Cross-talk between gut microbiota and brain: effects of probiotics on the endophenotype of R451C Neuroligin3 monogenic mouse model of autism

Autism spectrum disorders (ASDs) are neurodevelopmental syndromes characterized by repetitive behaviors and several social deficits. Recent studies have shown that beside genetics and environmental causes, the gut-brain axis plays an important role in the onset of these disorders. Indeed, immune dysfunction and gastrointestinal (GI) inflammation are common in individuals with ASDs and contribute to the severity of the disorder. Moreover, gut microbiota has been shown to modulate brain function and behavior. One of the most recurrent affected signaling in ASDs is the synaptic pathway and several mutations have been associated with risk genes encoding for the synaptic proteins Neuroligins (NLGNs) and Neurexins (NRXNs). Among the mutations in the NLGNs genes, the most studied is the R451C missense mutation in NLGN3. The monogenic model of autism expressing the human mutation R451C in the NLGN3 murine gene, shows an autistic-like phenotype characterized by behavioral deficits, imbalance between excitatory/inhibitory transmission, altered enteric neurons and GI dysfunctions. All these characteristics make the R451C NLGN3 mouse model suitable for studying alterations of the gut-brain axis. An increasingly holistic vision of autism has led to the search for alternative therapeutic strategies, aimed at recovering peripheral alterations, with indirect beneficial consequences on the central nervous system (CNS). One of the most innovative therapeutic approaches for ASDs is the use of personalized probiotic-based dietary interventions, since probiotics have been shown to simultaneously restore gut microbiota composition, by promoting beneficial taxa, and ameliorate behavioral deficits. The focus of my PhD project is to assess the effects of five probiotic or potential probiotic strains, a mix of Lactiplantibacillus (Lpb.) plantarum C9O4 and LT52, Lactobacillus (L.) reuteri АТСС РТА 6475, Lactiplantibacillus (Lpb.) plantarum H64 and Periweissella (P.) beninensis LMG 25373T on the impaired behavioral and endophenotype of the R451C NLGN3 knock-in (KI) mouse model of autism. The first are a mix of two food-borne strains, already known for their anti-inflammatory and antioxidant properties, as well as the ability to modulate compromised GI microbiota; the second one is already known for its positive effects on others murine models of autism and on autistic children; while Lpb. plantarum H64 has been characterized as a GABA-producing strain with well-established in vitro probiotic properties, and P. beninensis LMG 25373T is a novel exopolysaccharide (EPS)-producing strain recently identified as a promising probiotic candidate. The treatments started independently at weaning (postnatal day 21) and lasted five weeks on both wild-type and mutant mice. Behavioral improvements were assessed using the three-chambers sociability test, finding pro-social effects for all bacterial strains. At the molecular level, the impact of the treatments was evaluated in key brain regions implicated in ASDs pathophysiology, including the prefrontal cortex, hippocampus, and cerebellum, where neurotransmission alterations have been described for R451C NLGN3 KI mice. We found a potential restoration of the excitatory/inhibitory neurotransmission indicated by the rescue of protein levels of key synapses components and a reduction in the activation of the unfolded protein response (UPR). Gut and fecal samples were employed to evaluate the effect of strains administration on intestinal permeability, immune response and gut microbiota composition, finding that all the treatments promote the enrichment of beneficial, gut-supportive taxa and restore gut functions in the mutant mouse model. To further analyze aspects related to the gut-brain axis, plasma samples were analyzed to identify molecules that, carried by the blood stream, might act as messengers between the gut and the brain such as amino acids, neurotransmitters and endocannabinoids (ECs), observing slight modulations. This project supports innovative research into the biological underpinnings of autism and offers solid basis to develop safe, evidence-based interventions. By targeting the gut-brain axis, this study explores how specific bacterial strains can modulate both GI and neural dysfunction in a genetically validated mouse model. The use of well-characterized, bioactive strains with distinct mechanistic properties holds strong translational promise, offering a non-invasive, personalized therapeutic strategy that could alleviate core behavioral symptoms and comorbidity with GI disturbances. This integrated approach addresses the growing recognition of systemic, whole-body contributions to autism and reflects commitment to advancing holistic, biologically grounded treatments.