Unraveling the impact of bacterial peptidoglycans from gut microbiota on brain development, function and behavior

Over recent decades, the microbiota-gut-brain axis has emerged as a central regulator of neurodevelopment, influencing lifelong brain functions. Increasing preclinical and clinical evidence implicates the gut microbiota as a susceptibility factor for neurodevelopmental and psychiatric disorders, including autism spectrum disorder (ASD). Additionally, mounting evidence highlights the pivotal role of the maternal gut microbiota during pregnancy and lactation in shaping early-life neural circuits and influencing long-term brain health in offspring. However, the molecular mechanisms through which maternal microbial signals affect fetal brain development remain poorly understood. This thesis investigates how alterations in the maternal and adult gut microbiota influence brain development, function, and behavior, with a particular focus on bacterial peptidoglycan (PGN) fragments as key signaling molecules.

Paper I investigated whether disrupting the maternal gut microbiota with antibiotics during late pregnancy and early postnatal life affects early neurobehavioral outcomes relevant to ASD in offspring. Neonatal mice born to treated dams exhibited sex-specific alterations in ultrasonic vocalizations. As juveniles, these mice showed reduced social motivation, impaired social interactions, and altered anxiety-like behavior. These behavioral alterations were accompanied by reduced expression of oxytocin receptor and tight-junction proteins in the prefrontal cortex, low-grade colonic inflammation, and shifts in gut microbiota composition. These findings underscore the vulnerability of the maternal gut microbiota during the perinatal period and its critical role in shaping offspring social and emotional development.

Papers Il and Ill explored the role of PGN fragments-specifically muramyl dipeptide (MDP)-as maternal gut microbiota-derived signals that influence fetal brain development. PGN fragments administered during mid- or late pregnancy were detected in the amniotic fluid, confirming their translocation to the fetal compartment in the absence of overt inflammation. Mid-gestation PGN exposure induced widespread transcriptomic alterations in fetal brains, affecting genes involved in chromatin remodeling, estrogen receptor signaling, synaptic plasticity, and glutamatergic transmission. Male offspring exhibited altered neonatal vocalizations and impaired social behavior, accompanied by changes in gut microbiota composition and dysregulated expression of synaptic (e.g., Ppp1r9b, Bdnf, Oxtr) and glutamatergic (Gria1) genes in the prefrontal cortex.

In contrast, late gestational PGN exposure led to distinct, sex-specific behavioral outcomes. Male offspring displayed reduced social interaction, while females showed increased social engagement, reduced locomotion, and impaired social recognition. These effects were associated with altered expression of microglial (Trem2, Cx3cr1) and synaptic (Dlg4, Darpp-32) genes. Together, these findings highlight that the timing of PGN exposure and fetal sex critically shape neurodevelopmental responses, likely reflecting differences in developmental stage and hormonal milieu.

Paper IV extended these findings into adulthood, examining how short-term exposure to B-lactam antibiotics alters PGN translocation and impacts brain function and behavior. Adult male mice treated with ampicillin showed region- specific increases in brain PGN levels. Expression of PGN transporters (Slc15a4 and Slc46a3) in the brain varied in response to changes in PGN levels. Within 72 hours, antibiotic treatment triggered rapid changes in synaptic and microglial gene expression, disrupted functional brain connectivity, and impaired sociability and social recognition. These effects were accompanied by pronounced shifts in gut microbial composition. Notably, administration of DAP-type PGN fragments (i.e., iE-DAP) derived from Gram-negative bacteria recapitulated key behavioral and molecular effects observed in antibiotic-treated mice, suggesting a direct role of PGN in antibiotic-associated neurobehavioral alterations.

Collectively, this thesis identifies PGN as a critical molecular messenger in the microbiota-gut-brain axis, capable of influencing fetal brain programming and shaping long-term neurodevelopmental trajectories. It provides compelling evidence that maternal microbial signals-particularly during sensitive developmental windows-can shape neurodevelopment in a sex- and timing- dependent manner. By linking maternal microbiota alterations to specific molecular and behavioral outcomes, this work offers novel insights into how antibiotic use or dysregulated PGN levels during pregnancy may contribute to atypical neurodevelopment. Ultimately, these findings open new avenues for therapeutic strategies targeting PGN signaling to mitigate neurodevelopmental disorders and promote brain health.

List of scientific papers

I. Morel C, Martínez Sánchez I, Cherifi Y, Chartrel N, Diaz Heijtz R. Perturbation of maternal gut microbiota in mice during a critical perinatal window influences early neurobehavioral outcomes in offspring. Neuropharmacology. 2023 May 15;229:109479. https://doi.org/10.1016/j.neuropharm.2023.109479

II. Perego S#, Martínez Sánchez# I, Hökfelt T, Diaz Heijtz R. Maternal Peptidoglycan overexposure in mid-pregnancy alters neurodevelopmental trajectories and behavioral outcomes in mice. #: These authors contributed equally to the work. [Manuscript]

III. Martínez Sanchez I, Spielbauer J, Diaz Heijtz R. Maternal peptidoglycan overexposure during late pregnancy alters neurodevelopment and behavior in juvenile offspring. Brain Behavior and Immunity. 2025 Mar 7;127:96-102. https://doi.org/10.1016/j.bbi.2025.03.014

IV. Martínez Sánchez I, Kim WS, Heather C, Nylén S, Shapiro MG, Diaz Heijtz R. Short-term exposure to B-lactam antibiotics enhances peptidoglycan translocation to the brain, disrupting functional connectivity and social recognition in mice. [Submitted]