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

CYFIP1 overexpression amplifies IL-6/STAT3 and IFN-gamma/STAT1 signaling: potential implications for neuroinflammation and autism spectrum disorder | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results CYFIP1 overexpression amplifies IL-6/STAT3 and IFN-gamma/STAT1 signaling: potential implications for neuroinflammation and autism spectrum disorder View ORCID Profile Emily-Rose Martin , View ORCID Profile Josan G. Martin , Mark A. Russell , View ORCID Profile Asami Oguro-Ando doi: https://doi.org/10.1101/2025.10.23.683866 Emily-Rose Martin 1 University of Exeter Medical School, University of Exeter , Exeter, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Emily-Rose Martin Josan G. Martin 1 University of Exeter Medical School, University of Exeter , Exeter, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Josan G. Martin Mark A. Russell 1 University of Exeter Medical School, University of Exeter , Exeter, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Asami Oguro-Ando 1 University of Exeter Medical School, University of Exeter , Exeter, United Kingdom 2 Faculty of Pharmaceutical Sciences, Tokyo University of Science , Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Asami Oguro-Ando For correspondence: a.oguro-ando{at}exeter.ac.uk Abstract Full Text Info/History Metrics Preview PDF Abstract Autism spectrum disorder (ASD) encompasses a group of neurodevelopmental disorders characterised by impaired social interaction, delayed language development, and repetitive or restrictive behaviours. While both genetic and environmental factors contribute to ASD, the specific molecular mechanisms underlying these interactions remain unclear. In a recent preprint, we showed that cytokine signaling may be dysregulated in chromosome 15q-duplication syndrome (Dup(15q)), one of the most common syndromic forms of ASD. Specifically, Dup(15q) induced pluripotent stem cells (iPSC)-derived neurons exhibit an amplified Signal Transducer and Activator of Transcription-3 (STAT3) response to Interleukin-6 (IL-6), a proinflammatory cytokine often upregulated in ASD. To identify which gene in the 15q region may be responsible for modifying cytokine signalling responses in Dup(15q), we focus on the Cytoplasmic FMRP-Interacting Protein 1 (CYFIP1 ) gene, as increased CYFIP1 dosage in Dup(15q) is associated with increased severity of neurobehavioral symptoms in individuals with Dup(15q) and CYFIP1 dysregulation has been linked to altered expression of various genes involved in immunoregulatory signalling pathways. Here, we explore the eeects of CYFIP1 overexpression ( CYFIP1 -OE) on cytokine signaling, demonstrating that CYFIP1 -OE in HEK293 cells modifies the expression and activity of several cytokine signaling-related transcription factors, including STAT3 and STAT1. Additionally, we use SH-SY5Y neuroblastoma cells to assess neuron-related phenotypes, showing that CYFIP1 -OE alters IL-6-induced neurite outgrowth. These findings provide novel insights into how CYFIP1 may contribute to dysregulated cytokine responses in ASD, advancing our understanding of the molecular mechanisms that underlie neuroinflammatory processes in this disorder. Background Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental conditions in which both genetic variation and environmental exposures contribute to pathogenesis ( 1 ). 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 1,000 genes identified as potentially contributing to its onset and development ( 2 ). In addition to genetic predisposition, environmental risk factors, particularly maternal immune activation (MIA) and neuroinflammation, have been recognised as key contributors to ASD pathogenesis ( 3 , 4 ). 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 ( 3 ). 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 ( 5 ). JAKMIP1 expression was commonly dysregulated in Fragile X syndrome (FXS) and chromosome 15q-duplication syndrome (Dup(15q)), both genetic disorders often comorbid with ASD ( 5 ). 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 ( 5 ). 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 dysregulation in syndromic ASD could modify how cells respond to cytokines, we previously assessed IL-6-induced STAT3 signalling in a Dup(15q) induced pluripotent stem cell (iPSC)-derived neuronal model ( 6 ), as Dup(15q) is one of the most common forms of syndromic ASD, accounting for around 1% of ASD cases ( 7 ). Compared to a control iPSC line, we observed that JAKMIP1 expression fluctuates in Dup(15q) iPSC and iPSC-derived cortical neurons (iNeurons), being downregulated in Dup(15q) iPSC and upregulated in iNeurons. Despite these opposing trends, we found that STAT3 expression was consistently reduced in both the Dup(15q) iPSCs and iNeurons, indicating that Dup15q cells would likely display altered responses to IL-6. Curiously, though STAT3 expression was reduced, IL-6-induced STAT3 activity was significantly enhanced in both Dup(15q) iPSCs and iNeurons. These findings suggest that unlike in the SH-SY5Y cells, JAKMIP1 is likely not regulating STAT3 expression or activity in Dup(15q) and instead, another gene in the chromosome 15q region exerts stronger influence on STAT3 activity. Several genes are encoded within the 15q region of chromosome 15, however, a gene of particular interest in relation to ASD and cytokine signalling is Cytoplasmic FMRP-Interacting Protein 1 ( CYFIP1 ), as studies have demonstrated that duplications (or even deletions) encompassing CYFIP1 increase the severity of neurobehavioural phenotypes ( 8 ), and that CYFIP1 expression is upregulated in the brains of individuals with Dup(15q) and ASD ( 9 ). Although CYFIP1 is most known for its roles in repressing neuronal protein translation as part of Fragile X Messenger Ribonucleoprotein (FMRP)-containing ribonucleoprotein complexes ( 10 ) and regulating actin polymerisation as a core component of the WAVE regulatory complex (WRC) ( 11 ), there is evidence to suggest that CYFIP1 dysregulation may alter the expression of various genes involved in immunoregulatory signalling pathways ( 12 ). 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 gene targets of SMAD Family Member 3 (SMAD3), Nuclear Factor Kappa B (NF-κB) or Interferon-Stimulated Response Elements (ISRE) ( 12 ). Moreover, another study identified CYFIP1 as a potential hub gene that may coordinate Transforming Growth Factor Beta 1 (TGF-β1)/SMAD3 signalling ( 13 ). Therefore, we hypothesised that CYFIP1 may be responsible for the observed enhanced IL-6-induced activity in Dup(15q) iPSCs and iNeurons ( 6 ). This paper therefore explores the eeects of increased CYFIP1 expression (modelled by CYFIP1 -overexpression (OE)) on cytokine signalling. We provide the first evidence to directly support a novel role for CYFIP1 in modulating cytokine signalling, demonstrating that CYFIP1 -OE in HEK293 cells alters the expression and activity of cytokine signalling-related transcription factors, STAT1 and STAT3 . Taking this further, we show how alterations in the STAT3 signalling pathway due to CYFIP1 -OE aeects 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. Methods HEK293 and SH-SY5Y cell culture and treatment Human embryonic kidney (HEK)-293 cells (#CRL-1573™, American Type Culture Collection ) and human neuroblastoma cells (#CRL-2266™, American Type Culture Collection ) were thawed from frozen stocks stored in 10% dimethyl sulfoxide (#D2650-5X5ML, Sigma-Aldrich ®) at −80°C. HEK293 cells and SH-SY5Y cells were maintained in 10-cm diameter Petri dishes (#P7612-360EA, Sigma-Aldrich ®) with 10 mL 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% CO 2 , 95% humidity. Cells were passaged at 70-80% confluency using 3 mL of pre-warmed TrypLE™ (#10718463, Fisher Scientific ™) for 5 min and TrypLE™ was inactivated by dilution in an equal volume of DMEM/F-12 + 10% FBS. 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). 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. Cells were treated with IL-6 or IFN-γ for dieerent amounts of time depending on the purpose of the experiment. To assess STAT3 activation by Western blotting for phosphorylated STAT3 (P-STAT3 Y705 ), cells were treated with IL-6 for 30 minutes. To assess STAT3 or STAT1 transcriptional activity by Dual-Luciferase® Reporter (DLR™) assays, cells were treated with IL-6 or IFN-γ for 18 hours post-transfection. To measure mRNA expression of STAT3 or STAT1 responsive genes, cells were treated with IL-6 or IFN-γ for 4 hours. Finally, to assess IL-6-induced neurite outgrowth, cells were treated with IL-6 over a 48-hour period of dieerentiation. HEK293 cell transfection 24 hours prior to transfection, 300,000 HEK293 cells were seeded into Nunc cell-culture treated 6-well plates (#10469282, Fisher Scientific ™) in 3 mL DMEM/F-12 + 10% FBS. The following day, HEK293 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 (i.e., final amount of 2.5 μg of plasmid and 5 μL of Lipofectamine™ LTX per well). Media was replaced the following day. All experiments were performed 72 hours post-transfection. SH-SY5Y cell transfection 2,000,000 SH-SY5Y cells were transfected by Nucleofection™ (#V4XC-2012, Lonza Bioscience ) following manufacturer instructions with 5 μg of 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 eeects of CYFIP1 -OE on neuritogenesis. A 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. Protein extraction and Western blotting 72 hours post-transfection, cells were washed with ice-cold 1x phosphate bueered saline (1xPBS) (#P4417-50TAB, Sigma-Aldrich®) then collected with a cell scraper in 300 μL of ice-cold RIPA lysis bueer (150 mM NaCl, 50 mM Tris/Cl pH = 8.0, 0.5% Sodium Deoxycholate, 0.1% SDS, 1% Triton X-100) supplemented with 1 mM PMSF, 1% Phosphatase Inhibitor Cocktail 2 (#P5726-1ML, Sigma Aldrich®), 1% Phosphatase Inhibitor Cocktail 3 (#P0044-1ML, Sigma Aldrich®) and 1% Complete Protease Inhibitor Cocktail (#P8340-1ML, Sigma Aldrich®). Cell lysates were incubated on ice for 30 minutes, cleared by centrifugation at 17,000xg for 15 minutes at 4°C, then supernatants collected. Bicinchoninic Acid assays (#10678484, Thermo Scientific ) were performed to estimate protein concentrations and 50 μg of total protein was diluted with Laemmli bueer (final concentration of 60 mM Tris/Cl pH = 6.8, 10% Glycerol, 2% SDS, 5% 2-Mercaptoethanol, 0.02% Bromophenol Blue), then boiled for 5 minutes at 95°C. 50 μg of total protein for each sample was loaded into 4–15% precast polyacrylamide gels (#4561085, Bio-Rad Laboratories ) alongside a pre-stained protein ladder (#PL00001, Proteintech Group ). Electrophoresis was performed in the Mini-PROTEAN® Tetra Vertical Electrophoresis Cell (#1658004, Bio-Rad Laboratories ) with 1ξSDS-PAGE running bueer (2.5 mM tris-base; 19 mM glycine (#10070150, Fisher Chemical ™); 0.01% SDS; pH = 8.3 in ddH 2 O). Proteins were then transferred onto methanol-activated PVDF membranes (#IPVH304F0, Millipore ) by a wet transfer method in the Mini-PROTEAN® Tetra Vertical Electrophoresis Cell. PVDF membranes were then blocked for at least 1 hour at room temperature with 5 mL of 5% skim milk in 1x tris-bueered saline (TBS; 2 mM Tris-Base; 15 mM NaCl; pH = 7.6 in ddH 2 O) supplemented with 0.1% Tween® 20 (#10485733, Fisher BioReagents ™) (1xTBS-T). After blocking, membranes were sequentially incubated with 5 mL of primary antibody (gentle rocking for 2-3 hours at room temperature or overnight at 4°C), then secondary antibody (gentle rocking for 30 minutes-1 hour at room temperature). Primary and secondary antibodies were diluted in 5% skim milk in 1x TBS-T (or 5% BSA in 1x TBS-T for anti-P-STAT3 Y705 ). The dilution factors and specific antibodies used are listed in Table 1 . Three 5-minute washes with 1x TBS-T were performed between antibody incubations. GAPDH was used as a loading control to normalise expression of target proteins. 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. View this table: View inline View popup Download powerpoint Table 1: Antibodies used in this study RNA extraction, cDNA conversion and qRT-PCR 72 hours post-transfection, total RNA was isolated and purified using the Direct-zol™ RNA Miniprep kit following manufacturer instructions (#R2051, Zymo Research ). 500 ng of 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 Table 2 . mRNA expression levels were standardized against two reference genes, Glyceraldehyde-3-Phosphate Dehydrogenase ( GAPDH ) and RNA Polymerase II Subunit A ( POLR2A ) using the Pfael method ( 14 ) and normalised to the control cells. View this table: View inline View popup Download powerpoint Table 2: Primers used in this study Dual-luciferase reporter assays 50,000 HEK293 cells were seeded into 24-well plates and incubated overnight. The following day, cells were transfected with 2 μL of STAT3 Cignal Reporter plasmid (#336841, GeneGlobe ID: CCS-9028L, Qiagen ) or 2 μL of STAT1 (GAS) Cignal Reporter plasmid (#336841, GeneGlobe ID: CCS-009L, Qiagen ) using Lipofectamine™ LTX as previously described. Four hours post-transfection, cells were treated with DMEM/F-12 +10% FBS with or without IL-6- or IFN-γ and incubated overnight. 18 hours later, Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter (DLR™) Assay System (#E1960, Promega ) following manufacturer instructions. Luminescence was measured in white-bottomed LUMITRAC™ 96-well plates (#655074, Greiner Bio-One ) using the PHERAstar FS microplate reader ( BMG LABTECH ). SH-SY5Y diSerentiation and neurite outgrowth analysis Sterile 13-mm-diameter glass coverslips (#631-1578, VWR ®) were placed into 24-well plates and coated with 300 μL of 20 μg/mL poly-D-Lysine (PDL; #354210, Corning ®) by overnight incubation at 37°C. 24 hours post-transfection, 30,000 SH-SY5Y cells were seeded onto PDL-coated coverslips in DMEM/F-12 + 10% FBS. The following day, media was replaced with 20 ng/mL BDNF and 10 μM RA in DMEM/F-12 (without FBS). 48 hours later, SH-SY5Y cells were fixed with 4% paraformaldehyde (PFA), and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (#D9542, Sigma-Aldrich ®) for 15 minutes. Coverslips were mounted onto glass microscope slides with ProLong™ Diamond Antifade Mountant (#15468070, Fisher Scientific ™) and 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; ( 15 )). Prior to image analysis, a scale bar was used to set a pixel to μm ratio to each image, after which the “Segmented Line” tool was used to measure the distance from the edge of the nucleus to the furthest end of each neurite. 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. 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 dieerences in measurements between two groups (e.g., CYFIP1 -OE against control). When investigating the eeects of and interactions between two independent variables (e.g., the eeect of both CYFIP1 -OE and IL-6 treatment on STAT3 transcriptional activity), two-way analysis of variance (ANOVA) were performed. 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. Results 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 HEK293 cells, referred to as CYFIP1 -OE and control cells respectively ( Figures 1A and 1B ). We then confirmed increased CYFIP1 mRNA expression using qRT-PCR ( Figure 1C ) and protein expression using Western blotting ( Figures 1D and E ). Download figure Open in new tab Figure 1 | Modelling CYFIP1 overexpression in HEK293 cells A) HEK293 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 hours 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 hours post-transfection measured by qRT-PCR. D) Increased CYFIP1 protein expression in CYFIP1-OE cells 72 hours 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. CYFIP1 -OE leads to reduced JAKMIP1 expression It has been previously demonstrated that JAKMIP1 expression is reduced following CYFIP1 -OE in human neuroblastoma cells ( 5 ). Therefore, to confirm whether this is the case in HEK293 cells, we measured JAKMIP1 expression in CYFIP1 -OE HEK293 cells by qRT-PCR and Western blotting. Agreeing with Nishimura et al., we found that JAKMIP1 mRNA ( Figure 2A ) and protein expression ( Figure 2B and C ) is significantly reduced in CYFIP1 -OE HEK293 cells. Download figure Open in new tab Figure 2 | 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. CYFIP1 -OE alters the expression of several cytokine signalling-related transcription factors We have previously demonstrated that Dup(15q) iPSCs and iNeurons with increased CYFIP1 expression show reduced STAT3 expression ( 6 ). Furthermore, we have demonstrated that JAKMIP1 -knockout SH-SY5Y cells also show reduced STAT3 expression and that JAKMIP1 deficiency may alter other cytokine signalling pathways too ( 6 ). Therefore, to determine whether CYFIP1 -OE may also lead to altered expression of cytokine signalling-related transcription factors, qRT-PCR was performed to measure mRNA expression of STAT1 , STAT3 , STAT5B and NFKB1 . We observed that the expression of STAT1 ( Figure 3A ) and STAT3 ( Figure 3B ) were reduced in CYFIP1 -OE HEK293 cells, whereas the expression of STAT5B ( Figure 5C ) and NFKB1 ( Figure 5D ) were increased. This suggests that CYFIP1 may also modulate the expression of cytokine signalling-related transcription factors, likely via modulation of JAKMIP1 . Download figure Open in new tab Figure 3 | CYFIP1-OE alters the expression of other cytokine signal-regulated 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 < 0.0001. Download figure Open in new tab Figure 4 | CYFIP1-OE enhances STAT3 transcriptional activity but not STAT3 activation A) Western blotting of STAT3 expression and phosphorylation (P-STAT3 Y705 ) in Control and CYFIP1-OE cells following 30-minute 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-STAT3 relative to the GAPDH loading control or D) P-STAT3 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 hours post-transfection with Cignal Reporter plasmids. Firefly luminescence intensity is expressed as a ratio to Renilla luminescence intensity to adjust for transfection emiciency. 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 hours, 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 hours, 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 Dimerence tests when comparing multiple groups; ns – P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Download figure Open in new tab Figure 5 | 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 emects of CYFIP1-OE on IL-6-influenced neuritogenesis. Cells were dimerentiated for two days with 20 ng/mL BDNF and 10 μM RA under serum starvation with or without 20 ng/mL IL-6, fixed with 4% PFA and stained with DAPI. A) Fluorescence micrographs of dimerentiated SH-SY5Y cells. Images captured using an upright Leica DM4000 B LED microscope. Scale bar = 50 μm. 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 Dimerence tests; ns – P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001. CYFIP1 -OE enhances IL-6-induced STAT3 activity To investigate whether reduced STAT3 expression in CYFIP1 -OE HEK293 cells leads to altered cytokine-induced STAT3 responses we first performed Western blotting to assess IL-6-induced STAT3 Y705 phosphorylation (representative of STAT3 activation) ( Figure 4A ). Despite the reduction in STAT3 expression ( Figure 4B ), CYFIP1 -OE cells still show strong responses to IL-6, demonstrating no significant dieerence in IL-6-stimulated STAT3 Y705 phosphorylation levels compared to control cells, both at the ‘whole-cell level’ (when STAT3 Y705 phosphorylation levels are normalised to GAPDH) ( Figure 4C ) and upon closer examination of the phosphorylation kinetics of STAT3 itself (when STAT3 Y750 phosphorylation levels are normalised to total STAT3 protein levels) ( Figure 4D ). This suggests that although CYFIP1 -OE cells show reduced STAT3 expression, STAT3 activation following IL-6 stimulation is unaltered. STAT3 activity is modulated by various post-translational modifications in addition to phosphorylation at the Y705 residue. Therefore, despite observing no change in STAT3 Y705 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 ( Figure 4E ), 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 untreated control cells ( Figure 4F ). 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 ( Figure 4G ). 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 ( Figure 4H ), however the fold change in SOCS3 expression relative to unstimulated cells shows that IL-6-induced SOCS3 expression is magnified in CYFIP1 -OE cells ( Figure 4I ). Taken together, these findings complement the DLR™ assay results, suggesting that CYFIP1 -OE cells show enhanced STAT3 transcriptional activity following IL-6 stimulation. CYFIP1 -OE enhances IL-6-induced neuritogenesis To understand how this enhanced STAT3 response to IL-6 in CYFIP1 -OE cells observed at a molecular level aeects a broader IL-6-stimulated cellular process, we dieerentiated CYFIP1 -OE SH-SY5Y cells in the presence of IL-6 and measured the neurite lengths produced ( Figure 5A ), as we have previously shown that IL-6 induces neurite outgrowth in SH-SY5Y cells ( 6 ). As expected, control and CYFIP1 -OE cells dieerentiated in the presence of IL-6 exhibit longer LNL ( Figure 5B ) and TNL ( Figure 5C ) measurements than cells dieerentiated 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 ( Figure 5B ) and TNL ( Figure 5C ) measurements are no longer significantly dieerent between the two populations when dieerentiated with IL-6. These data appear to agree with the suggestion that CYFIP1 -OE leads to enhanced STAT3 responses to IL-6, and that this enhancement in IL-6-induced STAT3 activity leads to increased IL-6-stimulated neurite extension and production. CYFIP1 -OE enhances IFN-γ-induced STAT1 transcriptional activity Having determined that CYFIP1 -OE alters the expression of several cytokine signalling-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 signalling-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 HEK293 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 HEK293 cells show increased Firefly luminescence following IFN-γ stimulation compared to control cells ( Figure 6A ), suggesting that IFN-γ-induced STAT1 transcriptional activity is enhanced in CYFIP1 -OE cells, again regardless of the reduced STAT1 expression. Download figure Open in new tab Figure 6 | 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 20 ng/mL IFN-γ for 18 hours post-transfection with Cignal Reporter plasmids. Firefly luminescence intensity is expressed as a ratio to Renilla luminescence intensity to adjust for transfection emiciency. B) Relative mRNA expression of STAT1 in Control and CYFIP1-OE cells treated with 0 ng/mL or 20 ng/mL IFN-γ for 4 hours, measured by qRT-PCR. C) Relative mRNA expression of SOCS1 in Control and CYFIP1-OE cells treated with 0 ng/mL or 20 ng/mL IFN-γ for 4 hours, 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 Dimerence 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-response genes following IFN-g stimulation. CYFIP1 -OE cells show increased IFN-γ-induced STAT1 expression ( Figure 6B ), suggesting an enhanced transcription response to IFN-γ stimulation. Additionally, although not significantly dieerent at baseline, CYFIP1 -OE cells show increased SOCS1 expression following IFN-γ treatment ( Figure 6C ), again suggesting an enhanced STAT1 transcriptional response to IFN-γ stimulation. This also suggests that the observed alterations in IL-6/STAT3 signalling are not limited to STAT3 and that CYFIP1 may play a role in modulating a larger range of cytokine signalling pathways. Discussion A novel role for CYFIP1 in modulating IL-6/STAT3 signalling independent from JAKMIP1 Nishimura et al . ( 5 ) 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 ( 6 ). 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 HEK293 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 HEK293 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 signalling in both HEK293 and SH-SY5Y cells, and its upregulation could be responsible for the enhanced responsiveness to IL-6 exhibited by Dup(15q) iPSCs and neurons. 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 ( 6 ) may suggest that JAKMIP1, rather than CYFIP1, is responsible for regulating STAT3 expression. This could occur through its interactions with various RNA-binding proteins (RBPs) or splicing factors ( 6 ). However, it is important to note that CYFIP1 overexpression enhances IL-6-induced STAT3 transcriptional activity, whereas JAKMIP1 -knockout impairs it ( 6 ). 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 dieerence in the levels STAT3 Y705 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, STAT3 K685 acetylation may be necessary for IL-6-induced STAT3-responsive gene expression ( 16 ). 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) signalling ( 17 ). Importantly, both IFN-α and IFN-γ signalling 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 signalling, thereby enhancing the IFN response. Furthermore, STAT1 activity is also known to be altered by acetylation, similar to STAT3 ( 18 ). If CYFIP1-mediated regulation of STAT3 is dependent on post-translational modifications, the same mechanism(s) could be responsible for CYFIP1 regulation both STAT3 and STAT1 activity. Of course, all of these possibilities would require further validation. Relevance to ASD and neurodevelopment Cytokine signalling plays crucial roles in neurodevelopment beyond immune responses, with STAT3 and STAT1 signalling in particular regulating several key processes. For example, STAT3 signalling has been demonstrated to control the switch between neurogenesis and gliogenesis, promoting both neural progenitor cell self-renewal and astrocyte dieerentiation ( 19 ). Similarly, STAT1 signalling has been shown to be involved in neurogenesis, and promoting neurite outgrowth ( 20 ). Given how crucial STAT3 and STAT1 signalling are for neurodevelopment, it is unsurprising that alterations in STAT3 and STAT1 signalling are associated with NDDs such as ASD ( 21 , 22 ). MIA, where an immune response is triggered by infection of exposure to immunogenic material during pregnancy, is associated with increased incidence of ASD ( 23 ). 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 ( 23 , 24 ). 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 eeects of immune activation on foetal brain development. Amplification of both the IL-6/STAT3 and IFN-γ/STAT1 signalling 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 ( 25 ), and our results suggest that CYFIP1 overexpression 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. Conclusion In summary, this study identifies a novel role for CYFIP1 in regulating the expression of cytokine signalling-related transcription factors, as well as enhancing their activity following cytokine stimulation. By amplifying these inflammatory pathways, CYFIP1 could play a central role in exacerbating the neuroinflammatory environment, leading to altered neurodevelopment and increased susceptibility to ASD. Further research is required to understand the mechanism by which CYFIP1 modulates these cytokine signalling pathways and to investigate this in the context of ASD with MIA. Declarations 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. Competing interests The authors declare that they have no competing interests Funding This work was supported by a Wellcome Trust funded Translational Research Exchange @ Exeter (TREE) pump-priming fund and the Bristol Japanese Cultural Showcase Author’s contributions E-R.M. conceived and designed the research, performed the laboratory research, generated the data and figures, and contributed to manuscript writing and editing. J.G.M. provided critical input and contributed to manuscript editing. M.A.R. provided critical input and reviewed the manuscript. A.O-A. conceived and designed the research, provided critical input, reviewed the manuscript, and secured funding. All authors reviewed and approved the final manuscript. Supplementary Files Download figure Open in new tab Supplementary Figure 1 | Modelling CYFIP1 overexpression in HEK293 cells Increased CYFIP1 protein expression in CYFIP1-OE cells 72 hours post-transfection confirmed via Western blotting. Membrane probed with: Rabbit Anti-CYFIP1 (#ab156016, Abcam), Rabbit Anti-FMRP (#13755-1-AP, Proteintech), Mouse Anti-GAPDH (#sc-47724, Santa Cruz Biotechnology), Goat Anti-Mouse DyLight™-680 (#35519, Invitrogen) and Goat Anti-Rabbit DyLight™-800 (#SA5-10036, Invitrogen). Download figure Open in new tab Supplementary Figure 2 | CYFIP1-OE leads to reduced JAKMIP1 protein expression Western blotting of JAKMIP1 expression in Control and CYFIP1-OE cells. Membrane probed with: Rabbit Anti-JAKMIP1 (#13846-1-AP, Proteintech), Mouse Anti-GAPDH (#sc-47724, Santa Cruz Biotechnology), Goat Anti-Mouse DyLight™-680 (#35519, Invitrogen) and Goat Anti-Rabbit DyLight™-800 (#SA5-10036, Invitrogen). Known non-specific bands produced are indicated on the blot. Download figure Open in new tab Supplementary Figure 3 | CYFIP1-OE has no eYect on STAT3 activation Western blotting of STAT3 expression and phosphorylation (P-STAT3 Y705 ) in Control and CYFIP1-OE cells following 30-minute treatment with 0 ng/mL IL-6 or 20 ng/mL IL-6. Membrane probed with: Mouse Anti-STAT3 (#9139S, Cell Signaling Technology), Rabbit Anti-P-STAT3 Y705 (#9131l, Cell Signaling Technology), Mouse Anti-GAPDH (#sc-47724, Santa Cruz Biotechnology), Goat Anti-Mouse DyLight™-680 (#35519, Invitrogen) and Goat Anti-Rabbit DyLight™-800 (#SA5-10036, Invitrogen). Known non-specific bands are indicated on the blot. 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 generous support of the charitable organisations that contributed to this work, including the Wellcome Trust Translational Research Exchange @ Exeter (TREE) pump-priming fund and the Bristol Japanese Culture Showcase. We thank Liming Yang for valuable discussions during lab meetings, and we are also grateful to Dr. Kaiyven Afi Leslie for his technical advice and support. Funder Information Declared Wellcome Trust Translational Research Exchange @ Exeter (TREE) , Project 121528 Footnotes Full uncropped Western blots have been added to pages 37, 38 and 39. 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 iNeurons iPSC-derived neurons FMRP Fragile X messenger ribonuclear protein WRC WAVE regulatory complex SMAD3 SMAD family member 3 NF-kB Nuclear factor of κB ISRE Interferon response element TGF-β1 Transforming growth factor β1 GAPDH Glyceraldehyde-3-phosphate dehydrogenase DLR™ Dual-luciferase reporter™ SOCS3 Suppressor of cytokine signalling 3 LNL Longest neurite length TNL Total neurite length IFN-γ Interferon-γ RBP RNA-binding protein References 1. ↵ American Psychiatric Publishing . American Psychiatric Association Diagnostic and statistical manual of mental disorders, fifth edition [DSM-5®] . 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