Significant outcomes
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Identifying differentially expressed genes and proteins enhances our understanding of the molecular mechanisms behind amisulpride’s clinical effects.
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It offers potentially new pathogenic mechanisms and treatment targets for schizophrenia.
Limitations
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The sample size is small and was conducted using only the SH-SY5Y cell line.
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Other genes and proteins need to be verified to gain more insight into the molecular mechanism of amisulpride.
Introduction
The effectiveness of traditional antipsychotic medications is linked to their ability to block dopamine D2 receptors (Willner, Reference Willner1997). Haloperidol is a traditional antipsychotic drug that acts as a complete dopamine D2 receptor antagonist. It is effective in alleviating schizophrenia symptoms but may cause side effects, including extrapyramidal symptoms and elevated prolactin levels (Beasley et al., Reference Beasley, Dellva, Tamura, Morgenstern, Glazer, Ferguson and Tollefson1999). A broader spectrum of therapeutic efficacy of atypical antipsychotic drugs such as risperidone, olanzapine, and clozapine has been introduced to the market for treating schizophrenia (Newcomer, Reference Newcomer2005). An atypical antipsychotic drug known as amisulpride is a substituted benzamide derivative that possesses a unique pharmacological profile, characterised by a high affinity for dopaminergic D2/D3 and an affinity for 5-HT7 receptors (Perrault et al., Reference Perrault, Depoortere, Morel, Sanger and Scatton1997; Moller, Reference Moller2003). Amisulpride is more effective and safer than traditional and multi-receptor antipsychotics in improving global symptoms, including both positive and negative symptoms of schizophrenia (Komossa et al., Reference Komossa, Rummel-Kluge, Hunger, Schmid, Schwarz, Silveira Da Mota Neto, Kissling and Leucht2010). Currently, there is still a limited understanding of the molecular mechanisms that contribute to the clinical effectiveness of amisulpride in treating schizophrenia.
Proteogenomics is a powerful strategy for gene and protein expression profiles in neuroscience and neurology (Nesvizhskii, Reference Nesvizhskii2014). For example, transcriptomic and proteomic analyses of antipsychotic drugs have identified genes associated with schizophrenia. (Bortolasci et al., Reference Bortolasci, Spolding, Kidnapillai, Connor, Truong, Liu, Panizzutti, Richardson, Gray, Berk, Dean and Walder2020; Truong et al., Reference Truong, Bortolasci, Kidnapillai, Spolding, Panizzutti, Liu, Kim, Dean, Richardson, Berk and Walder2022). A recent genome-wide mRNA expression study showed that eight antipsychotic drugs downregulate the expression of genes related to the focal adhesions pathway (Panizzutti et al., Reference Panizzutti, Bortolasci, Spolding, Kidnapillai, Connor, Truong, Liu, Hernández, Gray, Kim, Dean, Berk and Walder2025). This suggests that adhesion pathways may play a role in the pathophysiology of bipolar disorder and schizophrenia.
However, many detailed molecular mechanisms of amisulpride’s action remain unknown. In this study, we performed RNA sequencing (RNA-seq) and LC-MS/MS analysis to identify the differentially expressed genes (DEGs) and proteins in SH-SY5Y neuroblastoma cells treated with amisulpride. A more precise understanding of the molecular effects of amisulpride on SH-SY5Y cells could offer insights into the molecular mechanisms underlying psychiatric disorders, potentially revealing novel treatment targets.
Materials and methods
Cell culture and amisulpride treatment
The human SH-SY5Y neuroblastoma cell line (Sigma catalogue no. 94030304) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. The SH-SY5Y cells were cultured in a humidified environment with 5% CO2 at 37°C, and the medium was refreshed every two to three days. Amisulpride was sourced from Sigma–Aldrich (A2729). A stock solution was made in dimethyl sulfoxide (DMSO) and subsequently diluted in the medium to achieve the desired final concentration. The concentrations of amisulpride used in this study were selected based on previous research examining its effects on the SH-SY5Y cells (Park et al., Reference Park, Seo, Cho, Lee, Lee, Seol and Kim2011). We administered doses of 0.4, 4, 20, and 40 μg/ml, which are within a pharmacologically relevant range known to modulate the activity of D2/D3 and 5-HT7 receptors without causing cytotoxicity. A 24–hour treatment period was chosen to allow sufficient time for amisulpride-induced changes in the transcriptome and proteome while minimising downstream secondary stress responses or cell death. To ensure statistical robustness and reproducibility of the transcriptomic and proteomic data, we performed three independent biological replicates per condition, with each set derived from separate cell culture passages.
MTT assay
The SH-SY5Y cells were cultured in 96-well plates at a density of 1.5 × 104 cells per well and were incubated for 24 h. After this initial incubation, the cells were treated with amisulpride at various concentrations for 24 h in serum-free DMEM. Following treatment, the cells were washed twice with phosphate-buffered saline (PBS) and then cultured in DMEM supplemented with 10% FBS for two days. Subsequently, the cells were incubated with 0.5 mg/ml of the MTT (Sigma Chemical Co., St Louis, MO, USA) in DMEM for 4 h. Viable cells converted MTT to formazan, which appears blue-purple when dissolved in DMSO. The intensity of the colour, measured as absorbance, is directly proportional to the number of live cells. Absorbance at 545 nm was recorded using a microplate reader (Varioskan Flash, Thermo Fisher Scientific, Vantaa, Finland). The percentage of cell survival was calculated by dividing the absorbance of the amisulpride-treated samples by the absorbance of the corresponding DMSO-treated controls.
Real-time cell viability assay
Real-time cell viability was conducted using the RealTime-Glo™ MT Cell Viability Assay (Cat.# G9711, Promega). The SH-SY5Y cells (1 × 104 per well) were plated in a 96-well plate and cultured in a 5% CO2 atmosphere at 37°C overnight to ensure thorough incubation conditions. The medium was then replaced with a growth medium containing, various dosages of amisulpride, the MT Cell Viability Substrate (Promega) and NanoLuc Enzyme (Promega), and the cells were cultured for 72 h. Luciferase activity was measured using a microplate reader (VANTAstarTM, BMG LABTECH, Germany).
Total RNA preparation, RNA-seq, and RT-qPCR
Total RNA preparation, RNA-seq, RT-qPCR, and DEG identification were performed following the methods described in a previous study (Wang et al., Reference Wang, Hsu, Tsai, Cheng and Cheng2022). The target gene, FOSB (Hs00171851_m1, FAM™ dye-labelled TaqMan™ MGB probe), and two endogenous genes, GAPDH (Hs02786624_g1, VIC™ dye-labelled TaqMan™ MGB probe) and 18S (Hs99999901_s1, VIC™ dye-labelled TaqMan™ MGB probe), were analysed using TaqMan™ gene expression assays according to the manufacturer’s protocol (ThermoFisher Scientific Inc.). The expression levels of ACTG1, ANP32E, CLTC, and IPO8 were assayed using SYBR Green detection (ThermoFisher Scientific Inc.), and the primer sequences are listed in the Supplementary Table S1. All tests were performed in six replicates. Statistically significant differences between the treated and control groups were determined using a p–value threshold of <0.05.
Protein sample preparation and LC-MS/MS analysis
To prepare protein samples, cells were washed twice with cold PBS and then resuspended in a lysis buffer that contain 20 mM HEPES (pH 7.6), 7.5-mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.1% TritonX-100, 50 mM NaF, 0.1 mM Na3VO4, and a protease inhibitor cocktail (one mini-tablet/10 ml; Roche Diagnostics GmbH). The homogenates were centrifuged at 13,000 r.p.m. for 30 minutes at 4°C, and the resulting supernatants were stored at -80°C until needed.
In the process of solution digestion, LC-MS/MS and protein identification were conducted by BIOTOOLS CO., LTD in Taiwan. The procedure began with diluting the protein solutions in 50 mM ammonium bicarbonate (ABC, Sigma). The samples were then reduced with 10 mM dithiothreitol (DTT, Merck) at 56°C for 45 minutes. Following this, cysteine residues were blocked with 50 mM iodoacetamide (IAM, Sigma) at 25°C for 30 minutes. Next, the samples were digested with sequencing-grade modified porcine trypsin (Promega) at 37°C for 16 h. After digestion, the peptides were desalted, dried by vacuum centrifugation, and stored at -80°C until further use.
In the meticulous LC-MS/MS analysis process, the digested peptides were diluted in HPLC buffer A (0.1% formic acid) and loaded onto a reverse-phase column (Zorbax 300 SB-C18, 0.3 × 5 mm; Agilent Technologies). The desalted peptides were separated using a column (Waters BEH 1.7 µm, 100 μm I.D. × 10 cm with a 15 μm tip) and a multi-step gradient of HPLC buffer B (99.9% acetonitrile/0.1% formic acid) over a period of 70 minutes, at a flow rate of 0.3 μl/min. The liquid chromatography apparatus was coupled with a 2D linear ion trap mass spectrometer (Orbitrap Elite ETD; Thermo Fisher), which was operated using Xcalibur 2.2 software (Thermo Fisher). The full-scan MS was performed in the Orbitrap over a range of 400 to 2,000 Da, with a a resolution of 120,000 at m/z 400. Internal calibration was achieved using the ion signal of protonated dodecamethylcyclohexasiloxane ion at m/z 536.165365 as the lock mass. The analysis included 20 data-dependent MS/MS scan events, each followed by one MS scan for the 20 most abundant precursor ions in the preview MS scan. The m/z values selected for MS/MS were dynamically excluded for 40 s with a relative mass window of 15 ppm. The electrospray voltage was set to 2.0 kV, and the capillary temperature was maintained at 200°C. Automatic gain control for MS and MS/MS was set to 1,000 ms (for full scan) and 200 ms (for MS/MS), or 3 × 106 ions (full scan) and 3,000 ions (MS/MS) for maximum accumulated time or ions, respectively.
The protein identification was conducted using Proteome Discoverer software (version 2.3, Thermo Fisher Scientific). The MS/MS spectra were searched against the UniProt database utilising the Mascot search engine (Matrix Science, London, UK; version 2.5). For peptide identification, a mass tolerance of 10 ppm was allowed for intact peptide masses and 0.5 Da for CID fragment ions, with allowance for two missed cleavages resulting from the trypsin digestion. The variable modifications included oxidised methionine and acetylation at the protein N-terminal, while carbamidomethylation of cysteine was used as a static modification. Peptide-spectrum matches (PSM) were filtered based on high confidence, and the Mascot search engine ranked the top identification for each peptide, ensuring that the overall false discovery rate below 0.01. Proteins identified by only a single peptide hit were excluded from the final results.
Bioinformatic analysis
For Gene Ontology (GO) enrichment and pathway analysis, differentially expressed proteins between amisulpride-treated and non-treated samples (P < 0.05) were subjected to the Database for Annotation, Visualisation, and Integrated Discovery (DAVID, https://davidbioinformatics.nih.gov/) using the official gene symbol method.
Immunoblotting
Immunoblotting was conducted following standard protocols, utilising the primary antibodies listed below. Rabbit anti-ACTG1 (11227–1-AP; Proteintech, Rosemont, IL, USA), rabbit anti-ANP32E (A17220; ABclonal, Woburn, MA, USA), rabbit anti-CLTC (A12423; ABclonal), rabbit anti-CRMP1 (A2705; ABclonal), rabbit anti-HSD17B10 (A5448; ABclonal), rabbit anti-IPO8 (A14679; ABclonal), rabbit anti-NPEPPS (A08129–2; Boster Biological Technology, Pleasanton, CA, USA), rabbit anti-OTUB1 (A11656; ABclonal), rabbit anti-PRMT1 (A1055; ABclonal), rabbit anti-RAC1 (A7720; ABclonal), rabbit anti-SPTAN1 (A0160; ABclonal), rabbit anti-SEPTIN11 (A12189; ABclonal), rabbit anti-TIA1 (A12523; ABclonal), and mouse anti-GAPDH (G8795; Sigma–Aldrich, Saint Louis, MO, USA). Horseradish peroxidase-conjugated donkey anti-rabbit IgG (NA934V, GE Healthcare Life Sciences, UK) and human anti-mouse IgG (5220–0341; KPL) were used as secondary antibodies. Chemiluminescence was visualised using an enhanced chemiluminescence detection system (GTX400006; GeneTex).
Results
Effects of amisulpride on SH-SY5Y cell viability
The effects of a 24–hour amisulpride treatment (0.4, 4, 20, and 40 μg/ml) on SH-SY5Y cell viability were examined using the MTT assay (Fig. 1). The survival rates compared to 0 μg/ml controls were 81% (p = 3E-06) after exposure to 4 μg/ml amisulpride, 66% (p = 1.4E-17) after exposure to 20 μg/ml, and 74% (p = 4.1E-09) after exposure to 40 μg/ml amisulpride.
Figure 1. Amisulpride exhibits concentration-dependent effects on the viability of SH-SY5Y cells. The MTT assay was conducted to estimate cell numbers after a 24–hour treatment with varying concentrations of amisulpride. The results were calculated as the optical density at 545 nm (OD545) of amisulpride-treated cultures compared to non-treated control cultures and expressed as means ± standard deviation. A statistically significant difference between amisulpride-treated and non-treated cultures was identified using ANOVA, with post hoc tests performed using by LSD method (*p < 0.01, n = 80).
We measured cell viability over the 72 h of amisulpride treatment using the RealTime-Glo™ MT Cell Viability Assay. Our results confirmed that amisulpride at concentrations of 4, 20, and 40 μg/ml) inhibited cell survival compared to the 0 μg/ml control group (Fig. 2).
Figure 2. Analysis of amisulpride-treated SH-SY5Y cells with RealTime-Glo™ MT Cell Viability. Real-time cell viability (luminescent signals) was monitored every 30 minutes for 72 h on a VANTAstarTM microplate reader with a gas control module (37°C and 5% CO2) (n = 6).
Identification of differentially expressed genes by transcriptome sequencing
Nine samples from three biological replicates (with doses of 0, 20, and 40 ug/ml of amisulpride) were used for transcriptome sequencing. The number of reads per sample ranged from 38,707,688 to 51,321,892 across the nine sequenced RNA samples (Supplementary Table S2). Differential gene expression analysis revealed that the FOSB gene was significantly downregulated in the group treated with 20 μg/ml amisulpride group, with a fold change greater than 2 and an adjusted p-value of 7.46E-11 (Supplementary Table S3 and S4). The mRNA expression level of FOSB was confirmed in biologically replicated SH-SY5Y cells using a real-time quantitative PCR (RT-qPCR) assay (Fig. 3).
Figure 3. The mRNA expression level of FOSB in amisulpride-treated SH-SY5Y cells. (A) a schematic representation of the FOSB gene genomic map, including primers and probes (pink rectangle) used for the TaqMan assay (Hs00171851_m1 from Thermo Fisher Scientific). (B) RT-qPCR assay results showing the expression of FOSB in SH-SY5Y cells treated with amisulpride. GAPDH was used as an endogenous gene for normalisation. Data are expressed as fold changes relative to 0 μg/ml of amisulpride ± standard deviation (* p < 0.05; n = 6).
Protein ID, bioinformatic analysis, immunoblot verification, and RT-qPCR analysis
We utilised a label-free LC-MS/MS shotgun proteomics strategy to identify proteins that were differentially expressed in the SH-SY5Y cells treated with amisulpride. We analysed nine samples from three biological replicates at three different doses of amisulpride: 0, 20, and 40 μg/ml. Normalised peptide-spectrum matches (PSMs) were calculated using the following formula: (PSM in sample A/total PSM in sample A) × average PSM across all nine samples. Supplementary Table S5 lists the protein accession numbers, descriptions, and normalised PSMs of the differentially expressed proteins between amisulpride-treated and non-treated samples. To evaluate the differences in normalised PSMs between the two groups, we conducted Student’s t-tests with a significance threshold of P < 0.05.
GO enrichment and pathway analysis revealed that the differentially expressed proteins between amisulpride-treated and non-treated samples were significantly associated with several GO terms and three pathways after applying the Bonferroni adjustment (Table 1, P<0.05). Thirteen proteins that exhibited differential expression between the groups were selected for further verification using immunoblot analysis on independent biological replicates of the SH-SY5Y cells (Fig. 4A). We determined the fold differences in the expression of the selected proteins (Fig. 4B), and confirmed the differential expression of four proteins: ACTG1, ANP32E, CLTC, and IPO8. We compared the mRNA expression levels of four genes (ACTG1, ANP32E, CLTC, and IPO8) in biologically replicated SH-SY5Y cells treated with amisulpride (0, 20, and 40 μg/ml) using RT-qPCR assay. We did not find significant differences in expression among these four genes across the three groups (Supplementary Figure S1).
Figure 4. Immunoblotting analysis to validate the differential expression for 13 proteins in amisulpride-treated biological replicated SH-SY5Y cells. (A) immunoblotting and (B) quantification of protein expression showing the fold differences of four proteins (ACTG1, ANP32E, CLTC, IPO8) between amisulpride-treated groups and control. GAPDH was a loading control. Data are expressed as fold change to 0 ug/ml of amisulpride ± standard deviation (*p < 0.01, n = 3).
Table 1. Summary of GO enrichment and pathway analyses in differentially expressed proteins between amisulpride-treated and non-treated samples
BP: biological process; CC: cellular component; MF: molecular function; KEGG: Kyoto Encyclopaedia of Genes and Genomes (https://www.genome.jp/kegg/).
Discussion
Antipsychotic medications are the preferred treatment of choice for patients experiencing psychotic symptoms, including delusions, hallucinations, bizarre behaviour, agitation, and aggression. Research indicates that changes in neural plasticity, driven by differential gene expression in the brain, may support the clinical effectiveness of antipsychotic medications (Hyman & Nestler, Reference Hyman and Nestler1996). In this study, we conducted a transcriptomic analysis to discover a downregulated expression of a transcriptional factor, the FOSB gene, in SH-SY5Y neuroblastoma cells treated with amisulpride. We also performed proteomic analysis to identify several proteins differentially expressed in the amisulpride-treated cells, which involved pathways related to synapse, extracellular exosome, structural constituent of ribosome, RNA and protein binding, and bacterial invasion of epithelial cells. We confirmed four specific proteins (ACTG1, ANP32E, CLTC, and IPO8) among the differentially expressed proteins, which showed significant changes in expression due to amisulpride administration, using an immunoblotting assay.
The activator protein 1 (AP-1) transcription factor family is a crucial class of transcriptional regulators that control various aspects of cell physiology in response to environmental changes, including stress, cytokines, infections, oncogenic stimuli, and growth factors (Durchdewald et al., Reference Durchdewald, Angel and Hess2009). Upon regulation, the AP-1 controls the expression of target genes associated with cell proliferation, differentiation, transformation, apoptotic cell death, and migration (Hisanaga et al., Reference Hisanaga, Sagar, Hicks, Swanson and Sharp1990; Durchdewald et al., Reference Durchdewald, Angel and Hess2009). The AP-1 family includes essential region leucine zipper (bZIP) domain proteins such as JUN, FOS, and FOSB. These proteins must dimerise to form the transcription factor complex AP-1 before binding to their DNA target sites (Wagner, Reference Wagner2001). Research suggests that the transcriptional activation of the AP-1 complex plays a vital role in the central nervous system, brain development, and psychiatry disorders (Pennypacker, Reference Pennypacker1995). For example, multiple studies have reported changes in fos and jun family genes as markers of transcriptional activity alterations caused by antipsychotic drug treatment (Kontkanen et al., Reference Kontkanen, Lakso, Wong and Castren2002; Kiss & Osacka, Reference Kiss and Osacka2020). A study found that FosB mutant mice lost the induction of Fos-related proteins by chronic cocaine exposures and exhibited abnormal locomotor and conditioned place preference responses (Hiroi et al., Reference Hiroi, Brown, Haile, Ye, Greenberg and Nestler1997). In this study, we observed a reduction in the gene expression of the FOSB gene in the SH-SY5Y cells treated with amisulpride. This suggests that the FOSB gene may play a role in the molecular mechanism of antipsychotic drugs and the pathophysiology of psychiatric disorders. Notably, two studies have reported an association between the FOSB gene and the pathogenesis of schizophrenia, although their findings are inconsistent. A genome-wide gene expression study has found that the FOSB gene was upregulated in fibroblast samples from individuals with schizophrenia (Huang et al., Reference Huang, Liu, Wang, Tang, Teng, Li, Qiu, Wu and Chen2019). In contrast, a transcriptomic study revealed that the FOSB gene is downregulated in the brain tissue of patients with schizophrenia (Chen et al., Reference Chen, Dai, Tang, Mikhailova, Liang, Li, Zhou, Kopp, Weickert, Chen and Liu2023). The discrepancy might be attributed to differences in antipsychotic treatment and the types of tissue collected. Taken together, we propose that amisulpride’s effects on the regulation of genes within the AP-1 transcription factor family are linked to mental illnesses that feature psychotic symptoms. Moreover, the FOSB gene was acceptable as a candidate gene for schizophrenia and may play a role in its pathogenesis.
Evidence indicates that abnormal synaptogenesis and synaptic dysfunction are crucial to the pathophysiology of schizophrenia, highlighting potential therapeutic targets for synaptic circuit modulation in psychiatric disorders (Howes & Onwordi, Reference Howes and Onwordi2023; Wolf & Abi-Dargham, Reference Wolf and Abi-Dargham2023). In this study, we found amisulpride treatment can increase the protein level of a synaptic vesicle-related protein, clathrin heavy chain (CLTC), in the SH-SY5Y cells. CLTC encodes clathrin heavy chain, a crucial component of clathrin-coated vesicles that mediate intracellular trafficking, especially endocytosis and membrane recycling (Narayana et al., Reference Narayana, Gadgil, Mote, Rajan and Subramanyam2019; Itagaki & Kamei, Reference Itagaki and Kamei2025). Recent studies have shown that CLTC is also involved in synaptic transmission and the cycling of synaptic vesicles (Pannone et al., Reference Pannone, Muto, Nardecchia, Di Rocco, Marchei, Tosato, Petrini, Onorato, Lanza, Bertuccini, Manti, Folli, Galosi, Di Schiavi, Leuzzi, Tartaglia and Martinelli2023; Yang et al., Reference Yang, Zheng, Zhuang, Xu, Li and Hu2025a). Furthermore, several studies indicate that CLTC mutations are associated with childhood-onset schizophrenia, neurodevelopmental disorders, epileptic encephalopathy, and Parkinsonism (DeMari et al., Reference Demari, Mroske, Tang, Nimeh, Miller and Lebel2016; Manti et al., Reference Manti, Nardecchia, Barresi, Venditti, Pizzi, Hamdan, Blau, Burlina, Tartaglia and Leuzzi2019, Nabais Sá et al., Reference Nabais Sá, Venselaar, Wiel, Trimouille, Lasseaux, Naudion, Lacombe, Piton, Vincent-Delorme, Zweier, Reis, Trollmann, Ruiz, Gabau, Vetro, Guerrini, Bakhtiari, Kruer, Amor, Cooper, Bijlsma, Barakat, Van Dooren, Van Slegtenhorst, Pfundt, Gilissen, Willemsen, De Vries, De Brouwer and Koolen2020, Usnich et al., Reference Usnich, Becker, Nagel, Bäumer and Münchau2024). Overall, dysfunction of CLTC may contribute to a wide range of neuropsychiatric risks. Therefore, CLTC could be a potential therapeutic target for treatment of psychiatric disorders.
Mounting evidence underscores the pivotal role of actin remodelling in synaptogenesis, synaptic plasticity, and the intricate development of neurites in burgeoning neurons (Matus, Reference Matus2000; Hotulainen & Hoogenraad, Reference Hotulainen and Hoogenraad2010). For instance, the dynamic nature of actin filaments is instrumental in forming dendritic spines during development, and they contribute significantly to the structural plasticity of mature synapses (Matus, Reference Matus2000). A growing body of research has illuminated the regulatory mechanisms that finely tune actin dynamics within dendritic spines (Mattila & Lappalainen, Reference Mattila and Lappalainen2008; Hotulainen & Hoogenraad, Reference Hotulainen and Hoogenraad2010). Notably, Kimoto and colleagues discovered that levels of transcripts associated with actin and mitochondrial oxidative phosphorylation are profoundly altered in individuals with schizophrenia (Kimoto et al., Reference Kimoto, Hashimoto, Berry, Tsubomoto, Yamaguchi, Enwright, Chen, Kawabata, Kikuchi, Kishimoto and Lewis2022). Furthermore, a study reports alterations in the dendritic spine across multiple brain regions in schizophrenia (Glausier & Lewis, Reference Glausier and Lewis2013). The compelling evidence suggests that the abnormal morphology of dendritic spines observed in schizophrenia may indeed be linked to disruptions in the delicate regulation of actin cytoskeletal dynamics. In this study, we found that amisulpride treatment can decrease the protein level of ACTG1 in the SH-SY5Y cells. The ACTG1 gene encodes cytoplasmic gamma actin, which is likely to play a vital role in cell morphology, motility, and other actin-related functions (Rivière et al., Reference Rivière, Van Bon, Hoischen, Kholmanskikh, O‘roak, Gilissen, Gijsen, Sullivan, Christian, Abdul-Rahman, Atkin, Chassaing, Drouin-Garraud, Fry, Fryns, Gripp, Kempers, Kleefstra, Mancini, Nowaczyk, Van Ravenswaaij-Arts, Roscioli, Marble, Rosenfeld, Siu, De Vries, Shendure, Verloes, Veltman, Brunner, Ross, Pilz and Dobyns2012). In neurons, ACTG1 is involved in shaping dendritic spines and synaptic structures (Schreiber et al., Reference Schreiber, Végh, Dawitz, Kroon, Loos, Labonté, Li, Van Nierop, Van Diepen, De Zeeuw, Kneussel, Meredith, Smit and Van Kesteren2015), indicating its contribution to learning, memory, and neuroplasticity by facilitating synaptic remodelling. Variants in the ACTG1 gene were observed in patients with Baraitser-Winter syndrome and agenesis of the corpus callosum and neuronal heterotopia (Vontell et al., Reference Vontell, Supramaniam, Davidson, Thornton, Marnerides, Holder-Espinasse, Lillis, Yau, Jansson, Hagberg and Rutherford2019). Besides, several reports show variants in the ACTG1 gene are associated with obsessive–compulsive disorder (Göbel et al., Reference Göbel, Berninger, Schlump, Feige, Runge, Nickel, Schiele, Van Elst, Hotz, Alter, Domschke, Tzschach and Endres2022) and autism spectrum disorder (Tuncay et al., Reference Tuncay, Parmalee, Khalil, Kaur, Kumar, Jimale, Howe, Goodspeed, Evans, Alzghoul, Xing, Scherer and Chahrour2022). Therefore, the processes associated with ACTG1 in actin cytoskeletal dynamics may represent novel therapeutic targets for schizophrenia. However, the relationship between the ACTG1 gene and the pathogenesis of schizophrenia requires further detailed elucidation.
Research indicates that antipsychotic medications can alter epigenomic patterns, including DNA methylation and histone modifications, affecting gene expression in the brain (Marques et al., Reference Marques, Vaziri, Greenway and Bousman2025). Understanding the biological functions of epigenetics in the field of psychiatry would facilitate the development of new therapies for psychiatric disorders. In this study, we found amisulpride treatment can increase the protein level of ANP32E in the SH-SY5Y cells. The ANP32E gene encodes acidic nuclear phosphoprotein 32 family member E, which enables histone binding, acts as a histone chaperone, and assists in protein folding (Obri et al., Reference Obri, Ouararhni, Papin, Diebold, Padmanabhan, Marek, Stoll, Roy, Reilly, Mak, Dimitrov, Romier and Hamiche2014). It plays important roles in regulating chromatin, controlling gene expression, and helping the cell response to DNA damage (Obri et al., Reference Obri, Ouararhni, Papin, Diebold, Padmanabhan, Marek, Stoll, Roy, Reilly, Mak, Dimitrov, Romier and Hamiche2014). Gilda Stefanelli discovered that ANP32E plays a crucial role in regulating memory formation, transcription, and dendritic morphology by controlling steady-state H2A.Z binding in neurons (Stefanelli et al., Reference Stefanelli, Makowski, Brimble, Hall, Reda, Creighton, Leonetti, TaB, Zakaria, Baumbach, Greer, Davidoff, Walters, Murphy and Zovkic2021). Schizophrenia is increasingly recognised as a neurodevelopmental disorder that involves epigenetic factors (Yang et al., Reference Yang, Sun, Li and Zhang2025b). We hypothesise that ANP32E impacts which neuronal genes are active or silenced during brain development by regulating chromatin structure through the eviction of H2A.Z. Taken together, we propose that amisulpride’s action on ANP32E may be linked to the transcriptional regulation of target genes associated with histone chaperones, which provides potentially novel pathogenic mechanisms and treatment targets for psychiatric disorders.
The IPO8 gene encodes importin-8, a nuclear transport belonging to the importin b family, which mediates the import of proteins into the nucleus (Görlich et al., Reference Görlich, Dabrowski, Bischoff, Kutay, Bork, Hartmann, Prehn and Izaurralde1997; Miyamoto et al., Reference Miyamoto, Yamada and Yoneda2016). It plays crucial roles in nucleocytoplasmic trafficking, gene regulation, and signal transduction (Liang et al., Reference Liang, Zhang, Wang, Li, Cong, Luo and Zhang2013; Wei et al., Reference Wei, Li, Wang, Zhang and Zen2014). A genetic study identified a single-nucleotide polymorphism in the IPO8 gene that is associated with the brain systems responsible for eye movement, which are known to be impaired in psychotic disorders (Lencer et al., Reference Lencer, Mills, Alliey-Rodriguez, Shafee, Lee, Reilly, Sprenger, Mcdowell, Mccarroll, Keshavan, Pearlson, Tamminga, Clementz, Gershon, Sweeney and Bishop2017). Notably, Nganou et al., demonstrated that IPO8 knockdown was associated with defects in neuronal migration (Nganou et al., Reference Nganou, Silva, Gladwyn-Ng, Engel, Coumans, Delgado-Escueta, Tanaka, Nguyen, Grisar, De Nijs and Lakaye2018). Numerous studies show that IPO8 is expressed in various tissues, including the adult brain, which is known to transport several proteins essential for brain development (Yao et al., Reference Yao, Chen, Cottonham and Xu2008; Weinmann et al., Reference Weinmann, Höck, Ivacevic, Ohrt, Mütze, Schwille, Kremmer, Benes, Urlaub and Meister2009; Volpon et al., Reference Volpon, Culjkovic-Kraljacic, Osborne, Ramteke, Sun, Niesman, Chook and Borden2016). Based on the above evidence, we hypothesise that dysfunction of IPO8 could disrupt nucleocytoplasmic trafficking, resulting in abnormal transcriptional regulation of neuronal genes associated with schizophrenia. The up-regulation of IPO8 induced by amisulpride, as demonstrated in this study, may be relevant to the clinical efficacy of antipsychotic medications.
To summarise, our data suggest that amisulpride may influence the differential expression of genes and proteins related to the AP-1 transcription factor family, cytoskeleton, histone binding activity, intracellular trafficking of receptors, endocytosis of various macromolecules, and nuclear localisation signals. This understanding of the molecular mechanisms underlying the clinical effectiveness of amisulpride and the pathogenesis of schizophrenia opens up exciting possibilities for further research. The detailed transcriptomic and proteomic analysis of amisulpride treatment provides a comprehensive understanding of cellular response at the molecular level, sparking curiosity and the need for more in-depth studies in the field.
This study has a major limitation. SH-SY5Y cells may not be the ideal standalone model for schizophrenia research. Although SH-SY5Y cells are a human-derived neuroblastoma cell line widely used in neuroscience due to their catecholaminergic properties and their ability to differentiate into neuron-like cells, researchers often prefer to use primary neuronal cells, co-culture systems, and rodent models (Park et al., Reference Park, Seo, Cho, Lee, Lee, Seol and Kim2011; Shipley et al., Reference Shipley, Mangold and Szpara2016). These alternatives are more effective for investigating synaptic function, morphology, neurotoxicity, neurotransmitter release, and disease modelling. Consequently, the findings of this study should be interpreted with caution.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/neu.2025.10040.
Financial support
This research was funded by Yuli Branch, Taipei Veterans General Hospital, Taiwan, grant number VHYL-112-09 and VHYL-113-01.
Author contributions
Conceptualisation, M.-C.C. and T.-M.H.; methodology, M.-C.C., S.-H.H., H.-Y.T.; formal analysis, M.-C.C., S.-H.H., and H.-Y.T.; investigation, M.-C.C., S.-H.H., H.-Y.T., and T.-M.H.; resources, M.-C.C. and T.-M.H.; writing – original draft preparation, M.-C.C.; writing – review and editing, M.-C.C.; funding acquisition, T.-M.H. All authors have read and agreed to the published version of the manuscript.
Competing interests
The authors declared no conflict of interest.
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