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.

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.

Diagnostic Potential of Neural Exosome Cargo as Biomarkers for Acute Brain Injury.

Objective: Neuronal exosomes purified from peripheral blood samples have been proposed as diagnostic tool in the setting of acute brain injury but never tested clinically. We hypothesized that exosome protein biomarkers would change over time following acute hypoxic brain injury and would predict response to therapy. Methods: Synaptopodin (SYNPO), an actin-associated protein present in postsynaptic spines, was evaluated as a potential biomarker as well as: synaptophysin, neuron-specific enolase, and mitochondrial cytochrome c oxidase. A secondary analysis was performed on neonatal samples collected at 8, 10, and 14 h after the initiation of therapeutic-controlled hypothermia for acute hypoxic-ischemic encephalopathy (n = 14). Neuronal exosomes were purified from serum and protein levels were quantified using standard ELISA methods. The primary study outcomes were length of stay (LOS), discharge on seizure medication (DCMED), and composite neuroimaging score (NIS). Results: The slope of change in neuronal exosome SYNPO between 8 and 14 h appeared to be the most promising biomarker for all three clinical study outcomes. SYNPO was highly correlated with LOS (-0.91, P < 0.001). SYNPO increased in 6/8 without DCMED and was worse or neutral in 5/5 with DCMED (P = 0.02). All four neonates with an abnormal NIS had neutral or decreasing SYNPO (P = 0.055). Other candidate biomarkers were not associated with outcomes. Interpretation: This report provides the first clinical evidence that neural exosomes turn over rapidly enough in the peripheral circulation to be used as a "troponin-like" test following acute brain injury. Optimal sampling and biomarkers likely vary with type of brain injury.