# Microbial metabolites alleviate Aß-induced alterations of intestinal epithelial barrier and enteric nervous system connectivity

Rodrigue Brossaud, Valentine S Moullé, Marina Giblaine, Sergine Even, Marie-Bernadette Maillard, Séverine Chevalier, Thibauld Oullier, Eric Guédon, Yves Le Loir, Moustapha Cissé, et al.

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## To cite this version:

Rodrigue Brossaud, Valentine S Moullé, Marina Giblaine, Sergine Even, Marie-Bernadette Maillard, et al. Microbial metabolites alleviate Aß-induced alterations of intestinal epithelial barrier and enteric nervous system connectivity. Journal of Functional Foods, 2025, 135, pp.107095. 10.1016/j.jff.2025.107095. hal-05490797

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Microbial metabolites alleviate Aß-induced alterations of intestinal
epithelial barrier and enteric nervous system connectivity☆

Rodrigue Brossaud<sup>a,1</sup>, Valentine S. Moullé<sup>a,1</sup>, Marina Giblaine<sup>b</sup>, Sergine Even<sup>b</sup>,
Marie-Bernadette Maillard<sup>b</sup>, Séverine Chevalier<sup>b</sup>, Thibauld Oullier<sup>a</sup>, Eric Guédon<sup>b</sup>,
Yves Le Loir<sup>b</sup>, Moustapha Cissé<sup>a,*</sup>, Hélène Falentin<sup>b,*</sup>, Michel Neunlist<sup>a</sup>

ARTICLE INFO

Keywords:

Enteric nervous system

Intestinal permeability

Amyloid-β

Microbial metabolites

Dairy product

A B S T R A C T

Amyloid-β (Aβ) induced alterations in gut functions, such as reduced motility and compromised barrier integrity contribute to age-related frailty. Therefore, developing fermentation-based products to prevent age-related gut disorders represents a major therapeutic interest. Primary cultures of enteric nervous system and Caco-2 monolayers were exposed to Aβ oligomers following pretreatment with individual or a combination of candidate microbial derived metabolites (butyrate, gamma-aminobutyric acid, and indole derivatives), or a candidate metabolite-enriched fermented dairy product that we subsequently developed. Results showed that individual metabolites significantly preserved markers of neuronal connectivity (beta-tubulin III and synaptophysin) and stabilized tight junction proteins (zonula occludens-1 and occludin), reducing Aβ-induced dysfunction. Furthermore, enhanced protective effects of the fermented dairy products enriched with microbial metabolites were observed, suggesting a promising dietary strategy for supporting gut health in aging and Alzheimer's disease, although in vivo validation is needed.

# 1. Introduction

The proper regulation of intestinal function is essential for overall
health, as the gut plays a role not only in nutrient absorption but also as a
protective barrier against potentially harmful agents. The gut also hosts
a vast microbiota, consisting of bacteria, viruses, and fungi that interact
to maintain intestinal homeostasis (Adak & Khan, 2019). This micro-
biota plays a key role in supporting the intestinal barrier by influencing
permeability and promoting the integrity of the enteric nervous system
(ENS), which coordinates motility and other digestive functions. Aging
is often associated with disruptions to these digestive functions and
microbiota, including a decrease in microbial diversity (Mariat et al.,
2009), increased intestinal epithelial barrier (IEB) permeability (Di
Vincenzo et al., 2024; Tran & Greenwood-Van Meerveld, 2013), and

disturbances in intestinal motility, such as constipation, likely caused by
alterations in the ENS (Deb et al., 2020; Morley, 2007). Indeed, aging
significantly impacts the ENS, with structural changes including reduced
neuronal density in the myenteric ganglia. Studies have shown
decreased ganglia size and increased intercellular spaces in the colon in
both elderly humans and animal models, leading to progressive
neuronal loss and impaired digestive motility (Nguyen et al., 2023;
Wade, 2002). These changes could contribute to an increased frailty in
older individuals, underscoring the importance of developing strategies
to preserve gut health in aging and prevent the onset of age-related
diseases.

Among the processes and mechanisms implicated in the decline of
gut function during aging, the accumulation of misfolded proteins, or
"proteinopathies," plays a central role (Berner et al., 2023). In

<footnote><sup>a</sup> Nantes Université, CHU Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France</footnote>

<footnote><sup>b</sup> Science et Technique du Lait et de l'Œuf (STLO), Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), L'Institut Agro, Rennes, France</footnote>

<footnote>* This article is part of a Special issue entitled: 'Anti-aging effects of functional foods' published in Journal of Functional Foods.</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: rodrigue.brossaud@univ-nantes.fr (R. Brossaud), valentine.moulle@univ-nantes.fr (V.S. Moullé), marina.giblaine@inrae.fr (M. Giblaine), Sergine.Even@inrae.fr (S. Even), Marie-Bernadette.Maillard@inrae.fr (M.-B. Maillard), severine.chevalier@inrae.fr (S. Chevalier), thibauld.oullier@univ-nantes.fr (T. Oullier), yves.le-loir@inrae.fr (Y. Le Loir), moustapha.cisse@univ-nantes.fr (M. Cissé), helene.falentin@inrae.fr (H. Falentin), michel.neunlist@univ-nantes.fr (M. Neunlist).</footnote>

<footnote n="1">¹ Authors contributed equally to the work</footnote>
particular, amyloid-β (Aβ) peptide, mainly known for its implication in Alzheimer's disease (AD) (Murphy & LeVine, 2010), is recognized for its involvement not only in brain alterations but also in intestinal dysfunction (Jin et al., 2023). Studies have shown the presence of Aβ in the intestines of Alzheimer's patients and AD mouse models is correlated with early digestive disorders such as constipation (Arai et al., 1991; Joachim et al., 1989; Liu et al., 2023; Puig et al., 2012). In AD murine models, Aβ accumulation leads to alterations in the ENS and IEB, disrupting intestinal motility and increasing intestinal permeability, which are typical features of age-related dysfunction and AD (He et al., 2023; Liu et al., 2023). Therefore, identifying candidates able to prevent Aβ-induced alteration of ENS or barrier functions may represent a major interest.

Gut microbiota and its metabolites are well-known key regulators of ENS and IEB functions. Studies in germ-free mice have shown that intestinal permeability, particularly paracellular permeability, is disrupted but can be restored with microbial recolonization (Hayes et al., 2018). This is linked to the direct influence of certain bacteria on the expression of tight junction proteins such as occludin and zonula occludens (ZO-1) (Peng et al., 2009). Additionally, microbiota depletion in rodents results in decreased intestinal motility and contractility, a phenomenon also observed when the microbiota from constipated patients is transferred to mice (Cao et al., 2017; Husebye et al., 2001; Vicentini et al., 2021). Among the microbiota components, gut-microbiota derived metabolites might participate in these alterations of gut homeostasis. Recent findings suggest that short chain fatty acids (SCFAs), indoles or gamma-aminobutyric acid (GABA) can regulate ENS and IEB functions (Duranti et al., 2020; Morrison & Preston, 2016; Zhao et al., 2018). Although modulating the microbiota and its metabolites appears promising for supporting health in aging populations, implementing this approach is challenging. One effective strategy for influencing an individual's microbiota composition is through diet. Notably, fermented foods are recognized for their potential health benefits, primarily due to microbial metabolites produced during fermentation-induced microbial growth and enzymatic conversion of food components (Gänzle et al., 2024; Kunyeit et al., 2024). However, the ability of key bacterial metabolites to prevent Aβ-induced alterations remains unknown.

In this study, we evaluated the effects of microbial metabolites, including those derived from a fermented dairy product enriched in beneficial compounds, to counteract Aβ-induced toxicity on the neuro-glio-epithelial unit in vitro. Among the various tested metabolites, we identified butyrate, GABA, and tryptophan derivatives (indole, indole-3-acetic acid, indole-3-lactic acid, and indole-3-carboxaldehyde) as potentially effective postbiotics in mitigating Aβ-induced impairments of IEB integrity and ENS connectivity. This research underscores the potential of dietary approaches, particularly those involving metabolite-enriched fermented products, in supporting gut and neurological health in the context of aging and AD.

## 2. Methods

### 2.1. Exogenous oligomeric Aβ preparation

Exogenous oligomeric Aβ preparation was performed as previously described (Stine et al., 2010). Briefly, recombinant human lyophilized synthetic Aβ<sub>1–42</sub> peptides (Sigma Aldrich, AG912) were reconstituted in hydroxyfluroisopropanol (HFIP; Sigma Aldrich, 18127), incubated at room temperature for 30 min and aliquoted to allow the HFIP to evaporate overnight. After resuspension of the peptide film in DMSO (Sigma Aldrich, D8418) at 1.1 mM, the preparation was diluted in neurobasal medium DMEM (Thermo Fisher, 21765–025)/F12Ham (Thermo Fisher, 21765–026) v/v with 1 % N2 (Thermo Fisher, 17402048) to 0.22 mM, incubated for 24 h at 4 °C and then stored at –80 °C until use. This preparation contained Aβ oligomers but no fibrils (Stine et al., 2003). Western-Blot analysis was performed to confirm the presence of

oligomers in the preparation.

### 2.2. Synthetic metabolite preparation

All metabolites were purchased from Sigma Aldrich and solubilized in culture medium as described in **Supplemental Data Table 1** (except for lactate, which was purchased in liquid form). Concentrations were determined based on our previous work (Soret et al., 2010) and according to the literature.

### 2.3. Production of dairy fermented fraction providing metabolites of interest

The strain type CLIB183 of *Yarrowia lipolytica* provided by the Biological Resource Center CIRM-Levures (Montpellier, France) was inoculated in 1 L of UHT whole milk (Delisse, France) at 1 % v/v to reach an initial concentration around $10^6$ CFU/mL in 5 L Erlenmeyer. The fermentation was performed in two steps: 48 h at 27 °C with shaking at 130 rpm in an incubator (Multitron, Infors, Germany), followed by a second step with the addition of fluid whole cream (Elle&Vire, France) to reach a fat concentration of 108 g/L, and 48 h incubation at 37 °C, with shaking at 130 rpm for lipolysis activation. At the end of the fermentation, the contents of the Erlenmeyer flasks were transferred into 500 mL Schott flasks and heat-treated at 72 °C for 10 min in a preheated water bath. After the heat treatment, the flasks were placed on ice for 3 h and then pooled, mixed, aliquoted, and frozen at –20 °C for subsequent use.

### 2.4. Enumeration of yeast in the fermented cream

1 mL of fermented cream was diluted in 9 mL of tryptone salt (0.1 % tryptone, AES Laboratoire, Combourg, France; 0.85 % NaCl, Labogros, Buchs, France). Serial decimal dilutions were then realized by micro-method as previously described (Baron et al., 2006). CFU were counted on Dico TM Brain Heart Infusion agar (Becton, Dickinson and Company, Maryland, USA) after incubation under aerobic conditions for 48 h at 30 °C.

### 2.5. Removal of protein from the fermented cream

After thawing the samples at room temperature, 4 g of sample was weighed into a 15mL tube (Falcon) and 4 g of sterile water for metabolite quantification or phosphate buffered saline for culture treatment heated at 43 °C was added. After homogenization by vortexing, the sample was centrifuged for 20 min at 20 °C and at 8500 g. Four grams of the supernatant were transferred in a Vivaspin 20 (Sartorius, VS0102) which was centrifuged for 15 min at 20 °C and at 6000 g. If necessary, centrifugation was repeated to obtain at least 2 mL of filtered sample. The deproteinized flow-through was stored at –80 °C until quantification.

### 2.6. Quantification of carboxylic acids

Fermentation in dairy matrix is characterized by an increase in lactic and acetic acid concentrations. *Y. lipolytica* is also well known to release carboxylic free fatty acids coming from the lipolysis of triglycerides from milk fat (Guerzoni et al., 2001). The deproteinized flow-through was thawed at room temperature and serially diluted by 10 and by 100 in 2 N H2SO4. Between each dilution, the sample was homogenized by inversion. Diluted samples were stored frozen at –20 °C. All expected carboxylic acids were quantified by HPLC separation and UV- or refractometry detection as described in (Harlé et al., 2020).

### 2.7. Quantification of GABA

The deproteinized flow-through was thawed at room temperature.
600 µL of sample was placed in a 1.5 mL microtube and mixed with 600 µL of 5 % sulfosalicylic acid +0.5 mM norleucine for use as an internal standard. After homogenization by vortexing, the sample was placed on ice for at least one hour. Then, the sample was centrifuged for 10 min at 10,000 g at room temperature. The supernatant was collected and filtered through a 0.2 µm filter (Chromafil Xtra PVDF 20/13, Magerey Nagel, Germany). The filtrate was collected in a vial and stored at -20 °C. Quantification of GABA was performed by ion chromatography and photometric detection as described in (Harlé et al., 2024).

## 2.8. Quantification of indolic compounds

Indole-3-lactic acid and indole-3-acetic acid concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). All solvents used were LC-MS grade and purchased from Bio-solve (Valkenswaard, Netherlands). Standard compounds were obtained from Sigma Aldrich (Saint-Quentin Fallavier, France). A pool of reference standard solutions was prepared and serially diluted in ultrapure water to obtain seven standard solutions ranging from 0.1 to 100 µmol/L. Exogenous $ ^{2}H_{3}-$indole-3-acetic acid (50 µL) diluted at 25 µmol/L in ultrapure water was added to 200 µL of standard solutions and biological samples. All samples were then treated with 600 µL ethyl acetate containing 0.5 % acetic acid, vortex-mixed, and centrifuged for 10 min at 10,000 g at 10 °C. Supernatants were then transferred to vials for LC-MS/MS analyses, performed on a Xevo® Absolute mass spectrometer with an electrospray interface and an Acquity Premier® UPLC™ device (Waters Corporation, Milford, MA, USA). Samples (10 µL) were injected onto an HSS T3 Premier column (1.7 µm, 2.1 × 100 mm, Waters Corporation) held at 60 °C. Compounds were separated using a linear gradient of mobile phase B (100 % acetonitrile, 0.1 % formic acid) in mobile phase A (5 % acetonitrile, 0.1 % formic acid) at a flow rate of 500 µL/min. Mobile phase B was kept constant for 0.5 min at 5 %, linearly increased from 5 % to 95 % for 8 min, kept constant for 1.5 min, returned to the initial condition over 0.5 min, and kept constant for 1 min before the next injection. Targeted compounds were then detected by the mass spectrometer with the electrospray interface operating in the positive ion mode (capillary voltage, 3.5 kV; desolvation gas ($N_{2}$) flow and temperature, 650 L/h and 350 °C; source temperature, 150 °C). The multiple reaction monitoring mode was applied for MS/MS detection: indole-3-lactic acid $m/z$ 206.1 → 118.1 (cone/collision: 30/20 V), indole-3-acetic acid $m/z$ 176.1 → 130.1 (cone/collision: 15/12 V), and $ ^{2}H_{3}-$indole-3-acetic acid $m/z$ 179.0 → 133.1 (cone/collision: 15/20 V). Chromatographic peak area ratios between unlabeled compounds and $ ^{2}H_{3}-$indole-3-acetic acid constituted the detector responses. Standard solutions were used to plot calibration curves for quantification (linear regression, 1/x weighting, origin excluded). Data acquisition and processing were achieved using MassLynx® and TargetLynx® software, respectively (version 4.0, Water Corporation).

## 2.9. Primary mixed culture of ENS

Primary culture of rat ENS was performed as previously described (Chevalier et al., 2008). Briefly, embryonic day 15 (E15) rat intestine was removed and finely diced in Hank's buffered salt solution and triturated mechanically using a scalpel. Tissue fragments were collected in DMEM/F12 v/v medium containing 50 µg/mL streptomycin and 50 U/mL penicillin and incubated for 15 min at 37 °C in the same medium containing 0.25 % trypsin (Sigma Aldrich, T1426). Trypsin was inactivated by addition of complete media containing 10 % FBS. Samples were incubated for 10 min at 37 °C with 0.1 % DNase I (Sigma Aldrich, 10104159001). After trituration and centrifugation for 10 min at 600 g, cells were plated in DMEM/F12 containing antibiotics and 10 % FBS at a density of 2.4 × 10⁵ cells/cm² on 24-well plastic culture plates (Sigma Aldrich, 3524) previously coated with 0.5 % gelatin (Sigma Aldrich, G5660) for 24 h. The next day, medium was replaced with fresh DMEM/F12Ham v/v medium without FBS but supplemented with 1 % N-2. Half

of the medium was replaced every 3 days, and primary cultures were maintained for 14 days.

## 2.10. Intestinal epithelial cell culture

The human IEC line Caco-2 was obtained from American Type Culture Collection (Manassas, VA) and cultured in DMEM containing 4.5 g/L glucose (Thermo Fisher, 11965092) supplemented with 10 % heat-inactivated FBS, 2 mM glutamine (Sigma Aldrich, 1294808), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Sigma Aldrich, P4333). For treatments and permeability experiments, Caco-2 were seeded in 12-well Transwell® filters (Sarstedt, 83–3931-041) at a density of 200,000 cells/insert and grown for 6 days. All cells were maintained in 95 % air, 5 % CO2 at 37 °C.

## 2.11. Experimental treatments

Both primary mixed ENS and IEC cultures were treated with metabolites (at concentrations mentioned in **Supplemental Data Table 1**), a metabolite cocktail containing acetate, butyrate, GABA, and tryptophane derivatives (at concentrations found in the deproteinized flow-through from the fermented cream), or a deproteinized flow-through from the fermented cream for 12 h before exposition to exogenous synthetic Aß oligomers preparation for 24 h. The metabolite cocktail and the deproteinized flow-through were diluted 10-fold before treatment of cell cultures. Based on unpublished dose-response experiments to evaluate Aß toxicity in vitro, primary mixed ENS cultures were treated with 2 µM of Aß oligomers directly added to the cell culture media, while Aß oligomers were added into the apical and basolateral compartments of IEC cultures at 10 µM. Cultures were then fixed for immunocytochemistry in 4 % paraformaldehyde (PFA) or lysed in adapted lysis buffer for biochemical analysis.

## 2.12. Immunocytochemistry

After treatments, primary mixed ENS cultures were fixed in PBS containing 4 % PFA for 15 min. Cells were permeabilized for 5 min at room temperature in 0.25 % 100× Triton in PBS, washed twice with PBS, and saturated for 30 min at 37 °C in PBS containing 10 % BSA. ENS cultures were incubated overnight at 4 °C with primary antibodies (**Supplemental Data Table 2**) diluted in PBS containing 3 % BSA. After washing, ENS cultures were incubated for 90 min at room temperature with appropriate FITC-conjugated, Cyanine 3-conjugated or Cyanine 5-conjugated secondary antibodies diluted in PBS containing 3 % BSA. Primary mixed ENS cultures were then washed and mounted with ProLong Gold Antifade Reagent (Thermo Fisher, P36930). Imaging of ENS cultures was performed using the confocal microscope Nikon A1 RSi (Nikon Instrument) with 20× or 60× dedicated oil immersion objectives (Nikon Instrument) using a digital camera driven by Nikon imaging software NIS-Elements. Then, analysis and quantification were performed using ImageJ software (ImageJ, version 2.1.0). To analyze immunostaining signals, ROI were drawn as delineated by the presence of the signal specific to the region and the integrated density was measured. Integrated densities were calculated per unit area and normalized to the control condition.

## 2.13. Western-blot

Cell cultures were lysed in RIPA lysis buffer (Sigma Aldrich, 20–188) diluted in PBS with a protease (Sigma Aldrich, S8820) and phosphatase inhibitor cocktail (Sigma Aldrich, P0044). Samples were sonicated (Diagenode, Bioruptor Plus), centrifuged at 10,000 g for 1 min and the protein concentration of the supernatant was determined using a Bradford assay (Thermo Fisher, 23236). Sample Reducing Agent (Thermo Fisher, NP0009) and NuPAGE Sample Buffer (Thermo Fisher, NP0008) were added to samples before denaturation at 95 °C for 5 min. For
Western-Blotting, 15 µg of protein from fresh or previously frozen lysates were separated using NuPAGE 4–12 % Bis-Tris gels (Thermo Fisher, WBT4122) with MES SDS NuPAGE running buffer (Thermo Fisher, NP0002) before electrophoretic transfer to nitrocellulose membranes (25V, 7 min) (Thermo Fisher, IB23001) with the iBlot2™ Dry Blotting System (Life Technologies). Membranes were then blocked for 2 h at room temperature in TBS with 0.1 % Tween-20 (TBS-T) (Sigma Aldrich, P1379) and 5 % non-fat dry milk and incubated with the primary antibodies (Supplemental Data Table 3) in TBS-T at 4 °C overnight. Membranes were washed three times in TBS-T and incubated with HorseRadish Peroxidase (HRP)-conjugated secondary antibodies in Tris-buffered saline (TBS) for 2 h at room temperature on an orbital shaker. Membranes were rinsed a further three times in TBS. A Bio-Rad ChemiDoc MP was used to detect HRP-conjugated secondary antibodies via Western Pierce ECL (Life Technologies, 32209), Supersignal™ West Pico (Life Technologies, 34580) or Supersignal™ West Femto (Life Technologies, 34096) substrate. Densitometry analysis was performed using Image-Lab Software (Bio-Rad, version 6.0.1).

## 2.14. Quantitative real-time polymerase chain reaction

Cell cultures were lysed in RA1 lysis buffer and RNA extraction was performed with NucleoSpin Triprep Kit (Macherey-Nagel, 740966) following the manufacturer's instructions. Potential genomic DNA contamination was removed by treatment with Turbo DNAse (Thermo Fisher, AM1907). Total RNA was quantified using an ND-1000 UV–Vis spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) and RNA quality was assessed by evaluating the 230/260 and the 260/280 ratio during quantification. cDNA was synthesized from 1 µg of purified and denatured mRNA using the Superscript III reverse transcriptase (Thermo Fisher, 19080093) and diluted to a final concentration of 8 ng eq RNA/µL. Quantitative Polymerase Chain Reaction (PCR) was performed using StepOne Plus (Thermo Fisher) detection system with Fast SYBR Green master mix (Thermo Fisher, 4385612) or Taqman® gene expression master mix (Thermo Fisher, 4369016). The PCR signal was normalized against RPS6 as reference gene to control for variability in the amount and quality of the RNA. The sense and antisense oligonucleotide primers used in this study are shown in supporting information (Supplemental Data Table 4).

## 2.15. Permeability measurement in Caco-2 culture

To determine the effect of these treatments on permeability, 50 µL of 1 mg/mL of sulfonic acid FITC (Thermo Fisher, F1130), 10 mg/mL of dextran 4 kDa TRITC (TdB Labs), and 3.75 mg/mL of HRP (Sigma Aldrich, P8250) were added to the apical compartment. The fluorescence level of basolateral aliquots (150 µL) was measured every 30 min for a period of 180 min using a fluorimeter (BioTek Synergy H1, microplate reader). Paracellular permeability was determined by averaging the slope change in fluorescence intensity over time by linear regression fit model. To assess transcellular permeability to HRP, an enzymatic activity assay with TMB (BD Biosciences, 555214) was performed.

## 2.16. Expression of data and statistical analysis

Data are expressed as mean ± SEM and individual values. The means of two groups were compared using unpaired Student's t-tests or Mann-Whitney test. Normally distributed continuous variables were compared using one-way ANOVA with Turkey's multiple comparisons post hoc test or Two-way ANOVA with Fisher's LSD post hoc test. Non-normally distributed data were analyzed using nonparametric Kruskal–Wallis's test followed by Dunn's multiple comparisons test. The statistical software package used was Prism 10 (GraphPad Prism 10, software). Differences between experimental groups were considered significant at \*p ≤ 0.05, \*\*p ≤ 0.01, \*\*\*p ≤ 0.001, or \*\*\*\*p ≤ 0.0001.

# 3. Results

## 3.1. Aß induces alterations in ENS connectivity and impairs intestinal permeability

Firstly, we aimed to determine the effects of primary ENS culture and Caco-2 cell monolayers exposure to Aß. Immunostaining of ENS culture revealed that 2 µM Aß treatment did not induce neuronal death, as the number of Hu + neurons remained unchanged between conditions (Fig. 1A & 1B). However, quantification of the neuronal marker TuJ1 showed a reduction in relative fluorescence intensity in Aß-treated ENS cultures, suggesting a disruption in enteric neuronal connectivity (Fig. 1A & 1C). This finding was further supported by a decrease in synaptophysin (SYN) protein expression in Aß-exposed ENS cultures, suggesting a compromised structural integrity of the neuronal network and a synaptic loss (Fig. 1D & E). However, actin was not affected by Aß treatment.

In parallel, both paracellular and transcellular permeability (respectively measured by sulfonic acid flux and HRP transport across the Caco-2 monolayer) were significantly increased following a 24 h treatment of Caco-2 monolayers with 10 µM Aß. No alteration in permeability was observed at lower Aß concentrations (Fig. 1F & G).

Altogether, our data showed that Aß exposure alters neuronal and epithelial functions by impairing neuronal connectivity and epithelial monolayer permeability.

## 3.2. Identification of microbial metabolites with beneficial effects against Aß-induced alterations in ENS connectivity

Since Aß alters key ENS processes involved in the neuronal regulation of gut functions, we investigated whether microbial metabolites (SCFAs, GABA, polyamines and tryptophane derivatives) could prevent Aß-induced alterations to neuronal connectivity in ENS cultures (Fig. 2A). Among the SCFAs tested, acetate and propionate pretreatments did not prevent Aß-induced decrease in the synaptic protein SYN (Fig. 2B & 2C) whereas butyrate fully prevented this depletion (Fig. 2D). In addition, neither lactate nor polyamines such as spermine or spermidine prevented Aß-induced reduction in SYN expression (Fig. 2E-G). Finally, pretreatment with GABA, a monoamine, or a combination of tryptophane derivatives (e.g. indole, indole-3-acetic acid, indole-3-lactic acid, indole-3-carboxaldehyde) prevented SYN decrease in Aß-treated ENS cultures (Fig. 2H & 2I).

To further confirm the beneficial effects of butyrate, GABA, and the tryptophane derivatives against Aß-induced alterations of enteric neuronal connectivity, we conducted immunostaining of TuJ1 (Fig. 2J). Quantification of fluorescence intensity revealed a significant decrease of TuJ1 in ENS cultures exposed to Aß (+Aß) (Fig. 2K-M). However, pretreatment of ENS cultures with each of the aforementioned metabolites prevented Aß-induced TuJ1 decrease. Therefore, pretreatment of ENS cultures with butyrate, GABA, or tryptophane derivatives before exposure to Aß appeared to significantly mitigate alterations of enteric neuronal connectivity in vitro.

## 3.3. Development of a fermented dairy product enriched with targeted bacterial metabolites

Next, we aimed to develop a functional food product enriched with previously identified beneficial metabolites. In particular, as this product is targeted at an older demographic, we developed a method to enrich a dairy product with our targeted microbial metabolites through a two-step fermentation process using *Y. lipolytica*, a yeast species of food interest, previously identified as being able to produce carboxylic acids and GABA (Wu et al., 2024). After the second step of lipolysis, cream fermented with the strain CLIB183 reached a concentration of 5.4 × 10⁸ CFU/mL and contained beneficial metabolites such as butyrate (12 mM) and GABA (1.6 mM) as well as indole-3-lactic acid and indole-
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Effect of Aβ treatment on ENS connectivity and IEB permeability. (A) Immunostaining of Hu and TuJ1 proteins in primary ENS cultures treated with 2 µM Aβ. (B) Number of Hu+ neurons per field in primary ENS cultures treated with 2 µM Aβ. Mann Whitney test (n=13 wells per condition). (C) Quantification of TuJ1 fluorescence intensity in primary ENS cultures treated with 2 µM Aβ. Mann Whitney test (n=13 wells per condition). (D) Representative immunoblots of indicated proteins in primary ENS cultures (E) Relative Actin and SYN protein levels in primary ENS cultures treated with 2 µM Aβ (% of controls). Mann Whitney test (n=11-13 wells per condition). (F,G) Paracellular and transcellular permeability in Caco-2 IEC culture model exposed to different concentrations of Aβ (0.1 µM, 1 µM and 10 µM). Kruskal-Wallis with Dunn's post hoc test (n=4 wells per condition). Statistical analysis: \*\*\*p≤0.001 and \*\*\*\*p≤0.0001.</figcaption></figure>

3-acetic acid at the respective concentrations of 1.8 and 0.4 µM. It should be noted that the fermented cream likely contained additional untargeted molecules derived from the dairy matrix or produced during the microbial fermentation, including lactate (1 mM) or acetate (24 mM).

### 3.4. Combined protective effects of a cocktail of microbial metabolites against Aβ-induced alterations in ENS connectivity and intestinal permeability

To assess the potential synergistic effects of the microbial metabolites, present in the fermented dairy product against Aβ-induced toxicity, we used a cocktail of selected metabolites at concentrations matching those in the dairy product, excluding other dairy components (e.g. lipids, proteins) that could have confounding effects. Interestingly, we showed that pretreatment of ENS cultures with this metabolite cocktail prevented Aβ-induced decrease in SYN protein expression (Fig. 3A & 3B). However, this cocktail did not mitigate Aβ-induced alterations of TuJ1 expression in the ENS (Fig. 3C & 3D).

Furthermore, we showed that the metabolite cocktail partially prevented Aβ-induced increase in paracellular permeability (Fig. 3E & 3F) without affecting transcellular permeability (Fig. 3G). Pretreatment of Caco-2 monolayers with the metabolite cocktail prior to Aβ exposure resulted in an increasing trend of caspase-3 mRNA expression compared with the Aβ-only condition (Fig. 3H). The metabolite cocktail and Aβ, alone or in combination, failed to significantly change ZO-1 and

occludin gene and protein expression, two tight junction proteins essential for maintaining IEB integrity (Fig. 3I-M).

### 3.5. A fermented dairy product enriched with microbial metabolites counteracts Aβ-induced ENS and IEB damage

Next, we evaluated the potential protective effects of the fermented dairy product enriched with beneficial bacterial metabolites against Aβ-induced disruptions of ENS connectivity and IEB integrity. Interestingly, pretreating ENS cultures with the dairy product did not counteract Aβ-induced alterations of SYN expression (Fig. 4A & 4B). However, it prevented Aβ-induced decrease in TuJ1 expression (Fig. 4C & 4D).

Then, we aimed to determine whether these protective effects of the dairy product towards the ENS extended to IEC functions upon Aβ-induced toxicity. We showed that, pretreatment of Caco-2 monolayers with the fermented dairy product for 12 h prior to Aβ exposure significantly reduced both paracellular and transcellular permeability increases induced by Aβ (Fig. 4E & 4F). In addition, caspase-3 mRNA expression, tended to be reduced following treatment with the dairy product alone. However, Aβ did not significantly alter caspase-3 mRNA levels in Caco-2 monolayers (Fig. 4G). Interestingly, while Aβ exposure tended to raise mRNA levels of ZO-1 and occludin, the dairy product alone significantly decreased occludin protein levels (Fig. 4H-L).

Altogether our findings indicate that pretreatment with a fermented dairy product, enriched with key microbial metabolites identified in our in vitro screening, offers protective effects against Aβ-induced neuronal
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Identification of microbial metabolites with protective effects against Aβ-induced alterations in enteric neuronal connectivity. (A) Experimental design diagram. (B-I) Representative immunoblots of indicated proteins and actin and SYN relative protein levels in primary ENS cultures pretreated with individual microbial metabolites (1 mM acetate, propionate, butyrate, lactate, spermine, spermidine; 50 µM GABA; 100 µM indole cocktail (i.e. indole and derivatives of indole) prior to 2 µM Aβ treatment. Kruskal-Wallis with Dunn's post hoc test (n = 9-18 wells per condition). (J) Immunostaining of Hu and TuJ1 proteins in primary ENS cultures pretreated with butyrate, GABA, or indole cocktail prior to 2 µM Aβ treatment. (K-M) Quantification of TuJ1 fluorescence intensity in primary ENS cultures pretreated with butyrate, GABA, or indole cocktail before 2 µM Aβ treatment. Kruskal-Wallis with Dunn's post hoc test (n = 15-18 wells per condition). Statistical analysis: \*p ≤ 0.05, \*\*p ≤ 0.01, \*\*\*p ≤ 0.001, and \*\*\*\*p ≤ 0.0001.</figcaption></figure>

and epithelial dysfunctions.

## 4. Discussion

Proteinopathies may play a role in age-related gastrointestinal dysfunctions and contribute to increased frailty in the elderly. Consequently, developing nutrition-based approaches to prevent or slow gastrointestinal dysfunctions related to aging could promote healthy aging. In this context, our study first demonstrated that Aβ impairs neuronal connectivity in the ENS and disrupts the IEB integrity, affecting two essential cellular components involved in gut homeostasis. We identified butyrate, GABA, and a mix of tryptophan derivatives as protective, either individually or in combination, against Aβ-induced toxicity in ENS cultures. Furthermore, a fermented dairy product enriched with these beneficial metabolites protects against Aβ-induced alteration of ENS connectivity and IEB, indicating significant protective

effects.

First, this study provides novel insights into the pathophysiological mechanisms of Aβ on the IEB and ENS connectivity. Previous research has shown that Aβ induces changes in inflammation-related genes and alters synaptic plasticity in the ENS (Lim et al., 2014; Liu et al., 2023). In line with these findings, we demonstrate that Aβ disrupts ENS connectivity by decreasing levels of SYN and TuJ1, in vitro. In addition, to the best of our knowledge, this study is the first to show that Aβ alters IEB integrity by increasing both paracellular and transcellular permeability in IEC Caco-2 cell cultures. Indeed, previous studies have shown intestinal permeability alterations with IEB damage in transgenic mouse models of AD without providing a mechanistic insight (He et al., 2023; Kim et al., 2020). Our findings also identify these permeability changes as promoted by an Aβ-dependent alteration of tight junction proteins ZO-1 and occludin. Despite the fact that gene and protein expressions of ZO-1 and occludin were not correlated with Aβ-dependent increase of
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Combined protective effects of a cocktail of microbial metabolites against Aβ-induced alterations in ENS connectivity and intestinal permeability.

(A) Representative immunoblots of indicated proteins. (B) Actin and SYN relative protein levels in primary ENS cultures pretreated with the metabolite (MT) cocktail prior to 2 µM Aβ exposure. Kruskal-Wallis with Dunn's post hoc test (n = 8 wells per condition). (C) Immunostaining of Hu and TuJ1 proteins in primary ENS cultures pretreated with the metabolite cocktail prior to 2 µM Aβ exposure. (D) Quantification of TuJ1 fluorescence intensity in primary ENS cultures pretreated with the metabolite cocktail before 2 µM Aβ exposure. Kruskal-Wallis with Dunn's post hoc test (n = 8 wells per condition). (E) Experimental design diagram. (F, G) Paracellular and transcellular permeability in IEC culture model Caco-2 pretreated with the metabolite cocktail prior to 10 µM Aβ exposure. Two-way ANOVA with Fisher's LSD post hoc test (n = 6–8 wells per condition). (H-M) Gene expression, representative immunoblots of indicated proteins, and protein expression levels of caspase 3, ZO-1, and Occludin analyzed by RT-qPCR and Western blot in IEC culture model Caco-2 pretreated with the metabolite cocktail prior to 10 µM Aβ exposure. Two-way ANOVA with Fisher's LSD post hoc test (n = 5–8 wells per condition). Statistical analysis: \*p ≤ 0.05, \*\*p ≤ 0.01, \*\*\*p ≤ 0.001, and \*\*\*\*p ≤ 0.0001.</figcaption></figure>

paracellular permeability, previous studies have established that the colocalization of these proteins in tight junctions is essential to maintain a barrier function (J. M. Anderson & Van Itallie, 1995; Fallon et al., 1993). Therefore, it will be important to also explore the cellular localization of these proteins in future studies. These findings add a complexity to Aβ pathology, which has been mostly studied in the context of the central nervous system in AD (Murphy & LeVine, 2010), but scarcely investigated in physiopathological condition in the gut, despite the fact that Aβ is constitutively present in both the gut and the brain.

Second, we demonstrate that specific microbial metabolites, either individually or in combination, exhibit protective effects against Aβ-induced alterations of ENS and IEB functions. Although the protective mechanism involved remains unknown, we hypothesize that the aryl hydrocarbon receptor (AhR) pathway may play a key role. Indeed, this pathway is known to be activated by butyrate and tryptophan derivatives, including indole compounds (Marinelli et al., 2019; Sun et al., 2020; Zhuang et al., 2023). Further supporting this hypothesis, previous studies have implicated the AhR pathway in AD by showing that its activation can reduce Aβ levels and improve cognitive deficits in AD

mouse models (Liu et al., 2023). However, AhR activation by candidate metabolites identified in our experimental paradigm remains to be shown in future studies.

Lastly, we demonstrate that the fermented dairy product enriched with beneficial metabolites fully reproduces the protective effects of the metabolite cocktail. Interestingly, its efficacy is enhanced compared to the cocktail alone, suggesting that other components in the dairy matrix, particularly lipids, may exert additional effects. Indeed, the fermented dairy product and metabolite combination tested in our culture models differ by two main components: lipids produced by the strain type CLIB183 of *Y. lipolytica* and other metabolites that were not measurable or not targeted but most likely present in the dairy product. For instance, other non-targeted molecules can be found in the deproteinized flow-through used in our experiments such as milk molecules (including lactose, oligosaccharides, citrate, sterols, vitamins A and B12) and metabolites resulting from the fermentation process (including amino acids, biogenic amines, small volatile compounds such as diacetyl, acetoin and, more generally, aldehydes, ketones, alcohol and esters) (McSweeney, 2004). These metabolites that we did not measure could also contribute to beneficial effects and explain the difference observed
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Protective effect of a fermented dairy product enriched with microbial metabolites on Aβ-induced ENS and IEB damage. (A) Representative immunoblots of indicated proteins. (B) Actin and SYN relative protein levels in primary ENS cultures pretreated with the dairy product prior to 2 µM Aβ exposure. Kruskal-Wallis with Dunn's post hoc test (n = 13 wells per condition) (C) Immunostaining of Hu and TuJ1 proteins in primary ENS cultures pretreated with the dairy product before 2 µM Aβ exposure. (D) Quantification of TuJ1 fluorescence intensity in primary ENS cultures pretreated the dairy product prior to 2 µM Aβ exposure. Kruskal-Wallis with Dunn's post hoc test (n = 13 wells per condition). (E, F) Paracellular and transcellular permeability in IEC culture model Caco-2 pretreated with the dairy product prior to 10 µM Aβ exposure. Two-way ANOVA with Fisher's LSD post hoc test (n = 9–11 wells per condition). (G-L) Gene expression, representative immunoblots of indicated proteins, and protein expression levels of caspase 3, ZO-1, and Occludin analyze by RT-qPCR and Western blot in IEC culture model Caco-2 pretreated with the dairy product prior to 10 µM Aβ exposure. Two-way ANOVA with Fisher's LSD post hoc test (n = 7–11 wells per condition). Statistical analysis: \*\*p ≤ 0.01, \*\*\*p ≤ 0.001, and \*\*\*\*p ≤ 0.0001.</figcaption></figure>

between the fermented dairy product and metabolite cocktail. For example, the food matrix components, including dairy fat, have been shown to enhance the protective effect of probiotics such as *P. freudenreichii* (Mantel et al., 2023). Moreover, dairy lipids per se can improve epithelial barrier integrity by maintaining ZO-1 and Occludin colocalization in Caco2 cells during inflammation challenge (R. C. Anderson et al., 2018). Thus, it is possible that, besides the probiotic, the metabolome in combination with the dairy fat can synergistically engage multiple pathways to improve neuronal connectivity and barrier integrity, highlighting the complexity of this area of expertise. Notably, our results reveal that the expression of tight junction protein Occludin is modulated by the fermented product. This effect has previously been reported for butyrate and tryptophane derivatives (Li et al., 2021; Xiao et al., 2022) and a previous study has shown that treatment with GABA decreases the expression of junctional adhesion molecule and Occludin in vitro in Caco-2 cells (Ma et al., 2018), suggesting that GABA might downregulate Occludin in our experimental paradigm. Thus, the dairy product may be potentially used therapeutically to strengthen the IEB

and ENS connectivity.

The development of this fermented dairy product enriched with key microbial metabolites is particularly relevant to the functional food sector. Compliant with rigorous health standards, this product could potentially be marketed in the medium-term. Its formulation is designed to meet the specific needs of seniors, especially in the context of aging-related pathologies such as reduced chewing ability and gastrointestinal disorders. This product could contribute to maintaining IEB integrity and supporting ENS connectivity, offering a promising nutritional approach to prevent or mitigate symptoms associated with intestinal and neuronal alterations in elderly populations.

A current limitation of our study is that it was exclusively conducted in vitro, which limits the extrapolation of the results in vivo. Thus, in vivo studies involving pre-clinical models are to be considered in the future to validate the efficacy of this product in a more complex biological context, considering the digestive processes that occur in the body which may modulate the product metabolism and biodisponibility. However, it is important to note that despite these limitations, in vitro
experiments can, to some extent, emulate in vivo conditions, as demonstrated by our previous study showing that butyrate administered to adult rats significantly increases the proportion of choline acetyl transferase-immunoreactive myenteric neurons, similarly to results obtained in primary ENS embryonic cultures (Soret et al., 2010).

In conclusion, this study highlights the potential of a fermented dairy product enriched with indole derivatives and SCFAs to mitigate Aβ-induced alterations of ENS connectivity and IEB integrity. Our findings demonstrate that integrating specific metabolites into functional products presents significant benefits for digestive and neurological health in seniors, in the context of aging and an increased risk of neurodegenerative diseases. Although studies conducted in pre-clinical models are needed to further validate our in vitro findings, this work paves the way for future research and applications in functional foods targeting gut and intestinal health.

### CRediT authorship contribution statement

**Rodrigue Brossaud:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization.
**Valentine S. Moullé:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization.
**Marina Giblaine:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization.
**Sergine Even:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization.
**Marie-Bernadette Maillard:** Investigation, **Séverine Chevalier:** Investigation, **Thibauld Oullier:** Investigation, **Eric Guédon:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization.
**Yves Le Loir:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization.
**Moustapha Cissé:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization.
**Hélène Falentin:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization.
**Michel Neunlist:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization.

### Ethics statement

None.

### Funding

This work was supported by the Regional Councils of Pays de la Loire (Grant No. 2019-013227 to MN) and Bretagne (Grant No. 19008213 to YLL) and by the dairy industry association Bba Milk Valley for their financial support through the interregional project PROLIFIC and by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Nantes Université, and the Institut National de Recherche pour l'Agri-culture, l'Alimentation et l'Environnement (INRAE).

### Declaration of competing interest

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

### Acknowledgement

We acknowledge the IBISA MicroPICell facility (Biogenouest), member of the national infrastructure France-Bioimaging supported by the French national research agency (ANR-10-INBS-04). We thank the CIRM-Levures (Doi: 10.15454/H4AT-ZC40) for kindly providing the yeast strain. We are very grateful to Mickaël Croyal for his advice and training on the quantification of indolic compounds and we thank the

Mass spectrometry core facility of UMS Biocore (M-shark, Nantes) for their help with data collection. We thank Jenny Greig for English editing. Finally, we thank Katy Fontaine and Karelle Smadja (TENS laboratory) for their involvement in budget management and administration.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jff.2025.107095.

### Data availability

Data will be made available on request.

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