Fetal Brain-Derived Exosomal miRNAs from Maternal Blood: Potential Diagnostic Biomarkers for Fetal Alcohol Spectrum Disorders (FASDs).

Fetal alcohol spectrum disorders (FASDs) are leading causes of neurodevelopmental disability but cannot be diagnosed early in utero. Because several microRNAs (miRNAs) are implicated in other neurological and neurodevelopmental disorders, the effects of EtOH exposure on the expression of these miRNAs and their target genes and pathways were assessed. In women who drank alcohol (EtOH) during pregnancy and non-drinking controls, matched individually for fetal sex and gestational age, the levels of miRNAs in fetal brain-derived exosomes (FB-Es) isolated from the mothers' serum correlated well with the contents of the corresponding fetal brain tissues obtained after voluntary pregnancy termination. In six EtOH-exposed cases and six matched controls, the levels of fetal brain and maternal serum miRNAs were quantified on the array by qRT-PCR. In FB-Es from 10 EtOH-exposed cases and 10 controls, selected miRNAs were quantified by ddPCR. Protein levels were quantified by ELISA. There were significant EtOH-associated reductions in the expression of several miRNAs, including miR-9 and its downstream neuronal targets BDNF, REST, Synapsin, and Sonic hedgehog. In 20 paired cases, reductions in FB-E miR-9 levels correlated strongly with reductions in fetal eye diameter, a prominent feature of FASDs. Thus, FB-E miR-9 levels might serve as a biomarker to predict FASDs in at-risk fetuses.

Effects of In Utero EtOH Exposure on 18S Ribosomal RNA Processing: Contribution to Fetal Alcohol Spectrum Disorder.

Fetal alcohol spectrum disorders (FASD) are leading causes of neurodevelopmental disability. The mechanisms by which alcohol (EtOH) disrupts fetal brain development are incompletely understood, as are the genetic factors that modify individual vulnerability. Because the phenotype abnormalities of FASD are so varied and widespread, we investigated whether fetal exposure to EtOH disrupts ribosome biogenesis and the processing of pre-ribosomal RNAs and ribosome assembly, by determining the effect of exposure to EtOH on the developmental expression of 18S rRNA and its cleaved forms, members of a novel class of short non-coding RNAs (srRNAs). In vitro neuronal cultures and fetal brains (11-22 weeks) were collected according to an IRB-approved protocol. Twenty EtOH-exposed brains from the first and second trimester were compared with ten unexposed controls matched for gestational age and fetal gender. Twenty fetal-brain-derived exosomes (FB-Es) were isolated from matching maternal blood. RNA was isolated using Qiagen RNA isolation kits. Fetal brain srRNA expression was quantified by ddPCR. srRNAs were expressed in the human brain and FB-Es during fetal development. EtOH exposure slightly decreased srRNA expression (1.1-fold; p = 0.03). Addition of srRNAs to in vitro neuronal cultures inhibited EtOH-induced caspase-3 activation (1.6-fold, p = 0.002) and increased cell survival (4.7%, p = 0.034). The addition of exogenous srRNAs reversed the EtOH-mediated downregulation of srRNAs (2-fold, p = 0.002). EtOH exposure suppressed expression of srRNAs in the developing brain, increased activity of caspase-3, and inhibited neuronal survival. Exogenous srRNAs reversed this effect, possibly by stabilizing endogenous srRNAs, or by increasing the association of cellular proteins with srRNAs, modifying gene transcription. Finally, the reduction in 18S rRNA levels correlated closely with the reduction in fetal eye diameter, an anatomical hallmark of FASD. The findings suggest a potential mechanism for EtOH-mediated neurotoxicity via alterations in 18S rRNA processing and the use of FB-Es for early diagnosis of FASD. Ribosome biogenesis may be a novel target to ameliorate FASD in utero or after birth. These findings are consistent with observations that gene-environment interactions contribute to FASD vulnerability.

Molecular Markers in Maternal Blood Exosomes Allow Early Detection of Fetal Alcohol Spectrum Disorders.

Prenatal alcohol exposure can cause developmental abnormalities (fetal alcohol spectrum disorders; FASD), including small eyes, face and brain, and neurobehavioral deficits. These cannot be detected early in pregnancy with available imaging techniques. Early diagnosis could facilitate development of therapeutic interventions. Banked human fetal brains and eyes at 9−22 weeks' gestation were paired with maternal blood samples, analyzed for morphometry, protein, and RNA expression, and apoptotic signaling. Alcohol (EtOH)-exposed (maternal self-report) fetuses were compared with unexposed controls matched for fetal age, sex, and maternal race. Fetal brain-derived exosomes (FB-E) were isolated from maternal blood and analyzed for protein, RNA, and apoptotic markers. EtOH use by mothers, assessed by self-report, was associated with reduced fetal eye diameter, brain size, and markers of synaptogenesis. Brain caspase-3 activity was increased. The reduction in eye and brain sizes were highly correlated with amount of EtOH intake and caspase-3 activity. Levels of several biomarkers in FB-E, most strikingly myelin basic protein (MBP; r > 0.9), correlated highly with morphological abnormalities. Reduction in FB-E MBP levels was highly correlated with EtOH exposure (p < 1.0 × 10−10). Although the morphological features of FAS appear long before they can be detected by live imaging, FB-E in the mother's blood may contain markers, particularly MBP, that predict FASD.

Ethanol-mediated alterations in oligodendrocyte differentiation in the developing brain.

INTRODUCTION: Alterations of white matter integrity and subsequent white matter structural deficits are consistent findings in Fetal Alcohol Syndrome (FAS), but knowledge regarding the molecular mechanisms underlying these abnormalities is incomplete. Experimental rodent models of FAS have shown dysregulation of cytokine expression leading to apoptosis of oligodendrocyte precursor cells (OPCs) and altered oligodendrocyte (OL) differentiation, but whether this is representative of human FAS pathogenesis has not been determined. METHODS: Fetal brain tissue (12.2-21.4 weeks gestation) from subjects undergoing elective termination of pregnancy was collected according to an IRB-approved protocol. Ethanol (EtOH) exposure status was classified based on a detailed face-to-face questionnaire adapted from the National Institute on Alcohol Abuse and Alcoholism Prenatal Alcohol and Sudden Infant Death Syndrome and Stillbirth (PASS) study. Twenty EtOH-exposed fetuses were compared with 20 gestational age matched controls. Cytokine and OPC marker mRNA expression was quantified by Real-Time Polymerase chain reaction (qRT-PCR). Patterns of protein expression of OPC markers and active Capase-3 were studied by Fluorescence Activated Cell Sorting (FACS). RESULTS: EtOH exposure was associated with reduced markers of cell viability, OPC differentiation, and OL maturation, while early OL differentiation markers were unchanged or increased. Expression of mRNAs for proteins specific to more mature forms of OL lineage (platelet-derived growth factor α (PDGFRα) and myelin basic protein (MBP) was lower in the EtOH group than in controls. Expression of the multifunctional growth and differentiation-promoting growth factor IGF-1, which is essential for normal development, also was reduced. Reductions were not observed for markers of early stages of OL differentiation, including Nuclear transcription factor NK-2 homeobox locus 2 (Nkx2.2). Expression of mRNAs for the proinflammatory cytokine, tumor necrosis factor-α (TNFα), and several proinflammatory chemokines was higher in the EtOH group compared to controls, including: Growth regulated protein alpha/chemokine (C-X-C motif) ligand 1 (GRO-α/CXCL1), Interleukin 8/chemokine (C-X-C motif) ligand 8 (IL8/CXCL8), Chemokine (C-X-C motif) ligand 6/Granulocyte chemotactic protein 2 (CXCL16/GCP2), epithelial-derived neutrophil-activating protein 78/chemokine (C-X-C motif) ligand 5 (ENA-78/CXCL5), monocyte chemoattractant protein-1 (MCP-1). EtOH exposure also was associated with an increase in the proportion of cells expressing markers of early stage OPCs, such as A2B5 and NG2. Finally, apoptosis (measured by caspase-3 activation) was increased substantially in the EtOH group compared to controls. CONCLUSION: Prenatal EtOH exposure is associated with excessive OL apoptosis and/or delayed OL maturation in human fetal brain. This is accompanied by markedly dysregulated expression of several chemokines and cytokines, in a pattern predictive of increased OL cytotoxicity and reduced OL differentiation. These findings are consistent with findings in animal models of FAS.

How do chemical patterns affect equilibrium droplet shapes?

By utilizing a proposed analytical model in combination with the phase-field method, we present a comprehensive study on the effect of chemical patterns on equilibrium droplet morphologies. Here, three influencing factors, the droplet sizes, contact angles, and the ratios of the hydrophilic area to the hydrophobic area, are contemplated. In the analytical model, chemical heterogeneities are described by different non-linear functions. By tuning these functions and the related parameters, the analytical model is capable of calculating the energy landscapes of the system. The chemically patterned surfaces display complex energy landscapes with chemical-heterogeneity-induced local minima, which correspond to the equilibrium morphologies of the droplets. Phase-field (PF) simulations are accordingly conducted and compared with the predicted equilibrium morphologies. In addition, we propose a modified Cassie-Baxter (CB) model to delineate the equilibrium droplet shapes. In contrast to the classic CB model, our extension is not only restricted to the shape with a spherical cap. Both the energy landscape method and the modified CB model are demonstrated to have a good agreement with the PF simulations.

Promoting Axon Regeneration in Adult CNS by Targeting Liver Kinase B1.

Liver kinase B1 (LKB1), a downstream effector of cyclic AMP (cAMP)/PKA and phosphatidylinositol 3-kinase (PI3K) pathways, is a determinant for migration and differentiation of many cells, but its role in CNS axon regeneration is unknown. Therefore, LKB1 was overexpressed in sensorimotor cortex of adult mice five days after mid-thoracic spinal cord injury, using an AAV2 vector. Regeneration of corticospinal axons was dramatically enhanced. Next, systemic injection of a mutant-AAV9 vector was used to upregulate LKB1 specifically in neurons. This promoted long-distance regeneration of injured corticospinal fibers into caudal spinal cord in adult mice and regrowth of descending serotonergic and tyrosine hydroxylase immunoreactive axons. Either intracortical or systemic viral delivery of LKB1 significantly improved recovery of locomotor functions in adult mice with spinal cord injury. Moreover, we demonstrated that LKB1 used AMPKα, NUAK1, and ERK as the downstream effectors in the cortex of adult mice. Thus, LKB1 may be a critical factor for enhancing the growth capacity of mature neurons and may be an important molecular target in the treatment of CNS injuries.

Investigation of Equilibrium Droplet Shapes on Chemically Striped Patterned Surfaces Using Phase-Field Method.

We systematically investigate the equilibrium shapes of droplets deposited on a set of chemically striped patterned surfaces by using an Allen?Cahn-type phase-field model. Varying the widths of the stripes d, the volume V, as well as the initial positions of the droplets, we release the droplets on the top of the surfaces and observe the final droplet shapes. It is found that there are either one or two equilibrium shapes for a fixed ratio of d/ V1/3 and each equilibrium shape corresponds to an energy minimum state. The aspect ratio of the droplets ? shows a periodic oscillation behavior with a decreasing amplitude as d/ V1/3 decreases, similar to the stick?slip?jump movement of a slowly condensing droplet on a chemically striped patterned surface. Additionally, by comparing the movements of slowly evaporating and condensing droplets, we have observed a hysteresis phenomenon, which reveals that the final shapes of droplets also rely on the moving paths. Through modifying the dynamic contact angle boundary condition, the contact line movements of droplets under condensation and evaporation, which are far from equilibrium, are addressed.

The role of RhoA in retrograde neuronal death and axon regeneration after spinal cord injury.

Paralysis following spinal cord injury (SCI) is due to interruption of axons and their failure to regenerate. It has been suggested that the small GTPase RhoA may be an intracellular signaling convergence point for several types of growth-inhibiting extracellular molecules. Even if this is true in vitro, it is not clear from studies in mammalian SCI, whether the effects of RhoA manipulations on axon growth in vivo are due to a RhoA-mediated inhibition of true regeneration or only of collateral sprouting from spared axons, since work on SCI generally is performed with partial injury models. RhoA also has been implicated in local neuronal apoptosis after SCI, but whether this reflects an effect on axotomy-induced cell death or an effect on other pathological mechanisms is not known. In order to resolve these ambiguities, we studied the effects of RhoA knockdown in the sea lamprey central nervous system (CNS), where after complete spinal cord transection (TX), robust but incomplete regeneration of large axons belonging to individually identified reticulospinal (RS) neurons occurs, and where some RS neurons show unambiguous delayed retrograde apoptosis after axotomy. RhoA protein was detected in neurons and axons of the lamprey brain and spinal cord, and its expression was increased post-TX. Knockdown of RhoA in vivo by retrogradely-delivered morpholino antisense oligonucleotides (MOs) to the RS neurons significantly reduced retrograde apoptosis signaling in identified RS neurons post-SCI, as indicated by Fluorochrome Labeled Inhibitor of Caspases (FLICA) in brain wholemounts. In individual RS neurons, the reduction of caspase activation by RhoA knockdown began at 2weeks post-TX and was still seen at 8weeks. RhoA knockdown slowed axon retraction and possibly increased early axon regeneration in the proximal stump. The number of axons regenerating beyond the lesion more than 5mm at 10weeks post-TX also was increased. Thus RhoA knockdown both enhanced true axon regeneration and inhibited retrograde apoptosis signaling after SCI.

Electric-field-induced lamellar to hexagonally perforated lamellar transition in diblock copolymer thin films: kinetic pathways.

Symmetric block-copolymers, hitherto, are well known to evolve into parallel, perpendicular and mixed lamellar morphologies under the concomitant influence of an electric field and substrate affinity. In the present work, we show that an additional imposed confinement can effectuate a novel parallel lamellar to hexagonally perforated lamellar (HPL) transition in monolayer and bilayer films. Three dimensional numerical studies are performed using the Ohta-Kawasaki functional, complemented with an exact solution of Maxwell's equation. HPL is shown to stabilize at large substrate affinity in a narrow region of the phase diagram between parallel and perpendicular lamellar transitions in ultra-thin films. Additionally, we also identify perforated lamellae as intermediate structures during parallel-to-perpendicular lamellar transition. A systematic analysis using Minkowski functionals yields deeper insights into the associated kinetic pathways.

Role of CSPG receptor LAR phosphatase in restricting axon regeneration after CNS injury.

Extracellular matrix molecule chondroitin sulfate proteoglycans (CSPGs) are highly upregulated in scar tissues and form a potent chemical barrier for CNS axon regeneration. Recent studies support that the receptor protein tyrosine phosphatase σ (PTPσ) and its subfamily member leukocyte common antigen related phosphatase (LAR) act as transmembrane receptors to mediate CSPG inhibition. PTPσ deficiency increased regrowth of ascending axons into scar tissues and descending corticospinal tract (CST) axons into the caudal spinal cord after spinal cord injury (SCI). Pharmacological LAR inhibition enhanced serotonergic axon growth in SCI mice. However, transgenic LAR deletion on axon growth in vivo and the role of LAR in regulating regrowth of other fiber tracts have not been studied. Here, we studied the role of LAR in restricting regrowth of injured descending CNS axons in deficient mice. LAR deletion increased regrowth of serotonergic axons into scar tissues and caudal spinal cord after dorsal over-hemitransection. LAR deletion also stimulated regrowth of CST fibers into the caudal spinal cord. LAR protein was upregulated days to weeks after injury and co-localized to serotonergic and CST axons. Moreover, LAR deletion improved functional recovery by increasing BMS locomotor scores and stride length and reducing grid walk errors. This is the first transgenic study that demonstrates the crucial role of LAR in restricting regrowth of injured CNS axons.

Neuronal endoplasmic reticulum stress in axon injury and neurodegeneration.

Injuries to central nervous system axons result not only in Wallerian degeneration of the axon distal to the injury, but also in death or atrophy of the axotomized neurons, depending on injury location and neuron type. No method of permanently avoiding these changes has been found, despite extensive knowledge concerning mechanisms of secondary neuronal injury. The autonomous endoplasmic reticulum (ER) stress pathway in neurons has recently been implicated in retrograde neuronal degeneration. In addition to the emerging role of ER morphology in axon maintenance, we propose that ER stress is a common neuronal response to disturbances in axon integrity and a general mechanism for neurodegeneration. Thus, manipulation of the ER stress pathway could have important therapeutic implications for neuroprotection.

A phase-field approach for wetting phenomena of multiphase droplets on solid surfaces.

We study the equilibrium wetting behavior of immiscible multiphase systems on a flat, solid substrate. We present numerical computations which are based on a vector-valued multiphase-field model of Allen-Cahn type, with a new boundary condition, based on appropriately designed surface energy contributions in order to ensure the right contact angles at multiphase junctions. Experimental investigations are carried out to validate the method and to support the numerical results.

Micro-computed tomography assessment of vertebral column defects in retinoic acid-induced rat model of myelomeningocele.

BACKGROUND: Myelomeningocele (MMC) is a common congenital malformation and the most severe form of spina bifida characterized by the protrusion of spinal cord and meninges through the spinal defect. Our objective was to improve the assessment of congenital vertebral defects in animal models of MMC using three-dimensional high resolution micro-computed tomography (micro-CT) imaging and quantitative digital analyses methods. METHODS: Lumbosacral MMC was induced in fetal rats by exposure of pregnant mothers at embryonic day 10 (E10) to all-trans retinoic acid, and rats were examined at term (embryonic day 22). The axial skeleton was examined in an MMC model for the first time using ex vivo micro-CT at 10 µm voxel resolution to allow high resolution two-dimensional and three-dimensional characterization of anomalies in lumbosacral vertebrae, and quantitative assessment of distances between dorsal vertebral arches in lumbosacral regions in MMC rats, compared with normal controls. RESULTS: We observed, in detail, skeletal defects in lumbosacral vertebra of MMC rats, including in the morphology of individual dorsal vertebral arches. Use of high resolution micro-CT has also enabled us to identify the delayed (nonfused) or absent ossification in vertebral bodies, increased fusion of adjacent lateral vertebral elements, and quantify the extent of dorsal arch widening. Distances between dorsal vertebral arches showed statistically significant increases from L5 through S4 in MMC rats, compared with normal controls. CONCLUSION: High-resolution micro-CT combined with digital quantification methods is a powerful technique ideally suited for precise assessment of complex congenital skeletal abnormalities such as examined in this rodent model of MMC.

Phase-field simulations at the atomic scale in comparison to molecular dynamics.

Early solidification is investigated using two different simulation techniques: the molecular dynamics (MD) and the phase-field (PF) methods. While the first describes the evolution of a system on the basis of motion equations of particles, the second grounds on the evolution of continuous local order parameter field. The aim of this study is to probe the ability of the mesoscopic phase-field method to make predictions of growth velocity at the nanoscopic length scale. For this purpose the isothermal growth of a spherical crystalline cluster embedded in a melt is considered. The system in study is Ni modeled with the embedded atom method (EAM). The bulk and interfacial properties required in the PF method are obtained from MD simulations. Also the initial configuration obtained from MD data is used in the PF as input. Results for the evolution of the cluster volume at high and moderate undercooling are presented.

Combined RhoA morpholino and ChABC treatment protects identified lamprey neurons from retrograde apoptosis after spinal cord injury.

Previously, we reported that RhoA knockdown by morpholino antisense oligonucleotides (MOs), and enzymatic digestion of chondroitin sulfate proteoglycans (CSPGs) at the site of injury with chondroitinase ABC (ChABC), each can reduce retrograde neuronal apoptosis after spinal cord transection in the lamprey. To elucidate the mechanisms in neuronal survival and axon regeneration, we have investigated whether these two effects are additive in vivo. We used lampreys as a spinal cord injury model. MOs were used to knockdown RhoA and Chondroitinase ABC (ChABC) was used to digest CSPGs in vivo. Retrograde labeling, fluorochrome-labeled inhibitor of caspase activity (FLICA), immunohistochemistry, and western blots were performed to assess axonal regeneration, neuronal apoptotic signaling and Akt activation. Four treatment combinations were evaluated at 2-, 4-, and 10-weeks post-transection: (1) Control MO plus enzyme buffer (Ctrl); (2) control MO plus ChABC; (3) RhoA MO plus enzyme buffer (RhoA MO); and (4) RhoA MO plus ChABC (RhoA MO + ChABC). Consistent with our previous findings, at 4-weeks post-transection, there was less caspase activation in the ChABC and RhoA MO groups than in the Ctrl group. Moreover, the RhoA MO plus ChABC group had the best protective effect on identified reticulospinal (RS) neurons among the four treatment combinations. At 2 weeks post-transection, when axons have retracted maximally in the rostral stump and are beginning to regenerate back toward the lesion, the axon tips in the three treatment groups each were closer to the transection than those in the Ctr MO plus enzyme buffer group. Long-term axon regeneration also was evaluated for the large, individually identified RS neurons at 10 weeks post-transection by retrograde labeling. The percent regenerated axons in the RhoA MO plus ChABC group was greater than that in any of the other groups. Akt phosphorylation levels at threonine 308 was quantified in the identified RS neurons by western blots and immunofluorescence. The RhoA MO plus ChABC treatment enhanced pAkt-308 phosphorylation more than any of the other treatment groups. Although some of the effects of CSPGs are mediated through RhoA activation, some growth-inhibiting mechanisms of RhoA and CSPGs are independent of each other, so combinatorial therapies may be warranted.

In utero ethanol exposure induces mitochondrial DNA damage and inhibits mtDNA repair in developing brain.

Introduction: Mitochondrial dysfunction is postulated to be a central event in fetal alcohol spectrum disorders (FASD). People with the most severe form of FASD, fetal alcohol syndrome (FAS) are estimated to live only 34 years (95% confidence interval, 31 to 37 years), and adults who were born with any form of FASD often develop early aging. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage, hallmarks of aging, are postulated central events in FASD. Ethanol (EtOH) can cause mtDNA damage, consequent increased oxidative stress, and changes in the mtDNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1). Studies of molecular mechanisms are limited by the absence of suitable human models and non-invasive tools. Methods: We compared human and rat EtOH-exposed fetal brain tissues and neuronal cultures, and fetal brain-derived exosomes (FB-Es) from maternal blood. Rat FASD was induced by administering a 6.7% alcohol liquid diet to pregnant dams. Human fetal (11-21 weeks) brain tissue was collected and characterized by maternal self-reported EtOH use. mtDNA was amplified by qPCR. OGG1 and Insulin-like growth factor 1 (IGF-1) mRNAs were assayed by qRT-PCR. Exosomal OGG1 was measured by ddPCR. Results: Maternal EtOH exposure increased mtDNA damage in fetal brain tissue and FB-Es. The damaged mtDNA in FB-Es correlated highly with small eye diameter, an anatomical hallmark of FASD. OGG1-mediated mtDNA repair was inhibited in EtOH-exposed fetal brain tissues. IGF-1 rescued neurons from EtOH-mediated mtDNA damage and OGG1 inhibition. Conclusion: The correlation between mtDNA damage and small eye size suggests that the amount of damaged mtDNA in FB-E may serve as a marker to predict which at risk fetuses will be born with FASD. Moreover, IGF-1 might reduce EtOH-caused mtDNA damage and neuronal apoptosis.

Identification of Lithium Compounds on Surfaces of Lithium Metal Anode with Machine-Learning-Assisted Analysis of ToF-SIMS Spectra.

Detailed knowledge about contamination and passivation compounds on the surface of lithium metal anodes (LMAs) is essential to enable their use in all-solid-state batteries (ASSBs). Time-of-flight secondary ion mass spectrometry (ToF-SIMS), a highly surface-sensitive technique, can be used to reliably characterize the surface status of LMAs. However, as ToF-SIMS data are usually highly complex, manual data analysis can be difficult and time-consuming. In this study, machine learning techniques, especially logistic regression (LR), are used to identify the characteristic secondary ions of 5 different pure lithium compounds. Furthermore, these models are applied to the mixture and LMA samples to enable identification of their compositions based on the measured ToF-SIMS spectra. This machine-learning-based analysis approach shows good performance in identifying characteristic ions of the analyzed compounds that fit well with their chemical nature. Moreover, satisfying accuracy in identifying the compositions of unseen new samples is achieved. In addition, the scope and limitations of such a strategy in practical applications are discussed. This work presents a robust analytical method that can assist researchers in simplifying the analysis of the studied lithium compound samples, offering the potential for broader applications in other material systems.

Biomarkers of Affective Dysregulation Associated with In Utero Exposure to EtOH.

INTRODUCTION: Children with fetal alcohol spectrum disorders (FASD) exhibit behavioral and affective dysregulation, including hyperactivity and depression. The mechanisms are not known, but they could conceivably be due to postnatal social or environmental factors. However, we postulate that, more likely, the affective dysregulation is associated with the effects of EtOH exposure on the development of fetal serotonergic (5-HT) and/or dopaminergic (DA) pathways, i.e., pathways that in postnatal life are believed to regulate mood. Many women who use alcohol (ethanol, EtOH) during pregnancy suffer from depression and take selective serotonin reuptake inhibitors (SSRIs), which might influence these monoaminergic pathways in the fetus. Alternatively, monoaminergic pathway abnormalities might reflect a direct effect of EtOH on the fetal brain. To distinguish between these possibilities, we measured their expressions in fetal brains and in fetal brain-derived exosomes (FB-Es) isolated from the mothers' blood. We hypothesized that maternal use of EtOH and/or SSRIs during pregnancy would be associated with impaired fetal neural development, detectable as abnormal levels of monoaminergic and apoptotic biomarkers in FB-Es. METHODS: Fetal brain tissues and maternal blood were collected at 9-23 weeks of pregnancy. EtOH groups were compared with unexposed controls matched for gestational age (GA). The expression of 84 genes associated with the DA and 5-HT pathways was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on microarrays. FB-Es also were assayed for serotonin transporter protein (SERT) and brain-derived neurotrophic factor (BDNF) by enzyme-linked immunosorbent assay (ELISA). RESULTS: Six EtOH-exposed human fetal brain samples were compared to SSRI- or polydrug-exposed samples and to unexposed controls. EtOH exposure was associated with significant upregulation of DA receptor D3 and 5-HT receptor HTR2C, while HTR3A was downregulated. Monoamine oxidase A (MAOA), MAOB, the serine/threonine kinase AKT3, and caspase-3 were upregulated, while mitogen-activated protein kinase 1 (MAPK1) and AKT2 were downregulated. ETOH was associated with significant upregulation of the DA transporter gene, while SERT was downregulated. There were significant correlations between EtOH exposure and (a) caspase-3 activation, (b) reduced SERT protein levels, and (c) reduced BDNF levels. SSRI exposure independently increased caspase-3 activity and downregulated SERT and BDNF. Early exposure to EtOH and SSRI together was associated synergistically with a significant upregulation of caspase-3 and a significant downregulation of SERT and BDNF. Reduced SERT and BDNF levels were strongly correlated with a reduction in eye diameter, a somatic manifestation of FASD. CONCLUSIONS: Maternal use of EtOH and SSRI during pregnancy each was associated with changes in fetal brain monoamine pathways, consistent with potential mechanisms for the affective dysregulation associated with FASD.

Maternal Blood Lipid Biomarkers of Oligodendrocyte Pathology to Predict Fetal Alcohol Spectrum Disorders.

Introduction: Up to 9.9% of children have fetal alcohol spectrum disorders (FASD), the most frequent cause of intellectual disability in the US. FASD may involve abnormal brain development, including dysmyelination, suggesting abnormal development of oligodendrocytes (OLs), which make myelin and are rich in lipids. Indeed, low serum levels of omega-3 fatty acids (ω-3) have been reported in FASD. Free fatty acids bind to specific receptors (FFARs). We have isolated cell type-specific fetal brain-derived exosomes (FB-E) from maternal blood and sampled their contents to search for lipid-related biomarkers that predict FASD. Methods: Blood samples were collected from two groups of pregnant women: 1) those who consumed EtOH during pregnancy, and 2) non-EtOH using controls, under an IRB-approved protocol. Serum and OL-derived exosomes (OL-Es) were used to assay myelin basic protein (MBP) and FFAR by ELISA and droplet digital PCR (ddPCR), respectively. Results: FFAR and MBP proteins were downregulated in the EtOH group compared to controls, and this difference was greatest in OL-Es from maternal blood compared maternal serum. Conclusion: MBP and FFAR levels were reduced in OL-Es from EtOH-consuming pregnant women. The data suggest potential therapeutic targets to predict which children are at risk for developing FASD and reduce dysmyelination in developing.

Exosomal Lipid Biomarkers of Oligodendrocyte Pathology to Predict Scoliosis in Children with Cerebral Palsy.

Introduction: Cerebral Palsy (CP), the most common cause of disability in children, is phenotypically heterogeneous. Approximately 20% of cases develop severe scoliosis. A pathological hallmark of CP is periventricular leukomalacia (PVL), which is due to dysmyelination, suggesting the possibility of a lipidomic abnormality. Risk factors for CP include perinatal hypoxia, prematurity, multiple gestation, ischemia, infection, and maternal alcohol consumption. There is evidence for low serum levels of omega-3 (ω-3) fatty acids in CP patients, and separately in idiopathic scoliosis. Many effects of free fatty acids (FFAs) are mediated via specific G protein-coupled free fatty acid receptors (FFARs), which play essential roles as nutritional and signaling molecules. FFAs, including ω-3, and their receptors are involved in the development and metabolism of oligodendrocytes (OLs), and are critical to myelination. Thus, the cases of CP that will develop severe scoliosis might be those in which there is a deficiency of ω-3, FFARs, or other lipidomic abnormality that is detectable early in the plasma. If so, we might be able to predict scoliosis and prevent it with dietary supplementation. Methods: Blood samples were collected from four groups of patients at the Philadelphia Shriners Children's Hospital (SCH-P): 1) patients with CP; 2) severe scoliosis (>40o); 3) CP plus scoliosis; and 4) non-impaired controls stratified by age (2-18 yrs), gender, and race/ethnicity, under an IRB-approved protocol. Serum proteins and RNA were purified, and OL-derived exosomes (OL-Es) isolated, using myelin basic protein (MBP) as a late OL marker. Protein was used for the detection of MBP and FFAR by enzyme-linked immunosorbent assays (ELISAs), and by flow cytometry. RNA was assayed by digital droplet polymerase chain reaction (ddPCR) for OL markers and FFAR expression. Results: FFAR and MBP proteins were downregulated in each of the three patient groups compared to controls, and this difference was greatest in both patients with CP plus scoliosis. Conclusion: Altogether, MBP and FFAR levels were reduced in OL-Es from both children with CP plus scoliosis. The lipid abnormalities specific to CP with scoliosis were concentrated in OLs. Our data might i) suggest therapeutic targets to reduce dysmyelination and scoliosis in CP, ii) predict which children are at risk for developing scoliosis, iii) lead to therapeutic trials of fatty acids for CP and other dysmyelinating neurological disorders.