CD24 Expression Identifies Teratogen-Sensitive Fetal Neural Stem Cell Subpopulations: Evidence from Developmental Ethanol Exposure and Orthotopic Cell Transfer Models

Abstract

BackgroundEthanol is a potent teratogen. Its adverse neural effects are partly mediated by disrupting fetal neurogenesis. The teratogenic process is poorly understood, and vulnerable neurogenic stages have not been identified. Identifying these is a prerequisite for therapeutic interventions to mitigate effects of teratogen exposures.MethodsWe used flow cytometry and qRT-PCR to screen fetal mouse-derived neurosphere cultures for ethanol-sensitive neural stem cell (NSC) subpopulations, to study NSC renewal and differentiation. The identity of vulnerable NSC populations was validated in vivo, using a maternal ethanol exposure model. Finally, the effect of ethanol exposure on the ability of vulnerable NSC subpopulations to integrate into the fetal neurogenic environment was assessed following ultrasound guided, adoptive transfer.ResultsEthanol decreased NSC mRNAs for c-kit, Musashi-1and GFAP. The CD24+ NSC population, specifically the CD24+CD15+ double-positive subpopulation, was selectively decreased by ethanol. Maternal ethanol exposure also resulted in decreased fetal forebrain CD24 expression. Ethanol pre-exposed CD24+ cells exhibited increased proliferation, and deficits in cell-autonomous and cue-directed neuronal differentiation, and following orthotopic transplantation into naïve fetuses, were unable to integrate into neurogenic niches. CD24depleted cells retained neurosphere regeneration capacity, but following ethanol exposure, generated increased numbers of CD24+ cells relative to controls.ConclusionsNeuronal lineage committed CD24+ cells exhibit specific vulnerability, and ethanol exposure persistently impairs this population’s cell-autonomous differentiation capacity. CD24+ cells may additionally serve as quorum sensors within neurogenic niches; their loss, leading to compensatory NSC activation, perhaps depleting renewal capacity. These data collectively advance a mechanistic hypothesis for teratogenesis leading to microencephaly.

The BAF (BRG1/BRM-Associated Factor) chromatin-remodeling complex exhibits ethanol sensitivity in fetal neural progenitor cells and regulates transcription at the miR-9-2 encoding gene locus

“…However, ethanol exposure did not increase binding of BRG1 to probed regions 1 and 4 of the pri-miR-9-2 locus which contained identified Oct4/Pou5f1 binding sites. This result is consistent with our previous data showing that ethanol exposure results in an overall loss of stem cell capacity in fetal neural epithelial cells (Santillano et al, 2005; Tingling et al, 2013). …”

“…However, surprisingly, research in ex vivo rodent models of fetal NSCs showed that ethanol exposure did not result in cell death (Prock and Miranda, 2007; Santillano et al, 2005), but rather NSC loss, due to increased proliferation associated with premature and aberrant maturation (Camarillo and Miranda, 2008; Miller and Nowakowski, 1991; Santillano et al, 2005; Tingling et al, 2013). We further determined that many effects of ethanol on fetal NSCs and early embryogenesis were mediated by repression of a class of non-protein-coding regulatory microRNAs (miRNAs, (Pappalardo-Carter et al, 2013; Sathyan et al, 2007; Tsai et al, 2014)).…”

MicroRNAs and Ethanol Toxicity

“…Graphs based on calculations from published sequencing data (Bar et al, 2008;Chiang et al, 2010) abundant miRNA in the adult brain, 16 positioning it, along with miR-124 as the key miRNA for maintaining neuronal identity and differentiation state in the adult as well. miR-9 expression was significantly decreased in the brains of presenilin-1 knockout mice, which exhibit premature neuronal differentiation and abnormal neuronal migration (Krichevsky et al, 2003), features that have been associated with fetal alcohol exposure in animal models (Mooney, Siegenthaler, & Miller, 2004;Santillano et al, 2005;Tingling et al, 2013). Krichevsky and colleagues subsequently showed that miR-9-5p and miR-9-3p (i.e., miR-9/9*) were part of a miRNA network that directed NSCs toward a neuronal lineage (Krichevsky, Sonntag, Isacson, & Kosik, 2006;Shibata et al, 2008), and others showed that suppression of miR-9 was required for NSCs to differentiate along an oligodendrocytic lineage .…”

Postnatal choline supplementation selectively attenuates hippocampal microRNA alterations associated with developmental alcohol exposure

“…Alcohol may alter cell proliferation, migration, differentiation, synaptogenesis, and myelination, depending on the developmental timing and other exposure parameters (Camarillo and Miranda, 2008; Gil-Mohapel et al, 2010; Kane et al, 2012; O'Leary-Moore et al, 2011; Santillano et al, 2005; Tingling et al, 2013; Wilhelm and Guizzetti, 2016). But alcohol may also lead to long-lasting changes in cell function, altering gene expression (Downing et al 2012; Downing et al, 2011) and compromising synaptic plasticity (Gil-Mohapel et al, 2010; Medina, 2011), all of which contribute to long-lasting pathology in neural structure and function.…”