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. 2026 Jun 22;55:101293. doi: 10.1016/j.bbih.2026.101293

CYFIP1 overexpression amplifies IL-6/STAT3 and IFN-γ/STAT1 signaling: potential implications for neuroinflammation and autism spectrum disorder

Emily-Rose Martin a, Josan G Martin a, Mark A Russell a, Asami Oguro-Ando a,b,⁎
PMCID: PMC13316639  PMID: 42382713

Abstract

Background and objectives

Autism spectrum disorder (ASD) encompasses a group of neurodevelopmental disorders influenced by genetic and environmental factors, although the molecular mechanisms underlying their interactions remain unclear. We previously reported dysregulated cytokine signalling in chromosome 15q-duplication syndrome (Dup(15q)), a common syndromic form of ASD. Dup(15q) induced pluripotent stem cell (iPSC)-derived neurons exhibit an amplified Signal Transducer and Activator of Transcription-3 (STAT3) response to Interleukin-6 (IL-6), a cytokine often upregulated in ASD. To identify candidate genes within the 15q region that may modify cytokine signalling, we investigated Cytoplasmic FMRP-Interacting Protein 1 (CYFIP1), as CYFIP1 dysregulation has been linked to altered expression of genes involved in immunoregulatory pathways.

Methods

CYFIP1 was overexpressed using a plasmid vector in human HEK-293 and SH-SY5Y neuroblastoma cells. Following stimulation with IL-6 or Interferon (IFN)-γ, a variety of biochemical assays (qRT-PCR, Western blotting and dual-luciferase reporter assays) and neurite tracing experiments were performed to assess the effects of increased CYFIP1 expression on IL-6/STAT3 and IFN-γ/STAT1 signaling responses.

Results

CYFIP1-overexpression HEK-293 cells display reduced STAT3 and STAT1 expression, but enhanced IL-6-induced/IFN-γ-induced STAT3/STAT1 transcriptional activity. Furthermore, CYFIP1-overexpression in SH-SY5Y cells was associated with reduced basal neurite outgrowth and altered IL-6-associated neurite outgrowth.

Conclusions

These findings suggest that CYFIP1-overexpression modifies cytokine-responsive transcriptional pathways in vitro and provides novel insight into how CYFIP1 dysregulation may contribute to dysregulated cytokine signaling in ASD, advancing our understanding of the molecular mechanisms underlying neuroinflammatory processes in this disorder.

Keywords: Autism spectrum disorder, CYFIP1, JAKMIP1, Cytokine signaling, IL-6, STAT3

Highlights

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    CYFIP1 -overexpression reduced the expression of STAT1 and STAT3 in HEK-293 cells.

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    Despite this, increased expression of CYFIP1 in HEK-293 cells enhances IL-6-induced STAT3 and IFN-γ-induced STAT1 activity.

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    CYFIP1-overexpressing SH-SY5Y cells show altered IL-6-associated neurite outgrowth.

1. Background

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental conditions in which both genetic variation and environmental exposures contribute to pathogenesis (American Psychiatric Publishing, 2016). Due to the highly heterogeneous nature of the disorder, both diagnosis and treatment remain challenging. Genetic factors, including both rare and common variants, are implicated in ASD, with over 1000 genes identified as potentially contributing to its onset and development (Abrahams et al., 2013). ASD heritability is estimated to be between 26 and 93% (Bourgeron, 2015), with concordance rates between siblings around 30%, dizygotic twins about 65%, and up to 99% in monozygotic twins (Ramaswami and Geschwind, 2018; Colvert et al., 2015; Hallmayer et al., 2011; Rosenberg et al., 2009; Bailey et al., 1995). In addition to genetic predisposition, environmental risk factors, particularly maternal immune activation (MIA) and neuroinflammation, have been recognised as key contributors to ASD pathogenesis (Modabbernia et al., 2017; Zawadzka et al., 2021). Therefore, it is likely that the interaction between an individual's genetic background and specific environmental exposures plays a crucial role in triggering the pathogenesis underlying ASD (Modabbernia et al., 2017). Consequently, elucidating the molecular mechanisms by which ASD-associated genes can alter neurodevelopmental processes in response to environmental stressors is critical for advancing our understanding of the disorder.

Janus Kinase and Microtubule-Interacting Protein 1 (JAKMIP1) was previously identified as a potential molecular link between syndromic forms of ASD and neuroinflammation (Nishimura et al., 2007). JAKMIP1 encodes a multi-functional protein that possesses microtubule- and RNA-binding capabilities, associates with Type B GABA Receptors and Janus Kinases (JAKs), and is reported to be involved in various cellular processes, including microtubule stabilisation, intracellular trafficking, neurite outgrowth, neuronal migration and local activity-dependent protein translation (Steindler et al., 2004a; Vidal et al., 2007, 2012; Berg et al., 2015; Couve et al., 2004). JAKMIP1 expression is commonly dysregulated in Fragile X syndrome (FXS) and chromosome 15q-duplication syndrome (Dup(15q)), both genetic disorders often comorbid with ASD (Nishimura et al., 2007). Furthermore, we discovered a role for JAKMIP1 in regulating Interleukin-6 (IL-6)-induced Signal Transducer and Activator of Transcription-3 (STAT3) signaling in neuronal cells, whereby deficiency of JAKMIP1 in SH-SY5Y neuroblastoma cells impairs STAT3 activation and neuritogenesis in response to IL-6 stimulation (Martin et al., 2025). These findings suggest that dysregulation of JAKMIP1 in ASD may alter the ability of neurons to respond to inflammatory cytokines, potentially leading to aberrations in neuronal morphology.

To investigate whether JAKMIP1-regulated IL-6/STAT3 signaling was relevant to syndromic forms of ASD, we previously assessed IL-6-induced STAT3 signaling in a Dup (15q) induced pluripotent stem cell (iPSC)-derived neuronal model (Martin et al., 2025). We discovered that whilst JAKMIP1 expression fluctuated in Dup(15q) iPSCs and iPSC-derived cortical neurons, IL-6-induced STAT3 activity was significantly enhanced in both Dup(15q) iPSCs and iNeurons (Martin et al., 2025). These findings suggested that some gene (or collection of genes) in the q arm of chromosome 15 likely exerts stronger influence on STAT3 activity in the context of ASD.

Several genes are encoded within the 15q region of chromosome 15, however, a gene of particular interest in relation to ASD and cytokine signaling is Cytoplasmic FMRP-Interacting Protein 1 (CYFIP1), as studies have demonstrated that duplications (or even deletions) encompassing CYFIP1 increase the severity of neurobehavioural phenotypes (Butler et al., 2004), and that CYFIP1 expression is upregulated in the brains of individuals with Dup(15q) and ASD (Oguro-Ando et al., 2015). Although CYFIP1 is most known for its roles in repressing neuronal protein translation as part of Fragile X Messenger Ribonucleoprotein (FMRP)-containing ribonucleoprotein complexes (Napoli et al., 2008) and regulating actin polymerisation as a core component of the WAVE regulatory complex (WRC) (DeRubeis et al., 2013), there is evidence to suggest that CYFIP1 dysregulation may alter the expression of various genes involved in immunoregulatory signaling pathways (Urraca et al., 2018). For example, a recent study profiling transcriptomic changes in Dup(15q) stem cell-derived neurons with upregulated CYFIP1 expression suggested that increased dosage of chromosome 15q11.2 genes could alter the expression of gene targets of SMAD Family Member 3 (SMAD3), Nuclear Factor Kappa B (NF-κB) or Interferons (IFN) (Urraca et al., 2018). Moreover, another study identified CYFIP1 as a potential hub gene that may coordinate Transforming Growth Factor Beta 1 (TGF-β1)/SMAD3 signaling (Erickson et al., 2019). Therefore, we hypothesised that CYFIP1 may be responsible for the observed enhanced IL-6-induced activity in Dup (15q) iPSCs and iNeurons (Martin et al., 2025).

This work explores the effects of increased CYFIP1 expression (modelled by CYFIP1-overexpression (OE)) on cytokine signaling. We provide the first evidence to directly support a novel role for CYFIP1 in modulating cytokine signaling, demonstrating that CYFIP1-OE in Human embryonic kidney (HEK)-293 cells alters the expression and activity of cytokine signaling-related transcription factors, STAT1 and STAT3. Taking this further, we show how alterations in the STAT3 signaling pathway due to CYFIP1-OE affects IL-6-induced neurite outgrowth in human SH-SY5Y neuroblastoma cells, illustrating that these findings continue to be relevant in a more neuronal-relevant model.

2. Methods

Full methodology details can be found in Supplementary File 1.

2.1. HEK-293 and SH-SY5Y cell culture

HEK-293 cells (#CRL-1573™, American Type Culture Collection) and human neuroblastoma SH-SY5Y cells (#CRL-2266™, American Type Culture Collection) were maintained in Dulbecco's modified eagle medium/nutrient mixture F-12 with GlutaMAX (referred to henceforth as DMEM/F-12) (#11524436, Fisher Scientific™) supplemented with 10% foetal bovine serum (FBS) (#11550356, Fisher Scientific™), and incubated at 37°C, 5% CO2, 95% humidity. Cells were passaged at 70-80% confluency using TrypLE™ (#10718463, Fisher Scientific™).

2.2. Cytokine treatments

Cells were stimulated with 20 ng/mL IL-6 (#7270IL-025, R&D Systems) or 20 ng/mL Interferon-γ (IFN-γ) (#285-IF-100, R&D Systems) diluted in DMEM/F-12 + FBS (or in DMEM/F-12 supplemented with Retinoic Acid (RA) and Brain-Derived Neurotrophic Factor (BDNF) for neurite tracing experiments). These cytokine concentrations and time-points were chosen as we have previously found them to be sufficient for inducing a STAT3 or STAT1 response (Martin et al., 2025; Dhayal et al., 2022). For all experiments using IL-6 or IFN-γ, control cells were used which were treated with corresponding media not containing IL-6 or IFN-γ. A schematic overview of the cytokine treatment conditions and time-points can be found in Supplementary Figure 1.

2.3. Plasmid transfection

For signalling pathway experiments, HEK-293 cells were transfected with either an RFP-T2A-CYFIP1 plasmid to induce CYFIP1-overexpression (these cells are referred to as ‘CYFIP1-OE cells'), or the backbone RFP-T2A plasmid as a control (these cells are referred to as ‘control cells’) using Lipofectamine™ LTX Reagent (#15338100, Fisher Scientific™), following manufacturer instructions. Media was replaced the following day following transfection and all experiments were performed 72 h post-transfection.

For neurite tracing experiments, SH-SY5Y cells were transfected by Nucleofection™ (#V4XC-2012, Lonza Bioscience), following manufacturer instructions, with either a mixture of the RFP-T2A backbone vector and pCAβ-YFP (encoding yellow fluorescent protein (YFP)) in a 1:1 ratio as the control condition; or a mixture of RFP-T2A-CYFIP1 and pCAβ-YFP (4:1 ratio) to assess the effects of CYFIP1-OE on neuritogenesis. The pCAβ-YFP plasmid was co-transfected to enable better visualisation of the cells for neurite tracing as the RFP encoded by the CYFIP1-OE plasmid photo-bleaches quickly. Media was replaced the following day after Nucleofection™.

2.4. Protein extraction and Western blotting

Cells were harvested in ice-cold RIPA lysis buffer supplemented with protease and phosphatase inhibitors. 50 μg of total protein was heated in Laemmli buffer for 5 min at 95°C and loaded into 4–15% precast polyacrylamide gels (#4561085, Bio-Rad Laboratories) for Western blotting alongside a pre-stained protein ladder (#PL00001, Proteintech Group). Following electrophoresis, proteins were transferred onto methanol-activated PVDF membranes (#IPVH304F0, Millipore), then sequentially incubated in blocking buffer and antibody solutions. Proteins were visualised using the Odyssey® CLx Imager (LI-COR Biosciences) and the Image Studio software (Version 5.2; LI-COR Biosciences) was used to perform densitometric analyses to compare the abundance of target proteins between samples. Full uncropped Western blot images for each primary antibody used can be found in Supplementary Figures 2-4, and antibody details and dilutions can be found in Supplementary File 1: Table 1.

2.5. RNA extraction, cDNA conversion and qRT-PCR

Total RNA was isolated and purified using the Direct-zol™ RNA Miniprep kit following manufacturer instructions (#R2051, Zymo Research). RNA was converted to cDNA using PrimeScript™ RT reagent kit (#RR037A, Takara Bio Europe) following manufacturer instructions. qRT-PCR was then performed using HOT FIREPol® EvaGreen® qPCR Master Mix with ROX (#01-02-00500, Solis BioDyne) with the QuantStudio 12K Flex qPCR machine (Thermo Fisher Scientific™). For primer sequences, see Supplementary File 1: Table 2. Each reaction was run in triplicate, and mRNA expression levels were normalised against the geometric mean of two reference genes using the Pfaffl method (Pfaffl, 2001). Gene expression ratios were then presented in figures relative to the control cells.

2.6. Dual-luciferase reporter assays

HEK-293 cells were transfected with STAT3 Cignal Reporter plasmid (#336841, GeneGlobe ID: CCS-9028L, Qiagen) or Gamma-ativated sequence (GAS) Cignal Reporter plasmid (#336841, GeneGlobe ID: CCS-009L, Qiagen) using Lipofectamine™ LTX Reagent. Six hours post-transfection, cells were treated with DMEM/F-12 + 10% FBS with or without 20 ng/mL IL-6 or IFN-γ and incubated for 18 h prior to the reporter assay. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter (DLR™) Assay System (#E1960, Promega), following manufacturer instructions, with the PHERAstar FS microplate reader (BMG LABTECH).

2.7. SH-SY5Y differentiation and neurite outgrowth analysis

SH-SY5Y cells were seeded onto sterile glass coverslips (#631-1578, VWR®; pre-coated with 20 μg/mL poly-D-Lysine (PDL; #354210, Corning®)) in DMEM/F-12 + 10% FBS. The following day, media was replaced with DMEM/F-12 (without FBS) containing 20 ng/mL BDNF and 10 μM RA. 48 h later, SH-SY5Y cells were fixed with 4% paraformaldehyde (PFA), and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (#D9542, Sigma-Aldrich®) prior to coverslip mounting onto microscope slides. Slides were imaged on the inverted DMi8 widefield microscope (Leica Microsystems).

All image analysis was performed manually using the FIJI software (ImageJ; Version 2.9.0 (Schindelin et al., 2012)). The length of the longest neurite produced by a single cell (referred to as longest neurite length (LNL); a measure of neurite extension) and the sum length of all neurites produced by a single cell (referred to as total neurite length (TNL); a measure of neuritogenesis) were measured.

2.8. Statistical analysis

Statistical analyses were performed using GraphPad Prism (GraphPad Prism Version 9.0.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com). Unpaired Student's t-Tests were used to assess statistical differences in measurements between two groups and two-way analysis of variance (ANOVA) were performed when assessing the effects of and interactions between two independent variables. Appropriate post hoc tests were also performed to account for multiple testing. Data are expressed as mean values ± standard error of the mean (SEM). P-values <0.05 were considered statistically significant.

3. Results

3.1. Modelling CYFIP1-overexpression

To model increased CYFIP1 expression, we utilised plasmid vectors encoding an RFP-T2A-CYFIP1 construct (to induce CYFIP1-OE) or RFP-T2A as a control, which we transfected into HEK-293 cells, referred to as CYFIP1-OE and control cells respectively (Fig. 1A and B). We then confirmed increased CYFIP1 mRNA expression using qRT-PCR (Fig. 1C) and protein expression using Western blotting (Fig. 1D and E). CYFIP1 mRNA expression was significantly increased in CYFIP1-OE cells (mean ± SEM = 472.30 ± 11.20 for CYFIP1-OE cells vs 1.00 ± 0.02 for control cells, P < 0.0001), as were CYFIP1 protein levels, compared to control cells (mean ± SEM = 3.68 ± 0.40 for CYFIP1-OE cells vs 1.00 ± 0.12 for control cells, P < 0.0001)

Fig. 1.

Fig. 1

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Modelling CYFIP1 overexpression in HEK-293 cells. A) HEK-293 cells were transfected with either RFP-T2A plasmid as a control (referred to as Control), or RFP-T2A-CYFIP1 to induce CYFIP1-overexpression (OE) (referred to as CYFIP1-OE). Created with BioRender.com. B) Control and CYFIP1-OE cells were imaged 72 h post-transfection live on the Leica DMi8 widefield microscope. Scale bar = 100 μm. C) Relative mRNA expression of CYFIP1 in Control and CYFIP1-OE cells 72 h post-transfection measured by qRT-PCR. D) Increased CYFIP1 protein expression in CYFIP1-OE cells 72 h post-transfection confirmed via Western blotting. E) Densitometric analysis of total CYFIP1 protein expression relative to the GAPDH loading control. All values are normalised to the Control cells. For all experiments, values are presented as mean ± SEM of N = 3 independent experiments, 2-3 technical replicates each. Statistical significance against Control was determined using Unpaired Student's T-Tests; ∗∗∗∗P < 0.0001.

3.2. CYFIP1-OE leads to reduced JAKMIP1 expression

It has been previously demonstrated that JAKMIP1 expression is reduced following CYFIP1-OE in human neuroblastoma cells (Nishimura et al., 2007). Therefore, to confirm whether this holds true in HEK-293 cells, we measured JAKMIP1 expression in CYFIP1-OE HEK-293 cells by qRT-PCR and Western blotting. Consistent with Nishimura et al. (2007), we found that JAKMIP1 mRNA expression was significantly reduced in CYFIP1-OE HEK293 cells (Fig. 2A) (mean ± SEM = 0.88 ± 0.03 for CYFIP1-OE cells vs 1.00 ± 0.02 for control cells, P = 0.0098), as was JAKMIP1 protein expression (Fig. 2B and C) (mean ± SEM = 0.80 ± 0.07 for CYFIP1-OE cells vs 1.00 ± 0.04 for control cells, P = 0.038).

Fig. 2.

Fig. 2

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CYFIP1-OE leads to reduced JAKMIP1 mRNA and protein expression. A) Relative mRNA expression of JAKMIP1 in Control and CYFIP1-OE cells measured by qRT-PCR. B) Western blotting of JAKMIP1 expression in Control and CYFIP1-OE cells. C) Densitometric analysis of total JAKMIP1 protein expression relative to the GAPDH loading control. For all experiments, values are presented as mean ± SEM of N = 3 independent experiments, 2-3 technical replicates each. Statistical significance against Control was determined using Unpaired Student's T-Tests; ∗P < 0.05; ∗∗P < 0.01.

3.3. CYFIP1-OE alters the expression of several cytokine signaling-related transcription factors

We have previously demonstrated that Dup(15q) iPSCs and iNeurons with increased CYFIP1 expression show reduced STAT3 expression (Martin et al., 2025). Furthermore, we have demonstrated that JAKMIP1-knockout SH-SY5Y cells also show reduced STAT3 expression and that JAKMIP1-deficiency may alter other cytokine signaling pathways too (Martin et al., 2025). Therefore, to determine whether CYFIP1-OE may also lead to altered expression of cytokine signaling-related transcription factors, qRT-PCR was performed to measure mRNA expression of STAT1, STAT3, STAT5B and NFKB1. We observed that the expression of STAT1 (Fig. 3A) and STAT3 (Fig. 3B) were reduced in CYFIP1-OE HEK-293 cells (STAT1: mean ± SEM = 0.94 ± 0.02 for CYFIP1-OE cells vs 1.00 ± 0.02 for control cells, P = 0.05, STAT3: mean ± SEM = 0.93 ± 0.01 for CYFIP1-OE cells vs 1.00 ± 0.02 for control cells, P = 0.0007), whereas the expression of STAT5B (Fig. 3C) and NFKB1 (Fig. 3D) were increased (STAT5B: mean ± SEM = 1.11 ± 0.031 for CYFIP1-OE cells vs 1 ± 0.022 for control cells, P = 0.006, NFKB1: mean ± SEM = 1.14 ± 0.02 for CYFIP1-OE cells vs 1.00 ± 0.02 for control cells, P = 0.0002). These findings indicate that CYFIP1-OE is associated with altered expression of several cytokine signalling-related transcription factors.

Fig. 3.

Fig. 3

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CYFIP1-OE alters the expression of other cytokine signaling-related transcription factors. Relative mRNA expression of A)STAT1, B)STAT3, C)STAT5B or D)NFKB1 in Control and CYFIP1-OE cells measured by qRT-PCR. For all experiments, values are presented as mean ± SEM of N = 3 independent experiments, 2-3 technical replicates each. Statistical significance against Control was determined using Unpaired Student's T-Tests; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

3.4. CYFIP1-OE enhances IL-6-induced STAT3 activity

To investigate whether reduced STAT3 expression in CYFIP1-OE HEK-293 cells leads to altered cytokine-induced STAT3 responses, we first performed Western blotting to assess IL-6-induced STAT3Y705 phosphorylation (representative of STAT3 activation) (Fig. 4A). Despite the reduction in STAT3 expression (Fig. 4B) (mean ± SEM = 0.74 ± 0.08 for CYFIP1-OE cells vs 1.00 ± 0.07 for control cells, P = 0.019), CYFIP1-OE cells still show strong responses to IL-6, demonstrating no significant difference in IL-6-stimulated STAT3Y705 phosphorylation levels compared to control cells, both at the ‘whole-cell level’ (when STAT3Y705 phosphorylation levels are normalised to GAPDH) (Fig. 4C) (mean difference = 7.61 for CYFIP1-OE cells with vs without IL-6, P = 0.012; mean difference = 11.86 for control cells with vs without IL-6, P < 0.0001), and upon closer examination of the phosphorylation kinetics of STAT3 itself (when STAT3Y705 phosphorylation levels are normalised to total STAT3 protein levels) (Fig. 4D) (mean difference = 10.08 for CYFIP1-OE cells with vs without IL-6, P < 0.0001; mean difference = 10.60 for control cells with vs without IL-6, P < 0.0001). This suggests that although CYFIP1-OE cells show reduced STAT3 expression, STAT3 activation following IL-6 stimulation is unaltered.

Fig. 4.

Fig. 4

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CYFIP1-OE enhances STAT3 transcriptional activity but not STAT3 activation. A) Western blotting of STAT3 expression and phosphorylation (P-STAT3Y705) in Control and CYFIP1-OE cells following 30-min treatment with 0 ng/mL IL-6 or 20 ng/mL IL-6. Densitometric analysis of B) total STAT3 protein expression relative to the GAPDH loading control, C) P-STAT3Y705 relative to the GAPDH loading control or D) P-STAT3Y705 relative to total STAT3 protein expression. E) Total transcriptional activity at a STAT3-regulated promoter in Control and CYFIP1-OE cells stimulated with or without 20 ng/mL IL-6 for 18 h post-transfection with Cignal Reporter plasmids. Firefly luminescence intensity is expressed as a ratio to Renilla luminescence intensity to adjust for transfection efficiency. F) Relative mRNA expression of STAT3 in Control and CYFIP1-OE cells treated with 0 ng/mL or 20 ng/mL IL-6 for 4 h, measured by qRT-PCR. G) Fold change in STAT3 mRNA expression following IL-6 treatment relative to untreated cells in part F). Values presented as mean ± SEM of N = 3 independent experiments, 3 technical replicates each. Statistical significance against Control was determined using Unpaired Student's T-Tests; ∗∗∗∗P < 0.0001. H) Relative mRNA expression of SOCS3 in Control and CYFIP1-OE cells treated with 0 ng/mL or 20 ng/mL IL-6 for 4 h, measured by qRT-PCR. I) Fold change in SOCS3 mRNA expression following IL-6 treatment relative to untreated cells in part H). For all experiments, values are presented as mean ± SEM of N = 3 independent experiments, 3-4 technical replicates each. Statistical significance against Control was determined using Unpaired Student's T-Tests when comparing two groups, or two-way ANOVAs with Tukey's Honest Significant Difference tests when comparing multiple groups; ns – P > 0.05; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.

STAT3 activity is modulated by various post-translational modifications in addition to phosphorylation at the Y705 residue. Therefore, despite observing no change in STAT3Y705 phosphorylation following CYFIP1-OE, we examined whether CYFIP1-OE cells show altered IL-6-induced transcriptional activity by performing a DLR™ assay. This assay places the control of a Firefly luciferase under a STAT3 response element, meaning that increased Firefly luminescence is indicative of increased STAT3 transcriptional activity. Using this DLR™ assay, we found that CYFIP1-OE cells show increased Firefly luminescence following IL-6 stimulation than control cells (Fig. 4E) (mean difference = 46.84 for CYFIP1-OE cells with vs without IL-6, P < 0.0001; mean difference = 28.77 for control cells with vs without IL-6, P < 0.0001; mean difference = 18.09 for CYFIP1-OE cells with IL-6 vs control cells with IL-6, P < 0.0001), suggesting that IL-6-induced STAT3 transcriptional activity is enhanced in CYFIP1-OE cells regardless of the reduced STAT3 expression.

To validate the DLR™ assay results, qRT-PCR was used to measure the mRNA expression of STAT3-responsive genes in CYFIP1-OE cells following IL-6 stimulation. Somewhat contradictory to the DLR™ assay, levels of STAT3 mRNA in IL-6-treated CYFIP1-OE cells were not higher than those in IL-6-treated control cells (Fig. 4F) (mean difference = 0.19 for CYFIP1-OE cells with vs without IL-6, P < 0.0001; mean difference = 0.10 for control cells with vs without IL-6, P = 0.026; mean difference = 0.01 for CYFIP1-OE cells with IL-6 vs control cells with IL-6, P = 0.99). However, considering that CYFIP1-OE cells already show reduced STAT3 mRNA expression at baseline, this is unsurprising. Therefore, after normalising the levels of IL-6-stimulated STAT3 expression to unstimulated STAT3 expression, CYFIP1-OE cells show an enhancement in IL-6-induced STAT3 mRNA expression compared to control cells (Fig. 4G) (mean ± SEM = 1.21 ± 0.01 for CYFIP1-OE cells vs 1.00 ± 0.02 for control cells, P < 0.0001). The expression of SOCS3 shows a similar phenomenon whereby IL-6-stimulated CYFIP1-OE cells appear to show lower SOCS3 mRNA levels compared to IL-6-treated control cells (Fig. 4H) (mean difference = 0.49 for CYFIP1-OE cells with vs without IL-6, P < 0.0001; mean difference = 0.34 for control cells with vs without IL-6, P = 0.0014; mean difference = 0.36 for CYFIP1-OE cells with IL-6 vs control cells with IL-6, P = 0.0009), however the fold change in SOCS3 expression relative to unstimulated cells shows that IL-6-induced SOCS3 expression is magnified in CYFIP1-OE cells (Fig. 4I) (mean ± SEM = 1.99 ± 0.007 for CYFIP1-OE cells vs 1.00 ± 0.05 for control cells, P < 0.0001). Taken together, these findings complement the DLR™ assay results, suggesting that CYFIP1-OE cells show enhanced STAT3 transcriptional activity following IL-6 stimulation.

3.5. CYFIP1-OE alters IL-6-associated neurite outgrowth in SH-SY5Y cells

To determine whether CYFIP1-OE is associated with altered IL-6-related neurite outgrowth, we differentiated control and CYFIP1-OE SH-SY5Y cells in the absence or presence of IL-6 and measured longest neurite length (LNL) and total neurite length (TNL) (Fig. 5A), as we have previously shown that IL-6 induces neurite outgrowth in SH-SY5Y cells (Martin et al., 2025). As expected, control and CYFIP1-OE cells differentiated in the presence of IL-6 exhibit longer LNL (Fig. 5B) and TNL (Fig. 5C) measurements than cells differentiated without IL-6. CYFIP1-OE cells appear to have enhanced neurite outgrowth responses to IL-6, as although CYFIP1-OE cells produce shorter neurites compared to the control as baseline, the LNL (Fig. 5B) and TNL (Fig. 5C) measurements are no longer significantly different between the two populations when differentiated with IL-6 (LNL: mean difference = 13.60 for CYFIP1-OE cells with vs without IL-6, P < 0.0001; mean difference = 7.47 for control cells with vs without IL-6, P = 0.025; mean difference = 7.12 for CYFIP1-OE cells without IL-6 vs control cells without IL-6, P = 0.039; mean difference = 0.99 for CYFIP1-OE cells with IL-6 vs control cells with IL-6, P = 0.98) (TNL: mean difference = 17.38 for CYFIP1-OE cells with vs without IL-6, P < 0.0001; mean difference = 12.47 for control cells with vs without IL-6, P = 0.0024; mean difference = −10.00 for CYFIP1-OE cells without IL-6 vs control cells without IL-6, P = 0.026; mean difference = -5.08 for CYFIP1-OE cells with IL-6 vs control cells with IL-6, P = 0.47). These data indicate that CYFIP1-OE is associated with reduced basal neurite outgrowth in SH-SY5Y cells, and while IL-6 treatment increases neurite outgrowth in both control and CYFIP1-OE cells, IL-6 treatment reduces the baseline difference between the two groups, suggesting that CYFIP1-OE may be enhancing IL-6-associated neurite outgrowth.

Fig. 5.

Fig. 5

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CYFIP1-OE enhances IL-6-induced neuritogenesis. SH-SY5Y cells were Nucleofected with either the RFP-T2A backbone vector and pCAβ-YFP as the control condition; or RFP-T2A-CYFIP1 and pCAβ-YFP to assess the effects of CYFIP1-OE on IL-6-influenced neuritogenesis. Cells were differentiated for two days with 20ng/mL BDNF and 10μM RA under serum starvation with or without 20ng/mL IL-6, fixed with 4% PFA and stained with DAPI. A) Fluorescence micrographs of differentiated SH-SY5Y cells. Images captured using an upright Leica DM4000 B LED microscope. Scale bar = 50μm. Neurite tracing analysis was performed using the FIJI software. B) The length of the longest neurite produced by the cell was measured for all conditions. C) The sum length of all neurites produced per cell is presented for each condition. For all experiments, values are presented as mean ± SEM of N = 4 independent experiments, 3 technical replicates each. Statistical significance against Control was determined using two-way ANOVAs with Tukey's Honest Significant Difference tests; ns – P > 0.05; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗∗P < 0.0001.

3.6. CYFIP1-OE enhances IFN-γ-induced STAT1 transcriptional activity

Having determined that CYFIP1-OE alters the expression of several cytokine signaling-related transcription factors, and that CYFIP1-OE enhances IL-6-induced STAT3 activity, we aimed to investigate whether CYFIP1-OE alters the activity of another cytokine signaling-related transcription factor. STAT1 was chosen to follow up with because similar to STAT3 expression, the expression of STAT1 was also reduced in CYFIP1-OE HEK-293 cells. A DLR™ assay which placed the control of a Firefly luciferase under a GAS element (the response element STAT1 binds to initiate transcription) was performed. We found that CYFIP1-OE HEK-293 cells show increased Firefly luminescence following IFN-γ stimulation compared to control cells (Fig. 6A) (mean difference = 0.97 for CYFIP1-OE cells with vs without IFN-γ, P < 0.0001; mean difference = 0.49 for control cells with vs without IFN-γ, P < 0.0001; mean difference = 0.72 for CYFIP1-OE cells with IFN-γ vs control cells with IFN-γ, P < 0.0001), suggesting that IFN-γ-induced STAT1 transcriptional activity is enhanced in CYFIP1-OE cells, again regardless of the reduced STAT1 expression.

Fig. 6.

Fig. 6

Open in a new tab

CYFIP1-OE leads to enhanced STAT1 transcriptional activity following IFN-γ treatment. A) Total transcriptional activity at a STAT1-regulated promoter in Control and CYFIP1-OE cells stimulated with or without 20ng/mL IFN-γ for 18 h post-transfection with Cignal Reporter plasmids. Firefly luminescence intensity is expressed as a ratio to Renilla luminescence intensity to adjust for transfection efficiency. B) Relative mRNA expression of STAT1 in Control and CYFIP1-OE cells treated with 0ng/mL or 20ng/mL IFN-γ for 4 h, measured by qRT-PCR. C) Relative mRNA expression of SOCS1 in Control and CYFIP1-OE cells treated with 0ng/mL or 20ng/mL IFN-γ for 4 h, measured by qRT-PCR. For all experiments, values are presented as mean ± SEM of N = 4 independent experiments, 3 technical replicates each. Statistical significance against Control was determined using two-way ANOVAs with Tukey's Honest Significant Difference tests; ns – P > 0.05; ∗P < 0.05; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.

As validation of the DLR™ assay, qRT-PCR was performed to measure the expression of STAT1-responsive genes following IFN- γ stimulation. CYFIP1-OE cells show increased IFN-γ-induced STAT1 expression (Fig. 6B) (mean difference = 2.41 for CYFIP1-OE cells with vs without IFN-γ, P < 0.0001; mean difference = 1.71 for control cells with vs without IFN-γ, P < 0.0001; mean difference = 0.54 for CYFIP1-OE cells with IFN-γ vs control cells with IFN-γ, P = 0.049), suggesting an enhanced transcription response to IFN-γ stimulation. Additionally, although not significantly different at baseline, CYFIP1-OE cells show increased SOCS1 expression following IFN-γ treatment (Fig. 6C) (mean difference = 0.74 for CYFIP1-OE cells with vs without IFN-γ, P < 0.0001; mean difference = 0.46 for control cells with vs without IFN-γ, P = 0.0003; mean difference = 0.31 for CYFIP1-OE cells with IFN-γ vs control cells with IFN-γ, P = 0.018), again suggesting an enhanced STAT1 transcriptional response to IFN-γ stimulation. This also suggests that the observed alterations in IL-6/STAT3 signaling are not limited to STAT3 and that CYFIP1 may play a role in modulating a larger range of cytokine signaling pathways.

4. Discussion

4.1. A novel role for CYFIP1 in modulating IL-6/STAT3 signaling independent from JAKMIP1

Nishimura et al. (2007) first linked JAKMIP1 to ASD and Dup(15q), reporting that JAKMIP1 expression was altered in Dup(15q) and that overexpression of the chromosome 15q gene CYFIP1 correspondingly resulted in JAKMIP1 downregulation. We expanded upon this in our previous work, identifying JAKMIP1 as a modulator of IL-6 cytokine signaling in SH-SY5Y cells, and demonstrating that Dup(15q) iPSCs and cortical neurons with elevated CYFIP1 dosage and expression exhibit enhanced IL-6-induced STAT3 activity (Martin et al., 2025). However, given that the magnified IL-6 signaling observed in these Dup(15q) cells did not correlate with JAKMIP1 expression levels, we hypothesised that another gene within the 15q region, likely CYFIP1, might be driving this increased responsiveness to IL-6.

To test this hypothesis, we induced CYFIP1-overexpression in HEK-293 cells and discovered that this resulted in the altered expression of several cytokine signaling-related transcription factors. In particular, STAT1 and STAT3 expression were reduced, whereas STAT5B and NFKB1 expression were raised, suggesting that CYFIP1 may potentially exert broad cellular functions that impact the signaling networks of multiple cytokines. Taking this forward, we measured IL-6-induced activation and activity of STAT3. Consistent with the Dup(15q) iPSCs and neurons from our previous work, we observed that despite the reduced STAT3 expression, IL-6-induced STAT3 responses were enhanced in CYFIP1-OE HEK-293 cells. Furthermore, CYFIP1-overexpression also potentiated IL-6-triggered neuritogenesis in SH-SY5Y cells. As such, this study provides the first evidence that CYFIP1 may play a role in regulating IL-6/STAT3 signaling in both HEK-293 and SH-SY5Y cells, and its upregulation could be responsible for the enhanced responsiveness to IL-6 exhibited by Dup(15q) iPSCs and neurons.

4.2. How does CYFIP1 influence STAT1 and STAT3 activity?

The mechanisms underlying CYFIP1-mediated regulation of STAT1 and STAT3 expression and activity remain unclear. Regarding STAT3, reduced STAT3 expression in JAKMIP1-deficient cells (Martin et al., 2025) may suggest that JAKMIP1, rather than CYFIP1, is responsible for regulating STAT3 expression. This could occur through its interactions with various RNA-binding proteins or splicing factors (Martin et al., 2025). However, it is important to note that CYFIP1-overexpression enhances IL-6-induced STAT3 transcriptional activity, whereas JAKMIP1-knockout impairs it (Martin et al., 2025). Thus, whilst JAKMIP1 could be responsible for controlling STAT3 expression, when considering STAT3 activity, our results indicate that it is more likely that CYFIP1 is responsible by a mechanism independent of JAKMIP1.

Interestingly, despite CYFIP1-overexpression leading to increased STAT3 transcriptional activity following IL-6 treatment, we observed no significant difference in the levels STAT3Y705 phosphorylation in CYFIP1-OE cells, suggesting that CYFIP1 does not alter STAT3 phosphorylation kinetics, and thus STAT3 activation. One reason for this discrepancy could be because of the influence of other post-translational modifications of STAT3. For example, STAT3K685 acetylation may be necessary for IL-6-induced STAT3-responsive gene expression (Wang et al., 2005). Rather than modulating STAT3 activation through phosphorylation, perhaps CYFIP1 may somehow control STAT3 activation through acetylation, however, the exact mechanism by which CYFIP1 could regulate acetylation is unclear.

In terms of STAT1 expression and activity, it would not be unreasonable to suggest that these changes in STAT1 activity may be mediated by the changes in JAKMIP1 expression. JAKMIP1 was previously reported to inhibit Interferon-α (IFN-α) signaling (Steindler et al., 2004b). Importantly, both IFN-α and IFN-γ signaling cascades involve the phosphorylation and activation of STAT1. It is therefore likely that CYFIP1-induced downregulation of JAKMIP1 may reduce the inhibiting influence of JAKMIP1 on IFN signaling, thereby enhancing the IFN response. Furthermore, STAT1 activity is also known to be altered by acetylation, similar to STAT3 (Leslie et al., 2024). If CYFIP1-mediated regulation of STAT3 is dependent on post-translational modifications, the same mechanism(s) could be responsible for CYFIP1 regulation of both STAT3 and STAT1 activity. Of course, all of these possibilities would require further validation.

4.3. Relevance to ASD and neurodevelopment

Cytokine signaling plays crucial roles in neurodevelopment beyond immune responses, with STAT3 and STAT1 signaling in particular regulating several key processes. For example, STAT3 signaling has been demonstrated to control the switch between neurogenesis and gliogenesis, promoting both neural progenitor cell self-renewal and astrocyte differentiation (Hong and Song, 2014). Similarly, STAT1 signaling has been shown to be involved in neurogenesis, and promoting neurite outgrowth (Warre-Cornish et al., 2020). Given how crucial STAT3 and STAT1 signaling are for neurodevelopment, it is unsurprising that alterations in STAT3 and STAT1 signaling are associated with Neurodevelopmental disorders such as ASD (Garay and McAllister, 2010; Majerczyk et al., 2022). MIA, where an immune response is triggered by infection or exposure to immunogenic material during pregnancy, is associated with increased incidence of ASD (Choudhury and Lennox, 2021). Both IL-6 and IFN-γ are pro-inflammatory cytokines that have been implicated in the pathogenesis of ASD, with IL-6 being a key player in MIA-induced ASD-like behaviours (Choudhury and Lennox, 2021; Samuelsson et al., 2006). Considering that CYFIP1-OE enhances STAT3 and STAT1 responses to IL-6 and IFN-γ respectively suggests that CYFIP1 may play a pivotal role in amplifying the inflammatory signals during MIA, potentially exacerbating the effects of immune activation on foetal brain development.

Amplification of both the IL-6/STAT3 and IFN-γ/STAT1 signaling pathways by CYFIP1 provides a potential molecular mechanism that explains the overlap between immune activation and ASD, particularly in cases where no obvious inflammatory trigger is identified. Elevated cytokine levels are frequently reported in individuals with ASD (Ashwood et al., 2011), and our results suggest that CYFIP1-overexpression in certain cases of syndromic ASD could act as a molecular convergence point, increasing the sensitivity of the developing brain to immune signals even under baseline inflammatory conditions. This could help to explain cases of ASD where immune activation is not readily apparent but inflammation-induced neurodevelopmental disruption still occurs.

4.4. Limitations to this study

HEK-293 cells were used in this study as they are easy to transfect and thus well suited to enable transient CYFIP1-overexpression (Thomas and Smart, 2005). As we are examining the role of CYFIP1 in the context of ASD and neural cytokine signaling, HEK-293 cells may not have been the most representative model cell line due to their renal origin. However, despite being an embryonic kidney cell line, there is some evidence to suggest that HEK-293 cells display neural characteristics, expressing many neuronal genes, including those encoding neurofilament proteins and neuronal receptors (He and Soderlund, 2010; Shaw et al., 2002). Therefore, although HEK-293 cells are non-neuronal, they can be used for their neuronal progenitor cell-like properties (He and Soderlund, 2010; Shaw et al., 2002). Importantly, we also tested the potential effect of altered IL-6/STAT3 signaling on a neuronal behaviour using differentiated SH-SY5Y cells, therefore validating our findings in a more neuronal-relevant model. It was also beneficial to use SH-SY5Y cells so that we could directly compare the effects of CYFIP1-OE on IL-6-stimulated neurite outgrowth to JAKMIP1 deficiency as per our previous work (Martin et al., 2025). Yet, it is also important to acknowledge that SH-SY5Y cells are not a perfect model (Kovalevich and Langford, 2013) and it is imperative that these findings are replicated in a more mature neuronal model to test direct relevancy for ASD.

In addition to choice of cell lines, there are also limitations to consider with the methodologies implemented to study cytokine signaling pathways. First is the choice of cytokine concentration. Cells were treated with 20 ng/mL IL-6 or IFN-γ, as we have previously found these concentrations to be sufficient to induce a robust STAT3 or STAT1 activation (Martin et al., 2025; Dhayal et al., 2022), yet these concentrations are higher even than the serum concentrations of IL-6 or IFN-γ observed in individuals with sepsis (Hou et al., 2015; Gharamti et al., 2022). Although supraphysiological, these concentrations ensure a strong signalling response, facilitating the detection of conditions that may modify these signalling pathways. Another important consideration when investigating cytokine signaling pathways is the choice of timepoints for cytokine treatments. All cytokine treatment timepoints were also chosen based on previous work (Martin et al., 2025; Dhayal et al., 2022). However, it is important to note that STAT responses to cytokine stimulation fluctuate, and that they tend to be self-propagating responses (Braun et al., 2013; Garbers et al., 2015). Hence, it is possible that varying cytokine concentrations or the duration of treatments may reveal additional insights into how these signaling pathways are altered in cases of ASD involving alterations to CYFIP1 expression and function (or its related pathways).

5. Conclusion

In summary, this study identifies a novel role for CYFIP1 in regulating the expression of cytokine signaling-related transcription factors, as well as enhancing their activity following cytokine stimulation. By amplifying these inflammatory pathways, CYFIP1 could potentially enhance neuroinflammatory signaling, altering neurodevelopment and increasing susceptibility in certain syndromic forms of ASD. Further research is required to understand the mechanism by which CYFIP1 modulates these cytokine signaling pathways and to investigate this in the context of ASD with MIA.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Funding

This work was supported by a Wellcome Wellcome Trust funded Translational Research Exchange @ Exeter (TREE) pump-priming fund and the Bristol Japanese Cultural Showcase.

CRediT authorship contribution statement

Emily-Rose Martin: Conceptualization, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Josan G. Martin: Conceptualization, Methodology, Writing – review & editing. Mark A. Russell: Supervision, Writing – review & editing. Asami Oguro-Ando: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The research was carried out at the National Institute for Health and Care Research (NIHR) Exeter Biomedical Research Centre (BRC). We gratefully acknowledge the RILD Imaging Facility, University of Exeter for their support and assistance with this work. We also extend our sincere gratitude to the generous support of the charitable organisations that contributed to this work, including the Wellcome Trust Translational Research Exchange @ Exeter (TREE) pump-priming fundand the Bristol Japanese Cultural Showcase. We thank Dr. Kaiyven Afi Leslie for his technical advice and support and are grateful for valuable discussions during lab meetings with Liming Yang and Madeline Eve.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbih.2026.101293.

List of abbreviations

ASD

Autism spectrum disorder

Dup(15q)

Chromosome 15q-duplication syndrome

CYFIP1

Cytoplasmic FMR1-intercating protein 1

iPSC

Induced pluripotent stem cell

IL-6

Interleukin-6

STAT3

Signal transducer and activator of transcription 3

OE

Overexpression

MIA

Maternal immune activation

JAKMIP1

Janus kinase and microtubule-interacting protein 1

FXS

Fragile X syndrome

FMRP

Fragile X messenger ribonuclear protein

WRC

WAVE regulatory complex

SMAD3

SMAD family member 3

NF-kB

Nuclear factor of κB

TGF-β1

Transforming growth factor β1

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

DLR™

Dual-luciferase reporter™

SOCS3

Suppressor of cytokine signaling 3

LNL

Longest neurite length

TNL

Total neurite length

IFN-γ

Interferon-γ

RA

Retinoic acid

BDNF

Brain-derived neurotrophic factor

HEK

Human embryonic kidney

FBS

Foetal bovine serum

JAK

Janus Kinase

RFP

Red fluorescent protein

YFP

Yellow fluorescent protein

GAS

Gamma-interferon-activated site

PDL

Poly-d-lysine

DAPI

4',6-diamidine-2-phenylindone

POLR2A

RNA polymerase II subunit A

Appendix A. Supplementary data

The following is/are the supplementary data to this article:

Multimedia component 1
mmc1.docx (38.3KB, docx)
Multimedia component 2
mmc2.docx (1.2MB, docx)

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
mmc1.docx (38.3KB, docx)
Multimedia component 2
mmc2.docx (1.2MB, docx)

Data Availability Statement

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Data will be made available on request.

Articles from Brain, Behavior, & Immunity - Health are provided here courtesy of Elsevier

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