Sex differences in the transcriptome of extracellular vesicles secreted by fetal neural stem cells and effects of chronic alcohol exposure.

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

Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure.

Accumulating evidence indicates that extracellular vesicles (EVs) mediate endocrine functions and also pathogenic effects of neurodevelopmental perturbagens like ethanol. We performed mass-spectrometry on EVs secreted by fetal murine cerebral cortical neural stem cells (NSCs), cultured ex-vivo as sex-specific neurosphere cultures, to identify overrepresented proteins and signaling pathways in EVs relative to parental NSCs in controls, and following exposure of parental NSCs to a dose range of ethanol. EV proteomes differ substantially from parental NSCs, and though EVs sequester proteins across sub-cellular compartments, they are enriched for distinct morphogenetic signals including the planar cell polarity pathway. Ethanol exposure favored selective protein sequestration in EVs and depletion in parental NSCs, and also resulted in dose-independent overrepresentation of cell-cycle and DNA replication pathways in EVs as well as dose-dependent overrepresentation of rRNA processing and mTor stress pathways. Transfer of untreated EVs to naïve cells resulted in decreased oxidative metabolism and S-phase, while EVs derived from ethanol-treated NSCs exhibited diminished effect. Collectively, these data show that NSCs secrete EVs with a distinct proteome that may have a general growth-inhibitory effect on recipient cells. Moreover, while ethanol results in selective transfer of proteins from NSCs to EVs, the efficacy of these exposure-derived EVs is diminished.

Gag-like proteins: Novel mediators of prenatal alcohol exposure in neural development.

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

Extracellular Vesicles in Premature Aging and Diseases in Adulthood Due to Developmental Exposures.

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

Toxic and Teratogenic Effects of Prenatal Alcohol Exposure on Fetal Development, Adolescence, and Adulthood.

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

Ethanol Exposure Increases miR-140 in Extracellular Vesicles: Implications for Fetal Neural Stem Cell Proliferation and Maturation.

BACKGROUND: Neural stem cells (NSCs) generate most of the neurons of the adult brain in humans, during the mid-first through second-trimester period. This critical neurogenic window is particularly vulnerable to prenatal alcohol exposure, which can result in diminished brain growth. Previous studies showed that ethanol (EtOH) exposure does not kill NSCs, but, rather, results in their depletion by influencing cell cycle kinetics and promoting aberrant maturation, in part, by altering NSC expression of key neurogenic miRNAs. NSCs reside in a complex microenvironment rich in extracellular vesicles, shown to traffic miRNA cargo between cells. METHODS: We profiled the miRNA content of extracellular vesicles from control and EtOH-exposed ex vivo neurosphere cultures of fetal NSCs. We subsequently examined the effects of one EtOH-sensitive miRNA, miR-140-3p, on NSC growth, survival, and maturation. RESULTS: EtOH exposure significantly elevates levels of a subset of miRNAs in secreted extracellular vesicles. Overexpression of one of these elevated miRNAs, miR-140-3p, and its passenger strand relative, miR-140-5p, significantly increased the proportion of S-phase cells while decreasing the proportion of G0 /G1 cells compared to controls. In contrast, while miR-140-3p knockdown had minimal effects on the proportion of cells in each phase of the cell cycle, knockdown of miR-140-5p significantly decreased the proportion of cells in G2 /M phase. Furthermore, miR-140-3p overexpression, during mitogen-withdrawal-induced NSC differentiation, favors astroglial maturation at the expense of neural and oligodendrocyte differentiation. CONCLUSIONS: Collectively, the dysregulated miRNA content of extracellular vesicles following EtOH exposure may result in aberrant neural progenitor cell growth and maturation, explaining brain growth deficits associated with prenatal alcohol exposure.

Nonprotein-coding RNAs in Fetal Alcohol Spectrum Disorders.

Early developmental exposure to ethanol, a known teratogen, can result in a range of neurodevelopmental disorders, collectively referred to as Fetal Alcohol Spectrum Disorders (FASDs). Changes in the environment, including exposure to teratogens, can result in long term alterations to the epigenetic landscape of a cell, thereby altering gene expression. Noncoding RNAs (ncRNAs) can affect transcription and translation of networks of genes. ncRNAs are dynamically expressed during development and have been identified as a target of alcohol. ncRNAs therefore make for attractive targets for novel therapeutics to address the developmental deficits associated with FASDs.