Psychiatric diseases have a strong heritable component known to not be restricted to DNA sequence-based genetic inheritance alone but to also involve epigenetic factors in germ cells. Initial evidence suggested that sperm RNA is causally linked to the transmission of symptoms induced by traumatic experiences. Here, we show that alterations in long RNA in sperm contribute to the inheritance of specific trauma symptoms. Injection of long RNA fraction from sperm of males exposed to postnatal trauma recapitulates the effects on food intake, glucose response to insulin and risk-taking in adulthood whereas the small RNA fraction alters body weight and behavioural despair. Alterations in long RNA are maintained after fertilization, suggesting a direct link between sperm and embryo RNA.

In the past decades, evidence supporting the transmission of acquired traits across generations has reshaped the field of genetics and the understanding of disease susceptibility. In humans, pioneer studies showed that exposure to famine, endocrine disruptors or trauma can affect descendants, and has led to a paradigm shift in thinking about heredity. Studies in humans have however been limited by the low number of successive generations, the different conditions that can be examined, and the lack of mechanistic insight they can provide. Animal models have been instrumental to circumvent these limitations and allowed studies on the mechanisms of inheritance of environmentally induced traits across generations in controlled and reproducible settings. However, most models available today are only intergenerational and do not demonstrate transmission beyond the direct offspring of exposed individuals. Here, we report transgenerational transmission of behavioral and metabolic phenotypes up to the 4th generation in a mouse model of paternal postnatal trauma (MSUS). Based on large animal numbers (up to 124 per group) from several independent breedings conducted 10 years apart by different experimenters, we show that depressive-like behaviors are transmitted to the offspring until the third generation, and risk-taking and glucose dysregulation until the fourth generation via males. The symptoms are consistent and reproducible, and persist with similar severity across generations. These results provide strong evidence that adverse conditions in early postnatal life can have transgenerational effects, and highlight the validity of MSUS as a solid model of transgenerational epigenetic inheritance.

30Psychiatric diseases have a strong heritable component known to not be restricted to 31 DNA sequence-based genetic inheritance alone but to also involve epigenetic factors in 32 germ cells 1,2 . Initial evidence suggested that sperm RNA is causally linked 2,3 to the 33 transmission of symptoms induced by traumatic experiences. Here we show that 34 alterations in long RNA in sperm contribute to the inheritance of specific trauma 35 symptoms. Injection of long RNA fraction from sperm of males exposed to postnatal 36 trauma recapitulates the effects on food intake, glucose response to insulin and risk-37 taking in adulthood whereas the small RNA fraction alters body weight and behavioral 38 despair. Alterations in long RNA are maintained after fertilization, suggesting a direct 39 link between sperm and embryo RNA. 40 41 42 43 Adverse experiences can have long-lasting transgenerational effects on mental and 46 physical health, and often increase disease risk 4,5 . Traumatic stress in early life in 47 particular, can induce pathologies like psychosis, depression and metabolic 48 dysfunctions in adulthood across generations 6 . To examine the biological factors 49 involved, we recapitulated heritable behavioural and metabolic effects of postnatal 50 trauma across several generations using a previously established model of 51 unpredictable maternal separation combined with unpredictable maternal stress 52(MSUS) in the mouse, that shows symptoms through up to three generations ( Fig.1) 2,7-53 13 . We have shown that such postnatal trauma alters small RNA in sperm and that 54 injection of total sperm RNA from exposed male mice into naïve fertilized oocytes elicits 55 symptoms reminiscent of those observed in natural offspring of exposed fathers 2 . Other 56 studies have demonstrated that adult stress 14,15 and environmental insults like altered 57 diet or vinclozolin exposure, or positive factors such as exercise or environmental 58 enrichment can affect small RNA in sperm 16-21 and somatic tissues 22 in the offspring.59Recently, tRNA fragments and their modifications were also found to be affected by 60 nutritional insult, and unmodified or modified sperm small RNA injected into fertilized 61 oocytes could mimic metabolic changes resulting from altered parental diet in the 62 progeny 18,23,24 . These studies therefore suggest that small RNA in sperm can be 63 carrier of heritable information. Here we sought to determine whether long RNA in 64 sperm also contributes to the transmission of the effects of previous exposure. 66 Materials and Methods 68Mice. C57BL/6J mice were housed in a temperature and humidity-controlled facility 69 under a reverse light-dark cycle, and food and water were provided ad libitum. 70 Experimental procedures were performed during the animals' active cycle. All71 experiments were approved by the cantonal veterinary office, Zurich (license 55/12 then 72 57/15).73 74 MSUS paradigm. C57BL/6J primiparous females and males were mated at 2.5-3 75 months of age. Randomly selected dams and litt...

Environmental factors can change phenotypes in exposed individuals and offspring and involve the germline, likely via biological signals in the periphery that communicate with germ cells. Here, using a mouse model of paternal exposure to traumatic stress, we identify circulating factors involving peroxisome proliferatoractivated receptor (PPAR) pathways in the effects of exposure to the germline. We show that exposure alters metabolic functions and pathways, particularly lipid-derived metabolites, in exposed fathers and their offspring. We collected data in a human cohort exposed to childhood trauma and observed similar metabolic alterations in circulation, suggesting conserved effects. Chronic injection of serum from trauma-exposed males into controls recapitulates metabolic phenotypes in the offspring. We identify lipid-activated nuclear receptors PPARs as potential mediators of the effects from father to offspring. Pharmacological PPAR activation in vivo reproduces metabolic dysfunctions in the offspring and grand-offspring of injected males and affects the sperm transcriptome in fathers and sons. In germ-like cells in vitro, both serum and PPAR agonist induce PPAR activation. Together, these results highlight the role of circulating factors as potential communication vectors between the periphery and the germline.

Adverse environmental and social conditions early in life have a strong impact on health. They are major risk factors for mental diseases in adulthood and, in some cases, their effects can be transmitted across generations. The consequences of detrimental stress conditions on brain metabolism across generations are not well known. Using high-field (14.1 T) magnetic resonance spectroscopy, we investigated the neurochemical profile of adult male mice exposed to traumatic stress in early postnatal life and of their offspring, and of undisturbed control mice. We found that, relative to controls, early life stress-exposed mice have metabolic alterations consistent with neuronal dysfunction, including reduced concentration of N-acetylaspartate, glutamate and γ-aminobutyrate, in the prefrontal cortex in basal conditions. Their offspring have normal neurochemical profiles in basal conditions. Remarkably, when challenged by an acute cold swim stress, the offspring has attenuated metabolic responses in the prefrontal cortex, hippocampus and striatum. In particular, the expected stress-induced reduction in the concentration of N-acetylaspartate, a putative marker of neuronal health, was prevented in the cortex and hippocampus. These findings suggest that paternal trauma can confer beneficial brain metabolism adaptations to acute stress in the offspring.

Chronic and excessive alcohol misuse results in changes in the expression of selected miRNAs and their mRNA targets in specific regions of the human brain. These expression changes likely underlie the cellular adaptations to long term alcohol misuse. In order to delineate the mechanism by which these expression changes occur, we have measured the expression of six miRNAs including miR-7, miR-153, miR-152, miR-15B, miR-203 and miR-144 in HEK293T, SH SY5Y and 1321 N1 cells following exposure to ethanol. These miRNAs are predicted to target key genes involved in the pathophysiology of alcoholism. Chronic and chronic-intermittent exposure to ethanol, and its removal, resulted in specific changes in miRNA expression in each cell line suggesting that different expression patterns can be elicited with different exposure paradigms and that the mechanism of ethanol’s effects is dependent on cell type. Specifically, chronic exposure to ethanol for five days followed by a five day withdrawal period resulted in up-regulation of several miRNAs in each of these cell lines similar to expression changes identified in post mortem human brain. Thus, this model can be used to elucidate the role of miRNAs in regulating gene expression changes that occur in response to ethanol exposure.

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