Hippocampal and peripheral blood DNA methylation signatures correlate at the gene and pathway level in a mouse model of autism.

Autism spectrum disorders (ASD) are polygenic multifactorial disorders influenced by environmental factors. ASD-related differential DNA methylation has been found in human peripheral tissues, such as placenta, paternal sperm, buccal epithelium, and blood. However, these data lack direct comparison of DNA methylation levels with brain tissue from the same individual to determine the extent that peripheral tissues are surrogates for behavior-related disorders. Here, whole genome methylation profiling at all the possible sites throughout the mouse genome (>25 million) from both brain and blood tissues revealed novel insights into the systemic contributions of DNA methylation to ASD. Sixty-six differentially methylated regions (DMRs) share the same genomic coordinates in these two tissues, many of which are linked to risk genes for neurodevelopmental disorders and intellectual disabilities (e.g. Prkch, Ptn, Hcfc1, Mid1, and Nfia). Gene ontological pathways revealed a significant number of common terms between brain and blood (N = 65 terms), and nearly half (30/65) were associated with brain/neuronal development. Furthermore, seven DMR-associated genes among these terms contain methyl-sensitive transcription factor sequence motifs within the DMRs of both tissues; four of them (Cux2, Kcnip2, Fgf13, and Mrtfa) contain the same methyl-sensitive transcription factor binding sequence motifs (HES1/2/5, TBX2 and TFAP2C), suggesting DNA methylation influences the binding of common transcription factors required for gene expression. Together, these findings suggest that peripheral blood is a good surrogate tissue for brain and support that DNA methylation contributes to altered gene regulation in the pathogenesis of ASD.

Oxidative stress associated with spatial memory impairment and social olfactory deterioration in female mice reveals premature aging aroused by perinatal protein malnutrition.

Early-life adversity, like perinatal protein malnutrition, increases the vulnerability to develop long-term alterations in brain structures and function. This study aimed to determine whether perinatal protein malnutrition predisposes to premature aging in a murine model and to assess the cellular and molecular mechanisms involved. To this end, mouse dams were fed either with a normal (NP, casein 20%) or a low-protein diet (LP, casein 8%) during gestation and lactation. Female offspring were evaluated at 2, 7 and 12 months of age. Positron emission tomography analysis showed alterations in the hippocampal CA3 region and the accessory olfactory bulb of LP mice during aging. Protein malnutrition impaired spatial memory, coinciding with higher levels of reactive oxygen species in the hippocampus and sirt7 upregulation. Protein malnutrition also led to higher senescence-associated ß-galactosidase activity and p21 expression. LP-12-month-old mice showed a higher number of newborn neurons that did not complete the maturation process. The social-odor discrimination in LP mice was impaired along life. In the olfactory bulb of LP mice, the senescence marker p21 was upregulated, coinciding with a downregulation of Sirt2 and Sirt7. Also, LP-12-month-old mice showed a downregulation of catalase and glutathione peroxidase, and LP-2-month-old mice showed a higher number of newborn neurons in the subventricular zone, which then returned to normal values. Our results show that perinatal protein malnutrition causes long-term impairment in cognitive and olfactory skills through an accelerated senescence phenotype accompanied by an increase in oxidative stress and altered sirtuin expression in the hippocampus and olfactory bulb.

Impaired social cognition caused by perinatal protein malnutrition evokes neurodevelopmental disorder symptoms and is intergenerationally transmitted.

Nutritional inadequacy before birth and during postnatal life can seriously interfere with brain development and lead to persistent deficits in learning and behavior. In this work, we asked if protein malnutrition affects domains of social cognition and if these phenotypes can be transmitted to the next generation. Female mice were fed with a normal or hypoproteic diet during pregnancy and lactation. After weaning, offspring were fed with a standard chow. Social interaction, social recognition memory, and dominance were evaluated in both sexes of F1 offspring and in the subsequent F2 generation. Glucose metabolism in the whole brain was analyzed through preclinical positron emission tomography. Genome-wide transcriptional analysis was performed in the medial prefrontal cortex followed by gene-ontology enrichment analysis. Compared with control animals, malnourished mice exhibited a deficit in social motivation and recognition memory and displayed a dominant phenotype. These altered behaviors, except for dominance, were transmitted to the next generation. Positron emission tomography analysis revealed lower glucose metabolism in the medial prefrontal cortex of F1 malnourished offspring. This brain region showed genome-wide transcriptional dysregulation, including 21 transcripts that overlapped with autism-associated genes. Our study cannot exclude that the lower maternal care provided by mothers exposed to a low-protein diet caused an additional impact on social cognition. Our results showed that maternal protein malnutrition dysregulates gene expression in the medial prefrontal cortex, promoting altered offspring behavior that was intergenerationally transmitted. These results support the hypothesis that early nutritional deficiency represents a risk factor for the emergence of symptoms associated with neurodevelopmental disorders.

Perinatal protein malnutrition results in genome-wide disruptions of 5-hydroxymethylcytosine at regions that can be restored to control levels by an enriched environment.

Maternal malnutrition remains one of the major adversities affecting brain development and long-term mental health outcomes, increasing the risk to develop anxiety and depressive disorders. We have previously shown that malnutrition-induced anxiety-like behaviours can be rescued by a social and sensory stimulation (enriched environment) in male mice. Here, we expand these findings to adult female mice and profiled genome-wide ventral hippocampal 5hmC levels related to malnutrition-induced anxiety-like behaviours and their rescue by an enriched environment. This approach revealed 508 differentially hydroxymethylated genes associated with protein malnutrition and that several genes (N = 34) exhibited a restored 5hmC abundance to control levels following exposure to an enriched environment, including genes involved in neuronal functions like dendrite outgrowth, axon guidance, and maintenance of neuronal circuits (e.g. Fltr3, Itsn1, Lman1, Lsamp, Nav, and Ror1) and epigenetic mechanisms (e.g. Hdac9 and Dicer1). Sequence motif predictions indicated that 5hmC may be modulating the binding of transcription factors for several of these transcripts, suggesting a regulatory role for 5hmC in response to perinatal malnutrition and exposure to an enriched environment. Together, these findings establish a role for 5hmC in early-life malnutrition and reveal genes linked to malnutrition-induced anxious behaviours that are mitigated by an enriched environment.

Exposure to enriched environment rescues anxiety-like behavior and miRNA deregulated expression induced by perinatal malnutrition while altering oligodendrocyte morphology.

Maternal malnutrition is one of the major early-life adversities affecting the development of newborn's brain and is associated with an increased risk to acquire cognitive and emotional deficiencies later in life. Studies in rodents have demonstrated that exposure to an enriched environment (EE) can reverse the negative consequences of early adversities. However, rescue of emotional disorders caused by perinatal malnutrition and the mechanisms involved has not been determined. We hypothesized that exposure to an EE may attenuate the anxiety-like disorders observed in mice subjected to perinatal protein malnutrition and that this could be mediated by epigenetic mechanisms. Male CF-1 mice were subject to perinatal protein malnutrition until weaning and then exposed to an EE for 5 weeks after which small RNA-seq was performed. In parallel, dark-light box and elevated plus maze tests were conducted to evaluate anxiety traits. We found that exposure to an EE reverses the anxiety-like behavior in malnourished mice. This reversal is paralleled by the expression of three miRNAs that become dysregulated by perinatal malnutrition (miR-187-3p, miR-369-3p and miR-132-3p). The predicted mRNA targets of these miRNAs are mostly related to axon guidance pathway. Accordingly, we also found that perinatal malnutrition leads to reduction in the cingulum size and altered oligodendrocyte morphology. These results suggest that EE-rescue of anxiety disorders derived from perinatal malnutrition is mediated by the modulation of miRNAs associated with the regulation of genes involved in axonal guidance.