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Abstract Autism Spectrum Disorder (ASD) is a sex-biased neurodevelopmental disorder with a male to female prevalence of 4:1, characterized by persistent deficits in social communication and interaction and restricted-repetitive patterns of behavior, interests or activities. Microbiota alterations as well as signs of neuroinflammation have been also reported in ASD. The involvement of immune dysregulation in ASD is further supported by evidence suggesting that maternal immune activation (MIA), especially during early pregnancy, may be a risk factor for ASD. The present study was aimed at characterizing the effects of MIA on behavior, gut microbiota and neuroinflammation in the mouse offspring also considering the impact of MIA in the two sexes. MIA offspring exhibited significant ASD-like behavioral alterations (i.e., deficits in sociability and sensorimotor gating, perseverative behaviors). The analysis of microbiota revealed changes in specific microbial taxa that recapitulated those seen in ASD children. In addition, molecular analyses indicated sex-related differences in the neuroinflammatory responses triggered by MIA, with a more prominent effect in the cerebellum. Our data suggest that both sexes should be included in the experimental designs of preclinical studies in order to identify those mechanisms that confer different vulnerability to ASD to males and females.

The Principle of the 3Rs is widely recognised as the methodological and ethical backbone of contemporary animal research. Different authors also stress the reciprocal links among the 3Rs, and how these often complement and reinforce each other. We very much agree with this point, but in this contribution we would like to raise some problems related to the application of the “3Rs”. There is an obvious link among “Replacement, “Reduction” and “Refinement”, but it is worth to notice also that each “R” has its own conceptual characteristics, as well as its own level of applicability. For example, a realistic “methodological inertia” has to be expected more in the case of “Replacement” than in the case of “Refinement”. This also leads to a second order of issues, and here we will offer our experience as projects evaluators. The “3Rs” differ also in the possibility to verify how are applied by the proponents of research protocols involving the use of animal models. Sometimes it appears that the application of the Principle still resolves itself in the use of formulaic sentences, from which it is difficult to really understand the reality of the laboratory decisional and procedural processes. However, the demanding characteristics of the “3Rs” can vary greatly, and this is something that has to be considered. We propose that a network, or a virtual platform, of evaluators could help both researchers and evaluators for a more satisfactory understanding and pragmatic application of the Principle of the 3Rs.

Maternal infections during pregnancy and the consequent maternal immune activation (MIA) are the major risk factors for autism spectrum disorder (ASD). Epidemiological evidence is corroborated by the preclinical models in which MIA leads to ASD-like behavioral abnormalities and altered neuroinflammatory profiles, with an increase in pro-inflammatory cytokines and microglial markers. In addition to neuroinflammatory response, an abnormal expression of endogenous retroviruses (ERVs) has been identified in neurodevelopmental disorders and have been found to correlate with disease severity. Our aim was to evaluate the transcriptional profile of several ERV families, ERV-related genes, and inflammatory mediators (by RT real-time PCR) in mouse offspring of both sexes, prenatally exposed to polyinosinic:polycytidylic acid (Poly I:C), a synthetic double-stranded RNA molecule targeting TLR-3 that mimics viral maternal infection during pregnancy. We found that prenatal exposure to Poly I:C deregulated the expression of some ERVs and ERV-related genes both in the prefrontal cortex (PFC) and hippocampus, while no changes were detected in the blood. Interestingly, sex-related differences in the expression levels of some ERVs, ERV-related genes, and inflammatory mediators that were higher in females than in males emerged only in PFC. Our findings support the tissue specificity of ERV and ERV-related transcriptional profiles in MIA mice.

Selenium (Se) is an essential trace element required for normal development as well as to counteract the adverse effects of environmental stressors. Conditions of low Se intake are present in some European countries. Our aim was to investigate the short- and long-term effects of early-life low Se supply on behavior and synaptic plasticity with a focus on the hippocampus, considering both suboptimal Se intake per se and its interaction with developmental exposure to lead (Pb). We established an animal model of Se restriction and low Pb exposure; female rats fed with an optimal (0.15 mg/kg) or suboptimal (0.04 mg/kg) Se diet were exposed from one month pre-mating until the end of lactation to 12.5 µg/mL Pb via drinking water. In rat offspring, the assessment of motor, emotional, and cognitive endpoints at different life stages were complemented by the evaluation of the expression and synaptic distribution of NMDA and AMPA receptor subunits at post-natal day (PND) 23 and 70 in the hippocampus. Suboptimal Se intake delayed the achievement of developmental milestones and induced early and long-term alterations in motor and emotional abilities. Behavioral alterations were mirrored by a drop in the expression of the majority of NMDA and AMPA receptor subunits analyzed at PND 23. The suboptimal Se status co-occurring with Pb exposure induced a transient body weight increase and persistent anxiety-like behavior. From the molecular point of view, we observed hippocampal alterations in NMDA (Glun2B and GluN1) and AMPA receptor subunit trafficking to the post-synapse in male rats only. Our study provides evidence of potential Se interactions with Pb in the developing brain.

Research in both animals and humans shows that some nutrients are important in pregnancy and during the first years of life to support brain and cognitive development. Our aim was to evaluate the role of selenium (Se) in supporting brain and behavioral plasticity and maturation. Pregnant and lactating female rats and their offspring up to postnatal day 40 were fed isocaloric diets differing in Se content—i.e., optimal, sub-optimal, and deficient—and neurodevelopmental, neuroinflammatory, and anti-oxidant markers were analyzed. We observed early adverse behavioral changes in juvenile rats only in sub-optimal offspring. In addition, sub-optimal, more than deficient supply, reduced basal glial reactivity in sex dimorphic and brain-area specific fashion. In female offspring, deficient and sub-optimal diets reduced the antioxidant Glutathione peroxidase (GPx) activity in the cortex and in the liver, the latter being the key organ regulating Se metabolism and homeostasis. The finding that the Se sub-optimal was more detrimental than Se deficient diet may suggest that maternal Se deficient diet, leading to a lower Se supply at earlier stages of fetal development, stimulated homeostatic mechanisms in the offspring that were not initiated by sub-optimal Se. Our observations demonstrate that even moderate Se deficiency during early life negatively may affect, in a sex-specific manner, optimal brain development.

Rett syndrome (RTT) is a rare neurological disorder caused by mutations in the X-linked MECP2 gene and a major cause of intellectual disability in females. No cure exists for RTT. We previously reported that the behavioural phenotype and brain mitochondria dysfunction are widely rescued by a single intracerebroventricular injection of the bacterial toxin CNF1 in a RTT mouse model carrying a truncating mutation of the MeCP2 gene (MeCP2-308 mice). Given the heterogeneity of MECP2 mutations in RTT patients, we tested the CNF1 therapeutic efficacy in a mouse model carrying a null mutation (MeCP2-Bird mice). CNF1 selectively rescued cognitive defects, without improving other RTT-related behavioural alterations, and restored brain mitochondrial respiratory chain complex activity in MeCP2-Bird mice. To shed light on the molecular mechanisms underlying the differential CNF1 effects on the behavioural phenotype, we compared treatment effects on relevant signalling cascades in the brain of the two RTT models. CNF1 provided a significant boost of the mTOR activation in MeCP2-308 hippocampus, which was not observed in the MeCP2-Bird model, possibly explaining the differential effects of CNF1. These results demonstrate that CNF1 efficacy depends on the mutation beared by MeCP2-mutated mice, stressing the need of testing potential therapeutic approaches across RTT models.

Transposable elements (TEs) are highly repetitive DNA sequences in the human genome that are the relics of previous retrotransposition events. Although the majority of TEs are transcriptionally inactive due to acquired mutations or epigenetic processes, around 8% of TEs exert transcriptional activity. It has been found that TEs contribute to somatic mosaicism that accounts for functional specification of various brain cells. Indeed, autonomous retrotransposition of long interspersed element-1 (LINE-1) sequences has been reported in the neural rat progenitor cells from the hippocampus, the human fetal brain and the human embryonic stem cells. Moreover, expression of TEs has been found to regulate immune-inflammatory responses, conditioning immunity against exogenous infections. Therefore, aberrant epigenetic regulation and expression of TEs emerged as a potential mechanism underlying the development of various mental disorders, including autism spectrum disorders (ASD), schizophrenia, bipolar disorder, major depression, and Alzheimer’s disease (AD). Consequently, some studies revealed that expression of some sequences of human endogenous retroviruses (HERVs) appears only in a certain group of patients with mental disorders (especially those with schizophrenia, bipolar disorder, and ASD) but not in healthy controls. In addition, it has been found that expression of HERVs might be related to subclinical inflammation observed in mental disorders. In this article, we provide an overview of detrimental effects of transposition on the brain development and immune mechanisms with relevance to mental disorders. We show that transposition is not the only mechanism, explaining the way TEs might shape the phenotype of mental disorders. Other mechanisms include the regulation of gene expression and the impact on genomic stability. Next, we review current evidence from studies investigating expression and epigenetic regulation of specific TEs in various mental disorders. Most consistently, these studies indicate altered expression of HERVs and methylation of LINE-1 sequences in patients with ASD, schizophrenia, and mood disorders. However, the contribution of TEs to the etiology of AD is poorly documented. Future studies should further investigate the mechanisms linking epigenetic processes, specific TEs and the phenotype of mental disorders to disentangle causal associations.

Among gliomas, primary tumors originating from glial cells, glioblastoma (GBM) identified as WHO grade IV glioma, is the most common and aggressive malignant brain tumor. We have previously shown that the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) is remarkably effective as an anti-neoplastic agent in a mouse model of glioma, reducing the tumor volume, increasing survival, and maintaining the functional properties of peritumoral neurons. However, being unable to cross the blood–brain barrier (BBB), CNF1 requires injection directly into the brain, which is a very invasive administration route. Thus, to overcome this pitfall, we designed a CNF1 variant characterized by the presence of an N-terminal BBB-crossing tag. The variant was produced and we verified whether its activity was comparable to that of wild-type CNF1 in GBM cells. We investigated the signaling pathways engaged in the cell response to CNF1 variants to provide preliminary data to the subsequent studies in experimental animals. CNF1 may represent a novel avenue for GBM therapy, particularly because, besides blocking tumor growth, it also preserves the healthy surrounding tissue, maintaining its architecture and functionality. This renders CNF1 the most interesting candidate for the treatment of brain tumors, among other potentially effective bacterial toxins.

Lead (Pb) exposure in early life affects brain development resulting in cognitive and behavioral deficits. Epidemiologic and experimental evidence of sex as an effect modifier of developmental Pb exposure is emerging. In the present study, we investigated Pb effects on behavior and mechanisms of neuroplasticity in the hippocampus and potential sex differences. To this aim, dams were exposed, from one month pre-mating to offspring weaning, to Pb via drinking water at 5 mg/kg body weight per day. In the offspring of both sexes, the longitudinal assessment of motor, emotional, and cognitive end points was performed. We also evaluated the expression and synaptic distribution of N-methyl-D-Aspartate receptor (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits at post-natal day (pnd) 23 and 70 in the hippocampus. Neonatal motor patterns and explorative behavior in offspring were affected in both sexes. Pb effects in emotional response and memory retention were observed in adult females only, preceded by increased levels of GluN2A and GluA1 subunits at the post-synapse at pnd 23. These data suggest that Pb exposure during development affects glutamatergic receptors distribution at the post-synaptic spine in females. These effects may contribute to alterations in selected behavioral domains.

A significant body of evidence supports the multifactorial etiology of neurodevelopmental disorders (NDDs) affecting children. The present review focuses on early exposure to environmental chemicals as a risk factor for neurodevelopment, and presents the major lines of evidence derived from epidemiological studies, underlying key uncertainties and research needs in this field. We introduce the exposome concept that, encompassing the totality of human environmental exposures to multiple risk factors, aims at explaining individual vulnerability and resilience to early chemical exposure. In this framework, we synthetically review the role of variable gene backgrounds, the involvement of epigenetic mechanisms as well as the function played by potential effect modifiers such as socioeconomic status. We describe laboratory rodent studies where the neurodevelopmental effects of environmental chemicals are assessed in the presence of either a “vulnerable” gene background or adverse pregnancy conditions (i.e., maternal stress). Finally, we discuss the need for more descriptive and “lifelike” experimental models of NDDs, to identify candidate biomarkers and pinpoint susceptible groups or life stages to be translated to large prospective studies within the exposome framework.

Rett syndrome (RTT) is a rare neurodevelopmental disorder usually caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). Several Mecp2 mutant mouse lines have been developed recapitulating part of the clinical features. In particular, Mecp2-308 female heterozygous mice, bearing a truncating mutation, are a validated model of the disease. While recent data suggest a role for inflammation in RTT, little information on the inflammatory status in murine models of the disease is available. Here, we investigated the inflammatory status by proteomic 2-DE/MALDI-ToF/ToF analyses in symptomatic Mecp2-308 female mice. Ten differentially expressed proteins were evidenced in the Mecp2-308 mutated plasma proteome. In particular, 5 positive acute-phase response (APR) proteins increased (i.e., kininogen-1, alpha-fetoprotein, mannose-binding protein C, alpha-1-antitrypsin, and alpha-2-macroglobulin), and 3 negative APR reactants were decreased (i.e., serotransferrin, albumin, and apolipoprotein A1). CD5 antigen-like and vitamin D-binding protein, two proteins strictly related to inflammation, were also changed. These results indicate for the first time a persistent unresolved inflammation of unknown origin in the Mecp2-308 mouse model.

Rett syndrome (RTT) is a rare neurodevelopmental disorder affecting almost exclusively females, caused in the overwhelming majority of the cases by loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). High circulating levels of oxidative stress (OS) markers in patients suggest the involvement of OS in the RTT pathogenesis. To investigate the occurrence of oxidative brain damage in Mecp2 mutant mouse models, several OS markers were evaluated in whole brains of Mecp2-null (pre-symptomatic, symptomatic, and rescued) and Mecp2-308 mutated (pre-symptomatic and symptomatic) mice, and compared to those of wild type littermates. Selected OS markers included non-protein-bound iron, isoprostanes (F2-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes) and 4-hydroxy-2-nonenal protein adducts. Our findings indicate that oxidative brain damage 1) occurs in both Mecp2-null (both −/y and stop/y) and Mecp2-308 (both 308/y males and 308/+ females) mouse models of RTT; 2) precedes the onset of symptoms in both Mecp2-null and Mecp2-308 models; and 3) is rescued by Mecp2 brain specific gene reactivation. Our data provide direct evidence of the link between Mecp2 deficiency, oxidative stress and RTT pathology, as demonstrated by the rescue of the brain oxidative homeostasis following brain-specifically Mecp2-reactivated mice. The present study indicates that oxidative brain damage is a previously unrecognized hallmark feature of murine RTT, and suggests that Mecp2 is involved in the protection of the brain from oxidative stress.

Autism spectrum disorders (ASD) are characterized by a high degree of genetic heterogeneity. Genomic studies identified common pathological processes underlying the heterogeneous clinical manifestations of ASD, and transcriptome analyses revealed that gene networks involved in synapse development, neuronal activity and immune function are deregulated in ASD. Mouse models provide unique tools to investigate the neurobiological basis of ASD; however, a comprehensive approach to identify transcriptional abnormalities in different ASD models has never been performed. Here we used two well-recognized ASD mouse models, BTBR T+ Itpr3tf/J (BTBR) and Engrailed-2 knockout (En2-/-), to identify conserved ASD-related molecular signatures. En2-/- mice bear a mutation within the EN2 transcription factor homeobox, while BTBR is an inbred strain with unknown genetic defects. Hippocampal RNA samples from BTBR, En2-/- and respective control (C57Bl/6J and En2+/+) adult mice were assessed for differential gene expression using microarrays. A total of 153 genes were similarly deregulated in the BTBR and En2-/- hippocampus. Mouse phenotype and gene ontology enrichment analyses were performed on BTBR and En2-/- hippocampal differentially expressed genes (DEGs). Pathways represented in both BTBR and En2-/- hippocampal DEGs included abnormal behavioral response and chemokine/MAP kinase signaling. Genes involved in abnormal function of the immune system and abnormal synaptic transmission/seizures were significantly represented among BTBR and En2-/- DEGs, respectively. Interestingly, both BTBR and En2-/- hippocampal DEGs showed a significant enrichment of ASD and schizophrenia (SCZ)-associated genes. Specific gene sets were enriched in the two models: microglial genes were significantly enriched among BTBR DEGs, whereas GABAergic/glutamatergic postsynaptic genes, FMRP-interacting genes and epilepsy-related genes were significantly enriched among En2-/- DEGs. Weighted correlation network analysis (WGCNA) performed on BTBR and En2-/- hippocampal transcriptomes together identified 6 modules significantly enriched in ASD-related genes. Each of these modules showed a specific enrichment profile in neuronal and glial genes, as well as in genes associated to ASD comorbidities such as epilepsy and SCZ. Our data reveal significant transcriptional similarities and differences between the BTBR and En2-/- hippocampus, indicating that transcriptome analysis of ASD mouse models may contribute to identify novel molecular targets for pharmacological studies.

Autism spectrum disorders are characterized by impaired social and communicative skills and repetitive behaviors. Emerging evidence supported the hypothesis that these neurodevelopmental disorders may result from a combination of genetic susceptibility and exposure to environmental toxins in early developmental phases. This study assessed the effects of prenatal exposure to chlorpyrifos (CPF), a widely diffused organophosphate insecticide endowed with developmental neurotoxicity at sub-toxic doses, in the BTBR T+tf/J mouse strain, a validated model of idiopathic autism that displays several behavioral traits relevant to the autism spectrum. To this aim, pregnant BTBR mice were administered from gestational day 14 to 17 with either vehicle or CPF at a dose of 6 mg/kg/bw by oral gavages. Offspring of both sexes underwent assessment of early developmental milestones, including somatic growth, motor behavior and ultrasound vocalization. To evaluate the potential long-term effects of CPF, two different social behavior patterns typically altered in the BTBR strain (free social interaction with a same-sex companion in females, or interaction with a sexually receptive female in males) were also examined in the two sexes at adulthood. Our findings indicate significant effects of CPF on somatic growth and neonatal motor patterns. CPF treated pups showed reduced weight gain, delayed motor maturation (i.e., persistency of immature patterns such as pivoting at the expenses of coordinated locomotion) and a trend to enhanced ultrasound vocalization. At adulthood, CPF associated alterations were found in males only: the altered pattern of investigation of a sexual partner, previously described in BTBR mice, was enhanced in CPF males, and associated to increased ultrasonic vocalization rate. These findings strengthen the need of future studies to evaluate the role of environmental chemicals in the etiology of neurodevelopment disorders.

BTBR T+ tf/J (BTBR) is an inbred mouse strain that displays social abnormalities and repetitive behaviors analogous to the first and third diagnostic symptoms of autism. Here we investigate ultrasonic vocalizations in BTBR, to address the second diagnostic symptom of autism, communication deficits. As compared to the commonly used C57BL/6J (B6) strain, BTBR pups called more loudly and more frequently when separated from their mothers and siblings. Detailed analysis of ten categories of calls revealed an unusual pattern in BTBR as compared to B6. BTBR emitted high levels of harmonics, two-syllable, and composite calls, but minimal numbers of chevron-shaped syllables, upward, downward, and short calls. Because body weights were higher in BTBR than B6 pups, one possible explanation was that larger thoracic size was responsible for the louder calls and different distribution of syllable categories. To test this possibility, we recorded separation calls from FVB/NJ, a strain with body weights similar to BTBR, and 129X1/SvJ, a strain with body weights similar to B6. BTBR remained the outlier on number of calls, displaying low numbers of complex, upward, chevron, short, and frequency steps calls, along with high harmonics and composites. Further, developmental milestones and growth rates were accelerated in BTBR, indicating an unusual neurodevelopmental trajectory. Overall, our findings demonstrate strain-specific patterns of ultrasonic calls that may represent different lexicons, or innate variations in complex vocal repertoires, in genetically distinct strains of mice. Particularly intriguing is the unusual pattern of vocalizations and the more frequent, loud harmonics evident in the BTBR mouse model of autism that may resemble the atypical vocalizations seen in some autistic infants.