β-sitosterol β-D-glucoside (BSSG) triggers intestinal inflammation in zebrafish and mouse models prior to neurodegeneration onset

β-sitosterol β-D-glucoside (BSSG) triggers intestinal inflammation in zebrafish and mouse models prior to neurodegeneration onset | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article β-sitosterol β-D-glucoside (BSSG) triggers intestinal inflammation in zebrafish and mouse models prior to neurodegeneration onset Francesca Terrin, Sofia Faggin, Edoardo Bizzotto, Davide Santinello, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9163005/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Glucosylated-sterols can be synthetized endogenously, absorbed through the diet or derive from bacterial infection. Their clinical relevance is currently underestimated, even though their imbalance has been associated with higher risk of undergoing neurodegeneration throughout life. We studied the detrimental effects elicited by dietary consumption of plant-derived β-sitosterol β-D-glucoside (BSSG), known to be associated with the occurrence of ALS-PDC, to decipher its possible mode of action. Methods Zebrafish larvae and adults, as well as mice, were treated with BSSG dissolved directly in the water or through customized food pellet, respectively. Being the first target tissue identified, morphological and functional characterization of the intestine were performed, together with transcriptional analysis and sequencing of gut microbiota. Ex vivo analysis of zebrafish gut contractility was applied to assess intestinal neuromuscular response. Mutant and transgenic zebrafish lines were used to explore a possible BSSG mechanism of action. Results BSSG caused intestinal inflammation in both zebrafish and mouse models. This previously unknown effect was evidenced by altered gut dysmotility and inflammatory response. Transcriptomic analyses revealed increased expression of inflammation-related genes in the intestine of both zebrafish and mice, while preliminary gut microbiota analyses suggested the onset of dysbiosis. Transgenic and mutant zebrafish lines depleted of genes involved in glucocorticoids synthesis and activity evidenced that BSSG likely interacts with the glucocorticoid receptor, potentially affecting its canonical anti-inflammatory activity. Conclusions We discovered a new set of pathways altered by dietary uptake of BSSG. This molecule introduced in the organism initially determines gut inflammation, altering intestinal morphology and functionality, and possibly leads to neurodegeneration through disruption of the well-known gut-brain axis. BSSG glucosylated sterols intestinal inflammation gut microbiota glucocorticoid receptor gut-brain axis zebrafish model mouse model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 BACKGROUND Amyotrophic Lateral Sclerosis - Parkinsonism Dementia complex (ALS-PDC) is a rare neurodegenerative disorder characterized by symptoms resembling amyotrophic lateral sclerosis (ALS), including motor neurons loss and progressive muscle wasting, as well as Parkinsonian features and dementia in later stages [ 1 ]. ALS-PDC was first identified in the early 1960s among the indigenous population of Guam, and later reported in the Kii peninsula of Honshu Island in Japan [ 2 ] and in the Western New Guinea [ 3 ]. Intriguingly, populations living in these regions consumed flour made from the seeds of cycad plants— Cycas micronesica , C. revoluta , and C. circinalis —as part of their diet and traditional medicine [ 4 ]. This shared dietary component was therefore considered a primary environmental trigger of the disorder. Notably, after World War II, with the increasing adoption of Western habits and reduced consumption of cycad-based products, the incidence of ALS-PDC declined significantly [ 5 ]. The key risk factor was identified in β-sitosterol β-D-glucoside (BSSG), a compound detected in considerable concentration in cycad seeds. This molecule was demonstrated neurotoxic both in vitro and in vivo , inducing glutamate-mediated excitotoxicity, promoting hyperphosphorylated tau accumulation in neurons, and exacerbating apoptosis in cultured astrocytes [ 6 , 7 ]. Therefore, high endogenous levels of BSSG, introduced through the diet, appeared to contribute to neurotoxicity and neuroinflammation, ultimately leading to the development of ALS-PDC [ 6 ]. BSSG is a glucosylated sterol consisting of a steroid backbone linked to a glucose moiety. Similar molecules are predominantly found in plants, fungi and algae and rarely in bacteria and animals as glucosylated forms of cholesterol [ 8 ]. Interestingly, increased levels of glucosyl-β-D-cholesterol (β-GlcChol), a glucosylated sterol endogenously synthetized by humans, have been found in patients harboring mutations in GBA1 gene, a major genetic risk factors for Parkinson’s disease (PD) [ 9 ]. Similarly, the glucosyl-α-D-cholesterol (α-GlcChol) is produced by the bacterium Helycobacter pylori during gastric infections, a condition regarded as an environmental factor associated with increased lifetime risk of developing PD [ 10 ]. Despite their clinical relevance, the mechanisms by which dysregulation of glucosylated sterols elicit deleterious effects in the nervous system remain undefined. In this study, we employed zebrafish and mouse models to elucidate how BSSG may contribute to neurotoxicity. Unexpectedly, our data indicate that the intestine represents the primary target tissue. We showed that this molecule induces a sustained gut inflammation, characterized by altered intestinal physiology, motility and gene expression in both animal models. Since BSSG-treated mice are an established pre-symptomatic model for ALS-PDC [ 11 ] and we observed a previously unreported intestinal inflammatory response, we propose that a dysregulation of the so-called gut-brain axis may affect the tight balance between the intestine and the brain. Finally, we discovered a potential interaction between BSSG and the glucocorticoid receptor (GR), likely mediated by its structural similarity to steroids, thus proposing a new mechanism of action. METHODS β-siteosterol β-D-glucoside synthesis β-siteosterol β-D-glucoside (BSSG) synthesis process and chemical characterization are extensively described in Additional File 1 . Zebrafish and mouse husbandry and treatment Zebrafish wild type lines used in this work, also in the generation of the stable mutant lines, derived from Tuebingen and Giotto strains matings. Embryos, larvae and adults, housed in the Zebrafish Facility of the University of Padova, were maintained according to standard procedures [ 12 ]. Embryos were obtained from natural mating of WT, mutant or transgenic adult fish and raised at 28,5°C in Petri dishes with Fish Water (50X: 25 g Instant Ocean, 39.25 g CaSO 4 and 5 g NaHCO 3 in 1L) maintained in a 12h light:12h dark (LD) cycle until 72 hours post fertilization (hpf). At this life stage, larvae were screened for fluorescence, when necessary, and exposed to treatment or control vehicle. Wild type C57BL/6 male mice, housed in the Animal Facility of the University of Padova, were fed for 15 weeks, following weaning (1 month after birth) and up to 6 months of age, with BSSG-enriched chow pellet or with commercial food as a control. Mice food pellet was prepared by Mucedola Srl. The treatment paradigm previously established in [ 11 ] consisted in feeding mice daily with 1 mg/day of BSSG for 5 days/week. At the end of the experimentation period, mice were sacrificed by cervical dislocation, weighed and dissected for organ extraction. All husbandry and experimental protocols were in accordance with national and EU guidelines for use of experimental animals and were approved by the Animal Care and Use Ethics Committee of the University of Padua and by the Italian Ministry of Health (Authorization n. 112/2015PR; Authorization n. 12/2023-PR; Authorization n. 690/2020-PR). Chemicals preparation BSSG powder (MW = 576,86 g/mol) was dissolved in DMSO (Sigma-Aldrich), β-sitosterol (β-Sito, MW = 414,71 g/mol; 85451, Sigma-Aldrich) powder was dissolved in ethanol (EtOH), both at a final concentration of 10 mM and sonicated until complete solubilization. Ultrasonic bath temperature was kept at around 26–27°C. Stock aliquots were stored at -20°C. All chemicals were administered to zebrafish larvae diluted to a final working concentration of 10 µM, while control larvae were exposed to the same volume of the vehicle solvent, that reached a maximum concentration of 0,1% v/v. Treatment of zebrafish larvae For acute treatment, zebrafish larvae were incubated in Fish Water containing either BSSG, β-sitosterol or vehicle from 3 to 5 dpf. Treatment was renewed every 24 h to avoid molecule deposits and changes in concentration. Chronic 15-days-long BSSG treatment was performed on zebrafish larvae from 15 to 30 dpf. Briefly, larvae obtained from three independent spawns were raised in the Zebrafish Facility of the University of Padova according to standard procedures until 15 dpf. They were then divided into 3 treatment and 3 control groups and raised during the experimentation period in glass beakers filled with 200 mL of Fish Water, maintained at 28,5°C with a 12:12 LD period and fed three times/day. Treatment was administered directly in the water to a final concentration of 10 µM and refreshed every two days. BSSG-enriched diet for adult zebrafish The experimental BSSG-enriched feed was produced at the Department of Veterinary Sciences of the University of Pisa by including the compound in a control feed, as described in [ 13 ]. Briefly, the control feed (a mixture of 50% TetraMin Flakes® and 50% GEMMA Micro 300®) was first finely ground (< 100 µm) and then mixed with the BSSG powder to achieve a uniform dispersion within the mixture. Afterward, distilled water was added to obtain a homogeneous dough that was then pelleted and dried at 40°C in a drying chamber for 24 h. Finally, dry pellets were re-ground to restore feed particle size and kept in sealed plastic tubes at -20°C until use. Commercial feed without BSSG was processed with an identical procedure, as a control feed. Male adult zebrafish at 4 months post fertilization (mpf) obtained from three independent spawns, were divided (N≥4) into treatment and control groups, homogeneous for animal body weight. Fish were fed 5% of their body weight once a day according to the indications in [ 14 ], to achieve a BSSG amount of about 15 µg/day/fish. The experiment lasted for 30 days. DNA extraction and genotyping of mutant zebrafish Genotyping of mutant zebrafish was performed at early stages from genomic DNA extracted using the HotSHOT protocol [ 15 ] and amplified with the locus-specific primers through PCR with 5x HOT FIREPol® Blend Master Mix (04-25-00125, Solis BioDyne). Primers used for genotyping are listed in Supplementary Table 1 . Morphological characterization of larval phenotype Treated and control larvae (N = 10 per condition in each replicate) were anesthetized with Tricaine, mounted in 2% methylcellulose in H 2 O on depression slides and imaged under a Leica M165 FC microscope with a Nikon DS-Fi2 digital camera. Scoring of the main morphological traits (standard length, eye area and area of the swimming bladder) was performed on Fiji-ImageJ software (Version 2.14.0/1.54p). The experiment was performed 3 times. Lipids extraction for mass spectrometry analysis (LC-MS) Heads and trunks of treated and control larvae (n = 30 in each condition) were separated with a sharp blade, pooled, snap frozen in liquid nitrogen and stored at -80°C until use. Folch’s method [ 16 ] was used to extract lipids. Briefly, frozen pellets were resuspended with Milli-Q water and homogenized with a Pellet Pestle® Motor (Kimble) for 30 sec alternated with 10 min incubation on ice (repeated for three times). After homogenization, the solvent solution composed of chloroform:methanol:water in a 8:4:3 volumetric ratio was added. Once obtained the organic phase, it was resuspended in the same solution and processed a second time to get rid of most contaminants. Finally, samples were dried with a lyophilizer, insufflated with nitrogen, sealed and stored at -80°C. Subsequent LC-MS measurements were carried out to calculate the achieved internal concentration of BSSG in the heads and trunks of treated larvae compared to controls. The lipid extracts were dissolved in 90:10 methanol/chloroform and 5 µL were injected into a Hewlett-Packard Model 1100 Series liquid chromatograph (Hewlett-Packard Development Company, CA) coupled to a photodiode array (PDA) detector (Agilent Technologies, Italy, Agilent 1100 Series) and to a Bruker Esquire-LC quadrupole ion-trap mass spectrometer (Bruker OptikGmbH, Germany) equipped with atmospheric pressure electrospray ion source. The LC-MS quantitative measurements were also performed in positive ion mode using a Triple Quadrupole (QQQ) mass spectrometer (Applied Biosystems, API 3000 QQQ) equipped with an electrospray ion source (ESI), and combined with a Shimadzu High Performance LC system (CBM-20 A, binary pump LC-20AB, Italy). Chromatographic analysis was carried out in both MS conditions at room temperature on a Kinetex -C8 100 × 4.6 mm, 2.6 µm column (Phenomenex, Italy). The eluent (1.0 mL/min) consisted of (A) methanol:water/10 mM ammonium acetate (70:30) and (B) methanol:isopropanol/10 mM ammonium acetate (90:10) using a linear gradient: 65%–100% B in 40 min, followed by isocratic B held for 10 min. Neutral red staining Zebrafish larvae treated with either BSSG, β-sito or the control vehicle, were incubated with Neutral red solution (553-24-2, Sigma-Aldrich) at a final concentration of 4 µg/mL for 3 h in the dark, and then briefly rinsed 3 times in Fish Water. Larvae were anesthetized with Tricaine and photographed under a Leica M165 FC microscope with a Nikon DS-Fi2 digital camera. Length of the stained portion of the mid-intestine was measured with Fiji-ImageJ. Each treatment was performed four times with at least 10 larvae per condition. Alcian blue staining in zebrafish larvae Treated and control WT and gr −/− zebrafish larvae were fixed in PFA (Sigma-Aldrich) 4% in PBS overnight at 4°C and then stored in PBS. Staining with alcian blue (Alcian blue 8GX – A5268, Sigma-Aldrich Sigma) was performed as described in [ 17 ]. Number of goblet cells was manually scored starting from the junction between the intestinal bulb and the mid-intestine. The experiment was performed three times, with at least 10 larvae per condition. Neutrophil enumeration in Tg(mpx:GFP) and fluorescence analysis in Tg(NFkB:GFP) , Tg(Stat3:EGFP) and cyp11c1; Tg(GRE:EGFP) larvae Transgenic larvae were obtained crossing Tg( mpx:GFP ) i114 [ 18 ], Tg(8xHs.NFκB:GFP,Luciferase) hdb5 (hereafter Tg(NFkB:GFP)) [ 19 ] or Tg(7xSRE-HSV.Ul23:EGFP) ia28 (hereafter Tg(Stat3:EGFP)) [ 20 ] x wild type zebrafish and cyp11c1 +/− x cyp11c1 +/− Tg(9xGCRE-HSV.Ul23:EGFP ia20 ) (hereafter Tg(GRE:EGFP)) [ 21 ]. At 3 dpf they were screened for specific fluorescence and only GFP-positive larvae were randomly divided into treated and control groups. After treatment, larvae were anesthetized and mounted in 1% low melting agarose. The same intestinal region, starting from the junction between the intestinal bulb and the mid-intestine and spanning at least 4 somites, was imaged by z-stacks (5 µm step size) under a 20X (for Tg(mpx: GFP ), Tg(NFkB:GFP) and cyp11c1; Tg(GRE:EGFP)) or 40X (for Tg(7xStat3:EGFP)) objective at Nikon C2 confocal microscope, using the software NIS ELEMENTS. Fluorescent neutrophils in Tg(mpx: GFP ) larvae were manually scored by scrolling through the z-stacks assisted by the Cell counter tool in Fiji-ImageJ to avoid repeated counting, as described in [ 22 ]. Only the neutrophils visible in the intestinal walls and inside the lumen along 5 somites were considered. To quantify the fluorescence signal in Tg(NFkB:GFP), Tg(Stat3:EGFP) and cyp11c1; Tg(GRE:EGFP) larvae, z-stack images were converted to maximum intensity z-projections and the mean fluorescence was measured in the same region of interest (ROI) with Fiji-ImageJ software. After the image acquisition, cyp11c1; Tg(GRE:EGFP) larvae were genotyped to discriminate cyp11c1 +/+ and cyp11c1 −/− ;Tg(GRE:EGFP) siblings. Each experiment was performed three times, with at least 10 larvae per condition. Fluorescence analysis of cyp11c1; Tg(GRE:EGFP) adults cyp11c1 +/+ ;Tg(GRE:EGFP) and cyp11c1 −/− Tg(GRE:EGFP) males of 4 mpf of age were randomly subdivided into treated (fed for 15 days with BSSG-enriched food) and control groups. At the end of the experimental period, fish were fasted 24 h prior to sacrifice to reduce the amount of food residues inside the intestine. After sacrifice, the intestine was extracted and mounted in 1% low-melting agarose for imaging. Samples were imaged by z-stacks (7 µm step size) under a 20X objective at Nikon C2 confocal microscope. To quantify the intestine fluorescence signal, z-stack images were converted to maximum intensity z-projections and the Mean fluorescence was measured after setting a threshold to isolate the fluorescent region. Values were then normalized on the untreated cyp11c1 −/− ;Tg(GRE:EGFP), considered as the background signal. Acridine Orange staining in zebrafish larvae Control and BSSG treated 5-dpf zebrafish larvae were incubated with Acridine Orange solution (A6014, Sigma-Aldrich) at a final concentration of 15 µg/mL for 10 min in the dark, and extensively rinsed in Fish Water. Larvae were then anesthetized and mounted in 1% low melting agarose. The same intestinal region was imaged by z-stacks (5 µm step size) under a 20X objective at Nikon C2 confocal microscope, using the software NIS ELEMENTS. To quantify the fluorescence signal, z-stack images were converted to maximum intensity z-projections and the mean fluorescence was measured in the same ROI spanning 4 somites with Fiji-ImageJ software. Peristalsis analysis According to the protocol described in [ 8 ], 5 dpf control and BSSG treated larvae were incubated with DCFH-DA (2’, 7’-Dichlorofluorescein diacetate, Sigma-Aldrich) at a final concentration of 1 mg/L overnight in the dark before the recording of peristaltic movements. Larvae were rinsed three times in Fish water, anesthetized for 1 min in Tricaine to limit its influence on muscular contraction, and mounted in 2% methylcellulose. Videos of 6–8 min were recorded under a Leica M165 FC fluorescence microscope. Each larva was then analyzed twice, scoring the number of peristaltic contractions considered when partial or total intestinal bulb invaginations were visible [ 23 ]. The average value was used to calculate the frequency of peristaltic movements in a period of 2 min. The experiment was performed three times, with at least 10 larvae per condition. Gastrointestinal transit assay According to the protocol described in [ 24 ] with slight modifications, gastrointestinal transit was evaluated as the progression of food bolus along the digestive tract for 24 h after feeding. Treated and control 5 dpf larvae were fed commercial food in the morning (8:00 a.m.) and left free to feed for 2 h. This was considered as “Time 0”. Larvae that presented the intestinal bulb filled with food bolus visible in brightfield were considered for the analysis and photographed under a Leica M165 FC microscope 4, 8 and 24 hours after feeding. The progression of food was then evaluated according to the scoring system described in [ 24 ]. N = 32 CTR and N = 24 treated larvae were examined. Ex vivo analysis of zebrafish gut contractility The intestinal contractility of adult zebrafish was analyzed ex vivo by measuring tension changes with the isolated organ bath technique, as previously described in mouse [ 25 , 26 ] and fish preparations [ 27 ]. Experiments were performed on the full-length intestine of zebrafish fasted for 24 h prior to the sacrifice, to reduce the amount of food residues in the lumen. Once extracted, the intestine was maintained in Krebs solution (NaCl 118 mM, KCl 4.7 mM, CaCl 2 ∙2H 2 O 2.5 mM, MgSO 4 ∙7H 2 O 1.2 mM, K 2 HPO 4 1.2 mM, NaHCO 3 25 mM, C 6 H 12 O 6 11 mM). Creating two loops with a silk thread, each intestine was mounted along the longitudinal axis of an organ bath containing 10 mL of oxygenated (95% O 2 + 5% CO 2 ) Krebs solution maintained at 28.5°C. Changes in muscle tension were recorded by isometric force transducers (World Precision Instruments, Berlin, Germany) to a PowerLab 4/30 data acquisition system using LabChart 8 software (ADInstruments, Besozzo, VA, Italy). Intestinal preparations were stretched to an initial tension of 0.1 g and left to equilibrate for 45 minutes to allow the development of rhythmic spontaneous contractions [ 25 , 26 ]. At the end of the equilibration period, samples were activated using 1 µM carbachol (CCh), a non-selective cholinergic receptor agonist, directly added to the Krebs solution. To evaluate smooth muscle contraction, each intestine was exposed to 40 mM KCl, a depolarizing agent that induces Ca 2+ release and subsequent smooth muscle contraction. To study the cholinergic receptor-mediated excitatory responses, samples were then exposed to increasing concentrations of CCh (0.001-100 µM) added in a cumulative manner in the organ baths obtaining concentration–effect curves. Neuronal-mediated contractions were evaluated using electrical field stimulation at increasing frequencies with constant voltage (EFS; 0–40 Hz; 1-ms pulse duration; 10-s pulse-trains, 80 V) through platinum electrodes connected to an S88 stimulator (Grass Instrument) to cause a change in the membrane potential of neurons and consequent release of neurotransmitters. Finally, to characterize the inhibitory neuromuscular response, zebrafish intestines were exposed to 0.1 µM isoprenaline, a non-selective β-adrenergic receptor agonist. Concentration-response curves were subjected to a nonlinear regression analysis (fitted to a sigmoidal equation) to calculate maximal tension (Emax) values. Contractile responses were expressed as gram tension/gram dry tissue weight [ 25 , 26 ]. N≥4 animals were analyzed. Microbiota sequencing Zebrafish gut microbiota sequencing Adult zebrafish treated for 30 days with BSSG-enriched food and their respective controls were sacrificed after 24 h of fasting, to allow gut clearing from food residues. The whole intestine was extracted, snap frozen in liquid nitrogen and stored at -80°C until used. Bacterial DNA extraction was performed with DNeasy® PowerSoil® Pro Kit (Qiagen) according to the manufacturer’s instructions, with slight modification: to obtain the optimal rupture of bacterial cell walls, samples were repeatedly homogenized on a BeadBug™ microtube homogenizer (Merck Life Sciences) and then continuously vortexed for 20’ until complete tissue destruction. The next steps were performed following the Kit protocol. DNA samples were quantified with a Qubit fluorometer (ThermoFisher) and diluted to 10 ng/µl. Bacterial DNA was then amplificated with a nested PCR protocol to obtain the bacterial 16S sequence. In the first PCR reaction (PCR I), DNA template is targeted with forward Eub8F and reverse 984yR primers and synthetized with Platinum TM Taq DNA Polymerase High Fidelity (ThermoFisher). 1 µl of this first PCR was used to perform the second reaction (PCR II), that exploits a different pair of internal oligonucleotides: 515F and 806R. They amplify the V4 hypervariable region of the bacterial 16S DNA, which allows to discriminate the different bacterial taxa [ 28 ]. PCR conditions varied according to the optimal annealing temperature of each primer pairs and were as follows: 94°C for 1 min to activate the DNA polymerase, 25 cycles of 94°C for 30 sec, 53°C (in PCR I)/57°C (in PCR II) for 30 sec, 68°C for 45 sec, one cycle at 68°C for 7 min to allow final elongation. Primer sequences are listed in Supplementary Table 2 . The sequencing of the 16S region was performed exploiting the Illumina sequencing platform to obtain pair-end sequences of 150 bp. The resulting reads were analyzed to assign the Amplicon Sequence Variants (ASVs) to the different bacterial taxa using the pipeline described in [ 79 ]. For these analyses, 3 WT and 3 gr −/− male controls were compared with 4 WT and 4 gr −/− treated male zebrafish. Mouse stool microbiota sequencing Bacterial DNA was extracted from few fecal samples using QIAamp Fast DNA Stool Mini Kit (51604, Qiagen) protocol for pathogen detection, optimized to maximize the ratio of non-mouse DNA. DNA was subsequently amplified with Phusion® High-Fidelity DNA Polymerase (BioLabs) using forward 515F and reverse 806R primers ( Supplementary Table 2 ) as follows: 98°C for 3 min followed by 25 cycles of 95°C for 45 sec, 58°C for 45 sec and 72°C for 50 sec, and a final cycle at 72°C for 5 min. The sequencing of the 16S region was performed by the sequencing facility of Biology Department at the University of Padova exploiting the Illumina sequencing platform to obtain pair-end sequences of 150 bp. Amplicon Sequence Variants (ASVs) were then assigned to the different bacterial taxa. The mean relative abundance of bacterial strains at family level was considered. Data were obtained from N = 2 male mice/condition. RNA extraction, reverse transcription and Real Time-quantitative PCR (RT-qPCR) Total RNA was extracted with TRIzol reagent (15596026, Thermo Fisher Scientific) from different tissue samples: pooled 5 dpf whole larvae (at least N = 15 in each sample); pooled 30 dpf whole larvae (N = 14 in each sample); single adult zebrafish intestine or brain; single mouse small intestine samples long around 1.5 cm. The quantity and quality of the isolated RNA were assessed using a NanoDrop 2000c (Thermo Fisher Scientific) and TapeStation System 4150 (Agilent). 1 µg (for zebrafish larvae and mouse tissue samples) or 500 ng (for adult zebrafish tissue samples) of total RNA were treated with RQ1 RNase-Free DNase (M6101, Promega) and further used for cDNA synthesis, employing High-Capacity cDNA Reverse Transcription Kit (4368814, ThermoFisher). cDNA was diluted 1:10 (or 1:5 in the case of mouse intestine samples) and amplified with 5x HOT FIREPol EvaGreen qPCR Mix Plus (08-36-00001, Solis BioDyne) and the locus-specific primers. Genes expression was measured with SybrGreen method on a CFX384 Touch Real-Time PCR Detection System (BioRad). Primers’ melting curves and threshold cycles (Ct) were automatically generated. Relative gene expression levels were calculated using the comparative Ct method (2 –ΔΔCt ) against a housekeeping gene ( β-actin for adult zebrafish tissues, ef1α for larval zebrafish samples; Tbp for mouse intestine samples). Primers used for RT-qPCR analysis are listed in Supplementary Table 3 . RNA sequencing (RNAseq) Library construction from RNA extracted from zebrafish larvae was performed using the QuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina (015UG009V0260, Lexogen) according to manufacturer’s protocol. Briefly, 500 ng of total RNA were retrotranscribed with an oligod(T) and, after RNA degradation, second cDNA strand was synthesized using random primers. dsDNA was amplified to include sequencing primers and library barcodes. Differently, libraries from mouse samples were constructed starting from at least 500 ng of total RNA to deplete ribosomal RNA by using Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina). rRNA-depleted RNA was subsequently purified through ethanol precipitation and employed for library sequencing construction. Briefly, after fragmentation, the first strand cDNA was synthesized using random hexamer primers (NEBNext Ultra II RNA First Strand RNA Synthesis Module; NEB). Then the second strand cDNA was synthesized, and dUTPs were replaced with dTTPs in the reaction buffer (NEBNext Ultra II Directional RNA Second Strand Synthesis Module; NEB). Double strand DNA was purified using magnetic beads, and the ends were repaired and A-tailed to facilitate adapter ligation. After gel size selection (250–800 bp), USER enzyme digestion (NEB) was employed to eliminate UTP-containing second strand cDNA filaments. Prior to sequencing, libraries were quantified using the Qbit (dsDNA Quantitation, High Sensitivity; Thermofisher). Subsequently, size distribution was assessed using the High Sensitivity DNA kit (Agilent Technologies). Zebrafish libraries were sequenced by the sequencing facility in the Department of Biology of University of Padova utilizing NextSeq 500 (Illumina) platform employing a single read approach. Conversely, mouse libraries were sequenced by Novogene facility using Novoseq X plus (Illumina) sequencer employing a paired end approach. Transcriptome data analysis After removing sequencing adapters with Cutadapt (version 4.7) [ 29 ], transcript expression was quantified using the Salmon method (version 1.10.3) [ 30 ]. The resulting count matrix was loaded in the R statistical environment, and the edgeR package (version 4.0.2) [ 31 ], was used for gene expression normalization and to identify differentially expressed genes (DEGs) between treated and control samples (FDR ≤ 0.05), reported in Additional files 4–5 . Zebrafish and murine DEGs were subsequently used for Gene Ontology (GO) enrichment analysis via enrichplot package on R software and bar chart were generated on ShinyGO [ 32 ]. The raw data of RNA sequencing have been deposited on the SRA database. Electron microscopy and ultrastructural analysis of mouse microvilli After sacrifice, mice small intestine was extracted and gently flushed with cold PBS. Small sections (0.5 cm long) of mouse small intestine were fixed with Karnovsky fixative (2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer) overnight at 4°C, washed with 0.1M cacodylate buffer, post-fixed with osmium tetroxide for 2 h and embedded in EMbed 812 (Electron Microscopy Sciences). Ultrathin sections, stained with uranyl acetate and lead citrate, were observed at a Philips M400 operating at 100 kV. The length of microvilli was measured from the tip to the base with Fiji-ImageJ software. Analyses were repeated on multiple images derived from 3 individuals for each condition. Alcian blue staining of mouse small intestine histological sections After sacrifice, mice small intestine was extracted and gently flushed with cold PBS. Small portions 1.5 cm long were fixed in Bouin fixative solution (30 mL saturated picric acid in ddH 2 O, 10 mL formaldehyde, 2 mL glacial acetic acid for 42 mL) for 24 h and then rinsed in 70% ethanol in distilled water. After dehydration of samples with graded ethanol series (80%-90%-100% in ddH2O) for 1 h at RT, they were infiltrated with xylene (Sigma-Aldrich), 1 xylene:1paraffin and finally 100% paraffin. Samples were embedded and sectioned with a Leica microtome with a 7 µm thickness. Tissues sections were deparaffinized, rehydrated to ddH 2 O, incubated in 3% glacial acetic acid solution for 3’, followed by 30’ incubation in alcian blue staining solution (1% Alcian blue 8GX – A5268, Sigma-Aldrich – in 3% glacial acetic acid; pH 2.5). Sections were rapidly rinsed in 3% glacial acetic acid solution, set for 5’ in running tap water, counter stained with Eosin Y (Sigma-Aldrich, USA), then rinsed in 100% ethanol and mounted for visualization. Alcian blue-stained goblet cells were counted along intestinal villi in 3 sections spaced 100 µm in 6 mice for each condition. Macrophages staining on mouse small intestine histological sections Mouse small intestines were collected, fixed, paraffin-embedded, sectioned as previously described, and mounted on Superfrost ® Plus microscope slides (J1800AMNZ, Thermo Scientific). Tissues sections were deparaffinized, rehydrated to ddH 2 O with graded ethanol series (100%-90%-70% in ddH2O) for 5 min each and incubated for 15 min in a quenching solution (NH 4 Cl 50 mM) to reduce autofluorescence. Antigen retrieval was performed by 10 min incubation in citrate buffer (citric acid 0.01 M, pH 6) followed by 10 min in TBS-1% Tween at RT. Slides were then incubated for 1h in saturating solution (15% goat serum, 2% BSA, 0.25% gelatine, 0.2% glycine in PBS supplemented with 0.5% Triton X-100). Histological sections were incubated overnight at 4°C in F4/80 monoclonal primary antibody (14-4801-82, Thermofisher Scientific) diluted 1:50. After 3 washes for 5 min in TBS-1% Tween, sections were incubated for 1 h at RT with goat anti-rat-Alexa Fluor 568 secondary antibody (A-11077, Thermofisher Scientific) at a final dilution of 1:200. To reduce autofluorescence, slides were immersed for 10 min in Sudan Black solution (0.1% in EtOH 70%), followed by extensive washing in TBS-1% Tween. Nuclei were stained with DAPI for 5 min and sections were mounted with SlowFade™ Diamond Antifade Mountant (S36967, Thermofisher Scientific). Histological sections were imaged using a Zeiss AXIO Zoom.V16 fluorescence microscope equipped with an Axiocam 305 mono camera. Three mice per condition were analysed by manual scoring of stained macrophages. Radioligand binding assay To verify the ability of BSSG to bind steroid hormone receptors at a concentration of 10 µM, a radio-ligand binding assay was performed by an external company using the NHR Binding Agonist Radioligand Assay (Eurofins) by Eurofins Panlabs Discovery Services. BSSG selectivity for human androgen receptor, estrogen receptor, glucocorticoid receptor, mineralocorticoid receptor and progesterone receptor were calculated as the percentage of inhibition for the binding of a radio-labeled ligand, specific for each receptor ([ 3 H]-methyltrienolone, [ 3 H]-estradiol, [ 3 H]-dexamethasone, [ 3 H]-aldosterone and [ 3 H]-progesterone, respectively). Zebrafish whole-mount HuC/D and Sox10 immunofluorescence After BSSG treatment, 5 dpf larvae were fixed overnight in 4% PFA (Sigma-Aldrich) in PBS at 4°C. Larvae were then dehydrated in 100% methanol and conserved at -20°C until used. After rehydration with graded methanol series (75-50-25% methanol in PBS) and PBT (0,2% Triton X-100 in PBS) for 5 min each at RT, larvae were depigmented (2% KOH and 3% H 2 O 2 in PBT for 5 min, followed by a 5 min wash in PBT) and permeabilized (15 min at -20°C in ice-cold 100% acetone, followed by washes in ddH 2 O and PBT). After a saturation step in a blocking solution (1% BSA and 5% sheep serum in PBT) for 4 h at RT, larvae were incubated over 2 days with pan-neuronal anti-HuC/D (A-21272, ThermoFisher) or Sox10 (GTX128374-S, GeneTex) primary antibody diluted 1:200 in the blocking solution at 4°C. Larvae were then washed four times in PBT for 20 min each at RT, saturated in the same blocking solution for 4 h and incubated overnight in the dark at 4°C with Streptavidin conjugate Alexa Fluor 555 secondary antibody (S32355, ThermoFisher) against HuC/D or goat anti-rabbit-Alexa Fluor 488 (A11034, Invitrogen) secondary antibody against Sox10 diluted 1:1000. After extensive washes in PBT, larvae were mounted in 1% low-melting agarose and imaged by z-stacks (3 µm step size) under a 20X objective at Nikon C2 confocal microscope. The same body region was imaged for all larvae. All HuC/D + enteric neurons and Sox10 + neuronal progenitors visible on the ventral side of the intestine were manually scored by scrolling through the z-stacks assisted by the Cell counter tool in Fiji-ImageJ to avoid repeated counting. N≥10 animals/condition. The experiment was repeated three times. Fish Embryo Acute Toxicity test FET test was performed according to the Organization for Economic Co-operation and Development (OECD, Paris, France) Guideline No. 236 (2013) [ 33 ] to define a non-lethal and non-toxic BSSG concentration to be used for zebrafish treatment in order to avoid teratogenic or developmental issues. Briefly, 6 hpf embryos were transferred singularly into 24 well plates (1 embryo in 1 mL solution/well) and incubated with increasing concentrations of BSSG (2.5-5-10-20-40 µM) and of the respective solvent control (DMSO at 0.1%-0.2%-0.4%). For each concentration, 20 embryos were individually incubated with BSSG, and the remaining 4 wells were used as internal negative controls (Fish Water). The negative and positive controls (1.5% EtOH) were also tested. The embryo medium was changed daily, and developmental status of zebrafish embryos and larvae was monitored until 4 dpf. Percentage of survival and hatching rates, presence of cardiac edema and of swimming bladder were determined from the total number of surviving embryos. Statistical analysis Statistical analyses were performed using Graph Pad Prism V10.2.3. Data are expressed as mean ± SEM and statistical significance was calculated with unpaired Student's t-test for two sample comparisons or one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Concerning ex vivo analysis of gut contractility, two-way ANOVA followed by Bonferroni post hoc test was used for multiple comparison. The differences between groups were considered significant when P < 0.05 . Post hoc tests were run only if F achieved P < 0.05 , and there was no significant variance inhomogeneity. For the gastrointestinal transit assay, differences in the distribution of food bolus in transit zones between control and treated larvae were assessed for each time point using Fisher’s exact test for 2x5 contingency tables on absolute larval counts. p -values are indicated with the following symbols: *P < 0.05; **P < 0.01; ***P < 0.001 ; ****P < 0.0001; ns, not significant . RESULTS BSSG administration triggers intestinal inflammation in zebrafish larvae Zebrafish larvae exposed to 10 µM BSSG presented a normal development ( Supplementary Fig. 1A-B ) and the mass spectrometry analysis of lipids extracted from larval heads and trunks confirmed the accumulation of this compound in the trunk region of treated animals ( Supplementary Fig. 1C ). The presence of dark aggregates in the intestine of nearly all larvae exposed to BSSG prompted us to hypothesize that the gut may represent its first target (Fig. 1 A). Consistently, in vivo analysis of acidified lysosomes evidenced a reduction in the number of lysosome-rich enterocytes (LREs) in treated larvae (Fig. 1 B). To determine whether these intestinal effects were specifically attributable to BSSG rather than to possible metabolites, we performed the same assay on larvae treated with β-sitosterol, that shares the chemical structure of BSSG but lacks the glucose moiety. This molecule did not affect LREs ( Supplementary Fig. 2A ), suggesting that detrimental effects only depend on BSSG consumption. Mucus-secreting goblet cells along the mid-intestine, essential for protecting the gut against digestive enzymes and external insults, were reduced after exposure to BSSG (Fig. 1 C). Conversely, the number of neutrophils infiltrating the mid-intestine was markedly increased in treated larvae (Fig. 1 D), providing an indication of intestinal inflammation onset in our model. This was further supported by the increased fluorescence in the intestine of BSSG-treated transgenic Tg(NFκB:GFP) larvae, indicating the activation of NF-κB pathway (Fig. 1 E). Consistently with the occurrence of an inflammatory phenotype, we also observed an increase in apoptosis in the mid-intestine (Fig. 1 F). Moreover, the reduction of Stat3-linked fluorescence specific of stem-like cells located at the base of the intestinal folds [ 20 ], which are analogous to crypt base columnar cells in mammals, may reflect a depletion of proliferating cells (Fig. 1 G). To further characterize the impact of BSSG on gut homeostasis, we performed analysis of inflammation-related markers expression on whole larvae following acute exposure. We observed a trend towards increased expression of mmp9 (matrix metallopeptidase 9), along with significant upregulation of stat3 (signal transducer and activator of transcription 3) and pept1 (peptide transporter 1). Reduced expression of agr2 (anterior gradient 2), involved in mucus production by goblet cells, further supported the presence of a defective intestinal epithelial barrier (Fig. 1 H). We also observed a reduction in the expression levels of autophagy-related genes atg5 (autophagy protein 5) and lc3b (microtubule associated protein 1 light chain 3b), suggesting a possible impairment of autophagy ( Supplementary Fig. 2B ). Taken together, these results depict a scenario in which BSSG administration leads to an intestinal inflammation disrupting essential cellular processes, potentially exacerbating gut dyshomeostasis. BSSG treatment alters gut motility and microbial composition To study the impact of BSSG on intestinal physiology, we firstly analyzed peristaltic frequency. Treated larvae exhibited a lower number of gut contractile waves compared to controls (Fig. 2 A). To corroborate this result, we monitored larval gastrointestinal transit along the digestive tract, ideally divided into transit zones [ 24 ] (Fig. 2 B, upper panel ). Over time, different localization of the food bolus indicated that treated larvae display delayed gastrointestinal transit (***, P < 0.001 with Fisher’s exact test 24 h after feeding) (Fig. 2 B, lower panel ). Since peristaltic activity is regulated by the enteric nervous system (ENS), we evaluated the number of both differentiated enteric neurons and neuronal precursors (labelled with HuC/D and Sox10, respectively), but we didn’t find any difference between treated and control larvae ( Supplementary Fig. 2C ), suggesting that BSSG may affect the functionality of enteric innervation rather than its density. Therefore, we investigated the involvement of enteric neurotransmission in gut dysmotility in adult zebrafish fed with a BSSG-enriched diet over a prolonged period, to mimic chronic exposure. For the first time, both receptor-mediated and non-receptor-mediated neuromuscular responses of isolated whole intestines were evaluated in zebrafish. Muscular contractile response was analysed exposing zebrafish intestine to KCl, a depolarizing agent that induces Ca 2+ release and subsequent smooth muscle contraction. A significant increase in the KCl-induced contraction was observed in intestinal preparations of treated individuals (Fig. 2 C). To further evaluate the excitatory cholinergic response, gut samples were exposed to increasing concentrations of carbachol (CCh), a non-selective cholinergic agonist. Cumulative concentration-response curves evidence a significant increase of the intestinal CCh-mediated contraction in treated intestines (Fig. 2 D), suggesting the presence of an altered cholinergic neurotransmission following BSSG treatment. Then, to verify the impact on ENS neuronal activity, intestinal preparations were subjected to EFS (electrical field stimulation) at increasing frequencies with constant voltage, to cause a change in the membrane potential of neurons and consequent release of neurotransmitters. Treated individuals displayed an increased excitatory neuromuscular response, determining a significant upward shift of the frequency-response curve to EFS (Fig. 2 E). Higher 10 Hz-EFS-mediated contraction confirmed alterations in excitatory cholinergic response. Conversely, muscular relaxation induced by isoprenaline, a non-selective β-adrenergic receptor agonist, was not affected by treatment (Fig. 2 F). Next, given the relevance of the microbiota in maintaining gut metabolic homeostasis and the well-established association between alterations in microbiota composition and intestinal inflammation in humans and animal models [ 34 ], we analyzed the microbiota of adult zebrafish following BSSG exposure. At the phylum level, Proteobacteria , Bacteroidota and Verrucomicrobiota increased, while Firmicutes and Actinobacteriota decreased (Fig. 2 G), determining a reduction of the Firmicutes/Bacteroidetes ratio, a widely recognized indicator of gut health [ 35 ]. At the family level, we observed an increase in Barnesiellaceae , Aeromonadaceae and Rubritaleaceae , and a reduction in the relative abundance of Erysipelotrichaceae, Enterobacteriaceae and Weeksellaceae (Fig. 2 H). Overall, although these results did not reach statistical significance, likely due to the limited sample size, they suggest that BSSG treatment may influence the composition of gut bacterial population, potentially contributing to the onset of dysbiosis. Transcriptome analysis of chronically treated larvae reveals intestinal dyshomeostasis, possibly prodromal to neurodegeneration RNAseq analysis of chronically treated zebrafish larvae revealed 261 differentially expressed genes (168 upregulated and 93 downregulated) compared to controls. Upregulated genes are involved in acute inflammatory response together with response to reactive oxygen species and defence response to bacterium (Fig. 3 A). The upregulation of both mmp9 and pept1 , in association with the increased expression of saa ( serum amyloid a ) and s100a10a ( s100 calcium binding protein a10 a ) (Fig. 3 B), corroborates previous findings in treated larvae (Fig. 1 H), indicating the presence of gut inflammation. BSSG treatment downregulated genes mainly associated with lateral line nerve development and oxygen transport (Fig. 3 A ’ ). Decrease of oxygen binding resulted also among downregulated molecular functions ( Supplementary Fig. 2D ). Notably, the entire group of hemoglobin ( hb ) genes exhibited a significant reduction in treated individuals (Fig. 3 B), a finding recently associated with the pathophysiology of different neurodegenerative diseases [ 36 – 38 ]. The decreased expression of mucin 5.3 (Fig. 3 B), secreted by goblet cells to neutralize digestive enzymes and pathogens, supported the reduction of goblet cells count observed in treated larvae (Fig. 1 C). To validate transcriptome results, we tested the expression of specific genes in the intestine of adult zebrafish confirming the upregulation of inflammation-related markers ( mmp9, mmp13 , il-4 , il-13 ) and cellular stress markers ( casp8 , caspase 8; nupr1 , nuclear protein 1) (Fig. 3 C, upper panel ). Since RNAseq analysis evidenced the alteration of genes potentially involved in neurodegeneration, we evaluated adult zebrafish brains. In contrast to our findings in the intestine, mmp9 expression in the brain exhibited a downregulation following treatment, potentially impacting its role in central nervous system (CNS) plasticity. We also observed the downmodulation of autophagy-related genes atg5 , lc3b and p62 (Fig. 3 C, lower panel ). BSSG-fed mice exhibit reduced weight and hallmarks of intestinal inflammation To investigate whether BSSG dietary consumption could determine intestinal alterations also in the mouse model, we provided WT mice with BSSG-enriched food for 15 weeks, and then we characterized intestinal phenotypes. We observed an increase in the number of macrophages in the small intestine of treated mice ( Supplementary Fig. 3A ). We then evaluated the number of goblet cells, finding a significant reduction in treated mice (Fig. 4 A), evidencing also in this animal model hallmarks of intestinal inflammation following BSSG dietary uptake. Moreover, ultrastructural analysis revealed that BSSG-fed mice exhibited significantly shorter microvilli compared to controls (Fig. 4 B), potentially implying impaired enteric absorption capacity. Consistently, they weighed less than controls (Fig. 4 C) despite consuming the same amount of food ( Supplementary Fig. 3B ). RNA-seq analysis of gut tissue revealed 1835 differentially expressed genes between treated and untreated mice (982 upregulated and 853 downregulated in treated animals). Upregulated genes are primarily associated with regulation of immune system process and immune response (Fig. 4 D-E, Supplementary Fig. 3C, left panel ) and are related with actin cytoskeleton , side of membrane , and microvillus ( Supplementary Fig. 3D ), thus confirming that BSSG directly impacts on genes involved in enterocytes’ brush border structure. Conversely, downregulated genes are involved in small molecules metabolic process, regulation of cell growth , heme / porphyrin-containing compound biosynthesis , and regulation of neuron projection development (Fig. 4 D ’ ) and are associated with glutathione transferase activity and transmembrane transporter activity ( Supplementary Fig. 3C, right panel ). RT-qPCR experiments confirmed the activation of inflammatory and immune response, evidencing the upregulation of Nod1 (NOD-like receptor 1), Tlr-2, Tlr-4, Tlr-6 (Toll-like receptor-2, -4, -6), Nlrp3 (NLR family pyrin domain containing 3), Il-1β (interleukin-1β) and Ifn-γ (interferon-γ) and the downregulation of Ikb-α (NF-kappa-B inhibitor alpha) and Reg3-γ (regenerating islet-derived protein 3-γ). We also observed the upregulation of Plp1 (proteolipid protein 1), which is highly expressed in enteric glia (Fig. 4 F). Finally, we found alterations in the composition of fecal microbiota in BSSG-fed mice. Treated mice exhibited a higher relative abundance of potentially pathogenic bacteria Bacteroidaceae, Helicobacteraceae and Prevotellaceae , typically associated with intestinal inflammation, and reduced recognized anti-inflammatory taxa like Lachnospiraceae , known to produce short chain fatty acids (SCFAs), highly beneficial for intestinal homeostasis, immune system modulation and energetic metabolism ( Supplementary Fig. 3E ). As in zebrafish, this preliminary microbiota analysis, although not reaching statistical significance, suggests the onset of dysbiosis in this pre-symptomatic ALS-PDC mouse model. BSSG interaction with the glucocorticoid receptor as a possible mechanism of action With the aim to discover a possible BSSG mode of action, we addressed whether it could interact with known receptors of steroid hormones, due to its structural similarity with these molecules. A radioligand binding assay demonstrated that BSSG determined a 12.5% inhibition of the binding between glucocorticoid receptor (GR) and its radiolabelled specific ligand ([ 3 H]-dexamethasone) and a 4.7% inhibition for the androgen receptor (AR) and its radiolabelled specific ligand ([ 3 H]-methyltrienolone). No interference with neither estrogen receptor (ER), mineralocorticoid receptor (MR) nor with progesterone receptor (PR) was observed ( Supplementary Fig. 2F ). To verify if BSSG effectively binds Gr in vivo , we exploited the cyp11c1 zebrafish mutant line recently generated in our laboratory. As other published cyp11c1 ( cytochrome P450 family 1 subfamily C member 1 ) zebrafish mutant lines, homozygous cyp11c1 −/− cannot synthetize active glucocorticoids (GCs), while retaining a functional Gr receptor [ 39 , 40 ]. To visualize Gr activity, this cyp11c1 mutant line was crossed with transgenic Tg(GRE:EGFP) line, which expresses GFP after Gr activation [ 21 ] (Fig. 5 A). Mutant cyp11c1 −/− ;Tg(GRE:EGFP), free of endogenous GCs, therefore, allow to visually discriminate the effective binding of BSSG with the Gr in vivo . We treated cyp11c1 −/− ;Tg(GRE:GFP) larvae and analysed their intestine. A significant increase in the fluorescent signal was observed, suggesting that BSSG can effectively modulate Gr activation (Fig. 5 B). The same analysis was performed on adult transgenic mutants exposed to BSSG-enriched diet, obtaining again a significant increase of the fluorescent signal in the intestine (Fig. 5 C). The analysis of gut fluorescence in cyp11c1 +/+ ; Tg(GRE:GFP) larvae and adults revealed, as expected, an higher baseline signal compared with cyp11c1 −/− ;Tg(GRE:GFP), since endogenous steroid hormones constitutively activate Gr [ 21 ]. Accordingly, BSSG treatment in cyp11c1 +/+ ;Tg(GRE:GFP) larvae failed to induce any detectable further increase of intestinal fluorescence. Gr deficiency reduces the negative effects of BSSG on gene expression To confirm the role of the Gr in mediating the action of BSSG, we exploited the nr3c1 ia30/ia30 zebrafish mutant line (hereafter called gr −/− ), previously generated in our laboratory, in which the gr gene has been knocked out [ 41 ] (Fig. 6 A). Interestingly, we couldn’t observe any difference in the number of goblet cells in gr −/− treated larvae compared to controls, in contrast to what occurs in treated WT individuals. Similarly, we observed no significant alteration in the expression of mmp9, il-4 and il-13 in the intestine of adult gr −/− zebrafish fed with BSSG-enriched diet. This suggests that BSSG likely interferes with anti-inflammatory Gr activity. Conversely, gene expression of casp8 and nupr1 appear significantly upregulated, similarly to what was observed in WT, conveying that BSSG likely interferes with multiple pathways and cellular targets beyond this receptor (Fig. 6 C, upper panel and 6C’ ). Interestingly, also mRNA levels of mmp9, p62, atg5 and lc3b do not differ between brains of treated and control gr −/− zebrafish (Fig. 6 C, lower panel and 6C’ ). After the in vivo evidence of a plausible BSSG interaction with Gr, we investigated if it could also impact on the fine-tuned regulation of gene expression commonly depending on GCs/Gr interaction. We therefore evaluated the expression of some Gr-target genes such as fkbp5 ( FKBP prolyl isomerase 5 ) and foxo3b ( forkhead box protein O 3b ) in the intestine of WT adults fed with BSSG. We observed a significant reduction in fkbp5 expression, while foxo3b displays a decreasing trend in treated individuals (Fig. 6 D), further corroborating our hypothesis. Gr deficiency mitigates the alteration of intestinal muscular contractility and the differences in microbiota composition To evaluate whether the impact of BSSG on intestinal neuromuscular function is modulated by Gr, we exploited the ex vivo approach in gr −/− adult zebrafish after administration of BSSG-enriched diet. We observed that gut samples from gr −/− do not show any variation in muscular- and neuronal-induced contractility when exposed to KCl, 1 µM CCh and 10 Hz EFSafter BSSG treatment, maintaining similar response compared to untreated gr −/− (Fig. 7 A-C). These data point out that defects in gut movements could be influenced by BSSG interaction with Gr and are abrogated in gr −/− mutant line. Moreover, the treatment seemed to determine fewer changes in the composition of gr −/− gut microbiota compared with those reported for WT (Fig. 7 D-E). At the phylum level, no evident alteration occurs, except for a slight increase in Firmicutes (Fig. 7 D, right side ). At the family level we observed a similar abundance of Barnesiellaceae , Erysipelotrichaceae and Rubritaleaceae , suggesting that BSSG exposure minimally affects these bacterial strains in gr −/− individuals (Fig. 7 E, right side ). The lack of the Gr determines some constitutive differences in gut microbiota, indeed Fusobacteriota are more abundant among gr −/− phyla whereas Firmicutes and Actinobacteriota are scarcely represented, in opposition to what observed in WT. Among families, Fusobacteriaceae, Comamonadaceae and Shewanellaceae are more present in gr −/− with respect to WT. Nevertheless, our data suggest that BSSG influences the gut microbial community of gr −/− differently than it does in WT, as emerges also from 2D PCoA and ASVs (Amplicon Sequence Variants) relative abundance calculated in all experimental groups ( Supplementary Fig. 4 ). DISCUSSION In this work we used the zebrafish and mouse models to characterize the link between BSSG intake and the occurrence of the complex neurodegenerative disorder known as ALS-PDC. We found that BSSG is effectively absorbed by zebrafish larvae, adults, and mice, where it triggers a wide range of effects related to intestinal dyshomeostasis. Damaged gut epithelial barrier, pro-inflammatory profile, impaired gut functionality and microbiota alteration are all characteristic hallmarks of a perturbed gut-brain axis, that connects the ENS and the CNS [ 42 ]. Therefore, we hypothesize that BSSG may affect this route, inducing gut inflammation that possibly predisposes to neurodegeneration. The pro-inflammatory activity seems to be caused specifically by BSSG, in agreement with the reported toxicity caused by the presence of a single glucidic group [ 43 ], since β-sitosterol did not elicited inflammatory effects on LREs, a marker of intestinal inflammation in different zebrafish models [ 44 , 45 ]. Gut dyshomeostasis was supported by a reduced number of goblet cells and decreased expression of agr2 , a gene essential for intestinal mucus production [ 46 ]. These alterations are known to promote intestinal inflammation through weakening of the epithelial barrier [ 47 ]. Furthermore, neutrophil recruitment to the mid-intestine, activation of the NF-κB pathway, increased apoptosis, reduced proliferative capacity of intestinal stem-like cells, and upregulation of immune-related genes collectively indicate an ongoing inflammatory response (Fig. 1 ). Indeed, mmp9 and stat3 play a role in the activation of the immune response, while enteric pept1 is stimulated by pro-inflammatory cytokines. Noteworthy, the expression of these genes is increased in inflammatory bowel disease (IBD) patients and related animal models [ 48 – 51 ]. Chronic administration of BSSG to zebrafish larvae and adults confirmed the inflammatory phenotype at the intestinal level, as emerged from transcriptomic analysis (Fig. 3 ). Together with mmp9 and pept1 , we highlighted the upregulation of saa , involved in the activation of NF-kB signalling, in the promotion of downstream genes like mmp9 itself and in the regulation of neutrophils migration [ 52 ]. Similarly, s100a10a , expressed in the digestive tract, drives neutrophil recruitment in a larval model of intestinal infection [ 53 ]. S100a10a protein is considered homolog of human CALPROTECTIN, another marker for IBD [ 54 ]. Interestingly, it is increased also in stool samples of PD and Alzheimer’s disease (AD) patients [ 55 ]. Based on these results and according to previous studies [ 11 ], we developed an ALS-PDC pre-symptomatic mouse model, focusing on the small intestine of BSSG-treated animals (Fig. 4 ). Similar to what we found in zebrafish, RNAseq of mouse small intestine revealed that most of the DEGs are involved in the modulation of immune response. Toll-like receptors (TLRs) are significantly upregulated: Tlr-2 , Tlr-4 and Tlr-6 , along with NOD-like receptors, have been associated with the activation of NF-kB pathway [ 56 ] and IL-1β production [ 57 ]. Moreover, the observed alteration of several players involved in NF-kB pathway, Nlrp3 assembly and inflammasome activation [ 58 ], together with the downregulation of Ikb-α , that usually guarantees a negative-feedback mechanism to modulate inflammatory response, suggests an impairment of this route after BSSG treatment, leading to chronic inflammation. Consistently, it is reported that patients and animal models with IBD present an increased expression of TLRs, persistent NF-kB and NLRP3 activation and higher levels of pro-inflammatory cytokines such as IL-1β and IFN-γ [ 56 ]. Interestingly, increased expression of the same pro-inflammatory genes has been observed in intestinal biopsies of PD patients [ 59 ], further corroborating the existence of a link between intestinal inflammation and the predisposition to this disease. Activation of the immune response following BSSG dietary uptake was further supported by the presence of more macrophages in the lamina propria of treated mice. Moreover, the antimicrobial peptide REG3-γ, involved in proper distribution of the mucus layer [ 60 ], is downregulated in our model. Coherently, we observed a reduction in the number of mucus-producing goblet cells, as already observed treated zebrafish larvae. Among upregulated DEGs we also found Lrrk2 ( Leucine-rich repeat kinase 2 ), one of the most relevant PD genetic risk factors. In the intestine, LRRK2 protein is involved in the activation of the immune system through positive regulation of NF-kB, and its increased expression has been associated with pro-inflammatory effects in IBD mouse models as well as in PD patients, as reviewed in [ 61 ]. Our zebrafish model provided evidence that this inflammatory condition is also associated with marked alterations in gut physiology, observed after both acute and chronic exposure to BSSG (Fig. 2 A-F). Deregulation of peristalsis and delayed gastrointestinal transit have been frequently recognized as hallmarks of IBD [ 62 ] and have emerged as prodromic symptoms in disorders related to the autistic spectrum [ 63 , 64 ] and in neurodegenerative diseases such as PD and ALS [ 65 , 66 ]. Constipation, indeed, can affect patients many years before the appearance of the typical motor symptoms. The innovative ex vivo analysis of gut contractility, applied for the first time on adult zebrafish intestines, gave us solid evidence that ENS functionality is affected after BSSG consumption. Several findings link intestinal muscular hypertrophy and hypercontractility with infection and with increased expression of pro-inflammatory cytokines. Among them, IL-4 and IL-13 are responsible of higher intestinal smooth muscle contraction in mice during gut inflammation or enteric infection [ 67 , 68 ]. Moreover, a mouse model of DSS (dextran sulphate sodium)-induced colitis showed increased neuromuscular contraction upon CCh stimulation [ 69 ]. Collectively, these data demonstrate that BSSG administration affects both muscular and neuronal districts in the gut, altering gastrointestinal motility and possibly damaging enteric neurons. Preliminary analyses of gut microbiota in treated adult zebrafish (Fig. 2 G-H) revealed a higher abundance of Proteobacteria and a reduction in Firmicutes , in agreement with data from TNBS (trinitrobenzene sulfonic acid)-induced intestinal inflammation [ 70 ] and IBD patients [ 71 , 72 ]. Furthermore, a reduction of the Firmicutes / Bacteroidetes ratio has been already linked with IBD [ 35 ], while alterations in the relative abundance of bacterial phyla/families similar to the ones presented in this work were described for zebrafish exposed to contaminants leading to dysbiosis [ 73 ]. Noteworthy, similar gut microbiota alterations have been found also in neurodegenerative diseases such as AD, ALS and PD, where changes of bacterial communities can be prodromal to the onset of the disease [ 74 ]. According to the hypothesis of a disturbance of the gut-brain axis, we investigated if BSSG exerted detrimental effects in the CNS of our zebrafish model. In the brains of treated adult zebrafish we found decreased expression of autophagy-related genes, possibly suggesting an impairment of the autophagic mechanism, which alone can induce neurodegeneration determining the accumulation of neurotoxic proteinaceous aggregates [ 75 ]. This aspect, however, deserves more extensive and tailored analyses. Data obtained using zebrafish mutant lines knocked out for genes relevant to GCs synthesis ( cyp11c1 ) and activity ( gr ), showed that BSSG could in part exert its effects by interacting with the Gr. In particular, increased fluorescence in cyp11c1 −/− ;Tg(GRE:GFP) zebrafish intestine (Fig. 5 ) suggests that this sterol-derived molecule can interfere with Gr nuclear translocation and activity. Moreover, the absence of significant changes in goblet cells number in gr − I− treated larvae, the presence of only minor alterations of gene expression in intestines and brains of gr −/− adult zebrafish fed with BSSG and their unchanged intestinal neuromuscular activity observed ex vivo (Fig. 7 A-C), evidenced that inflammatory and neuromuscular signalling could be, at least partially, regulated by BSSG interaction with Gr. Both fkbp5 and foxo3b are directly regulated by Gr, modulating sensitivity to GCs and resolving inflammation through the inhibition of NF-kB pathway [ 76 ], respectively. Their reduced expression in the intestine of treated WT zebrafish (Fig. 6 D), indeed, could be caused by local impairment of Gr function, supporting the idea of a reduction of glucocorticoids anti-inflammatory effect after BSSG exposure. Of note, recent evidence showed that absence of intestinal GR in DSS-treated mice exacerbated inflammatory response, emphasizing the protective role exerted by GR activity against IBD [ 77 ]. However, despite our extensive in vivo findings, this compelling hypothesis requires further in vitro validation due to the complexity of steroid nuclear activity. Nevertheless, altered GCs levels and impaired Gr regulation have already been linked with pathogenesis and progression of neurodegenerative diseases like ALS, PD and AD [ 78 ], further endorsing the proposed mechanism of action through which BSSG, targeting the Gr, contributes to the etiology of ALS-PDC. CONCLUSIONS To conclude, this work revealed that increased levels of dietary BSSG determine a marked intestinal inflammation previously unknown. Our results suggest that this molecule affects the enteric district at first and lately the CNS, inducing neurodegeneration culminating in ALS-PDC occurrence. Such interrelation between the intestine and the CNS, therefore, highlights the relevance of the gut-brain axis. Furthermore, BSSG interaction with the glucocorticoid receptor implies a possible modulation of its anti-inflammatory activity, offering novel hints on the importance of physiologic activity of GCs in the intestine, where they regulate immune homeostasis and inflammatory responses. Following the gut-brain axis paradigm, interference with such functions can exacerbate inflammation, possibly leading to neurodegeneration. Therefore, a possible first line of defence could account for the recovery of intestinal homeostasis before the irreversible spread of the disease. Abbreviations AD Alzheimer’s disease ALS Amyotrophic Lateral Sclerosis ALS-PDC Amyotrophic Lateral Sclerosis-Parkinsonism Dementia complex AR androgen receptor BSSG β-sitosterol β-D-glucoside CCh carbachol cyp11c1 Cytochrome P450 Family 1 Subfamily C Member 1 CNS central nervous system DMSO dimethyl sulfoxide DSS dextran sulphate sodium EFS electrical field stimulation ENS enteric nervous system ER estrogen receptor GCs glucocorticoids Gr glucocorticoid receptor IBD inflammatory bowel disease KCl potassium chloride LRE lysosome-rich enterocyte MR mineralcorticoid receptor PCoA Principal Component Analysis PD Parkinson’s disease PR progesterone receptor TNBS trinitrobenzene sulfonic acid WT wild type α-GlcChol glucosyl-α-D-cholesterol β-GlcChol glucosyl-β-D-cholesterol. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials Transcriptional data generated during the current study have been deposited in SRA database ( URL will be made available for reviewers ). Other data supporting the findings of this study are available on reasonable request to the corresponding authors. Competing interests The authors declare that they have no competing interests. Funding This work was supported by grants from the University of Padova to FT (MUR/University of Padova PhD Fellowship 2020 and Department of Biology PostDoc Fellowship 2024), SCa (Department of Biology Intramural Grant Seed 2020), LDV (Department of Biology Intramural Grant Seed 2022), MCG (San Camillo Hospital Grant, Treviso (Italy); UNIPD-DSF-PRID-2023), SF (MUR/University of Padova PhD Fellowship 2020 and Department of Pharmaceutical and Pharmacological Sciences PostDoc Fellowship ARD-B 2023) and to SCe (Department of Pharmaceutical and Pharmacological Sciences PostDoc Fellowship ARD-B 2020). This study was also funded by the project entitled “National Center For Gene Therapy And Drugs Based On RNA Technology Neurodegeneration” (Project ID: CN00000041 - SP. 3) granted to LB. Authors' contributions FT: study conceptualization and design, main data collection, analysis and interpretation of data, drafting and review of the original manuscript; SF: ex vivo experiments data collection and analysis, contributed to original drafting; EB: data curation on zebrafish gut microbiota; DS: data curation on zebrafish gut microbiota and mouse fecal microbiota; SCe: contributed to define ex vivo methodology; GB, FF, AS, RL: provided experimental materials, contributed to original drafting; GG: performed LC-MS analysis; GS: RNAseq data analysis; SCa: RNAseq data analysis, contributed to original drafting and review of the manuscript, funding acquisition; LT: review of the manuscript; LB: review of the manuscript, funding acquisition; MCG: ex vivo experiments data curation, contributed to original drafting and review of the manuscript, funding acquisition; NP, LDV: study conceptualization and design, data curation and interpretation, supervision, drafting and review of the original manuscript, funding acquisition. All authors read and approved the final manuscript. Acknowledgements The authors acknowledge the Zebrafish Facility and its facility manager Martina Milanetto, the Imaging Facility and the Sequencing Facility at Biology Department of the University of Padova. Illustrations were created with BioRender.com. References Hirano A, Kurland LT, Krooth RS, Lessell S. Parkinsonism-dementia complex, an endemic disease on the island of Guam. I. Clinical features. Brain. 1961;84:642–61. Morimoto S, Ishikawa M, Watanabe H, Isoda M, Takao M, Nakamura S, et al. Brain transcriptome analysis links deficiencies of stress-responsive proteins to the pathomechanism of Kii ALS/PDC. Antioxidants. 2020;9:1–16. Gajdusek DC, Salazar AM. Amyotrophic lateral sclerosis and parkinsonian syndromes in high incidence among the Auyu and Jakai people of West New Guinea. Neurology. 1982;32:107–26. Spencer PS, Palmer VS, Kisby GE. Western Pacific ALS-PDC: Evidence implicating cycad genotoxins. 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Supplementary Files Additionalfile1BSSGsynthesis.docx BSSG synthesis Additionalfile1BSSGsynthesis.docx BSSG synthesis Additionalfile3Supplementaryfiguresandlegends.docx Supplementary figures Additionalfile4ListofzebrafishDEGs.xlsx List of zebrafish DEGs Additionalfile5ListoofmouseDEGs.xlsx List of mouse DEGs ADDITIONALFILESLIST.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9163005","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":609526637,"identity":"2d25f85e-cb3d-45c4-9354-c3754bb69475","order_by":0,"name":"Francesca Terrin","email":"","orcid":"https://orcid.org/0000-0002-3484-1946","institution":"Department of Biology, University of Padova, 35131, Padova, Italy","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Terrin","suffix":""},{"id":609533758,"identity":"c3ef80fa-aef9-4e07-9f0b-37b9a7a3c176","order_by":1,"name":"Sofia Faggin","email":"","orcid":"","institution":"Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131, Padova, Italy; 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CIR-Myo Myology Center, University of Padova, 35131, Padova, Italy","correspondingAuthor":false,"prefix":"","firstName":"Stefano","middleName":"","lastName":"Cagnin","suffix":""},{"id":609533769,"identity":"67a780fb-e822-44ee-9168-87447b8b776f","order_by":12,"name":"Laura Treu","email":"","orcid":"","institution":"Department of Biology, University of Padova, 35131, Padova, Italy","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Treu","suffix":""},{"id":609533770,"identity":"f21569e4-3322-4c37-9a7a-bcec736c8c8b","order_by":13,"name":"Luigi Bubacco","email":"","orcid":"","institution":"Department of Biology, University of Padova, 35131, Padova, Italy","correspondingAuthor":false,"prefix":"","firstName":"Luigi","middleName":"","lastName":"Bubacco","suffix":""},{"id":609533771,"identity":"83e08401-5ef1-43ca-9e4c-fe24ee3c8f69","order_by":14,"name":"Maria Cecilia Giron","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYJCCAw8MgCQziFnBwMDGwGDAkEBISwJcyxmYFkJ64PKMbWDKgAGfNfztzQ8PJBTYMci78z58XDjvcB6fdPMGhoc/cGuROHPMAOiwZAbDw+zGxjO3HS5mkzlWgNdhBhI5IL8wMxg2s7FJ8247nNgmkYPfLwbyb0Ba6qFa5hCjRYIHpOUwgzwzSEsDEVokzqSB/HKcx4CZjdmY51h6YhvQLwcS0nBr4W8//PjDhz/VcvL9xxgf89RYJ86f3bzx4Q8b3FpggMfgANxiYOQS1gAE8g1IWkbBKBgFo2AUIAMAQ6RKdup4TXIAAAAASUVORK5CYII=","orcid":"","institution":"Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131, Padova, Italy","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"Cecilia","lastName":"Giron","suffix":""},{"id":609533772,"identity":"d4b4b7b8-61ee-4964-86c4-37a2aa3dcac2","order_by":15,"name":"Nicoletta Plotegher","email":"data:image/png;base64,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","orcid":"","institution":"Department of Biology, University of Padova, 35131, Padova, Italy","correspondingAuthor":true,"prefix":"","firstName":"Nicoletta","middleName":"","lastName":"Plotegher","suffix":""},{"id":609533773,"identity":"d0d80a0d-b699-4189-a7bf-4966269b9b76","order_by":16,"name":"Luisa Dalla Valle","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYBACPgbGBiDFzMDHDuIWWDDwA6kDDGwMPLi0sMG0sDGDuAYSDJINUC249LBBKCQtBgeg4ji1SCQ3f/jBYC3Pxsz8TOKHgYSc8Y3cg4cLyhhk7HFqSWyT7GFIN2xjZjOT7DGQMDa7kZdweMY5PA4DagE64TBjGzODsQGPgUTiths5Bod52/Bqaf74h+GwfRsz+2fDPwYS9ZtnENbSIA20JbGNmcfwMdCWBAMJQlp4HrZJyxikJwO1FD6WMZAwnHHmjQHQLxI8PAewa+FnT3/88U2FtW0/e/uGg28qbOT523OMPxeU2dizN+CwBgwM0PjAOJLApx4LYCZR/SgYBaNgFAxvAAAJD0hrYO5XRwAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Biology, University of Padova, 35131, Padova, Italy","correspondingAuthor":true,"prefix":"","firstName":"Luisa","middleName":"Dalla","lastName":"Valle","suffix":""}],"badges":[],"createdAt":"2026-03-18 21:21:32","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-9163005/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9163005/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105268178,"identity":"7b2215f4-9749-45aa-acca-f49aaa3dd939","added_by":"auto","created_at":"2026-03-24 07:58:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9258685,"visible":true,"origin":"","legend":"\u003cp\u003eEvidence of intestinal inflammation in zebrafish larvae. A) Representative intestine morphology of 5-dpf zebrafish larva and related magnification of CTR (left) and BSSG-treated (right). Arrowhead indicates dark clumps in the mid-intestine of a BSSG-treated larvae. \u0026nbsp;B) Analysis of the mid-intestine region stained with neutral red and quantification of its length. C) Analysis of goblet cells number stained with alcian blue in the mid-intestine of CTR and BSSG-treated larvae. D) Representative micrographs of the distribution and number of fluorescent neutrophils in the mid-intestine of CTR and BSSG-treated Tg(mpx:GFP) larvae. E) Analysis of fluorescent signal in the mid-intestine of Tg(NFkB:GFP) CTR and BSSG-treated larvae. F) Analysis of fluorescent signal in Acridine Orange-stained CTR and BSSG-treated larvae G) Representative micrographs of the distribution and number of fluorescent stem-like cells labelled by Stat3 expression in the mid-intestine of CTR and BSSG-treated Tg(Stat3:EGFP) larvae. Dotted lines evidence the analyzed regions. H) RT-qPCR analysis of inflammation-related genes in whole tissue of pooled CTR and BSSG-treated larvae. n≥3 biological replicates. Data are expressed as mean ± SEM. Statistical analysis was performed using unpaired Student’s t-test: * P \u0026lt; 0.05; **P \u0026lt; 0.01. Scale bar: 200 µm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/3ac0afb32ebfe4b8d736506a.png"},{"id":105268100,"identity":"78ce4af3-a85e-4101-92bd-3e675ef76a68","added_by":"auto","created_at":"2026-03-24 07:58:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2651107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpairment of intestinal motility and changes in gut microbiota A\u003c/strong\u003e) Upper panel: video frames showing intestinal peristalsis. Asterisk evidences the point where muscle contraction originates. Arrowhead indicates intestinal bulb invagination. sb, swimming bladder; a, anus. Lower panel: quantification of intestinal peristaltic movements scored in live imaging videos and then calculated as a frequency every 2 minutes. Data were generated from of n=3 biological replicates. Box and whiskers graph shows the median and the min/max values. \u003cstrong\u003eB\u003c/strong\u003e) Upper scheme: regions subdividing zebrafish larval intestine defined as “Zones”. 1: portion of the intestinal bulb rostral to the sb; 2: intestinal bulb below the sb; 3: junction between the intestinal bulb and the mid-intestine; 4: mid- and posterior intestine. When the intestine is empty, it is referred as E. Lower panel: bar graphs represent the profile of gastrointestinal transit reported as percentage of larvae that show the most rostral extent of the food bolus in one of the defined zones at different time points, based on visual analysis of alive individuals. Statistical significance refers to global differences assessed using Fisher’s exact test on absolute counts. \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e. N=32 CTR and 24 BSSG-treated larvae deriving from different spawns. \u003cstrong\u003eC-F\u003c/strong\u003e) \u003cem\u003eEx vivo \u003c/em\u003eanalysis of intestinal neuromuscular contraction elicited by: 40 mM KCl (\u003cstrong\u003eC\u003c/strong\u003e); concentration-response curves to CCh (0.001-100 μM) (\u003cstrong\u003eD\u003c/strong\u003e); electric field stimulation (EFS; 0–40 Hz, 80V) (\u003cstrong\u003eE\u003c/strong\u003e); intestinal relaxation to 0.1 μM isoprenaline (\u003cstrong\u003eF\u003c/strong\u003e), of isolated intestinal preparations of adult zebrafish with or without \u003cem\u003ein vivo\u003c/em\u003e BSSG treatment. Each dot represents a tissue sample from a single adult individual. N³8 animals/condition. Data are expressed as mean ± SEM. Statistical analysis was performed using unpaired Student’s t-test for two-sample comparisons, or a two-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for multiple comparisons; *\u003cem\u003eP \u0026lt; 0.05; \u003c/em\u003e**\u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e; *** \u003cem\u003eP \u0026lt; 0.001; ns, \u003c/em\u003enot significant\u003cem\u003e \u003c/em\u003e\u003cstrong\u003eG-H\u003c/strong\u003e) Analysis of adult zebrafish gut microbiota. The bar graphs show the percentage of the relative abundance in bacterial phyla (\u003cstrong\u003eG\u003c/strong\u003e) and families (\u003cstrong\u003eH\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/1742408db937afccff89c488.png"},{"id":105268179,"identity":"6b62fd40-0fd6-4360-97f7-939949bd66b7","added_by":"auto","created_at":"2026-03-24 07:58:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2718445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZebrafish transcriptome analysis. A\u003c/strong\u003e) Bar charts of RNAseq analysis of RNA samples from pooled 30 dpf chronically treated whole zebrafish larvae. GO enrichment for the upregulated genes (GO Biological process). \u003cstrong\u003eA’\u003c/strong\u003e) GO enrichment for the downregulated genes (GO Biological process). \u003cstrong\u003eB\u003c/strong\u003e) Volcano plot of differentially expressed genes in chronically treated larvae. Blue tones refer to downregulated DEGs, red tones to upregulated DEGs. Grey tones indicate not significant DEGs below the statistical threshold of \u003cem\u003eP\u0026lt;\u003c/em\u003e0.01. Log2(Fold Change)=±0.5 are indicated with a dotted line. \u003cstrong\u003eC\u003c/strong\u003e) RT-qPCR analysis of different inflammation-, cellular stress- and autophagy-related genes in RNA samples from adult zebrafish intestines (upper panel) and brains (lower panel). Each dot represents a tissue sample from a single individual. N³3 animals/condition. Data are expressed as mean ± SEM. Statistical analysis was performed using unpaired Student’s t-test. *\u003cem\u003eP \u0026lt;0 .05; \u003c/em\u003e**\u003cem\u003eP \u0026lt;0 \u003c/em\u003e.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/c248aba873b79b2da1cdd9bd.png"},{"id":105268090,"identity":"da52b354-a4fe-42ce-93fa-c39e94f7dd5a","added_by":"auto","created_at":"2026-03-24 07:58:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7451178,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvidence of gut inflammation in mouse model and intestinal transcriptome analysis. A\u003c/strong\u003e) Magnification of mouse small intestine histological sections stained with alcian blue and quantification of goblet cells number (3 images from N=6 animals/condition). Scale bar: 200 µm. \u003cstrong\u003eB\u003c/strong\u003e) Transmission electron microscopy of microvilli at the apical surface of mouse enterocytes. Scale bar: 1 µm. In the magnification box, microvilli of a treated mouse. Scale bar: 500 nm. Quantification of microvilli length. N=3 animals/condition. \u003cstrong\u003eC\u003c/strong\u003e) Mice body weight after 15 weeks of BSSG-enriched food diet. \u003cstrong\u003eD\u003c/strong\u003e) Bar charts of RNAseq analysis of RNA samples extracted from mouse small intestine. GO enrichment of upregulated genes (GO Biological process). \u003cstrong\u003eD’\u003c/strong\u003e) GO enrichment of downregulated genes (GO Biological process). \u003cstrong\u003eE\u003c/strong\u003e) Volcano plot of differentially expressed genes. Blue tones refer to downregulated DEGs, red tones to upregulated DEGs. Grey tones indicate not significant DEGs below the statistical threshold of \u003cem\u003eP\u0026lt;\u003c/em\u003e0.01. Log2(Fold Change)=±0.5 are indicated with a dotted line. \u003cstrong\u003eF\u003c/strong\u003e) RT-qPCR analysis of different inflammation-related genes in RNA samples from mouse small intestine. Each dot represents a tissue sample from a single individual. Bar graphs show the mean ± SEM. N³3 animals/condition. Statistical analysis was performed using unpaired Student’s t-test. * \u003cem\u003eP \u0026lt;0 .05; \u003c/em\u003e**\u003cem\u003eP \u0026lt;0 \u003c/em\u003e.01; *** \u003cem\u003eP \u0026lt;0 .001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/83ac298611886b368db450e1.png"},{"id":105268075,"identity":"ce09ccbb-be40-4ffa-8cc5-a3499dfbdae5","added_by":"auto","created_at":"2026-03-24 07:58:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4771205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eBSSG\u003c/strong\u003e \u003cstrong\u003einteraction with the glucocorticoid receptor.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA\u003c/strong\u003e) Schematic representation of the mutant and transgenic zebrafish lines crossed to obtain \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;\u003c/em\u003eTg(GRE:EGFP) individuals. \u003cstrong\u003eB\u003c/strong\u003e) Representative magnification of the mid-intestine of \u003cem\u003ecyp11c\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+ \u003c/em\u003e\u003c/sup\u003eand\u003csup\u003e\u003cem\u003e \u003c/em\u003e\u003c/sup\u003e\u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;\u003c/em\u003eTg(GRE:EGFP) zebrafish larvae and mean fluorescence quantification in treated larvae compared to controls. Dotted lines evidence the analyzed region. n=3 biological replicates. \u003cstrong\u003eC\u003c/strong\u003e) Representative 20X confocal acquisitions of GC-responsive intestine in adult UT (untreated) \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;\u003c/em\u003eTg(GRE:EGFP) after one-month feeding with BSSG-enriched food and Integrated Density quantification. N³3 animals/condition. Bar graphs show the mean ± SEM. Statistical analysis was performed using one-way ANOVA. *\u003cem\u003eP \u0026lt;0 \u003c/em\u003e.05; ***\u003cem\u003eP \u0026lt;0 \u003c/em\u003e.001; **** \u003cem\u003eP \u0026lt;0 .0001\u003c/em\u003e. Scale bar: 200 µm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/1ab4f8fd5e803eb0bc892ddf.png"},{"id":105268091,"identity":"c79be167-a130-41b4-80ae-97bbc78faed0","added_by":"auto","created_at":"2026-03-24 07:58:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3674853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGoblet cell analysis and gene expression in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003egr\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e\u003cstrong\u003ezebrafish line.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e) Schematic representation of the experimental setup exploiting \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003eadult zebrafish. \u003cstrong\u003eB\u003c/strong\u003e) Analysis of goblet cells number stained with alcian blue in the mid-intestine of \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003eCTR and BSSG-treated larvae. \u003cstrong\u003eC\u003c/strong\u003e) RT-qPCR analysis of different inflammation-, stress- and autophagy-related genes in RNA samples from adult \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003ezebrafish intestines (Upper panel) and brains (Lower panel). \u003cstrong\u003eC'\u003c/strong\u003e) Schematic summary of the differential expression of key markers in the intestine and brain of WT and \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003eadult zebrafish. Cell numbers indicate the amount of mRNA and the statistical significance (asterisks) relative to the respective untreated WT or \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e, which were both set to 1. \u003cstrong\u003eD\u003c/strong\u003e) RT-qPCR analysis of Gr-modulated genes in the intestine of WT adult zebrafish. Each dot represents a tissue sample from a single individual. N³3 animals/condition. Bar graphs show the mean ± SEM. Statistical analysis was performed using unpaired Student’s t-test. *\u003cem\u003eP \u0026lt; 0.05; \u003c/em\u003e**\u003cem\u003eP \u0026lt;0 \u003c/em\u003e.01; \u003cem\u003ens, \u003c/em\u003enot significant.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/0178a08f23648b5d0643d4bf.png"},{"id":105268103,"identity":"2c3815b5-dac8-498f-9041-0880b0199f6d","added_by":"auto","created_at":"2026-03-24 07:58:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1928465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of intestinal motility and gut microbiota in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003egr\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e\u003cstrong\u003ezebrafish line.\u003c/strong\u003e \u003cstrong\u003eA-C\u003c/strong\u003e) \u003cem\u003eEx vivo \u003c/em\u003eanalysis of intestinal contractile responses elicited by 40 mM KCl (\u003cstrong\u003eA\u003c/strong\u003e); 1 µM carbachol (CCh) (\u003cstrong\u003eB\u003c/strong\u003e); 10 Hz EFS (\u003cstrong\u003eC\u003c/strong\u003e), in isolated intestinal preparations of WT (left side of the graphs) and \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (right side of the graphs) adult zebrafish with or without BSSG treatment. Data are reported as mean ± SEM. Each dot represents a tissue sample from a single individual. N³4 animals/condition. Statistical analysis was performed using one-way ANOVA. *\u003cem\u003eP \u0026lt;0 \u003c/em\u003e.05; **\u003cem\u003eP \u0026lt;0 \u003c/em\u003e.01; ns, not significant. \u003cstrong\u003eD-E\u003c/strong\u003e) Analysis of adult zebrafish gut microbiota. Bar graphs show the percentage of the relative abundance in bacterial phyla (\u003cstrong\u003eD\u003c/strong\u003e) and families (\u003cstrong\u003eE\u003c/strong\u003e) in both WT (left side of the graphs) and \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003e(right side of the graphs).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/901acf89e1431933a0700267.png"},{"id":105728099,"identity":"18286cfb-3479-4241-8654-c8ab0b69365e","added_by":"auto","created_at":"2026-03-30 11:09:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":34976241,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/85f84019-ff10-4ee7-b253-a26e6c0d76b2.pdf"},{"id":105268175,"identity":"1e5a4bd8-e5ed-4b6a-a2ce-a56d7163192e","added_by":"auto","created_at":"2026-03-24 07:58:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3580601,"visible":true,"origin":"","legend":"\u003cp\u003eBSSG synthesis\u003c/p\u003e","description":"","filename":"Additionalfile1BSSGsynthesis.docx","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/ed51adefd5518dca76a28734.docx"},{"id":105268190,"identity":"4a520e87-f436-4881-bdd0-421ebfc6cc4d","added_by":"auto","created_at":"2026-03-24 07:58:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3580601,"visible":true,"origin":"","legend":"\u003cp\u003eBSSG synthesis\u003c/p\u003e","description":"","filename":"Additionalfile1BSSGsynthesis.docx","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/0a53214a62b7fc43c90a95ce.docx"},{"id":105268104,"identity":"9ce5b6f0-8a6c-4b88-878a-e473b748d1ca","added_by":"auto","created_at":"2026-03-24 07:58:18","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":33807014,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figures\u003c/p\u003e","description":"","filename":"Additionalfile3Supplementaryfiguresandlegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/634cdfee7b41f5ff67dd97ae.docx"},{"id":105564290,"identity":"63884df3-b445-41e6-b571-b92a266a7b89","added_by":"auto","created_at":"2026-03-27 12:49:11","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":86709,"visible":true,"origin":"","legend":"\u003cp\u003eList of zebrafish DEGs\u003c/p\u003e","description":"","filename":"Additionalfile4ListofzebrafishDEGs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/1814325bb28a64db82e2f4fe.xlsx"},{"id":105268099,"identity":"29e26111-9072-421e-841f-e681467a0161","added_by":"auto","created_at":"2026-03-24 07:58:16","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":366988,"visible":true,"origin":"","legend":"\u003cp\u003eList of mouse DEGs\u003c/p\u003e","description":"","filename":"Additionalfile5ListoofmouseDEGs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/3b19a1efc6096005d7508855.xlsx"},{"id":105268192,"identity":"520a6f41-e03b-4922-9a36-d6a2c2d735f6","added_by":"auto","created_at":"2026-03-24 07:58:31","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":17219,"visible":true,"origin":"","legend":"","description":"","filename":"ADDITIONALFILESLIST.docx","url":"https://assets-eu.researchsquare.com/files/rs-9163005/v1/c4de9e301ed61e8b0451f18b.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eβ-sitosterol β-D-glucoside (BSSG) triggers intestinal inflammation in zebrafish and mouse models prior to neurodegeneration onset\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eAmyotrophic Lateral Sclerosis - Parkinsonism Dementia complex (ALS-PDC) is a rare neurodegenerative disorder characterized by symptoms resembling amyotrophic lateral sclerosis (ALS), including motor neurons loss and progressive muscle wasting, as well as Parkinsonian features and dementia in later stages [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. ALS-PDC was first identified in the early 1960s among the indigenous population of Guam, and later reported in the Kii peninsula of Honshu Island in Japan [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e] and in the Western New Guinea [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. Intriguingly, populations living in these regions consumed flour made from the seeds of cycad plants\u0026mdash;\u003cem\u003eCycas micronesica\u003c/em\u003e, \u003cem\u003eC. revoluta\u003c/em\u003e, and \u003cem\u003eC. circinalis\u003c/em\u003e\u0026mdash;as part of their diet and traditional medicine [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. This shared dietary component was therefore considered a primary environmental trigger of the disorder. Notably, after World War II, with the increasing adoption of Western habits and reduced consumption of cycad-based products, the incidence of ALS-PDC declined significantly [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. The key risk factor was identified in \u0026beta;-sitosterol \u0026beta;-D-glucoside (BSSG), a compound detected in considerable concentration in cycad seeds. This molecule was demonstrated neurotoxic both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, inducing glutamate-mediated excitotoxicity, promoting hyperphosphorylated tau accumulation in neurons, and exacerbating apoptosis in cultured astrocytes [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, high endogenous levels of BSSG, introduced through the diet, appeared to contribute to neurotoxicity and neuroinflammation, ultimately leading to the development of ALS-PDC [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eBSSG is a glucosylated sterol consisting of a steroid backbone linked to a glucose moiety. Similar molecules are predominantly found in plants, fungi and algae and rarely in bacteria and animals as glucosylated forms of cholesterol [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. Interestingly, increased levels of glucosyl-\u0026beta;-D-cholesterol (\u0026beta;-GlcChol), a glucosylated sterol endogenously synthetized by humans, have been found in patients harboring mutations in \u003cem\u003eGBA1\u003c/em\u003e gene, a major genetic risk factors for Parkinson\u0026rsquo;s disease (PD) [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. Similarly, the glucosyl-\u0026alpha;-D-cholesterol (\u0026alpha;-GlcChol) is produced by the bacterium \u003cem\u003eHelycobacter pylori\u003c/em\u003e during gastric infections, a condition regarded as an environmental factor associated with increased lifetime risk of developing PD [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. Despite their clinical relevance, the mechanisms by which dysregulation of glucosylated sterols elicit deleterious effects in the nervous system remain undefined.\u003c/p\u003e\n\u003cp\u003eIn this study, we employed zebrafish and mouse models to elucidate how BSSG may contribute to neurotoxicity. Unexpectedly, our data indicate that the intestine represents the primary target tissue. We showed that this molecule induces a sustained gut inflammation, characterized by altered intestinal physiology, motility and gene expression in both animal models. Since BSSG-treated mice are an established pre-symptomatic model for ALS-PDC [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e] and we observed a previously unreported intestinal inflammatory response, we propose that a dysregulation of the so-called gut-brain axis may affect the tight balance between the intestine and the brain.\u003c/p\u003e\n\u003cp\u003eFinally, we discovered a potential interaction between BSSG and the glucocorticoid receptor (GR), likely mediated by its structural similarity to steroids, thus proposing a new mechanism of action.\u003c/p\u003e\n"},{"header":"METHODS","content":"\u003cp\u003e \u003cb\u003eβ-siteosterol β-D-glucoside synthesis\u003c/b\u003e \u003c/p\u003e\u003cp\u003eβ-siteosterol β-D-glucoside (BSSG) synthesis process and chemical characterization are extensively described in \u003cb\u003eAdditional File 1\u003c/b\u003e.\u003c/p\u003e\u003ch3\u003eZebrafish and mouse husbandry and treatment\u003c/h3\u003e\u003cp\u003eZebrafish wild type lines used in this work, also in the generation of the stable mutant lines, derived from Tuebingen and Giotto strains matings. Embryos, larvae and adults, housed in the Zebrafish Facility of the University of Padova, were maintained according to standard procedures [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. Embryos were obtained from natural mating of WT, mutant or transgenic adult fish and raised at 28,5°C in Petri dishes with Fish Water (50X: 25 g Instant Ocean, 39.25 g CaSO\u003csub\u003e4\u003c/sub\u003e and 5 g NaHCO\u003csub\u003e3\u003c/sub\u003e in 1L) maintained in a 12h light:12h dark (LD) cycle until 72 hours post fertilization (hpf). At this life stage, larvae were screened for fluorescence, when necessary, and exposed to treatment or control vehicle.\u003c/p\u003e\u003cp\u003eWild type C57BL/6 male mice, housed in the Animal Facility of the University of Padova, were fed for 15 weeks, following weaning (1 month after birth) and up to 6 months of age, with BSSG-enriched chow pellet or with commercial food as a control. Mice food pellet was prepared by Mucedola Srl. The treatment paradigm previously established in [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e] consisted in feeding mice daily with 1 mg/day of BSSG for 5 days/week. At the end of the experimentation period, mice were sacrificed by cervical dislocation, weighed and dissected for organ extraction. All husbandry and experimental protocols were in accordance with national and EU guidelines for use of experimental animals and were approved by the Animal Care and Use Ethics Committee of the University of Padua and by the Italian Ministry of Health (Authorization n. 112/2015PR; Authorization n. 12/2023-PR; Authorization n. 690/2020-PR).\u003c/p\u003e\u003ch2\u003eChemicals preparation\u003c/h2\u003e\u003cp\u003eBSSG powder (MW = 576,86 g/mol) was dissolved in DMSO (Sigma-Aldrich), β-sitosterol (β-Sito, MW = 414,71 g/mol; 85451, Sigma-Aldrich) powder was dissolved in ethanol (EtOH), both at a final concentration of 10 mM and sonicated until complete solubilization. Ultrasonic bath temperature was kept at around 26–27°C. Stock aliquots were stored at -20°C. All chemicals were administered to zebrafish larvae diluted to a final working concentration of 10 µM, while control larvae were exposed to the same volume of the vehicle solvent, that reached a maximum concentration of 0,1% v/v.\u003c/p\u003e\u003ch3\u003eTreatment of zebrafish larvae\u003c/h3\u003e\u003cp\u003eFor acute treatment, zebrafish larvae were incubated in Fish Water containing either BSSG, β-sitosterol or vehicle from 3 to 5 dpf. Treatment was renewed every 24 h to avoid molecule deposits and changes in concentration.\u003c/p\u003e\u003cp\u003eChronic 15-days-long BSSG treatment was performed on zebrafish larvae from 15 to 30 dpf. Briefly, larvae obtained from three independent spawns were raised in the Zebrafish Facility of the University of Padova according to standard procedures until 15 dpf. They were then divided into 3 treatment and 3 control groups and raised during the experimentation period in glass beakers filled with 200 mL of Fish Water, maintained at 28,5°C with a 12:12 LD period and fed three times/day. Treatment was administered directly in the water to a final concentration of 10 µM and refreshed every two days.\u003c/p\u003e\u003ch3\u003eBSSG-enriched diet for adult zebrafish\u003c/h3\u003e\u003cp\u003eThe experimental BSSG-enriched feed was produced at the Department of Veterinary Sciences of the University of Pisa by including the compound in a control feed, as described in [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Briefly, the control feed (a mixture of 50% TetraMin Flakes® and 50% GEMMA Micro 300®) was first finely ground (\u0026lt; 100 µm) and then mixed with the BSSG powder to achieve a uniform dispersion within the mixture. Afterward, distilled water was added to obtain a homogeneous dough that was then pelleted and dried at 40°C in a drying chamber for 24 h. Finally, dry pellets were re-ground to restore feed particle size and kept in sealed plastic tubes at -20°C until use. Commercial feed without BSSG was processed with an identical procedure, as a control feed. Male adult zebrafish at 4 months post fertilization (mpf) obtained from three independent spawns, were divided (N≥4) into treatment and control groups, homogeneous for animal body weight. Fish were fed 5% of their body weight once a day according to the indications in [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e], to achieve a BSSG amount of about 15 µg/day/fish. The experiment lasted for 30 days.\u003c/p\u003e\u003ch3\u003eDNA extraction and genotyping of mutant zebrafish\u003c/h3\u003e\u003cp\u003eGenotyping of mutant zebrafish was performed at early stages from genomic DNA extracted using the HotSHOT protocol [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e] and amplified with the locus-specific primers through PCR with 5x HOT FIREPol® Blend Master Mix (04-25-00125, Solis BioDyne). Primers used for genotyping are listed in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e\u003ch3\u003eMorphological characterization of larval phenotype\u003c/h3\u003e\u003cp\u003eTreated and control larvae (N = 10 per condition in each replicate) were anesthetized with Tricaine, mounted in 2% methylcellulose in H\u003csub\u003e2\u003c/sub\u003eO on depression slides and imaged under a Leica M165 FC microscope with a Nikon DS-Fi2 digital camera. Scoring of the main morphological traits (standard length, eye area and area of the swimming bladder) was performed on Fiji-ImageJ software (Version 2.14.0/1.54p). The experiment was performed 3 times.\u003c/p\u003e\u003ch2\u003eLipids extraction for mass spectrometry analysis (LC-MS)\u003c/h2\u003e\u003cp\u003eHeads and trunks of treated and control larvae (n = 30 in each condition) were separated with a sharp blade, pooled, snap frozen in liquid nitrogen and stored at -80°C until use. Folch’s method [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e] was used to extract lipids. Briefly, frozen pellets were resuspended with Milli-Q water and homogenized with a Pellet Pestle® Motor (Kimble) for 30 sec alternated with 10 min incubation on ice (repeated for three times). After homogenization, the solvent solution composed of chloroform:methanol:water in a 8:4:3 volumetric ratio was added. Once obtained the organic phase, it was resuspended in the same solution and processed a second time to get rid of most contaminants. Finally, samples were dried with a lyophilizer, insufflated with nitrogen, sealed and stored at -80°C. Subsequent LC-MS measurements were carried out to calculate the achieved internal concentration of BSSG in the heads and trunks of treated larvae compared to controls. The lipid extracts were dissolved in 90:10 methanol/chloroform and 5 µL were injected into a Hewlett-Packard Model 1100 Series liquid chromatograph (Hewlett-Packard Development Company, CA) coupled to a photodiode array (PDA) detector (Agilent Technologies, Italy, Agilent 1100 Series) and to a Bruker Esquire-LC quadrupole ion-trap mass spectrometer (Bruker OptikGmbH, Germany) equipped with atmospheric pressure electrospray ion source. The LC-MS quantitative measurements were also performed in positive ion mode using a Triple Quadrupole (QQQ) mass spectrometer (Applied Biosystems, API 3000 QQQ) equipped with an electrospray ion source (ESI), and combined with a Shimadzu High Performance LC system (CBM-20 A, binary pump LC-20AB, Italy). Chromatographic analysis was carried out in both MS conditions at room temperature on a Kinetex -C8 100 × 4.6 mm, 2.6 µm column (Phenomenex, Italy). The eluent (1.0 mL/min) consisted of (A) methanol:water/10 mM ammonium acetate (70:30) and (B) methanol:isopropanol/10 mM ammonium acetate (90:10) using a linear gradient: 65%–100% B in 40 min, followed by isocratic B held for 10 min.\u003c/p\u003e\u003ch3\u003eNeutral red staining\u003c/h3\u003e\u003cp\u003eZebrafish larvae treated with either BSSG, β-sito or the control vehicle, were incubated with Neutral red solution (553-24-2, Sigma-Aldrich) at a final concentration of 4 µg/mL for 3 h in the dark, and then briefly rinsed 3 times in Fish Water. Larvae were anesthetized with Tricaine and photographed under a Leica M165 FC microscope with a Nikon DS-Fi2 digital camera. Length of the stained portion of the mid-intestine was measured with Fiji-ImageJ. Each treatment was performed four times with at least 10 larvae per condition.\u003c/p\u003e\u003ch3\u003eAlcian blue staining in zebrafish larvae\u003c/h3\u003e\u003cp\u003eTreated and control WT and \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e zebrafish larvae were fixed in PFA (Sigma-Aldrich) 4% in PBS overnight at 4°C and then stored in PBS. Staining with alcian blue (Alcian blue 8GX – A5268, Sigma-Aldrich Sigma) was performed as described in [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. Number of goblet cells was manually scored starting from the junction between the intestinal bulb and the mid-intestine. The experiment was performed three times, with at least 10 larvae per condition.\u003c/p\u003e\u003cp\u003e \u003cb\u003eNeutrophil enumeration in Tg(mpx:GFP) and fluorescence analysis in Tg(NFkB:GFP)\u003c/b\u003e, \u003cb\u003eTg(Stat3:EGFP) and\u003c/b\u003e \u003cb\u003ecyp11c1;\u003c/b\u003e\u003cb\u003eTg(GRE:EGFP) larvae\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTransgenic larvae were obtained crossing Tg(\u003cem\u003empx:GFP\u003c/em\u003e)\u003csup\u003ei114\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e], Tg(8xHs.NFκB:GFP,Luciferase)\u003csup\u003ehdb5\u003c/sup\u003e (hereafter Tg(NFkB:GFP)) [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e] or Tg(7xSRE-HSV.Ul23:EGFP)\u003csup\u003eia28\u003c/sup\u003e (hereafter Tg(Stat3:EGFP)) [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e] x wild type zebrafish and \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/−\u003c/em\u003e\u003c/sup\u003e x \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/−\u003c/em\u003e\u003c/sup\u003e Tg(9xGCRE-HSV.Ul23:EGFP \u003csup\u003eia20\u003c/sup\u003e) (hereafter Tg(GRE:EGFP)) [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. At 3 dpf they were screened for specific fluorescence and only GFP-positive larvae were randomly divided into treated and control groups. After treatment, larvae were anesthetized and mounted in 1% low melting agarose. The same intestinal region, starting from the junction between the intestinal bulb and the mid-intestine and spanning at least 4 somites, was imaged by z-stacks (5 µm step size) under a 20X (for Tg(mpx:\u003cem\u003eGFP\u003c/em\u003e), Tg(NFkB:GFP) and \u003cem\u003ecyp11c1;\u003c/em\u003eTg(GRE:EGFP)) or 40X (for Tg(7xStat3:EGFP)) objective at Nikon C2 confocal microscope, using the software NIS ELEMENTS. Fluorescent neutrophils in Tg(mpx:\u003cem\u003eGFP\u003c/em\u003e) larvae were manually scored by scrolling through the z-stacks assisted by the Cell counter tool in Fiji-ImageJ to avoid repeated counting, as described in [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Only the neutrophils visible in the intestinal walls and inside the lumen along 5 somites were considered.\u003c/p\u003e\u003cp\u003eTo quantify the fluorescence signal in Tg(NFkB:GFP), Tg(Stat3:EGFP) and \u003cem\u003ecyp11c1;\u003c/em\u003eTg(GRE:EGFP) larvae, z-stack images were converted to maximum intensity z-projections and the mean fluorescence was measured in the same region of interest (ROI) with Fiji-ImageJ software. After the image acquisition, \u003cem\u003ecyp11c1;\u003c/em\u003eTg(GRE:EGFP) larvae were genotyped to discriminate \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e;Tg(GRE:EGFP) siblings.\u003c/p\u003e\u003cp\u003eEach experiment was performed three times, with at least 10 larvae per condition.\u003c/p\u003e\u003cp\u003e \u003cb\u003eFluorescence analysis of\u003c/b\u003e \u003cb\u003ecyp11c1;\u003c/b\u003e\u003cb\u003eTg(GRE:EGFP) adults\u003c/b\u003e\u003c/p\u003e\u003cp\u003e \u003cem\u003ecyp11c1\u003c/em\u003e \u003csup\u003e \u003cem\u003e+/+\u003c/em\u003e \u003c/sup\u003e;Tg(GRE:EGFP) and \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e Tg(GRE:EGFP) males of 4 mpf of age were randomly subdivided into treated (fed for 15 days with BSSG-enriched food) and control groups. At the end of the experimental period, fish were fasted 24 h prior to sacrifice to reduce the amount of food residues inside the intestine. After sacrifice, the intestine was extracted and mounted in 1% low-melting agarose for imaging. Samples were imaged by z-stacks (7 µm step size) under a 20X objective at Nikon C2 confocal microscope. To quantify the intestine fluorescence signal, z-stack images were converted to maximum intensity z-projections and the Mean fluorescence was measured after setting a threshold to isolate the fluorescent region. Values were then normalized on the untreated \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e;Tg(GRE:EGFP), considered as the background signal.\u003c/p\u003e\u003ch2\u003eAcridine Orange staining in zebrafish larvae\u003c/h2\u003e\u003cp\u003eControl and BSSG treated 5-dpf zebrafish larvae were incubated with Acridine Orange solution (A6014, Sigma-Aldrich) at a final concentration of 15 µg/mL for 10 min in the dark, and extensively rinsed in Fish Water. Larvae were then anesthetized and mounted in 1% low melting agarose. The same intestinal region was imaged by z-stacks (5 µm step size) under a 20X objective at Nikon C2 confocal microscope, using the software NIS ELEMENTS. To quantify the fluorescence signal, z-stack images were converted to maximum intensity z-projections and the mean fluorescence was measured in the same ROI spanning 4 somites with Fiji-ImageJ software.\u003c/p\u003e\u003ch2\u003ePeristalsis analysis\u003c/h2\u003e\u003cp\u003eAccording to the protocol described in [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e], 5 dpf control and BSSG treated larvae were incubated with DCFH-DA (2’, 7’-Dichlorofluorescein diacetate, Sigma-Aldrich) at a final concentration of 1 mg/L overnight in the dark before the recording of peristaltic movements. Larvae were rinsed three times in Fish water, anesthetized for 1 min in Tricaine to limit its influence on muscular contraction, and mounted in 2% methylcellulose. Videos of 6–8 min were recorded under a Leica M165 FC fluorescence microscope. Each larva was then analyzed twice, scoring the number of peristaltic contractions considered when partial or total intestinal bulb invaginations were visible [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. The average value was used to calculate the frequency of peristaltic movements in a period of 2 min. The experiment was performed three times, with at least 10 larvae per condition.\u003c/p\u003e\u003ch2\u003eGastrointestinal transit assay\u003c/h2\u003e\u003cp\u003eAccording to the protocol described in [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e] with slight modifications, gastrointestinal transit was evaluated as the progression of food bolus along the digestive tract for 24 h after feeding. Treated and control 5 dpf larvae were fed commercial food in the morning (8:00 a.m.) and left free to feed for 2 h. This was considered as “Time 0”. Larvae that presented the intestinal bulb filled with food bolus visible in brightfield were considered for the analysis and photographed under a Leica M165 FC microscope 4, 8 and 24 hours after feeding. The progression of food was then evaluated according to the scoring system described in [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. N = 32 CTR and N = 24 treated larvae were examined.\u003c/p\u003e\u003cp\u003e \u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003eanalysis of zebrafish gut contractility\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe intestinal contractility of adult zebrafish was analyzed \u003cem\u003eex vivo\u003c/em\u003e by measuring tension changes with the isolated organ bath technique, as previously described in mouse [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e] and fish preparations [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Experiments were performed on the full-length intestine of zebrafish fasted for 24 h prior to the sacrifice, to reduce the amount of food residues in the lumen. Once extracted, the intestine was maintained in Krebs solution (NaCl 118 mM, KCl 4.7 mM, CaCl\u003csub\u003e2\u003c/sub\u003e∙2H\u003csub\u003e2\u003c/sub\u003eO 2.5 mM, MgSO\u003csub\u003e4\u003c/sub\u003e∙7H\u003csub\u003e2\u003c/sub\u003eO 1.2 mM, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 1.2 mM, NaHCO\u003csub\u003e3\u003c/sub\u003e 25 mM, C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e 11 mM). Creating two loops with a silk thread, each intestine was mounted along the longitudinal axis of an organ bath containing 10 mL of oxygenated (95% O\u003csub\u003e2\u003c/sub\u003e + 5% CO\u003csub\u003e2\u003c/sub\u003e) Krebs solution maintained at 28.5°C. Changes in muscle tension were recorded by isometric force transducers (World Precision Instruments, Berlin, Germany) to a PowerLab 4/30 data acquisition system using LabChart 8 software (ADInstruments, Besozzo, VA, Italy). Intestinal preparations were stretched to an initial tension of 0.1 g and left to equilibrate for 45 minutes to allow the development of rhythmic spontaneous contractions [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. At the end of the equilibration period, samples were activated using 1 µM carbachol (CCh), a non-selective cholinergic receptor agonist, directly added to the Krebs solution. To evaluate smooth muscle contraction, each intestine was exposed to 40 mM KCl, a depolarizing agent that induces Ca\u003csup\u003e2+\u003c/sup\u003e release and subsequent smooth muscle contraction. To study the cholinergic receptor-mediated excitatory responses, samples were then exposed to increasing concentrations of CCh (0.001-100 µM) added in a cumulative manner in the organ baths obtaining concentration–effect curves. Neuronal-mediated contractions were evaluated using electrical field stimulation at increasing frequencies with constant voltage (EFS; 0–40 Hz; 1-ms pulse duration; 10-s pulse-trains, 80 V) through platinum electrodes connected to an S88 stimulator (Grass Instrument) to cause a change in the membrane potential of neurons and consequent release of neurotransmitters. Finally, to characterize the inhibitory neuromuscular response, zebrafish intestines were exposed to 0.1 µM isoprenaline, a non-selective β-adrenergic receptor agonist. Concentration-response curves were subjected to a nonlinear regression analysis (fitted to a sigmoidal equation) to calculate maximal tension (Emax) values. Contractile responses were expressed as gram tension/gram dry tissue weight [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. N≥4 animals were analyzed.\u003c/p\u003e\u003ch2\u003eMicrobiota sequencing\u003c/h2\u003e\u003ch2\u003eZebrafish gut microbiota sequencing\u003c/h2\u003e\u003cp\u003eAdult zebrafish treated for 30 days with BSSG-enriched food and their respective controls were sacrificed after 24 h of fasting, to allow gut clearing from food residues. The whole intestine was extracted, snap frozen in liquid nitrogen and stored at -80°C until used. Bacterial DNA extraction was performed with DNeasy® PowerSoil® Pro Kit (Qiagen) according to the manufacturer’s instructions, with slight modification: to obtain the optimal rupture of bacterial cell walls, samples were repeatedly homogenized on a BeadBug™ microtube homogenizer (Merck Life Sciences) and then continuously vortexed for 20’ until complete tissue destruction. The next steps were performed following the Kit protocol. DNA samples were quantified with a Qubit fluorometer (ThermoFisher) and diluted to 10 ng/µl. Bacterial DNA was then amplificated with a nested PCR protocol to obtain the bacterial 16S sequence. In the first PCR reaction (PCR I), DNA template is targeted with forward Eub8F and reverse 984yR primers and synthetized with Platinum TM Taq DNA Polymerase High Fidelity (ThermoFisher). 1 µl of this first PCR was used to perform the second reaction (PCR II), that exploits a different pair of internal oligonucleotides: 515F and 806R. They amplify the V4 hypervariable region of the bacterial 16S DNA, which allows to discriminate the different bacterial taxa [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. PCR conditions varied according to the optimal annealing temperature of each primer pairs and were as follows: 94°C for 1 min to activate the DNA polymerase, 25 cycles of 94°C for 30 sec, 53°C (in PCR I)/57°C (in PCR II) for 30 sec, 68°C for 45 sec, one cycle at 68°C for 7 min to allow final elongation. Primer sequences are listed in \u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e. The sequencing of the 16S region was performed exploiting the Illumina sequencing platform to obtain pair-end sequences of 150 bp. The resulting reads were analyzed to assign the Amplicon Sequence Variants (ASVs) to the different bacterial taxa using the pipeline described in [\u003cspan class=\"CitationRef\"\u003e79\u003c/span\u003e]. For these analyses, 3 WT and 3 \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e male controls were compared with 4 WT and 4 \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e treated male zebrafish.\u003c/p\u003e\u003ch2\u003eMouse stool microbiota sequencing\u003c/h2\u003e\u003cp\u003eBacterial DNA was extracted from few fecal samples using QIAamp Fast DNA Stool Mini Kit (51604, Qiagen) protocol for pathogen detection, optimized to maximize the ratio of non-mouse DNA. DNA was subsequently amplified with Phusion® High-Fidelity DNA Polymerase (BioLabs) using forward 515F and reverse 806R primers (\u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e) as follows: 98°C for 3 min followed by 25 cycles of 95°C for 45 sec, 58°C for 45 sec and 72°C for 50 sec, and a final cycle at 72°C for 5 min. The sequencing of the 16S region was performed by the sequencing facility of Biology Department at the University of Padova exploiting the Illumina sequencing platform to obtain pair-end sequences of 150 bp. Amplicon Sequence Variants (ASVs) were then assigned to the different bacterial taxa. The mean relative abundance of bacterial strains at family level was considered. Data were obtained from N = 2 male mice/condition.\u003c/p\u003e\u003ch2\u003eRNA extraction, reverse transcription and Real Time-quantitative PCR (RT-qPCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted with TRIzol reagent (15596026, Thermo Fisher Scientific) from different tissue samples: pooled 5 dpf whole larvae (at least N = 15 in each sample); pooled 30 dpf whole larvae (N = 14 in each sample); single adult zebrafish intestine or brain; single mouse small intestine samples long around 1.5 cm. The quantity and quality of the isolated RNA were assessed using a NanoDrop 2000c (Thermo Fisher Scientific) and TapeStation System 4150 (Agilent). 1 µg (for zebrafish larvae and mouse tissue samples) or 500 ng (for adult zebrafish tissue samples) of total RNA were treated with RQ1 RNase-Free DNase (M6101, Promega) and further used for cDNA synthesis, employing High-Capacity cDNA Reverse Transcription Kit (4368814, ThermoFisher). cDNA was diluted 1:10 (or 1:5 in the case of mouse intestine samples) and amplified with 5x HOT FIREPol EvaGreen qPCR Mix Plus (08-36-00001, Solis BioDyne) and the locus-specific primers. Genes expression was measured with SybrGreen method on a CFX384 Touch Real-Time PCR Detection System (BioRad). Primers’ melting curves and threshold cycles (Ct) were automatically generated. Relative gene expression levels were calculated using the comparative Ct method (2\u003csup\u003e–ΔΔCt\u003c/sup\u003e) against a housekeeping gene (\u003cem\u003eβ-actin\u003c/em\u003e for adult zebrafish tissues, \u003cem\u003eef1α\u003c/em\u003e for larval zebrafish samples; \u003cem\u003eTbp\u003c/em\u003e for mouse intestine samples). Primers used for RT-qPCR analysis are listed in \u003cb\u003eSupplementary Table\u0026nbsp;3\u003c/b\u003e.\u003c/p\u003e\u003ch2\u003eRNA sequencing (RNAseq)\u003c/h2\u003e\u003cp\u003eLibrary construction from RNA extracted from zebrafish larvae was performed using the QuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina (015UG009V0260, Lexogen) according to manufacturer’s protocol. Briefly, 500 ng of total RNA were retrotranscribed with an oligod(T) and, after RNA degradation, second cDNA strand was synthesized using random primers. dsDNA was amplified to include sequencing primers and library barcodes. Differently, libraries from mouse samples were constructed starting from at least 500 ng of total RNA to deplete ribosomal RNA by using Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina). rRNA-depleted RNA was subsequently purified through ethanol precipitation and employed for library sequencing construction. Briefly, after fragmentation, the first strand cDNA was synthesized using random hexamer primers (NEBNext Ultra II RNA First Strand RNA Synthesis Module; NEB). Then the second strand cDNA was synthesized, and dUTPs were replaced with dTTPs in the reaction buffer (NEBNext Ultra II Directional RNA Second Strand Synthesis Module; NEB). Double strand DNA was purified using magnetic beads, and the ends were repaired and A-tailed to facilitate adapter ligation. After gel size selection (250–800 bp), USER enzyme digestion (NEB) was employed to eliminate UTP-containing second strand cDNA filaments. Prior to sequencing, libraries were quantified using the Qbit (dsDNA Quantitation, High Sensitivity; Thermofisher). Subsequently, size distribution was assessed using the High Sensitivity DNA kit (Agilent Technologies). Zebrafish libraries were sequenced by the sequencing facility in the Department of Biology of University of Padova utilizing NextSeq 500 (Illumina) platform employing a single read approach. Conversely, mouse libraries were sequenced by Novogene facility using Novoseq X plus (Illumina) sequencer employing a paired end approach.\u003c/p\u003e\u003ch2\u003eTranscriptome data analysis\u003c/h2\u003e\u003cp\u003eAfter removing sequencing adapters with Cutadapt (version 4.7) [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e], transcript expression was quantified using the Salmon method (version 1.10.3) [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. The resulting count matrix was loaded in the R statistical environment, and the edgeR package (version 4.0.2) [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e], was used for gene expression normalization and to identify differentially expressed genes (DEGs) between treated and control samples (FDR ≤ 0.05), reported in \u003cb\u003eAdditional files 4–5\u003c/b\u003e. Zebrafish and murine DEGs were subsequently used for Gene Ontology (GO) enrichment analysis via \u003cem\u003eenrichplot\u003c/em\u003e package on R software and bar chart were generated on ShinyGO [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. The raw data of RNA sequencing have been deposited on the SRA database.\u003c/p\u003e\u003ch2\u003eElectron microscopy and ultrastructural analysis of mouse microvilli\u003c/h2\u003e\u003cp\u003eAfter sacrifice, mice small intestine was extracted and gently flushed with cold PBS. Small sections (0.5 cm long) of mouse small intestine were fixed with Karnovsky fixative (2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer) overnight at 4°C, washed with 0.1M cacodylate buffer, post-fixed with osmium tetroxide for 2 h and embedded in EMbed 812 (Electron Microscopy Sciences). Ultrathin sections, stained with uranyl acetate and lead citrate, were observed at a Philips M400 operating at 100 kV. The length of microvilli was measured from the tip to the base with Fiji-ImageJ software. Analyses were repeated on multiple images derived from 3 individuals for each condition.\u003c/p\u003e\u003ch2\u003eAlcian blue staining of mouse small intestine histological sections\u003c/h2\u003e\u003cp\u003eAfter sacrifice, mice small intestine was extracted and gently flushed with cold PBS. Small portions 1.5 cm long were fixed in Bouin fixative solution (30 mL saturated picric acid in ddH\u003csub\u003e2\u003c/sub\u003eO, 10 mL formaldehyde, 2 mL glacial acetic acid for 42 mL) for 24 h and then rinsed in 70% ethanol in distilled water. After dehydration of samples with graded ethanol series (80%-90%-100% in ddH2O) for 1 h at RT, they were infiltrated with xylene (Sigma-Aldrich), 1 xylene:1paraffin and finally 100% paraffin. Samples were embedded and sectioned with a Leica microtome with a 7 µm thickness. Tissues sections were deparaffinized, rehydrated to ddH\u003csub\u003e2\u003c/sub\u003eO, incubated in 3% glacial acetic acid solution for 3’, followed by 30’ incubation in alcian blue staining solution (1% Alcian blue 8GX – A5268, Sigma-Aldrich – in 3% glacial acetic acid; pH 2.5). Sections were rapidly rinsed in 3% glacial acetic acid solution, set for 5’ in running tap water, counter stained with Eosin Y (Sigma-Aldrich, USA), then rinsed in 100% ethanol and mounted for visualization. Alcian blue-stained goblet cells were counted along intestinal villi in 3 sections spaced 100 µm in 6 mice for each condition.\u003c/p\u003e\u003ch2\u003eMacrophages staining on mouse small intestine histological sections\u003c/h2\u003e\u003cp\u003eMouse small intestines were collected, fixed, paraffin-embedded, sectioned as previously described, and mounted on Superfrost ® Plus microscope slides (J1800AMNZ, Thermo Scientific). Tissues sections were deparaffinized, rehydrated to ddH\u003csub\u003e2\u003c/sub\u003eO with graded ethanol series (100%-90%-70% in ddH2O) for 5 min each and incubated for 15 min in a quenching solution (NH\u003csub\u003e4\u003c/sub\u003eCl 50 mM) to reduce autofluorescence. Antigen retrieval was performed by 10 min incubation in citrate buffer (citric acid 0.01 M, pH 6) followed by 10 min in TBS-1% Tween at RT. Slides were then incubated for 1h in saturating solution (15% goat serum, 2% BSA, 0.25% gelatine, 0.2% glycine in PBS supplemented with 0.5% Triton X-100). Histological sections were incubated overnight at 4°C in F4/80 monoclonal primary antibody (14-4801-82, Thermofisher Scientific) diluted 1:50. After 3 washes for 5 min in TBS-1% Tween, sections were incubated for 1 h at RT with goat anti-rat-Alexa Fluor 568 secondary antibody (A-11077, Thermofisher Scientific) at a final dilution of 1:200. To reduce autofluorescence, slides were immersed for 10 min in Sudan Black solution (0.1% in EtOH 70%), followed by extensive washing in TBS-1% Tween. Nuclei were stained with DAPI for 5 min and sections were mounted with SlowFade™ Diamond Antifade Mountant (S36967, Thermofisher Scientific). Histological sections were imaged using a Zeiss AXIO Zoom.V16 fluorescence microscope equipped with an Axiocam 305 mono camera. Three mice per condition were analysed by manual scoring of stained macrophages.\u003c/p\u003e\u003ch2\u003eRadioligand binding assay\u003c/h2\u003e\u003cp\u003eTo verify the ability of BSSG to bind steroid hormone receptors at a concentration of 10 µM, a radio-ligand binding assay was performed by an external company using the NHR Binding Agonist Radioligand Assay (Eurofins) by Eurofins Panlabs Discovery Services. BSSG selectivity for human androgen receptor, estrogen receptor, glucocorticoid receptor, mineralocorticoid receptor and progesterone receptor were calculated as the percentage of inhibition for the binding of a radio-labeled ligand, specific for each receptor ([\u003csup\u003e3\u003c/sup\u003eH]-methyltrienolone, [\u003csup\u003e3\u003c/sup\u003eH]-estradiol, [\u003csup\u003e3\u003c/sup\u003eH]-dexamethasone, [\u003csup\u003e3\u003c/sup\u003eH]-aldosterone and [\u003csup\u003e3\u003c/sup\u003eH]-progesterone, respectively).\u003c/p\u003e\u003ch2\u003eZebrafish whole-mount HuC/D and Sox10 immunofluorescence\u003c/h2\u003e\u003cp\u003eAfter BSSG treatment, 5 dpf larvae were fixed overnight in 4% PFA (Sigma-Aldrich) in PBS at 4°C. Larvae were then dehydrated in 100% methanol and conserved at -20°C until used. After rehydration with graded methanol series (75-50-25% methanol in PBS) and PBT (0,2% Triton X-100 in PBS) for 5 min each at RT, larvae were depigmented (2% KOH and 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in PBT for 5 min, followed by a 5 min wash in PBT) and permeabilized (15 min at -20°C in ice-cold 100% acetone, followed by washes in ddH\u003csub\u003e2\u003c/sub\u003eO and PBT). After a saturation step in a blocking solution (1% BSA and 5% sheep serum in PBT) for 4 h at RT, larvae were incubated over 2 days with pan-neuronal anti-HuC/D (A-21272, ThermoFisher) or Sox10 (GTX128374-S, GeneTex) primary antibody diluted 1:200 in the blocking solution at 4°C. Larvae were then washed four times in PBT for 20 min each at RT, saturated in the same blocking solution for 4 h and incubated overnight in the dark at 4°C with Streptavidin conjugate Alexa Fluor 555 secondary antibody (S32355, ThermoFisher) against HuC/D or goat anti-rabbit-Alexa Fluor 488 (A11034, Invitrogen) secondary antibody against Sox10 diluted 1:1000. After extensive washes in PBT, larvae were mounted in 1% low-melting agarose and imaged by z-stacks (3 µm step size) under a 20X objective at Nikon C2 confocal microscope. The same body region was imaged for all larvae. All HuC/D\u003csup\u003e+\u003c/sup\u003e enteric neurons and Sox10\u003csup\u003e+\u003c/sup\u003e neuronal progenitors visible on the ventral side of the intestine were manually scored by scrolling through the z-stacks assisted by the Cell counter tool in Fiji-ImageJ to avoid repeated counting. N≥10 animals/condition. The experiment was repeated three times.\u003c/p\u003e\u003ch2\u003eFish Embryo Acute Toxicity test\u003c/h2\u003e\u003cp\u003eFET test was performed according to the Organization for Economic Co-operation and Development (OECD, Paris, France) Guideline No. 236 (2013) [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e] to define a non-lethal and non-toxic BSSG concentration to be used for zebrafish treatment in order to avoid teratogenic or developmental issues.\u003c/p\u003e\u003cp\u003eBriefly, 6 hpf embryos were transferred singularly into 24 well plates (1 embryo in 1 mL solution/well) and incubated with increasing concentrations of BSSG (2.5-5-10-20-40 µM) and of the respective solvent control (DMSO at 0.1%-0.2%-0.4%). For each concentration, 20 embryos were individually incubated with BSSG, and the remaining 4 wells were used as internal negative controls (Fish Water). The negative and positive controls (1.5% EtOH) were also tested. The embryo medium was changed daily, and developmental status of zebrafish embryos and larvae was monitored until 4 dpf. Percentage of survival and hatching rates, presence of cardiac edema and of swimming bladder were determined from the total number of surviving embryos.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using Graph Pad Prism V10.2.3. Data are expressed as mean ± SEM and statistical significance was calculated with unpaired Student's t-test for two sample comparisons or one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Concerning \u003cem\u003eex vivo\u003c/em\u003e analysis of gut contractility, two-way ANOVA followed by Bonferroni post hoc test was used for multiple comparison. The differences between groups were considered significant when \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e. Post hoc tests were run only if F achieved \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e, and there was no significant variance inhomogeneity. For the gastrointestinal transit assay, differences in the distribution of food bolus in transit zones between control and treated larvae were assessed for each time point using Fisher’s exact test for 2x5 contingency tables on absolute larval counts. \u003cem\u003ep\u003c/em\u003e-values are indicated with the following symbols: \u003cem\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001\u003c/em\u003e; \u003cem\u003e****P \u0026lt; 0.0001; ns, not significant\u003c/em\u003e.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eBSSG administration triggers intestinal inflammation in zebrafish larvae\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eZebrafish larvae exposed to 10 \u0026micro;M BSSG presented a normal development (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A-B\u003c/b\u003e) and the mass spectrometry analysis of lipids extracted from larval heads and trunks confirmed the accumulation of this compound in the trunk region of treated animals (\u003cb\u003eSupplementary Fig.\u0026nbsp;1C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe presence of dark aggregates in the intestine of nearly all larvae exposed to BSSG prompted us to hypothesize that the gut may represent its first target (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Consistently, \u003cem\u003ein vivo\u003c/em\u003e analysis of acidified lysosomes evidenced a reduction in the number of lysosome-rich enterocytes (LREs) in treated larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To determine whether these intestinal effects were specifically attributable to BSSG rather than to possible metabolites, we performed the same assay on larvae treated with β-sitosterol, that shares the chemical structure of BSSG but lacks the glucose moiety. This molecule did not affect LREs (\u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e), suggesting that detrimental effects only depend on BSSG consumption.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMucus-secreting goblet cells along the mid-intestine, essential for protecting the gut against digestive enzymes and external insults, were reduced after exposure to BSSG (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Conversely, the number of neutrophils infiltrating the mid-intestine was markedly increased in treated larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), providing an indication of intestinal inflammation onset in our model. This was further supported by the increased fluorescence in the intestine of BSSG-treated transgenic Tg(NFκB:GFP) larvae, indicating the activation of NF-κB pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Consistently with the occurrence of an inflammatory phenotype, we also observed an increase in apoptosis in the mid-intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eMoreover, the reduction of Stat3-linked fluorescence specific of stem-like cells located at the base of the intestinal folds [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], which are analogous to crypt base columnar cells in mammals, may reflect a depletion of proliferating cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eTo further characterize the impact of BSSG on gut homeostasis, we performed analysis of inflammation-related markers expression on whole larvae following acute exposure. We observed a trend towards increased expression of \u003cem\u003emmp9\u003c/em\u003e (matrix metallopeptidase 9), along with significant upregulation of \u003cem\u003estat3\u003c/em\u003e (signal transducer and activator of transcription 3) and \u003cem\u003epept1\u003c/em\u003e (peptide transporter 1). Reduced expression of \u003cem\u003eagr2\u003c/em\u003e (anterior gradient 2), involved in mucus production by goblet cells, further supported the presence of a defective intestinal epithelial barrier (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eWe also observed a reduction in the expression levels of autophagy-related genes \u003cem\u003eatg5\u003c/em\u003e (autophagy protein 5) and \u003cem\u003elc3b\u003c/em\u003e (microtubule associated protein 1 light chain 3b), suggesting a possible impairment of autophagy (\u003cb\u003eSupplementary Fig.\u0026nbsp;2B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTaken together, these results depict a scenario in which BSSG administration leads to an intestinal inflammation disrupting essential cellular processes, potentially exacerbating gut dyshomeostasis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eBSSG treatment alters gut motility and microbial composition\u003c/h2\u003e \u003cp\u003eTo study the impact of BSSG on intestinal physiology, we firstly analyzed peristaltic frequency. Treated larvae exhibited a lower number of gut contractile waves compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To corroborate this result, we monitored larval gastrointestinal transit along the digestive tract, ideally divided into transit zones [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003eupper panel\u003c/b\u003e). Over time, different localization of the food bolus indicated that treated larvae display delayed gastrointestinal transit (***, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001 with Fisher\u0026rsquo;s exact test 24 h after feeding) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003elower panel\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince peristaltic activity is regulated by the enteric nervous system (ENS), we evaluated the number of both differentiated enteric neurons and neuronal precursors (labelled with HuC/D and Sox10, respectively), but we didn\u0026rsquo;t find any difference between treated and control larvae (\u003cb\u003eSupplementary Fig.\u0026nbsp;2C\u003c/b\u003e), suggesting that BSSG may affect the functionality of enteric innervation rather than its density.\u003c/p\u003e \u003cp\u003eTherefore, we investigated the involvement of enteric neurotransmission in gut dysmotility in adult zebrafish fed with a BSSG-enriched diet over a prolonged period, to mimic chronic exposure. For the first time, both receptor-mediated and non-receptor-mediated neuromuscular responses of isolated whole intestines were evaluated in zebrafish. Muscular contractile response was analysed exposing zebrafish intestine to KCl, a depolarizing agent that induces Ca\u003csup\u003e2+\u003c/sup\u003e release and subsequent smooth muscle contraction. A significant increase in the KCl-induced contraction was observed in intestinal preparations of treated individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To further evaluate the excitatory cholinergic response, gut samples were exposed to increasing concentrations of carbachol (CCh), a non-selective cholinergic agonist. Cumulative concentration-response curves evidence a significant increase of the intestinal CCh-mediated contraction in treated intestines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), suggesting the presence of an altered cholinergic neurotransmission following BSSG treatment. Then, to verify the impact on ENS neuronal activity, intestinal preparations were subjected to EFS (electrical field stimulation) at increasing frequencies with constant voltage, to cause a change in the membrane potential of neurons and consequent release of neurotransmitters. Treated individuals displayed an increased excitatory neuromuscular response, determining a significant upward shift of the frequency-response curve to EFS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Higher 10 Hz-EFS-mediated contraction confirmed alterations in excitatory cholinergic response. Conversely, muscular relaxation induced by isoprenaline, a non-selective β-adrenergic receptor agonist, was not affected by treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eNext, given the relevance of the microbiota in maintaining gut metabolic homeostasis and the well-established association between alterations in microbiota composition and intestinal inflammation in humans and animal models [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], we analyzed the microbiota of adult zebrafish following BSSG exposure. At the phylum level, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eBacteroidota\u003c/em\u003e and \u003cem\u003eVerrucomicrobiota\u003c/em\u003e increased, while \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eActinobacteriota\u003c/em\u003e decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), determining a reduction of the \u003cem\u003eFirmicutes/Bacteroidetes\u003c/em\u003e ratio, a widely recognized indicator of gut health [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. At the family level, we observed an increase in \u003cem\u003eBarnesiellaceae\u003c/em\u003e, \u003cem\u003eAeromonadaceae\u003c/em\u003e and \u003cem\u003eRubritaleaceae\u003c/em\u003e, and a reduction in the relative abundance of \u003cem\u003eErysipelotrichaceae, Enterobacteriaceae\u003c/em\u003e and \u003cem\u003eWeeksellaceae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Overall, although these results did not reach statistical significance, likely due to the limited sample size, they suggest that BSSG treatment may influence the composition of gut bacterial population, potentially contributing to the onset of dysbiosis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTranscriptome analysis of chronically treated larvae reveals intestinal dyshomeostasis, possibly prodromal to neurodegeneration\u003c/h3\u003e\n\u003cp\u003eRNAseq analysis of chronically treated zebrafish larvae revealed 261 differentially expressed genes (168 upregulated and 93 downregulated) compared to controls. Upregulated genes are involved in \u003cem\u003eacute inflammatory response\u003c/em\u003e together with \u003cem\u003eresponse to reactive oxygen species\u003c/em\u003e and \u003cem\u003edefence response to bacterium\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The upregulation of both \u003cem\u003emmp9\u003c/em\u003e and \u003cem\u003epept1\u003c/em\u003e, in association with the increased expression of \u003cem\u003esaa\u003c/em\u003e (\u003cem\u003eserum amyloid a\u003c/em\u003e) and \u003cem\u003es100a10a\u003c/em\u003e (\u003cem\u003es100 calcium binding protein a10 a\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), corroborates previous findings in treated larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), indicating the presence of gut inflammation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBSSG treatment downregulated genes mainly associated with \u003cem\u003elateral line nerve development\u003c/em\u003e and \u003cem\u003eoxygen transport\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e\u0026rsquo;\u003c/b\u003e). Decrease of \u003cem\u003eoxygen binding\u003c/em\u003e resulted also among downregulated molecular functions (\u003cb\u003eSupplementary Fig.\u0026nbsp;2D\u003c/b\u003e). Notably, the entire group of hemoglobin (\u003cem\u003ehb\u003c/em\u003e) genes exhibited a significant reduction in treated individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), a finding recently associated with the pathophysiology of different neurodegenerative diseases [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The decreased expression of \u003cem\u003emucin 5.3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), secreted by goblet cells to neutralize digestive enzymes and pathogens, supported the reduction of goblet cells count observed in treated larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo validate transcriptome results, we tested the expression of specific genes in the intestine of adult zebrafish confirming the upregulation of inflammation-related markers (\u003cem\u003emmp9, mmp13\u003c/em\u003e, \u003cem\u003eil-4\u003c/em\u003e, \u003cem\u003eil-13\u003c/em\u003e) and cellular stress markers (\u003cem\u003ecasp8\u003c/em\u003e, caspase 8; \u003cem\u003enupr1\u003c/em\u003e, nuclear protein 1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cb\u003eupper panel\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eSince RNAseq analysis evidenced the alteration of genes potentially involved in neurodegeneration, we evaluated adult zebrafish brains. In contrast to our findings in the intestine, \u003cem\u003emmp9\u003c/em\u003e expression in the brain exhibited a downregulation following treatment, potentially impacting its role in central nervous system (CNS) plasticity. We also observed the downmodulation of autophagy-related genes \u003cem\u003eatg5\u003c/em\u003e, \u003cem\u003elc3b\u003c/em\u003e and \u003cem\u003ep62\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cb\u003elower panel\u003c/b\u003e).\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eBSSG-fed mice exhibit reduced weight and hallmarks of intestinal inflammation\u003c/h2\u003e \u003cp\u003eTo investigate whether BSSG dietary consumption could determine intestinal alterations also in the mouse model, we provided WT mice with BSSG-enriched food for 15 weeks, and then we characterized intestinal phenotypes. We observed an increase in the number of macrophages in the small intestine of treated mice (\u003cb\u003eSupplementary Fig.\u0026nbsp;3A\u003c/b\u003e). We then evaluated the number of goblet cells, finding a significant reduction in treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), evidencing also in this animal model hallmarks of intestinal inflammation following BSSG dietary uptake. Moreover, ultrastructural analysis revealed that BSSG-fed mice exhibited significantly shorter microvilli compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), potentially implying impaired enteric absorption capacity. Consistently, they weighed less than controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) despite consuming the same amount of food (\u003cb\u003eSupplementary Fig.\u0026nbsp;3B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRNA-seq analysis of gut tissue revealed 1835 differentially expressed genes between treated and untreated mice (982 upregulated and 853 downregulated in treated animals). Upregulated genes are primarily associated with \u003cem\u003eregulation of immune system process\u003c/em\u003e and \u003cem\u003eimmune response\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E, \u003cb\u003eSupplementary Fig.\u0026nbsp;3C, left panel\u003c/b\u003e) and are related with \u003cem\u003eactin cytoskeleton\u003c/em\u003e, \u003cem\u003eside of membrane\u003c/em\u003e, and \u003cem\u003emicrovillus\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;3D\u003c/b\u003e), thus confirming that BSSG directly impacts on genes involved in enterocytes\u0026rsquo; brush border structure. Conversely, downregulated genes are involved in \u003cem\u003esmall molecules metabolic process, regulation of cell growth\u003c/em\u003e, \u003cem\u003eheme\u003c/em\u003e/\u003cem\u003eporphyrin-containing compound biosynthesis\u003c/em\u003e, and \u003cem\u003eregulation of neuron projection development\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e\u0026rsquo;\u003c/b\u003e) and are associated with \u003cem\u003eglutathione transferase activity\u003c/em\u003e and \u003cem\u003etransmembrane transporter activity\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;3C, right panel\u003c/b\u003e). RT-qPCR experiments confirmed the activation of inflammatory and immune response, evidencing the upregulation of \u003cem\u003eNod1\u003c/em\u003e (NOD-like receptor 1), \u003cem\u003eTlr-2, Tlr-4, Tlr-6\u003c/em\u003e (Toll-like receptor-2, -4, -6), \u003cem\u003eNlrp3\u003c/em\u003e (NLR family pyrin domain containing 3), \u003cem\u003eIl-1β\u003c/em\u003e (interleukin-1β) and \u003cem\u003eIfn-γ\u003c/em\u003e (interferon-γ) and the downregulation of \u003cem\u003eIkb-α\u003c/em\u003e (NF-kappa-B inhibitor alpha) and \u003cem\u003eReg3-γ\u003c/em\u003e (regenerating islet-derived protein 3-γ). We also observed the upregulation of \u003cem\u003ePlp1\u003c/em\u003e (proteolipid protein 1), which is highly expressed in enteric glia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eFinally, we found alterations in the composition of fecal microbiota in BSSG-fed mice. Treated mice exhibited a higher relative abundance of potentially pathogenic bacteria \u003cem\u003eBacteroidaceae, Helicobacteraceae and Prevotellaceae\u003c/em\u003e, typically associated with intestinal inflammation, and reduced recognized anti-inflammatory taxa like \u003cem\u003eLachnospiraceae\u003c/em\u003e, known to produce short chain fatty acids (SCFAs), highly beneficial for intestinal homeostasis, immune system modulation and energetic metabolism (\u003cb\u003eSupplementary Fig.\u0026nbsp;3E\u003c/b\u003e). As in zebrafish, this preliminary microbiota analysis, although not reaching statistical significance, suggests the onset of dysbiosis in this pre-symptomatic ALS-PDC mouse model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eBSSG interaction with the glucocorticoid receptor as a possible mechanism of action\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eWith the aim to discover a possible BSSG mode of action, we addressed whether it could interact with known receptors of steroid hormones, due to its structural similarity with these molecules. A radioligand binding assay demonstrated that BSSG determined a 12.5% inhibition of the binding between glucocorticoid receptor (GR) and its radiolabelled specific ligand ([\u003csup\u003e3\u003c/sup\u003eH]-dexamethasone) and a 4.7% inhibition for the androgen receptor (AR) and its radiolabelled specific ligand ([\u003csup\u003e3\u003c/sup\u003eH]-methyltrienolone). No interference with neither estrogen receptor (ER), mineralocorticoid receptor (MR) nor with progesterone receptor (PR) was observed (\u003cb\u003eSupplementary Fig.\u0026nbsp;2F\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo verify if BSSG effectively binds Gr \u003cem\u003ein vivo\u003c/em\u003e, we exploited the \u003cem\u003ecyp11c1\u003c/em\u003e zebrafish mutant line recently generated in our laboratory. As other published \u003cem\u003ecyp11c1\u003c/em\u003e (\u003cem\u003ecytochrome P450 family 1 subfamily C member 1\u003c/em\u003e) zebrafish mutant lines, homozygous \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e cannot synthetize active glucocorticoids (GCs), while retaining a functional Gr receptor [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. To visualize Gr activity, this \u003cem\u003ecyp11c1\u003c/em\u003e mutant line was crossed with transgenic Tg(GRE:EGFP) line, which expresses GFP after Gr activation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Mutant \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e;Tg(GRE:EGFP), free of endogenous GCs, therefore, allow to visually discriminate the effective binding of BSSG with the Gr \u003cem\u003ein vivo\u003c/em\u003e. We treated \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e;Tg(GRE:GFP) larvae and analysed their intestine. A significant increase in the fluorescent signal was observed, suggesting that BSSG can effectively modulate Gr activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The same analysis was performed on adult transgenic mutants exposed to BSSG-enriched diet, obtaining again a significant increase of the fluorescent signal in the intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The analysis of gut fluorescence in \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e; Tg(GRE:GFP) larvae and adults revealed, as expected, an higher baseline signal compared with \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e;Tg(GRE:GFP), since endogenous steroid hormones constitutively activate Gr [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Accordingly, BSSG treatment in \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e;Tg(GRE:GFP) larvae failed to induce any detectable further increase of intestinal fluorescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eGr deficiency reduces the negative effects of BSSG on gene expression\u003c/h2\u003e \u003cp\u003eTo confirm the role of the Gr in mediating the action of BSSG, we exploited the \u003cem\u003enr3c1\u003c/em\u003e\u003csup\u003e\u003cem\u003eia30/ia30\u003c/em\u003e\u003c/sup\u003e zebrafish mutant line (hereafter called \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e), previously generated in our laboratory, in which the \u003cem\u003egr\u003c/em\u003e gene has been knocked out [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, we couldn\u0026rsquo;t observe any difference in the number of goblet cells in \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e treated larvae compared to controls, in contrast to what occurs in treated WT individuals. Similarly, we observed no significant alteration in the expression of \u003cem\u003emmp9, il-4\u003c/em\u003e and \u003cem\u003eil-13\u003c/em\u003e in the intestine of adult \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e zebrafish fed with BSSG-enriched diet. This suggests that BSSG likely interferes with anti-inflammatory Gr activity. Conversely, gene expression of \u003cem\u003ecasp8\u003c/em\u003e and \u003cem\u003enupr1\u003c/em\u003e appear significantly upregulated, similarly to what was observed in WT, conveying that BSSG likely interferes with multiple pathways and cellular targets beyond this receptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cb\u003eupper panel and 6C\u0026rsquo;\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, also mRNA levels of \u003cem\u003emmp9, p62, atg5\u003c/em\u003e and \u003cem\u003elc3b\u003c/em\u003e do not differ between brains of treated and control \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cb\u003elower panel and 6C\u0026rsquo;\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAfter the \u003cem\u003ein vivo\u003c/em\u003e evidence of a plausible BSSG interaction with Gr, we investigated if it could also impact on the fine-tuned regulation of gene expression commonly depending on GCs/Gr interaction. We therefore evaluated the expression of some Gr-target genes such as \u003cem\u003efkbp5\u003c/em\u003e (\u003cem\u003eFKBP prolyl isomerase 5\u003c/em\u003e) and \u003cem\u003efoxo3b\u003c/em\u003e (\u003cem\u003eforkhead box protein O 3b\u003c/em\u003e) in the intestine of WT adults fed with BSSG. We observed a significant reduction in \u003cem\u003efkbp5\u003c/em\u003e expression, while \u003cem\u003efoxo3b\u003c/em\u003e displays a decreasing trend in treated individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), further corroborating our hypothesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eGr deficiency mitigates the alteration of intestinal muscular contractility and the differences in microbiota composition\u003c/h2\u003e \u003cp\u003eTo evaluate whether the impact of BSSG on intestinal neuromuscular function is modulated by Gr, we exploited the \u003cem\u003eex vivo\u003c/em\u003e approach in \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e adult zebrafish after administration of BSSG-enriched diet. We observed that gut samples from \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e do not show any variation in muscular- and neuronal-induced contractility when exposed to KCl, 1 \u0026micro;M CCh and 10 Hz EFSafter BSSG treatment, maintaining similar response compared to untreated \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). These data point out that defects in gut movements could be influenced by BSSG interaction with Gr and are abrogated in \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mutant line.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, the treatment seemed to determine fewer changes in the composition of \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e gut microbiota compared with those reported for WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-E). At the phylum level, no evident alteration occurs, except for a slight increase in \u003cem\u003eFirmicutes\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cb\u003eright side\u003c/b\u003e). At the family level we observed a similar abundance of \u003cem\u003eBarnesiellaceae\u003c/em\u003e, \u003cem\u003eErysipelotrichaceae\u003c/em\u003e and \u003cem\u003eRubritaleaceae\u003c/em\u003e, suggesting that BSSG exposure minimally affects these bacterial strains in \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, \u003cb\u003eright side\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe lack of the Gr determines some constitutive differences in gut microbiota, indeed \u003cem\u003eFusobacteriota\u003c/em\u003e are more abundant among \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e phyla whereas \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eActinobacteriota\u003c/em\u003e are scarcely represented, in opposition to what observed in WT. Among families, \u003cem\u003eFusobacteriaceae, Comamonadaceae\u003c/em\u003e and \u003cem\u003eShewanellaceae\u003c/em\u003e are more present in \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e with respect to WT. Nevertheless, our data suggest that BSSG influences the gut microbial community of \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e differently than it does in WT, as emerges also from 2D PCoA and ASVs (Amplicon Sequence Variants) relative abundance calculated in all experimental groups (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this work we used the zebrafish and mouse models to characterize the link between BSSG intake and the occurrence of the complex neurodegenerative disorder known as ALS-PDC.\u003c/p\u003e \u003cp\u003eWe found that BSSG is effectively absorbed by zebrafish larvae, adults, and mice, where it triggers a wide range of effects related to intestinal dyshomeostasis. Damaged gut epithelial barrier, pro-inflammatory profile, impaired gut functionality and microbiota alteration are all characteristic hallmarks of a perturbed gut-brain axis, that connects the ENS and the CNS [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Therefore, we hypothesize that BSSG may affect this route, inducing gut inflammation that possibly predisposes to neurodegeneration.\u003c/p\u003e \u003cp\u003eThe pro-inflammatory activity seems to be caused specifically by BSSG, in agreement with the reported toxicity caused by the presence of a single glucidic group [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], since β-sitosterol did not elicited inflammatory effects on LREs, a marker of intestinal inflammation in different zebrafish models [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Gut dyshomeostasis was supported by a reduced number of goblet cells and decreased expression of \u003cem\u003eagr2\u003c/em\u003e, a gene essential for intestinal mucus production [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. These alterations are known to promote intestinal inflammation through weakening of the epithelial barrier [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Furthermore, neutrophil recruitment to the mid-intestine, activation of the NF-κB pathway, increased apoptosis, reduced proliferative capacity of intestinal stem-like cells, and upregulation of immune-related genes collectively indicate an ongoing inflammatory response (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Indeed, \u003cem\u003emmp9\u003c/em\u003e and \u003cem\u003estat3\u003c/em\u003e play a role in the activation of the immune response, while enteric \u003cem\u003epept1\u003c/em\u003e is stimulated by pro-inflammatory cytokines. Noteworthy, the expression of these genes is increased in inflammatory bowel disease (IBD) patients and related animal models [\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChronic administration of BSSG to zebrafish larvae and adults confirmed the inflammatory phenotype at the intestinal level, as emerged from transcriptomic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Together with \u003cem\u003emmp9\u003c/em\u003e and \u003cem\u003epept1\u003c/em\u003e, we highlighted the upregulation of \u003cem\u003esaa\u003c/em\u003e, involved in the activation of NF-kB signalling, in the promotion of downstream genes like \u003cem\u003emmp9\u003c/em\u003e itself and in the regulation of neutrophils migration [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Similarly, \u003cem\u003es100a10a\u003c/em\u003e, expressed in the digestive tract, drives neutrophil recruitment in a larval model of intestinal infection [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. S100a10a protein is considered homolog of human CALPROTECTIN, another marker for IBD [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Interestingly, it is increased also in stool samples of PD and Alzheimer\u0026rsquo;s disease (AD) patients [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on these results and according to previous studies [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], we developed an ALS-PDC pre-symptomatic mouse model, focusing on the small intestine of BSSG-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similar to what we found in zebrafish, RNAseq of mouse small intestine revealed that most of the DEGs are involved in the modulation of immune response. Toll-like receptors (TLRs) are significantly upregulated: \u003cem\u003eTlr-2\u003c/em\u003e, \u003cem\u003eTlr-4\u003c/em\u003e and \u003cem\u003eTlr-6\u003c/em\u003e, along with NOD-like receptors, have been associated with the activation of NF-kB pathway [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] and IL-1β production [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Moreover, the observed alteration of several players involved in NF-kB pathway, Nlrp3 assembly and inflammasome activation [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], together with the downregulation of \u003cem\u003eIkb-α\u003c/em\u003e, that usually guarantees a negative-feedback mechanism to modulate inflammatory response, suggests an impairment of this route after BSSG treatment, leading to chronic inflammation. Consistently, it is reported that patients and animal models with IBD present an increased expression of TLRs, persistent NF-kB and NLRP3 activation and higher levels of pro-inflammatory cytokines such as IL-1β and IFN-γ [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Interestingly, increased expression of the same pro-inflammatory genes has been observed in intestinal biopsies of PD patients [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], further corroborating the existence of a link between intestinal inflammation and the predisposition to this disease. Activation of the immune response following BSSG dietary uptake was further supported by the presence of more macrophages in the lamina propria of treated mice.\u003c/p\u003e \u003cp\u003eMoreover, the antimicrobial peptide REG3-γ, involved in proper distribution of the mucus layer [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], is downregulated in our model. Coherently, we observed a reduction in the number of mucus-producing goblet cells, as already observed treated zebrafish larvae. Among upregulated DEGs we also found \u003cem\u003eLrrk2\u003c/em\u003e (\u003cem\u003eLeucine-rich repeat kinase 2\u003c/em\u003e), one of the most relevant PD genetic risk factors. In the intestine, LRRK2 protein is involved in the activation of the immune system through positive regulation of NF-kB, and its increased expression has been associated with pro-inflammatory effects in IBD mouse models as well as in PD patients, as reviewed in [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur zebrafish model provided evidence that this inflammatory condition is also associated with marked alterations in gut physiology, observed after both acute and chronic exposure to BSSG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-F). Deregulation of peristalsis and delayed gastrointestinal transit have been frequently recognized as hallmarks of IBD [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] and have emerged as prodromic symptoms in disorders related to the autistic spectrum [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] and in neurodegenerative diseases such as PD and ALS [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Constipation, indeed, can affect patients many years before the appearance of the typical motor symptoms. The innovative \u003cem\u003eex vivo\u003c/em\u003e analysis of gut contractility, applied for the first time on adult zebrafish intestines, gave us solid evidence that ENS functionality is affected after BSSG consumption. Several findings link intestinal muscular hypertrophy and hypercontractility with infection and with increased expression of pro-inflammatory cytokines. Among them, IL-4 and IL-13 are responsible of higher intestinal smooth muscle contraction in mice during gut inflammation or enteric infection [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Moreover, a mouse model of DSS (dextran sulphate sodium)-induced colitis showed increased neuromuscular contraction upon CCh stimulation [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Collectively, these data demonstrate that BSSG administration affects both muscular and neuronal districts in the gut, altering gastrointestinal motility and possibly damaging enteric neurons.\u003c/p\u003e \u003cp\u003ePreliminary analyses of gut microbiota in treated adult zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H) revealed a higher abundance of \u003cem\u003eProteobacteria\u003c/em\u003e and a reduction in \u003cem\u003eFirmicutes\u003c/em\u003e, in agreement with data from TNBS (trinitrobenzene sulfonic acid)-induced intestinal inflammation [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] and IBD patients [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Furthermore, a reduction of the \u003cem\u003eFirmicutes\u003c/em\u003e/\u003cem\u003eBacteroidetes\u003c/em\u003e ratio has been already linked with IBD [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], while alterations in the relative abundance of bacterial phyla/families similar to the ones presented in this work were described for zebrafish exposed to contaminants leading to dysbiosis [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Noteworthy, similar gut microbiota alterations have been found also in neurodegenerative diseases such as AD, ALS and PD, where changes of bacterial communities can be prodromal to the onset of the disease [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to the hypothesis of a disturbance of the gut-brain axis, we investigated if BSSG exerted detrimental effects in the CNS of our zebrafish model. In the brains of treated adult zebrafish we found decreased expression of autophagy-related genes, possibly suggesting an impairment of the autophagic mechanism, which alone can induce neurodegeneration determining the accumulation of neurotoxic proteinaceous aggregates [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. This aspect, however, deserves more extensive and tailored analyses.\u003c/p\u003e \u003cp\u003eData obtained using zebrafish mutant lines knocked out for genes relevant to GCs synthesis (\u003cem\u003ecyp11c1\u003c/em\u003e) and activity (\u003cem\u003egr\u003c/em\u003e), showed that BSSG could in part exert its effects by interacting with the Gr. In particular, increased fluorescence in \u003cem\u003ecyp11c1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e;Tg(GRE:GFP) zebrafish intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) suggests that this sterol-derived molecule can interfere with Gr nuclear translocation and activity. Moreover, the absence of significant changes in goblet cells number in \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;I\u0026minus;\u003c/em\u003e\u003c/sup\u003e treated larvae, the presence of only minor alterations of gene expression in intestines and brains of \u003cem\u003egr\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e adult zebrafish fed with BSSG and their unchanged intestinal neuromuscular activity observed \u003cem\u003eex vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C), evidenced that inflammatory and neuromuscular signalling could be, at least partially, regulated by BSSG interaction with Gr. Both \u003cem\u003efkbp5\u003c/em\u003e and \u003cem\u003efoxo3b\u003c/em\u003e are directly regulated by Gr, modulating sensitivity to GCs and resolving inflammation through the inhibition of NF-kB pathway [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e], respectively. Their reduced expression in the intestine of treated WT zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), indeed, could be caused by local impairment of Gr function, supporting the idea of a reduction of glucocorticoids anti-inflammatory effect after BSSG exposure. Of note, recent evidence showed that absence of intestinal GR in DSS-treated mice exacerbated inflammatory response, emphasizing the protective role exerted by GR activity against IBD [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. However, despite our extensive \u003cem\u003ein vivo\u003c/em\u003e findings, this compelling hypothesis requires further \u003cem\u003ein vitro\u003c/em\u003e validation due to the complexity of steroid nuclear activity. Nevertheless, altered GCs levels and impaired Gr regulation have already been linked with pathogenesis and progression of neurodegenerative diseases like ALS, PD and AD [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], further endorsing the proposed mechanism of action through which BSSG, targeting the Gr, contributes to the etiology of ALS-PDC.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eTo conclude, this work revealed that increased levels of dietary BSSG determine a marked intestinal inflammation previously unknown. Our results suggest that this molecule affects the enteric district at first and lately the CNS, inducing neurodegeneration culminating in ALS-PDC occurrence. Such interrelation between the intestine and the CNS, therefore, highlights the relevance of the gut-brain axis. Furthermore, BSSG interaction with the glucocorticoid receptor implies a possible modulation of its anti-inflammatory activity, offering novel hints on the importance of physiologic activity of GCs in the intestine, where they regulate immune homeostasis and inflammatory responses. Following the gut-brain axis paradigm, interference with such functions can exacerbate inflammation, possibly leading to neurodegeneration. Therefore, a possible first line of defence could account for the recovery of intestinal homeostasis before the irreversible spread of the disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlzheimer\u0026rsquo;s disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmyotrophic Lateral Sclerosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALS-PDC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmyotrophic Lateral Sclerosis-Parkinsonism Dementia complex\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eandrogen receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBSSG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eβ-sitosterol β-D-glucoside\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCh\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbachol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecyp11c1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCytochrome P450 Family 1 Subfamily C Member 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCNS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecentral nervous system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edimethyl sulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDSS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edextran sulphate sodium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEFS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eelectrical field stimulation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eENS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenteric nervous system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eestrogen receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglucocorticoids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGr\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglucocorticoid receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIBD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einflammatory bowel disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKCl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epotassium chloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLRE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elysosome-rich enterocyte\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emineralcorticoid receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCoA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrincipal Component Analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eParkinson\u0026rsquo;s disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprogesterone receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etrinitrobenzene sulfonic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewild type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eα-GlcChol\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglucosyl-α-D-cholesterol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eβ-GlcChol\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglucosyl-β-D-cholesterol.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTranscriptional data generated during the current study have been deposited in SRA database (\u003cem\u003eURL will be made available for reviewers\u003c/em\u003e). Other data supporting the findings of this study are available on reasonable request to the corresponding authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the University of Padova to FT (MUR/University of Padova PhD Fellowship 2020 and Department of Biology PostDoc Fellowship 2024), SCa (Department of Biology Intramural Grant Seed 2020), LDV (Department of Biology Intramural Grant Seed 2022),\u0026nbsp;MCG (San Camillo Hospital Grant, Treviso (Italy); UNIPD-DSF-PRID-2023), SF (MUR/University of Padova PhD Fellowship 2020 and Department of Pharmaceutical and Pharmacological Sciences PostDoc Fellowship ARD-B 2023) and to SCe (Department of Pharmaceutical and Pharmacological Sciences PostDoc Fellowship ARD-B 2020).\u0026nbsp;This study was also funded by the project entitled “National Center For Gene Therapy And Drugs Based On RNA Technology Neurodegeneration” (Project ID: CN00000041 - SP. 3) granted to LB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFT: study conceptualization and design, main data collection, analysis and interpretation of data, drafting and review of the original manuscript; SF: \u003cem\u003eex vivo\u0026nbsp;\u003c/em\u003eexperiments data collection and analysis, contributed to original drafting; EB: data curation on zebrafish gut microbiota; DS: data curation on zebrafish gut microbiota and mouse fecal microbiota; SCe: contributed to define \u003cem\u003eex vivo\u0026nbsp;\u003c/em\u003emethodology; GB, FF, AS, RL: provided experimental materials, contributed to original drafting; GG: performed LC-MS analysis; GS: RNAseq data analysis; SCa: RNAseq data analysis, contributed to original drafting and review of the manuscript, funding acquisition; LT: review of the manuscript; LB: review of the manuscript, funding acquisition; MCG: \u003cem\u003eex vivo\u0026nbsp;\u003c/em\u003eexperiments data curation, contributed to original drafting and review of the manuscript, funding acquisition; NP, LDV: study conceptualization and design, data curation and interpretation, supervision, drafting and review of the original manuscript, funding acquisition. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the Zebrafish Facility and its facility manager Martina Milanetto, the Imaging Facility and the Sequencing Facility at Biology Department of the University of Padova. Illustrations were created with BioRender.com.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHirano A, Kurland LT, Krooth RS, Lessell S. Parkinsonism-dementia complex, an endemic disease on the island of Guam. I. Clinical features. Brain. 1961;84:642\u0026ndash;61.\u003c/li\u003e\n \u003cli\u003eMorimoto S, Ishikawa M, Watanabe H, Isoda M, Takao M, Nakamura S, et al. Brain transcriptome analysis links deficiencies of stress-responsive proteins to the pathomechanism of Kii ALS/PDC. Antioxidants. 2020;9:1\u0026ndash;16.\u003c/li\u003e\n \u003cli\u003eGajdusek DC, Salazar AM. 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J Neurochem. 2002;82:516\u0026ndash;28.\u003c/li\u003e\n \u003cli\u003eBorenstein AR, Mortimer JA, Schofield E, Wu Y, Salmon DP, Gamst A, et al. Cycad exposure and risk of dementia, MCI, and PDC in the Chamorro population of Guam. Neurology. 2007;68:1764\u0026ndash;71.\u003c/li\u003e\n \u003cli\u003eShimamura M. Structure, metabolism and biological functions of steryl glycosides in mammals. Biochem J. 2020;477:4243\u0026ndash;61.\u003c/li\u003e\n \u003cli\u003eAkiyama H, Hirabayashi Y. A novel function for glucocerebrosidase as a regulator of sterylglucoside metabolism. Biochim Biophys acta Gen Subj. 2017;1861:2507\u0026ndash;14.\u003c/li\u003e\n \u003cli\u003eSchulz JD, Hawkes EL, Shaw CA. Cycad toxins, Helicobacter pylori and parkinsonism: Cholesterol glucosides as the common denomenator. Med Hypotheses. 2006;66:1222\u0026ndash;6.\u003c/li\u003e\n \u003cli\u003eTabata RC, Wilson JMB, Ly P, Zwiegers P, Kwok D, Van Kampen JM, et al. 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J Cell Sci. 2017;130:648\u0026ndash;57.\u003c/li\u003e\n \u003cli\u003ePeron M, Dinarello A, Meneghetti G, Martorano L, Facchinello N, Vettori A, et al. The stem-like Stat3-responsive cells of zebrafish intestine are Wnt/\u0026beta;-catenin dependent. Development. 2020;147.\u003c/li\u003e\n \u003cli\u003eBenato F, Colletti E, Skobo T, Moro E, Colombo L, Argenton F, et al. A living biosensor model to dynamically trace glucocorticoid transcriptional activity during development and adult life in zebrafish. Mol Cell Endocrinol. 2014;392:60\u0026ndash;72.\u003c/li\u003e\n \u003cli\u003eLiao X, Lan Y, Shao R, Liu J, Liang S, Yin Z, et al. Vitamin D Enhances Neutrophil Generation and Function in Zebrafish (Danio rerio). J Innate Immun. 2022;14:229\u0026ndash;42.\u003c/li\u003e\n \u003cli\u003eShi Y, Zhang Y, Zhao F, Ruan H, Huang H, Luo L, et al. Acetylcholine serves as a derepressor in Loperamide-induced Opioid-Induced Bowel Dysfunction (OIBD) in zebrafish. Sci Rep. 2014;4:1\u0026ndash;12.\u003c/li\u003e\n \u003cli\u003eField HA, Kelley KA, Martell L, Goldstein AM, Serluca FC. Analysis of gastrointestinal physiology using a novel intestinal transit assay in zebrafish. Neurogastroenterol Motil. 2009;21:304\u0026ndash;12.\u003c/li\u003e\n \u003cli\u003eCerantola S, Faggin S, Annaloro G, Mainente F, Filippini R, Savarino EV, et al. Influence of Tilia tomentosa Moench Extract on Mouse Small Intestine Neuromuscular Contractility. Nutrients. 2021;13.\u003c/li\u003e\n \u003cli\u003eFaggin S, Cerantola S, Caputi V, Tietto A, Stocco E, Bosi A, et al. Toll-like receptor 4 deficiency ameliorates experimental ileitis and enteric neuropathy: Involvement of nitrergic and 5-hydroxytryptaminergic neurotransmission. Br J Pharmacol. 2025;182:1803\u0026ndash;22.\u003c/li\u003e\n \u003cli\u003eBosi A, Banfi D, Moroni F, Ceccotti C, Giron MC, Antonini M, et al. 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Nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor. Sci Rep. 2017;7:1\u0026ndash;13.\u003c/li\u003e\n \u003cli\u003eYadav H, Jaldhi, Bhardwaj R, Anamika, Bakshi A, Gupta S, et al. Unveiling the role of gut-brain axis in regulating neurodegenerative diseases: A comprehensive review. Life Sci. 2023;330:122022.\u003c/li\u003e\n \u003cli\u003eTakechi M, Tanaka Y. Structure-activity relationships of synthetic digitoxigenyl glycosides. Phytochemistry. 1994;37:1421\u0026ndash;3.\u003c/li\u003e\n \u003cli\u003eChuang L-S, Morrison J, Hsu N-Y, Labrias PR, Nayar S, Chen E, et al. Zebrafish modeling of intestinal injury, bacterial exposures and medications defines epithelial in vivo responses relevant to human inflammatory bowel disease. Dis Model Mech. 2019;12.\u003c/li\u003e\n \u003cli\u003eCarnovali M, Banfi G, Porta G, Mariotti M. Soybean Meal‐Dependent Acute Intestinal Inflammation Delays Osteogenesis in Zebrafish Larvae. 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Compr Physiol. 2018;8:731\u0026ndash;60.\u003c/li\u003e\n \u003cli\u003eIngersoll SA, Ayyadurai S, Charania MA, Laroui H, Yan Y, Merlin D. The role and pathophysiological relevance of membrane transporter PepT1 in intestinal inflammation and inflammatory bowel disease. Am J Physiol Gastrointest Liver Physiol. 2012;302:G484-92.\u003c/li\u003e\n \u003cli\u003eKanther M, Tomkovich S, Xiaolun S, Grosser MR, Koo J, Flynn EJ 3rd, et al. Commensal microbiota stimulate systemic neutrophil migration through induction of serum amyloid A. Cell Microbiol. 2014;16:1053\u0026ndash;67.\u003c/li\u003e\n \u003cli\u003eFarr D, Nag D, Chazin WJ, Harrison S, Thummel R, Luo X, et al. Neutrophil-Associated Responses to Vibrio cholerae Infection in a Natural Host Model. Infect Immun. 2022;90:e0046621.\u003c/li\u003e\n \u003cli\u003eNag D, Farr D, Raychaudhuri S, Withey JH. 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Int J Mol Sci. 2020;21.\u003c/li\u003e\n \u003cli\u003eMetabioinfomicsLab/16S_pipe_metabioinfomics:https://github.com/MetabioinfomicsLab/16S_pipe_metabioinfomics\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Padua","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"BSSG, glucosylated sterols, intestinal inflammation, gut microbiota, glucocorticoid receptor, gut-brain axis, zebrafish model, mouse model","lastPublishedDoi":"10.21203/rs.3.rs-9163005/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9163005/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eGlucosylated-sterols can be synthetized endogenously, absorbed through the diet or derive from bacterial infection. Their clinical relevance is currently underestimated, even though their imbalance has been associated with higher risk of undergoing neurodegeneration throughout life. We studied the detrimental effects elicited by dietary consumption of plant-derived β-sitosterol β-D-glucoside (BSSG), known to be associated with the occurrence of ALS-PDC, to decipher its possible mode of action.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eZebrafish larvae and adults, as well as mice, were treated with BSSG dissolved directly in the water or through customized food pellet, respectively. Being the first target tissue identified, morphological and functional characterization of the intestine were performed, together with transcriptional analysis and sequencing of gut microbiota. \u003cem\u003eEx vivo\u003c/em\u003e analysis of zebrafish gut contractility was applied to assess intestinal neuromuscular response. Mutant and transgenic zebrafish lines were used to explore a possible BSSG mechanism of action.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBSSG caused intestinal inflammation in both zebrafish and mouse models. This previously unknown effect was evidenced by altered gut dysmotility and inflammatory response. Transcriptomic analyses revealed increased expression of inflammation-related genes in the intestine of both zebrafish and mice, while preliminary gut microbiota analyses suggested the onset of dysbiosis. Transgenic and mutant zebrafish lines depleted of genes involved in glucocorticoids synthesis and activity evidenced that BSSG likely interacts with the glucocorticoid receptor, potentially affecting its canonical anti-inflammatory activity.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWe discovered a new set of pathways altered by dietary uptake of BSSG. This molecule introduced in the organism initially determines gut inflammation, altering intestinal morphology and functionality, and possibly leads to neurodegeneration through disruption of the well-known gut-brain axis.\u003c/p\u003e","manuscriptTitle":"β-sitosterol β-D-glucoside (BSSG) triggers intestinal inflammation in zebrafish and mouse models prior to neurodegeneration onset","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 07:55:58","doi":"10.21203/rs.3.rs-9163005/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"719ed395-9758-4a31-80e2-cf8b1d7cdda3","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T07:55:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 07:55:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9163005","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9163005","identity":"rs-9163005","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}