Paul D. Drew

Background/Objectives: Fetal alcohol spectrum disorders (FASDs) occur in nearly 5% of children in the United States and have been associated with alterations in neurological functions, neuroanatomical changes, and behavioral deficits encompassing an individual’s lifetime. Alterations in myelination have been reported in both rodent models and humans. The cerebellum is a heavily myelinated brain region, and oligodendrocyte and myelination transcripts have been reported to be altered in the cerebellum following early-life ethanol (EtOH) exposure in a mouse model. In this study, we investigated cerebellar-recruited behaviors in adult female mice that were exposed to EtOH from postnatal day (P) 4 to P9. We investigated whether changes in oligodendrocyte lineage markers were present in adulthood. Methods: C57BL/6J offspring received a total of 5.0 g/kg/day of either ethanol (EtOH) or saline in two separate doses delivered subcutaneously two hours apart from P4 to P9. On P21, offspring were weaned and housed with same-sex littermates throughout the duration of the study. From P60 to P90, females underwent behavioral testing including an open field test (OFT), rotarod, and balance beam. Behavior naïve littermates were euthanized on P105, and cerebella were collected for qPCR to assess oligodendrocyte lineage transcripts. Results: We reported that, following EtOH exposure from P4 to P9, adult female mice had increased ambulatory behaviors in the OFT and subtle changes in behavior in the rotarod and balance beam compared to saline-exposed controls. Despite the behavioral changes observed in adulthood, we found that alterations in oligodendrocyte lineage transcripts present on P10 did not persist into adulthood. Conclusions: Subcutaneous injection of EtOH from P4 to P9 resulted in long-term consequences in locomotor and cerebellar-recruited behaviors in female mice.

Fetal alcohol spectrum disorders (FASD) caused by developmental ethanol exposure lead to cerebellar impairments, including motor problems, decreased cerebellar weight, and cell death. Alterations in the sole output of the cerebellar cortex, Purkinje cells, and central nervous system immune cells, microglia, have been reported in animal models of FASD. To determine how developmental ethanol exposure affects adult cerebellar microglia and Purkinje cells, we used a human third-trimester binge exposure model in which mice received ethanol or saline from postnatal (P) days 4–9. In adolescence, cerebellar cranial windows were implanted and mice were aged to young adulthood for examination of microglia and Purkinje cells in vivo with two-photon imaging or in fixed tissue. Ethanol had no effect on microglia density, morphology, dynamics, or injury response. However, Purkinje cell linear frequency was reduced by ethanol. Microglia–Purkinje cell interactions in the Purkinje Cell Layer were altered in females compared to males. Overall, developmental ethanol exposure had few effects on cerebellar microglia in young adulthood and Purkinje cells appeared to be more susceptible to its effects.

Ethanol induces neuroinflammation, which is believed to contribute to the pathogenesis of alcohol use disorder (AUD). Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) expressed on both immune cells, including microglia and astrocytes, and non-immune cells in the central nervous system (CNS). Studies have shown that alcohol activates TLR4 signaling, resulting in the induction of pro-inflammatory cytokines and chemokines in the CNS. However, the effect of alcohol on signaling pathways downstream of TLR4, such as MyD88 and TRIF (TICAM) signaling, has not been evaluated extensively. In the current study, we treated male wild-type, TLR4-, MyD88-, and TRIF-deficient mice using a chronic plus binge mouse model of AUD. Evaluation of mRNA expression by qRT-PCR revealed that ethanol increased IL-1β, TNF-α, CCL2, COX2, FosB, and JunB in the cerebellum in wild-type and TRIF-deficient mice, while ethanol generally did not increase the expression of these molecules in TLR4- and MyD88-deficient mice. Furthermore, IRF3, IRF7, and IFN-β1, which are associated with the TRIF-dependent signaling cascade, were largely unaffected by alcohol. Collectively, these results suggest that the TLR4 and downstream MyD88-dependent signaling pathways are essential in ethanol-induced neuroinflammation in this mouse model of AUD.

Fetal alcohol spectrum disorders (FASD) are a group of neurodevelopmental disorders caused by ethanol exposure in utero, which can result in neurocognitive and behavioral impairments, growth defects, and craniofacial anomalies. FASD affects up to 1-5% of school-aged children in the United States, and there is currently no cure. The underlying mechanisms involved in ethanol teratogenesis remain elusive and need greater understanding to develop and implement effective therapies. Using a third trimester human equivalent postnatal mouse model of FASD, we evaluate the transcriptomic changes induced by ethanol exposure in the cerebellum on P5 and P6, after only 1 or 2 days of ethanol exposure, with the goal of shedding light on the transcriptomic changes induced early during the onset and development of FASD. We have highlighted key pathways and cellular functions altered by ethanol exposure, which include pathways related to immune function and cytokine signaling as well as the cell cycle. Additionally, we found that ethanol exposure resulted in an increase in transcripts associated with a neurodegenerative microglia phenotype, and acute- and pan-injury reactive astrocyte phenotypes. Mixed effects on oligodendrocyte lineage cell associated transcripts and cell cycle associated transcripts were observed. These studies help to elucidate the underlying mechanisms that may be involved with the onset of FASD and provide further insights that may aid in identifying novel targets for interventions and therapeutics.

Alcohol use disorder (AUD) is one of the most common preventable mental health disorders and can result in pathology within the CNS, including the cerebellum. Cerebellar alcohol exposure during adulthood has been associated with disruptions in proper cerebellar function. However, the mechanisms regulating ethanol-induced cerebellar neuropathology are not well understood. High-throughput next generation sequencing was performed to compare control versus ethanol-treated adult C57BL/6J mice in a chronic plus binge model of AUD. Mice were euthanized, cerebella were microdissected, and RNA was isolated and submitted for RNA-sequencing. Down-stream transcriptomic analyses revealed significant changes in gene expression and global biological pathways in control versus ethanol-treated mice that included pathogen-influenced signaling pathways and cellular immune response pathways. Microglial-associated genes showed a decrease in homeostasis-associated transcripts and an increase in transcripts associated with chronic neurodegenerative diseases, while astrocyte-associated genes showed an increase in transcripts associated with acute injury. Oligodendrocyte lineage cell genes showed a decrease in transcripts associated with both immature progenitors as well as myelinating oligodendrocytes. These data provide new insight into the mechanisms by which ethanol induces cerebellar neuropathology and alterations to the immune response in AUD.

Background: Hippocampal and cerebellar neuropathology occurs in individuals with alcohol use disorders (AUD), resulting in impaired cognitive and motor function. Objectives: Evaluate the effects of ethanol on the expression of pro- and anti-inflammatory molecules, as well as the effects of the anti-inflammatory PPAR-γ agonist pioglitazone in suppressing ethanol-induced neuroinflammation. Methods: Adult male and female mice were treated chronically with ethanol for just under a month followed by a single acute binge dose of ethanol. Animals were provided liquid diet in the absence of ethanol (Control; n = 18, 9 M/9F), liquid diet containing ethanol (ethanol; n = 22, 11 M/11F), or liquid diet containing ethanol plus gavage administration of 30.0 mg/kg pioglitazone (ethanol + pioglitazone; n = 20, 10 M/10F). The hippocampus and cerebellum were isolated 24 h following the binge dose of ethanol, mRNA was isolated, and pro- and anti-inflammatory molecules were quantified by qRT-PCR. Results: Ethanol significantly (p < .05) increased the expression of pro-inflammatory molecules IL-1β, TNF-α, CCL2, and COX2; increased the expression of inflammasome-related molecules NLRP3 and Casp1 but decreased IL-18; and altered the expression of anti-inflammatory molecules including TGFβR1 in the hippocampus and cerebellum, though some differences were observed between males and females and the two brain regions. The anti-inflammatory pioglitazone inhibited ethanol-induced alterations in the expression of most, but not all, inflammation-related molecules. Conclusion: Chronic plus binge administration of ethanol induced the expression of inflammatory molecules in adult mice and pioglitazone suppressed ethanol-induced neuroinflammation.

Background: The developing hippocampus and cerebellum, unique among brain regions, exhibit a secondary surge in neurogenesis during the third trimester of pregnancy. Ethanol (EtOH) exposure during this period is results in a loss of tissue volume and associated neurobehavioral deficits. However, mechanisms that link EtOH exposure to teratology in these regions are not well understood. We therefore analyzed transcriptomic adaptations to EtOH exposure to identify mechanistic linkages.Methods: Hippocampi and cerebella were microdissected at postnatal day (P)10, from control C57BL/6J mouse pups, and pups treated with 4 g/kg of EtOH from P4 to P9. RNA was isolated and RNA‐seq analysis was performed. We compared gene expression in EtOH‐ and vehicle‐treated control neonates and performed biological pathway‐overrepresentation analysis.Results: While EtOH exposure resulted in the general induction of genes associated with the S‐phase of mitosis in both cerebellum and hippocampus, overall there was little overlap in differentially regulated genes and associated biological pathways between these regions. In cerebellum, EtOH additionally induced gene expression associated with the G2/M‐phases of the cell cycle and sonic hedgehog signaling, while in hippocampus, EtOH‐induced the pathways for ribosome biogenesis and protein translation. Moreover, EtOH inhibited the transcriptomic identities associated with inhibitory interneuron subpopulations in the hippocampus, while in the cerebellum there was a more pronounced inhibition of transcripts across multiple oligodendrocyte maturation stages.Conclusions: These data indicate that during the delayed neurogenic period, EtOH may stimulate the cell cycle, but it otherwise results in widely divergent molecular effects in the hippocampus and cerebellum. Moreover, these data provide evidence for region‐ and cell‐type‐specific vulnerability, which may contribute to the pathogenic effects of developmental EtOH exposure.