Marina Guizzetti, Ph.D. - US grants

DESCRIPTION (provided by applicant): This proposal for an "exploratory/developmental research grant" (R21) will address a novel aspect of ethanol's developmental neurotoxicity i.e. its effect on cholesterol homeostasis in astrocytes. While too much cholesterol may be deleterious as in case of atherosclerosis and Alzheimer's disease, too little can engender birth defects. Different degrees of mental retardation are often observed in documented inborn errors of cholesterol synthesis such as Smith-Lemli-Opitz syndrome, as well as in the maternal phenylketonuria, were the accumulating metabolite, phenylacetate, is an inhibitor of cholesterol synthesis. Lack of cholesterol during brain development as a consequence of these genetic defects leads, among others, to severe brain damage including microcephaly and mental retardation, both of which are hallmarks of fetal alcohol syndrome. The effect of ethanol on cholesterol homeostasis in the developing brain has not been investigated. Astrocytes produce most of the brain cholesterol which is used for proliferation or is released, via astrocyte-secreted high density lipoprotein-like particles containing apolipoprotein E, outside the cell where it is taken up and utilized by neurons for dendrite outgrowth and to form synapses. Specific aims of the proposal are to investigate the effects of ethanol on the following aspects of cholesterol homeostasis in rat E16, E21, and P6 astrocytes: 1. The effect of ethanol on the "de novo" synthesis and esterification of cholesterol. 2. The effect of ethanol on cholesterol trafficking. 3. The effect of ethanol on apolipoprotein E levels and release and on lipoprotein release and composition. Altogether these studies will provide indication of whether ethanol affects cholesterol homeostasis in astrocytes. If one or more of these end-points are affected by ethanol, further studies will be directed to better understand the mechanism(s) affected by ethanol and to explore the functional consequences of this effect on the developing brain.

DESCRIPTION (provided by applicant): This proposal will address a novel aspect of ethanol's developmental neurotoxicity, i.e. its effect on cholesterol homeostasis in the central nervous system. While too much cholesterol may be deleterious, as in case of atherosclerosis and Alzheimer's disease, too little can produce birth defects. Different degrees of mental retardation are often observed in inborn errors of cholesterol synthesis such as Smith-Lemli-Opitz syndrome. Lack of cholesterol during brain development as a consequence of these genetic defects leads to severe brain damage, including microcephaly and mental retardation, both of which are hallmarks of the fetal alcohol syndrome. The effects of ethanol on cholesterol homeostasis in the developing brain have not been investigated. In the brain, cholesterol is mostly produced endogenously, and its homeostasis is regulated by endogenously produced lipoproteins and cholesterol transporters. This proposal will investigate the hypothesis that ethanol, by upregulating cholesterol transporters and lipoprotein production by central nervous system cells, will increase the clearance of cholesterol from the brain, causing cholesterol depletion. Low levels of cholesterol are consistent with some of the effects caused by in utero alcohol exposure such as inhibition of the sonic hedgehog pathway, neuronal development and survival, and cell proliferation. Specific Aims of the proposal are: 1) The investigation of the role of ABCA1 and ABCG1 in ethanol-induced cholesterol efflux. The effect of ethanol on cholesterol efflux and cholesterol levels will be investigated in cortical neurons in vitro;in addition, the role of ABCA1, ABCG1, and phospholipase D in ethanol-induced upregulation of cholesterol efflux and cholesterol transporter induction in astrocytes and, possibly, in neurons will be examined by selectively removing these proteins form astrocyte and neuron cultures. 2) The investigation of the effect of ethanol on astrocyte-generated lipoproteins and their interaction with neurons. The effect of ethanol on the composition of astrocyte-produced lipoproteins separated by gel filtration, and their role on cholesterol efflux from neurons will be analyzed. Furthermore, the effect of ethanol on lipoprotein composition and cholesterol efflux will be examined in an astrocyte/neuron co- culture system. 3) The investigation of the effect of in vivo ethanol administration on ABC cholesterol transporters and on cholesterol levels in the developing brain. The effect of in vivo exposure to ethanol during gestation on the cholesterol transporters ABCA1 and ABCG1 transcription and expression in neurons and astrocytes and on cholesterol levels will be assessed in the neocortex of rat fetuses at gestational day 21. Altogether, these studies are directed at characterizing a possible novel mechanism involved in ethanol-induced neurodevelopmental effects, i.e. its effects on cholesterol homeostasis in the brain. PUBLIC HEALTH RELEVANCE The proposed studies on the effect of ethanol on cholesterol homeostasis in the developing brain were prompted by the observation that several of the neurodevelopmental effects caused by ethanol are consistent with effects caused by lack of cholesterol. Cholesterol is indeed necessary for various aspects of brain development. We hypothesized that ethanol, by mechanisms to be investigated in the proposed project, may affect cholesterol homoeostasis and reduce cholesterol levels in the developing brain. The most recent discoveries in the field of cholesterol trafficking will be applied to the investigation of the effects of ethanol on cholesterol transporters, lipoprotein generation and, ultimately, cholesterol levels in the developing brain. As cholesterol is a regular component of the diet, our studies could potentially lead to the revision of dietary guidelines for pregnant women at risk. It may also help understanding why in some very poor regions of South Africa the prevalence of FAS is much higher than in Western countries as these populations also experience severe malnutrition.

Ethanol exerts profound effects on the developing brain, which range from structural abnormalities to functional anomalies, resulting in compromised cognitive and behavioral functions characteristic of fetal alcohol spectrum disorders (FASD). Neuronal maturation is an essential process in the development of the nervous system. Brain-specific chondroitin sulfate proteoglycans (CSPGs) neurocan and brevican constitute inhibitory cues that prevent the extension of neurites in improper directions therefore contributing to the proper formation of neuronal architecture. Neurocan and brevican inhibit neurite outgrowth and are highly produced by glial cells, and astrocytes in particular, both in vitro and in vivo. CSPG are glycoproteins consisting of core-proteins attached to linear chains of glycosaminoglycans (GAGs) called chondroitin sulfates (CS), which consists in chondroitin-4 sulfate (C4S) and chondroitin-6 sulfate (C6S); the inhibitory properties of CSPGs depend on their core-protein and their GAG chains. Several enzymes are involved in the biosynthesis and degradation of GAG chains; arylsulfatase B (ARSB) and galactose-6-sulfatase (GALNS) remove sulfate groups from C4S and C6S respectively and are required for the degradation of CS-GAGs. An unscheduled increase in CSPGs in the still developing brain may lead to altered brain connectivity and to premature decrease in neuronal plasticity. In this proposal we hypothesize that ethanol-treated astrocytes inhibits neuritogenesis by increasing CSPGs. More specifically, we hypothesize that ethanol, through the inhibition of ARSB and GALNS activity in astrocytes, increases CS-GAG and CSPG core-protein neurocan and brevican levels in these cells leading to the inhibition of hippocampal neuron neuritogenesis. Specific aim 1 will investigate the effect of ethanol on ARSB and GALNS activity and expression, on CS-GAG, neurocan, and brevican levels, and on the role of ARSB and GALNS in modulating ethanol-induced inhibition of astrocyte-mediated hippocampal neuritogenesis in vitro. Specific Aim 2 will investigate the effect of in vivo neonatal alcohol exposure on ARSB and GALNS activity and protein and mRNA expression, CS-GAG, C4S-GAG, and neurocan- and brevican-bound sGAG levels, and neurocan and brevican protein and mRNA expression in the hippocampus.

? DESCRIPTION (provided by applicant): Fetal Alcohol Spectrum Disorders (FASD) are characterized by structural brain abnormalities and compromised cognitive and behavioral functions. Neuronal connectivity and plasticity are affected by developmental alcohol exposure. The exposure of astrocytes to ethanol inhibits neuritogenesis in hippocampal neurons co-cultured with ethanol-treated astrocytes. However, a lack of understanding of the mechanisms by which ethanol affects neuronal structural plasticity in the developing brain persists. Filling tis gap will open the path to the identification of highly effective treatments to prevent or ameliorat the developmental effects of ethanol. Lecticans are inhibitory proteoglycans that, in the brain, prevent the extension of axons and dendrites. Lecticans consist of a core-protein moiety covalently bound to linear chains of disaccharides, chondroitin sulfates glycosaminoglycans (CS-GAGs), which are comprised mostly of chondroitin-4 sulfate (C4S) and chondroitin-6 sulfate (C6S); the inhibitory properties of lecticans depend on their core-protein and their sGAG side-chains. Two sulfatase enzymes, arylsulfatase B (ARSB) and galactose-6-sulfatase (GALNS) remove sulfate groups from C4S and C6S respectively and are required for the degradation of CS-GAGs. We find that ethanol inhibits the activity of ARSB and GALNS and that ethanol and ARSB silencing increase the expression of the lectican neurocan and C4S-GAG content in astrocytes in vitro. ARSB activity is inhibited, neurocan and sGAG levels are upregulated, and pyramidal neuron dendritic arborization is reduced also in the hippocampus of neonatal rats after in vivo ethanol exposure. Reduced ARSB activity and dendritic arborization persist up to post-natal day 36 and is correlated with reduced performance in spatial learning and memory tasks. In this proposal we hypothesize that the inhibition of ARSB and GALNS activity by ethanol in the developing hippocampus induces an unscheduled increase in lectican (neurocan, brevican, and versican) core-protein and CS-GAG levels leading to decreased in neuronal structural plasticity; these effects are long-lasting and causally linked to reduced performance in learning and memory tasks and are rescued by recombinant (r)ARSB. We plan to test our hypothesis by pursuing the following three specific aims: 1) To investigate the mechanisms of ethanol-induced sulfatase inhibition and the consequences on lectican core-proteins and sGAG levels and on CS disaccharide levels and composition in astrocytes. 2) To investigate the role of: sulfatases, SUMF1, lecticans, and oxidative stress on ethanol-treated astrocyte inhibition of neurite outgrowth. 3) To investigate the effects of in vivo neonatal ethanol exposure and of rARSB injections on sulfatase activity, lectican expression, sGAG levels and CS disaccharide levels, and dendritic arborization in the hippocampus and on spatial learning and memory. The proposed work is innovative and significant as it explores a novel mechanism underlying ethanol-induced inhibition of neuronal plasticity and will yield strategies aimed at sulfatase enzyme activation for the treatment of alcohol teratogenesis therefore advancing FASD research.

Alcohol dependence can cause depression by affecting hippocampal functions. Animal models of depression display dendrific atrophy and decreased spine density in hippocampal CA3 pyramidal neurons. Astrocytes have been shown to play key roles in regulafing structural and functional plasticity in neurons. Epigenetic regulation of gene expression can be modulated by histone acetylation and DNA methylation through the acfivity histone deacetylases (HDACs) and DNA methyltrasferases (DNMTs) respectively. We hypothesize that chronic ethanol and /or withdrawal induce epigenetic changes in astrocytes, affect the release of ECM proteins, inhibit neuronal plasficity in the hippocampus, and cause depressive-like behavior, effects that may be rescued by HDAC inhibifion.

DESCRIPTION (provided by applicant): Both genetic and environmental contributions have crucial roles in the development of a complex disease such as alcoholism. Unfortunately, little progress has been made in identifying the underlying molecular mechanisms altered during abstinence to aid development of novel therapeutics for the maintenance of sobriety. We propose a combined genetic, molecular, pharmacological and behavioral strategy to identify pathways that are altered after a period of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic alcohol abuse, but the stability of these expression differences in the abstinent alcoholic is controversial. We have previously reported identification of pathways altered in prefrontal cortex (PFC), a brain region associated with cognitive dysfunction and damage in alcoholics, during a defined period of abstinence. To characterize genetic contributions, both sexes of an animal model with widely divergent responses to alcohol derived by selective breeding, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines, were analyzed. During a sustained period of abstinence, the transcriptional response correlated with withdrawal phenotype rather than sex. Bioinformatic analysis showed that among the major pathways altered that were the most dimorphic between WSR and WSP mice were 'acetylation' and 'histone deacetylase complex'. Data shows a complex phenotype-specific regulation during abstinence indicating widespread epigenetic reprogramming in the low response WSR but not the high response WSP mice exposed to the same ethanol concentrations. We will identify phenotype-specific regulatory mechanisms in the low response animal model in three specific aims by integrating data from high-throughput targeting technologies including expression profiling, DNaseI-seq and ChIP-seq, with confirmation of involvement of pathways to modulate relapse using pharmacological intervention in our established dependence-induced relapse drinking model. We hypothesize that targetable epigenetic mechanisms maintain expression differences during abstinence and that these differences increase the risk of relapse in the low response to alcohol endophenotype. These studies have high impact because of the morbidity/mortality associated with alcohol abuse, the high incidence of alcohol use disorders in the general population, and the tremendous impact these maladies have on human health. In addition, neuroadaptive changes and altered expression patterns may also play a role in persistent neurotoxicity and brain damage during abstinence with detrimental consequences for learning and memory functions, to play a role in the down-ward cycle of addiction and the self-sustaining nature of alcoholism. Thus, successful completion of these aims will aid in our understanding of the mechanism(s) underlying the risk for relapse and advance our ability to provide therapy for alcohol abuse targeted to the low response endophenotype, through identification of novel pharmacotherapies or to enhance translational applications for currently available therapeutics with previously unrecognized utility.

PROJECT SUMMARY The effects of developmental alcohol exposure on astrocytes remains largely unknown, despite the extensive and growing evidence of the roles played by these cells both in the developing and adult brain. This gap in knowledge is due in part to the challenges of studying these cells in vivo. We propose to employ new technologies allowing the study of astrocytes in vivo to gain mechanistic insights into astrocytic functions altered by developmental alcohol exposure to advance the pace of our discoveries of the roles played by astrocytes in Fetal Alcohol Spectrum Disorders (FASD). We propose to use the Aldh1l1-EGFP-Rpl10a mice that allow for the selective pull down of actively translating RNA from astrocytes by the translating ribosome affinity purification (TRAP) method and for the isolation of astrocyte-specific nuclei by Fluorescent-Activated Cell Sorting (FACS). Hence, this system allows to analyze changes in both astrocyte-specific nuclear RNA expression and astrocyte-specific RNA translation by RNA-seq. We hypothesize that neonatal alcohol exposure induces extensive changes in the translation of genes involved in several astrocyte-mediated processes in vivo and on molecular stuctures mainly contributed by astrocytes, such as the extracellular matrix (ECM) that modulate some of these processes. We also hypothesize that changes in translation are in part driven by changes in transcription and in part independent from transcription. Additionally, the proposed studies will assess astrocyte heterogeneity in their response to developmental alcohol exposure across developmental stages, sexes, and brain regions. The cell type-specificity of the proposed studies will help to disentangle astrocyte function and dysfunction in FASD from contributions of other cell types. We are particularly interested in alterations involving the ECM as we have reported that several proteins of the ECM play important roles in neuronal development and are dysregulated by ethanol. We expect the results of our proposed Aims to be very impactful to the FASD field. We will study the prefrontal cortex (PFC) and hippocampus (HPC) of developing (PD7) and adult (PD90) female and male Aldh1l1-EGFP-Rpl10a mice. Aim 1: To identify changes in the astrocyte nuclear transcriptome induced by neonatal ethanol exposure by FACS sorting of astrocyte nuclei followed by RNA-seq and pathway analysis. Aim 2: To identify changes in the astrocyte translatome induced by neonatal ethanol exposure by TRAP-RNA-seq and integrate these findings with transcriptome data. Aim 3: To explore the dysregulation of the astrocyte ECM network that underlies some of the developmental effects of ethanol by TRAP-qPCR, Fluorescence In Situ Hybridization (FISH)-RNAscope, Western blot, and immunohistochemistry to validate at both mRNA and protein levels ethanol-induced changes in ECM proteins. The proposed studies address NIH/NIAA priorities as they will provide mechanistic insights into astrocyte functions altered by developmental alcohol exposure in the developing and adult brain which are likely involved in behavioral abnormalities and mental illnesses developed by adults with FASD.