Kazue Hashimoto-Torii - US grants

This application is a part of the competitive renewal for the Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CIFASD) in response to RFA-AA-17-012. Pathological outcomes of Fetal Alcohol Spectrum Disorder (FASD) stemming from prenatal alcohol exposure (PAE) are devastating and highly variable, especially in regards to cognitive and learning deficits apparent in later life. Early intervention for such deficits is imperative for optimal outcomes; however, the pattern and magnitude of these deficits are not predictive even when accounting for the level of alcohol exposure, which by itself is difficult to accurately assess. Therefore, early, and precise biomarkers for predicting the risk of cognitive and behavior problems are crucial for establishing an effective treatment. This project aims at establishing a novel approach to identifying such biomarkers for predicting the risk of children afflicted with FASD. Based on our preliminary data, we hypothesize that single-cell level epigenetic changes detectable in blood cell samples serve as biomarkers in predicting risks of cognitive and learning deficits before their symptomatic manifestations. By employing cutting-edge cellular droplet technology, we will test this hypothesis using the mouse model of PAE (Aim 1), and examine whether these biomarkers are applicable for human patients with a history of PAE (Aim 2). The Hashimoto-Torii lab will perform the single-cell droplet digital PCR- based biomarker analyses (drop-PCR) with both human and mouse blood samples. The Torii lab will collect the mouse blood samples, perform comprehensive mouse behavior analyses, and statistically evaluate potential correlations between the animal behaviors and drop-PCR results. The Chambers lab will collect the human blood samples, perform neurocognitive tests, and statistically evaluation of potential correlations between these test scores and the drop-PCR results. This project will allow for critical assessment in linking biomarkers with comprehensive evaluations of neurocognitive deficits, brain structural abnormalities and facial dysmorphology. In addition, these studies maximize the potential our collaborations with other CIFASD research including the neurobehavioral (Chambers), genetic (Foroud) and dysmorphology core (Jones) projects. Cross-sectional approaches using controlled animal studies (Eberhart and Parnell) will provide essential mechanistic insights. Our identified biomarkers and those obtained through studies using cytokine (Chambers) and miRNA (Weinberg) panels generated for the same PAE patients will provide a rare opportunity to test this combined biomarker strategy for accurate prediction of PAE outcomes. By capitalizing on CIFASD infrastructure, this project will develop innovative single-cell biomarkers that impact FASD research and translational science at large.

Summary One of the most devastating consequences of alcohol misuse is its teratogenic effects on fetal development, particularly in the development of the brain and the formation of its complex circuitry. These permanent developmental deficits caused by alcohol misuse are referred to as Fetal Alcohol Spectrum Disorder (FASD). Recent findings have demonstrated that fetal alcohol exposure results in wide-ranging and adaptive alterations of gene expression within the developing cerebral cortex. Such coordinated gene expression changes are regulated mainly by highly specified transcription factors and miRNAs. In addition, recent studies on budding yeast and fruit fly have demonstrated that RNA binding proteins coordinately regulate the post-transcriptional modification of specific types of mRNAs in response to rapid environmental changes. This post-transcriptional regulatory complex of specific mRNAs and RNA binding proteins, known as RNA-operons, are suggested to have important roles in cellular adaptation to environment and diseases. However, the response of RNA-operons as a result of alcohol exposure within the mammalian brain remains unknown. We have recently found that the RNA binding protein, Rbm39, is upregulated in response to ethanol exposure in both the human and mouse embryonic cortex. Our preliminary data suggests that the upregulation of Rbm39 is required for protecting neural progenitor cells from both cell death and cell cycle arrest in embryonic mouse cortices exposed to ethanol. However, alterations in Rbm39 expression, whether due to loss- or gain-of- function, do not show obvious effects on normal cortical development. These results suggest that the Rbm39 plays specific roles resulting from ethanol exposure. In addition, we have found that the Rbm39 regulates the post-transcriptional modifications of specific mRNAs that are required for adaptive cellular protection from ethanol. Therefore, we hypothesize that the Rbm39 coordinates post-transcriptional modification through RNA- operons after exposure to ethanol, providing an immediate and adaptive system in protecting cells within the embryonic cortex. We will examine this hypothesis through three discrete aims. Aim 1 will define the requirement of Rbm39 for neural protection by knocking down Rbm39 in embryonic cortices exposed to ethanol. Aim 2 will determine whether exogenous increased of Rbm39 activity on RNA- operons can further reduce the brain damages by ethanol. Aim3 will examine whether Rbm39 orchestrates immediate post-transcriptional modification of specific genes in response to ethanol exposure.

Project Summary/Abstract Prenatal alcohol exposure can lead to devastating effects on the developing cerebral cortex, thereby causing a wide spectrum of neurological and psychiatric conditions after birth. One of the promising intervention approaches lies in enhancing intrinsic protective mechanisms to overwhelm the triggered pathological mechanisms. Therefore, understanding the defense mechanisms deployed by neural cells is imperative for the development of potential therapies for alcohol-induced neuropsychiatric conditions. The goal of the proposed study is to characterize a new pathway by which primary cilia could suppress the adverse impact of alcohol on developing cortical neurons. The primary cilium is a unique organelle which is known to be essential for a cell to sense and respond to environmental changes. However, the role of cilia during brain development, particularly, in harsh prenatal environment such as exposure to alcohol or other environmental stressors remains largely unknown. By combining a mouse model of cilia deficiency specifically in the cerebral cortex and alcohol exposure during the brain sprout stage, we found evidences that support our hypothesis; cilia may play a critical role in protecting cortical neurons from alcohol-inducible dendritic/spine degeneration or/and other permanent morphological alterations. We will test this hypothesis by characterizing cortical phenotypes of cilia-deficient conditional knockout mice exposed to alcohol (Aim1), and testing candidate molecular mechanisms which may mediate cilia-dependent inhibition of dendritic/spine degeneration in vivo (Aim2).

Prenatal alcohol (ethanol) exposure (PAE) significantly impacts cognitive and behavioral abilities of the offspring. These conditions are defined as Fetal Alcohol Spectrum Disorders (FASD). Early intervention of such abnormalities is imperative for optimal outcomes; however, specific therapeutic targets and effective treatments are yet unavailable. The goal of this project is to elucidate the mechanisms underlying the long-term impacts of prenatal alcohol exposure and find effective treatments for the symptoms caused by such impacts in FASD. We have recently shown that the activation of heat shock signaling, which protects young neurons in the alcohol-exposed embryonic brain, can instead cause neuronal migration delay when it is hyperactivated. Our preliminary analysis has identified novel, long-term changes in gene expression associated with this prenatal hyperactivation of heat shock signaling, at the single-cell level within the brains of adolescent mice. These mice show gross and fine motor skills impairment, one of the earliest problems in FASD patients noticed by caregivers. Some of these changes were negatively correlated with the motor learning ability of these mice, and remarkably, reverting one of such altered factors, increased Kcnn2 (a calcium-activated potassium channel) function, improved the motor learning deficits. In addition, preliminary data suggested that overexpression of Kcnn2 in the motor cortex alone can cause motor learning defects. Based on these findings and preliminary data, we hypothesize that epigenetic changes associated with acute high-level activation of heat shock signaling in the fetal brain by PAE are involved in motor learning defects in later life. To test this hypothesis, we will first define the postnatal epigenetic traits specifically associated with the prenatal acute activation of heat shock signaling in the motor cortex of PAE mice, which display motor learning deficits (Aim 1). By investigating the specific effects of Kcnn2 overexpression in untreated mice and those of Kcnn2 knockdown in PAE mice, we will then define how increased Kcnn2 expression contributes to the learning deficits of PAE mice (Aim 2). We also test whether reverting the increased Kcnn2 function can be a novel therapeutic target to improve the deficits (Aim 3). Our multidisciplinary team puts our expertise to achieve these aims, by employing in vivo Kcnn2 manipulation, in vivo imaging and behavior analysis (Torii lab.), electrophysiology and epigenetic analyses (Hashimoto-Torii lab.). By combining a unique reporter system that we developed with these cutting-edge techniques, we will uncover hitherto unknown epigenetic mechanisms leading to neurobehavioral problems in FASD, and develop potentially novel interventions.