Dennis R. Grayson, Ph.D. - US grants

The major objective of the proposed research is to determine the effects of chronic exposure to cocaine (in rats) on the function of neural circuitry involving the substantia nigra. In particular, dynamic in vivo interactions between dopaminergic transmission and GABA-containing outputs from the basal ganglia will be examined in order to define how chronic exposure to cocaine modifies these interactions. For these studies, we will make measurements indicative of the turnover rate a) of GABA in specific pathways (nigrotectal and nigrotegmental) which are known to mediate the expression of certain behaviors elicited by dopamine stimulants such as cocaine and b) of dopamine in the nigrostriatal pathway. The neurotransmitter turnover in these pathways will be manipulated using the following probes: a) directly and indirectly-acting dopamine agonists given either systemically or directly applied to specific intracerebral sites and b) serotonergic agonists and antagonists applied directly into substantia nigra. The effect of these probes on transmitter turnover will be evaluated in rats chronically exposed to cocaine in comparison to drug-naive rats. Using turnover as an index of the functional responsiveness of the nigral outputs to selected neurotransmitters in the nigrostriatal circuit, we hope to identify those synaptic links that are functionally altered by chronic exposure to cocaine. An attempt will be made to relate neurochemical changes observed to each other and to changes in behavioral responses. In particular, we hope to identify functional neurochemical alterations that may account for the behavioral sensitization to cocaine in chronically exposed animals. As sensitization to psychomotor stimulants in animals may be a model of exaggerated psychotogenic actions of these drugs in chronic stimulant abusers (8), it is possible that the information we obtain may provide insights into the neuropathological basis for behavioral disorders induced by stimulant abuse.

DESCRIPTION (provided by applicant): During embryonic brain development, reelin is secreted by Cajal-Retzius (CR) cells into the extracellular matrix (ECM) and serves to orchestrate the positioning of migrating neurons in the developing cortex, hippocampus and cerebellum. In the cortex of adult rat, reelin is secreted from a subpopulation of GABAergic neurons into the ECM by a constitutive mechanism. In adult non-human primates, reelin, present in the ECM, is contiguous with alpha3-containing integrin receptors in the vicinity of dendritic spines. This raises the possibility that adult reelin-integrin interactions may contribute to synaptic plasticity by modulating the expression of cytoskeletal proteins that facilitate the trophism of dendritic spines. In the post-mortem brains of schizophrenia patients, basal dendritic spine density is decreased. Moreover, in brains obtained from patients diagnosed with schizophrenia and bipolar illness with psychosis, reelin mRNA and protein levels are reduced by approximately 50 percent. Cytosine methylation of genomic DNA influences a panorama of cellular processes, including gene transcription, genomic imprinting, and genome stability. Experiments outlined in this application will expand upon our preliminary data that support our hypothesis that the human reelin promoter is modulated by DNA methylation, which determines transcription factor accessibility through alterations in chromatin structure. Our goal is to obtain information relevant to mechanisms responsible for appropriate temporal and spatial patterns of reelin gene expression. We will define those parts of the promoter operative in modulating reelin expression in primary neuronal cell cultures (cortical neurons and glia and cerebellar granule neurons). Using this information, we will identify trans-acting factors that interact with these regulatory regions (Aim 1). Secondly, we will also focus on the role that DNA methylation plays in defining reelin gene (RELN) expression in neuronal precursors differentiating in vitro (Aim 2). Finally, we will generate transgenic animals using the reelin promoter to drive expression of a lacZ reporter gene and examine the role of these regulatory sequences in targeting neuronal expression of RELN (Aim 1). We will examine the methylation pattern of the human promoter transgene in various primary cultures derived from these transgenic mice (Aim 2) and will compare the patterns of methylation of the endogenous gene with the human reelin transgene. Information obtained from the proposed experiments will provide the framework for formulating hypotheses relevant to mechanisms by which reelin expression may be functionally compromised in psychiatric diseases. Ultimately, an understanding of events responsible for modulating reelin expression may provide preliminary clues as to how the gene might be manipulated in the future as one potential therapeutic approach to these complex mental disorders.

Project Summary The goal of the Epigenetics Core is to provide a robust common platform for the identification and analysis of genes affected by withdrawal from chronic ethanol exposure in the brain. During the current funding period, the Epigenetics Core identified gene networks both induced and repressed by alcohol exposure or withdrawal in the amygdala, hippocampus, ventral tegmental area and limbic cortex and specific target genes that showed changes in RNA levels, as well as, in H3K9/14ac levels were functionally validated. In the current submission, the proposed Aims will allow preclinical CARE Research Projects (Projects 1-3) to obtain novel information regarding genome-wide changes in chromatin accessibility (ATAC-seq) and histone methylation (H3K27me3 ChIP-seq). These findings will be integrated with data obtained during the current funding period (RNA-seq and H3K9/14ac ChIP-seq). We will also perform ATAC-seq and RNA-seq in the prefrontal cortex (PFC) of the alcohol use disorder (AUD)/control cohort obtained from the New South Wales Tissue Resource Center (Project 4). In addition, we will investigate the across genome levels of 5- hydroxymethylcytosine (5hmC) in the same tissue using Illumina?s EPIC Methylation Array. Results from these studies will reveal brain region-selective changes in genes that are likely to contribute to alcohol use disorders. The above studies constitute Aim 1 of the Core. Aim 2 proposes bioinformatics pipelines for the analyses of the genome-wide datasets and the 5hmC array and integrating the large datasets for both preclinical and post- mortem studies. The integration of datasets from multiple non-biased approaches is innovative and will provide context for interpreting changes in the gene regulatory landscape in response to withdrawal from alcohol. In Aim 3, we propose to employ a novel dCas9:P300 acetyl transferase fusion protein to alter acetylation levels at specific genomic loci. This will allow us to activate expression of key genes in specific brain regions by altering acetylation selectively at their corresponding promoters. Similarly, we will use a dCas9:Tet1 fusion protein to alter 5mC and 5hmC levels at glucocorticoid receptor (Nr3c1) to modulate mRNA expression. The Epigenetics Core is using state-of-the-art technology to obtain genome-wide information to provide additional resources for each CARE component to address their specific hypotheses. The Core will perform quality control measures at many of the key steps of the protocols described above. In addition, key findings in the genome-wide measures will be validated by Core personnel in consult with Project leaders. The Epigenetics Core is an important platform, not only for individual CARE Projects, but also for University-wide NIH-funded epigenetics investigators.