C-section and systemic inflammation synergize to disrupt the neonatal gut microbiota and brain development in a model of prematurity.

Infants born very preterm (below 28 weeks of gestation) are at high risk of developing neurodevelopmental disorders, such as intellectual deficiency, autism spectrum disorders, and attention deficit. Preterm birth often occurs in the context of perinatal systemic inflammation due to chorioamnionitis and postnatal sepsis. In addition, C-section is often performed for very preterm neonates to avoid hypoxia during a vaginal delivery. We have developed and characterized a mouse model based on intraperitoneal injections of IL-1ß between postnatal days one and five to reproduce perinatal systemic inflammation. This model replicates several neuropathological, brain imaging, and behavioral deficits observed in preterm infants. We hypothesized that C-sections could synergize with systemic inflammation to induce more severe brain abnormalities. We observed that C-sections significantly exacerbated the deleterious effects of IL-1ß on reduced gut microbial diversity, increased levels of circulating peptidoglycans, abnormal microglia/macrophage reactivity, impaired myelination, and reduced functional connectivity in the brain relative to vaginal delivery plus intraperitoneal saline. These data demonstrate the deleterious synergistic effects of C-section and neonatal systemic inflammation on brain maldevelopment and malfunction, two conditions frequently observed in very preterm infants, who are at high risk of developing neurodevelopmental disorders.

Multispecies probiotic intake during pregnancy modulates neurodevelopmental trajectories of offspring: Aiming towards precision microbial intervention.

Recent research highlights the pivotal role of the maternal gut microbiome during pregnancy in shaping offspring neurodevelopment. In this study, we investigated the impact of maternal intake of a multispecies probiotic formulation during a critical prenatal window (from gestational day 6 until birth) on neurodevelopmental trajectories in mice. Our findings demonstrate significant and persistent benefits in emotional behavior, gut microbiota composition, and expression of tight junction-related genes, particularly in male offspring, who exhibited heightened sensitivity to the probiotic intervention compared to females. Additionally, we observed elevated gene expression levels of the anti-inflammatory cytokine IL-10 and the oxytocin receptor (Oxtr) in the prefrontal cortex (PFC) of exposed juvenile offspring; however, these changes persisted only in the adult male offspring. Furthermore, the sustained increase in the expression of the proton-coupled oligopeptide transporter 1 (PepT1), which is involved in the transport of bacterial peptidoglycan motifs, in the PFC of exposed male offspring suggests a potential mechanistic pathway underlying the observed sex-dependent effects on behavior and gene expression. These results underscore the potential of prenatal multispecies probiotic interventions to promote long-term neurodevelopmental outcomes, with implications for precision microbial reconstitution aimed at promoting healthy neurodevelopment and behavior.

Bacterial peptidoglycan signalling in microglia: Activation by MDP via the NF-κB/MAPK pathway.

Bacterial peptidoglycan (PGN) fragments are commonly studied in the context of bacterial infections. However, PGN fragments recently gained recognition as signalling molecules from the commensal gut microbiota in the healthy host. Here we focus on the minimal bioactive PGN motif muramyl dipeptide (MDP), found in both Gram-positive and Gram-negative commensal bacteria, which signals through the Nod2 receptor. MDP from the gut microbiota translocates to the brain and is associated with changes in neurodevelopment and behaviour, yet there is limited knowledge about the underlying mechanisms. In this study we demonstrate that physiologically relevant doses of MDP induce rapid changes in microglial gene expression and lead to cytokine and chemokine secretion. In immortalised microglial (IMG) cells, C-C Motif Chemokine Ligand 5 (CCL5/RANTES) expression is acutely sensitive to the lowest physiologically prevalent dose (0.1 µg/ml) of MDP. As CCL5 plays an important role in memory formation and synaptic plasticity, microglial CCL5 might be the missing link in elucidating MDP-induced alterations in synaptic gene expression. We observed that a higher physiological dose of MDP elevates the expression of cytokines TNF-α and IL-1ß, indicating a transition toward a pro-inflammatory phenotype in IMG cells, which was validated in primary microglial cultures. Furthermore, MDP induces the translocation of NF-κB subunit p65 into the nucleus, which is blocked by MAPK p38 inhibitor SB202190, suggesting that an interplay of both the NF-κB and MAPK pathways is responsible for the MDP-specific microglial phenotype. These findings underscore the significance of different MDP levels in shaping microglial function in the CNS and indicate MDP as a potential mediator for early inflammatory processes in the brain. It also positions microglia as an important target in the gut microbiota-brain-axis pathway through PGN signalling.

Food, nutrition, and autism: from soil to fork.

Food and nutrition-related factors have the potential to impact development of autism spectrum disorder (ASD) and quality of life for people with ASD, but gaps in evidence exist. On 10 November 2022, Tufts University's Friedman School of Nutrition Science and Policy and Food and Nutrition Innovation Institute hosted a 1-d meeting to explore the evidence and evidence gaps regarding the relationships of food and nutrition with ASD. This meeting report summarizes the presentations and deliberations from the meeting. Topics addressed included prenatal and child dietary intake, the microbiome, obesity, food-related environmental exposures, mechanisms and biological processes linking these factors and ASD, food-related social factors, and data sources for future research. Presentations highlighted evidence for protective associations with prenatal folic acid supplementation and ASD development, increases in risk of ASD with maternal gestational obesity, and the potential for exposure to environmental contaminants in foods and food packaging to influence ASD development. The importance of the maternal and child microbiome in ASD development or ASD-related behaviors in the child was reviewed, as was the role of discrimination in leading to disparities in environmental exposures and psychosocial factors that may influence ASD. The role of child diet and high prevalence of food selectivity in children with ASD and its association with adverse outcomes were also discussed. Priority evidence gaps identified by participants include further clarifying ASD development, including biomarkers and key mechanisms; interactions among psychosocial, social, and biological determinants; interventions addressing diet, supplementation, and the microbiome to prevent and improve quality of life for people with ASD; and mechanisms of action of diet-related factors associated with ASD. Participants developed research proposals to address the priority evidence gaps. The workshop findings serve as a foundation for future prioritization of scientific research to address evidence gaps related to food, nutrition, and ASD.

Sex Differences in the Allele Distribution of PGLYRP2 Variant rs892145 in Parkinson's Disease.

Introduction: Parkinson's disease (PD) is a complex multifactorial disease, involving genetic susceptibility, environmental risk factors, and gene-environmental interactions. The microbiota-gut-brain axis is hypothesized to play a role in the pathophysiology of PD, and peptidoglycan recognition proteins (PGLYRPs), which modulate the gut microbiota, are, therefore, relevant candidate genes for PD. Methods: Using quantitative real-time PCR, we genotyped three PGLYRP variants (rs892145, rs959117, and rs10888557) and performed an association analysis in 508 PD patients and 585 control individuals. We further conducted a meta-analysis of rs892145 and analyzed PGLYRP2 gene expression in lymphocytes from patients with PD and controls. Results: Although initial analysis of the three variants rs892145, rs959117, and rs10888557 and a meta-analysis of rs892145 did not reveal any association between the selected variants and PD, we found an interaction between sex and genotype for rs892145, with a marked difference in the allele distribution of rs892145 between male and female patients. As compared to controls, the T allele was less common in female patients (odds ratio = 0.76, P = 0.04) and more common in male patients (odds ratio = 1.29, P = 0.04). No difference was found in PGLYRP2 gene expression between PD patients and controls (P = 0.38), nor between sexes (P = 0.07). Discussion. Overall, this genetic screening in Swedish PD patients does not support previous results demonstrating associations of PGLYRP variants with the risk of PD. Meta-analysis of rs892145 revealed pronounced heterogeneity between previously published studies which is likely to have influenced the results. Taken together, the genetic and gene expression analyses suggest a possible link between genetic variants in PGLYRP2 and sex differences in PD. Because of the limited sample size in our study, these results need to be verified in independent cohorts before concluding.

Early-life differences in the gut microbiota composition and functionality of infants at elevated likelihood of developing autism spectrum disorder.

Evidence from cross-sectional human studies, and preliminary microbial-based intervention studies, have implicated the microbiota-gut-brain axis in the neurobiology of autism spectrum disorder (ASD). Using a prospective longitudinal study design, we investigated the developmental profile of the fecal microbiota and metabolome in infants with (n = 16) and without (n = 19) a family history of ASD across the first 36 months of life. In addition, the general developmental levels of infants were evaluated using the Mullen Scales of Early Learning (MSEL) test at 5 and 36 months of age, and with ADOS-2 at 36 months of age. At 5 months of age, infants at elevated-likelihood of ASD (EL) harbored less Bifidobacterium and more Clostridium and Klebsiella species compared to the low-likelihood infants (LL). Untargeted metabolic profiling highlighted that LL infants excreted a greater amount of fecal γ-aminobutyric acid (GABA) at 5 months, which progressively declined with age. Similar age-dependent patterns were not observed in the EL group, with GABA being consistently low across all timepoints. Integrated microbiome-metabolome analysis showed a positive correlation between GABA and Bifidobacterium species and negative associations with Clostridium species. In vitro experiments supported these observations demonstrating that bifidobacteria can produce GABA while clostridia can consume it. At the behavioral level, there were no significant differences between the EL and LL groups at 5 months. However, at 36 months of age, the EL group had significantly lower MSEL and ADOS-2 scores compared to the LL group. Taken together, the present results reveal early life alterations in gut microbiota composition and functionality in infants at elevated-likelihood of ASD. These changes occur before any behavioral impairments can be detected, supporting a possible role for the gut microbiota in emerging behavioral variability later in life.

Perturbation of maternal gut microbiota in mice during a critical perinatal window influences early neurobehavioral outcomes in offspring.

The gut microbiota is increasingly recognized as a key environmental factor that shapes host development and physiology, including neural circuits formation and function. Concurrently, there has been growing concern that early-life antibiotic exposure may alter brain developmental trajectories, increasing the risk for neurodevelopmental disorders such as autism spectrum disorder (ASD). Here, we assessed whether perturbation of the maternal gut microbiota in mice during a narrow critical perinatal window (last week of pregnancy and first three postnatal days), induced by exposure to a commonly used broad-spectrum oral antibiotic (ampicillin), influences offspring neurobehavioral outcomes relevant to ASD. Our results demonstrate that neonatal offspring from antibiotic-treated dams display an altered pattern of ultrasonic communication, which was more pronounced in males. Moreover, juvenile male, but not female, offspring from antibiotic-treated dams showed reduced social motivation and social interaction, as well as context-dependent anxiety-like behavior. However, no changes were observed in locomotor or exploratory activity. This behavioral phenotype of exposed juvenile males was associated with reduced gene expression of the oxytocin receptor (OXTR) and several tight-junction proteins in the prefrontal cortex, a key region involved in the regulation of social and emotional behaviors, as well as a mild inflammatory response in the colon. Further, juvenile offspring from exposed dams also showed distinct alterations in several gut bacterial species, including, Lactobacillus murinus, and Parabacteroides goldsteinii. Overall, this study highlights the importance of the maternal microbiome in early-life, and how its perturbation by a widely used antibiotic could contribute to atypical social and emotional development of offspring in a sex-dependent manner.

Bacterial Peptidoglycans from Microbiota in Neurodevelopment and Behavior.

It is increasingly recognized that the gut microbiota profoundly influences many aspects of host development and physiology, including the modulation of brain development and behavior. However, the precise molecular mechanisms and signaling pathways involved in communication between the microbiota and the developing brain remain to be fully elucidated. Germline-encoded pattern-recognition receptors (PRRs) that recognize conserved microbial molecular signatures such as bacterial surface molecules (e.g., peptidoglycans, PGNs) have emerged as potential key regulators of gut microbiota-brain interactions. We highlight current evidence supporting multiple and essential roles for PGNs and their sensing molecules beyond innate immunity, extending to neurodevelopment and behavior. In addition, the possible implications of the PGN signaling pathway for the pathogenesis of neurodevelopmental disorders such as autism spectrum disorder (ASD) are considered.

Developmental Signatures of Microbiota-Derived Metabolites in the Mouse Brain.

The gut microbiome is recognized to exert a wide-ranging influence on host health and disease, including brain development and behavior. Commensal bacteria can produce bioactive molecules that enter the circulation and impact host physiology and homeostasis. However, little is known about the potential for these metabolites to cross the blood-brain barrier and enter the developing brain under normal physiological conditions. In this study, we used a liquid chromatography-mass spectrometry-based metabolomic approach to characterize the developmental profiles of microbial-derived metabolites in the forebrains of mice across three key postnatal developmental stages, co-occurring with the maturation of the gut microbiota. We demonstrate that direct metabolites of the gut microbiome (e.g., imidazole propionate) or products of the combinatorial metabolism between the microbiome and host (e.g., 3-indoxyl-sulfate, trimethylamine-N-oxide, and phenylacetylglycine) are present in the forebrains of mice as early as the neonatal period and remain into adulthood. These findings demonstrate that microbial-associated molecules can cross the BBB either in their detected form or as precursor molecules that undergo further processing in the brain. These chemical messengers are able to bind receptors known to be expressed in the brain. Alterations in the gut microbiome may therefore influence neurodevelopmental trajectories via the regulation of these microbial-associated metabolites.

Bacterial peptidoglycans as novel signaling molecules from microbiota to brain.

Mounting evidence indicates that gut microbiota exerts a broad range of effects on host physiology and development beyond the gastrointestinal tract, including the modulation of brain development. However, the mechanisms mediating the interactions between the microbiota and the developing brain are still poorly understood. Pattern recognition receptors of the innate immune system that recognize microbial products, such as peptidoglycans have emerged as potential key regulators of gut microbiome-brain interactions. Peptidoglycan-sensing molecules are expressed in the placenta and brain during specific time windows of development. Moreover, peptidoglycans are ubiquitously present in circulation and can cross the blood brain barrier. This review brings together the current evidence supporting a broad function of peptidoglycans well beyond host's immunity, extending to neurodevelopment and behavior.

Correction to: Can Neonatal Systemic Inflammation and Hypoxia Yield a Cerebral Palsy-Like Phenotype in Periadolescent Mice?

The original version of this article unfortunately contained a mistake in Author name. In Rochellys Diaz Heijtz, "Diaz" should be classified as Familyname.

Genetic Variation in the Dopamine System Influences Intervention Outcome in Children with Cerebral Palsy.

BACKGROUND: There is large variation in treatment responses in children with cerebral palsy. Experimental and clinical results suggest that dopamine neurotransmission and brain-derived neurotrophic factor (BDNF) signalling are involved in motor learning and plasticity, which are key factors in modern habilitation success. We examined whether naturally occurring variations in dopamine and BDNF genes influenced the treatment outcomes. METHODS: Thirty-three children (18-60months of age) with spastic unilateral cerebral palsy were enrolled in the study. Each child had participated in a training programme consisting of active training of the involved hand for 2h every day during a 2-month training period. The training outcome was measured using Assisting Hand Assessment before and after the training period. Saliva was collected for genotyping of COMT, DAT, DRD1, DRD2, DRD3, and BDNF. Regression analyses were used to examine associations between genetic variation and training outcome. FINDINGS: There was a statistically significant association between variation in dopamine genes and treatment outcome. Children with a high polygenic dopamine gene score including polymorphisms of five dopamine genes (COMT, DAT, DRD1, DRD2, and DRD3), and reflecting higher endogenous dopaminergic neurotransmission, had the greatest functional outcome gains after intervention. INTERPRETATION: Naturally occurring genetic variation in the dopamine system can influence treatment outcomes in children with cerebral palsy. A polygenic dopamine score might be valid for treatment outcome prediction and for designing individually tailored interventions for children with cerebral palsy.

Neuropeptide and Small Transmitter Coexistence: Fundamental Studies and Relevance to Mental Illness.

Neuropeptides are auxiliary messenger molecules that always co-exist in nerve cells with one or more small molecule (classic) neurotransmitters. Neuropeptides act both as transmitters and trophic factors, and play a role particularly when the nervous system is challenged, as by injury, pain or stress. Here neuropeptides and coexistence in mammals are reviewed, but with special focus on the 29/30 amino acid galanin and its three receptors GalR1, -R2 and -R3. In particular, galanin's role as a co-transmitter in both rodent and human noradrenergic locus coeruleus (LC) neurons is addressed. Extensive experimental animal data strongly suggest a role for the galanin system in depression-like behavior. The translational potential of these results was tested by studying the galanin system in postmortem human brains, first in normal brains, and then in a comparison of five regions of brains obtained from depressed people who committed suicide, and from matched controls. The distribution of galanin and the four galanin system transcripts in the normal human brain was determined, and selective and parallel changes in levels of transcripts and DNA methylation for galanin and its three receptors were assessed in depressed patients who committed suicide: upregulation of transcripts, e.g., for galanin and GalR3 in LC, paralleled by a decrease in DNA methylation, suggesting involvement of epigenetic mechanisms. It is hypothesized that, when exposed to severe stress, the noradrenergic LC neurons fire in bursts and release galanin from their soma/dendrites. Galanin then acts on somato-dendritic, inhibitory galanin autoreceptors, opening potassium channels and inhibiting firing. The purpose of these autoreceptors is to act as a 'brake' to prevent overexcitation, a brake that is also part of resilience to stress that protects against depression. Depression then arises when the inhibition is too strong and long lasting - a maladaption, allostatic load, leading to depletion of NA levels in the forebrain. It is suggested that disinhibition by a galanin antagonist may have antidepressant activity by restoring forebrain NA levels. A role of galanin in depression is also supported by a recent candidate gene study, showing that variants in genes for galanin and its three receptors confer increased risk of depression and anxiety in people who experienced childhood adversity or recent negative life events. In summary, galanin, a neuropeptide coexisting in LC neurons, may participate in the mechanism underlying resilience against a serious and common disorder, MDD. Existing and further results may lead to an increased understanding of how this illness develops, which in turn could provide a basis for its treatment.

Sex-dependent alterations in motor and anxiety-like behavior of aged bacterial peptidoglycan sensing molecule 2 knockout mice.

Peptidoglycan recognition proteins (PGRPs) are key sensing-molecules of the innate immune system that specifically detect bacterial peptidoglycan (PGN) and its derivates. PGRPs have recently emerged as potential key regulators of normal brain development and behavior. To test the hypothesis that PGRPs play a role in motor control and anxiety-like behavior in later life, we used 15-month old male and female peptidoglycan recognition protein 2 (Pglyrp2) knockout (KO) mice. Pglyrp2 is an N-acetylmuramyl-l-alanine amidase that hydrolyzes PGN between the sugar backbone and the peptide chain (which is unique among the mammalian PGRPs). Using a battery of behavioral tests, we demonstrate that Pglyrp2 KO male mice display decreased levels of anxiety-like behavior compared with wild type (WT) males. In contrast, Pglyrp2 KO female mice show reduced rearing activity and increased anxiety-like behavior compared to WT females. In the accelerated rotarod test, however, Pglyrp2 KO female mice performed better compared to WT females (i.e., they had longer latency to fall off the rotarod). Further, Pglyrp2 KO male mice exhibited decreased expression levels of synaptophysin, gephyrin, and brain-derived neurotrophic factor in the frontal cortex, but not in the amygdala. Pglyrp2 KO female mice exhibited increased expression levels of spinophilin and alpha-synuclein in the frontal cortex, while exhibiting decreased expression levels of synaptophysin, gephyrin and spinophilin in the amygdala. Our findings suggest a novel role for Pglyrp2asa key regulator of motor and anxiety-like behavior in late life.

Brief Report: Association Between Autism Spectrum Disorder, Gastrointestinal Problems and Perinatal Risk Factors Within Sibling Pairs.

Autism spectrum disorder (ASD) has been associated with gastrointestinal (GI) problems, but the nature of this association is unclear. Parents to siblings, concordant or discordant for ASD (N = 217), participated in a web survey covering mother's weight gain during pregnancy, maternal viral/bacterial infection and use of antibiotics, duration of breastfeeding, mode of delivery, birth weight and child GI problems. ASD was associated with GI problems and perinatal environmental risk, based on a summation of maternal infection and antibiotic use during pregnancy and/or the breastfeeding period. The association between GI problems and ASD remained within the sibling pairs (ß = 1.23; p < .001) in the adjusted model. Our results indicate non-shared environmental effects on the ASD/GI association, but none of the factors examined explained the link.