition in five-familial AD (5xFAD) mouse retina 25 , 26 , and apoptosis pathway and photoreceptor degeneration induced by Aβ 27 – 30 have been reported. The retina can be considered a basic model of the brain, as certain pathological alterations and therapeutic approaches observed in the retina may have relevance or applicability to the brain. In this study, we performed RNA sequencing (RNA-seq) analysis to compare the retina transcriptomes of 1.5- and 6-month-old C57BL/6J and 5xFAD transgenic mice. We observed that the photoreceptor cell-maintenance and AD-related genes were upregulated in 1.5-month-old 5xFAD mouse retinas. Interestingly, we detected a global increase in the RI events in 1.5-month-old 5xFAD mouse retinas. The elevated RI genes correlated with visual perception and the maintenance process of photoreceptors. Furthermore, physiological retina functions of phototransduction were impaired in 1.5-month-old 5xFAD mouse retinas. Our results suggest that a global increase of RI may be a transcriptional signature of early onset, AD-like amyloidosis and support other clinical studies regarding visual dysfunction in patients with early-stage AD. Methods Animals The male C57BL/6J wild type mice and 5xFAD mice (20–27g, 1.5-6 months old) including human APP and presenilin 1 with the following mutations (APP K670N/M671L + I716V + V717I and PS1 M146L + L286V) were acquired from Jackson Laboratories (Tg6799; Jackson Laboratory, MMRRC Stock #034848). According to JAX, this strain has been maintained through backcrossing to C57BL/6J over 7 generation. The mice were kept in a controlled environment with a 12-hour cycle of light and darkness. The lights were turned off at 20:00. The mice had unrestricted access to food (5053 PicoLab® Rodent Diet 20, Labdiet, USA) and drink. Weaning occurred at 3 weeks of age, and same-sex groups of 1–6 mice were housed per polyethylene cages (30 × 19 × 13 cm) containing a wood-chip bedding and covered by a metal cage top and micro-isolator filter. The mice were anesthetized with isoflurane (CAS 26675-46-7), and cervical dislocation was performed 5 minutes later. Retinas were then harvested for immunohistochemical analysis and total RNA sequencing. Ethics declarations The experimental research protocol on animals was approved by Chung-Ang University granted approval for the experimental protocol (2020-00061) and followed the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines 31 . All procedures were performed in compliance with relevant guidelines and regulations, as stated in the Methods section. Immunohistochemistry In brief, retinas from 5xFAD mice were rapidly excised by removing the lenses on a cold plate with phosphate-buffered saline (PBS). Retinal tissues were then fixed in 4% paraformaldehyde (#15710; Electron Microscopy Sciences, Hatfield, PA, USA) and subsequently incubated in 10%, 20%, and 30% sucrose-PBS solutions for 1, 3, and 12 hours, respectively. The fixed retinas were embedded in either 7% agarose gel or OCT compound (4583, SAKURA, CA, USA), followed by sectioning with a vibratome (7000SMZ, Campden Instruments, England) or cryotome (CM1850, Leica, Germany), respectively. Following permeabilization in a solution of PBS containing 0.1% Triton X-100 for a duration of 10 minutes, the sections were subsequently blocked for 1 hour in a solution consisting of 5% normal goat serum in PBS with 0.1% Triton X-100. Specimens were then incubated overnight at 4°C with Aβ (1–42) primary antibodies (Cell Signaling, Danvers, USA). After washing with PBS, specimens were incubated with secondary antibodies for 1 hour at room temperature (23–25°C) in the dark. Nuclear counterstaining was performed using 5 µg/ml 4',6-diamidino-2-phenylindole (DAPI) for 3 minutes in the dark. Fluorescence images were captured using a Zeiss Axio Observer Z1 LSM 700 confocal microscope and processed with the ZEN program (ZEN lite 2011) (Carl Zeiss, Oberkochen, Germany). Electroretinogram (ERG) Mice underwent full-field flash electroretinography (ERG) to evaluate retinal function under scotopic and photopic conditions using the Espion e2 Visual Electrophysiology System (Diagnosys, Lowell, MA, USA). Briefly, for scotopic measurements, mice were dark-adapted overnight following anesthesia with 2X avertin solution (200 µL per 20 g mouse) containing tribromoethanol (T48402, SIGMA) and tert-amyl alcohol (240486; SIGMA). Prior to recording, pupil dilation was achieved using isoptoatropine (Alcon, Republic of Korea), followed by the application of a small drop of 2% hypromellose (Samil, Republic of Korea) to each eye. Ground and reference subdermal electrodes were placed subcutaneously near the hindquarters and between the eyes, respectively, while the mice were positioned on a heated pad (37 ℃). Recording electrodes were then placed on the cornea, and ERG was conducted simultaneously under scotopic conditions for both eyes, using progressively increasing green light stimulus intensities. After a 2-minute adaptation to 1.6 log cd·s/m², photopic responses were recorded. Total RNA isolation and reverse transcription-quantitative PCR (RT-qPCR) One hundred milligrams of mouse retina were used to extract total RNA using TRIzol solution (15596018, Invitrogen, CA, USA) following the directions provided by the manufacturer. To remove any contaminated genomic DNA, 10 µg of total RNA was incubated with 20 units of RNase-free DNase I and 4 units of RNase inhibitor (both from New England Biolabs) in DEPC-treated water. The reaction mixture was incubated for 1 hour at 37°C, followed by 10 minutes at 60°C. The RNA samples were then quantified spectrophotometrically at 260 nm, with an OD260:OD280 ratio ranging from 1.8 and 2.0, confirming their suitability for downstream analyses. cDNA synthesis was performed using Oligo-dT primers (6110A, Takara). 1 µg of total RNA was mixed 1 µL of oligo-dT, and the mixture was preheated at 70°C for 10 minutes to denature RNA secondary structures. The mixture was rapidly cooled to 4°C, after which 2 µL of 10X reverse-transcriptase buffer, 10 mM DTT, 200 units of reverse transcriptase (18064022, Invitrogen, CA, USA) were added to reach a final volume of 20 µL. The reverse-transcription reaction was carried out at 40°C for 60 minutes and terminated by heating at 94°C for 20 seconds. The resulting cDNA was stored at -20°C. Expression levels of mouse Cngb1 , Ppef2 , Rpgrip1 , Whrn , Prom1 , and Crocc mRNAs were assessed by RT-qPCR using specific primers listed in Supplementary Table 1. Primers were designed with Primer-BLAST and validated via a BLAST search in the NCBI database. The specificity of each amplified product was confirmed through melting curve analysis. Following the manufacturer's instructions, RT-qPCR was carried out using a Bio-Rad CFX96 Real-Time PCR Detection System using iQ SYBR Green PCR Supermix (#1708880, Bio-Rad, Hercules, CA, USA). The reference gene utilized for normalization was Gapdh , and the relative mRNA expression levels were computed using the 2 −(ΔΔCt) technique. All PCR reactions were conducted in duplicate for each experimental condition. Library preparation and RNA-seq The RNA-seq library was prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina, Inc., San Diego, CA, cat. no. RS-122–2002). Briefly, 100 ng of total RNA from each sample was utilized. Poly-A mRNA was isolated by binding to poly-T oligo-attached magnetic beads, followed by mRNA fragmentation. The cleaved RNA fragments were then reverse transcribed into double-stranded cDNA. Following the degradation of the RNA template, a random primer with an Illumina-compatible linker sequence at the 5' end was used to start second-strand synthesis. The resulting double-stranded cDNA library was purified with AMPure XP beads (A63881; Beckman Coulter, CA, USA) to remove all reaction components. The library was then amplified to add complete adapter sequences necessary for cluster generation, followed by another round of purification to remove PCR components. The quality and size distribution of the libraries were evaluated using an Agilent 2100 Bioanalyzer equipped with a High-Sensitivity DNA Chip (Agilent Technologies, Santa Clara, CA) Finally, the cDNA libraries were sequenced using paired-end 75 sequencing on an Illumina NextSeq 500 system (Illumina, Inc., San Diego, CA). Bioinformatical analysis for RNA-seq mRNA-seq data quality was assessed using FastQC (v.0.11.9). The low quality and TruSeq adapter sequence in mRNA-seq reads were trimmed using TrimGalore (v.0.6.6). Indices were generated from the GRCm38.p6.101 genome assembly sequence to align the representative transcript sequences to both the genome and transcriptome. The alignment of sequences to the reference genome was performed using STAR (v.2.7.1a) with the ENCODE v3 parameters. Alignment sequences were applied for transcript assembly and estimation of their abundances using RSEM (v.1.3.3). The statistics for differentially expressed transcripts (or genes) and fragments per kilobase of transcripts per million mapped reads (FPKM) were calculated using DESeq2 (v.1.32.0). Identification of differential RI events To detect and quantify RI events in all known introns, rMATS (v.4.1.2), IRFinder (v.1.3.0), and iREAD (v.0.8.9) were used. The analysis involved performing rMATS on trimmed FASTQ files with default parameters, and the resulting junction and exon counts were used for RI detection. IRFinder was performed from trimmed FASTQ with default parameters. Differentially expressed RI events were calculated using a generalized linear model in DESeq2. In the case of iREAD, bam files generated from the STAR aligner were used with the ENCODE v3 parameter. The read count output from iREAD was used as an input to DESeq2 for detecting RI events. Results Profiling of transcriptomic dynamics in the retinas of 5xFAD mice In instances of certain AD, Aβ was found to be deposited in entire retinal layers, encompassing the outer nuclear, outer plexiform, inner nuclear, inner plexiform, and ganglion cell layers. Consistently, we detected the accumulation of Aβ plaques in both 2- and 6-month-old 5xFAD mouse retinas (Fig. 1 A). The number and signal intensity of Aβ plaques in inner and outer segment (IS/OS) were significantly higher in 2-month-old 5xFAD than in wild type (WT) mouse retinas (Fig. 1 A). In 5xFAD brain tissues, aging was associated with a global increase in Aβ plaques (supplementary Fig. S1 A-C). To identify differentially expressed genes (DEGs) in 5xFAD mouse retinas, RNA-seq analysis was conducted on 1.5-month-old mice to examine early onset AD-like amyloidosis and on 6-month-old mice to assess AD-like amyloidosis progression (Fig. 1 B) 32 . Principal component analysis was employed to validate the most substantial variances among groups and to ensure reproducibility among biological replicates (Fig. 1 C, supplementary Fig. S1 D). The volcano plots depict the statistical significance and corresponding expression levels of differentially expressed transcripts (DETs) under each experimental condition (DESeq2 P value < 0.05, absolute log2 fold change abs (log 2 FC) ≥ 1 and variance-to-mean ratio ≤ 1) (supplementary Fig. S1 E). Compared with the retinas of 1.5-month-old WT mice, the retinas of 1.5-month-old 5xFAD mice exhibited 2,566 upregulated transcripts and 1,331 downregulated transcripts (supplementary Fig. S1 F, left). In 6-month-old mice, the DET analysis revealed 1,108 upregulated transcripts and 2,015 downregulated transcripts (supplementary Fig. S1 F, right). The integrated mean expression patterns of significant DETs are depicted in a scatter plot, with log2 fold-change for 5xFAD mouse retinas plotted along the x-axis (1.5-month-old) and y-axis (6-month-old), respectively (Fig. 1 D). We applied k-means clustering (k = 6) to DETs, with the value determined using the elbow method (supplementary Fig. S1 G). A heatmap was generated to visualize the expression patterns of the six clusters identified using k-means clustering (Fig. 1 E, supplementary Fig. S1 G). Given the observed structural regeneration and visual dysfunction in the early stages of 5xFAD mouse retinas, 21 , 22 we focused on clusters 1 and 4, which were specifically down- or upregulated in the retinas of 1.5-month-old 5xFAD mice. To identify transcriptomic pathways relevant to the 1.5-month-old group, gene ontology (GO) analysis was conducted on upregulated (n = 217) and downregulated (n = 188) genes. The upregulated DEGs were categorized into several functional groups, including “negative regulation of axonogenesis”, “photoreceptor cell maintenance”, “nervous system development”, and “apoptotic process” (Fig. 1 F, left panel). GO of downregulated DEGs was classified as “retinal rod cell differentiation” and “neuromuscular synaptic transmission” (Fig. 1 F, right panel). Interestingly, 22% of the upregulated genes in cluster 4 were found to overlap with the curated AD gene list from the Disease Gene Network (DisGeNet) database 33 (P value < 0.05, two-tailed \(\:{\chi\:}^{2}\) test with Yates’ correction) (Fig. 1 G). Previous studies have raised concerns about early retinal changes in 5xFAD mice due to the potential influence of retinal degeneration-associated alleles, such as Pde6b and Trem2 34–36 . To address this, we performed sequencing analysis of Pde6b rd 1 and Trem2 S148E —genes implicated in retinal degeneration—at 1.5 and 6 months of age in both WT and 5xFAD mice. Additionally, we examined other relevant alleles, including Prph2 and Rd3 . Our results confirmed that both control (C57BL/6J WT) and 5xFAD mice do not carry any mutations in these retinal degeneration-associated alleles (supplementary Fig. S2). These findings suggest that the gene expression changes observed in the early stages of 5xFAD may be related to the maintenance of retinal function and cell death. Furthermore, the altered gene expression patterns in the 5xFAD retina show a significant correlation with genes associated with Alzheimer's disease in the retina. Dysregulation of AS in the retina of 5xFAD mice As we previously found that changes in the cellular microenvironment can affect the splicing function in the cytotoxic responses and diseased tissues 37 , the cytotoxic effect of Aβ plaque in AS events was analyzed. To examine AS events in the retinas of 5xFAD mice, rMATS 38 was applied to identify splice site variants. Considering the retinas of 1.5-month-old 5xFAD mice, statistically significant (P value < 0.05) splice site variants in protein-coding genes were as follows: skipped exons (SE), 2,100 genes; RI, 745 genes; an alternative 5’-splice site (A5SS), 388 genes; an alternative 3’-splice site (A3SS), 588 genes; and mutually exclusive exons (MXE), 210 genes. Considering the retinas of the 6-month-old 5xFAD mice, significant splice site variants were as follows: SE, 2,496 genes; RI, 831 genes; A5SS, 428 genes; A3SS, 678 genes; and MXE, 446 genes (Fig. 2 A). Given the presence of increased RI during aging and in AD brain tissues 12 , we analyzed and compared RI in mouse retinas using three algorithms: IRFinder 39 , iREAD 40 , and rMATS. IRFinder calculates an intron retention (IR) ratio of intronic and exonic abundance to identify RI events. iREAD introduces the Shannon entropy score to measure the uniformity of the distribution patterns of mapped reads in RI regions. rMATS focuses on uniquely mapping read-in splice sites and uses Bayesian statistical frameworks. In the retina of 1.5-month-old 5xFAD mice, IRFinder detected 2,960 statistically significant events (P value < 0.05), whereas iREAD identified 489 events. In the retina of 6-month-old 5xFAD mice, 3,871 and 423 events were detected using IRFinder and iREAD, respectively (Fig. 2 B). IRFinder was used in the following analysis because it not only verified statistically significant RI events but also detected overlapped events using other algorithms. To identify only the RI effect, we focused on RI events in genes that showed non-significantly different expressions between WT and 5xFAD mouse retinas. Intron-retaining transcripts have lower expression levels owing to nonsense-mediated mRNA decay, which directly impacts the detectability of RI 41 . Therefore, we analyzed RI events identified in protein-coding genes that exhibited no differences in the mRNA expression level. The differential RI was extracted through DESeq2 P value < 0.05 and the difference of the IR ratio over 0.1 between the 1.5-month-old WT and 5xFAD mouse retinas (Fig. 2 C). Upon performing a pairwise comparison between the retinas of 5xFAD and WT mice, we identified 766 upregulated and 210 downregulated RI events in 5xFAD mice (Fig. 2 C). The same conditions were applied to the 6-month-old group to identify differential RI, yielding 208 and 741 RI events in the retinas of 5xFAD mice and WT mice, respectively (Fig. 2 D). FPKM of RI-enriched regions was significantly increased in the retina of 1.5-month-old 5xFAD mice than in WT mouse retinas when FPKM exon regions showed similar expression in both groups (Fig. 2 E). The profile plots of RI-enriched regions in 6-month-old 5xFAD mouse retina showed similar patterns (Fig. 2 F). Interestingly, the RI-enriched region in the 1.5-month-old group showed a more significant difference between groups and lower variance in replicates than those in the 6-month-old group. Taken together, AS events were identified in the early stages of 5xFAD mouse retinas, with notable and significant changes observed in RIs. Increased RI events in 5xFAD mouse retina We next attempted to identify specific genes in 5xFAD where RI can be prominently detected by performing additional analyses. Applying the criteria of FPKM > 1.0 in the RI region and variance-to-mean ratio < 1 across all groups, we acquired differentially enriched RIs. A total of 613 differentially RI-enriched regions were identified in the 1.5-month-old 5xFAD mouse retina, while 607 RI regions were identified in the 6-month-old 5xFAD mouse retina. Next, differentially expressed RI regions for each group were identified through the application of \(\:k\) -means algorithms (with k = 2), revealing 543 RI-enriched regions in the retina of 1.5-month-old 5xFAD mice (Fig. 3 A). The same algorithm was applied to identify 75 specific RI-enriched regions in 6-month-old 5xFAD mouse retina (Fig. 3 B). To further assess the functional importance of RI-enriched genes, we conducted the GO analysis. The genes of 5xFAD-specific RI regions in 1.5-month-old mice (n = 362) were involved in “photoreceptor cell maintenance”, “visual perception”, “cell projection organization,” and “DNA repair” (Fig. 3 C), while “ubiquitin-dependent protein catabolic process” and “protein deubiquitination” were enriched in the retina of 6-month-old 5xFAD mice (supplementary Fig. S3A). We observed an overlap of 22% between upregulated genes and curated AD genes in 1.5-month-old 5xFAD mice, as described in Fig. 1 G. Consistently, our results further revealed that approximately 18 and 19% of differential RI genes in the retinas of 1.5- and 6-month-old 5xFAD mice overlapped with curated AD genes (supplementary Fig. S3B), respectively. Notably, these differential RI genes demonstrated significant overlap with retinal marker genes, accounting for 32.81 and 39% in the retinas of 1.5- and 6-month-old 5xFAD mice, respectively (supplementary Fig. S3B). All RI regions and mRNA expressions are visualized using a circos plot to substantiate the presence of RI patterns in the retinas of 1.5-month-old 5xFAD mice (Fig. 3 D). The outmost circle displayed a scatter depicting log10(-P values) of significantly upregulated RI in the retina of 1.5-month-old mice. The second and third circles represent the expression of all protein-coding genes as log2 scale. The last circle shows retinal marker genes associated with crucial retinal functions, including retinal homeostasis, retinal development, and visual perception. A subset of RI events persisted within genes related to retinal functions, including Rpgrip1 , Pde6b , Gnb1 , and Cacna1f . These findings emphasize the occurrence of RI in early-stage 5xFAD mouse retinas, particularly in genes associated with retinal markers, including those involved in functional homeostasis. Effects of RIs of the retina of 5xFAD mice in physiological impairment To validate whether the RIs were enriched in retinal markers ( Cngb1 , Ppef2 , Rpgrip1 , Whrn , Prom1 , and Crocc ) in early-stage 5xFAD mouse retinas, we performed qPCR using a primer pair that recognizes the exon-intron junction and the RI region (Fig. 4 ). Integrated Genome View represented the level of the RI in 1.5-month-old WT (blue) and 5xFAD mouse retinas (red). The reduced signal from the RI indicated a lower presence of mRNA transcripts containing this intron, compared to the abundant exon signal (grey). Consistent with RNA-seq data, we detected significant differences in the level of RI of photoreceptor-specific genes between the retinas of WT and 5xFAD mice. To determine the physiological dysfunction of phototransduction by RI events, we analyzed the ERGs of 1.5- and 6-month-old WT and 5xFAD mouse retinas under dark-adapted (scotopic) and light-adapted (photopic) conditions. The magnitude of the scotopic ERG a-wave and b-wave corresponded to processes involving phototransduction in the OS of rod cells and the synaptic transmission between rod spherules and rod bipolar cells. Similarly, the photopic ERGs reflect both phototransduction in the OS of cone cells and the synaptic transmission between cone pedicles and cone bipolar cells. Interestingly, the ERG responses of 1.5-month-old 5xFAD mouse declined in shape compared with those of WT mouse under scotopic conditions (Fig. 5 A). At stimulus luminance, the scotopic a-wave decreased significantly in the retinas of 1.5-month-old 5xFAD mice (54.70 ± 5.374 versus 28.37 ± 1.767 µV at 0.7 log cdžsec/m 2 , P value = 0.0331 / 48.05 ± 2.475 versus 25.22 ± 7.665 µV at 1.0 log cdžsec/m 2 , P value = 0.0035) (Fig. 5 a, Fig. S3a, left). In addition, the scotopic b-wave amplitude decreased at both low and high stimuli in the retinas of 1.5-month-old 5xFAD mice (169.4 ± 18.88 versus 98.23 ± 20.39 µV at 0.7 log cdžsec/m 2 , P value = 0.0296), which can be attributed to compromised phototransduction between rod and bipolar cells (Fig. 5 A, supplementary Fig. S4A, left). Conversely, the amplitudes of both a-and b-waves under photopic conditions showed no significant differences between the retinas of 1.5- and 6-month-old WT and 5xFAD mice, indicating that cone function was not significantly affected in this context (Fig. 5 B, supplementary Fig. S4A, right). In addition, there was no significant alternation in the implicit time of a- and b‑waves between the 5xFAD and control conditions (supplementary Fig. S4B). These findings suggest the presence of an impaired phototransduction process from rod cells to rod-bipolar cells in the retinas of 1.5-month-old 5xFAD mice. This impairment appears to be linked to RI accumulation in photoreceptor-specific genes. Discussion With the rising global incidence of AD, largely driven by the aging population, extensive efforts have been dedicated to devising effective methods for screening high-risk individuals before the onset of AD symptoms or detecting AD in its early stages. In recent decades, substantial advancements have been made in comprehending the pathophysiology of AD and formulating intervention approaches. Nonetheless, there remains a lack of effective treatments and sufficient diagnostic tools for AD. Therefore, there is an urgent need for biomarkers for AD to enable early diagnosis and risk assessment for this condition. In the last decade, our comprehension of AD has expanded substantially beyond its well-established definitive indicators, namely cerebral Aβ plaques and neurofibrillary tangles. The presence of Aβ accumulation in the brain has been extensively documented, while the identification of these specific biomarkers in the retina has only recently been detected. In 2008, Ning and collaborators were the pioneers in demonstrating that the accumulation of Aβ in the 5xFAD mouse retina was linked to neurodegeneration 42 . Among various AD-related mouse models, we employed 5xFAD mice, well known for their early onset and the initiation of amyloid plaque formations. Our observations distinctly revealed the presence of AD in the retina, hippocampus, and cerebral cortex between 1.5- and 6-months of age (Fig. 1 a and Fig. S1 a, b). The retina functions as the primary sensory organ for vision and plays a crucial role within the central nervous system. The retina is regarded as an extension of the brain and a potential biomarker for AD. Recently, retinal biomarkers for the early detection of patients at high risk of AD have been discovered 43 – 46 . Considering that the retina serves as a “window” to the brain and offers a valuable avenue for exploring AD pathophysiology, the examination of retinal changes holds considerable potential in the early detection of AD 47 . However, precise mechanisms underlying the pathophysiology and alteration of gene expressions in AD remain poorly understood. AS is a critical factor influencing the transcriptome complexity in the AD transgenic mouse (5xFAD). Notably, AS governs a distinct group of genes that are differentially regulated at the transcriptional level. Consequently, AS is an additional and highly adaptable mechanism for understanding proper regulation of gene expression. Our research has identified elevated IR as a crucial result of dysregulated splicing in the context of aging and AD pathogenesis in the retina. Changes in splicing and IR at specific genes during aging may underlie AD progression from a normal state to different stages of AD. To investigate this hypothesis, it is crucial to analyze the IR patterns derived from the retina of WT mice (1.5- and 6- months) and mice with mild cognitive impairment and advanced stages of AD (1.5- and 6- months of 5xFAD) using RNA-seq data. In the present study, we clearly represented that IR occurs during the pre-disease state in the retina when pathological alterations could be observed in the brain (Fig. 2 ). Importantly, examining quantitative changes in transcription patterns during disease could reveal new candidate biomarkers for neurodegenerative diseases. Here, we observed the augmentation of the RI in six particular genes within the retina of 1.5-month-old 5xFAD mice. Among them, Cngb1 , Ppef2 , and Rpgrip1 are known to be involved in the phototransduction process and are crucial for the viability and functionality of photoreceptor cells. Cngb1 is a subunit of the cGMP-gated channel found in rod photoreceptors. Its primary function is to regulate the flow of ions into the OS of rod photoreceptors in response to light-induced changes in intracellular cGMP levels 48 , 49 . Ppef2 encodes a photoreceptor protein that also plays a role in rod phototransduction and dephosphorylates photoactivated rhodopsin 50 . Rpgrip1 encoded protein interacts with GTPase regulator protein and is a key component of cone and rod photoreceptors. Rpgrip1 is required for normal disk morphogenesis and disk organization in the OS of photoreceptor cells and for survival of photoreceptor cells 51 – 53 . Furthermore, Whrn , Prom1I , and Crocc are involved in the maintenance of the cilium of photoreceptors that include the periciliary membrane complex, ciliogenesis, and key regulators of disk morphogenesis 45 , 54 , 55 . ERG assesses the function of the retinal cells by exposing the eye to light stimuli and measuring the resulting electrical responses of these cells. In 6-month-old 5xFAD mice, amyloid plaques had expanded across the hippocampus and neocortical areas, and spatial learning deficiencies were noticeable around 4 months of age 32 , 56 ; synapse degeneration and dysfunction along with neuronal loss were detected in the hippocampus at approximately 6 months 57 . Interestingly, we observed that the ERG response of 1.5-month-old 5xFAD mice decreased significantly under scotopic conditions, indicating a defect in rod photoreceptors (Fig. 4 ). Comprehensive results between transcriptomic analysis and physiology alteration suggest that abnormal RI accumulation on retinal photoreceptor marker genes leads to early detectable ERG defects. In this context, the ERG pattern demonstrates a robust phenotype of AD that can be utilized for the timely identification of AD using non-invasive diagnostic techniques. Additional investigations using more notable cohorts of independent peripheral blood mononuclear cells from individuals with AD are essential to acquiring a comprehensive understanding of the intricate AS networks involved in this neurodegenerative disorder. Pursuing this objective in future academic endeavors is a formidable challenge. The functional roles of several AS isoforms and their potential influence on AD remain uncertain. Examining splicing dysregulation in AD could improve our current understanding of this complicated condition. Additionally, identifying novel disease biomarkers has been associated with the pathophysiology of other neurodegenerative diseases. This study suggests the potential application of a newly discovered biological marker in the retina for the early detection of AD. Declarations Acknowledgements The study was supported by Korea Environment Industry & Technology Institute (KEITI) through ‘‘Digital Infrastructure Building Project for Monitoring, Surveying and Evaluating the Environmental Health Program’’ (grant and award number: 2021003330007) funded by Korea Ministry of Environment (MOE). This work also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (Basic Research Laboratory: NRF-RS-2023-00220089; NRF-2023R1A2C1007657). Author contributions Overall scientific conceptualization, J.-W.K.; methodology and investigation, H.-M.L., J.-Y.K., J.K., G.-S.S., A.-R.J., Y.P., C.-H.K., Y.H., J.-H.L. and J.-W.K.; data analysis and scientific comments, H.-M.L., J.-Y.K., J.K., G.-S.S., A.-R.J., Y.P., C.-H.K., Y.H., J.-H.L., K. H. L., S. R. and J.-W.K.; statistical and bioinformatical analysis, J.K., Y.H., J.-Y.K., and J.-W.K.; writing – original draft, H.-M.L., J.-Y.K., J.K., and J.-W.K.; funding acquisition, J.-W.K.; supervision and project administration, J.-W.K. Competing interests The authors declare no competing interests Ethics approval The animal study protocol was approved by the Institutional Review Board of Chung-Ang University (2020-00061), and the approval date is July 27, 2020. Consent for publication All authors agree to the publication of this manuscript References Bertram, L., Lill, C. M. & Tanzi, R. E. The genetics of Alzheimer disease: back to the future. 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(\u003cstrong\u003eA\u003c/strong\u003e) Immunofluorescent confocal images of amyloid-β (red), 4′,6-diamidino-2-phenylindole (DAPI) (Blue)-stained retina of 2- and 6- month-old wild type (WT) and 5xFAD mice. (\u003cstrong\u003eB\u003c/strong\u003e) Experimental design and transcriptome analysis pipeline. (\u003cstrong\u003eC\u003c/strong\u003e) Principal component analysis plot of RNA sequencing data from the retinas of 1.5- and 6-month WT and 5xFAD mice. (\u003cstrong\u003eD\u003c/strong\u003e) Scatter plot illustrating transcript expression pattern changes in 1.5-month and 6-month groups. Upregulated transcripts are highlighted in pink (1.5-month-old) and red (6-month-old), while downregulated transcripts are highlighted in light blue (1.5-month-old) and blue (6-month-old). Filtering criteria used: (abs(log\u003csub\u003e2\u003c/sub\u003eFC) \u0026gt; 1.0, P value \u0026lt; 0.05 VMR ≤ 1) in each 1.5- and 6-month group. (\u003cstrong\u003eE\u003c/strong\u003e) Clustering of transcripts using k-means clustering from differentially expressed transcripts (a total of 7,020 transcripts). Upregulated transcripts are highlighted in red (cluster 4), while downregulated transcripts are highlighted in blue (cluster 1) in the 1.5-month-old group only.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eF\u003c/strong\u003e) Gene Ontology (GO) terms of biological process analysis in cluster 1 and cluster 4. (\u003cstrong\u003eG\u003c/strong\u003e) Overlap of DETs in 5xFAD groups with curated AD genes from DisGeNet. Statistical significance was assessed using a two-tailed chi-square test with Yates's continuity correction. AD, Alzheimer’s disease; DETs, differentially expressed transcripts\u003c/p\u003e","description":"","filename":"FinalFigures71.png","url":"https://assets-eu.researchsquare.com/files/rs-6392160/v1/65a30bfa15a339264bdd2b38.png"},{"id":82295112,"identity":"5b58e5d3-f93a-4c30-b700-400a9fdf8fcc","added_by":"auto","created_at":"2025-05-08 19:10:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":387726,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential RI mark in 5xFAD mouse retinas. (\u003cstrong\u003eA\u003c/strong\u003e) Alternative splicing events in protein-coding genes were assessed using the rMATS (P value \u0026lt; 0.05) for 1.5-month (blue) and 6-month (orange) old mice. (\u003cstrong\u003eB\u003c/strong\u003e) Venn diagram illustrating the significant RI events using IRFinder, iREAD, and rMATS in 1.5- and 6-month-old mice. (\u003cstrong\u003eC, D\u003c/strong\u003e) Scatter plots representing the difference of IR ratio in relation to log2 fold-change of gene expression for (\u003cstrong\u003eC\u003c/strong\u003e) of 1.5-month and (\u003cstrong\u003eD\u003c/strong\u003e) 6-month-old mice. Upregulated and downregulated RI events with strict filtering criteria (difference of IR ratio over 0.1, P value of RI \u0026lt; 0.05) in non-regulated genes (log2FC under 1 and P value \u0026gt; 0.05) are represented by red dots and blue dots, respectively. (\u003cstrong\u003eE, F\u003c/strong\u003e) Profile plots showing the upregulated (upper panel) and downregulated (lower panel) RI regions in (\u003cstrong\u003eE\u003c/strong\u003e) 1.5-month-old and (\u003cstrong\u003eF\u003c/strong\u003e) 6-month-old mice. Error regions indicate the mean \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;SD. RI, retained intron.\u003c/p\u003e","description":"","filename":"FinalFigures72.png","url":"https://assets-eu.researchsquare.com/files/rs-6392160/v1/9bdd68be1807a720c5742433.png"},{"id":82295113,"identity":"f81ac35d-bcfd-4273-be29-6ef48b068f47","added_by":"auto","created_at":"2025-05-08 19:10:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1366764,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis dynamics of RI patterns in 5xFAD mouse retina.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA, B\u003c/strong\u003e) Clustered heatmap of (\u003cstrong\u003eA\u003c/strong\u003e) 613 RIs in the retinas of 1.5-month-old and (\u003cstrong\u003eB\u003c/strong\u003e) 607 RIs in 6-month-old mice using k-means method. (\u003cstrong\u003eC\u003c/strong\u003e) \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe scatter plot illustrates confidence scores for enriched gene ontologies associated with clustering based on functional similarity in the semantic space. Gene Ontology (GO) analysis of biological processes was performed using the DAVID tool, highlighting upregulated regulatory interactions (RIs) in 1.5-month-old mice (362 genes in 543 events). (\u003cstrong\u003eD\u003c/strong\u003e) Circos plot displaying the integrated graphical views of transcriptomic and RI dynamics in the retina of 1.5-month-old 5xFAD mice. RI, retained intron.\u003c/p\u003e","description":"","filename":"FinalFigures73.png","url":"https://assets-eu.researchsquare.com/files/rs-6392160/v1/ac4a3fa2ad08fc1371db2309.png"},{"id":82294727,"identity":"62d69705-d5ba-4f74-beeb-b05479623a62","added_by":"auto","created_at":"2025-05-08 19:02:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44509,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental validation of RI at retinal marker genes in 5xFAD mouse retina. (\u003cstrong\u003eA, B\u003c/strong\u003e) Genomic region representation of RT-qPCR on Integrative Genome Browser (IGV) (left). The transcript expression level of RI at retinal marker genes between wild type (WT) and 5xFAD mice (right). RT-qPCR, reverse transcription-quantitative PCR. RI, retained intron.\u003c/p\u003e","description":"","filename":"FinalFigures74.png","url":"https://assets-eu.researchsquare.com/files/rs-6392160/v1/22ecf9ac3c9331467f7d2de4.png"},{"id":82294730,"identity":"a628f1a6-c4b1-4a31-a7b3-f8c6b88d60d5","added_by":"auto","created_at":"2025-05-08 19:02:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":59453,"visible":true,"origin":"","legend":"\u003cp\u003eEarly detection of physiological changes in 5xFAD mouse retina using electroretinogram (ERG). (\u003cstrong\u003eA, B\u003c/strong\u003e) Waterfall plot of ERGs at 5 green light stimulus points (OFF, -0.8, 0.7, 1.6, 2.8 log cd.sec/m2) (left). Each scotopic and photopic ERG measured using green light stimuli from -1.7 to 1.3 and -0.2 to 3.1 log cd.sec/m2, respectively, in 1.5-month-old wild type (WT) and 5xFAD mouse (Significant difference wave P value = 0.0035, each representative of n \u0026gt; 2) (right).\u003c/p\u003e","description":"","filename":"FinalFigures75.png","url":"https://assets-eu.researchsquare.com/files/rs-6392160/v1/df09622bb3bdc4a416a1bb9a.png"},{"id":87219358,"identity":"bec91d17-81c5-46f0-b502-bd33244f2b61","added_by":"auto","created_at":"2025-07-21 16:04:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3108953,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6392160/v1/41c2f236-2136-4eec-9781-166aed0fe91f.pdf"},{"id":82295475,"identity":"674e8153-f104-4c20-af03-4fa988736908","added_by":"auto","created_at":"2025-05-08 19:18:31","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":862932,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6392160/v1/29a3ff49ca77c76877878787.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Retained introns in phototransduction genes of 5xFAD mouse retina suggest vision impairment as an early diagnostic marker for Alzheimer’s disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is a complex neurodegenerative disorder that leads to a gradual decline in cognitive functions, such as memory and behavior\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Although aging is the most notable risk factor for AD, the rate and extent of cognitive decline vary between individuals, influenced by environmental factors, personal experiences, and genetic predispositions\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Epigenetic changes and abnormal RNA splicing events at various AD susceptibility loci may contribute to the onset of AD in healthy brains\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. With advances in transcriptomic and epigenomic technologies, potential neurodegenerative alterations in the AD brain can be detected based on evidence such as downregulation of neuronal functions and upregulation of the innate immune response\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Employing these approaches could aid in comprehensively clarifying alterations in gene expression regulation in the AD brain, revealing pathophysiology and potential therapeutic options\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Identifying molecular alterations as a biomarker in the brain of a living individual prior to AD development remains challenging. Nonetheless, ongoing efforts to develop non-invasive methods for early AD diagnosis without causing damage to the brain tissue are hindered by considerable hurdles.\u003c/p\u003e \u003cp\u003eAlternative splicing (AS) is a regulatory mechanism that can markedly enhance proteome diversity by generating multiple mRNA transcripts from a single gene\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Disruptions in splicing patterns are frequently observed during normal aging and may be linked to susceptibility to various diseases, including cancers, cardiovascular disease, and several neuropathies\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Retained intron (RI) is a type of AS that is garnering interest in human health and disease investigations. RI occurs when an intron is inappropriately removed from the pre-mRNA transcript, resulting in the retention of an intron sequence in the mature mRNA. RI in the brain affects gene expression and protein production and has been implicated in the development of age-related diseases such as Parkinson\u0026rsquo;s disease and AD\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Amyloid beta (Aβ) deposition is not limited to the brain and can occur in various tissues and organs, including the bone, heart, and retina, contributing to a range of diseases and conditions beyond AD\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, the correlation between splicing patterns and physiological functions in Aβ-deposited tissues remains poorly understood.\u003c/p\u003e \u003cp\u003eThe retina is an extension of the central nervous system, and its structure and functions share several similarities with those of the brain\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Owing to the homology between the retina and the brain, AD researchers have focused on the retina as a \u0026ldquo;window into the brain\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Some classic ocular diseases exhibit neurodegenerative mechanisms similar to those in central nervous system disorders\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. For instance, structural degeneration in the retinal nerve fiber layer at an early stage of AD\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, visual dysfunction in AD\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, Aβ deposition in five-familial AD (5xFAD) mouse retina\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and apoptosis pathway and photoreceptor degeneration induced by Aβ\u003csup\u003e\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e have been reported. The retina can be considered a basic model of the brain, as certain pathological alterations and therapeutic approaches observed in the retina may have relevance or applicability to the brain.\u003c/p\u003e \u003cp\u003eIn this study, we performed RNA sequencing (RNA-seq) analysis to compare the retina transcriptomes of 1.5- and 6-month-old C57BL/6J and 5xFAD transgenic mice. We observed that the photoreceptor cell-maintenance and AD-related genes were upregulated in 1.5-month-old 5xFAD mouse retinas. Interestingly, we detected a global increase in the RI events in 1.5-month-old 5xFAD mouse retinas. The elevated RI genes correlated with visual perception and the maintenance process of photoreceptors. Furthermore, physiological retina functions of phototransduction were impaired in 1.5-month-old 5xFAD mouse retinas. Our results suggest that a global increase of RI may be a transcriptional signature of early onset, AD-like amyloidosis and support other clinical studies regarding visual dysfunction in patients with early-stage AD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eThe male C57BL/6J wild type mice and 5xFAD mice (20\u0026ndash;27g, 1.5-6 months old) including human APP and presenilin 1 with the following mutations (APP K670N/M671L\u0026thinsp;+\u0026thinsp;I716V\u0026thinsp;+\u0026thinsp;V717I and PS1 M146L\u0026thinsp;+\u0026thinsp;L286V) were acquired from Jackson Laboratories (Tg6799; Jackson Laboratory, MMRRC Stock #034848). According to JAX, this strain has been maintained through backcrossing to C57BL/6J over 7 generation. The mice were kept in a controlled environment with a 12-hour cycle of light and darkness. The lights were turned off at 20:00. The mice had unrestricted access to food (5053 PicoLab\u0026reg; Rodent Diet 20, Labdiet, USA) and drink. Weaning occurred at 3 weeks of age, and same-sex groups of 1\u0026ndash;6 mice were housed per polyethylene cages (30 \u0026times; 19 \u0026times; 13 cm) containing a wood-chip bedding and covered by a metal cage top and micro-isolator filter. The mice were anesthetized with isoflurane (CAS 26675-46-7), and cervical dislocation was performed 5 minutes later. Retinas were then harvested for immunohistochemical analysis and total RNA sequencing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEthics declarations\u003c/h3\u003e\n\u003cp\u003eThe experimental research protocol on animals was approved by Chung-Ang University granted approval for the experimental protocol (2020-00061) and followed the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. All procedures were performed in compliance with relevant guidelines and regulations, as stated in the Methods section.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eIn brief, retinas from 5xFAD mice were rapidly excised by removing the lenses on a cold plate with phosphate-buffered saline (PBS). Retinal tissues were then fixed in 4% paraformaldehyde (#15710; Electron Microscopy Sciences, Hatfield, PA, USA) and subsequently incubated in 10%, 20%, and 30% sucrose-PBS solutions for 1, 3, and 12 hours, respectively. The fixed retinas were embedded in either 7% agarose gel or OCT compound (4583, SAKURA, CA, USA), followed by sectioning with a vibratome (7000SMZ, Campden Instruments, England) or cryotome (CM1850, Leica, Germany), respectively. Following permeabilization in a solution of PBS containing 0.1% Triton X-100 for a duration of 10 minutes, the sections were subsequently blocked for 1 hour in a solution consisting of 5% normal goat serum in PBS with 0.1% Triton X-100. Specimens were then incubated overnight at 4\u0026deg;C with Aβ (1\u0026ndash;42) primary antibodies (Cell Signaling, Danvers, USA). After washing with PBS, specimens were incubated with secondary antibodies for 1 hour at room temperature (23\u0026ndash;25\u0026deg;C) in the dark. Nuclear counterstaining was performed using 5 \u0026micro;g/ml 4',6-diamidino-2-phenylindole (DAPI) for 3 minutes in the dark. Fluorescence images were captured using a Zeiss Axio Observer Z1 LSM 700 confocal microscope and processed with the ZEN program (ZEN lite 2011) (Carl Zeiss, Oberkochen, Germany).\u003c/p\u003e\n\u003ch3\u003eElectroretinogram (ERG)\u003c/h3\u003e\n\u003cp\u003eMice underwent full-field flash electroretinography (ERG) to evaluate retinal function under scotopic and photopic conditions using the Espion e2 Visual Electrophysiology System (Diagnosys, Lowell, MA, USA). Briefly, for scotopic measurements, mice were dark-adapted overnight following anesthesia with 2X avertin solution (200 \u0026micro;L per 20 g mouse) containing tribromoethanol (T48402, SIGMA) and tert-amyl alcohol (240486; SIGMA). Prior to recording, pupil dilation was achieved using isoptoatropine (Alcon, Republic of Korea), followed by the application of a small drop of 2% hypromellose (Samil, Republic of Korea) to each eye. Ground and reference subdermal electrodes were placed subcutaneously near the hindquarters and between the eyes, respectively, while the mice were positioned on a heated pad (37 ℃). Recording electrodes were then placed on the cornea, and ERG was conducted simultaneously under scotopic conditions for both eyes, using progressively increasing green light stimulus intensities. After a 2-minute adaptation to 1.6 log cd\u0026middot;s/m\u0026sup2;, photopic responses were recorded.\u003c/p\u003e\n\u003ch3\u003eTotal RNA isolation and reverse transcription-quantitative PCR (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eOne hundred milligrams of mouse retina were used to extract total RNA using TRIzol solution (15596018, Invitrogen, CA, USA) following the directions provided by the manufacturer. To remove any contaminated genomic DNA, 10 \u0026micro;g of total RNA was incubated with 20 units of RNase-free DNase I and 4 units of RNase inhibitor (both from New England Biolabs) in DEPC-treated water. The reaction mixture was incubated for 1 hour at 37\u0026deg;C, followed by 10 minutes at 60\u0026deg;C. The RNA samples were then quantified spectrophotometrically at 260 nm, with an OD260:OD280 ratio ranging from 1.8 and 2.0, confirming their suitability for downstream analyses. cDNA synthesis was performed using Oligo-dT primers (6110A, Takara). 1 \u0026micro;g of total RNA was mixed 1 \u0026micro;L of oligo-dT, and the mixture was preheated at 70\u0026deg;C for 10 minutes to denature RNA secondary structures. The mixture was rapidly cooled to 4\u0026deg;C, after which 2 \u0026micro;L of 10X reverse-transcriptase buffer, 10 mM DTT, 200 units of reverse transcriptase (18064022, Invitrogen, CA, USA) were added to reach a final volume of 20 \u0026micro;L. The reverse-transcription reaction was carried out at 40\u0026deg;C for 60 minutes and terminated by heating at 94\u0026deg;C for 20 seconds. The resulting cDNA was stored at -20\u0026deg;C. Expression levels of mouse \u003cem\u003eCngb1\u003c/em\u003e, \u003cem\u003ePpef2\u003c/em\u003e, \u003cem\u003eRpgrip1\u003c/em\u003e, \u003cem\u003eWhrn\u003c/em\u003e, \u003cem\u003eProm1\u003c/em\u003e, and \u003cem\u003eCrocc\u003c/em\u003e mRNAs were assessed by RT-qPCR using specific primers listed in Supplementary Table\u0026nbsp;1. Primers were designed with Primer-BLAST and validated via a BLAST search in the NCBI database. The specificity of each amplified product was confirmed through melting curve analysis. Following the manufacturer's instructions, RT-qPCR was carried out using a Bio-Rad CFX96 Real-Time PCR Detection System using iQ SYBR Green PCR Supermix (#1708880, Bio-Rad, Hercules, CA, USA). The reference gene utilized for normalization was \u003cem\u003eGapdh\u003c/em\u003e, and the relative mRNA expression levels were computed using the 2\u003csup\u003e\u0026minus;(ΔΔCt)\u003c/sup\u003e technique. All PCR reactions were conducted in duplicate for each experimental condition.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLibrary preparation and RNA-seq\u003c/h2\u003e \u003cp\u003eThe RNA-seq library was prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina, Inc., San Diego, CA, cat. no. RS-122\u0026ndash;2002). Briefly, 100 ng of total RNA from each sample was utilized. Poly-A mRNA was isolated by binding to poly-T oligo-attached magnetic beads, followed by mRNA fragmentation. The cleaved RNA fragments were then reverse transcribed into double-stranded cDNA. Following the degradation of the RNA template, a random primer with an Illumina-compatible linker sequence at the 5' end was used to start second-strand synthesis. The resulting double-stranded cDNA library was purified with AMPure XP beads (A63881; Beckman Coulter, CA, USA) to remove all reaction components. The library was then amplified to add complete adapter sequences necessary for cluster generation, followed by another round of purification to remove PCR components. The quality and size distribution of the libraries were evaluated using an Agilent 2100 Bioanalyzer equipped with a High-Sensitivity DNA Chip (Agilent Technologies, Santa Clara, CA) Finally, the cDNA libraries were sequenced using paired-end 75 sequencing on an Illumina NextSeq 500 system (Illumina, Inc., San Diego, CA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBioinformatical analysis for RNA-seq\u003c/h3\u003e\n\u003cp\u003emRNA-seq data quality was assessed using FastQC (v.0.11.9). The low quality and TruSeq adapter sequence in mRNA-seq reads were trimmed using TrimGalore (v.0.6.6). Indices were generated from the GRCm38.p6.101 genome assembly sequence to align the representative transcript sequences to both the genome and transcriptome. The alignment of sequences to the reference genome was performed using STAR (v.2.7.1a) with the ENCODE v3 parameters. Alignment sequences were applied for transcript assembly and estimation of their abundances using RSEM (v.1.3.3). The statistics for differentially expressed transcripts (or genes) and fragments per kilobase of transcripts per million mapped reads (FPKM) were calculated using DESeq2 (v.1.32.0).\u003c/p\u003e\n\u003ch3\u003eIdentification of differential RI events\u003c/h3\u003e\n\u003cp\u003eTo detect and quantify RI events in all known introns, rMATS (v.4.1.2), IRFinder (v.1.3.0), and iREAD (v.0.8.9) were used. The analysis involved performing rMATS on trimmed FASTQ files with default parameters, and the resulting junction and exon counts were used for RI detection. IRFinder was performed from trimmed FASTQ with default parameters. Differentially expressed RI events were calculated using a generalized linear model in DESeq2. In the case of iREAD, bam files generated from the STAR aligner were used with the ENCODE v3 parameter. The read count output from iREAD was used as an input to DESeq2 for detecting RI events.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProfiling of transcriptomic dynamics in the retinas of 5xFAD mice\u003c/h2\u003e \u003cp\u003eIn instances of certain AD, Aβ was found to be deposited in entire retinal layers, encompassing the outer nuclear, outer plexiform, inner nuclear, inner plexiform, and ganglion cell layers. Consistently, we detected the accumulation of Aβ plaques in both 2- and 6-month-old 5xFAD mouse retinas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The number and signal intensity of Aβ plaques in inner and outer segment (IS/OS) were significantly higher in 2-month-old 5xFAD than in wild type (WT) mouse retinas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In 5xFAD brain tissues, aging was associated with a global increase in Aβ plaques (supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify differentially expressed genes (DEGs) in 5xFAD mouse retinas, RNA-seq analysis was conducted on 1.5-month-old mice to examine early onset AD-like amyloidosis and on 6-month-old mice to assess AD-like amyloidosis progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Principal component analysis was employed to validate the most substantial variances among groups and to ensure reproducibility among biological replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). The volcano plots depict the statistical significance and corresponding expression levels of differentially expressed transcripts (DETs) under each experimental condition (DESeq2 P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, absolute log2 fold change abs (log\u003csub\u003e2\u003c/sub\u003eFC)\u0026thinsp;\u0026ge;\u0026thinsp;1 and variance-to-mean ratio\u0026thinsp;\u0026le;\u0026thinsp;1) (supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE). Compared with the retinas of 1.5-month-old WT mice, the retinas of 1.5-month-old 5xFAD mice exhibited 2,566 upregulated transcripts and 1,331 downregulated transcripts (supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF, left). In 6-month-old mice, the DET analysis revealed 1,108 upregulated transcripts and 2,015 downregulated transcripts (supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF, right). The integrated mean expression patterns of significant DETs are depicted in a scatter plot, with log2 fold-change for 5xFAD mouse retinas plotted along the x-axis (1.5-month-old) and y-axis (6-month-old), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). We applied k-means clustering (k\u0026thinsp;=\u0026thinsp;6) to DETs, with the value determined using the elbow method (supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG). A heatmap was generated to visualize the expression patterns of the six clusters identified using k-means clustering (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG). Given the observed structural regeneration and visual dysfunction in the early stages of 5xFAD mouse retinas, \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e we focused on clusters 1 and 4, which were specifically down- or upregulated in the retinas of 1.5-month-old 5xFAD mice. To identify transcriptomic pathways relevant to the 1.5-month-old group, gene ontology (GO) analysis was conducted on upregulated (n\u0026thinsp;=\u0026thinsp;217) and downregulated (n\u0026thinsp;=\u0026thinsp;188) genes. The upregulated DEGs were categorized into several functional groups, including \u0026ldquo;negative regulation of axonogenesis\u0026rdquo;, \u0026ldquo;photoreceptor cell maintenance\u0026rdquo;, \u0026ldquo;nervous system development\u0026rdquo;, and \u0026ldquo;apoptotic process\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, left panel). GO of downregulated DEGs was classified as \u0026ldquo;retinal rod cell differentiation\u0026rdquo; and \u0026ldquo;neuromuscular synaptic transmission\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, right panel). Interestingly, 22% of the upregulated genes in cluster 4 were found to overlap with the curated AD gene list from the Disease Gene Network (DisGeNet) database\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e (P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, two-tailed \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\chi\\:}^{2}\\)\u003c/span\u003e\u003c/span\u003e test with Yates\u0026rsquo; correction) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003ePrevious studies have raised concerns about early retinal changes in 5xFAD mice due to the potential influence of retinal degeneration-associated alleles, such as \u003cem\u003ePde6b\u003c/em\u003e and \u003cem\u003eTrem2\u003c/em\u003e\u003csup\u003e34\u0026ndash;36\u003c/sup\u003e. To address this, we performed sequencing analysis of \u003cem\u003ePde6b\u003c/em\u003e\u003csup\u003e\u003cem\u003erd\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTrem2\u003c/em\u003e\u003csup\u003e\u003cem\u003eS148E\u003c/em\u003e\u003c/sup\u003e \u0026mdash;genes implicated in retinal degeneration\u0026mdash;at 1.5 and 6 months of age in both WT and 5xFAD mice. Additionally, we examined other relevant alleles, including \u003cem\u003ePrph2\u003c/em\u003e and \u003cem\u003eRd3\u003c/em\u003e. Our results confirmed that both control (C57BL/6J WT) and 5xFAD mice do not carry any mutations in these retinal degeneration-associated alleles (supplementary Fig. S2).\u003c/p\u003e \u003cp\u003eThese findings suggest that the gene expression changes observed in the early stages of 5xFAD may be related to the maintenance of retinal function and cell death. Furthermore, the altered gene expression patterns in the 5xFAD retina show a significant correlation with genes associated with Alzheimer's disease in the retina.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDysregulation of AS in the retina of 5xFAD mice\u003c/h2\u003e \u003cp\u003eAs we previously found that changes in the cellular microenvironment can affect the splicing function in the cytotoxic responses and diseased tissues \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, the cytotoxic effect of Aβ plaque in AS events was analyzed. To examine AS events in the retinas of 5xFAD mice, rMATS\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e was applied to identify splice site variants. Considering the retinas of 1.5-month-old 5xFAD mice, statistically significant (P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) splice site variants in protein-coding genes were as follows: skipped exons (SE), 2,100 genes; RI, 745 genes; an alternative 5\u0026rsquo;-splice site (A5SS), 388 genes; an alternative 3\u0026rsquo;-splice site (A3SS), 588 genes; and mutually exclusive exons (MXE), 210 genes. Considering the retinas of the 6-month-old 5xFAD mice, significant splice site variants were as follows: SE, 2,496 genes; RI, 831 genes; A5SS, 428 genes; A3SS, 678 genes; and MXE, 446 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Given the presence of increased RI during aging and in AD brain tissues\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, we analyzed and compared RI in mouse retinas using three algorithms: IRFinder\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, iREAD\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and rMATS. IRFinder calculates an intron retention (IR) ratio of intronic and exonic abundance to identify RI events. iREAD introduces the Shannon entropy score to measure the uniformity of the distribution patterns of mapped reads in RI regions. rMATS focuses on uniquely mapping read-in splice sites and uses Bayesian statistical frameworks. In the retina of 1.5-month-old 5xFAD mice, IRFinder detected 2,960 statistically significant events (P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas iREAD identified 489 events. In the retina of 6-month-old 5xFAD mice, 3,871 and 423 events were detected using IRFinder and iREAD, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). IRFinder was used in the following analysis because it not only verified statistically significant RI events but also detected overlapped events using other algorithms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify only the RI effect, we focused on RI events in genes that showed non-significantly different expressions between WT and 5xFAD mouse retinas. Intron-retaining transcripts have lower expression levels owing to nonsense-mediated mRNA decay, which directly impacts the detectability of RI\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Therefore, we analyzed RI events identified in protein-coding genes that exhibited no differences in the mRNA expression level. The differential RI was extracted through DESeq2 P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and the difference of the IR ratio over 0.1 between the 1.5-month-old WT and 5xFAD mouse retinas (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Upon performing a pairwise comparison between the retinas of 5xFAD and WT mice, we identified 766 upregulated and 210 downregulated RI events in 5xFAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The same conditions were applied to the 6-month-old group to identify differential RI, yielding 208 and 741 RI events in the retinas of 5xFAD mice and WT mice, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). FPKM of RI-enriched regions was significantly increased in the retina of 1.5-month-old 5xFAD mice than in WT mouse retinas when FPKM exon regions showed similar expression in both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The profile plots of RI-enriched regions in 6-month-old 5xFAD mouse retina showed similar patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Interestingly, the RI-enriched region in the 1.5-month-old group showed a more significant difference between groups and lower variance in replicates than those in the 6-month-old group. Taken together, AS events were identified in the early stages of 5xFAD mouse retinas, with notable and significant changes observed in RIs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIncreased RI events in 5xFAD mouse retina\u003c/h2\u003e \u003cp\u003eWe next attempted to identify specific genes in 5xFAD where RI can be prominently detected by performing additional analyses. Applying the criteria of FPKM\u0026thinsp;\u0026gt;\u0026thinsp;1.0 in the RI region and variance-to-mean ratio\u0026thinsp;\u0026lt;\u0026thinsp;1 across all groups, we acquired differentially enriched RIs. A total of 613 differentially RI-enriched regions were identified in the 1.5-month-old 5xFAD mouse retina, while 607 RI regions were identified in the 6-month-old 5xFAD mouse retina. Next, differentially expressed RI regions for each group were identified through the application of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:k\\)\u003c/span\u003e\u003c/span\u003e-means algorithms (with k\u0026thinsp;=\u0026thinsp;2), revealing 543 RI-enriched regions in the retina of 1.5-month-old 5xFAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The same algorithm was applied to identify 75 specific RI-enriched regions in 6-month-old 5xFAD mouse retina (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To further assess the functional importance of RI-enriched genes, we conducted the GO analysis. The genes of 5xFAD-specific RI regions in 1.5-month-old mice (n\u0026thinsp;=\u0026thinsp;362) were involved in \u0026ldquo;photoreceptor cell maintenance\u0026rdquo;, \u0026ldquo;visual perception\u0026rdquo;, \u0026ldquo;cell projection organization,\u0026rdquo; and \u0026ldquo;DNA repair\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), while \u0026ldquo;ubiquitin-dependent protein catabolic process\u0026rdquo; and \u0026ldquo;protein deubiquitination\u0026rdquo; were enriched in the retina of 6-month-old 5xFAD mice (supplementary Fig. S3A). We observed an overlap of 22% between upregulated genes and curated AD genes in 1.5-month-old 5xFAD mice, as described in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG. Consistently, our results further revealed that approximately 18 and 19% of differential RI genes in the retinas of 1.5- and 6-month-old 5xFAD mice overlapped with curated AD genes (supplementary Fig. S3B), respectively. Notably, these differential RI genes demonstrated significant overlap with retinal marker genes, accounting for 32.81 and 39% in the retinas of 1.5- and 6-month-old 5xFAD mice, respectively (supplementary Fig. S3B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll RI regions and mRNA expressions are visualized using a circos plot to substantiate the presence of RI patterns in the retinas of 1.5-month-old 5xFAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The outmost circle displayed a scatter depicting log10(-P values) of significantly upregulated RI in the retina of 1.5-month-old mice. The second and third circles represent the expression of all protein-coding genes as log2 scale. The last circle shows retinal marker genes associated with crucial retinal functions, including retinal homeostasis, retinal development, and visual perception. A subset of RI events persisted within genes related to retinal functions, including \u003cem\u003eRpgrip1\u003c/em\u003e, \u003cem\u003ePde6b\u003c/em\u003e, \u003cem\u003eGnb1\u003c/em\u003e, and \u003cem\u003eCacna1f\u003c/em\u003e. These findings emphasize the occurrence of RI in early-stage 5xFAD mouse retinas, particularly in genes associated with retinal markers, including those involved in functional homeostasis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects of RIs of the retina of 5xFAD mice in physiological impairment\u003c/h2\u003e \u003cp\u003eTo validate whether the RIs were enriched in retinal markers (\u003cem\u003eCngb1\u003c/em\u003e, \u003cem\u003ePpef2\u003c/em\u003e, \u003cem\u003eRpgrip1\u003c/em\u003e, \u003cem\u003eWhrn\u003c/em\u003e, \u003cem\u003eProm1\u003c/em\u003e, and \u003cem\u003eCrocc\u003c/em\u003e) in early-stage 5xFAD mouse retinas, we performed qPCR using a primer pair that recognizes the exon-intron junction and the RI region (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Integrated Genome View represented the level of the RI in 1.5-month-old WT (blue) and 5xFAD mouse retinas (red). The reduced signal from the RI indicated a lower presence of mRNA transcripts containing this intron, compared to the abundant exon signal (grey). Consistent with RNA-seq data, we detected significant differences in the level of RI of photoreceptor-specific genes between the retinas of WT and 5xFAD mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the physiological dysfunction of phototransduction by RI events, we analyzed the ERGs of 1.5- and 6-month-old WT and 5xFAD mouse retinas under dark-adapted (scotopic) and light-adapted (photopic) conditions. The magnitude of the scotopic ERG a-wave and b-wave corresponded to processes involving phototransduction in the OS of rod cells and the synaptic transmission between rod spherules and rod bipolar cells. Similarly, the photopic ERGs reflect both phototransduction in the OS of cone cells and the synaptic transmission between cone pedicles and cone bipolar cells. Interestingly, the ERG responses of 1.5-month-old 5xFAD mouse declined in shape compared with those of WT mouse under scotopic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At stimulus luminance, the scotopic a-wave decreased significantly in the retinas of 1.5-month-old 5xFAD mice (54.70\u0026thinsp;\u0026plusmn;\u0026thinsp;5.374 versus 28.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.767 \u0026micro;V at 0.7 log cd\u0026#158;sec/m\u003csup\u003e2\u003c/sup\u003e, P value\u0026thinsp;=\u0026thinsp;0.0331 / 48.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.475 versus 25.22\u0026thinsp;\u0026plusmn;\u0026thinsp;7.665 \u0026micro;V at 1.0 log cd\u0026#158;sec/m\u003csup\u003e2\u003c/sup\u003e, P value\u0026thinsp;=\u0026thinsp;0.0035) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, Fig. S3a, left). In addition, the scotopic b-wave amplitude decreased at both low and high stimuli in the retinas of 1.5-month-old 5xFAD mice (169.4\u0026thinsp;\u0026plusmn;\u0026thinsp;18.88 versus 98.23\u0026thinsp;\u0026plusmn;\u0026thinsp;20.39 \u0026micro;V at 0.7 log cd\u0026#158;sec/m\u003csup\u003e2\u003c/sup\u003e, P value\u0026thinsp;=\u0026thinsp;0.0296), which can be attributed to compromised phototransduction between rod and bipolar cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, supplementary Fig. S4A, left). Conversely, the amplitudes of both a-and b-waves under photopic conditions showed no significant differences between the retinas of 1.5- and 6-month-old WT and 5xFAD mice, indicating that cone function was not significantly affected in this context (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, supplementary Fig. S4A, right). In addition, there was no significant alternation in the implicit time of a- and b‑waves between the 5xFAD and control conditions (supplementary Fig. S4B). These findings suggest the presence of an impaired phototransduction process from rod cells to rod-bipolar cells in the retinas of 1.5-month-old 5xFAD mice. This impairment appears to be linked to RI accumulation in photoreceptor-specific genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWith the rising global incidence of AD, largely driven by the aging population, extensive efforts have been dedicated to devising effective methods for screening high-risk individuals before the onset of AD symptoms or detecting AD in its early stages. In recent decades, substantial advancements have been made in comprehending the pathophysiology of AD and formulating intervention approaches. Nonetheless, there remains a lack of effective treatments and sufficient diagnostic tools for AD. Therefore, there is an urgent need for biomarkers for AD to enable early diagnosis and risk assessment for this condition.\u003c/p\u003e \u003cp\u003eIn the last decade, our comprehension of AD has expanded substantially beyond its well-established definitive indicators, namely cerebral Aβ plaques and neurofibrillary tangles. The presence of Aβ accumulation in the brain has been extensively documented, while the identification of these specific biomarkers in the retina has only recently been detected. In 2008, Ning and collaborators were the pioneers in demonstrating that the accumulation of Aβ in the 5xFAD mouse retina was linked to neurodegeneration\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Among various AD-related mouse models, we employed 5xFAD mice, well known for their early onset and the initiation of amyloid plaque formations. Our observations distinctly revealed the presence of AD in the retina, hippocampus, and cerebral cortex between 1.5- and 6-months of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea, b). The retina functions as the primary sensory organ for vision and plays a crucial role within the central nervous system. The retina is regarded as an extension of the brain and a potential biomarker for AD. Recently, retinal biomarkers for the early detection of patients at high risk of AD have been discovered\u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Considering that the retina serves as a \u0026ldquo;window\u0026rdquo; to the brain and offers a valuable avenue for exploring AD pathophysiology, the examination of retinal changes holds considerable potential in the early detection of AD\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, precise mechanisms underlying the pathophysiology and alteration of gene expressions in AD remain poorly understood.\u003c/p\u003e \u003cp\u003eAS is a critical factor influencing the transcriptome complexity in the AD transgenic mouse (5xFAD). Notably, AS governs a distinct group of genes that are differentially regulated at the transcriptional level. Consequently, AS is an additional and highly adaptable mechanism for understanding proper regulation of gene expression. Our research has identified elevated IR as a crucial result of dysregulated splicing in the context of aging and AD pathogenesis in the retina. Changes in splicing and IR at specific genes during aging may underlie AD progression from a normal state to different stages of AD. To investigate this hypothesis, it is crucial to analyze the IR patterns derived from the retina of WT mice (1.5- and 6- months) and mice with mild cognitive impairment and advanced stages of AD (1.5- and 6- months of 5xFAD) using RNA-seq data. In the present study, we clearly represented that IR occurs during the pre-disease state in the retina when pathological alterations could be observed in the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Importantly, examining quantitative changes in transcription patterns during disease could reveal new candidate biomarkers for neurodegenerative diseases. Here, we observed the augmentation of the RI in six particular genes within the retina of 1.5-month-old 5xFAD mice. Among them, \u003cem\u003eCngb1\u003c/em\u003e, \u003cem\u003ePpef2\u003c/em\u003e, and \u003cem\u003eRpgrip1\u003c/em\u003e are known to be involved in the phototransduction process and are crucial for the viability and functionality of photoreceptor cells. \u003cem\u003eCngb1\u003c/em\u003e is a subunit of the cGMP-gated channel found in rod photoreceptors. Its primary function is to regulate the flow of ions into the OS of rod photoreceptors in response to light-induced changes in intracellular cGMP levels \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePpef2\u003c/em\u003e encodes a photoreceptor protein that also plays a role in rod phototransduction and dephosphorylates photoactivated rhodopsin\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eRpgrip1\u003c/em\u003e encoded protein interacts with GTPase regulator protein and is a key component of cone and rod photoreceptors. \u003cem\u003eRpgrip1\u003c/em\u003e is required for normal disk morphogenesis and disk organization in the OS of photoreceptor cells and for survival of photoreceptor cells\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Furthermore, \u003cem\u003eWhrn\u003c/em\u003e, \u003cem\u003eProm1I\u003c/em\u003e, and \u003cem\u003eCrocc\u003c/em\u003e are involved in the maintenance of the cilium of photoreceptors that include the periciliary membrane complex, ciliogenesis, and key regulators of disk morphogenesis\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eERG assesses the function of the retinal cells by exposing the eye to light stimuli and measuring the resulting electrical responses of these cells. In 6-month-old 5xFAD mice, amyloid plaques had expanded across the hippocampus and neocortical areas, and spatial learning deficiencies were noticeable around 4 months of age\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e; synapse degeneration and dysfunction along with neuronal loss were detected in the hippocampus at approximately 6 months\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Interestingly, we observed that the ERG response of 1.5-month-old 5xFAD mice decreased significantly under scotopic conditions, indicating a defect in rod photoreceptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Comprehensive results between transcriptomic analysis and physiology alteration suggest that abnormal RI accumulation on retinal photoreceptor marker genes leads to early detectable ERG defects. In this context, the ERG pattern demonstrates a robust phenotype of AD that can be utilized for the timely identification of AD using non-invasive diagnostic techniques.\u003c/p\u003e \u003cp\u003eAdditional investigations using more notable cohorts of independent peripheral blood mononuclear cells from individuals with AD are essential to acquiring a comprehensive understanding of the intricate AS networks involved in this neurodegenerative disorder. Pursuing this objective in future academic endeavors is a formidable challenge. The functional roles of several AS isoforms and their potential influence on AD remain uncertain. Examining splicing dysregulation in AD could improve our current understanding of this complicated condition. Additionally, identifying novel disease biomarkers has been associated with the pathophysiology of other neurodegenerative diseases. This study suggests the potential application of a newly discovered biological marker in the retina for the early detection of AD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by Korea Environment Industry \u0026amp; Technology Institute (KEITI) through \u0026lsquo;\u0026lsquo;Digital Infrastructure Building Project for Monitoring, Surveying and Evaluating the Environmental Health Program\u0026rsquo;\u0026rsquo; (grant and award number: 2021003330007) funded by Korea Ministry of Environment (MOE). This work also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (Basic Research Laboratory: NRF-RS-2023-00220089; NRF-2023R1A2C1007657).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall scientific conceptualization, J.-W.K.; methodology and investigation, H.-M.L., J.-Y.K., J.K., G.-S.S., A.-R.J., Y.P., C.-H.K., Y.H., J.-H.L. and J.-W.K.; data analysis and scientific comments, H.-M.L., J.-Y.K., J.K., G.-S.S., A.-R.J., Y.P., C.-H.K., Y.H., J.-H.L., K. H. L., S. R. and J.-W.K.; statistical and bioinformatical analysis, J.K., Y.H., J.-Y.K., and J.-W.K.; writing \u0026ndash; original draft, H.-M.L., J.-Y.K., J.K., \u0026nbsp;and J.-W.K.; funding acquisition, J.-W.K.; supervision and project administration, J.-W.K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study protocol was approved by the Institutional Review Board of Chung-Ang University (2020-00061), and the approval date is July 27, 2020.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All authors agree to the publication of this manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBertram, L., Lill, C. 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Neurodegener\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e, 2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1750-1326-8-2\u003c/span\u003e\u003cspan address=\"10.1186/1750-1326-8-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer's disease (AD), Retina, Alternative splicing, Retained intron, 5xFAD","lastPublishedDoi":"10.21203/rs.3.rs-6392160/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6392160/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is an age-related neurodegenerative disorder characterized by neuronal and synaptic loss in the brain, which leads to cognitive impairment and dementia. Therefore, early diagnosis by employing various biomarkers is crucial for preventing and treating AD. Although retinal pathology is an emerging biomarker associated with AD, detailed molecular mechanisms of retinal impairments remain unclear. Herein, we identified genome-wide dysfunction of alternative splicing in the early stage of 5xFAD transgenic mouse retina by performing RNA sequencing analysis. Notably, retained intron, highly enriched in phototransduction and retinal genes of 1.5-month-old 5xFAD mouse retina, was significantly associated with retinal physiological impairment of rod photoreceptors, as evidenced by electroretinogram (ERG) analysis. These results indicate that the abnormal scotopic ERG associated with global splicing impairment may be valuable as an early detection biomarker for AD.\u003c/p\u003e","manuscriptTitle":"Retained introns in phototransduction genes of 5xFAD mouse retina suggest vision impairment as an early diagnostic marker for Alzheimer’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-08 19:02:26","doi":"10.21203/rs.3.rs-6392160/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-04T06:16:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-02T19:44:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136836380987175887693194401466721848185","date":"2025-05-07T15:52:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-06T13:12:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240084565594141823651237124635289676038","date":"2025-05-05T18:34:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-05T15:23:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T17:11:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-27T07:25:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-27T07:24:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4e4dbf59-59fd-4407-b625-247597e9bd3f","owner":[],"postedDate":"May 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48219918,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":48219919,"name":"Biological sciences/Molecular biology/Transcriptomics"}],"tags":[],"updatedAt":"2025-07-21T16:01:59+00:00","versionOfRecord":{"articleIdentity":"rs-6392160","link":"https://doi.org/10.1038/s41598-025-11065-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-15 15:57:10","publishedOnDateReadable":"July 15th, 2025"},"versionCreatedAt":"2025-05-08 19:02:26","video":"","vorDoi":"10.1038/s41598-025-11065-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-11065-z","workflowStages":[]},"version":"v1","identity":"rs-6392160","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6392160","identity":"rs-6392160","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}