Neuronal membrane glycoprotein (nmgp-1) gene deficiency affects chemosensation-related behaviors, dauer exit and egg-laying in Caenorhabditis elegans.

The nervous system monitors the environment to maintain homeostasis, which can be affected by stressful conditions. Using mammalian models of chronic stress, we previously observed altered brain levels of GPM6A, a protein involved in neuronal morphology. However, GPM6A's role in systemic stress responses remains unresolved. The nematode Caenorhabditis elegans expresses a GPM6A ortholog, the neuronal membrane glycoprotein 1 (NMGP-1). Because of the shared features between nematode and mammalian nervous systems and the vast genetic tools available in C. elegans, we used the worm to elucidate the role of GPM6A in the stress response. We first identified nmgp-1 expression in different amphid and phasmid neurons. To understand the nmgp-1 role, we characterized the behavior of nmgp-1(RNAi) animals and two nmgp-1 mutant alleles. Compared to control animals, mutant and RNAi-treated worms exhibited increased recovery time from the stress-resistant dauer stage, altered SDS chemosensation and reduced egg-laying rate resulting in egg retention (bag-of-worms phenotype). Silencing of nmgp-1 expression induced morphological abnormalities in the ASJ sensory neurons, partly responsible for dauer exit. These results indicate that nmgp-1 is required for neuronal morphology and for behaviors associated with chemosensation. Finally, we propose nmgp-1 mutants as a tool to screen drugs for human nervous system pathologies.

Prenatal restraint stress decreases the expression of alpha-7 nicotinic receptor in the brain of adult rat offspring.

Prenatal stress (PS) strongly impacts fetal brain development and function in adulthood. In normal aging and Alzheimer's disease, there is hypothalamic-pituitary-adrenal axis dysfunction and loss of cholinergic neurons and neuronal nicotinic acetylcholine receptors (nAChRs). This study investigated whether prenatal restraint stress affects nAChR expression in the brain of adult offspring. For PS, pregnant dams were placed in a plastic restrainer for 45 min, three times daily during the last week of pregnancy; controls were undisturbed. Male offspring were analyzed at postnatal day (PND) 60 (n = 4 rats per group). Western blot (WB) and fluorescence microscopy showed that PS decreased α7-AChR subunit expression (∼50%) in the frontal cortex in the adult offspring. PS decreased significantly the number of α7-AChR-expressing cells in the medial prefrontal cortex (by ∼25%) and in the sensory-motor cortex (by ∼20%) without affecting the total cell number in those areas. No alterations were found in the hippocampus by quantitative polymerase chain reaction (qPCR), or WB analysis, but a detailed fluorescence microscopy analysis showed that PS affected α7-AChR mainly in the CA3 and dentate gyrus subfields: PS decreased α7-AChR subunit expression by ∼25 and ∼30%, respectively. Importantly, PS decreased the number of α7-AChR-expressing cells and the total cell number (by ∼15 and 20%, respectively) in the dentate gyrus. PS differently affected α4-AChR: PS impaired its mRNA expression in the frontal cortex (by ∼50%), without affecting protein levels. These results demonstrate that disturbances during gestation produce long-term alterations in the expression pattern of α7-AChR in rat brain.

LAT1-dependent placental methionine uptake is a key player in fetal programming of metabolic disease.

The Developmental Origins of Health and Disease hypothesis sustains that exposure to different stressors during prenatal development prepares the offspring for the challenges to be encountered after birth. We studied the gestational period as a particularly vulnerable window where different stressors can have strong implications for fetal programming of the offspring's life-long metabolic status via alterations of specific placentally expressed nutrient transporters. To study this mechanism, we used a murine prenatal stress model, human preeclampsia, early miscarriage, and healthy placental tissue samples, in addition to in vitro models of placental cells. In stressed mice, placental overexpression of L-type amino acid transporter 1 (Lat1) and subsequent global placental DNA hypermethylation was accompanied by fetal and adult hypothalamic dysregulation in global DNA methylation and gene expression as well as long-term metabolic abnormalities exclusively in female offspring. In human preeclampsia, early miscarriage, and under hypoxic conditions, placental LAT1 was significantly upregulated, leading to increased methionine uptake and global DNA hypermethylation. Remarkably, subgroups of healthy term placentas with high expression of stress-related genes presented increased levels of placental LAT1 mRNA and protein, DNA and RNA hypermethylation, increased methionine uptake capacity, one-carbon metabolic pathway disruption, higher methionine concentration in the placenta and transport to the fetus specifically in females. Since LAT1 mediates the intracellular accumulation of methionine, global DNA methylation, and one-carbon metabolism in the placenta, our findings hint at a major sex-specific global response to a variety of prenatal stressors affecting placental function, epigenetic programming, and life-long metabolic disease and provide a much-needed insight into early-life factors predisposing females/women to metabolic disorders.

Prenatal maternal restraint stress exposure alters the reproductive hormone profile and testis development of the rat male offspring.

Several studies have demonstrated that the presence of stressors during pregnancy induces adverse effects on the neuroendocrine system of the offspring later in life. In the present work, we investigated the effects of early programming on the male reproductive system, employing a prenatal stress (PS) paradigm. This study found that when pregnant dams were placed in a plastic restrainer three times a day during the last week of pregnancy, the offspring showed reduced anogenital distance and delayed testicular descent. Serum luteinising hormone (LH) and follicle-stimulating hormone (FSH) levels were decreased at postnatal day (PND) 28 and testosterone was decreased at PND 75. Increased testosterone plus dihydrotestosterone (T + DHT) concentrations correlated with increased testicular 5α Reductase-1 (5αR-1) mRNA expression at PND 28. Moreover, PS accelerated spermatogenesis at PND 35 and 60, and increased mean seminiferous tubule diameter in pubertal offspring and reduced Leydig cell number was observed at PND 35 and 60. PS offspring had increased androgen receptor (AR) mRNA level at PND 28, and at PND 35 had increased the numbers of Sertoli cells immunopositive for AR. Overall, the results confirm that stress during gestation can induce long-term effects on the male offspring reproductive system. Of particular interest is the pre-pubertal imbalance of circulating hormones that probably trigger accelerated testicular development, followed by an increase in total androgens and a decrease in testosterone concentration during adulthood. Exposure to an unfavourable intrauterine environment might prepare for harsh external conditions by triggering early puberty, increasing reproductive potential.

Filopodial protrusions induced by glycoprotein M6a exhibit high motility and aids synapse formation.

M6a is a neuronal membrane glycoprotein whose expression diminishes during chronic stress. M6a overexpression in rat primary hippocampal neurons induces the formation of filopodial protrusions that could be spine precursors. As the filopodium and spine motility has been associated with synaptogenesis, we analysed the motility of M6a-induced protrusions by time-lapse imaging. Our data demonstrate that the motile protrusions formed by the neurons overexpressing M6a were more abundant and moved faster than those formed in control cells. When different putative M6a phosphorylation sites were mutated, the neurons transfected with a mutant lacking intracellular phosphorylation sites bore filopodia, but these protrusions did not move as fast as those formed by cells overexpressing wild-type M6a. This suggests a role for M6a phosphorylation state in filopodium motility. Furthermore, we show that M6a-induced protrusions could be stabilized upon contact with presynaptic region. The motility of filopodia contacting or not neurites overexpressing synaptophysin was analysed. We show that the protrusions that apparently contacted synaptophysin-labeled cells exhibited less motility. The behavior of filopodia from M6a-overexpressing cells and control cells was alike. Thus, M6a-induced protrusions may be spine precursors that move to reach presynaptic membrane. We suggest that M6a is a key molecule for spine formation during development.

Conserved cellular function and stress-mediated regulation among members of the proteolipid protein family.

Chronic stress causes morphological alterations in the hippocampus of rodents and tree shrews, including atrophy of CA3 dendrites and loss of synapses. The molecular mechanisms underlying these structural changes remain largely unknown. We have previously identified M6a as a stress responsive gene and shown that M6a is involved in filopodium/spine outgrowth and, likely, synapse formation. M6a belongs to the proteolipid protein (PLP) family, all of their members having four transmembrane domains that allow their localization at the plasma membrane. In the present work, we analyzed other members of this family, the closely related M6b as well as PLP and its splice variant DM20. We found that chronic restraint stress in mice reduces M6b and DM20, but not PLP, mRNA levels in the hippocampus. In addition, M6b and DM20, but again not PLP, induce filopodium formation in primary cultures of hippocampal neurons. Several M6b protein isoforms were studied, all of them having similar effects except for the one lacking the transmembrane domains. Our results reveal a conserved cellular function and a stress-mediated regulation among members of the proteolipid protein family, suggesting an involvement of proteolipid proteins in the stress response.

Non-invasive biomarkers of fetal brain development reflecting prenatal stress: An integrative multi-scale multi-species perspective on data collection and analysis.

Prenatal stress (PS) impacts early postnatal behavioural and cognitive development. This process of 'fetal programming' is mediated by the effects of the prenatal experience on the developing hypothalamic-pituitary-adrenal (HPA) axis and autonomic nervous system (ANS). We derive a multi-scale multi-species approach to devising preclinical and clinical studies to identify early non-invasively available pre- and postnatal biomarkers of PS. The multiple scales include brain epigenome, metabolome, microbiome and the ANS activity gauged via an array of advanced non-invasively obtainable properties of fetal heart rate fluctuations. The proposed framework has the potential to reveal mechanistic links between maternal stress during pregnancy and changes across these physiological scales. Such biomarkers may hence be useful as early and non-invasive predictors of neurodevelopmental trajectories influenced by the PS as well as follow-up indicators of success of therapeutic interventions to correct such altered neurodevelopmental trajectories. PS studies must be conducted on multiple scales derived from concerted observations in multiple animal models and human cohorts performed in an interactive and iterative manner and deploying machine learning for data synthesis, identification and validation of the best non-invasive detection and follow-up biomarkers, a prerequisite for designing effective therapeutic interventions.

In Vivo and In Vitro Neuronal Plasticity Modulation by Epigenetic Regulators.

Prenatal stress (PS) induces molecular changes that alter neural connectivity, increasing the risk for neuropsychiatric disorders. Here we analyzed -in the hippocampus of adult rats exposed to PS- the epigenetic signature mediating the PS-induced neuroplasticity changes. Furthermore, using cultured hippocampal neurons, we investigated the effects on neuroplasticity of an epigenetic modulator. PS induced significant modifications in the mRNA levels of stress-related transcription factor MEF2A, SUV39H1 histone methyltransferase, and TET1 hydroxylase, indicating that PS modifies gene expression through chromatin remodeling. In in vitro analysis, histone acetylation inhibition with apicidin increased filopodium density, suggesting that the external regulation of acetylation levels might modulate neuronal morphology. These results offer a way to enhance neural connectivity that could be considered to revert PS effects.

Neural glycoprotein M6a is released in extracellular vesicles and modulated by chronic stressors in blood.

Membrane neuronal glycoprotein M6a is highly expressed in the brain and contributes to neural plasticity promoting neurite growth and spine and synapse formation. We have previously showed that chronic stressors alter hippocampal M6a mRNA levels in rodents and tree shrews. We now show that M6a glycoprotein can be detected in mouse blood. M6a is a transmembrane glycoprotein and, as such, unlikely to be free in blood. Here we demonstrate that, in blood, M6a is transported in extracellular vesicles (EVs). It is also shown that M6a-containing EVs are delivered from cultured primary neurons as well as from M6a-transfected COS-7 cells. Released EVs containing M6a can be incorporated into COS-7 cells changing its phenotype through formation of membrane protrusions. Thus, M6a-containing EVs might contribute to maintain cellular plasticity. M6a presence in blood was used to monitor stress effects. Chronic restraint stress modulated M6a protein level in a sex dependent manner. Analysis of individual animals indicated that M6a level variations depend on the stressor applied. The response to stressors in blood makes M6a amenable to further studies in the stress disorder field.

Interfering polysialyltransferase ST8SiaII/STX mRNA inhibits neurite growth during early hippocampal development.

Polysialic acid (PSA) attached to NCAM is involved in cell-cell interactions participating in structural and functional plasticity of neuronal circuits. Two polysialyltransferases, ST8SiaII/STX and ST8SiaIV/PST, polysialylate NCAM. We previously suggested that ST8SiaII/STX is the key enzyme for polysialylation in hippocampus. Here, polysialyltransferase mRNA interference experiments showed that, knock down of ST8SiaIV/PST transcripts did not affect PSA expression, but PSA was almost absent from neuronal surfaces when ST8SiaII/STX mRNA was interfered. Non-polysialylated neurons bore a similar number of neurites per cell than polysialylated neurons. However, non-polysialylated processes were shorter and a lower density of synaptophysin clusters accompanied this reduced neuritic growth. Therefore, ST8SiaII/STX expression is essential to allow a correct neuritic development at initial stages of hippocampus ontogeny.

Glutamate neurotransmission is affected in prenatally stressed offspring.

Previous studies from our laboratory have shown that male adult offspring of stressed mothers exhibited higher levels of ionotropic and metabotropic glutamate receptors than control rats. These offspring also showed long-lasting astroglial hypertrophy and a reduced dendritic arborization with synaptic loss. Since metabolism of glutamate is dependent on interactions between neurons and surrounding astroglia, our results suggest that glutamate neurotransmitter pathways might be impaired in the brain of prenatally stressed rats. To study the effect of prenatal stress on the metabolism and neurotransmitter function of glutamate, pregnant rats were subjected to restrain stress during the last week of gestation. Brains of the adult offspring were used to assess glutamate metabolism, uptake and release as well as expression of glutamate receptors and transporters. While glutamate metabolism was not affected it was found that prenatal stress (PS) changed the expression of the transporters, thus, producing a higher level of vesicular vGluT-1 in the frontal cortex (FCx) and elevated levels of GLT1 protein and messenger RNA in the hippocampus (HPC) of adult male PS offspring. We also observed increased uptake capacity for glutamate in the FCx of PS male offspring while no such changes were observed in the HPC. The results show that changes mediated by PS on the adult glutamatergic system are brain region specific. Overall, PS produces long-term changes in the glutamatergic system modulating the expression of glutamate transporters and altering synaptic transmission of the adult brain.

Neurotrophic factors and depolarization do not enhance viability and process regrowth on a purified set of chick embryo retinal ganglion cells.

As retinal ganglion cells (RGCs) develop, the amount of membrane glycoprotein Thy-1 increases, consequently, the RGC binding to Thy-1 antibody enlarges. This phenomenon sustained Thy-1 panning purification of two sets: loose bound cells (LBCs); and tight bound cells (TBCs). Thy-1, neurofilament and growth associated protein-43 characterized both sets as RGCs, but the expression of RA4 antigen and gangliotetraosylgangliosides distinguished LBCs as immature and TBCs as mature. The cell composition in 4-day TBC cultures remained unchanged as immunocharacterization indicated. These cells survived and regrew their processes in plain medium, however, trophic factors (TFs) and depolarization did not enhance their development. This peculiar behavior might be due to their neural maturity, whereas in LBC cultures, TFs produced positive effects.

Prenatal stress changes the glycoprotein GPM6A gene expression and induces epigenetic changes in rat offspring brain.

Prenatal stress (PS) exerts strong impact on fetal brain development and on adult offspring brain functions. Previous work demonstrated that chronic stress alters the mRNA expression of GPM6A, a neuronal glycoprotein involved in filopodium extension. In this work, we analyzed the effect of PS on gpm6a expression and the epigenetic mechanisms involved. Pregnant Wistar rats received restraint stress during the last week of gestation. Male offspring were sacrificed on postnatal days 28 and 60. Hippocampus and prefrontal cortex samples were analyzed for gene expression (qPCR for mRNAs and microRNAs), methylation status (bisulfite conversion) and protein levels. Hippocampal neurons in culture were used to analyze microRNA overexpression effects. Prenatal stress induced changes in gpm6a levels in both tissues and at both ages analyzed, indicating a persistent effect. Two CpG islands in the gpm6a gene were identified. Variations in the methylation pattern at three specific CpGs were found in hippocampus, but not in PFC samples from PS offspring. microRNAs predicted to target gpm6a were identified in silico. qPCR measurements showed that PS modified the expression of several microRNAs in both tissues, being microRNA-133b the most significantly altered. Further studies overexpressing this microRNA in neuronal cultures showed a reduction in gmp6a mRNA and protein level. Moreover filopodium density was also reduced, suggesting that GPM6A function was affected. Gestational stress affected gpm6a gene expression in offspring likely through changes in methylation status and in posttranscriptional regulation by microRNAs. Thus, our findings propose gpm6a as a novel target for epigenetic regulation during prenatal stress.

Retinal ganglion cells are autonomous circadian oscillators synthesizing N-acetylserotonin during the day.

Retinal ganglion cells send visual and circadian information to the brain regarding the environmental light-dark cycles. We investigated the capability of retinal ganglion cells of synthesizing melatonin, a highly reliable circadian marker that regulates retinal physiology, as well as the capacity of these cells to function as autonomous circadian oscillators. Chick retinal ganglion cells presented higher levels of melatonin assessed by radioimmunoassay during both the subjective day in constant darkness and the light phase of a light-dark cycle. Similar changes were observed in mRNA levels and activity of arylalkylamine N-acetyltransferase, a key enzyme in melatonin biosynthesis, with the highest levels of both parameters during the subjective day. These daily variations were preceded by the elevation of cyclic-AMP content, the second messenger involved in the regulation of melatonin biosynthesis. Moreover, cultures of immunopurified retinal ganglion cells at embryonic day 8 synchronized by medium exchange synthesized a [3H]melatonin-like indole from [3H]tryptophan. This [3H]indole was rapidly released to the culture medium and exhibited a daily variation, with levels peaking 8 h after synchronization, which declined a few hours later. Cultures of embryonic retinal ganglion cells also showed self-sustained daily rhythms in arylalkylamine N-acetyltransferase mRNA expression during at least three cycles with a period near 24 h. These rhythms were also observed after the application of glutamate. The results demonstrate that chick retinal ganglion cells may function as autonomous circadian oscillators synthesizing a melatonin-like indole during the day.