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.