Prenatal alcohol exposure (PAE) can result in decreased neurogenic capacity of neural stem cells (NSCs), which, during the early-to-mid gestation period, give rise to most neurons of the adult brain. This loss of neurogenic capacity explains, in part, neurobehavioral and brain growth deficits associated with Fetal Alcohol Spectrum Disorders (FASD). However, the mechanisms that mediate the persistent effects of episodic PAE on fetal NSCs are unknown. We and others have recently identified a novel class of sub-200 nanometer-sized extracellular vesicles (EVs) secreted by NSCs, as a means for intercellular transfer of biomolecules including proteins and nucleic acids. I hypothesize that EVs constitute endocrine-like organelles that propagate the effects of ethanol throughout the stem cell microenvironment in time and space. My preliminary and published data support the hypotheses that ethanol (i) alters the sequestration of proteins into EVs released by fetal NSCs, in a dose-dependent manner, and, by interfering with protein expression in EVs, (ii) results in loss of stem cell renewal and diminished neurogenesis. Using mass spectrometric and transcriptomic tools and cell and molecular biological analyses of EV function in a murine model, planned and completed studies address the following specific aims:
Aim 1 seeks to identify and validate the effects of alcohol exposure on proteins and RNAs in EVs and associated NSCs. My studies show that EVs from parent NSCs exposed to moderate doses of ethanol (Mod-EtoHEVs) exhibit a significant increase in proteins of the Nonsense- Mediated Decay (NMD), whereas EVs obtained following NSC exposure to higher levels of ethanol (Hi-EtoHEVs) overexpress mitochondrial proteins that constitute a Danger-Associated Molecular Pattern (mito-DAMP) signal. These data will be validated and compared to the ethanol-affected intracellular proteome of parent NSCs.
Aim 2 will evaluate the mediating role of Mod-EtoHEV-derived NMD on NSC proliferation, differentiation, and death, under basal and ethanol-exposure conditions. Our working hypothesis is that Mod-EtoHEVs transfer NMD components to recipient cells to confer compensatory neuroprotection.
Aim 3 will evaluate the mediating role of Hi-EtoHEV-derived mito-DAMP on NSC proliferation, differentiation, and death. Our working hypothesis is that the secretion of a ?danger? signal in response to high levels of ethanol exposure triggers a pro-inflammatory cytokine response in recipient NSCs, resulting in impaired neurogenesis. My studies are expected to uncover novel intercellular communication pathways that mediate the effects of PAE on early neural maturation. This training plan will equip me with technical and research methodology skills and develop specific competency with data analytic and statistical approaches for proteome and transcriptome assessment. The plan?s focus on dissecting and disrupting the connection between PAE and FASD integrates well with my career goal to become a scientist with a focus on the role of early environmental mediators of developmental disability.
Prenatal alcohol exposure (PAE) is the leading non-genetic cause of intellectual and other brain disabilities. This project seeks to characterize the basal and ethanol-mediated alterations of extracellular vesicle proteins and RNAs in neural stem cell (NSC) during neural development. The goal of these studies is to uncover novel intercellular mechanisms of neural development that will lead to interventions capable of mitigating the effects of PAE and consequently diminish the impact of Fetal Alcohol Spectrum Disorders (FASD). This is a goal consistent with the mission of the National Institute of Alcohol Abuse and Alcoholism, which is to find ways to ?reduce alcohol-related problems? by research in ?prevention and treatment?.
