Epigenetic mechanisms linking early-life adversities and mental health.

Early-life adversities, whether prenatal or postnatal exposure, have been linked to adverse mental health outcomes later in life increasing the risk of several psychiatric disorders. Research on its neurobiological consequences demonstrated an association between exposure to adversities and persistent alterations in the structure, function, and connectivity of the brain. Consistent evidence supports the idea that regulation of gene expression through epigenetic mechanisms are involved in embedding the impact of early-life experiences in the genome and mediate between social environments and later behavioral phenotypes. In addition, studies from rodent models and humans suggest that these experiences and the acquired risk factors can be transmitted through epigenetic mechanisms to offspring and the following generations potentially contributing to a cycle of disease or disease risk. However, one of the important aspects of epigenetic mechanisms, unlike genetic sequences that are fixed and unchangeable, is that although the epigenetic markings are long-lasting, they are nevertheless potentially reversible. In this review, we summarize our current understanding of the epigenetic mechanisms involved in the mental health consequences derived from early-life exposure to malnutrition, maltreatment and poverty, adversities with huge and pervasive impact on mental health. We also discuss the evidence about transgenerational epigenetic inheritance in mammals and experimental data suggesting that suitable social and pharmacological interventions could reverse adverse epigenetic modifications induced by early-life negative social experiences. In this regard, these studies must be accompanied by efforts to determine the causes that promote these adversities and that result in health inequity in the population.

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.

Early life stress induces visual dysfunction and retinal structural alterations in adult mice.

Early life stress (ELS) is defined as a period of severe and/or chronic trauma, as well as environmental/social deprivation or neglect in the prenatal/early postnatal stage. Presently, the impact of ELS on the retina in the adult stage is unknown. The long-term consequences of ELS at retinal level were analyzed in an animal model of maternal separation with early weaning (MSEW), which mimics early life maternal neglect. For this purpose, mice were separated from the dams for 2 h at postnatal days (PNDs) 4-6, for 3 h at PNDs 7-9, for 4 h at PNDs 10-12, for 6 h at PNDs 13-16, and weaned at PND17. At the end of each separation period, mothers were subjected to movement restriction for 10 min. Control pups were left undisturbed from PND0, and weaned at PND21. Electroretinograms, visual evoked potentials, vision-guided behavioral tests, retinal anterograde transport, and retinal histopathology were examined at PNDs 60-80. MSEW induced long-lasting functional and histological effects at retinal level, including decreased retinal ganglion cell function and alterations in vision-guided behaviors, likely associated to decreased synaptophysin content, retina-superior colliculus communication deficit, increased microglial phagocytic activity, and retinal ganglion cell loss through a corticoid-dependent mechanism. A treatment with mifepristone, injected every 3 days between PNDs 4 and16, prevented functional and structural alterations induced by MSEW. These results suggest that retinal alterations might be included among the childhood adversity-induced threats to life quality, and that an early intervention with mifepristone avoided ELS-induced retinal disturbances.

Perinatal protein malnutrition induces the emergence of enduring effects and age-related impairment behaviors, increasing the death risk in a mouse model.

BACKGROUND: Early-life adversity impacts on the offspring's brain development and is associated with a higher risk of developing age-associated diseases. In particular, perinatal protein malnutrition appears to be one of the most critical nutritional deficiencies affecting the individual's health and survival, but little is known about its effects on the persistence of behavioral alterations throughout life. Thus, the aim of the present study was to investigate how perinatal protein malnutrition impacts on age-related changes in the neuromuscular, cognitive and behavioral functions throughout life in a mouse model. METHODS: One group of CF-1 dams received a normal-protein diet (NP: 20% casein) during gestation and lactation, whereas another group received a low-protein diet (LP: 10% casein). The offspring of both groups were analyzed by means of several behavioral tests at four different ages (young: 6-10 weeks old, mature: 22-26 weeks old, middle age: 39-43 weeks old, and old: 55-59 weeks old). RESULTS: Regarding neuromuscular functions, LP mice showed an early deterioration in muscular strength and a reduction in the body weight throughout life. Regarding behavior, while NP mice showed an age-related reduction of exploratory behavior, LP mice showed a constantly low level of this behavior, as well as high anxiety-like behavior, which remained at high levels throughout life. Regarding cognitive functions, LP mice showed deteriorated working memory at middle age. Finally, LP mice died 3.4 times earlier than NP mice. Analysis of the sex-related vulnerability showed that females and males were equally affected by perinatal protein malnutrition throughout life. CONCLUSION: Our results demonstrate that perinatal protein malnutrition induces enduring and age-related impairment behaviors, which culminate in higher death risk, affecting males and females equally.

Nutritional stress timing differentially programs cognitive abilities in young adult male mice.

Objectives: The impact of chronic exposure to environmental adversities on brain regions involved in cognition and mental health depends on whether it occurs during the perinatal period, childhood, adolescence or adulthood. The effects of these adversities on the brain and behavior arise as a function of the timing of the exposure and their co-occurrence with the development of specific regions. Here we aimed to explore the behavioral phenotypes derived from two nutritional stress paradigms which differed in the timing of exposure: a low-protein perinatal diet during gestation and lactation and a low-protein diet during adolescence.Methods: Locomotor and exploratory activity, recognition memory and aversive memory were measured in CF-1 8-week-old male mice subjected to perinatal malnutrition (LP-P) or adolescent malnutrition (LP-A), and their respective controls with normal protein diet (NP-P and NP-A).Results: By using the open field test, we found that LP-P and LP-A mice showed reduced exploratory activity compared to controls, but no alterations in their locomotor activity. Recognition memory was impaired only in LP-P mice. Interestingly, aversive memory was not altered in LP-P mice but was enhanced in LP-A mice. Considering the stress-inoculation theory, we hypothesized that protein malnutrition during adolescence represents a challenging but still moderate stressful environment, which promotes active coping in face of later adversity.Conclusion: Our results indicate that while perinatal malnutrition impairs recognition memory, adolescent malnutrition enhances aversive memory, showing dissimilar adaptive responses.

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.

Limited contextual memory and transcriptional dysregulation in the medial prefrontal cortex of mice exposed to early protein malnutrition are intergenerationally transmitted.

Memory contextualization is vital for the subsequent retrieval of relevant memories in specific situations and is a critical dimension of social cognition. The inability to properly contextualize information has been described as characteristic of psychiatric disorders like autism spectrum disorders, schizophrenia, and post-traumatic stress disorder. The exposure to early-life adversities, such as nutritional deficiency, increases the risk to trigger alterations in different domains of cognition related to those observed in mental diseases. In this work, we explored the consequences of exposure to perinatal protein malnutrition on contextual memory in a mouse model and assessed whether these consequences are transmitted to the next generation. Female mice were fed with a normal or hypoproteic diet during pregnancy and lactation. To evaluate contextual memory, the object-context mismatch test was performed in both sexes of F1 offspring and in the subsequent F2 generation. We observed that contextual memory was altered in mice of both sexes that had been subjected to maternal protein malnutrition and that the deficit in contextual memory was transmitted to the next generation. The basis of this alteration seems to be a transcriptional dysregulation of genes involved in the excitatory and inhibitory balance and immediate-early genes within the medial prefrontal cortex (mPFC) of both generations. The expression of genes encoding enzymes that regulate H3K27me3 levels was altered in the mPFC and partially in sperm of F1 malnourished mice. These results support the hypothesis that early nutritional deficiency represents a risk factor for the emergence of symptoms associated with mental 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.

Intergenerational transmission of maternal care deficiency and offspring development delay induced by perinatal protein malnutrition.

Objectives: Early life represents a sensitive and critical period for an individual. Nutrition plays a crucial role in the maturation and functional development of the central nervous system. Inadequate nutrition before birth and during the postnatal life can seriously interfere with brain development and lead to behavioral and neurological disorders such as learning disabilities and psychiatric diseases. In addition, the quality of mother-infant interactions represents an important adaptive pathway that prepares offspring for the conditions of life. In this work, we asked if protein malnutrition alters maternal care and offspring development and if these phenotypes can be transmitted to next generation.Methods: Female mice were fed with a normal or hypoproteic diet during pregnancy and lactation. Nurturing behaviors, i.e. arched, blanket and passive nursing, and liking and grooming of the pups, were evaluated from postnatal day 1 (PD1) to postnatal day 7 (PD7). The same protocol was employed to evaluate maternal behavior for filial generation 1 (F1) and filial generation 2 (F2) dams. Offspring development was evaluated for F1, F2, and F3 generations. Developmental landmarks and neurological reflexes were assessed from PD8 until complete development of the landmark or acquisition of the reflex.Results: Our results show that malnourished dams provide a lesser and more fragmented maternal care than their normally fed counterparts. This altered maternal behavior as well as the delay in the physical and neurological development observed in the offspring from malnourished mothers was transmitted up to two generations at least.Conclusion: These results highlight the harmful effects of protein malnutrition even for generations that are not directly exposed to this environmental adversity.

Effects of cocaine base paste on anxiety-like behavior and immediate-early gene expression in nucleus accumbens and medial prefrontal cortex of female mice.

RATIONALE: Cocaine base paste (CBP) is an illegal drug of abuse usually consumed by adolescents in a socio-economically vulnerable situation. Repeated drug use targets key brain circuits disrupting the processes that underlie emotions and cognition. At the basis of such neuroadaptations lie changes in the expression of immediate-early genes (IEGs). Nevertheless, changes in transcriptional regulation associated with CBP consumption remain unknown. OBJECTIVES: We aimed to describe behavioral phenotype related to locomotion, anxiety-like behavior, and memory of CBP-injected mice and to study IEGs expression after an abstinence period. METHODS: Five-week-old female CF-1 mice were i.p. injected daily with vehicle or CBP (40 mg/kg) for 10 days and subjected to a 10-day period of abstinence. Open field and novel object recognition tests were used to evaluate locomotion and anxiety-like behaviors and recognition memory, respectively, during chronic administration and after abstinence. After abstinence, prefrontal cortex (mPFC) and nucleus accumbens (NAc) were isolated and gene expression analysis performed through real-time PCR. RESULTS: We found an increase in locomotion and anxiety-like behavior during CBP administration and after the abstinence period. Furthermore, the CBP group showed impaired recognition memory after abstinence. Egr1, FosB, ΔFosB, Arc, Bdnf, and TrkB expression was upregulated in CBP-injected mice in NAc and FosB, ΔFosB, Arc, and Npas4 expression was downregulated in mPFC. We generated an anxiety score and found positive and negative correlations with IEGs expression in NAc and mPFC, respectively. CONCLUSION: Our results suggest that chronic CBP exposure induced alterations in anxiety-like behavior and recognition memory. These changes were accompanied by altered IEGs expression.

Multimodal neurocognitive markers of interoceptive tuning in smoked cocaine.

Contemporary neurocognitive models of drug addiction have associated this condition with changes in interoception -namely, the sensing and processing of body signals that fulfill homeostatic functions relevant for the onset and maintenance of addictive behavior. However, most previous evidence is inconsistent, behaviorally unspecific, and virtually null in terms of direct electrophysiological and multimodal markers. To circumvent these limitations, we conducted the first assessment of the relation between cardiac interoception and smoked cocaine dependence (SCD) in a sample of (a) 25 participants who fulfilled criteria for dependence on such a drug, (b) 22 participants addicted to insufflated clorhidrate cocaine (only for behavioral assessment), and (c) 25 healthy controls matched by age, gender, education, and socioeconomic status. We use a validated heartbeat-detection (HBD) task and measured modulations of the heart-evoked potential (HEP) during interoceptive accuracy and interoceptive learning conditions. We complemented this behavioral and electrophysiological data with offline structural (MRI) and functional connectivity (fMRI) analysis of the main interoceptive hubs. HBD and HEP results convergently showed that SCD subjects presented ongoing psychophysiological measures of enhanced interoceptive accuracy. This pattern was associated with a structural and functional tuning of interoceptive networks (reduced volume and specialized network segregation). Taken together, our findings provide the first evidence of an association between cardiac interoception and smoked cocaine, partially supporting models that propose hyper-interoception as a key aspect of addiction. More generally, our study shows that multimodal assessments of interoception could substantially inform the clinical and neurocognitive characterization of psychophysiological and neurocognitive adaptations triggered by addiction.

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.

Perinatal protein malnutrition alters expression of miRNA biogenesis genes Xpo5 and Ago2 in mice brain.

Due to its widespread incidence, maternal malnutrition remains one of the major non-genetic factors affecting the development of newborn's brain. While all nutrients have certain influence on brain maturation, proteins appear to be the most critical for the development of neurological functions. An increasing number of studies point out that the effects of early-life nutritional inadequacy has long lasting effects on the brain and lead to permanent deficits in learning and behavior. Epigenetic mechanisms provide a potential link between the nutrition status during critical periods and changes in gene expression that may lead to disease phenotypes. Among those epigenetic mechanisms microRNAs (miRNAs) emerge as promising molecules for the link between nutrition and gene expression due to their relevance in many central nervous system functions. The objective of the current study was to evaluate the impact of perinatal protein malnutrition on the development of male and female mice offspring and to analyze the expression of the genes involved in the miRNA biogenesis pathway in different mouse brain structures. We demonstrated that early nutritional stress such as exposition to a protein-deficient diet during gestation and lactation reduced the hippocampal weight, delayed offspring's development and deregulated the expression of Xpo5 and Ago2 genes in hippocampus and hypothalamus of weanling mice. Moreover, an overall increase in mature miRNAs was consistent with the induction of Xpo5 mRNA. Altered miRNA biogenesis could modify the availability and functionality of miRNA becoming a causal factor of the adverse effects of protein malnutrition.

Oxidative stress-induced CREB upregulation promotes DNA damage repair prior to neuronal cell death protection.

cAMP response element-binding (CREB) protein is a cellular transcription factor that mediates responses to different physiological and pathological signals. Using a model of human neuronal cells we demonstrate herein, that CREB is phosphorylated after oxidative stress induced by hydrogen peroxide. This phosphorylation is largely independent of PKA and of the canonical phosphoacceptor site at ser-133, and is accompanied by an upregulation of CREB expression at both mRNA and protein levels. In accordance with previous data, we show that CREB upregulation promotes cell survival and that its silencing results in an increment of apoptosis after oxidative stress. Interestingly, we also found that CREB promotes DNA repair after treatment with hydrogen peroxide. Using a cDNA microarray we found that CREB is responsible for the regulation of many genes involved in DNA repair and cell survival after oxidative injury. In summary, the neuroprotective effect mediated by CREB appears to follow three essential steps following oxidative injury. First, the upregulation of CREB expression that allows sufficient level of activated and phosphorylated protein is the primordial event that promotes the induction of genes of the DNA Damage Response. Then and when the DNA repair is effective, CREB induces detoxification and survival genes. This kinetics seems to be important to completely resolve oxidative-induced neuronal damages.

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells.

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.

Nuclear translocation of p19INK4d in response to oxidative DNA damage promotes chromatin relaxation.

DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site.

CDK5-mediated phosphorylation of p19INK4d avoids DNA damage-induced neurodegeneration in mouse hippocampus and prevents loss of cognitive functions.

DNA damage, which perturbs genomic stability, has been linked to cognitive decline in the aging human brain, and mutations in DNA repair genes have neurological implications. Several studies have suggested that DNA damage is also increased in brain disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. However, the precise mechanisms connecting DNA damage with neurodegeneration remain poorly understood. CDK5, a critical enzyme in the development of the central nervous system, phosphorylates a number of synaptic proteins and regulates dendritic spine morphogenesis, synaptic plasticity and learning. In addition to these physiological roles, CDK5 has been involved in the neuronal death initiated by DNA damage. We hypothesized that p19INK4d, a member of the cell cycle inhibitor family INK4, is involved in a neuroprotective mechanism activated in response to DNA damage. We found that in response to genotoxic injury or increased levels of intracellular calcium, p19INK4d is transcriptionally induced and phosphorylated by CDK5 which provides it with greater stability in postmitotic neurons. p19INK4d expression improves DNA repair, decreases apoptosis and increases neuronal survival under conditions of genotoxic stress. Our in vivo experiments showed that decreased levels of p19INK4d rendered hippocampal neurons more sensitive to genotoxic insult resulting in the loss of cognitive abilities that rely on the integrity of this brain structure. We propose a feedback mechanism by which the neurotoxic effects of CDK5-p25 activated by genotoxic stress or abnormal intracellular calcium levels are counteracted by the induction and stabilization of p19INK4d protein reducing the adverse consequences on brain functions.

Early protein malnutrition negatively impacts physical growth and neurological reflexes and evokes anxiety and depressive-like behaviors.

Malnutrition is a worldwide problem affecting millions of unborn and young children during the most vulnerable stages of their development. In humans, poor maternal nutrition is a major cause of intrauterine growth restriction which is associated with an increased risk of perinatal mortality and long-term morbidity. In addition, intrauterine growth restriction correlates with neurodevelopmental delays and alterations of brain structure and neurochemistry. While there is no doubt that maternal malnutrition is a principal cause of perturbed development of the fetal brain and that all nutrients have certain influence on brain maturation, proteins appear to be the most critical for the development of neurological functions. In the present study we assessed male and female mouse offspring, born to dams protein restricted during pregnancy and lactation, in physical growth and neurobehavioral development and also in social interaction, motivation, anxiety and depressive behaviors. Moreover, we evaluate the impact of the low protein diet on dams in relation to their maternal care and anxiety-related behavior given that these clearly affect pups development. We observed that maternal protein restriction during pregnancy and lactation delayed the physical growth and neurodevelopment of the offspring in a sex-independent manner. In addition, maternal undernutrition negatively affected offspring's juvenile social play, motivation, exploratory activity and risk assessment behaviors. These findings show that protein restriction during critical periods of development detrimentally program progeny behavior.

p19INK4d is involved in the cellular senescence mechanism contributing to heterochromatin formation.

BACKGROUND: During evolution, organisms with renewable tissues have developed mechanisms to prevent tumorigenesis, including cellular senescence and apoptosis. Cellular senescence is characterized by a permanent cell cycle arrest triggered by both endogenous stress and exogenous stress. The p19INK4d, a member of the family of cyclin-dependent kinase inhibitors (INK4), plays an important role on cell cycle regulation and in the cellular DNA damage response. We hypothesize that p19INK4d is a potential factor involved in the onset and/or maintenance of the senescent state. METHODS: Senescence was confirmed by measuring the cell cycle arrest and the senescence-associated ß-galactosidase activity. Changes in p19INK4d expression and localization during senescence were determined by Western blot and immunofluorescence assays. Chromatin condensation was measured by microccocal nuclease digestion and histone salt extraction. RESULTS: The data presented here show for the first time that p19INK4d expression is up-regulated by different types of senescence. Changes in senescence-associated hallmarks were driven by modulation of p19 expression indicating a direct link between p19INK4d induction and the establishment of cellular senescence. Following a senescence stimulus, p19INK4d translocates to the nucleus and tightly associates with chromatin. Moreover, reduced levels of p19INK4d impair senescence-related global genomic heterochromatinization. Analysis of p19INK4d mRNA and protein levels in tissues from differently aged mice revealed an up-regulation of p19INK4d that correlates with age. CONCLUSION: We propose that p19INK4d participates in the cellular mechanisms that trigger senescence by contributing to chromatin compaction. GENERAL SIGNIFICANCE: This study provides novel insights into the dynamics process of cellular senescence, a central tumor suppressive mechanism.

Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies.