The short-chain fatty acid butyrate prevents gut-brain amyloid-β pathology and neuroiNnflammation in an Alzheimer mouse model
Molecular Psychiatry (2026)
Abstract
Amyloid-β (Aβ) plays a critical role in Alzheimer’s disease (AD) and its accumulation in the brain is pivotal to disease progression and precedes memory and neuronal loss. Besides the severely handicapping brain symptoms, AD patients display early gastro-intestinal (GI) manifestations such as upper and lower GI dysmotility, in particular constipation. Although there is increasing evidence of Aβ accumulation in the gut, its pathogenic effects on enteric nervous system (ENS) connectivity and gut function as well as underlying pathophysiological mechanisms are poorly understood. Furthermore, studies have reported a gut to brain transmission of Aβ that causes memory deficits in mice. Therefore, identifying therapeutics which can reduce Aβ accumulation in the gut at an early stage of the disease could have the advantage of slowing or even reversing disease progression before severe alterations or irreversible damages at both intestinal and brain levels. Hence, in this study, we investigated the capacity of the short-fatty acid butyrate to restore Aβ-driven alteration of ENS connectivity and gut-brain functions in the SAMP8 mouse model of AD. Here we show that SAMP8 mice display a gut amyloid pathology, an alteration of ENS connectivity and gut defects prior to memory decline. BACE1, an Aβ-producing enzyme, expression and activity are increased whereas neprilysin, an Aβ-degrading enzyme, is decreased in the gut of SAMP8 mice, indicating a rise in the Amyloid Precursor Protein (APP) holoprotein processing and a reduction of Aβ clearance which promote an amyloidosis. In primary ENS cultures, Aβ causes a degradation of synaptic-associated proteins EphB2 and synaptophysin, leading to an alteration of ENS connectivity. In wild-type mice, intra-colon delivery of Aβ alters ENS connectivity and causes subsequent GI symptoms, recapitulating the phenotype of the SAMP8 mouse model of aging and AD. Moreover, Aβ impairs ENS connectivity in human induced pluripotent stem cell (iPSC)-derived intestinal organoids and explant cultures of human colon, indicating that Aβ causes ENS lesions in models of the human gut. Butyrate, a short-chain fatty acid derived from bacterial metabolism, reduces Aβ secretion and preserves enteric neuronal connectivity in vitro and in vivo, and blocks Aβ accumulation in the gut, brain and plasma in SAMP8 mice. In addition, butyrate ameliorates neuroinflammation and prevents gut dysfunction and memory deficit. Collectively, these findings suggest that Aβ promotes gut symptoms through alteration of ENS connectivity and butyrate counteracts these impairments with an amelioration of neuroinflammation and memory function in AD model.
Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disease mostly associated with cognitive decline and has no effective treatment to date. A major histological hallmark of AD pathogenesis is the formation of amyloid plaques caused by deposition of the amyloid-β (Aβ) peptide, which is produced by the proteolytic cleavage by β- and γ-secretase activities of amyloid precursor protein (APP) holoprotein that localizes to the plasma membrane [1]. Besides cognitive deficits, Alzheimer’s patients often suffer from other comorbidities [2, 3], including gastro-intestinal (GI) symptoms, such as upper and lower bowel dysmotility, in particular constipation, which have recently drawn attention due to early manifestations, sometimes decades before memory loss [4]. Interestingly, recent studies indicate that patients with constipation are at greater risk of developing AD [5] and are more likely to display a faster progression of brain symptoms [6]. Similarly to Parkinson’s disease [7], these findings support a change in the long-held dogma that AD is a brain centered disease and an evolution towards a more complex notion of a multi-organ disease, in which peripheric organs, in particular the gut, play a pivotal role [8]. Therefore, better understanding AD-associated pathophysiological mechanisms and identifying therapeutic strategies to restore gut functions and memory in AD, although fraught with challenges, would constitute a significant step forward in AD research. Thus, the gut represents not only an organ to better understand AD pathophysiology, but also a therapeutic target to ameliorate GI symptoms.
The enteric nervous system (ENS), a critical regulator of gut functions [9], is likely involved in GI symptoms in AD [10]. Previous studies indicate that Aβ accumulates in the gut of AD mice and patients with AD [11, 12]. Because Aβ affects brain plasticity to cause memory deficits [13], it might also alter gut connectivity to cause GI symptoms. In the brain, Aβ interacts functionally and structurally with several distinct types of plasma membrane–anchored receptors, including EphB2 receptor tyrosine kinase, a master regulator of neuronal plasticity [14, 15]. We have recently shown that EphB2 receptor is an important regulator of ENS connectivity [16]. Whether Aβ harms enteric neurons in a similar fashion to neurons in the brain by impairing synaptic proteins such as EphB2 remains to be established. Furthermore, whether alterations of ENS connectivity and GI dysfunction can be prevented through pharmacological intervention in AD context is unknown.
The short chain fatty acid (SCFA) butyrate is a natural product of bacterial fermentation of dietary fiber in the colon [17], a major source of energy for colonocytes. It also exerts beneficial effects via the maintenance of intestinal epithelium barrier integrity, gut motility and anti-inflammatory effects. In addition, butyrate has also the capacity to regulate central nervous system (CNS) functions such as the blood brain barrier and has been shown to protect against Aβ toxicity to CNS neurons both in vitro [18] and in vivo [19, 20]. However, butyrate capacity to counteract amyloid pathology in the gut and in particular its toxicity in the ENS remains unexplored in AD.
Here, we performed a longitudinal characterization of gut and brain functions in the SAMP8 model of aging and sporadic AD. We explored gut amyloidogenesis in early stages, prior to memory deficit. We examined ENS connectivity and measured APP amyloidogenic processing, Aβ levels and clearance. Furthermore, we investigated the causal role of Aβ in ENS lesions and GI dysfunction in different in vitro and in vivo models, including ENS cultures, iPSC-derived intestinal organoids, human colon explants and mice by using electrophysiology, imaging, biochemistry, gut function exploration and behavior. Finally, we explored butyrate capacity to block Aβ-driven ENS lesions and prevent gut and brain dysfunctions in SAMP8 mice. Our results show that butyrate alleviates amyloid pathology, neuroinflammation and prevents gut dysmotility and memory loss in AD model.
Materials and methods
Ethics statement
All experimental procedures involving animals were approved by the Ethics Committee of Nantes University and Inserm for Animal Use in Research, and all methods were performed in accordance with the relevant guidelines and regulations.
Animals
SAMP8 mice and control littermates were bred in-house and were previously described [21]. Animals were housed on a reverse light–dark cycle. Care and experimental manipulation of animals were in accordance with French standard ethical guidelines for laboratory animals. For each time course experiment, two cohorts of animals were used, with littermates randomized to the appropriate groups before manipulation. Mice were randomly assigned to experimental groups and all the experiments were performed double-blinded. Animals were used according to “3Rs” principles (Replacement, Reduction and Refinement) in all experimental procedures. Mice were euthanized at the indicated time points to collect tissue for analyses and comparison. Data collection and analyses were performed blinded to the conditions of the experiments and subsequently reported to genotype and/or treatment. Tg2576 mice [22] tissues were provided by Université Toulouse III – Paul Sabatier Centre de Biologie Intégrative Centre de Recherches sur la Cognition Animale – CNRS UMR 5169. Sample sizes were determined based on previous animal studies to ensure adequate power to detect significant differences (p < 0.05).
Oral butyrate supplementation
In a previous study, 8 g/liter of butyrate was administered via drinking water for 8 weeks to modulate neuroinflammation in mice [23]. Here, we used half of that concentration because butyrate was chronically administered to mice in drinking water for a longer period of time. Briefly, 4 groups of mice were set-up at weaning (3-week-old, n = 10–15 mice per group). Sodium butyrate was given for 5 months in drinking water (ad libidum) at a concentration of 4.4 g/liter (control groups received water only). After 5 months of continuous butyrate supplementation, mice were tested in vivo for gut functions, then subjected to behavior tests. After euthanasia, blood and tissues from colon and hippocampus were collected for ex vivo analysis. Body weight was recorded 3 days a week.
Tissue dissection and immunohistochemistry procedures
Gut histology—Segments of mouse proximal colon were fixed in 1X PBS containing 4% paraformaldehyde for 3 h at room temperature or at 4 °C overnight. Whole mounts of longitudinal muscle and myenteric plexus were obtained by microdissection and were first permeabilized with PBS-NaN3 1% sodium azide containing 4% horse serum and 0.5% Triton X-100. Tissues were then incubated with primary antibodies (Supplementary Table 1) overnight. After several washes in PBS, tissues were incubated for 1 h at room temperature with the appropriate FITC-conjugated or Alexa 568-conjugated secondary antibodies diluted in PBS-NaN3 4% horse serum 0.5%Triton X100. Tissues were washed with PBS and mounted with ProLong Gold Antifade Reagents with DAPI (Molecular Probes, Carlsbad, CA, USA).
Brain histology—Brain left hemisphere were extracted from mice and post-fixed in 4% PFA in PBS for 16–20 h at 4 °C then transferred to a 30% sucrose solution in 1X PBS until saturation. Serial coronal sections 35-μm thick were cut using a cryostat (Microm HM 560 MV) and preserved in a cryoprotectant (25% glycerol /25% ethylen glycol /50% 0.2 M PBS) at -20 °C until use. Coronal sections were rinsed three times with 1X PBS. Floating sections were blocked with 3.5% Hose Serum and 0.5% 100X Triton in 1X PBS for 2 h at room temperature and incubated in primary antibody (Supplementary Table 1) in the same buffer overnight at 4 °C on a shaker. Sections were washed three times with PBS and then incubated with appropriate secondary antibodies diluted in PBS with 3.5% Hose Serum/ 0.5% 100X Triton in 1X PBS. Sections were washed three times in PBS, mounted with ProLong Gold Antifade Reagents (Thermo Fisher, P36930) on coverslips for confocal imaging.
Primary cultures
ENS culture—Primary cultures of rat enteric nervous system (ENS) were performed as previously described [24]. Briefly, embryonic day 15 (E15) rat intestine were removed and finely diced in Hank’s buffered salt solution and triturated mechanically using a scalpel. Tissue fragments were collected in Dulbecco’s modified Eagle medium (DMEM)/F12 (1:1) medium (Life Technologies, Carlsbad, CA, USA) containing 50 μg ml−1 streptomycin and 50 U ml−1 penicillin and incubated for 15 min at 37 °C in the same medium containing 0.25% trypsin (Invitrogen). After 15 min, the reaction was inactivated by adding 10% fetal bovine serum (FBS) for 5 min. Samples were incubated for 10 min at 37 °C with 0.1% DNase I (Sigma, St Louis, MO, USA). After trituration and centrifugation for 10 min at 84 g, cells were plated in DMEM (Thermofisher #1966025) /F12 (Thermofisher #11765054) containing antibiotics and 10% 1X FBS at a density of 2.4×105 cells cm2 on 24-well plates previously coated with 0.5% gelatin (Sigma) for 24 h. Medium was replaced with fresh DMEM/F12 without serum and supplemented with 1% N2 (Invitrogen #17502048). Half of the medium was replaced every 3 days, and primary cultures were maintained for up to 14 days.
Pure enteric neuron culture—Isolation and dissection of rat intestines have been previously described [25]. Briefly, embryonic day 15 (E15) rat intestines were collected and dissected in cold Hank’s buffered salt solution (HBSS). Each whole intestine (duodenum to sigmoid colon) was individually placed in a drop of cold HBSS in a Petri dish and cut into 8 pieces of equal length. Pieces were placed in a 24-well plastic culture plate (Corning®-115 Costar®, ref 3524, Merck KGaA, Darmstadt, Germany) previously coated with type I collagen/20 mM acetic acid and containing an enteric glial cell (EGC)-conditioned culture medium with 50 ng/mL GDNF (R&D Systems–bio-techne, 512-GF-050/CF). EGC cultures were obtained from enteric nervous system (ENS) primary cultures derived from rat embryonic intestines [26]. EGC-conditioned medium was filtered through a sterile 0.22 μM PolyEhterSulfone membrane before storage at -20 °C until use. After 5 days of culture (D5), many cells migrated from the explants and covered a large part of the well. Explants were removed using a P1000 pipette, and cells were isolated with 300 μL of Accutase per well for 5 min. Accutase was inactivated by adding 4.7 mL of DMEM + FBS and cell suspensions were centrifuged at 1500 rpm for 5 min at room temperature. Cell pellets were suspended in EGC-conditioned medium + GDNF (50 ng/mL) and seeded in previously poly-L-lysine (0.1 mg/mL)-coated P24 plate for immunofluorescence, on 18 mm diameter coverslip (Neuvitro corp., Vancouver, WA, USA) for patch-clamp, Ibidi 8-well plate for Ca2+ and imaging. At D6, an antimitotic (cytosine arabinoside: AraC, 5 μM) was added to the medium to eliminate the remaining glial and muscle cells. Purified neurons were used for functional and morphological tests at DIV11 and DIV12.