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Background Ethanol ( EtOH) and nicotine are often co-abused. However, their combined effects on fetal neural development, particularly on fetal neural stem cells ( NSCs), which generate most neurons of the adult brain during the second trimester of pregnancy, are poorly understood. We previously showed that EtOH influenced NSC maturation in part, by suppressing the expression of specific micro RNAs (mi RNAs). Here, we tested in fetal NSCs the extent to which EtOH and nicotine coregulated known EtOH-sensitive (mi R-9, mi R-21, mi R-153, and mi R-335), a nicotine-sensitive mi RNA (mi R-140-3p), and mRNAs for nicotinic acetylcholine receptor ( nAChR) subunits. Additionally, we tested the extent to which these effects were nAChR dependent. Methods Gestational day 12.5 mouse fetal murine cerebral cortical-derived neurosphere cultures were exposed to EtOH, nicotine, and mecamylamine, a noncompetitive nAChR antagonist, individually or in combination, for short (24 hour) and long (5 day) periods, to mimic exposure during the in vivo period of neurogenesis. Levels of mi RNAs, mi RNA-regulated transcripts, and nAChR subunit mRNAs were assessed by quantitative reverse transcription polymerase chain reaction. Results EtOH suppressed the expression of known EtOH-sensitive mi RNAs and miR-140-3p, while nicotine at concentrations attained by cigarette smokers induced a dose-related increase in these mi RNAs. Nicotine's effect was blocked by EtOH and by mecamylamine. Finally, EtOH decreased the expression of nAChR subunit mRNAs and, like mecamylamine, prevented the nicotine-associated increase in α4 and β2 nAChR transcripts. Conclusions EtOH and nicotine exert mutually antagonistic, nAChR-mediated effects on teratogen-sensitive mi RNAs in fetal NSCs. These data suggest that concurrent exposure to EtOH and nicotine disrupts mi RNA regulatory networks that are important for NSC maturation. [ABSTRACT FROM AUTHOR]

Neurons within the spinal cord can support several forms of plasticity, including response- outcome (instrumental) learning. After a complete spinal transection, experimental subjects are capable of learning to hold the hindlimb in a flexed position (response) if shock (outcome) is delivered to the tibialis anterior muscle when the limb is extended. This response-contingent shock produces a robust learning that is mediated by ionotropic glutamate receptors (iGluRs). Exposure to nociceptive stimuli that are independent of limb position (e.g., uncontrollable shock; peripheral inflammation) produces a long-term (>24 h) inhibition of spinal learning. This inhibition of plasticity in spinal learning is itself a form of plasticity that requires iGluR activation and protein synthesis. Plasticity of plasticity (metaplasticity) in the CNS has been linked to group I metabotropic glutamate receptors (subtypes mGluR1 and mGluR5) and activation of protein kinase C (PKC). The present study explores the role of mGluRs and PKC in the metaplastic inhibition of spinal cord learning using a combination of behavioral, pharmacological, and biochemical techniques. Activation of group I mGluRs was found to be both necessary and sufficient for metaplastic inhibition of spinal learning. PKC was activated by stimuli that inhibit spinal learning, and inhibiting PKC activity restored the capacity for spinal learning. Finally, a PKC inhibitor blocked the metaplastic inhibition of spinal learning produced by a group I mGluR agonist. The data strongly suggest that group I mGluRs control metaplasticity of spinal learning through a PKC-dependent mechanism, providing a potential therapeutic target for promoting use-dependent plasticity after spinal cord injury. [ABSTRACT FROM AUTHOR]

The fetal brain is sensitive to a variety of teratogens, including ethanol. We showed previously that ethanol induced mitosis and stem cell maturation, but not death, in fetal cerebral cortex-derived progenitors. We tested the hypothesis that micro-RNAs (miRNAs) could mediate the teratogenic effects of ethanol in a fetal mouse cerebral cortex-derived neurosphere culture model. Ethanol, at a level attained by alcoholics, significantly suppressed the expression of four miRNAs, miR-21, -335, -9, and -153, whereas a lower ethanol concentration, attainable during social drinking, induced miR-335 expression. A GABAA receptor-dependent mechanism mediated miR-21, but not miR-335 suppression, suggesting that divergent mechanisms regulate ethanol-sensitive miRNAs. Antisense-mediated suppression of miR-21 expression resulted in apoptosis, suggesting that miR-21 is an antiapoptotic factor. miR-335 knockdown promoted cell proliferation and prevented death induced by concurrently suppressing miR-21, indicating that miR-335 is a proapoptotic, antimitogenic factor whose actions are antagonistic to miR-21. Computational analyses identified two genes, Jagged-1, a Notch-receptor ligand, and embryonic-lethal abnormal vision, Drosophila-like 2 (ELAVL2), a brain-specific regulator of RNA stability, as presumptive targets of three of four ethanol-sensitive micro-RNAs. Combined knockdown of miR-335, -21, and -153 significantly increased Jagged-1 mRNA. Furthermore, ethanol induced both Jagged-1 and ELAVL2 mRNA. The collective suppression of micro-RNAs is consistent with ethanol induction of cell cycle and neuroepithelial maturation in the absence of apoptosis. These data identify a role for micro-RNAs as epigenetic intermediaries, which permit teratogens to shape complex, divergent developmental processes, and additionally demonstrate that coordinately regulated miRNAs exhibit both functional synergy and antagonism toward each other. [ABSTRACT FROM AUTHOR]

cell bioBackground: Apoptosis is important for normal cerebral cortical development. We previously showed that the Fas suicide receptor was expressed within the developing cerebral cortex, and that in vitro Fas activation resulted in caspase-dependent death. Alterations in cell-surface Fas expression may significantly influence cortical development. Therefore, in the following studies, we sought to identify developmentally relevant cell biological processes that regulate cell-surface Fas expression and reciprocal consequences of Fas receptor activation. Results: Flow-cytometric analyses identified two distinct neural sub-populations that expressed Fas on their cell surface at high (FasHi) or moderate (FasMod) levels. The anti-apoptotic protein FLIP further delineated a subset of Fas-expressing cells with potential apoptosis-resistance. FasMod precursors were mainly in G0, while FasHi precursors were largely apoptotic. However, birth-date analysis indicated that neuroblasts express the highest levels of cell-surface Fas at the end of S-phase, or after their final round of mitosis, suggesting that Fas expression is induced at cell cycle checkpoints or during interkinetic nuclear movements. FasHi expression was associated with loss of cell-matrix adhesion and anoikis. Activation of the transcription factor p53 was associated with induction of Fas expression, while the gonadal hormone estrogen antagonistically suppressed cell-surface Fas expression. Estrogen also induced entry into S-phase and decreased the number of Fas-expressing neuroblasts that were apoptotic. Concurrent exposure to estrogen and to soluble Fas-ligand (sFasL) suppressed p21/waf-1 and PCNA. In contrast, estrogen and sFasL, individually and together, induced cyclin-A expression, suggesting activation of compensatory survival mechanisms. Conclusions: Embryonic cortical neuronal precursors are intrinsically heterogeneous with respect to Fas suicide-sensitivity. Competing intrinsic (p53, cell cycle, FLIP expression), proximal (extra-cellular matrix) and extrinsic factors (gonadal hormones) collectively regulate Fas suicide-sensitivity either during neurogenesis, or possibly during neuronal migration, and may ultimately determine which neuroblasts successfully contribute neurons to the differentiating cortical plate. [ABSTRACT FROM AUTHOR]

Background: Prenatal alcohol exposure (PAE) is a significant risk factor for developmental disability, although its health consequences across the lifespan are poorly understood. Here, we hypothesized that latent brain and systemic consequences of PAE influence resiliency to adult‐onset neurological disease, specifically, cerebrovascular ischemic stroke. Methods: Pregnant Sprague–Dawley rats were exposed episodically to ethanol during the fetal neurogenic period. Adult (5 months) male and female PAE and control offspring were subjected to endothelin‐1‐induced unilateral middle cerebral artery occlusion. In the acute injury phase outcomes including stroke volume and neurological, endocrine, and gut permeability markers were assessed. Because the effects of stroke in human populations evolve over months to years, we also assessed hippocampal‐ and amygdala‐dependent memory function and social interaction preference up to 6 months following a stroke, in middle‐aged offspring. Results: Prenatal alcohol exposure did not alter infarct volume, but significantly increased neurological deficits in both sexes, and impaired interhemispheric sensorimotor integration in PAE females. The IGF‐1/IGFBP3 ratio, a measure of bioavailable IGF‐1, was significantly reduced, while circulating levels of bacterial lipopolysaccharide, an inflammagen, were significantly increased in PAE males. In PAE females, the circulating IGF‐1/IGFBP3 ratio was significantly increased and estradiol‐17b levels were significantly reduced. The intestinal fatty acid binding protein, a surrogate marker of gut permeability was also significantly increased in PAE females. Longer‐term deficits in hippocampal‐associated memory and social interactions were observed in PAE males, while deficits in amygdala‐dependent memory were observed in PAE females. Conclusions: PAE contributes to adverse effects on brain health and decreased resiliency in response to a common adult‐onset neurovascular disease, cerebrovascular ischemic stroke. [ABSTRACT FROM AUTHOR]

Most persons with fetal alcohol spectrum disorders (FASDs) remain undiagnosed or are diagnosed in later life. To address the need for earlier diagnosis, we previously assessed miRNAs in the blood plasma of pregnant women who were classified as unexposed to alcohol (UE), heavily exposed with affected infants (HEa), or heavily exposed with apparently unaffected infants (HEua). We reported that maternal miRNAs predicted FASD-related growth and psychomotor deficits in infants. Here, we assessed whether fetal sex influenced alterations in maternal circulating miRNAs following prenatal alcohol exposure (PAE). To overcome the loss of statistical power due to disaggregating maternal samples by fetal sex, we adapted a strategy of iterative bootstrap resampling with replacement to assess the stability of statistical parameter estimates. Bootstrap estimates of parametric and effect size tests identified male and female fetal sex-associated maternal miRNA responses to PAE that were not observed in the aggregated sample. Additionally, we observed, in HEa mothers of female, but not male fetuses, a network of co-secreted miRNAs whose expression was linked to miRNAs encoded on the X-chromosome. Interestingly, the number of significant miRNA correlations for the HEua group mothers with female fetuses was intermediate between HEa and UE mothers at mid-pregnancy, but more similar to UE mothers by the end of pregnancy. Collectively, these data show that fetal sex predicts maternal circulating miRNA adaptations, a critical consideration when adopting maternal miRNAs as diagnostic biomarkers. Moreover, a maternal co-secretion network, predominantly in pregnancies with female fetuses, emerged as an index of risk for adverse birth outcomes due to PAE. [ABSTRACT FROM AUTHOR]

Circulating miRNAs the in blood are promising biomarkers for predicting pregnancy complications and adverse birth outcomes. Previous work identified 11 gestationally elevated maternal circulating miRNAs (HEamiRNAs) that predicted infant growth deficits following prenatal alcohol exposure and regulated epithelial–mesenchymal transition in the placenta. Here we show that a single intravascular administration of pooled murine-conserved HEamiRNAs to pregnant mice on gestational day 10 (GD10) attenuates umbilical cord blood flow during gestation, explaining the observed intrauterine growth restriction (IUGR), specifically decreased fetal weight, and morphometric indices of cranial growth. Moreover, RNAseq of the fetal portion of the placenta demonstrated that this single exposure has lasting transcriptomic changes, including upregulation of members of the Notch pathway (Dll4, Rfng, Hey1), which is a pathway important for trophoblast migration and differentiation. Weighted gene co-expression network analysis also identified chemokine signaling, which is responsible for regulating immune cell-mediated angiogenesis in the placenta, as an important predictor of fetal growth and head size. Our data suggest that HEamiRNAs perturb the expression of placental genes relevant for angiogenesis, resulting in impaired umbilical cord blood flow and subsequently, IUGR. [ABSTRACT FROM AUTHOR]

Prenatal alcohol exposure can result in a range of physical, neuropathological, and behavioral alterations, collectively termed fetal alcohol spectrum disorders (FASD). We have shown that supplementation with the nutrient choline reduces the severity of developmental alcohol-associated deficits in hippocampal-dependent behaviors and normalizes some aspects of hippocampal cholinergic development and DNA methylation patterns. Alcohol's developmental effects may also be mediated, in part, by altering microRNAs (miRNAs) that serve as negative regulators of gene translation. To determine whether choline supplementation alters ethanol's long-lasting effects on miRNAs, Sprague-Dawley rats were exposed to 5.25 g/kg/day ethanol from postnatal days (PD) 4-9 via intubation; controls received sham intubations. Subjects were treated with choline chloride (100 mg/kg/day) or saline vehicle subcutaneously (s.c.) from PD 4-21. On PD 22, subjects were sacrificed, and RNA was isolated from the hippocampus. MiRNA expression was assessed with TaqMan Human MicroRNA Panel Low-Density Arrays. Ethanol significantly increased miRNA expression variance, an effect that was attenuated with choline supplementation. Cluster analysis of stably expressed miRNAs that exceeded an ANOVA p < 0.05 criterion indicated that for both male and female offspring, control and ethanol-exposed groups were most dissimilar from each other, with choline-supplemented groups in between. MiRNAs that expressed an average 2-fold change due to ethanol exposure were further analyzed to identify which ethanol-sensitive miRNAs were protected by choline supplementation. We found that at a false discovery rate (FDR)-adjusted criterion of p < 0.05, miR-200c was induced by ethanol exposure and that choline prevented this effect. Collectively, our data show that choline supplementation can normalize disturbances in miRNA expression following developmental alcohol exposure and can protect specific miRNAs from induction by ethanol. These findings have important implications for the mechanisms by which choline may serve as a potential treatment for FASD. [ABSTRACT FROM AUTHOR]

Background: Prenatal alcohol (ethanol) exposure (PAE) results in brain growth restriction, in part, by reprogramming self-renewal and maturation of fetal neural stem cells (NSCs) during neurogenesis. We recently showed that ethanol resulted in enrichment of both proteins and pro-maturation microRNAs in sub-200-nm-sized extracellular vesicles (EVs) secreted by fetal NSCs. Moreover, EVs secreted by ethanol-exposed NSCs exhibited diminished efficacy in controlling NSC metabolism and maturation. Here we tested the hypothesis that ethanol may also influence the packaging of RNAs into EVs from cell-of-origin NSCs. Methods: Sex-specified fetal murine iso-cortical neuroepithelia from three separate pregnancies were maintained ex vivo, as neurosphere cultures to model the early neurogenic niche. EVs were isolated by ultracentrifugation from NSCs exposed to a dose range of ethanol. RNA from paired EV and cell-of-origin NSC samples was processed for ribosomal RNA-depleted RNA sequencing. Differential expression analysis and exploratory weighted gene co-expression network analysis (WGCNA) identified candidate genes and gene networks that were drivers of alterations to the transcriptome of EVs relative to cells. Results: The RNA content of EVs differed significantly from cell-of-origin NSCs. Biological sex contributed to unique transcriptome variance in EV samples, where > 75% of the most variant transcripts were also sex-variant in EVs but not in cell-of-origin NSCs. WGCNA analysis also identified sex-dependent enrichment of pathways, including dopamine receptor binding and ectoderm formation in female EVs and cell-substrate adhesion in male EVs, with the top significant DEGs from differential analysis of overall individual gene expressions, i.e., Arhgap15, enriched in female EVs, and Cenpa, enriched in male EVs, also serving as WCGNA hub genes of sex-biased EV WGCNA clusters. In addition to the baseline RNA content differences, ethanol exposure resulted in a significant dose-dependent change in transcript expression in both EVs and cell-of-origin NSCs that predominantly altered sex-invariant RNAs. Moreover, at the highest dose, ~ 73% of significantly altered RNAs were enriched in EVs, but depleted in NSCs. Conclusions: The EV transcriptome is distinctly different from, and more sex-variant than, the transcriptome of cell-of-origin NSCs. Ethanol, a common teratogen, results in dose-dependent sorting of RNA transcripts from NSCs to EVs which may reprogram the EV-mediated endocrine environment during neurogenesis. Highlights: The RNA content of EVs differ significantly from their cell-of-origin NSCs. Biological sex contributes to significant differences in the transcriptome of EVs. Most sex differences observed in EVs were not present in parent-of-origin NSCs, suggesting sex-dependent sorting of RNA into EVs. Ethanol exposure resulted in specific RNA transcripts being enriched in secreted EVs, while being depleting in cell-of-origin NSCs, suggesting that cell stress may alter the sorting of RNA from cells to EVs. Transcripts enriched in EVs, following ethanol exposure of NSCs, encode proteins selectively overrepresented in biological pathways like mRNA splicing and transport. [ABSTRACT FROM AUTHOR]

Background: Prenatal alcohol exposure (PAE) has been shown to alter fetal blood flow in utero and is also associated with placental insufficiency and intrauterine growth restriction (IUGR), suggesting an underlying connection between perturbed circulation and pregnancy outcomes. Methods: Timed‐pregnant C57/BL6NHsd mice, bred in‐house, were exposed by gavage on gestational day 10 (GD10) to ethanol (3 g/kg) or purified water, as a control. Pulse‐wave Doppler ultrasound measurements for umbilical arteries and ascending aorta were obtained post‐gavage (GD12, GD14, GD18) on 2 fetuses/litter. RNA from the non‐decidual (labyrinthine and junctional zone) portion of placentas was isolated and processed for RNA‐seq and subsequent bioinformatic analyses, and the association between transcriptomic changes and fetal phenotypes assessed. Results: Exposure to ethanol in pregnant mice on GD10 attenuates umbilical cord blood flow transiently during gestation, and is associated with indices of IUGR, specifically decreased fetal weight and morphometric indices of cranial growth. Moreover, RNA‐seq of the fetal portion of the placenta demonstrated that this single exposure has lasting transcriptomic changes, including upregulation of Tet3, which is associated with spontaneous abortion. Weighted gene co‐expression network analysis (WGCNA) identified erythrocyte differentiation and homeostasis as important pathways associated with improved umbilical cord blood flow as gestation progresses. WGCNA also identified sensory perception of chemical stimulus/odorant and receptor activity as important pathways associated with cranial growth. Conclusion: Our data suggest that PAE perturbs the expression of placental genes relevant for placental hematopoiesis and environmental sensing, resulting in transient impairment of umbilical cord blood flow and, subsequently, IUGR. [ABSTRACT FROM AUTHOR]

Prenatal opioid exposure (POE) is commonly associated with neonatal opioid withdrawal syndrome (NOWS), which is characterized by a broad variability in symptoms and severity. Currently there are no diagnostic tools to reliably predict which infants will develop severe NOWS, while risk stratification would allow for proactive decisions about appropriate clinical monitoring and interventions. The aim of this prospective cohort study was to assess if extracellular microRNAs (miRNAs) in umbilical cord plasma of infants with POE could predict NOWS severity. Participants (n = 58) consisted of pregnant women receiving medications for opioid use disorder and their infants. NOWS severity was operationalized as the need for pharmacologic treatment and prolonged hospitalization (≥ 14 days). Cord blood miRNAs were assessed using semi-quantitative qRT-PCR arrays. Receiver operating characteristic curves and area under the curve (AUC) were estimated. The expression of three miRNAs (miR-128-3p, miR-30c-5p, miR-421) predicted need for pharmacologic treatment (AUC: 0.85) and prolonged hospitalization (AUC: 0.90). Predictive validity improved after two miRNAs (let-7d-5p, miR-584-5p) were added to the need for pharmacologic treatment model (AUC: 0.94) and another two miRNAs (let-7b-5p, miR-10-5p) to the prolonged hospitalization model (AUC: 0.99). Infant cord blood extracellular miRNAs can proactively identify opioid-exposed neonates at high-risk for developing severe NOWS. [ABSTRACT FROM AUTHOR]

Background: We previously showed that ethanol did not kill fetal neural stem cells (NSCs), but that their numbers nevertheless are decreased due to aberrant maturation and loss of self‐renewal. To identify mechanisms that mediate this loss of NSCs, we focused on a family of Gag‐like proteins (GLPs), derived from retroviral gene remnants within mammalian genomes. GLPs are important for fetal development, though their role in brain development is virtually unexplored. Moreover, GLPs may be transferred between cells in extracellular vesicles (EVs) and thereby transfer environmental adaptations between cells. We hypothesized that GLPs may mediate some effects of ethanol in NSCs. Methods: Sex‐segregated male and female fetal murine cortical NSCs, cultured ex vivo as nonadherent neurospheres, were exposed to a dose range of ethanol and to mitogen‐withdrawal‐induced differentiation. We used siRNAs to assess the effects of NSC‐expressed GLP knockdown on growth, survival, and maturation and in silico GLP knockout, in an in vivo single‐cell RNA‐sequencing dataset, to identify GLP‐mediated developmental pathways that were also ethanol‐sensitive. Results: PEG10 isoform‐1, isoform‐2, and PNMA2 were identified as dominant GLP species in both NSCs and their EVs. Ethanol‐exposed NSCs exhibited significantly elevated PEG10 isoform‐2 and PNMA2 protein during differentiation. Both PEG10 and PNMA2 were mediated apoptosis resistance and additionally, PEG10 promoted neuronal and astrocyte lineage maturation. Neither GLP influenced metabolism nor cell cycle in NSCs. Virtual PEG10 and PNMA2 knockout identified gene transcription regulation and ubiquitin‐ligation processes as candidate mediators of GLP‐linked prenatal alcohol effects. Conclusions: Collectively, GLPs present in NSCs and their EVs may confer apoptosis resistance within the NSC niche and contribute to the abnormal maturation induced by ethanol. [ABSTRACT FROM AUTHOR]

Abstract: The annual meeting of the Fetal Alcohol Spectrum Disorders Study Group (FASDSG) was held on June 26, 2010 in San Antonio, TX, as a satellite of the Research Society on Alcoholism meeting. The FASDSG membership includes clinical, basic, and social scientists who meet to discuss recent advances and issues in Fetal Alcohol Spectrum Disorder (FASD) research. The central theme of the meeting was “Glia and Neurons: Teamwork in Pathology and Therapy.” Alcohol disruption of neuron development and alcohol-induced neurodegeneration is central to the pathology and clinical expression of FASD. The active role of glia as perpetrator, victim, or bystander in neurotoxicology and neurodegenerative processes has emerged at the forefront of adult central nervous system (CNS) disorders and therapy. Glia– and neuron–glial interactions hold the potential to elucidate causes and offer treatment of FASD as well. Growing evidence indicates that neurons and glia are direct targets of alcohol, but may also be vulnerable to molecules produced in peripheral systems as a result of alcohol exposure. Diagnostics and therapies can take advantage of these processes and biomarkers, and these may be applicable to CNS pathology in FASD. Two keynote speakers, Howard E. Gendelman, M.D., and Ernest M. Graham, M.D, addressed the role of glia and neuroinflammation in brain development and neurodegeneration. The invited speakers and FASDSG members discussed new paradigms in CNS development and discuss new strategies for understanding and treating neurodegenerative disease. Members of the FASDSG provided updates on new findings through presentation of breaking research in the FASt data sessions. Representatives of national agencies provided updates on programs, activities, and funding priorities. The Henry Rosett Award was presented to R. Louise Floyd, R.N., D.S.N., for her career contributions to the field of fetal alcohol research. The Student and Postdoctoral Fellow Research Merit Award was presented to Shonagh O’Leary-Moore, Ph.D., for her contributions to the field as a young investigator. [Copyright &y& Elsevier]

Six-day ‘binge’ ethanol intoxication postnatal days (PD) 4–9 delays up-regulation of γ-aminobutyric acid type A receptors (GABAARs) in developing rat septal neurons [Dev. Brain Res. 130 (2001) 25]. This distortion occurs during synaptogenesis and could contribute to cognitive dysfunction in fetal alcohol syndrome (FAS). Here, we asked two questions concerning requirements for vulnerability to GABAAR blunting by ethanol. First, we asked whether receptor blunting required PD 4–9 ethanol exposure in rat pups and found that just a brief 2-day exposure (PD 8–9) was as effective as all 6 days. However, 2-day exposure on PD 4–5 was ineffective, showing that ‘binge’ timing was important. We also asked whether ‘binge’ exposure directly inhibited intrinsic processes of septal neurons and could blunt GABAARs on cells maturing outside the brain. Embryonic septal neurons grown in serum-free dispersed culture developed extensive dendritic arborizations, spontaneous synaptic activity and robust whole-cell GABAAR function, but surprisingly, did not show developmental up-regulation of GABAARs like septal neurons maturing in vivo [Brain Res. 810 (1998) 100]. Furthermore, age-matched 6-day ‘binge’ ethanol exposure did not blunt GABAAR function in septal neurons in vitro. These results suggest developmental mechanisms driving up-regulation of GABAAR function in septal neurons in vivo briefly becomes vulnerable to ethanol insult in early postnatal life. While septal neurons express comparable functional GABAARs whether maturing in vivo or in vitro, vulnerability to ethanol-induced receptor blunting requires elements of an intact brain environment not replicated in culture. [Copyright &y& Elsevier]

Nociceptive input diminishes recovery and increases lesion area after a spinal cord injury (SCI). Recent work has linked these effects to the expansion of hemorrhage at the site of injury. The current article examines whether these adverse effects are linked to a pain-induced rise in blood pressure (BP) and/or flow. Male rats with a low-thoracic SCI were treated with noxious input (electrical stimulation [shock] or capsaicin) soon after injury. Locomotor recovery and BP were assessed throughout. Tissues were collected 3 h, 24 h, or 21 days later. Both electrical stimulation and capsaicin undermined locomotor function and increased the area of hemorrhage. Changes in BP/flow varied depending on type of noxious input, with only shock producing changes in BP. Providing behavioral control over the termination of noxious stimulation attenuated the rise in BP and hemorrhage. Pretreatment with the α-1 adrenergic receptor inverse agonist, prazosin, reduced the stimulation-induced rise in BP and hemorrhage. Prazosin also attenuated the adverse effect that noxious stimulation has on long-term recovery. Administration of the adrenergic agonist, norepinephrine 1 day after injury induced an increase in BP and disrupted locomotor function, but had little effect on hemorrhage. Further, inducing a rise in BP/flow using norepinephrine undermined long-term recovery and increased tissue loss. Mediational analyses suggest that the pain-induced rise in blood flow may foster hemorrhage after SCI. Increased BP appears to act through an independent process to adversely affect locomotor performance, tissue sparing, and long-term recovery. [ABSTRACT FROM AUTHOR]

• Prenatal alcohol exposure (PAE) impairs glucose metabolism in female offspring. • PAE causes sex-dependent changes in peripheral immune cells and cytokines. • PAE impairs cognition in both sexes with sex-specific effect on affective behaviors. • Changes in peripheral immune cells and cytokines predict behavioral outcomes in PAE. Prenatal alcohol exposure (PAE) can result in neurobehavioral anomalies, that may be exacerbated by co-occurring metabolic and immune system deficits. To test the hypothesis that the peripheral inflammation in adult PAE offspring is linked to poor glucose metabolism and neurocognitive deficits, pregnant Sprague-Dawley rats were exposed to ethanol vapor or ambient air during the latter half of gestation. We assessed, in adult offspring of both sexes, performance on a battery of neurocognitive behaviors, glucose tolerance, circulating and splenic immune cells by flow-cytometry, and circulating and tissue (liver, mesenteric adipose, and spleen) cytokines by multiplexed assays. PAE reduced both the ratio of spleen to body weight and splenic regulatory T-cell (Treg) numbers. PAE males, but not females exhibited an increase in circulating monocytes. Overall, PAE males exhibited a suppression of cytokine levels, while PAE females exhibited elevated cytokines in mesenteric adipose tissue (IL-6 and IL1α) and liver (IFN-γ, IL-1β, IL-13, IL-18, IL-12p70, and MCP-1), along with increased glucose intolerance. Behavioral analysis also showed sex-dependent PAE effects. PAE-males exhibited increased anxiety-like behavior while PAE-females showed decreased social interaction. PAE offspring of both sexes exhibited impaired recognition of novel objects. Multilinear regression modeling to predict the association between peripheral immune status, glucose intolerance and behavioral outcomes, showed that in PAE offspring, higher levels of adipose leptin and liver TNF- α predicted higher circulating glucose levels. Lower liver IL-1 α and higher plasma fractalkine predicted more time spent in the center of an open-field with sex being an additional predictor. Higher circulating and splenic Tregs predicted better social interaction in the PAE-offspring. Collectively, our data show that peripheral immune status is a persistent, sex-dependent predictor of glucose intolerance and neurobehavioral function in adult PAE offspring. [ABSTRACT FROM AUTHOR]

The developmental origins of health and disease (DOHaD) is a paradigm that links prenatal and early life exposures that occur during crucial periods of development to health outcome and risk of disease later in life. Maternal exposures to stress, some psychoactive drugs and alcohol, and environmental chemicals, among others, may result in functional changes in developing fetal tissues, creating a predisposition for disease in the individual as they age. Extracellular vesicles (EVs) may be mediators of both the immediate effects of exposure during development and early childhood as well as the long-term consequences of exposure that lead to increased risk and disease severity later in life. Given the prevalence of diseases with developmental origins, such as cardiovascular disease, neurodegenerative disorders, osteoporosis, metabolic dysfunction, and cancer, it is important to identify persistent mediators of disease risk. In this review, we take this approach, viewing diseases typically associated with aging in light of early life exposures and discuss the potential role of EVs as mediators of lasting consequences. [ABSTRACT FROM AUTHOR]

Background: The developing hippocampus and cerebellum, unique among brain regions, exhibit a secondary surge in neurogenesis during the third trimester of pregnancy. Ethanol (EtOH) exposure during this period is results in a loss of tissue volume and associated neurobehavioral deficits. However, mechanisms that link EtOH exposure to teratology in these regions are not well understood. We therefore analyzed transcriptomic adaptations to EtOH exposure to identify mechanistic linkages. Methods: Hippocampi and cerebella were microdissected at postnatal day (P)10, from control C57BL/6J mouse pups, and pups treated with 4 g/kg of EtOH from P4 to P9. RNA was isolated and RNA‐seq analysis was performed. We compared gene expression in EtOH‐ and vehicle‐treated control neonates and performed biological pathway‐overrepresentation analysis. Results: While EtOH exposure resulted in the general induction of genes associated with the S‐phase of mitosis in both cerebellum and hippocampus, overall there was little overlap in differentially regulated genes and associated biological pathways between these regions. In cerebellum, EtOH additionally induced gene expression associated with the G2/M‐phases of the cell cycle and sonic hedgehog signaling, while in hippocampus, EtOH‐induced the pathways for ribosome biogenesis and protein translation. Moreover, EtOH inhibited the transcriptomic identities associated with inhibitory interneuron subpopulations in the hippocampus, while in the cerebellum there was a more pronounced inhibition of transcripts across multiple oligodendrocyte maturation stages. Conclusions: These data indicate that during the delayed neurogenic period, EtOH may stimulate the cell cycle, but it otherwise results in widely divergent molecular effects in the hippocampus and cerebellum. Moreover, these data provide evidence for region‐ and cell‐type‐specific vulnerability, which may contribute to the pathogenic effects of developmental EtOH exposure. [ABSTRACT FROM AUTHOR]

Prenatal alcohol exposure (PAE) can result in cognitive and behavioral disabilities and growth deficits. Because alcohol-related neurobehavioral deficits may occur in the absence of overt dysmorphic features or growth deficits, there is a need to identify biomarkers of PAE that can predict neurobehavioral impairment. In this study, we assessed infant plasma extracellular, circulating miRNAs (exmiRNAs) obtained from a heavily exposed Cape Town cohort to determine whether these can be used to predict PAE-related growth restriction and cognitive impairment. PAE, controlling for smoking as a covariate, altered 27% of expressed exmiRNAs with clinically-relevant effect sizes (Cohen's d ≥ 0.4). Moreover, at 2 weeks, PAE increased correlated expression of exmiRNAs across chromosomes, suggesting potential co-regulation. In confirmatory factor analysis, the variance in expression for PAE-altered exmiRNAs at 2 weeks and 6.5 months was best described by three-factor models. Pathway analysis found that factors at 2 weeks were associated with (F1) cell maturation, cell cycle inhibition, and somatic growth, (F2) cell survival, apoptosis, cardiac development, and metabolism, and (F3) cell proliferation, skeletal development, hematopoiesis, and inflammation, and at 6.5 months with (F1) neurodevelopment, neural crest/mesoderm-derivative development and growth, (F2) immune system and inflammation, and (F3) somatic growth and cardiovascular development. Factors F3 at 2 weeks and F2 at 6.5 months partially mediated PAE-induced growth deficits, and factor F3 at 2 weeks partially mediated effects of PAE on infant recognition memory at 6.5 months. These findings indicate that infant exmiRNAs can help identify infants who will exhibit PAE-related deficits in growth and cognition. [ABSTRACT FROM AUTHOR]

• Prenatal alcohol exposure (PAE) impacts health outcomes across the lifespan. • Distinct profiles of secondary conditions develop during adolescence and adulthood. • Sex-specific effects of PAE are measurable in utero and evolve with age. • Additional work is needed to better understand sexually dimorphic effects of PAE. Prenatal alcohol exposure (PAE) can reprogram the development of cells and tissues, resulting in a spectrum of physical and neurobehavioral teratology. PAE immediately impacts fetal growth, but its effects carry forward post-parturition, into adolescence and adulthood, and can result in a cluster of disabilities, collectively termed Fetal Alcohol Spectrum Disorders. Emerging preclinical and clinical research investigating neurological and behavioral outcomes in exposed offspring point to genetic sex as an important modifier of the effects of PAE. In this review, we discuss the literature on sex differences following PAE, with studies spanning the fetal period through adulthood, and highlight gaps in research where sex differences are likely, but currently under-investigated. Understanding how sex and PAE interact to affect offspring health outcomes across the lifespan is critical for identifying the full complement of PAE-associated secondary conditions, and for refining targeted interventions to improve the quality of life for individuals with PAE. [ABSTRACT FROM AUTHOR]

Maternal exposure to drugs during pregnancy is known to have detrimental effects on the fetus. Alcohol (ethanol) and nicotine are two of the most commonly co-abused substances during pregnancy, and prenatal poly-drug exposure is common due, in part, to the prevalence of unplanned pregnancies. The second trimester is a critical period for fetal neurogenesis and angiogenesis. When drug exposure occurs during this time, fetal brain development is affected. Several behavioral, morphological, and functional studies have evaluated the changes in fetal brain development due to exposure to these drugs individually. However, research on the combined effects of ethanol and nicotine is far more limited, specifically on fetal vasculature changes and development. We use correlation mapping optical coherence angiography (cm-OCA) to evaluate acute changes in fetal brain vasculature caused by maternal exposure to a combination of ethanol and nicotine. Ethanol (16.6% v/v, at a dose of 0.75g / kg) and nicotine (at a dose of 0.1 mg / kg) were administered to pregnant mice after initial cm-OCA measurements in utero. Subsequent measurements were taken at 5-min intervals for a total period of 45 min. Results from these experiments were compared to results from our previous studies in which the mother was exposed to only ethanol (dose: 0.75 g / kg) or nicotine (dose: 0.1 mg / kg). While results from exposure to ethanol or nicotine independently showed vasoconstriction, no significant change in vasculature was observed with combined exposure. Results suggested antagonistic effects of ethanol and nicotine on fetal brain vasculature. [ABSTRACT FROM AUTHOR]

Fetal alcohol spectrum disorders are a leading cause of intellectual disability worldwide. Previous studies have shown that developmental ethanol exposure results in loss of microRNAs (miRNAs), including miR-9, and loss of these miRNAs, in turn, mediates some of ethanol's teratogenic effects in the developing brain. We previously found that ethanol increased methylation at the miR-9-2 encoding gene locus in mouse fetal neural stem cells (NSC), advancing a mechanism for epigenetic silencing of this locus and consequently, miR-9 loss in NSCs. Therefore, we assessed the role of the BAF (BRG1/BRM-Associated Factor) complex, which disassembles nucleosomes to facilitate access to chromatin, as an epigenetic mediator of ethanol's effects on miR-9. Chromatin immunoprecipitation and DNAse I-hypersensitivity analyses showed that the BAF complex was associated with both transcriptionally accessible and heterochromatic regions of the miR-9-2 locus, and that disintegration of the BAF complex by combined knockdown of BAF170 and BAF155 resulted in a significant decrease in miR-9. We hypothesized that ethanol exposure would result in loss of BAF-complex function at the miR-9-2 locus. However, ethanol exposure significantly increased mRNA transcripts for maturation-associated BAF-complex members BAF170, SS18, ARID2, BAF60a, BRM/BAF190b, and BAF53b. Ethanol also significantly increased BAF-complex binding within an intron containing a CpG island and in the terminal exon encoding precursor (pre)-miR-9-2. These data suggest that the BAF complex may adaptively respond to ethanol exposure to protect against a complete loss of miR-9-2 in fetal NSCs. Chromatin remodeling factors may adapt to the presence of a teratogen, to maintain transcription of critical miRNA regulatory pathways. [ABSTRACT FROM AUTHOR]

Background Prenatal alcohol exposure (PAE) can result in physical and neurocognitive deficits that are collectively termed 'fetal alcohol spectrum disorders' (FASD). Although FASD is associated with lifelong intellectual disability, the mechanisms mediating the emergence of secondary mental health and physical disabilities are poorly understood. Based on our previous data showing that maternal ethanol (EtOH) exposure in mice resulted in an immediate reduction in cranially directed fetal blood flow, we hypothesized that such exposure would also result in persistent alterations in cranially directed blood flow in the prenatally alcohol-exposed (PAE) adult. We also hypothesized that PAE adults exposed to an acute cerebrovascular insult would exhibit more brain damage and neurobehavioral impairment compared to non-PAE adult controls. Methods Pregnant C57BL/6 mice were exposed to EtOH, 3 g/kg, or water by intragastric gavage. Blood flow in carotid, renal, and femoral arteries was assessed by ultrasound imaging in PAE and control adults at 3, 6, and 12 months of age. To mimic ischemic stroke in young adult populations, 3-month-old PAE and control animals were subject to transient middle cerebral artery occlusion (MCAo) and subsequently assessed for behavioral recovery, stroke infarct volume, and brain cytokine profiles. Results PAE resulted in a significant age-related decrease in blood acceleration in adult mice, specifically in the carotid artery. A unilateral transient MCAo resulted in equivalent cortico-striatal damage in both PAE and control adults. However, PAE adult mice exhibited significantly decreased poststroke behavioral recovery compared to controls. Conclusions Our data collectively show that PAE adult mice exhibit a persistent, long-term loss of cranially directed blood flow, and decreased capacity to compensate for brain trauma due to acute-onset adult diseases like ischemic stroke. [ABSTRACT FROM AUTHOR]

Alcohol and marijuana are two of the most consumed psychoactive substances by pregnant people, and independently, both substances have been associated with lifelong impacts on fetal neurodevelopment. Importantly, individuals of child-bearing age are increasingly engaging in simultaneous alcohol and cannabinoid (SAC) use, which amplifies each drug's pharmacodynamic effects and increases craving for both substances. However, to date, investigations of prenatal polysubstance use are notably limited in both human and non-human populations. In this review paper, we will address what is currently known about combined exposure to these substances, both directly and prenatally, and identify shared prenatal targets from single-exposure paradigms that may highlight susceptible neurobiological mechanisms for future investigation and therapeutic intervention. Finally, we conclude this manuscript by discussing factors that we feel are essential in the consideration and experimental design of future preclinical SAC studies. • Young adults of child-bearing age are increasingly engaging in simultaneous alcohol and cannabinoid (SAC) use. • SAC users demonstrate distinct preferences and drug-seeking behaviors from single-drug users. • Studies investigating prenatal alcohol (PAE) & prenatal cannabinoid exposure (PCE) share common neurodevelopmental targets. • Prenatal SAC exposure may augment offspring deficits from single-drug exposure across several neurodevelopmental domains. • SAC is an under-investigated area of research, and more controlled studies of prenatal polysubstance exposure are mandated. [ABSTRACT FROM AUTHOR]

Fetal alcohol spectrum disorders (FASD) are difficult to diagnose since many heavily exposed infants, at risk for intellectual disability, do not exhibit craniofacial dysmorphology or growth deficits. Consequently, there is a need for biomarkers that predict disability. In both animal models and human studies, alcohol exposure during pregnancy resulted in significant alterations in circulating microRNAs (miRNAs) in maternal blood. In the current study, we asked if changes in plasma miRNAs in alcohol-exposed pregnant mothers, either alone or in conjunction with other clinical variables, could predict infant outcomes. Sixty-eight pregnant women at two perinatal care clinics in western Ukraine were recruited into the study. Detailed health and alcohol consumption histories, and 2nd and 3rd trimester blood samples were obtained. Birth cohort infants were assessed by a geneticist and classified as unexposed (UE), heavily prenatally exposed and affected (HEa) or heavily exposed but apparently unaffected (HEua). MiRNAs were assessed in plasma samples using qRT-PCR arrays. ANOVA models identified 11 miRNAs that were all significantly elevated in maternal plasma from the HEa group relative to HEua and UE groups. In a random forest analysis classification model, a combination of high variance miRNAs, smoking history and socioeconomic status classified membership in HEa and UE groups, with a misclassification rate of 13%. The RFA model also classified 17% of the HEua group as UE-like, whereas 83% were HEa-like, at least at one stage of pregnancy. Collectively our data indicate that maternal plasma miRNAs predict infant outcomes, and may be useful to classify difficult-to-diagnose FASD subpopulations. [ABSTRACT FROM AUTHOR]

Increased pain sensitivity is a common sequela to spinal cord injury (SCI). Moreover, drugs like morphine, though critical for pain management, elicit pro-inflammatory effects that exacerbate chronic pain symptoms. Previous reports showed that SCI results in the induction and suppression of several microRNAs (miRNAs), both at the site of injury, as well as in segments of the spinal cord distal to the injury site. We hypothesized that morphine would modulate the expression of these miRNAs, and that expression of these SCI-sensitive miRNAs may predict adaptation of distal nociceptive circuitry following SCI. To determine whether morphine treatment further dysregulates SCI-sensitive miRNAs, their expression was examined by qRT-PCR in sham controls and in response to vehicle and morphine treatment following contusion in rats, at either 2 or 15 days post-SCI. Our data indicated that expression of miR1, miR124, and miR129-2 at the injury site predicted the nociceptive response mediated by spinal regions distal to the lesion site, suggesting a molecular mechanism for the interaction of SCI with adaptation of functionally intact distal sensorimotor circuitry. Moreover, the SCI-induced miRNA, miR21 was induced by subsequent morphine administration, representing an alternate, and hitherto unidentified, maladaptive response to morphine exposure. Contrary to predictions, mRNA for the pro-inflammatory interleukin-6 receptor (IL6R), an identified target of SCI-sensitive miRNAs, was also induced following SCI, indicating dissociation between miRNA and target gene expression. Moreover, IL6R mRNA expression was inversely correlated with locomotor function suggesting that inflammation is a predictor of decreased spinal cord function. Collectively, our data indicate that miR21 and other SCI-sensitive miRNAs may constitute therapeutic targets, not only for improving functional recovery following SCI, but also for attenuating the effects of SCI on pain sensitivity. [ABSTRACT FROM AUTHOR]

We previously showed that peripheral noxious input after spinal cord injury (SCI) inhibits beneficial spinal plasticity and impairs recovery of locomotor and bladder functions. These observations suggest that noxious input may similarly affect the development and maintenance of chronic neuropathic pain, an important consequence of SCI. In adult rats with a moderate contusion SCI, we investigated the effect of noxious tail stimulation, administered 1 day after SCI on mechanical withdrawal responses to von Frey stimuli from 1 to 28 days after treatment. In addition, because the proinflammatory cytokine tumor necrosis factor alpha (TNFα) is implicated in numerous injury-induced processes including pain hypersensitivity, we assessed the temporal and spatial expression of TNFα, TNF receptors, and several downstream signaling targets after stimulation. Our results showed that unlike sham surgery or SCI only, nociceptive stimulation after SCI induced mechanical sensitivity by 24 h. These behavioral changes were accompanied by increased expression of TNFα. Cellular assessments of downstream targets of TNFα revealed that nociceptive stimulation increased the expression of caspase 8 and the active subunit (12 kDa) of caspase 3, indicative of active apoptosis at a time point consistent with the onset of mechanical allodynia. In addition, immunohistochemical analysis revealed distinct morphological signs of apoptosis in neurons and microglia at 24 h after stimulation. Interestingly, expression of the inflammatory mediator NFκB was unaltered by nociceptive stimulation. These results suggest that noxious input caudal to the level of SCI can increase the onset and expression of behavioral responses indicative of pain, potentially involving TNFα signaling. [ABSTRACT FROM AUTHOR]

Background From studies using a diverse range of model organisms, we now acknowledge that epigenetic changes to chromatin structure provide a plausible link between environmental teratogens and alterations in gene expression leading to disease. Observations from a number of independent laboratories indicate that ethanol (EtOH) has the capacity to act as a powerful epigenetic disruptor and potentially derail the coordinated processes of cellular differentiation. In this study, we sought to examine whether primary neurospheres cultured under conditions maintaining stemness were susceptible to alcohol-induced alterations in the histone code. We focused our studies on trimethylated histone 3 lysine 4 and trimethylated histone 3 lysine 27, as these are 2 of the most prominent posttranslational histone modifications regulating stem cell maintenance and neural differentiation. Methods Primary neurosphere cultures were maintained under conditions promoting the stem cell state and treated with EtOH for 5 days. Control and EtOH-treated cellular extracts were examined using a combination of quantitative RT- PCR and chromatin immunoprecipitation techniques. Results We find that the regulatory regions of genes controlling both neural precursor cell identity and processes of differentiation exhibited significant declines in the enrichment of the chromatin marks examined. Despite these widespread changes in chromatin structure, only a small subset of genes including Dlx2, Fabp7, Nestin, Olig2, and Pax6 displayed EtOH-induced alterations in transcription. Unexpectedly, the majority of chromatin-modifying enzymes examined including members of the Polycomb Repressive Complex displayed minimal changes in expression and localization. Only transcripts encoding Dnmt1, Uhrf1, Ehmt1, Ash2 l, Wdr5, and Kdm1b exhibited significant differences. Conclusions Our results indicate that primary neurospheres maintained as stem cells in vitro are susceptible to alcohol-induced perturbation of the histone code and errors in the epigenetic program. These observations indicate that alterations to chromatin structure may represent a crucial component of alcohol teratogenesis and progress toward a better understanding of the developmental origins of fetal alcohol spectrum disorders. [ABSTRACT FROM AUTHOR]

Identification of the transcriptional networks disrupted by prenatal ethanol exposure remains a core requirement to better understanding the molecular mechanisms of alcohol-induced teratogenesis. In this regard, quantitative reverse-transcriptase polymerase chain reaction (qPCR) has emerged as an essential technique in our efforts to characterize alterations in gene expression brought on by exposure to alcohol. However, many publications continue to report the utilization of inappropriate methods of qPCR normalization, and for many in vitro models, no consistent set of empirically tested normalization controls have been identified. In the present study, we sought to identify a group of candidate reference genes for use within studies of alcohol exposed embryonic, placental, and neurosphere stem cells under both conditions maintaining stemness as well as throughout in vitro differentiation. To this end, we surveyed the recent literature and compiled a short list of fourteen candidate genes commonly used as normalization controls in qPCR studies of gene expression. This list included: Actb, B2m, Gapdh, Gusb, H2afz, Hk2, Hmbs, Hprt, Mrpl1, Pgk1, Ppia, Sdha, Tbp, and Ywhaz. From these studies, we find no single candidate gene was consistently refractory to the influence of alcohol nor completely stable throughout in vitro differentiation. Accordingly, we propose normalizing qPCR measurements to the geometric mean CT values obtained for three independent reference mRNAs as a reliable method to accurately interpret qPCR data and assess alterations in gene expression within alcohol treated cultures. Highlighting the importance of careful and empirical reference gene selection, the commonly used reference gene Actb was often amongst the least stable candidate genes tested. In fact, it would not serve as a valid normalization control in many cases. Data presented here will aid in the design of future experiments using stem cells to study the transcriptional processes driving differentiation, and model the developmental impact of teratogens. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are members of a large class of non-protein-coding RNA (ncRNA) molecules that represent a significant, but until recently unappreciated, layer of cellular regulation. Assessment of the generation and function of miRNAs suggests that these ncRNAs are vulnerable to interference from genetic, epigenetic, and environmental factors. A small but rapidly expanding body of studies using a variety of animal- and cell culture-based experimental models also has shown that miRNAs are important targets of alcohol during fetal development and that their dysregulation likely plays a significant role in the etiology of fetal alcohol spectrum disorders (FASD). Accordingly, an analysis of the regulation and function of these miRNAs may yield important clues to the management of FASD. [ABSTRACT FROM AUTHOR]

Prenatal alcohol exposure (PAE) can have immediate and long-lasting toxic and teratogenic effects on an individual's development and health. As a toxicant, alcohol can lead to a variety of physical and neurological anomalies in the fetus that can lead to behavioral and other impairments which may last a lifetime. Recent studies have focused on identifying mechanisms that mediate the immediate teratogenic effects of alcohol on fetal development and mechanisms that facilitate the persistent toxic effects of alcohol on health and predisposition to disease later in life. This review focuses on the contribution of epigenetic modifications and intercellular transporters like extracellular vesicles to the toxicity of PAE and to immediate and long-term consequences on an individual's health and risk of disease. [ABSTRACT FROM AUTHOR]

Prenatal ethanol exposure can result in loss of neural stem cells (NSCs) and decreased brain growth. Here, we assessed whether a noncoding RNA (ncRNA) related to the NSC self-renewal factor Oct4/Pou5f1, and transcribed from a processed pseudogene locus on mouse chromosome 9 (mOct4pg9), contributed to the loss of NSCs due to ethanol. Mouse fetal cortical-derived NSCs, cultured ex vivo to mimic the early neurogenic environment of the fetal telencephalon, expressed m Oct4pg9 ncRNA at significantly higher levels than the parent Oct4/Pou5f1 mRNA. Ethanol exposure increased expression of m Oct4pg9 ncRNA, but decreased expression of Oct4/Pou5f1. Gain- and loss-of-function analyses indicated that m Oct4pg9 overexpression generally mimicked effects of ethanol exposure, resulting in increased proliferation and expression of transcripts associated with neural maturation. Moreover, m Oct4pg9 associated with Ago2 and with miRNAs, including the anti-proliferative miR-328-3p, whose levels were reduced following m Oct4pg9 overexpression. Finally, m Oct4pg9 inhibited Oct4/Pou5f1–3'UTR-dependent protein translation. Consistent with these observations, data from single-cell transcriptome analysis showed that m Oct4pg9-expressing progenitors lack Oct4/Pou5f1, but instead overexpress transcripts for increased mitosis, suggesting initiation of transit amplification. Collectively, these data suggest that the inhibitory effects of ethanol on brain development are explained, in part, by a novel ncRNA which promotes loss of NSC identity and maturation. • Oct4pg9 is a novel mouse pseudogene-encoded noncoding RNA (ncRNA), related to the gene encoding the transcription factor Oct4/Pou5f1, is expressed in neural stem cells (NSCs), and is induced by alcohol (ethanol) exposure • Oct4pg9 ncRNA and Oct4/Pou5f1 mRNA are expressed in non-overlapping sub-populations of neural stem cells. • Oct4pg9 associates with miRNA-chaperone Ago2, and with the antiproliferative miRNA, miR-328-3p. • Oct4pg9 expression results in inhibition of Oct4/Pou5f1 3'UTR-dependent translation. • Gain- and loss-of-function studies indicate that Oct4pg9 upregulation explains pro-maturation and teratogenic effects of ethanol on NSCs. [ABSTRACT FROM AUTHOR]

A hallmark of fetal alcohol spectrum disorders (FASD) is neurobehavioral deficits that still do not have effective treatment. Here, we present that reduction of Apolipoprotein E (APOE) is critically involved in neurobehavioral deficits in FASD. We show that prenatal alcohol exposure (PAE) changes chromatin accessibility of Apoe locus, and causes reduction of APOE levels in both the brain and peripheral blood in postnatal mice. Of note, postnatal administration of an APOE receptor agonist (APOE-RA) mitigates motor learning deficits and anxiety in those mice. Several molecular and electrophysiological properties essential for learning, which are altered by PAE, are restored by APOE-RA. Our human genome-wide association study further reveals that the interaction of PAE and a single nucleotide polymorphism in the APOE enhancer which chromatin is closed by PAE in mice is associated with lower scores in the delayed matching-to-sample task in children. APOE in the plasma is also reduced in PAE children, and the reduced level is associated with their lower cognitive performance. These findings suggest that controlling the APOE level can serve as an effective treatment for neurobehavioral deficits in FASD., (© 2024. The Author(s).)

Background: Children exposed prenatally to alcohol or cannabinoids individually can exhibit growth deficits and increased risk for adverse birth outcomes. However, these drugs are often co-consumed and their combined effects on early brain development are virtually unknown. The blood vessels of the fetal brain emerge and mature during the neurogenic period to support nutritional needs of the rapidly growing brain, and teratogenic exposure during this gestational window may therefore impair fetal cerebrovascular development., Study Design: To determine whether prenatal polysubstance exposure confers additional risk for impaired fetal-directed blood flow, we performed high resolution in vivo ultrasound imaging in C57Bl/6J pregnant mice. After pregnancy confirmation, dams were randomly assigned to one of four groups: drug-free control, alcohol-exposed, cannabinoid-exposed or alcohol-and-cannabinoid-exposed. Drug exposure occurred daily between Gestational Days 12-15, equivalent to the transition between the first and second trimesters in humans. Dams first received an intraperitoneal injection of either cannabinoid agonist CP-55,940 (750 µg/kg) or volume-equivalent vehicle. Then, dams were placed in vapor chambers for 30 min of inhalation of either ethanol or room air. Dams underwent ultrasound imaging on three days of pregnancy: Gestational Day 11 (pre-exposure), Gestational Day 13.5 (peri-exposure) and Gestational Day 16 (post-exposure)., Results: All drug exposures decreased fetal cranial blood flow 24-hours after the final exposure episode, though combined alcohol and cannabinoid co-exposure reduced internal carotid artery blood flow relative to all other exposures. Umbilical artery metrics were not affected by drug exposure, indicating a specific vulnerability of fetal cranial circulation. Cannabinoid exposure significantly reduced cerebroplacental ratios, mirroring prior findings in cannabis-exposed human fetuses. Post-exposure cerebroplacental ratios significantly predicted subsequent perinatal mortality (p = 0.019, area under the curve, 0.772; sensitivity, 81%; specificity, 85.70%) and retroactively diagnosed prior drug exposure (p = 0.005; AUC, 0.861; sensitivity, 86.40%; specificity, 66.7%)., Conclusions: Fetal cerebrovasculature is significantly impaired by exposure to alcohol or cannabinoids, and co-exposure confers additional risk for adverse birth outcomes. Considering the rising potency and global availability of cannabis products, there is an imperative for research to explore translational models of prenatal drug exposure, including polysubstance models, to inform appropriate strategies for treatment and care in pregnancies affected by drug exposure., (© 2024. The Author(s).)

Prenatal alcohol exposure (PAE) impairs recovery from cerebrovascular ischemic stroke in adult rodents. Since the gut becomes dysbiotic following stroke, we assessed links between PAE and enteric portal inflammation. Adult control and PAE rat offspring received a unilateral endothelin-1-induced occlusion of the middle cerebral artery. Post-stroke behavioral disabilities and brain cytokines were assessed. Mesenteric adipose and liver transcriptomes were assessed from stroke-exposed and stroke-naive offspring. We identified, in the liver of stroke-naive animals, a moderate correlation between PAE and a gene network for inflammatory necroptosis. PAE inhibited the acute-phase brain inflammatory cytokine response to stroke. Post-stroke neurological function was correlated with an adipose gene network associated with B-lymphocyte differentiation and nuclear factor κB (NF-κB) signaling and with a liver pro-inflammatory gene network. Collectively, PAE inhibits brain inflammation but results in an inflammatory signature in enteric portal tissues after stroke, suggesting that PAE persistently and adversely impacts the gut-brain axis following adult-onset disease., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Alcohol exposure in adulthood can result in inflammation, malnutrition, and altered gastroenteric microbiota, which may disrupt efficient nutrient extraction. Clinical and preclinical studies have documented convincingly that prenatal alcohol exposure (PAE) also results in persistent inflammation and nutrition deficiencies, though research on the impact of PAE on the enteric microbiota is in its infancy. Importantly, other neurodevelopmental disorders, including autism spectrum and attention deficit/hyperactivity disorders, have been linked to gut microbiota dysbiosis. The combined evidence from alcohol exposure in adulthood and from other neurodevelopmental disorders supports the hypothesis that gut microbiota dysbiosis is likely an etiological feature that contributes to negative developmental, including neurodevelopmental, consequences of PAE and results in fetal alcohol spectrum disorders. Here, we highlight published data that support a role for gut microbiota in healthy development and explore the implication of these studies for the role of altered microbiota in the lifelong health consequences of PAE., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Upreti, Rouzer, Bowring, Labbe, Kumar, Miranda and Mahnke.)

Alcohol use disorder (AUD) is one of the most common preventable mental health disorders and can result in pathology within the CNS, including the cerebellum. Cerebellar alcohol exposure during adulthood has been associated with disruptions in proper cerebellar function. However, the mechanisms regulating ethanol-induced cerebellar neuropathology are not well understood. High-throughput next generation sequencing was performed to compare control versus ethanol-treated adult C57BL/6J mice in a chronic plus binge model of AUD. Mice were euthanized, cerebella were microdissected, and RNA was isolated and submitted for RNA-sequencing. Down-stream transcriptomic analyses revealed significant changes in gene expression and global biological pathways in control versus ethanol-treated mice that included pathogen-influenced signaling pathways and cellular immune response pathways. Microglial-associated genes showed a decrease in homeostasis-associated transcripts and an increase in transcripts associated with chronic neurodegenerative diseases, while astrocyte-associated genes showed an increase in transcripts associated with acute injury. Oligodendrocyte lineage cell genes showed a decrease in transcripts associated with both immature progenitors as well as myelinating oligodendrocytes. These data provide new insight into the mechanisms by which ethanol induces cerebellar neuropathology and alterations to the immune response in AUD.