Effects of the spi1a gene on Alzheimer's disease-related phenotypes in a zebrafish model

Effects of the spi1a gene on Alzheimer's disease-related phenotypes in a zebrafish model | 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 Research Article Effects of the spi1a gene on Alzheimer's disease-related phenotypes in a zebrafish model xia sun, zhixing wu, kangkang ge, yuying wang, lili tian, jiayu wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6961277/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Genome-wide sequence analysis has identified the SPI1 gene as a genetic risk factor for Alzheimer's disease (AD). SPI1 encodes the PU.1 protein, which plays a critical role in microglial development and immune responses, primarily studied in mouse models. However, no studies have yet reported the impact of the SPI1 gene on AD-related phenotypes in zebrafish. Therefore, this study utilized CRISPR/Cas9 gene editing to generate spi1a knockout zebrafish mutants, investigating the effects of spi1a loss-of-function on AD-associated phenotypes. The results showed that spi1a knockout led to reduced locomotor activity and increased brain cell apoptosis in larvae, while working memory, acetylcholinesterase (AChE) activity and Aβ1–42 levels remained unchanged. In contrast, adult spi1a knockout zebrafish exhibited significant cognitive decline, upregulated apoptosis-related genes, elevated AChE activity and increased Aβ1–42 accumulation. Transcriptomic analysis further revealed that spi1a knockout altered the expression of multiple AD-related genes and affected immune and inflammation-related signaling pathways. In conclusion, spi1a deficiency induced AD-like phenotypes in adult zebrafish. This study demonstrates the role of spi1a in modulating AD-related phenotypes in both larval and adult zebrafish, providing crucial insights into AD pathogenesis and establishing a valuable model for future high-throughput drug screening and therapeutic development. spi1a gene zebrafish model Alzheimer's disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights First demonstration of SPI1/spi1a ’s role in a zebrafish AD model. Age-dependent phenotypes and mechanistic insights. A versatile platform for high-throughput drug discovery. 1. Introduction Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder characterized by progressive cognitive dysfunction and behavioral impairments, representing the most prevalent form of dementia in the elderly population [1] . With the accelerating trend of global aging, the number of AD patients worldwide is projected to reach 78 million by 2030 and surge to 139 million by 2050, imposing tremendous psychological and socioeconomic burdens on patients, families, and society [2] . The clinical manifestations of AD primarily include progressive cognitive decline, memory deficits, mood disorders, aphasia, language dysfunction, and impaired ability to perform daily living activities. The hallmark pathological features of AD consist of extracellular amyloid-β (Aβ) plaque deposition and intracellular neurofibrillary tangles formed by hyperphosphorylated tau protein, which are closely associated with neuronal and synaptic loss, ultimately leading to the cognitive impairments observed in AD [3] . The zebrafish ( Danio rerio ), which shares high biological, structural, functional and genetic homology with humans, has been widely used in human disease modeling and represents a valuable model for Alzheimer's disease (AD) research [4] . Zebrafish possess orthologous genes involved in human AD-related pathways, enabling them to recapitulate specific features of AD-associated pathological processes observed in humans [5] . While lacking clearly defined cortical or hippocampal structures characteristic of mammalian brains, zebrafish exhibit comparable neuroanatomical organization to humans and can perform learning and memory tasks through brain regions functionally equivalent to these structures [6] . Both larval and adult zebrafish serve as excellent behavioral models that effectively simulate the complex behavioral alterations seen in AD patients [5, 7] . These distinctive characteristics establish zebrafish as an ideal model for AD research, not only facilitating elucidation of disease pathogenesis but also providing a robust platform for drug screening and therapeutic development that is comparable to mammalian in vivo and in vitro drug screening models. Genome-wide association studies (GWASs) have identified the SPI1 gene (encoding PU.1) as being associated with AD [8] . In mice, this gene is also known as Spi1 or Sfpi1 . PU.1, an E26 transformation-specific (ETS) family transcription factor, is primarily expressed in monocytes/macrophages, microglia, neutrophils, mast cells, B cells and early erythroid cells [9, 10] . Within the central nervous system (CNS), SPI1 is exclusively expressed in microglia, where its expression levels influence microglial transcription, activation and phenotype. In primary human microglia, PU.1 silencing alters the expression of AD-related genes and genes involved in antigen presentation and phagocytosis, suggesting that attenuating PU.1 expression may represent an effective therapeutic approach to limit microglia-mediated inflammatory responses in AD [11] . Studies in the murine BV-2 microglial cell line demonstrate that PU.1 knockout activates protein translation, antioxidant responses, and cholesterol/lipid metabolism pathways while reducing pro-inflammatory gene expression, conferring protective effects against AD; conversely, increased PU.1 expression enhances zymosan phagocytosis and promotes inflammatory responses, thereby elevating AD risk [12] . In mouse models with Spi1 knockdown or overexpression, Spi1 deficiency exacerbates multiple pathological features including insoluble amyloid-β (Aβ) levels, amyloid plaque deposition and gliosis, aggravating AD symptoms, whereas Spi1 overexpression significantly ameliorates these phenotypes and dystrophic neurites [13] . These findings collectively reveal the complex role of the Spi1 gene in AD pathogenesis. This study employed CRISPR/Cas9 gene editing technology to generate spi1a (the zebrafish ortholog of human SPI1 gene) mutant zebrafish, investigating the role of spi1a in AD pathogenesis using this model system. Through comprehensive behavioral analyses including light/dark transition tests, Y-maze, and T-maze experiments in both larval and adult zebrafish, we evaluated AD-associated learning and memory capabilities. The research further examined expression patterns of apoptosis-related genes, acetylcholinesterase activity and amyloid-β (Aβ1–42) levels in zebrafish. Transcriptomic profiling was conducted to elucidate spi1a's regulatory effects on AD-related genes and signaling pathways at the molecular level, thereby establishing a valuable zebrafish model for advancing Alzheimer's disease treatment research and facilitating novel drug development. 2. Materials and methods 2.1 Maintenance of Zebrafish All zebrafish used in this study were wild-type AB strain adults obtained from the China Zebrafish Resource Center. Both wild-type and mutant zebrafish were maintained at 27–29°C under a 14-hour light/10-hour dark cycle. Sexually mature adult males and females that had not mated for at least one week were selected for breeding at 1:1 or 2:1 ratios to obtain embryos, which were staged by hours post-fertilization (hpf) or days post-fertilization (dpf) or months post-fertilisation (mpf). Fertilized eggs were cultured in E3 medium at 28.5°C. All zebrafish experiments were conducted in compliance with the Zhejiang Laboratory Animal Welfare Guidelines and were approved by the Institutional Animal Care and Use Committee of Hangzhou Normal University. 2.2 Generation of spi1a mutant zebrafish The CRISPR/Cas9 gene editing in zebrafish was performed following established protocols [14] . The zebrafish orthologs of human SPI1 gene, spi1a and spi1b , were identified through the ZFIN (The Zebrafish Information Network). Using CRISPR/Cas9 technology, we first generated spi1a knockout zebrafish mutants. The target site for spi1a knockout (5′-GGGTAGAATGGTCCCCATGGCGG-3′) was designed using (CHOPCHOP). A mixture of sgRNA (50 ng/µL) and Cas9 protein (0.2 µg/µL) was microinjected into single-cell stage embryos. Mutations were verified by comparing with the wild-type sequence. Genotyping of offspring was performed using primers listed in supplementary Table 1. 2.3 Light-dark transition test in larval zebrafish The light-dark transition test was conducted as previously described to evaluate zebrafish locomotor activity and responsiveness [15] . Behavioral measurements were performed using the Danio Vision tracking system (Noldus Information Technology, Wageningen, the Netherlands). At 6 dpf, zebrafish larvae were individually placed in a 96-well plate (one larva per well). Following 20 minutes of dark adaptation, the test consisted of four alternating light-dark cycles (5 minutes light/5 minutes dark each). The video tracking system recorded the total swimming distance and velocity changes during the light-dark transitions for subsequent analysis. 2.4 Y-maze test in larval zebrafish To evaluate working memory capacity in zebrafish larvae, the free movement pattern (FMP) Y-maze test was performed according to previously reported experimental protocols [16] . The Y-maze apparatus consisted of three white acrylic walls arranged at 120° angles with a transparent acrylic base, measuring 1.2 cm × 0.4 cm × 0.3 cm (length × width × height). During testing, the Y-maze was filled with aquarium water to allow sufficient swimming depth. Prior to FMP Y-maze testing, no training or habituation was required, and larvae were randomly selected from the tank. Eighteen 6 dpf wild-type and spi1a -/- mutant larvae per group were allowed to freely explore the Y-maze for 1 hour without introducing changes in environment, novelty, or food rewards. The sequence of left and right turn choices made by larvae during the 1-hour free exploration period was recorded to investigate working memory alterations. 2.5 T-maze test in adult zebrafish The T-maze test was employed to evaluate learning and memory capabilities in adult zebrafish [17] , utilizing a color-biased and food-rewarded protocol following established methodology [18] . The apparatus consisted of an acrylic T-shaped maze with one long arm (50 cm×10 cm×10 cm) and two short arms (red and green arms, 20 cm×10 cm×10 cm). The long arm featured a starting compartment (10 cm×10 cm×10 cm) at its end, while one short arm contained a target chamber with brine shrimp, designated as the enriched chamber (EC) (10 cm×10 cm×10 cm) with green walls. The 5-day testing protocol (4 training days, 1 test day) involved groups of 17 zebrafish. Prior to behavioral testing, fish were acclimated in the T-maze for 1 hour. During trials, zebrafish were placed in the starting compartment for 1 minute before the sliding door opened. Fish received punishment when entering the red arm, while those entering the green arm could obtain brine shrimp. Each zebrafish underwent one daily trial for 4 consecutive training days. If a fish failed to locate the EC within 4 minutes, it was guided there and remained for 1 minute. Learning ability was assessed by recording the time required to swim from the start box to the EC. On the fifth test day, despite having been trained to preferentially enter the green arm, no punishment or food reward was provided. Memory retention was evaluated by comparing cumulative time spent in the EC. All behaviors were recorded for 4 minutes using the Danio Vision tracking system (Noldus), with analysis of distance traveled, time spent, and entry frequency for each zone. 2.6 Quantification of Aβ1–42 levels in zebrafish The Aβ1–42 levels in both spi1a -/- mutant larvae and adult brain tissues were measured using a Zebrafish Aβ1–42 ELISA Kit (202308, Shanghai Enzyme-linked Biotechnology Co., Ltd, China) following the manufacturer's instructions. For sample preparation, 6 dpf spi1a -/- larvae and 6 mpf spi1a -/- adult brain tissues were homogenized in cold physiological saline at a 1:9 (mass:volume) ratio in 1.5 mL tubes using ice-bath homogenization, followed by centrifugation at 5000 rpm for 10 minutes. The supernatants were collected for Aβ1–42 quantification. Absorbance was measured at 450 nm using a Spark multimode microplate reader (ThermoFisher, USA), and the optical density (OD) values were recorded to calculate sample concentrations. The Aβ1–42 concentrations were expressed as ng/mg of total protein. 2.7 Determination of Acetylcholinesterase (AChE) activity in zebrafish Following behavioral studies of spi1a -/- mutant larvae and adults, we further evaluated the effects on acetylcholinesterase (AChE) activity using an AChE Activity Assay Kit (D799813-0050, Sangon Biotech, China). For AChE determination, whole-body homogenates from 6 dpf spi1a -/- larvae and brain tissues from 6 mpf spi1a -/- adults were prepared by adding AChE extraction buffer at a 1:9 (mass:volume) ratio, followed by ice-bath homogenization and centrifugation at 8000 rpm for 10 minutes at 4°C to obtain supernatants. AChE activity was then measured according to the manufacturer's instructions. Absorbance at 412 nm was determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, USA) to calculate AChE activity. 2.8 Apoptosis staining in larval zebrafish Acridine orange (AO) staining was used to specifically label apoptotic cells with green fluorescence [19] . Twenty randomly selected 6 dpf wild-type and spi1a -/- mutant larvae were washed three times with distilled water (5 minutes each) and subsequently incubated in 5 µg/mL AO solution at 37°C in the dark for 60 minutes. Following staining, larvae were anesthetized with 0.03% MS-222 and immobilized using 3% methylcellulose. Apoptotic cells in zebrafish embryos were identified as bright, punctate green spots under fluorescence microscopy (Zeiss AxioObserver V12). 2.9 RNA Extraction and Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted from zebrafish using TRIzol reagent (Invitrogen, CA, USA), and complementary DNA (cDNA) was synthesized using the HiFiScript gDNA Removal RT Master Mix reverse transcription kit (CW Bio, Beijing, China). Quantitative real-time PCR was performed on a CFX96 real-time PCR detection system (Bio-Rad, USA) with 2 µL cDNA in 20 µL SYBR reaction mixture, with all samples run in triplicate. The primer sequences used in this experiment are listed in supplementary table 1 . 2.10 Transcriptome analysis Total RNA was extracted from 6 dpf spi1a -/- larvae and 6 mpf spi1a -/- adult brain tissues using TRIzol reagent (Invitrogen, CA, USA), with three independent biological replicates performed for each group. The concentration and purity of the RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), while RNA integrity was evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Transcriptome libraries were constructed following the manufacturer’s instructions of the VAHTS Universal V6 RNA-seq Library Prep Kit. Sequencing was performed on the Illumina Novaseq 6000 platform, generating 150 bp paired-end reads. Differential expression gene (DEG) analysis was conducted using DESeq2 software, with genes meeting the thresholds of q-value 2 or fold change < 0.5 defined as differentially expressed genes [20] . Subsequently, Gene Ontology (GO) and KEGG Pathway enrichment analyses based on the hypergeometric distribution algorithm were performed on the DEGs to identify significantly enriched functional terms [21, 22] . 2.11 Statistical analyses All experiments were performed with at least three independent biological replicates, and the data were analyzed using two-way ANOVA and paired two-tailed t-tests with GraphPad Prism 8.0 software. All experimental data are presented as mean ± standard error of the mean (SEM), and a p-value < 0.05 was considered statistically significant. 3. Results 3.1 Obtaining the spi1a-knockout zebrafish mutant line using CRISPR/Cas9 gene editing To investigate the function of the spi1a gene in zebrafish, we generated a spi1a -/- mutant line using the CRISPR/Cas9 system. A target site was designed on the third exon of zebrafish spi1a (Figure 1 A). By co-injecting Cas9 mRNA and gRNA into wild-type zebrafish embryos at the one-cell stage, we obtained founders carrying the desired mutation. After intercrossing the F1 generation, we identified homozygous mutants with a 14-bp deletion in the third exon (Figure 1 B-C). Sequence alignment analysis revealed that this mutation causes a frameshift, altering the amino acid sequence and disrupting the functional domain of the spi1a protein (Figure 1 D). These results demonstrate that we successfully established the spi1a -/- mutant model, which was subsequently used for further studies. 3.2 The effect of the spi1a gene on locomotor behavior and response ability in zebrafish larvae We found that knockout of the spi1a gene showed no visible impact on zebrafish growth and development (Figure S1) , and further investigated its impact on larval motor function. Zebrafish larvae exhibit distinct locomotor patterns in light and dark conditions, which are closely associated with anxiety, learning, memory, and defensive behaviors [23] . In this study, following the experimental protocol established by MacPhail et al. [24] , we observed that wild-type larvae moved slower in light and faster in dark conditions, whereas spi1a -/- mutants showed significantly reduced average speed during light-dark transitions (Figure 2 A). Quantitative analysis revealed significantly attenuated speed variations during light-dark alternations in spi1a -/- mutants (Figure 2 B), indicating impaired responsiveness. Furthermore, spi1a -/- mutants exhibited significantly decreased total swimming distance compared to wild-type controls during all light-dark cycles (Figure 2 C). These results demonstrate that spi1a knockout compromises both responsiveness and locomotor capacity in zebrafish. 3.3 The effect of the spi1a gene on learning and memory in the zebrafish This study evaluated learning and memory capabilities in zebrafish using the free-movement pattern (FMP) Y-maze for larvae and T-maze for adults. In larval Y-maze tests, compared to WT, spi1a knockout did not alter the search strategies of 6 dpf larvae, with alternating patterns (LRLR, RLRL) remaining predominant in exploration (Figure 3 A). No significant differences were observed between spi1a -/- and WT groups in either repetitive strategy usage frequency or alternating strategy occurrence (Figure 3 B-C), indicating that spi1a deletion does not affect larval working memory. However, in adult T-maze tests, although both WT and spi1a -/- adults showed decreased latency periods with training days, the spi1a -/- group exhibited significantly prolonged latency compared to WT (Figure 3 D), along with significantly reduced cumulative time spent in the EC zone (Figure 3 E), demonstrating substantial impairment in learning and memory capacities in spi1a -/- adults. These results demonstrate stage-specific regulatory roles of the spi1a gene in cognitive functions: while minimally impacting working memory in larvae, it becomes crucial for learning and memory in adult zebrafish. 3.4 The effect of the spi1a gene on Aβ1-42 plaques in zebrafish Aβ1-42 is a toxic amyloid peptide that accumulates extensively in the brains of AD patients, leading to amyloid deposition and subsequent neuronal damage [25] . Compared to 6 dpf WT, no significant changes in Aβ1-42 levels were observed in spi1a -/- larvae (Figure 4 A). Both WT and spi1a -/- showed relatively low Aβ1-42 levels at this stage, suggesting insufficient accumulation during larval development. We further examined Aβ1-42 in 6 mpf adult brain tissues, revealing significantly elevated Aβ1-42 levels in spi1a -/- adults compared to WT (Figure 4 B). These results demonstrate that spi1a knockout promotes Aβ1-42 accumulation specifically in adult zebrafish brains. 3.5 The effect of the spi1a gene on Acetylcholinesterase (AChE) Activity in Zebrafish Acetylcholine (ACh), a crucial neurotransmitter in brain tissue, is closely associated with learning and memory performance. Acetylcholinesterase (AChE) hydrolyzes acetylcholine and induces cholinergic neuronal dysfunction, serving as a primary therapeutic target for Alzheimer's disease (AD) [26] . In this experiment, we measured AChE activity of 6 dpf larvae and 6 mpf adult brain tissues to determine whether spi1a gene knockout affects neurotransmitters related to zebrafish learning and memory capabilities. Results showed no significant difference in AChE levels between spi1a -/- larvae and wild-type (WT) controls at 6 dpf (Figure 5 A). However, in 6 mpf adult brain, spi1a -/- zebrafish exhibited significantly increased AChE content in brain tissues compared to WT (Figure 5 B). These findings demonstrate that spi1a gene knockout leads to elevated acetylcholinesterase levels specifically in adult zebrafish brains. 3.6 The effect of the spi1a gene on brain cell apoptosis in zebrafish To investigate the effects of spi1a deficiency on brain cell apoptosis in zebrafish, we conducted systematic experimental analyses. Acridine orange (AO) staining in 6 dpf larvae revealed significantly increased apoptotic cells (manifested as granular green dots) in the brain regions of spi1a -/- group (Figure 6 A-B). Molecular analyses demonstrated consistent apoptosis-related gene expression patterns in both 6 dpf larvae (Figure 6 C-G) and 6 mpf adult brain tissues of spi1a -/- group (Figure S2): significant upregulation of pro-apoptotic genes ( p53 , bax , caspase3 , and caspase9 ) along with marked downregulation of the anti-apoptotic gene bcl2 . These results demonstrate that spi1a deletion activates the p53/caspase apoptotic pathway, leading to increased neuronal apoptosis in both larval and adult zebrafish brains. 3.7 The effect of the spi1a gene on Alzheimer's Disease-Associated Genes in Zebrafish Through transcriptome sequencing analysis, we investigated the impact of spi1a knockout on AD-related genes in zebrafish. Based on 787 AD-associated genes from the ZFIN database, we identified 18 AD-related DEGs (929 upregulated and 577 downregulated) in 6 dpf spi1a -/- larvae (Figure S3 A、C), and 21 AD-related DEGs (738 upregulated and 287 downregulated) in 6 mpf spi1a -/- adult brain tissues (Figure S3 B、D). Cross-analysis identified six core AD-related genes ( grna , ctss2.1 , casp23 , ccl35.1 , fas , tap2a ). qRT-PCR validation revealed: in larvae, grna , casp23 , tap2a and fas were significantly upregulated while ccl35.1 and ctss2.1 showed no significant changes (Figure S3 E-J); in adult brain, all genes except tap2a (downregulated) were significantly upregulated (Figure S4). These findings demonstrate that spi1a influences zebrafish AD-like phenotypes through stage-specific regulation of AD-related gene expression. 3.8 The effect of the spi1a gene on the gene expression profile To investigate the potential pathways through which the loss of the spi1a gene affects AD-related phenotypes, we performed GO functional enrichment analysis on all differentially expressed genes (DEGs) in 6 dpf spi1a -/- larvae. This study selected the top 30 most significant pathways, and the GO enrichment analysis revealed that the genes were enriched in three representative categories: biological processes, cellular components, and molecular functions. The DEGs were primarily enriched in biological processes such as negative regulation of NIK/NF-kappaB signaling, negative regulation of innate immune response, negative regulation of phosphatidylinositol 3-kinase signaling, negative regulation of interferon-alpha production, and negative regulation of NLRP3 inflammasome complex assembly (Figure 7 A). We further conducted GO enrichment analysis on all DEGs in 6 mpf spi1a -/- adult brain, which showed that the DEGs were mainly enriched in biological processes including immune system process, inflammatory response, defense response to virus, immune response, and innate immune response (Figure 7 B). In both larvae and adult brain, the DEGs were predominantly enriched in functions related to inflammatory and immune responses. We performed KEGG enrichment analysis on all DEGs from 6 dpf spi1a -/- larvae zebrafish, selecting the top 30 most significant pathways. The analysis revealed significant enrichment of DEGs in Toll-like/NOD-like receptor signaling pathways, cytokine-cytokine interactions, and necroptosis (Figure 8 A). Similarly, KEGG enrichment analysis of all DEGs from 6 mpf spi1a -/- adult zebrafish brain showed significant enrichment in apoptosis, Toll-like/NOD-like receptor signaling pathways, p53 signaling pathway, cytokine-cytokine interactions, and necroptosis (Figure 8 B). These results demonstrate that spi1a gene knockout affects signaling pathways related to inflammatory and immune responses in zebrafish. 4. Discussion In zebrafish, the SPI1 gene has two homologs, spi1a and s pi1b . This study employed CRISPR/Cas9 technology to first knockout the spi1a gene in zebrafish, investigating its impact on AD-related phenotypes to establish a foundation for developing a zebrafish AD drug screening model. The symptoms of human AD primarily include cognitive deficits along with a series of behavioral and emotional issues, such as aggression, anxiety, social interaction, object discrimination, and color preference, all of which have been comprehensively characterized in zebrafish [27, 28] . Notably, various behavioral tests, including T/Y maze tests, novel object recognition tests, light-dark tests, and novel diving tests, can be employed to assess cognitive ability, locomotion, exploratory tendency, and anxiety in zebrafish AD models [29] . In our study, we assessed cognitive function and motor ability in spi1a -/- zebrafish using T/Y-maze and light/dark transition tests. The results demonstrated that while larvae exhibited decreased locomotor activity in light/dark tests, their working memory remained intact in Y-maze tests. In contrast, adult fish showed significant learning and memory impairments in T-maze tests, characterized by prolonged latency and reduced dwelling time in target zones. The age factor may be a key reason for the differences in cognitive function between spi1a -/- zebrafish larvae and adults. In the larval stage, although locomotor activity was reduced in the light-dark transition test, working memory remained unaffected in the Y-maze test, suggesting that developmental compensatory mechanisms (such as synaptic remodeling) may maintain basic cognitive functions. However, with aging, cumulative damage—similar to that observed during human aging, including cellular senescence, oxidative stress, and chronic inflammation—gradually disrupts neural homeostasis, leading to significant learning and memory impairments in spi1a -/- adult zebrafish. This may stem from age-related pathological protein accumulation (such as β-amyloid), mitochondrial dysfunction, or exacerbated neuroinflammation, all of which synergistically impair neural plasticity and cognitive function in the zebrafish brain. Mechanistic investigations revealed that spi1a -/- adult zebrafish brain (but not larvae) displayed significantly increased Aβ1–42 deposition along with elevated AChE activity and reduced acetylcholine levels, consistent with the cholinergic hypothesis of AD and explaining their cognitive dysfunction. Furthermore, apoptosis assays demonstrated increased brain cell apoptosis in both larvae and adult spi1a -/- zebrafish, evidenced by upregulation of pro-apoptotic genes ( p53 , bax , caspase3 , and caspase9 ) and downregulation of anti-apoptotic gene ( bcl2 ). These findings demonstrated that spi1a deficiency may induce brain cell apoptosis through p53/caspase-dependent apoptotic pathways, which could also contribute to the observed motor deficits in larvae. To obtain a more comprehensive understanding of the biological pathways potentially regulated by spi1a , we extracted RNA from 6 dpf wild-type and spi1a -/- larvae as well as the brain tissues of 6 mpf wild-type and spi1a -/- adult zebrafish for transcriptome sequencing. Based on transcriptome data analysis, we identified six differentially expressed genes (DEGs) associated with AD: grna , casp23 , tap2a , fas , ccl35.1 and ctss2.1 . In larvae, spi1a knockout did not significantly alter ctss2.1 expression; however, in adult zebrafish brain, spi1a knockout led to a significant upregulation of ctss2.1 . Studies have shown elevated CTSS expression in multiple brain regions of AD patients, including the hippocampus. In aged and AD model mice, CTSS levels were significantly increased in neurons, exacerbating neuroinflammation and contributing to cognitive decline via the CX3CL1-CX3CR1 and JAK2-STAT3 pathways [30] . In both larvae and adult zebrafish brain, spi1a knockout significantly upregulated fas expression. The FAS gene, a member of the tumor necrosis factor (TNF) receptor superfamily, has been implicated in cell-mediated apoptosis. Since apoptosis is considered a major contributor to cell loss in neurodegenerative diseases, FAS -mediated apoptosis is believed to be linked to AD [31] . Research has demonstrated increased FAS mRNA expression in the brains of AD patients, along with significantly higher FAS levels in their cerebrospinal fluid compared to controls [32] . Notably, the progranulin (PGRN) gene ( GRN ), a key regulator of inflammation, has been reported to exhibit loss-of-function effects that enhance phagocytosis and proinflammatory cytokine production in microglia and macrophages, promoting frontotemporal dementia (FTD) development [33] . However, in our study, spi1a knockout led to significant upregulation of grna expression, contrary to previous reports. These results suggest that spi1a may regulate multiple AD-related genes and pathways, including inflammation and apoptosis. To elucidate the role of spi1a in Alzheimer's disease pathogenesis, we performed transcriptome analysis on spi1a -/- zebrafish. GO enrichment revealed that differentially expressed genes were significantly enriched in immune-inflammatory pathways, including negative regulation of NF-KB signaling, innate immune response, PI3K signaling, and NLRP3 inflammasome assembly. KEGG analysis identified significant activation of Toll-like/NOD-like receptor pathways, cytokine-cytokine interactions, and cell death pathways. In AD pathology, Aβ and Tau aggregates function as DAMPs that are recognized by various PRRs (, TLRs, NLRs e.g.), triggering downstream inflammatory cascades that promote proinflammatory cytokine/chemokine production and induce cell death [34–36] . Notably, transcriptomic studies in Spi1 knockdown and overexpressing mice demonstrated Spi1's interaction with microglial phagocytosis genes ( Tyrobp , Laptm5 , Fcer1g , Trem2 ), complement genes ( C1qa , C1qc ), and inflammatory response genes ( CD74 , C1qa , C1qc , Ctss , Trem2 ), confirming Spi1's involvement in microglial phagocytosis and inflammatory regulation [13] .Collectively, these findings suggest that spi1a deficiency may dysregulate immune-inflammatory signaling pathways in zebrafish, impairing microglial function. This could lead to either microglial hyperactivation or compromised Aβ clearance capacity, ultimately exacerbating neuroinflammation and accelerating AD progression. In summary, our findings demonstrate that genetic knockout of spi1a in zebrafish induces AD-related phenotypic alterations at behavioral, cellular, and molecular levels, which not only provides insights into AD pathogenesis but also establishes a foundation for developing high-throughput drug screening platforms in future research. Declarations Funding This work was supported by the Natural Science Foundation of Zhejiang Province (grant number: LY20H060003, LY20H090007) and the Zhejiang Province Traditional Chinese Medicine Science and Technology Project Foundation (No. 2022ZA006). Author Contribution Xia sun: Data curation, Formal analysis, Investigation, Methodology. Zhixing wu: Investigation, Methodology, Supervision, Validation. kangkang ge: Data curation, Formal analysis, Writing – original draft. 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Nat Rev Immunol, 2014, 14(7): 463-77. HENEKA M T, KUMMER M P, STUTZ A, et al. NLRP3 is activated in Alzheimer's disease and contributes to pathology in APP/PS1 mice [J]. Nature, 2013, 493(7434): 674-8. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Gene structure of zebrafish \u003cem\u003espi1a\u003c/em\u003e, with the sgRNA target site indicated by a red arrow, the specific target sequence shown in black, and the PAM (NGG) sequence in red. (B). DNA sequence alignment between \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand wild-type (WT), showing a 14-bp deletion in the corresponding genomic region of \u003cem\u003espi1a\u003c/em\u003e. (C). DNA sequencing chromatogram of \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e. (D). The 14-bp deletion in \u003cem\u003espi1a\u003c/em\u003e results in a frameshift mutation that alters the coding sequence of the protein.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6961277/v1/d4bd884685cfd03b801404d7.png"},{"id":93049281,"identity":"77fa7ebc-f807-4db2-b65e-e9bcc7d8e234","added_by":"auto","created_at":"2025-10-08 13:54:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":248384,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of larval response capability and locomotor behavior in light-dark alternation test. \u003c/strong\u003e(A). Movement patterns and average speed of zebrafish under 5-minute light 5-minute dark alternating stimulation. (B). Quantitative analysis of average speed changes during light-dark alternation (n=12). (C). Quantitative analysis of total swimming distance during light-dark alternation phases (n=20). Data are presented as mean±SEM; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6961277/v1/2bfa48462da78cc71e34cf06.png"},{"id":93049302,"identity":"907ea4b8-fef1-406a-92a2-2bf2f60952ab","added_by":"auto","created_at":"2025-10-08 13:54:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":212994,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe learning and memory abilities of zebrafish. \u003c/strong\u003e(A). Frequency distribution of global quadruplet strategies during 1-hour exploration in the FMP Y-maze. The dashed line indicates the 6.25% random selection level. (B). Percentage of repetitive strategy usage in global 1-hour patterns. (C). Percentage of alternating strategy usage in global 1-hour patterns. Data are presented as mean±SEM (n=18, ns: \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). (D). Latency periods of 6 mpf WT and \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e groups; (E). Cumulative time spent in the EC zone by 6 mpf WT and \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e groups. Data are presented as mean±SEM (n=17, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6961277/v1/d74c6626ebaf7800d7bbe9ce.png"},{"id":93049278,"identity":"4af760ad-9b95-4d5a-b7e3-9fec6754c418","added_by":"auto","created_at":"2025-10-08 13:54:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of Aβ1-42 plaques in zebrafish. \u003c/strong\u003e(A). Quantitative analysis of Aβ1-42 levels in 6 dpf zebrafish larvae. (B). Quantitative analysis of Aβ1-42 levels in 6 mpf adult zebrafish brain. Data are presented as mean±SEM (n=6, ns: \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6961277/v1/f04973253dfeb8c1e9568fc2.png"},{"id":93049314,"identity":"1d97f468-207a-4660-abb9-592211d4a9a7","added_by":"auto","created_at":"2025-10-08 13:54:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":127616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMeasurement of AChE activity in zebrafish. \u003c/strong\u003e(A). Quantitative analysis of AChE levels in 6 dpf zebrafish larvae. (B). Quantitative analysis of AChE levels in 6 mpf adult zebrafish brain. Data are presented as mean±SEM (n=8, ns: \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6961277/v1/17453c8f7a82b5f5c4946db3.png"},{"id":93049286,"identity":"d9b4358d-ab75-4670-848d-54deb4fdcb58","added_by":"auto","created_at":"2025-10-08 13:54:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":197956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003espi1a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockout on brain apoptosis in zebrafish larvae. \u003c/strong\u003e(A-B). AO staining showing apoptotic cells in brain regions of 6 dpf WT and \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e larvae (dashed lines outline brain areas, red arrows indicate apoptotic cells; scale bar: 100μm). (C-G). qRT-PCR analysis of apoptosis-related genes (\u003cem\u003ebax\u003c/em\u003e, \u003cem\u003ebcl2\u003c/em\u003e, \u003cem\u003ep53\u003c/em\u003e, \u003cem\u003ecaspase3\u003c/em\u003e and \u003cem\u003ecaspase9\u003c/em\u003e) expression. Data are presented as mean±SEM (n=20, ns: \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6961277/v1/532fa21bacd7323175ae9a86.png"},{"id":93049287,"identity":"1c690986-ed7e-479c-a60d-054a68933d8e","added_by":"auto","created_at":"2025-10-08 13:54:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":351088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGO functional enrichment analysis of larvae and adult zebrafish brain.\u003c/strong\u003e (A). GO functional enrichment analysis of 6 dpf \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e larvae; (B). GO functional enrichment analysis of 6 mpf \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e adult brain. The horizontal axis represents the -log\u003csub\u003e10\u003c/sub\u003e(p-value), indicating the significance level of GO term enrichment, while the vertical axis displays the GO term names, with biological processes (BP), cellular components (CC), and molecular functions (MF) labeled in green, orange, and blue, respectively.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6961277/v1/0da797acc3f13ac33ee2f388.png"},{"id":93049307,"identity":"f9adcf64-740f-4b67-b05f-ea08f20b8e30","added_by":"auto","created_at":"2025-10-08 13:54:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":238328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKEGG pathway enrichment analysis of larvae and adult zebrafish brain.\u003c/strong\u003e (A). KEGG pathway enrichment analysis of 6 dpf \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e larvae; (B). KEGG pathway enrichment analysis of 6 mpf \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e adult zebrafish brain. The horizontal axis represents the enrichment score while the vertical axis displays KEGG pathways. The bubble size is proportional to the number of differentially expressed protein-coding genes, and the color gradient (blue→white→yellow→red) reflects the variation in enrichment p-values, with redder colors indicating higher significance levels\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6961277/v1/b61d313e81e5dce60a2f97d8.png"},{"id":107705295,"identity":"5f810706-9162-482f-b295-dfb520e8a4d8","added_by":"auto","created_at":"2026-04-24 09:11:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2122408,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6961277/v1/da612891-5de7-49a9-b978-fdc800508df4.pdf"},{"id":93049283,"identity":"c3d125f0-8a1d-4262-9159-fc94dcf93e54","added_by":"auto","created_at":"2025-10-08 13:54:14","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1314412,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6961277/v1/6cc90d2ceacd8f4125b21414.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of the spi1a gene on Alzheimer's disease-related phenotypes in a zebrafish model","fulltext":[{"header":"Highlights","content":"\u003cul start=\"50\"\u003e\n \u003cli\u003eFirst demonstration of \u003cem\u003eSPI1/spi1a\u003c/em\u003e\u0026rsquo;s role in a zebrafish AD model.\u003c/li\u003e\n \u003cli\u003eAge-dependent phenotypes and mechanistic insights.\u003c/li\u003e\n \u003cli\u003eA versatile platform for high-throughput drug discovery.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAlzheimer's disease (AD) is a multifactorial neurodegenerative disorder characterized by progressive cognitive dysfunction and behavioral impairments, representing the most prevalent form of dementia in the elderly population\u003csup\u003e[1]\u003c/sup\u003e. With the accelerating trend of global aging, the number of AD patients worldwide is projected to reach 78\u0026nbsp;million by 2030 and surge to 139\u0026nbsp;million by 2050, imposing tremendous psychological and socioeconomic burdens on patients, families, and society\u003csup\u003e[2]\u003c/sup\u003e. The clinical manifestations of AD primarily include progressive cognitive decline, memory deficits, mood disorders, aphasia, language dysfunction, and impaired ability to perform daily living activities. The hallmark pathological features of AD consist of extracellular amyloid-β (Aβ) plaque deposition and intracellular neurofibrillary tangles formed by hyperphosphorylated tau protein, which are closely associated with neuronal and synaptic loss, ultimately leading to the cognitive impairments observed in AD\u003csup\u003e[3]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e), which shares high biological, structural, functional and genetic homology with humans, has been widely used in human disease modeling and represents a valuable model for Alzheimer's disease (AD) research \u003csup\u003e[4]\u003c/sup\u003e. Zebrafish possess orthologous genes involved in human AD-related pathways, enabling them to recapitulate specific features of AD-associated pathological processes observed in humans\u003csup\u003e[5]\u003c/sup\u003e. While lacking clearly defined cortical or hippocampal structures characteristic of mammalian brains, zebrafish exhibit comparable neuroanatomical organization to humans and can perform learning and memory tasks through brain regions functionally equivalent to these structures\u003csup\u003e[6]\u003c/sup\u003e. Both larval and adult zebrafish serve as excellent behavioral models that effectively simulate the complex behavioral alterations seen in AD patients\u003csup\u003e[5, 7]\u003c/sup\u003e. These distinctive characteristics establish zebrafish as an ideal model for AD research, not only facilitating elucidation of disease pathogenesis but also providing a robust platform for drug screening and therapeutic development that is comparable to mammalian in vivo and in vitro drug screening models.\u003c/p\u003e\u003cp\u003eGenome-wide association studies (GWASs) have identified the \u003cem\u003eSPI1\u003c/em\u003e gene (encoding PU.1) as being associated with AD\u003csup\u003e[8]\u003c/sup\u003e. In mice, this gene is also known as \u003cem\u003eSpi1\u003c/em\u003e or \u003cem\u003eSfpi1\u003c/em\u003e. PU.1, an E26 transformation-specific (ETS) family transcription factor, is primarily expressed in monocytes/macrophages, microglia, neutrophils, mast cells, B cells and early erythroid cells\u003csup\u003e[9, 10]\u003c/sup\u003e. Within the central nervous system (CNS), \u003cem\u003eSPI1\u003c/em\u003e is exclusively expressed in microglia, where its expression levels influence microglial transcription, activation and phenotype. In primary human microglia, PU.1 silencing alters the expression of AD-related genes and genes involved in antigen presentation and phagocytosis, suggesting that attenuating PU.1 expression may represent an effective therapeutic approach to limit microglia-mediated inflammatory responses in AD\u003csup\u003e[11]\u003c/sup\u003e. Studies in the murine BV-2 microglial cell line demonstrate that PU.1 knockout activates protein translation, antioxidant responses, and cholesterol/lipid metabolism pathways while reducing pro-inflammatory gene expression, conferring protective effects against AD; conversely, increased PU.1 expression enhances zymosan phagocytosis and promotes inflammatory responses, thereby elevating AD risk\u003csup\u003e[12]\u003c/sup\u003e. In mouse models with \u003cem\u003eSpi1\u003c/em\u003e knockdown or overexpression, \u003cem\u003eSpi1\u003c/em\u003e deficiency exacerbates multiple pathological features including insoluble amyloid-β (Aβ) levels, amyloid plaque deposition and gliosis, aggravating AD symptoms, whereas \u003cem\u003eSpi1\u003c/em\u003e overexpression significantly ameliorates these phenotypes and dystrophic neurites\u003csup\u003e[13]\u003c/sup\u003e. These findings collectively reveal the complex role of the \u003cem\u003eSpi1\u003c/em\u003e gene in AD pathogenesis.\u003c/p\u003e\u003cp\u003eThis study employed CRISPR/Cas9 gene editing technology to generate \u003cem\u003espi1a\u003c/em\u003e (the zebrafish ortholog of human \u003cem\u003eSPI1\u003c/em\u003e gene) mutant zebrafish, investigating the role of \u003cem\u003espi1a\u003c/em\u003e in AD pathogenesis using this model system. Through comprehensive behavioral analyses including light/dark transition tests, Y-maze, and T-maze experiments in both larval and adult zebrafish, we evaluated AD-associated learning and memory capabilities. The research further examined expression patterns of apoptosis-related genes, acetylcholinesterase activity and amyloid-β (Aβ1\u0026ndash;42) levels in zebrafish. Transcriptomic profiling was conducted to elucidate spi1a's regulatory effects on AD-related genes and signaling pathways at the molecular level, thereby establishing a valuable zebrafish model for advancing Alzheimer's disease treatment research and facilitating novel drug development.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Maintenance of Zebrafish\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAll zebrafish used in this study were wild-type AB strain adults obtained from the China Zebrafish Resource Center. Both wild-type and mutant zebrafish were maintained at 27\u0026ndash;29\u0026deg;C under a 14-hour light/10-hour dark cycle. Sexually mature adult males and females that had not mated for at least one week were selected for breeding at 1:1 or 2:1 ratios to obtain embryos, which were staged by hours post-fertilization (hpf) or days post-fertilization (dpf) or months post-fertilisation (mpf). Fertilized eggs were cultured in E3 medium at 28.5\u0026deg;C. All zebrafish experiments were conducted in compliance with the Zhejiang Laboratory Animal Welfare Guidelines and were approved by the Institutional Animal Care and Use Committee of Hangzhou Normal University.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Generation of \u003cem\u003espi1a\u003c/em\u003e mutant zebrafish\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe CRISPR/Cas9 gene editing in zebrafish was performed following established protocols\u003csup\u003e[14]\u003c/sup\u003e. The zebrafish orthologs of human \u003cem\u003eSPI1\u003c/em\u003e gene, \u003cem\u003espi1a\u003c/em\u003e and \u003cem\u003espi1b\u003c/em\u003e, were identified through the ZFIN (The Zebrafish Information Network). Using CRISPR/Cas9 technology, we first generated \u003cem\u003espi1a\u003c/em\u003e knockout zebrafish mutants. The target site for \u003cem\u003espi1a\u003c/em\u003e knockout (5\u0026prime;-GGGTAGAATGGTCCCCATGGCGG-3\u0026prime;) was designed using (CHOPCHOP). A mixture of sgRNA (50 ng/\u0026micro;L) and Cas9 protein (0.2 \u0026micro;g/\u0026micro;L) was microinjected into single-cell stage embryos. Mutations were verified by comparing with the wild-type sequence. Genotyping of offspring was performed using primers listed in supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Light-dark transition test in larval zebrafish\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe light-dark transition test was conducted as previously described to evaluate zebrafish locomotor activity and responsiveness\u003csup\u003e[15]\u003c/sup\u003e. Behavioral measurements were performed using the Danio Vision tracking system (Noldus Information Technology, Wageningen, the Netherlands). At 6 dpf, zebrafish larvae were individually placed in a 96-well plate (one larva per well). Following 20 minutes of dark adaptation, the test consisted of four alternating light-dark cycles (5 minutes light/5 minutes dark each). The video tracking system recorded the total swimming distance and velocity changes during the light-dark transitions for subsequent analysis.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Y-maze test in larval zebrafish\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo evaluate working memory capacity in zebrafish larvae, the free movement pattern (FMP) Y-maze test was performed according to previously reported experimental protocols\u003csup\u003e[16]\u003c/sup\u003e. The Y-maze apparatus consisted of three white acrylic walls arranged at 120\u0026deg; angles with a transparent acrylic base, measuring 1.2 cm \u0026times; 0.4 cm \u0026times; 0.3 cm (length \u0026times; width \u0026times; height). During testing, the Y-maze was filled with aquarium water to allow sufficient swimming depth. Prior to FMP Y-maze testing, no training or habituation was required, and larvae were randomly selected from the tank. Eighteen 6 dpf wild-type and \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mutant larvae per group were allowed to freely explore the Y-maze for 1 hour without introducing changes in environment, novelty, or food rewards. The sequence of left and right turn choices made by larvae during the 1-hour free exploration period was recorded to investigate working memory alterations.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 T-maze test in adult zebrafish\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe T-maze test was employed to evaluate learning and memory capabilities in adult zebrafish\u003csup\u003e[17]\u003c/sup\u003e, utilizing a color-biased and food-rewarded protocol following established methodology\u003csup\u003e[18]\u003c/sup\u003e. The apparatus consisted of an acrylic T-shaped maze with one long arm (50 cm\u0026times;10 cm\u0026times;10 cm) and two short arms (red and green arms, 20 cm\u0026times;10 cm\u0026times;10 cm). The long arm featured a starting compartment (10 cm\u0026times;10 cm\u0026times;10 cm) at its end, while one short arm contained a target chamber with brine shrimp, designated as the enriched chamber (EC) (10 cm\u0026times;10 cm\u0026times;10 cm) with green walls.\u003c/p\u003e\u003cp\u003eThe 5-day testing protocol (4 training days, 1 test day) involved groups of 17 zebrafish. Prior to behavioral testing, fish were acclimated in the T-maze for 1 hour. During trials, zebrafish were placed in the starting compartment for 1 minute before the sliding door opened. Fish received punishment when entering the red arm, while those entering the green arm could obtain brine shrimp. Each zebrafish underwent one daily trial for 4 consecutive training days. If a fish failed to locate the EC within 4 minutes, it was guided there and remained for 1 minute. Learning ability was assessed by recording the time required to swim from the start box to the EC. On the fifth test day, despite having been trained to preferentially enter the green arm, no punishment or food reward was provided. Memory retention was evaluated by comparing cumulative time spent in the EC. All behaviors were recorded for 4 minutes using the Danio Vision tracking system (Noldus), with analysis of distance traveled, time spent, and entry frequency for each zone.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Quantification of Aβ1\u0026ndash;42 levels in zebrafish\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe Aβ1\u0026ndash;42 levels in both \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mutant larvae and adult brain tissues were measured using a Zebrafish Aβ1\u0026ndash;42 ELISA Kit (202308, Shanghai Enzyme-linked Biotechnology Co., Ltd, China) following the manufacturer's instructions. For sample preparation, 6 dpf \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e larvae and 6 mpf \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e adult brain tissues were homogenized in cold physiological saline at a 1:9 (mass:volume) ratio in 1.5 mL tubes using ice-bath homogenization, followed by centrifugation at 5000 rpm for 10 minutes. The supernatants were collected for Aβ1\u0026ndash;42 quantification. Absorbance was measured at 450 nm using a Spark multimode microplate reader (ThermoFisher, USA), and the optical density (OD) values were recorded to calculate sample concentrations. The Aβ1\u0026ndash;42 concentrations were expressed as ng/mg of total protein.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Determination of Acetylcholinesterase (AChE) activity in zebrafish\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFollowing behavioral studies of \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mutant larvae and adults, we further evaluated the effects on acetylcholinesterase (AChE) activity using an AChE Activity Assay Kit (D799813-0050, Sangon Biotech, China). For AChE determination, whole-body homogenates from 6 dpf \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e larvae and brain tissues from 6 mpf \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e adults were prepared by adding AChE extraction buffer at a 1:9 (mass:volume) ratio, followed by ice-bath homogenization and centrifugation at 8000 rpm for 10 minutes at 4\u0026deg;C to obtain supernatants. AChE activity was then measured according to the manufacturer's instructions. Absorbance at 412 nm was determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, USA) to calculate AChE activity.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Apoptosis staining in larval zebrafish\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAcridine orange (AO) staining was used to specifically label apoptotic cells with green fluorescence\u003csup\u003e[19]\u003c/sup\u003e. Twenty randomly selected 6 dpf wild-type and \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mutant larvae were washed three times with distilled water (5 minutes each) and subsequently incubated in 5 \u0026micro;g/mL AO solution at 37\u0026deg;C in the dark for 60 minutes. Following staining, larvae were anesthetized with 0.03% MS-222 and immobilized using 3% methylcellulose. Apoptotic cells in zebrafish embryos were identified as bright, punctate green spots under fluorescence microscopy (Zeiss AxioObserver V12).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTotal RNA was extracted from zebrafish using TRIzol reagent (Invitrogen, CA, USA), and complementary DNA (cDNA) was synthesized using the HiFiScript gDNA Removal RT Master Mix reverse transcription kit (CW Bio, Beijing, China). Quantitative real-time PCR was performed on a CFX96 real-time PCR detection system (Bio-Rad, USA) with 2 \u0026micro;L cDNA in 20 \u0026micro;L SYBR reaction mixture, with all samples run in triplicate. The primer sequences used in this experiment are listed in supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Transcriptome analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTotal RNA was extracted from 6 dpf \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e larvae and 6 mpf \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e adult brain tissues using TRIzol reagent (Invitrogen, CA, USA), with three independent biological replicates performed for each group. The concentration and purity of the RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), while RNA integrity was evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Transcriptome libraries were constructed following the manufacturer\u0026rsquo;s instructions of the VAHTS Universal V6 RNA-seq Library Prep Kit. Sequencing was performed on the Illumina Novaseq 6000 platform, generating 150 bp paired-end reads. Differential expression gene (DEG) analysis was conducted using DESeq2 software, with genes meeting the thresholds of q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and fold change\u0026thinsp;\u0026gt;\u0026thinsp;2 or fold change\u0026thinsp;\u0026lt;\u0026thinsp;0.5 defined as differentially expressed genes\u003csup\u003e[20]\u003c/sup\u003e. Subsequently, Gene Ontology (GO) and KEGG Pathway enrichment analyses based on the hypergeometric distribution algorithm were performed on the DEGs to identify significantly enriched functional terms\u003csup\u003e[21, 22]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Statistical analyses\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAll experiments were performed with at least three independent biological replicates, and the data were analyzed using two-way ANOVA and paired two-tailed t-tests with GraphPad Prism 8.0 software. All experimental data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM), and a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Obtaining the spi1a-knockout zebrafish mutant line using CRISPR/Cas9 gene editing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the function of the \u003cem\u003espi1a\u003c/em\u003e gene in zebrafish, we generated a \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mutant line using the CRISPR/Cas9 system. A target site was designed on the third exon of zebrafish spi1a (Figure 1 A). By co-injecting Cas9 mRNA and gRNA into wild-type zebrafish embryos at the one-cell stage, we obtained founders carrying the desired mutation. After intercrossing the F1 generation, we identified homozygous mutants with a 14-bp deletion in the third exon (Figure 1 B-C). Sequence alignment analysis revealed that this mutation causes a frameshift, altering the amino acid sequence and disrupting the functional domain of the \u003cem\u003espi1a\u003c/em\u003e protein (Figure 1 D). These results demonstrate that we successfully established the \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mutant model, which was subsequently used for further studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 The effect of the \u003cem\u003espi1a\u003c/em\u003e gene on locomotor behavior and response ability in zebrafish larvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe found that knockout of the \u003cem\u003espi1a\u003c/em\u003e gene showed no visible impact on zebrafish growth and development (Figure S1)\u003cem\u003e,\u003c/em\u003e and further investigated its impact on larval motor function. Zebrafish larvae exhibit distinct locomotor patterns in light and dark conditions, which are closely associated with anxiety, learning, memory, and defensive behaviors\u003csup\u003e[23]\u003c/sup\u003e. In this study, following the experimental protocol established by MacPhail et al.\u003csup\u003e[24]\u003c/sup\u003e, we observed that wild-type larvae moved slower in light and faster in dark conditions, whereas \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mutants showed significantly reduced average speed during light-dark transitions (Figure 2 A). Quantitative analysis revealed significantly attenuated speed variations during light-dark alternations in \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mutants (Figure 2 B), indicating impaired responsiveness. Furthermore, \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mutants exhibited significantly decreased total swimming distance compared to wild-type controls during all light-dark cycles (Figure 2 C). These results demonstrate that\u003cem\u003e\u0026nbsp;spi1a\u003c/em\u003e knockout compromises both responsiveness and locomotor capacity in zebrafish.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 The effect of the \u003cem\u003espi1a\u003c/em\u003e gene on learning and memory in the zebrafish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study evaluated learning and memory capabilities in zebrafish using the free-movement pattern (FMP) Y-maze for larvae and T-maze for adults. In larval Y-maze tests, compared to WT, \u003cem\u003espi1a\u0026nbsp;\u003c/em\u003eknockout did not alter the search strategies of 6 dpf larvae, with alternating patterns (LRLR, RLRL) remaining predominant in exploration (Figure 3 A). No significant differences were observed between \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e and WT groups in either repetitive strategy usage frequency or alternating strategy occurrence (Figure 3 B-C), indicating that \u003cem\u003espi1a\u003c/em\u003e deletion does not affect larval working memory. However, in adult T-maze tests, although both WT and \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e adults showed decreased latency periods with training days, the \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e group exhibited significantly prolonged latency compared to WT (Figure 3 D), along with significantly reduced cumulative time spent in the EC zone (Figure 3 E), demonstrating substantial impairment in learning and memory capacities in \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e adults. These results demonstrate stage-specific regulatory roles of the \u003cem\u003espi1a\u003c/em\u003e gene in cognitive functions: while minimally impacting working memory in larvae, it becomes crucial for learning and memory in adult zebrafish.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 The effect of the \u003cem\u003espi1a\u003c/em\u003e gene on A\u0026beta;1-42 plaques in zebrafish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA\u0026beta;1-42 is a toxic amyloid peptide that accumulates extensively in the brains of AD patients, leading to amyloid deposition and subsequent neuronal damage\u003csup\u003e[25]\u003c/sup\u003e. Compared to 6 dpf WT, no significant changes in A\u0026beta;1-42 levels were observed in \u003cem\u003espi1a\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003elarvae (Figure 4 A). Both WT and \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e showed relatively low A\u0026beta;1-42 levels at this stage, suggesting insufficient accumulation during larval development. We further examined A\u0026beta;1-42 in 6 mpf adult brain tissues, revealing significantly elevated A\u0026beta;1-42 levels in \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e adults compared to WT (Figure 4 B). These results demonstrate that \u003cem\u003espi1a\u003c/em\u003e knockout promotes A\u0026beta;1-42 accumulation specifically in adult zebrafish brains.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 The effect of the \u003cem\u003espi1a\u003c/em\u003e gene on Acetylcholinesterase (AChE) Activity in Zebrafish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAcetylcholine (ACh), a crucial neurotransmitter in brain tissue, is closely associated with learning and memory performance. Acetylcholinesterase (AChE) hydrolyzes acetylcholine and induces cholinergic neuronal dysfunction, serving as a primary therapeutic target for Alzheimer\u0026apos;s disease (AD)\u003csup\u003e[26]\u003c/sup\u003e. In this experiment, we measured AChE activity of 6 dpf larvae and 6 mpf adult brain tissues to determine whether \u003cem\u003espi1a\u003c/em\u003e gene knockout affects neurotransmitters related to zebrafish learning and memory capabilities. Results showed no significant difference in AChE levels between \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e larvae and wild-type (WT) controls at 6 dpf (Figure 5 A). However, in 6 mpf adult brain, \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish exhibited significantly increased AChE content in brain tissues compared to WT (Figure 5 B). These findings demonstrate that \u003cem\u003espi1a\u003c/em\u003e gene knockout leads to elevated acetylcholinesterase levels specifically in adult zebrafish brains.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 The effect of the \u003cem\u003espi1a\u003c/em\u003e gene on brain cell apoptosis in zebrafish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of\u003cem\u003e\u0026nbsp;spi1a\u003c/em\u003e deficiency on brain cell apoptosis in zebrafish, we conducted systematic experimental analyses. Acridine orange (AO) staining in 6 dpf larvae revealed significantly increased apoptotic cells (manifested as granular green dots) in the brain regions of \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003egroup (Figure 6 A-B). Molecular analyses demonstrated consistent apoptosis-related gene expression patterns in both 6 dpf larvae (Figure 6 C-G) and 6 mpf adult brain tissues of \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e group (Figure S2): significant upregulation of pro-apoptotic genes (\u003cem\u003ep53\u003c/em\u003e, \u003cem\u003ebax\u003c/em\u003e, \u003cem\u003ecaspase3\u003c/em\u003e, and \u003cem\u003ecaspase9\u003c/em\u003e) along with marked downregulation of the anti-apoptotic gene \u003cem\u003ebcl2\u003c/em\u003e. These results demonstrate that \u003cem\u003espi1a\u003c/em\u003e deletion activates the p53/caspase apoptotic pathway, leading to increased neuronal apoptosis in both larval and adult zebrafish brains.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 The effect of the \u003cem\u003espi1a\u003c/em\u003e gene on Alzheimer\u0026apos;s Disease-Associated Genes in Zebrafish\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough transcriptome sequencing analysis, we investigated the impact of \u003cem\u003espi1a\u003c/em\u003e knockout on AD-related genes in zebrafish. Based on 787 AD-associated genes from the ZFIN database, we identified 18 AD-related DEGs (929 upregulated and 577 downregulated) in 6 dpf \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e larvae (Figure S3 A、C), and 21 AD-related DEGs (738 upregulated and 287 downregulated) in 6 mpf \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e adult brain tissues (Figure S3 B、D). Cross-analysis identified six core AD-related genes (\u003cem\u003egrna\u003c/em\u003e, \u003cem\u003ectss2.1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;casp23\u003c/em\u003e, \u003cem\u003eccl35.1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;fas\u003c/em\u003e, \u003cem\u003etap2a\u003c/em\u003e). qRT-PCR validation revealed: in larvae, \u003cem\u003egrna\u003c/em\u003e, \u003cem\u003ecasp23\u003c/em\u003e, \u003cem\u003etap2a\u003c/em\u003e and\u003cem\u003e\u0026nbsp;fas\u003c/em\u003e were significantly upregulated while \u003cem\u003eccl35.1\u003c/em\u003e and \u003cem\u003ectss2.1\u003c/em\u003e showed no significant changes (Figure S3 E-J); in adult brain, all genes except \u003cem\u003etap2a\u0026nbsp;\u003c/em\u003e(downregulated) were significantly upregulated (Figure S4). These findings demonstrate that \u003cem\u003espi1a\u003c/em\u003e influences zebrafish AD-like phenotypes through stage-specific regulation of AD-related gene expression.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 The effect of the \u003cem\u003espi1a\u003c/em\u003e gene on the gene expression profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the potential pathways through which the loss of the \u003cem\u003espi1a\u003c/em\u003e gene affects AD-related phenotypes, we performed GO functional enrichment analysis on all differentially expressed genes (DEGs) in 6 dpf \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e larvae. This study selected the top 30 most significant pathways, and the GO enrichment analysis revealed that the genes were enriched in three representative categories: biological processes, cellular components, and molecular functions. The DEGs were primarily enriched in biological processes such as negative regulation of NIK/NF-kappaB signaling, negative regulation of innate immune response, negative regulation of phosphatidylinositol 3-kinase signaling, negative regulation of interferon-alpha production, and negative regulation of NLRP3 inflammasome complex assembly (Figure 7 A). We further conducted GO enrichment analysis on all DEGs in 6 mpf \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e adult brain, which showed that the DEGs were mainly enriched in biological processes including immune system process, inflammatory response, defense response to virus, immune response, and innate immune response (Figure 7 B). In both larvae and adult brain, the DEGs were predominantly enriched in functions related to inflammatory and immune responses.\u003c/p\u003e\n\u003cp\u003eWe performed KEGG enrichment analysis on all DEGs from 6 dpf \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e larvae zebrafish, selecting the top 30 most significant pathways. The analysis revealed significant enrichment of DEGs in Toll-like/NOD-like receptor signaling pathways, cytokine-cytokine interactions, and necroptosis (Figure 8 A). Similarly, KEGG enrichment analysis of all DEGs from 6 mpf \u003cem\u003espi1a\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e adult zebrafish brain showed significant enrichment in apoptosis, Toll-like/NOD-like receptor signaling pathways, p53 signaling pathway, cytokine-cytokine interactions, and necroptosis (Figure 8 B). These results demonstrate that \u003cem\u003espi1a\u003c/em\u003e gene knockout affects signaling pathways related to inflammatory and immune responses in zebrafish.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn zebrafish, the \u003cem\u003eSPI1\u003c/em\u003e gene has two homologs, \u003cem\u003espi1a\u003c/em\u003e and s\u003cem\u003epi1b\u003c/em\u003e. This study employed CRISPR/Cas9 technology to first knockout the \u003cem\u003espi1a\u003c/em\u003e gene in zebrafish, investigating its impact on AD-related phenotypes to establish a foundation for developing a zebrafish AD drug screening model. The symptoms of human AD primarily include cognitive deficits along with a series of behavioral and emotional issues, such as aggression, anxiety, social interaction, object discrimination, and color preference, all of which have been comprehensively characterized in zebrafish\u003csup\u003e[27, 28]\u003c/sup\u003e. Notably, various behavioral tests, including T/Y maze tests, novel object recognition tests, light-dark tests, and novel diving tests, can be employed to assess cognitive ability, locomotion, exploratory tendency, and anxiety in zebrafish AD models\u003csup\u003e[29]\u003c/sup\u003e. In our study, we assessed cognitive function and motor ability in \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e zebrafish using T/Y-maze and light/dark transition tests. The results demonstrated that while larvae exhibited decreased locomotor activity in light/dark tests, their working memory remained intact in Y-maze tests. In contrast, adult fish showed significant learning and memory impairments in T-maze tests, characterized by prolonged latency and reduced dwelling time in target zones. The age factor may be a key reason for the differences in cognitive function between \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e zebrafish larvae and adults. In the larval stage, although locomotor activity was reduced in the light-dark transition test, working memory remained unaffected in the Y-maze test, suggesting that developmental compensatory mechanisms (such as synaptic remodeling) may maintain basic cognitive functions. However, with aging, cumulative damage\u0026mdash;similar to that observed during human aging, including cellular senescence, oxidative stress, and chronic inflammation\u0026mdash;gradually disrupts neural homeostasis, leading to significant learning and memory impairments in \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e adult zebrafish. This may stem from age-related pathological protein accumulation (such as β-amyloid), mitochondrial dysfunction, or exacerbated neuroinflammation, all of which synergistically impair neural plasticity and cognitive function in the zebrafish brain.\u003c/p\u003e\u003cp\u003eMechanistic investigations revealed that \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e adult zebrafish brain (but not larvae) displayed significantly increased Aβ1\u0026ndash;42 deposition along with elevated AChE activity and reduced acetylcholine levels, consistent with the cholinergic hypothesis of AD and explaining their cognitive dysfunction. Furthermore, apoptosis assays demonstrated increased brain cell apoptosis in both larvae and adult \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e zebrafish, evidenced by upregulation of pro-apoptotic genes (\u003cem\u003ep53\u003c/em\u003e, \u003cem\u003ebax\u003c/em\u003e, \u003cem\u003ecaspase3\u003c/em\u003e, and \u003cem\u003ecaspase9\u003c/em\u003e) and downregulation of anti-apoptotic gene (\u003cem\u003ebcl2\u003c/em\u003e). These findings demonstrated that \u003cem\u003espi1a\u003c/em\u003e deficiency may induce brain cell apoptosis through p53/caspase-dependent apoptotic pathways, which could also contribute to the observed motor deficits in larvae.\u003c/p\u003e\u003cp\u003eTo obtain a more comprehensive understanding of the biological pathways potentially regulated by \u003cem\u003espi1a\u003c/em\u003e, we extracted RNA from 6 dpf wild-type and \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e larvae as well as the brain tissues of 6 mpf wild-type and \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e adult zebrafish for transcriptome sequencing. Based on transcriptome data analysis, we identified six differentially expressed genes (DEGs) associated with AD: \u003cem\u003egrna\u003c/em\u003e, \u003cem\u003ecasp23\u003c/em\u003e, \u003cem\u003etap2a\u003c/em\u003e, \u003cem\u003efas\u003c/em\u003e, \u003cem\u003eccl35.1\u003c/em\u003e and \u003cem\u003ectss2.1\u003c/em\u003e. In larvae, \u003cem\u003espi1a\u003c/em\u003e knockout did not significantly alter \u003cem\u003ectss2.1\u003c/em\u003e expression; however, in adult zebrafish brain, \u003cem\u003espi1a\u003c/em\u003e knockout led to a significant upregulation of \u003cem\u003ectss2.1\u003c/em\u003e. Studies have shown elevated \u003cem\u003eCTSS\u003c/em\u003e expression in multiple brain regions of AD patients, including the hippocampus. In aged and AD model mice, \u003cem\u003eCTSS\u003c/em\u003e levels were significantly increased in neurons, exacerbating neuroinflammation and contributing to cognitive decline via the CX3CL1-CX3CR1 and JAK2-STAT3 pathways\u003csup\u003e[30]\u003c/sup\u003e. In both larvae and adult zebrafish brain, \u003cem\u003espi1a\u003c/em\u003e knockout significantly upregulated \u003cem\u003efas\u003c/em\u003e expression. The \u003cem\u003eFAS\u003c/em\u003e gene, a member of the tumor necrosis factor (TNF) receptor superfamily, has been implicated in cell-mediated apoptosis. Since apoptosis is considered a major contributor to cell loss in neurodegenerative diseases, \u003cem\u003eFAS\u003c/em\u003e-mediated apoptosis is believed to be linked to AD\u003csup\u003e[31]\u003c/sup\u003e. Research has demonstrated increased \u003cem\u003eFAS\u003c/em\u003e mRNA expression in the brains of AD patients, along with significantly higher \u003cem\u003eFAS\u003c/em\u003e levels in their cerebrospinal fluid compared to controls\u003csup\u003e[32]\u003c/sup\u003e. Notably, the progranulin (PGRN) gene (\u003cem\u003eGRN\u003c/em\u003e), a key regulator of inflammation, has been reported to exhibit loss-of-function effects that enhance phagocytosis and proinflammatory cytokine production in microglia and macrophages, promoting frontotemporal dementia (FTD) development\u003csup\u003e[33]\u003c/sup\u003e. However, in our study, \u003cem\u003espi1a\u003c/em\u003e knockout led to significant upregulation of \u003cem\u003egrna\u003c/em\u003e expression, contrary to previous reports. These results suggest that \u003cem\u003espi1a\u003c/em\u003e may regulate multiple AD-related genes and pathways, including inflammation and apoptosis.\u003c/p\u003e\u003cp\u003eTo elucidate the role of \u003cem\u003espi1a\u003c/em\u003e in Alzheimer's disease pathogenesis, we performed transcriptome analysis on \u003cem\u003espi1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e zebrafish. GO enrichment revealed that differentially expressed genes were significantly enriched in immune-inflammatory pathways, including negative regulation of NF-KB signaling, innate immune response, PI3K signaling, and NLRP3 inflammasome assembly. KEGG analysis identified significant activation of Toll-like/NOD-like receptor pathways, cytokine-cytokine interactions, and cell death pathways. In AD pathology, Aβ and Tau aggregates function as DAMPs that are recognized by various PRRs (, TLRs, NLRs e.g.), triggering downstream inflammatory cascades that promote proinflammatory cytokine/chemokine production and induce cell death\u003csup\u003e[34\u0026ndash;36]\u003c/sup\u003e. Notably, transcriptomic studies in \u003cem\u003eSpi1\u003c/em\u003e knockdown and overexpressing mice demonstrated \u003cem\u003eSpi1's\u003c/em\u003e interaction with microglial phagocytosis genes (\u003cem\u003eTyrobp\u003c/em\u003e, \u003cem\u003eLaptm5\u003c/em\u003e, \u003cem\u003eFcer1g\u003c/em\u003e, \u003cem\u003eTrem2\u003c/em\u003e), complement genes (\u003cem\u003eC1qa\u003c/em\u003e, \u003cem\u003eC1qc\u003c/em\u003e), and inflammatory response genes (\u003cem\u003eCD74\u003c/em\u003e, \u003cem\u003eC1qa\u003c/em\u003e, \u003cem\u003eC1qc\u003c/em\u003e, \u003cem\u003eCtss\u003c/em\u003e, \u003cem\u003eTrem2\u003c/em\u003e), confirming \u003cem\u003eSpi1's\u003c/em\u003e involvement in microglial phagocytosis and inflammatory regulation\u003csup\u003e[13]\u003c/sup\u003e.Collectively, these findings suggest that \u003cem\u003espi1a\u003c/em\u003e deficiency may dysregulate immune-inflammatory signaling pathways in zebrafish, impairing microglial function. This could lead to either microglial hyperactivation or compromised Aβ clearance capacity, ultimately exacerbating neuroinflammation and accelerating AD progression.\u003c/p\u003e\u003cp\u003eIn summary, our findings demonstrate that genetic knockout of \u003cem\u003espi1a\u003c/em\u003e in zebrafish induces AD-related phenotypic alterations at behavioral, cellular, and molecular levels, which not only provides insights into AD pathogenesis but also establishes a foundation for developing high-throughput drug screening platforms in future research.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Natural Science Foundation of Zhejiang Province (grant number: LY20H060003, LY20H090007) and the Zhejiang Province Traditional Chinese Medicine Science and Technology Project Foundation (No. 2022ZA006).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXia sun: Data curation, Formal analysis, Investigation, Methodology. Zhixing wu: Investigation, Methodology, Supervision, Validation. kangkang ge: Data curation, Formal analysis, Writing \u0026ndash; original draft. Yuying wang: Investigation, Methodology, Writing \u0026ndash; review \u0026amp; editing. Lili tian: Data curation, Formal analysis. Jiayu Wang, Methodology. Guoqing liang: Writing \u0026ndash; original draft. Donglai sheng: Funding acquisition, Supervision, Validation, Conceptualization, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLANE C A, HARDY J, SCHOTT J M. Alzheimer\u0026apos;s disease [J]. Eur J Neurol, 2018, 25(1): 59-70.\u003c/li\u003e\n\u003cli\u003eTHAKRAL S, YADAV A, SINGH V, et al. Alzheimer\u0026apos;s disease: Molecular aspects and treatment opportunities using herbal drugs [J]. Ageing Res Rev, 2023, 88: 101960.\u003c/li\u003e\n\u003cli\u003eRAHMAN M M, LENDEL C. 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NLRP3 is activated in Alzheimer\u0026apos;s disease and contributes to pathology in APP/PS1 mice [J]. Nature, 2013, 493(7434): 674-8.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"spi1a gene, zebrafish model, Alzheimer's disease","lastPublishedDoi":"10.21203/rs.3.rs-6961277/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6961277/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGenome-wide sequence analysis has identified the \u003cem\u003eSPI1\u003c/em\u003e gene as a genetic risk factor for Alzheimer's disease (AD). \u003cem\u003eSPI1\u003c/em\u003e encodes the PU.1 protein, which plays a critical role in microglial development and immune responses, primarily studied in mouse models. However, no studies have yet reported the impact of the \u003cem\u003eSPI1\u003c/em\u003e gene on AD-related phenotypes in zebrafish. Therefore, this study utilized CRISPR/Cas9 gene editing to generate \u003cem\u003espi1a\u003c/em\u003e knockout zebrafish mutants, investigating the effects of \u003cem\u003espi1a\u003c/em\u003e loss-of-function on AD-associated phenotypes. The results showed that \u003cem\u003espi1a\u003c/em\u003e knockout led to reduced locomotor activity and increased brain cell apoptosis in larvae, while working memory, acetylcholinesterase (AChE) activity and Aβ1\u0026ndash;42 levels remained unchanged. In contrast, adult \u003cem\u003espi1a\u003c/em\u003e knockout zebrafish exhibited significant cognitive decline, upregulated apoptosis-related genes, elevated AChE activity and increased Aβ1\u0026ndash;42 accumulation. Transcriptomic analysis further revealed that \u003cem\u003espi1a\u003c/em\u003e knockout altered the expression of multiple AD-related genes and affected immune and inflammation-related signaling pathways. In conclusion, \u003cem\u003espi1a\u003c/em\u003e deficiency induced AD-like phenotypes in adult zebrafish. This study demonstrates the role of \u003cem\u003espi1a\u003c/em\u003e in modulating AD-related phenotypes in both larval and adult zebrafish, providing crucial insights into AD pathogenesis and establishing a valuable model for future high-throughput drug screening and therapeutic development.\u003c/p\u003e","manuscriptTitle":"Effects of the spi1a gene on Alzheimer's disease-related phenotypes in a zebrafish model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 13:54:05","doi":"10.21203/rs.3.rs-6961277/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"728fe0c2-c878-486c-b029-2ff6458c1705","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-18T03:39:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 13:54:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6961277","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6961277","identity":"rs-6961277","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}