small molecules

2026-09-26 | Plasma and Brain-Derived Extracellular Vesicle Biomarkers Following a Randomized Controlled Trial of Choline for Neurodevelopment in Fetal Alcohol Spectrum Disorder: A Pilot Analysis.

Postnatal choline supplementation improves memory, non-verbal IQ, executive function, and white-matter microstructure in children with fetal alcohol spectrum disorder (FASD) across randomized controlled trials; however, peripheral and brain biomarkers of these effects are uncharacterized. We measured eight immune and neurotrophic biomarkers (TNFα, transferrin receptor 1 [TfR1], BDNF, ferritin, MCP-1, RANTES, CRP, and Eotaxin) in plasma and brain-derived extracellular vesicles (BDEVs) from 24 children with FASD randomized to choline (n = 13; 7 male, 6 female) or placebo (n = 11; 7 male, 4 female), before and approximately nine months after treatment (23 with paired pre/post samples). Values were log10-transformed to correct for non-normal distribution. We modeled Group × Time effects with linear mixed models, adjusted for age, sex, and sample storage duration, cross-checked with cluster-robust ordinary least squares, and tested plasma-to-BDEV concordance for each analyte with linear regression adjusted for age and sex. Multiplicity was corrected by Benjamini-Hochberg FDR. Plasma MCP-1 showed a nominally significant Group × Time interaction (p = 0.035, q = 0.28), rising over time in the placebo but not the choline group. Ferritin and CRP showed similar patterns that did not reach significance (interaction p = 0.099 and p = 0.118; q = 0.31 for both). RANTES and BDNF rose significantly in BDEVs over time, without evidence of a differential change by treatment group, and survived FDR correction (both q ≤ 0.02); ferritin also rose significantly (p = 0.021) but its Time effect did not survive FDR correction (q = 0.06). BDEV TfR1 was nominally lower in the choline group than placebo at both timepoints (p = 0.023; q = 0.13). Plasma levels were associated with BDEV levels for five markers (ferritin, TfR1, TNFα, Eotaxin, RANTES; all p < 0.05), independent of age, sex, time, and group; all five associations survived FDR correction (q ≤ 0.01). The plasma-BDEV association for MCP-1 became non-significant when adjusted for time and group (p = 0.134, q = 0.13). CRP and BDNF showed a between-person, but not within-person, plasma-BDEV association. In this small, exploratory sample of children with FASD, a condition characterized by brain iron deficiency and inflammation, choline was associated with an attenuated rise in pro-inflammatory plasma MCP-1 relative to placebo, generating a new hypothesized candidate biomarker for further study. Most BDEV markers changed over time (Pre-to-Post), not with treatment. Plasma and BDEV BDNF were not significantly associated overall, although a between-person association was present without a corresponding within-person signal, suggesting that inferring central nervous system function from plasma BDNF should proceed with caution. These hypothesis-generating findings support further, larger-scale investigation of BDEV content analysis as a candidate complement to this trial program's cognitive and neuroimaging outcomes. Replication and expansion of the BDEV panel of analytes in a larger sample are needed. ClinicalTrials.Gov [NCT02735473]; registered 2 April 2016.

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2026-09-13 | Developmental Deficiency of Oligodendrocytes and Myelination in the Central Nervous System of the Fetal Alcohol Syndrome Mouse Model.

Fetal alcohol spectrum disorder (FASD) is a severe condition characterized by physical and neurodevelopmental abnormalities in the fetus resulting from prenatal alcohol exposure (PAE) or prenatal and lactation alcohol exposure (PLAE). Behavioral abnormalities associated with white matter damage have been extensively investigated in FASD patients and animal models, which suggested the involvement of myelination in FASD pathology. However, the impact of FASD on oligodendrocytes (OLs) and myelination within the central nervous system (CNS) and the underlying mechanisms are little discovered. This study aimed to reveal the effect and role of PLAE on CNS myelination in a FASD mouse model. We set up a PLAE approach to generate the FASD mouse model to explore the effects of FASD on CNS myelination. Open-field test, elevated plus maze, and tail suspension test were used to evaluate possible behavior deficiency of the FASD mice. Myelination in the cortex and hippocampus of the FASD mice was detected at postnatal days 14, 21, and 90 (P14, P21, and P 90). Then we counted the number of oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (mOLs) to reveal the cell fates of oligodendrocyte lineage cells in the FASD mice. Next, we set up BLBP+/GFAP+ double-staining to monitor the possible dysfunction of radial glial cells, which may contribute to the myelination deficiency in FASD mice. Finally, an FDA-approved pro-myelination drug, clemastine fumarate (CF), was used to validate the possible advantages of myelin restoration and behavior defects in the FASD mice. Impaired myelination was observed in the cortex and hippocampus of the male FASD offspring, associated with hyperactivity-like behaviors, whereas the female FASD mice exhibited delayed myelin formation and normal behaviors, indicating a gender-specific manner of FASD pathology. Furthermore, the different myelination deficiency between male and female FASD mice is due to the fact that only the male FASD mice exhibited a failure of oligodendrocyte differentiation. Additionally, FASD exerts an impact on the quantity and differentiation of radial glia cells, which may contribute to the myelination defect. Finally, CF-treated male FASD mice showed myelin regeneration, leading to partial alleviation of behavioral abnormality in the male FASD mice. Our data reveal a gender-related deficiency of CNS myelination in the FASD mouse model, suggesting a potential therapy target for FASD.

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2026-09-08 | Prenatal Alcohol Exposure Produces Selective Changes in Neuroimmune Gene Expression Across Brain Regions of Adult Mice.

An overwhelming body of evidence suggests neuroimmune dysfunction as a key underlying mechanism of fetal alcohol spectrum disorder (FASD)-associated adverse central nervous system (CNS) outcomes. While few studies have highlighted the lingering effects of prenatal alcohol exposure (PAE) on producing specific immune factors, others suggest a primed neuroimmune state in adulthood, in which a proinflammatory bias is unmasked following subsequent immune activation in later life. However, the PAE-induced neuroimmune landscape in adulthood remains poorly defined. We hypothesized that PAE induces long-term changes in gene expression linked to neuroimmune function that may be brain region-specific. Using long-read next-generation RNA sequencing of brain tissues from a previously established model of a moderate PAE in mice, we compared across six regions: medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), hypothalamus, hippocampus, midbrain, and medulla. A comprehensive bioinformatics analysis investigated PAE-induced changes, dysregulated gene pathways, and transcriptional regulators with a focus on neuroimmune function. Our data identified at least 60 differentially expressed genes per brain region, many of which were associated with neuroimmune function. Upregulation of multiple pro-inflammatory factors and pathways was observed, suggesting ongoing baseline neuroimmune activation, potentially involving PXR, TNF, TLR4, the complement pathway, and various cytokine and chemokine signaling. A comparative analysis identified multiple upstream transcriptional regulators across multiple brain regions, including MECP2, TCF7L2, and IL-4. Importantly, this unbiased analysis revealed heterogeneity across brain regions in the activation of canonical immune pathways and highlighted previously unprecedented roles of pathways such as PXR, matrix metalloproteases, and cytokine signaling (e.g., IL-15, IL-27, IL-17) in PAE. PAE creates a unique inflammatory signature in the adult brain, even in the absence of secondary injury, with novel patterns of region-specific changes in genes implicated in glial-immune function. These data identify potential immune targets to elucidate the mechanisms underlying behavioral dysfunction and provide a framework for future therapeutic interventions.

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2026-08-27 | The Gut-Brain Axis in Fetal Alcohol Spectrum Disorder (FASD): Why the Gut Shapes Behavior, Depression, and Self-Injurious Behavior in Children with Prenatal Alcohol Exposure-A Narrative Review with a Proposal for Staged Nutritional and Microbiological Intervention.

Prenatal alcohol exposure (PAE) leads to fetal alcohol spectrum disorder (FASD), the most common preventable cause of neurodevelopmental impairment. The classical narrative attributes the clinical picture of FASD exclusively to direct ethanol-induced brain injury. In the present review, we argue that this perspective is incomplete and leads to diagnostic errors, most often to the misdiagnosis of ADHD in children who in fact have FASD. We propose that, alongside the direct neurotoxicity of ethanol, an important and clinically under-recognized complementary mechanism is gut-brain axis dysfunction: alcohol damages the enteric nervous system and enteric glial cells, induces dysbiosis with deep deficits of butyrate and other short-chain fatty acids (SCFAs), damages the enterochromaffin cells responsible for 90% of peripheral serotonin production, and-through translocation of lipopolysaccharide (LPS) and activation of the Toll-like receptor 4 (TLR4)-sustains a neuroinflammatory brain signature. This cascade-superimposed on direct ethanol neurotoxicity-may account for the high rates of depression, anxiety, self-injurious behavior, and suicide attempts observed in individuals with FASD and for the limited efficacy of traditional interventions focused solely on the central nervous system. The 2024 Polish Institute of Mother and Child (Okulicz-Kozaryn et al.) study showed that 50.3% of pregnant women consumed alcohol, and 11% did so regularly, against only 7% who admitted so in questionnaires. The real clinical picture of children with FASD is further complicated by three factors to which we devote separate sections in this paper: prenatal co-exposure to nicotine, cannabinoids, and opioids; the loss of vertical microbiota transmission and breastfeeding in children transferred to foster care (where the prevalence of FASD is 18.8% and in children's homes in some regions reaches up to 80%); and the substantial over-representation of preterm and small-for-gestational-age (SGA) infants (in the Hasken et al. cohort, 18.4% of children with FASD were born preterm and 51.4% were born SGA). In the final section, we present a structured, staged protocol for nutritional and microbiological intervention grounded in a hierarchy of evidence: from interventions supported by randomized controlled trials (RCT-level; choline) through interventions supported by strong mechanistic rationale and RCTs in related populations (sodium butyrate, Lactobacillus rhamnosus GG, GOS/FOS prebiotics-galacto-oligosaccharides and fructo-oligosaccharides, and omega-3 fatty acids) to experimental interventions. The protocol also covers the window before 2 years of age: we argue that, given the over-representation of preterm and SGA infants among children with FASD, the analogy to preterm infants on parenteral nutrition and to post-institutional infants applies in substantial part to the same patients, which justifies extending the indications for choline and other nutritional interventions. The paper includes a compact table of dosing proposals for each age window (from pregnancy to school-age child) and provides clinicians with concrete answers: where to start, what to avoid, and what to monitor, with explicit signposting of regulatory limitations for individual substances in Poland and the European Union.

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2026-08-06 | Maternal gut and vaginal microbiota, alcohol use during pregnancy, and infant fetal alcohol spectrum disorder diagnosis in a South African cohort

Abstract Background The detrimental impact of alcohol consumption on the gut microbiome is well-established. However, less is known about how alcohol exposure during pregnancy affects the maternal gut and vaginal microbiota, or how these microbial changes relate to subsequent infant diagnosis of fetal alcohol spectrum disorder (FASD). We therefore investigated associations between self-reported alcohol use during pregnancy, infant FASD diagnosis, and maternal gut and vaginal microbiota. Methods Fecal samples (n = 207) and vaginal swabs (n = 28) from pregnant participants recruited through antenatal clinics in the Western Cape Province of South Africa were profiled by 16S rRNA V1-V2 amplicon sequencing. Maternal alcohol use was assessed using Alcohol Use Disorder Identification Test (AUDIT) scores, and FASD was diagnosed in infants using revised Institute of Medicine criteria. Microbial diversity, taxonomic profiles and PICRUSt2 -predicted functional pathways were analyzed using vegan , phyloseq and MaAsLin3 . Results Maternal AUDIT scores were negatively associated with maternal gut microbiota richness, Shannon, and Inverse Simpson diversity ( p < 0.05). Observed richness was also reduced in participants whose infants were diagnosed with FASD ( p = 0.046). Gut microbiota community structure was not significantly associated with alcohol use or infant FASD diagnosis. However, taxonomic and functional analysis suggested gram-positive taxa depletion with alcohol use and FASD diagnosis, and impaired one-carbon metabolism among participants with infants diagnosed with FASD. Vaginal microbiota diversity and composition were not associated with alcohol use or infant FASD diagnostic group. Conclusions This is the first human study to investigate the maternal gut and vaginal microbiota in relation to alcohol use during pregnancy and infant FASD outcomes. Our findings suggest that alcohol use is associated with disruption of the maternal gut microbiota, with potential implications for FASD development in exposed infants. Further investigation of alcohol-associated maternal microbial disturbances may inform microbiota-targeted strategies to improve maternal and infant health linked to alcohol use.

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proteins

2026-09-02 | Cloning of human DING: Developmental expression and downregulation by EtOH in-utero.

An estimated 15-20% of women consume alcohol (EtOH) during pregnancy. Women with alcohol use in early pregnancy are likely to have a child with fetal alcohol spectrum disorders (FASD). Recently, we reported neuroprotective effects of human DING (a member of the DING family of phosphatases) against EtOH-mediated toxicity in rats and in human fetal cortical neurons in vitro. Now, we report the sequencing and developmental expression patterns of endogenous DING in human fetal brain. DING cDNA was cloned from human U87MG astrocytoma cells with primers specific to the plant DING gene and known prokaryotic DING genes. This cDNA was used to prepare antibodies. The full-length human DING gene p38hu (1095 nucleotide bases) is flanked by the first initiating codon, ATG, and the last, stop codon, TAA. Post-mortem fetal tissues and maternal blood were collected during pregnancy between 8 and 37 weeks' gestation. The developmental, spatial, and temporal expression of DING protein in fetal brain tissue was analyzed by immunohistochemistry. Developmental expression of DING in fetal brain and placenta was quantified by qWestern blots. DING promoter expression was assayed by ddPCR. Statistical analysis included ANOVA. Sequencing revealed different-sized genomic DNA clones. The anti-DING antibody detected proteins ranging in size from 35 to 40 kDa, and high molecular weight precursor protein in fetal brain and placenta. DING protein was present in fetal brain at early stages and its level was increased at later gestational ages. The DING promoter was expressed in fetal brain, neurospheres, and fetal brain-derived exosomes. DING levels were reduced in samples exposed to maternally consumed alcohol. Because DING is neuroprotective, its reduced expression in fetuses exposed to alcohol may suggest a mechanism that contributes to the pathogenesis of FASD, which could lead to the development of therapeutic tools aimed at preventing, ameliorating or reversing this prevalent group of syndromes that are implicated in as many as 5% of births world-wide.

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2026-04-02 | Growth hormone therapy in a patient with short stature due to fetal alcohol syndrome: seven-year follow-up.

Fetal alcohol syndrome (FAS) is associated with persistent growth retardation, but the long-term efficacy of GH therapy for FAS-related short stature remains unclear. A Japanese girl born at 37 wk with birth weight 2,064 g (-2.1 SD) and birth height 41.5 cm (-2.7 SD) was diagnosed with FAS based on characteristic facial features, growth failure, developmental delay, and documented maternal alcohol consumption (120 g/d during pregnancy). She scored 4434 on the FASD 4-Digit Diagnostic Code. At 3 yr of age, her height was 78 cm (-4.18 SD) with poor growth velocity. GH therapy was initiated at 0.19 mg/kg/wk, increased to 0.23 mg/kg/wk, and gradually increased to 0.43 mg/kg/wk. After 7 yr of treatment, serum IGF-1 levels increased significantly from 59 ng/mL (-2.5 SD) at baseline to 306 ng/mL (+2.0 SD) at 8 yr, but height SD scores remained around -4 SD with growth velocity fluctuating between -3 and 0 SD. No adverse effects were observed. While GH therapy appears safe in FAS patients, its efficacy for improving linear growth is limited, suggesting that growth impairment in FAS involves mechanisms beyond GH deficiency, including growth plate dysfunction and peripheral GH resistance.

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2025-12-30 | Incretin-Related Pathology and Serum Exosome Detection in Experimental Alcohol-Related Brain Damage.

Alcohol's chronic neurotoxic and degenerative effects mediate alcohol-related brain damage (ARBD), which is marked by neurobehavioral, cognitive, and motor deficits. Major underlying abnormalities include impairments in signaling through the insulin and insulin-like growth factor (IGF) pathways, which regulate energy metabolism. This study examined the potential role of dysregulated incretin network-related mechanisms as mediators of ARBD and evaluated a non-invasive serum exosome (S-EV)-based approach for detecting brain abnormalities. Frontal lobe tissue and S-EVs isolated from Long-Evans adolescent rats maintained for 2 weeks on control or 24% ethanol (caloric) containing liquid diets (n = 8/group) were analyzed using multiplex magnetic bead-based enzyme-linked immunosorbent assays (ELISAs). ARBD was associated with significantly reduced insulin, C-peptide, glucagon, ghrelin, leptin, GIP, and amylin levels in the frontal lobe and/or S-EV samples. In contrast, chronic ethanol exposure had no significant effects on PP, PYY, or GLP-1, and it did not increase proinflammatory cytokine expression. Chronic ethanol feeding broadly affected (primarily inhibiting) the expression of metabolic hormones linked to insulin/IGF signaling. The reductions in GIP and amylin suggest potential targets for therapeutic intervention to enhance brain energy metabolism via insulin networks. On the other hand, the findings suggest that GLP-1 receptor agonists may have limited efficacy in remediating the effects of ARBD. Finally, the results support the use of non-invasive S-EV assays to detect and guide treatment for metabolic brain dysfunction in ARBD.

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2025-11-21 | Neuroprotective Role of DING protein in Fetal Alcohol Spectrum Disorders and Depression.

An estimated 20% of women consume alcohol during pregnancy, and 10% of women receive antidepressants during their pregnancy. Women with depression are more likely to use alcohol (EtOH) in early pregnancy and are more likely to have a child with one of the fetal alcohol spectrum disorders (FASD). Previously, we provided evidence in rat neurons in vitro that DING phosphatase (p38SJ, a member of the DINGGG family of proteins that has neuroprotective effects under conditions of cellular stress) was neuroprotective against EtOH-mediated toxicity. Now, we examine the effects of DING, alone or in combination with EtOH and serotonergic (5-HT) pathway molecules and drugs, on EtOH-mediated neurotoxicity in human fetal tissues in vitro. Human fetal brain tissue was collected between 9 and 23 weeks' gestation. Primary neurons and neurospheres were prepared from a 16-week fetal brain. Exposures to EtOH and SSRI were assessed by detailed questionnaires. Neurotoxicity/apoptosis was assessed in synaptosome extracts with the Caspase-Glo 3/7 assay and a neurotoxicity microarray. Developmental expressions of DING, serotonin transporter (SERT), and 5-HT1A receptor (5HT1A) protein levels were quantified in the brain, placenta, synaptosomes, serum, and "fetal brain-derived exosomes" (FB-Es) by quantitative western blotting and digital droplet PCR. Increasing the levels of activated DING, either by addition to culture media or by intracellular overexpression, was associated with increased cell survival. Developmental expressions of DING, in the presence of EtOH- or SSRI-exposure, were investigated in brain and placenta tissues. While 5HT1A, 5-hydroxytryptamine serotonin receptor 1A (5-HT1A) was strongly inhibited under EtOH stress conditions (>10-fold), DING was not only able to reverse the negative effects of stress, as measured by the synaptic plasticity array, but also upregulated the 5-HT receptor (5-fold compared to untreated control) upon treatment of neurons. Maternal EtOH use is associated with decreased 5-HT receptor and DING protein expression in the fetal brain and with increased neuronal apoptosis. Combined exposure to EtOH and SSRIs may have greater toxic effects than either one alone. DING reversed the toxic effects of EtOH on serotonin receptors in neurons and neurospheres. These findings provide potential mechanisms for the neuroprotective effects of DING, which might suggest neuroprotective approaches to preventing fetal alcohol spectrum disorders (FASD).

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2025-03-21 | Neuroprotective Effects of Erythropoietin in Fetal Alcohol Spectrum Disorder: BDNF Upregulation, Reduced Astrogliosis, and Anxiety Relief.

The term fetal alcohol spectrum disorder (FASD) refers to a variety of negative consequences that may develop in children born to women who have consumed alcohol during pregnancy. Anxiety disorders are common in FASD. Animals exposed to alcohol exhibit anxiety-like traits. Erythropoietin (EPO) is produced by the kidneys and liver. The synthesis of EPO by immature neurons also plays a crucial role in the embryonic stage. In addition, EPO inhibits astrogliosis and cell death while increasing the number of brain-derived neurotrophic factor (BDNF)-positive cells, which is linked to reductions in anxiety-like behavior and neuronal damage. This study investigated the protective effects of EPO on ethanol-induced neurotoxicity in the hippocampus of rat pups. The intubation of the total daily dose of ethanol (5/27 g/kg/day) was started from Postnatal Day 2 (PD2) to PD10 (corresponding to the third trimester of pregnancy in humans). After intubation, 1,000 and 2,000 U/kg EPO were injected subcutaneously. The elevated plus maze was performed 39 days after the birth of the pups to determine the levels of anxiety. Immunohistochemical staining was then performed to determine GFAP and BDNF levels 40 days after birth. Nissl staining was done to measure necrotic cell death. EPO administration significantly improved the anxious behavior associated with FASD (p < .001). EPO significantly increased BDNF levels (p < .001), decreased glial fibrillary acidic protein expression (p < .001), and attenuated alcohol neurotoxicity-induced necrotic cell death (p < .001). EPO treatment provides protection against ethanol neurotoxicity, which consequently leads to lower anxiety levels.

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gene therapies

2022-11-04 | The pro-apoptotic Bax gene modifies susceptibility to craniofacial dysmorphology following gastrulation-stage alcohol exposure.

During early development, alcohol exposure causes apoptotic cell death in discrete regions of the embryo which are associated with distinctive patterns of later-life abnormalities. In gastrulation, which occurs during the third week of human pregnancy, alcohol targets the ectoderm, the precursor of the eyes, face, and brain. This midline tissue loss leads to the craniofacial dysmorphologies, such as microphthalmia and a smooth philtrum, which define fetal alcohol syndrome (FAS). An important regulator of alcohol-induced cell death is the pro-apoptotic protein Bax. The current study determines if mice lacking the Bax gene are less susceptible to the pathogenic effects of gastrulation-stage alcohol exposure. Male and female Bax+/- mice mated to produce embryos with full (-/- ) or partial (+/- ) Bax deletions, or Bax+/+ wild-type controls. On Gestational Day 7 (GD 7), embryos received two alcohol (2.9 g/kg, 4 hr apart), or control exposures. A subset of embryos was collected 12 hr later and examined for the presence of apoptotic cell death, while others were examined on GD 17 for the presence of FAS-like facial features. Full Bax deletion reduced embryonic apoptotic cell death and the incidence of fetal eye and face malformations, indicating that Bax normally facilitates the development of alcohol-induced defects. An RNA-seq analysis of GD 7 Bax+/+ and Bax-/- embryos revealed 63 differentially expressed genes, some of which may interact with the Bax deletion to further protect against apoptosis. Overall, these experiments identify that Bax is a primary teratogenic mechanism of gastrulation-stage alcohol exposure.

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2022-07-19 | Exosome-shuttled miR-126 mediates ethanol-induced disruption of neural crest cell-placode cell interaction by targeting SDF1

Abstract During embryonic development, two populations of multipotent stem cells, cranial neural crest cells (NCCs) and epibranchial placode cells (PCs), are anatomically adjacent to each other. The coordinated migration of NCCs and PCs plays a major role in the morphogenesis of craniofacial skeletons and cranial nerves. It’s known that ethanol-induced dysfunction of NCCs and PCs is a key contributor to the defects of craniofacial skeletons and cranial nerves implicated in Fetal Alcohol Spectrum Disorder (FASD). However, how ethanol disrupts the coordinated interaction between NCCs and PCs was not elucidated. To fill in this gap, we established a well-designed cell co-culture system to investigate the reciprocal interaction between human NCCs (hNCCs) and human PCs (hPCs), and also monitored the migration behavior of NCCs and PCs in zebrafish embryos. We found that ethanol exposure resulted in a disruption of coordinated hNCCs-hPCs interaction, as well as in zebrafish embryos. Treating hNCCs-hPCs with exosomes derived from ethanol-exposed hNCCs (Exo EtOH ) mimicked ethanol-induced impairment of hNCCs-hPCs interaction. We also observed that a chemoattractant, SDF1, was downregulated in ethanol-treated hPCs and zebrafish embryos. Meanwhile, miR-126 level in Exo EtOH was significantly higher than that in control exosomes (Exo Con ). We further validated that Exo EtOH -encapsulated miR-126 from hNCCs can be transferred to hPCs to suppress SDF1 expression in hPCs. Knockdown of SDF1 replicated ethanol-induced abnormalities either in vitro or in zebrafish embryos. On the contrary, overexpression of SDF1 or inhibiting miR-126 strongly rescued ethanol-induced impairment of hNCCs-hPCs interaction and developmental defects.

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2022-03-19 | mRNA expression analysis of the hippocampus in a vervet monkey model of fetal alcohol spectrum disorder

Abstract Background Fetal alcohol spectrum disorders (FASD) are common, yet preventable developmental disorders that stem from prenatal exposure to alcohol. This exposure leads to a wide array of behavioural and physical problems with a complex and poorly defined biological basis. Molecular investigations to date predominantly use rodent animal models, but because of genetic, developmental and social behavioral similarity, primate models are more relevant. We previously reported reduced cortical and hippocampal neuron levels in an Old World monkey ( Chlorocebus sabaeus ) model with ethanol exposure targeted to the period of rapid synaptogenesis and report here an initial molecular study of this model. The goal of this study was to evaluate mRNA expression of the hippocampus at two different behavioural stages (5 months, 2 years) corresponding to human infancy and early childhood. Methods Offspring of alcohol-preferring or control dams drank a maximum of 3.5 g ethanol per kg body weight or calorically matched sucrose solution 4 days per week during the last 2 months of gestation. Total mRNA expression was measured with the Affymetrix GeneChip Rhesus Macaque Genome Array in a 2 × 2 study design that interrogated two independent variables, age at sacrifice, and alcohol consumption during gestation. Results and discussion Statistical analysis identified a preferential downregulation of expression when interrogating the factor ‘alcohol’ with a balanced effect of upregulation vs. downregulation for the independent variable ‘age’. Functional exploration of both independent variables shows that the alcohol consumption factor generates broad functional annotation clusters that likely implicate a role for epigenetics in the observed differential expression, while the variable age reliably produced functional annotation clusters predominantly related to development. Furthermore, our data reveals a novel connection between EFNB1 and the FASDs; this is highly plausible both due to the role of EFNB1 in neuronal development as well as its central role in craniofrontal nasal syndrome (CFNS). Fold changes for key genes were subsequently confirmed via q RT-PCR. Conclusion Prenatal alcohol exposure leads to global downregulation in mRNA expression. The cellular interference model of EFNB1 provides a potential clue regarding how genetically susceptible individuals may develop the phenotypic triad generally associated with classic fetal alcohol syndrome.

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2022-03-01 | Loss of tumor protein 53 protects against alcohol-induced facial malformations in mice and zebrafish.

Alcohol exposure during the gastrulation stage of development causes the craniofacial and brain malformations that define fetal alcohol syndrome. These malformations, such as a deficient philtrum, are exemplified by a loss of midline tissue and correspond, at least in part, to regionally selective cell death in the embryo. The tumor suppressor protein Tp53 is an important mechanism for cell death, but the role of Tp53 in the consequences of alcohol exposure during the gastrulation stage has yet to be examined. The current studies used mice and zebrafish to test whether genetic loss of Tp53 is a conserved mechanism to protect against the effects of early developmental stage alcohol exposure. Female mice, heterozygous for a mutation in the Tp53 gene, were mated with Tp53 heterozygous males, and the resulting embryos were exposed during gastrulation on gestational day 7 (GD 7) to alcohol (two maternal injections of 2.9 g/kg, i.p., 4 h apart) or a vehicle control. Zebrafish mutants or heterozygotes for the tp53zdf1  M214K mutation and their wild-type controls were exposed to alcohol (1.5% or 2%) beginning 6 h postfertilization (hpf), the onset of gastrulation. Examination of GD 17 mice revealed that eye defects were the most common phenotype among alcohol-exposed fetuses, occurring in nearly 75% of the alcohol-exposed wild-type fetuses. Tp53 gene deletion reduced the incidence of eye defects in both the heterozygous and mutant fetuses (to about 35% and 20% of fetuses, respectively) and completely protected against alcohol-induced facial malformations. Zebrafish (4 days postfertilization) also demonstrated alcohol-induced reductions of eye size and trabeculae length that were less common and less severe in tp53 mutants, indicating a protective effect of tp53 deletion. These results identify an evolutionarily conserved role of Tp53 as a pathogenic mechanism for alcohol-induced teratogenesis.

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2020-02-27 | Hippocampal DNA Methylation in a Mouse Model of Fetal Alcohol Spectrum Disorder That Includes Maternal Separation Stress Only Partially Explains Changes in Gene Expression

Fetal alcohol spectrum disorder (FASD) is characterized by developmental and behavioral deficits caused by maternal drinking during pregnancy. Children born with FASD often face additional stresses, including maternal separation, that add yet additional deficits. The mechanism associated with this interaction is not known. We have used a mouse model for prenatal ethanol exposure and maternal separation to demonstrate that the combination of the two treatments results in more than additive deficits. Further, the behavioral deficits are associated with changes in hippocampal gene expression that persist into adulthood. What initiates and maintains these changes remains to be established and forms the focus of this report. Specifically, MeDIP-Seq was used to assess if changes in promoter DNA methylation are affected by exposure to prenatal ethanol and maternal separation including its relationship to gene expression. The novel results show that different sets of genes implicated by promoter DNA methylation are affected by both treatments independently, and a relatively unique set of genes are affected by the combination of the two treatments. Prenatal ethanol exposure leads to altered promoter DNA methylation at genes important for transcriptional regulation. Maternal separation leads to changes at genes important for histone methylation and immune response, and the combination of two treatments results in DNA methylation changes at genes important for neuronal migration and immune response. Our dual results from the same hippocampal samples suggest there is minimal complementarity between changes in promoter DNA methylation and gene expression, although genes involved tend to be critical for brain development and function. While remaining to be validated, such results argue that mechanisms beyond promoter DNA methylation must be involved in lasting gene expression alterations leading to behavioral deficits implicated in FASD. They may facilitate early and reliable diagnosis, as well as novel strategies for the amelioration of FASD-related deficits.

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oligonucleotides

2025-03-25 | Recent Advances in the Role of Non-coding RNAs in Fetal Alcohol Spectrum Disorders.

Despite numerous preclinical studies modeling fetal alcohol spectrum disorder (FASD)-associated neurodevelopmental deficits to date, a comprehensive molecular landscape dictating these deficits remains poorly understood. Non-coding RNAs constitute a substantial layer of epigenetic regulation of gene expression at the transcriptional, post-transcriptional, translational, and post-translational levels. Yet, little is known about the differential expression of non-coding RNAs in the context of prenatal alcohol exposure (PAE) that are mechanistically linked with FASD-related neurobehavior deficits. This chapter reviews our current knowledge from preclinical studies in non-coding RNA-mediated molecular mechanisms that may underlie FASD pathophysiology. This chapter also summarizes relevant clinical evidence and current efforts in utilizing these non-coding RNA molecules as biomarkers of PAE-associated deficits impacting central nervous system (CNS) function. Unraveling the diverse roles of various species of non-coding RNAs is critical to enhancing our comprehension of these intricate molecular pathways. Understanding these pathways would likely contribute to identifying critical molecular target(s) for developing efficient treatment strategies and prognostic and diagnostic markers fostering advancements in treating and managing FASD-related CNS dysfunction.

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2022-12-05 | MicroRNA-150-5p is upregulated in the brain microvasculature during prenatal alcohol exposure and inhibits the angiogenic factor Vezf1.

Fetal alcohol spectrum disorders (FASD) occur in children who were exposed to alcohol in utero and are manifested in a wide range of neurocognitive deficits. These deficits could be caused by alterations to the cortical microvasculature that are controlled by post-transcriptional regulators such as microRNAs. Using an established mouse model of moderate prenatal alcohol exposure (PAE), we isolated cortices (CTX) and brain microvascular endothelial cells (BMVECs) at embryonic day 18 (E18) and examined the expression of miR-150-5p and potential downstream targets. Cellular transfections and intrauterine injections with LNA™ mimics or inhibitors were used to test miR-150-5p regulation of novel target vascular endothelial zinc finger 1 (Vezf1). Dual-luciferase assays were used to assess the direct binding of miR-150-5p to the Vezf1 3'UTR. The effects of miR-150-5p and Vezf1 on endothelial cell function were determined by in vitro migration and tube formation assays. We found that miR-150-5p was upregulated and Vezf1 was downregulated during PAE in the E18 CTX and BMVECs. Transfection with miR-150-5p mimics resulted in decreased Vezf1 expression in BMVECs, while miR-150-5p inhibition did the opposite. Dual-luciferase assays revealed direct binding of miR-150-5p with the Vezf1 3'UTR. Intrauterine injections showed that miR-150-5p regulates the expression of Vezf1 in vivo during PAE. miR-150-5p overexpression decreased BMVEC migration and tube formation, while miR-150-5p inhibition enhanced migration and tube formation. Vezf1 overexpression rescued the effects of the miR-150-5p mimic. Alcohol treatment of BMVECs increased miR-150-5p expression and inhibited migration and tube formation. Finally, miR-150-5p inhibition and Vezf1 overexpression rescued the negative effects of alcohol on migration and tube formation. miR-150-5p regulation of Vezf1 results in altered endothelial cell function during alcohol exposure. Further, miR-150-5p inhibition of Vezf1 may adversely alter the development of the cortical microvasculature during PAE and contribute to deficits seen in patients with FASD.

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2022-10-13 | Ethanol Effects on Early Developmental Stages Studied Using the Zebrafish

Fetal alcohol spectrum disorder (FASD) results from prenatal ethanol exposure. The zebrafish (Danio rerio) is an outstanding in vivo FASD model. Early development produced the three germ layers and embryonic axes patterning. A critical pluripotency transcriptional gene circuit of sox2, pou5f1 (oct4; recently renamed pou5f3), and nanog maintain potency and self-renewal. Ethanol affects sox2 expression, which functions with pou5f1 to control target gene transcription. Various genes, like elf3, may interact and regulate sox2, and elf3 knockdown affects early development. Downstream of the pluripotency transcriptional circuit, developmental signaling activities regulate morphogenetic cell movements and lineage specification. These activities are also affected by ethanol exposure. Hedgehog signaling is a critical developmental signaling pathway that controls numerous developmental events, including neural axis specification. Sonic hedgehog activities are affected by embryonic ethanol exposure. Activation of sonic hedgehog expression is controlled by TGF-ß family members, Nodal and Bmp, during dorsoventral (DV) embryonic axis establishment. Ethanol may perturb TGF-ß family receptors and signaling activities, including the sonic hedgehog pathway. Significantly, experiments show that activation of sonic hedgehog signaling rescues some embryonic ethanol exposure effects. More research is needed to understand how ethanol affects early developmental signaling and morphogenesis.

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2022-05-03 | Up-regulation of microRNA-34a mediates ethanol-induced impairment of neural crest cell migration in vitro and in zebrafish embryos through modulating epithelial-mesenchymal transition by targeting Snail1.

Prenatal ethanol exposure can impair neural crest cell (NCC) development, including NCC survival, differentiation and migration, contributing to the craniofacial dysmorphology in Fetal Alcohol Spectrum Disorders (FASD). Epithelial-mesenchymal transition (EMT) plays an important role in regulating the migration of NCCs. The objective of this study is to determine whether ethanol exposure can suppress NCC migration through inhibiting EMT and whether microRNA-34a (miR-34a) is involved in the ethanol-induced impairment of EMT in NCCs. We found that exposure to 100 mM ethanol significantly inhibited the migration of NCCs. qRT-PCR and Western Blot analysis revealed that exposure to ethanol robustly reduced the mRNA and protein expression of Snail1, a critical transcriptional factor that has a pivotal role in the regulation of EMT. Ethanol exposure also significantly increased the mRNA expression of the Snail1 target gene E-cadherin1 and inhibited EMT in NCCs. We also found that exposure to ethanol significantly elevated the expression of miR-34a that targets Snail1 in NCCs. In addition, down-regulation of miR-34a prevented ethanol-induced repression of Snail1 and diminished ethanol-induced upregulation of Snail1 target gene E-cadherin1 in NCCs. Inhibition of miR-34a restored EMT and prevented ethanol-induced inhibition of NCC migration in vitro and in zebrafish embryos in vivo. These results demonstrate that ethanol-induced upregulation of miR-34a contributes to the impairment of NCC migration through suppressing EMT by targeting Snail1.

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2022-01-31 | Ethanol modulation of hippocampal neuroinflammation, myelination, and neurodevelopment in a postnatal mouse model of fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) are alarmingly common and result in significant personal and societal loss. Neuropathology of the hippocampus is common in FASD leading to aberrant cognitive function. In the current study, we evaluated the effects of ethanol on the expression of a targeted set of molecules involved in neuroinflammation, myelination, neurotransmission, and neuron function in the developing hippocampus in a postnatal model of FASD. Mice were treated with ethanol from P4-P9, hippocampi were isolated 24 h after the final treatment at P10, and mRNA levels were quantitated by qRT-PCR. We evaluated the effects of ethanol on both pro-inflammatory and anti-inflammatory molecules in the hippocampus and identified novel mechanisms by which ethanol induces neuroinflammation. We further demonstrated that ethanol decreased expression of molecules associated with mature oligodendrocytes and greatly diminished expression of a lacZ reporter driven by the first half of the myelin proteolipid protein (PLP) gene (PLP1). In addition, ethanol caused a decrease in genes expressed in oligodendrocyte progenitor cells (OPCs). Together, these studies suggest ethanol may modulate pathogenesis in the developing hippocampus through effects on cells of the oligodendrocyte lineage, resulting in altered oligodendrogenesis and myelination. We also observed differential expression of molecules important in synaptic plasticity, neurogenesis, and neurotransmission. Collectively, the molecules evaluated in these studies may play a role in ethanol-induced pathology in the developing hippocampus and contribute to cognitive impairment associated with FASD. A better understanding of these molecules and their effects on the developing hippocampus may lead to novel treatment strategies for FASD.

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2025-03-13 | Prenatal Alcohol Exposure Disrupts CXCL16 Expression in Rat Hippocampus: Temporal and Sex Differences.

Prenatal alcohol exposure (PAE) affects around 40,000 newborns every year and poses a significant health risk. Although much is already known about the neurotoxic mechanisms of PAE, new findings continue to emerge. Studies with mouse models show that PAE leads to overexpression of proinflammatory cytokines and chemokines in the brain, which disrupts important neurodevelopmental processes such as cell migration, survival and proliferation of neurons. The chemokine CXCL16 is overexpressed in the brain following various impairments, including PAE. This study shows that CXCL16 expression varies by developmental stage and sex, consistent with known sexual dimorphism in immune responses. In females, CXCL16 expression may be influenced by estrogen-related mechanisms, possibly related to the alcohol-mediated rebound effect described here. In contrast, the male hippocampus shows greater resilience to PAE-induced CXCL16 changes. Furthermore, the presence of CXCL16 in neuronal nuclei suggests a role in gene regulation, similar to other chemokines such as CCL5 and CXCL4. These findings shed light on the role of chemokines in hippocampal neuroplasticity and may pave the way for better treatment of fetal alcohol spectrum disorder (FASD).

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2024-09-26 | Neuroimmune Interactions in Fetal Alcohol Spectrum Disorders: Potential Therapeutic Targets and Intervention Strategies.

Fetal alcohol spectrum disorders (FASD) are a set of abnormalities caused by prenatal exposure to ethanol and are characterized by developmental defects in the brain that lead to various overt and non-overt physiological abnormalities. Growing evidence suggests that in utero alcohol exposure induces functional and structural abnormalities in gliogenesis and neuron-glia interactions, suggesting a possible role of glial cell pathologies in the development of FASD. However, the molecular mechanisms of neuron-glia interactions that lead to the development of FASD are not clearly understood. In this review, we discuss glial cell pathologies with a particular emphasis on microglia, primary resident immune cells in the brain. Additionally, we examine the involvement of several neuroimmune molecules released by glial cells, their signaling pathways, and epigenetic mechanisms responsible for FASD-related alteration in brain functions. Growing evidence suggests that extracellular vesicles (EVs) play a crucial role in the communication between cells via transporting bioactive cargo from one cell to the other. This review emphasizes the role of EVs in the context of neuron-glia interactions during prenatal alcohol exposure. Finally, some potential applications involving nutritional, pharmacological, cell-based, and exosome-based therapies in the treatment of FASD are discussed.

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2024-01-10 | Expression of placental CD146 is dysregulated by prenatal alcohol exposure and contributes in cortical vasculature development and positioning of vessel-associated oligodendrocytes

Recent data showed that prenatal alcohol exposure (PAE) impairs the “placenta–brain” axis controlling fetal brain angiogenesis in human and preclinical models. Placental growth factor (PlGF) has been identified as a proangiogenic messenger between these two organs. CD146, a partner of the VEGFR-1/2 signalosome, is involved in placental angiogenesis and exists as a soluble circulating form. The aim of the present study was to investigate whether placental CD146 may contribute to brain vascular defects described in fetal alcohol spectrum disorder. At a physiological level, quantitative reverse transcription polymerase chain reaction experiments performed in human placenta showed that CD146 is expressed in developing villi and that membrane and soluble forms of CD146 are differentially expressed from the first trimester to term. In the mouse placenta, a similar expression pattern of CD146 was found. CD146 immunoreactivity was detected in the labyrinth zone and colocalized with CD31-positive endothelial cells. Significant amounts of soluble CD146 were quantified by ELISA in fetal blood, and the levels decreased after birth. In the fetal brain, the membrane form of CD146 was the majority and colocalized with microvessels. At a pathophysiological level, PAE induced marked dysregulation of CD146 expression. The soluble form of CD146 decreased in both placenta and fetal blood, whereas it increased in the fetal brain. Similarly, the expression of several members of the CD146 signalosome, such as VEGFR2 and PSEN, was differentially impaired between the two organs by PAE. At a functional level, targeted repression of placental CD146 by in utero electroporation (IUE) of CRISPR/Cas9 lentiviral plasmids resulted in (i) a decrease in cortical vessel density, (ii) a loss of radial vascular organization, and (iii) a reduced density of oligodendrocytes. Statistical analysis showed that the more the vasculature was impaired, the more the cortical oligodendrocyte density was reduced. Altogether, these data support that placental CD146 contributes to the proangiogenic “placenta–brain” axis and that placental CD146 dysfunction contributes to the cortical oligo-vascular development. Soluble CD146 would represent a promising placental biomarker candidate representative of alcohol-induced neurovascular defects in neonates, as recently suggested by PlGF (patents WO2016207253 and WO2018100143).

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2023-09-29 | Autocrine positive feedback of tumor necrosis factor from activated microglia proposed to be of widespread relevance in chronic neurological disease.

Over a decade's experience of post-stroke rehabilitation by administering the specific anti-TNF biological, etanercept, by the novel perispinal route, is consistent with a wide range of chronically diminished neurological function having been caused by persistent excessive cerebral levels of TNF. We propose that this TNF persistence, and cerebral disease chronicity, largely arises from a positive autocrine feedback loop of this cytokine, allowing the persistence of microglial activation caused by the excess TNF that these cells produce. It appears that many of these observations have never been exploited to construct a broad understanding and treatment of certain chronic, yet reversible, neurological illnesses. We propose that this treatment allows these chronically activated microglia to revert to their normal quiescent state, rather than simply neutralizing the direct harmful effects of this cytokine after its release from microglia. Logically, this also applies to the chronic cerebral aspects of various other neurological conditions characterized by activated microglia. These include long COVID, Lyme disease, post-stroke syndromes, traumatic brain injury, chronic traumatic encephalopathy, post-chemotherapy, post-irradiation cerebral dysfunction, cerebral palsy, fetal alcohol syndrome, hepatic encephalopathy, the antinociceptive state of morphine tolerance, and neurogenic pain. In addition, certain psychiatric states, in isolation or as sequelae of infectious diseases such as Lyme disease and long COVID, are candidates for being understood through this approach and treated accordingly. Perispinal etanercept provides the prospect of being able to treat various chronic central nervous system illnesses, whether they are of infectious or non-infectious origin, through reversing excess TNF generation by microglia.

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2022-03-25 | Prenatal Drugs and Their Effects on the Developing Brain: Insights From Three-Dimensional Human Organoids

Decades of research have unequivocally demonstrated that fetal exposure to both recreational and prescription drugs in utero negatively impacts the developing brain. More recently, the application of cutting-edge techniques in neurodevelopmental research has attempted to identify how the fetal brain responds to specific environmental stimuli. Meanwhile, human fetal brain studies still encounter ethical considerations and technical limitations in tissue collection. Human-induced pluripotent stem cell (iPSC)-derived brain organoid technology has emerged as a powerful alternative to examine fetal neurobiology. In fact, human 3D organoid tissues recapitulate cerebral development during the first trimester of pregnancy. In this review, we aim to provide a comprehensive summary of fetal brain metabolic studies related to drug abuse in animal and human models. Additionally, we will discuss the current challenges and prospects of using brain organoids for large-scale metabolomics. Incorporating cutting-edge techniques in human brain organoids may lead to uncovering novel molecular and cellular mechanisms of neurodevelopment, direct novel therapeutic approaches, and raise new exciting questions.

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