Amy Sabrina Gardiner

Fetal alcohol spectrum disorders (FASD) are caused by prenatal alcohol exposure (PAE), which leads to developmental defects during embryogenesis. Angiogenesis is one facet of development that is heavily dysregulated during PAE and where such impairment is poorly understood. Here we found that expression levels of focal adhesion molecules Tensin 1 (Tns1) and Tensin 2 (Tns2) were significantly reduced by alcohol exposure in brain microvascular endothelial cells (BMVECs). We also found that miR-150-5p, which we previously found was increased by PAE, regulates the expression of Tns1 and Tns2. Dual-luciferase assays demonstrated direct binding of miR-150-5p to the 3’UTRs of Tns1 and Tns2. Additionally, we utilized sprouting angiogenesis bead assays to evaluate endothelial cell sprouting in response to ethanol (EtOH) treatment and following Tns1 and/or Tns2 overexpression as well as miR-150-5p modulation. Tns1 and Tns2 rescued both EtOH- and miR-150-5p-mediated deficits in sprouting angiogenesis. Overall, we found that miR-150-5p negatively regulates Tns1 and Tns2 in BMVECs and that augmenting Tns1 and Tns2 expression may reverse the negative effects of EtOH, and subsequent miR-150-5p elevation, thus improving endothelial cell sprouting during cerebrovascular development.

Fetal alcohol spectrum disorders (FASD) constitute a wide range of developmental, cognitive, and behavioral impairments caused by prenatal alcohol exposure (PAE). Although neuronal and vascular consequences of PAE have been studied, how alcohol affects the cerebrovasculature within the framework of the neurovascular unit (NVU) across development remains poorly understood. At minimum, the NVU comprises neurons, astrocyte endfeet, and endothelial cells (ECs), which coordinate to maintain brain homeostasis. Here, we used NanoString’s Digital Spatial Profiling platform to characterize spatial transcriptomic data from neurons, astrocytes, and ECs from PAE and saccharin (SAC) control cortices at embryonic day 18 (E18) and postnatal day 28 (P28). Differentially expressed genes were then used for Ingenuity Pathway Analysis (IPA) to identify altered biological pathways and perform comparison analyses across developmental time points, while CellChat was used to infer cell–cell communication networks. We uncovered thousands of differentially expressed genes and numerous altered pathways and biological processes in PAE cortices across development. Both IPA and CellChat analyses implicated dysregulation of vascular and extracellular matrix (ECM) remodeling, cell adhesion, and neuroinflammatory signaling. CellChat further predicted the loss of several key bidirectional relationships and altered ligand-receptor interactions among neurovascular cell types at E18 and P28. Overall, these findings identify PAE-associated alterations in neurovascular gene expression and intercellular signaling across development, providing potential mechanisms by which PAE may disrupt neurodevelopment.

Elevation of miR-150-5p in the brain vasculature during prenatal alcohol exposure occurs partially through increased transcription. This increase in transcription is mediated by the upregulation of Tet1; the elevated TET1 protein partially demethylates the miR-150 promoter into a 5hmC-rich sequence. Consequently, transcription factor binding to the promoter sequence is altered, resulting in increased miR-150 transcription.

This review focuses on the function of HuD (ELAVL4) from the perspective of the mRNA targets and biological pathways it regulates from early brain development to the adult nervous system. HuD is a member of the Hu family of RNA-binding proteins (RBPs), which are critical post-transcriptional regulators and one of the earliest neuronal markers. HuD knockout (KO) mice exhibit deficits in motoneuron development and learning and memory, and multiple studies demonstrate HuD is essential for neuronal differentiation, nerve regeneration, and synaptic plasticity. In previous studies, we identified the repertoire of mRNAs bound to HuD in both embryonic and adult mouse brain tissues. Of the close to 4000 HuD-bound mRNAs, half are expressed in both developing and mature neurons. These shared mRNA targets are enriched in several networks and biological pathways, including proliferation of neural cell progenitors, quantity of synapses, and regeneration of the nervous system. Common target mRNAs are also involved in signaling from GABA, opioid, and ephrin receptors. Targets expressed only in embryonic brains contribute to several developmental pathways, including RHO GTPase activation, WNT/β-catenin signaling, and semaphorin interactions, while those expressed exclusively in adult tissues participate in synaptogenesis, JAK-STAT signaling, regulation of stress-induced responses, and hypoxia signaling. Comparative analyses of HuD-regulated pathways across the E18, adult, and common sets of targets reveal that, despite differences in specific mRNA components, these sets share 15 canonical pathways and 31 diseases and functions. Shared functional networks include axon guidance, neuritogenesis, and netrin signaling. These findings underscore that HuD-mediated target regulation in mature neurons often recapitulates processes used for neurite remodeling in early neuronal differentiation.

Background: Fetal alcohol spectrum disorders (FASD) occur in children who were exposed to alcohol in utero and are manifested in a wide range of neurocognitive deficits. These deficits could be caused by alterations to the cortical microvasculature that are controlled by post‐transcriptional regulators such as microRNAs.Methods: Using an established mouse model of moderate prenatal alcohol exposure (PAE), we isolated cortices (CTX) and brain microvascular endothelial cells (BMVECs) at embryonic day 18 (E18) and examined the expression of miR‐150‐5p and potential downstream targets. Cellular transfections and intrauterine injections with LNA™ mimics or inhibitors were used to test miR‐150‐5p regulation of novel target vascular endothelial zinc finger 1 (Vezf1). Dual‐luciferase assays were used to assess the direct binding of miR‐150‐5p to the Vezf1 3′UTR. The effects of miR‐150‐5p and Vezf1 on endothelial cell function were determined by in vitro migration and tube formation assays.Results: We found that miR‐150‐5p was upregulated and Vezf1 was downregulated during PAE in the E18 CTX and BMVECs. Transfection with miR‐150‐5p mimics resulted in decreased Vezf1 expression in BMVECs, while miR‐150‐5p inhibition did the opposite. Dual‐luciferase assays revealed direct binding of miR‐150‐5p with the Vezf1 3′UTR. Intrauterine injections showed that miR‐150‐5p regulates the expression of Vezf1 in vivo during PAE. miR‐150‐5p overexpression decreased BMVEC migration and tube formation, while miR‐150‐5p inhibition enhanced migration and tube formation. Vezf1 overexpression rescued the effects of the miR‐150‐5p mimic. Alcohol treatment of BMVECs increased miR‐150‐5p expression and inhibited migration and tube formation. Finally, miR‐150‐5p inhibition and Vezf1 overexpression rescued the negative effects of alcohol on migration and tube formation.Conclusions: miR‐150‐5p regulation of Vezf1 results in altered endothelial cell function during alcohol exposure. Further, miR‐150‐5p inhibition of Vezf1 may adversely alter the development of the cortical microvasculature during PAE and contribute to deficits seen in patients with FASD.

The neuronal Hu/ELAV-like proteins HuB, HuC and HuD are a class of RNA-binding proteins that are crucial for proper development and maintenance of the nervous system. These proteins bind to AU-rich elements (AREs) in the untranslated regions (3′-UTRs) of target mRNAs regulating mRNA stability, transport and translation. In addition to these cytoplasmic functions, Hu proteins have been implicated in alternative splicing and alternative polyadenylation in the nucleus. The purpose of this study was to identify transcriptome-wide effects of HuD deletion on both of these nuclear events using RNA sequencing data obtained from the neocortex of Elavl4^–/– (HuD KO) mice. HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of proximal polyadenylation signals (PAS), resulting in shorter 3′-UTRs. None of these genes overlapped with those showing alternative splicing events. Overall, HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.

The KH-type splicing regulatory protein (KHSRP) is an RNA-binding protein linked to decay of AU-rich element containing mRNAs. We have previously shown that KHSRP destabilizes the mRNA encoding the growth-associated protein GAP-43 and decreases neurite growth in cultured embryonic neurons. In contrast, loss of KHSRP stabilizes Gap43 mRNA and increases neurite growth. Here, we have tested functions of neural KHSRP in vivo . We find upregulation of 1460 mRNAs in the neocortex of adult Khsrp ^−/− mice, of which 527 bind to KHSRP with high specificity. These KHSRP targets are involved in pathways for neuronal morphology, axon guidance, neurotransmission and long-term memory. Neocortical neurons show increased axon growth and dendritic spine density in Khsrp ^−/− mice. Analyses of neuronal cultures from embryonic Khsrp ^−/− mice point to a neuron-intrinsic alteration in axonal and dendritic growth and elevations in KHSRP-target mRNAs, including subcellularly localized mRNAs. Hippocampus and infralimbic cortex of Khsrp ^−/− mice show presynaptic elevations in neurotransmission. The Khsrp ^−/− mice have significant deficits in both trace conditioning and attention set-shifting tasks compared Khsrp ^+/+ mice, indicating impaired prefrontal- and hippocampal-dependent memory consolidation with loss of KHSRP. Overall, our results indicate that prenatal deletion of KHSRP impairs neuronal development resulting in alterations in neuronal morphology and function by changing post-transcriptional control of neuronal gene expression.

Background: Brain microvascular endothelial cells form a highly selective blood brain barrier regulated by the endothelial tight junctions. Cerebral ischemia selectively targets tight junction protein complexes, which leads to significant damage to cerebral microvasculature. Short noncoding molecules called micro RNA s are implicated in the regulation of various pathological states, including endothelial barrier dysfunction. In the present study, we investigated the influence of micro RNA‐155 (miR‐155) on the barrier characteristics of human primary brain microvascular endothelial cells ( HBMEC s). Methods and Results: Oxygen‐glucose deprivation was used as an in vitro model of ischemic stroke. HBMEC s were subjected to 3 hours of oxygen‐glucose deprivation, followed by transfections with miR‐155 inhibitor, mimic, or appropriate control oligonucleotides. Intact normoxia control HBMEC s and 4 oxygen‐glucose deprivation–treated groups of cells transfected with appropriate nucleotide were subjected to endothelial monolayer electrical resistance and permeability assays, cell viability assay, assessment of NO and human cytokine/chemokine release, immunofluorescence microscopy, Western blot, and polymerase chain reaction analyses. Assessment of endothelial resistance and permeability demonstrated that miR‐155 inhibition improved HBMEC s monolayer integrity. In addition, miR‐155 inhibition significantly increased the levels of major tight junction proteins claudin‐1 and zonula occludens protein‐1, while its overexpression reduced these levels. Immunoprecipitation and colocalization analyses detected that miR‐155 inhibition supported the association between zonula occludens protein‐1 and claudin‐1 and their stabilization at the HBMEC membrane. Luciferase reporter assay verified that claudin‐1 is directly targeted by miR‐155. Conclusions: Based on these results, we conclude that miR‐155 inhibition–induced strengthening of endothelial tight junctions after oxygen‐glucose deprivation is mediated via its direct target protein claudin‐1.

HuD protein (also known as ELAVL4) has been shown to stabilize mRNAs with AU-rich elements (ARE) in their 3′ untranslated regions (UTRs), including Gap43, which has been linked to axon growth. HuD also binds to neuritin (Nrn1) mRNA, whose 3′UTR contains ARE sequences. Although the Nrn1 3′UTR has been shown to mediate its axonal localization in embryonic hippocampal neurons, it is not active in adult dorsal root ganglion (DRG) neurons. Here, we asked why the 3′UTR is not sufficient to mediate the axonal localization of Nrn1 mRNA in DRG neurons. HuD overexpression increases the ability of the Nrn1 3′UTR to mediate axonal localizing in DRG neurons. HuD binds directly to the Nrn1 ARE with about a two-fold higher affinity than to the Gap43 ARE. Although the Nrn1 ARE can displace the Gap43 ARE from HuD binding, HuD binds to the full 3′UTR of Gap43 with higher affinity, such that higher levels of Nrn1 are needed to displace the Gap43 3′UTR. The Nrn1 3′UTR can mediate a higher level of axonal localization when endogenous Gap43 is depleted from DRG neurons. Taken together, our data indicate that endogenous Nrn1 and Gap43 mRNAs compete for binding to HuD for their axonal localization and activity of the Nrn1 3′UTR.