Gregory A. Kapatos - US grants

DESCRIPTION (provided by applicant): GTP cyclohydrolase I (GCH1) catalyzes the first and rate-limiting step in the synthesis of tetrahydrobiopterin, the essential cofactor for tyrosine hydroxylase and the production of dopamine (DA) within nigrostriatal DA (NSDA) neurons. A complete analysis of the cis-acting elements in the GCH1 proximal promoter necessary for basal and cAMP-dependent transcription is near and the goal of Aim 1 is to identify cognate binding proteins for the GC-box and determine their role in basal and cAMP-dependent GCH1 transcription. The trans-acting factors recruited by these c/selements are also important and the goal of Aim 2 is to forge a link between phosphorylation of C/EBPbeta and NF-Y and cAMP-dependent GCH1 transcription. Little is known about how NSDA neurons regulate GCH1 gene expression. The goal of Aim 3 is to understand the temporal changes in protein-promoter DNA interactions that take place during cAMP-dependent GCH1 transcription in NSDA neurons and to test the hypothesis that GCH1 transcription is negatively coupled to somatodendritic D2 autoreceptor tone. Heterozygous mutations in GCH1 can cause DOPA-responsive dystonia (DRD), an autosomal dominant disorder with partial penetrance that selectively decreases DA synthesis within NSDA neurons and presents in childhood as a dystonia and in adulthood as Parkinson's disease (PD). Half of DRD patients have no mutation in the GCH1 open reading frame and presumably have mutations in GCH1 gene regulatory regions. The conserved genomic cis-elements we have already described are therefore likely sites for mutations associated with GCH1 deficiency. Unaffected first-degree relatives of DRD patients are also known to have a 23-fold higher incidence of parkinsonism than do normal controls, suggesting a link between DRD, GCH1 and PD. With the hypothesis that background genetic variability in GCH1 may promote susceptibility to familial parkinsonism and idiopathic PD, we propose in Aim 4 to sequence and functionally characterize mutations in GCH1 proximal promoter and coding regions in familial parkinsonism. Because association mapping is potentially a more powerful strategy for identifying genetic variability additional studies in Aim 4 will assess genetic variability within the GCH1 gene in PD cases versus controls. The goal of Aim 5 is to determine whether genetic variability in the human GCH1 gene influences GCH1 transcription or GCH1 enzyme activity. We expect that this multidisciplinary approach will yield important new information on the role of GCH1 in NSDA neuron function and will lead to a new understanding of DRD and familial and idiopathic PD.

DESCRIPTION(Adapted from applicant's abstract): The enzyme GTP cyclohydrolase I (GTPCH) catalyzes the first and limiting step in the synthesis of tetrahydrobiopterin (BH4), the required cofactor for monoamine (MA) neurotransmitter production. BH4 availability is a control point for MA synthesis and is regulated by changes in GTPCH gene expression. GTPCH gene expression is controlled by signal transduction pathways that converge on the GTPCH promoter. Some of these signaling pathways utilize the second messenger cAMP and protein kinase A (PKA). In order to better understand how cAMP and PKA control GTPCH gene transcription we have cloned, sequenced and begun to characterize 5.8 kb of the rat GTPCH promoter. We have focused on the cis-acting CRE and adjacent CCAAT-box elements within the GTPCH core promoter because we have already shown them to be critical for basal and cAMP-dependent transcription. The overall goal of this application is to firmly establish that the trans-acting factors ATF-4 and NF-Y are bound by these DNA response elements and, through the actions of PKA, serve to mediate the cAMP-dependent enhancement of GTPCH transcription that we have observed in PC12 cells. Specific Aim I will use recombinant ATF-4 and NF-Y, EMSA, supershift, and in vitro and in vivo DNA footprinting techniques to analyze the protein complexes recruited by the CRE and CCAAT-box elements. This Aim will also investigate the spatial relationship between the CRE and CCAAT-box as well as the role of the non-canonical TATA-box in transcription. Specific i Aim 2 will use TPCH promoter reporter constructs combined with foreed expression of wild type and dominant negative forms of ATF-4 and NF-Y to test the hypothesis that these proteins and the eo-activator CBP play an essential role in basal and cAMP-dependent transcription. Specifie Aim 3 will use GTPCH promoter reporter eonstruets along with foreed expression of wild type and dominant negative subunits of PKA, a peptide inhibitor of PKA and a PKA-defieient eell line to establish the role of PKA in the control of transeription. This Aim will also investigate whether ATF-4 and NF-Y are phosphorylated in vitro and in vivo by PKA. This researeh is important because dopamine (DA) synthesis by human nigrostriatal neurons is selectively vulnerable to genetic mutations in GTPCH that produce hereditary progressive dystonia (HPD), a disorder of movement that primarily effects young girls. Knowledge of how eAMP-eoupled signal transduetion pathways eontrol GTPCH gene transeription within DA neurons is therefore critical to our understanding of the underlying causes of the HPD phenotype and possibly other illnesses that involve nigrostriatal DA neurons, such as Parkinson's Disease.

This research team is conducting a genome-wide search for advantageous genetic changes in humankind's ancestry. A focus is on discovering the genetic changes that shaped humankind's enlarged brain and complex cognitive abilities. The first step in the method of discovery is to identify the many thousands of genes encoding proteins that function in the cerebral cortex of humans and other catarrhine primates such as chimpanzees, gorillas, and rhesus monkeys. Next, for these cerebral cortical brain expressed genes, determine which ones show species differences in expression patterns, especially identify those genes with expression patterns that differ between humans and the other catarrhines. Finally, by data mining take full advantage not only at the completed DNA sequence of the human genome but also of the soon to be completed chimpanzee genome and, for many of the genes of interest, the growing bodies of DNA sequence data from a range of primates. With this mined sequence, data for genes of interest (those that cerebral cortical cells express) identify by phylogenetic analysis the actual mutational changes that occurred either in gene regulatory regions or in protein encoding regions and that were favored by natural selection. Results obtained so far indicate that both types of mutational changes, gene regulatory and protein-encoding, shaped the distinctive features of the human phenotype. By combining the considerable expertise of geneticists, biochemists, neuroscientists, and anthropologists, this research will bridge diverse disciplines in order to elucidate the linked genotypic-phenotypic evolution of the human brain and thereby contribute to studies that seek a fundamental understanding of human origins.
This research should have broad societal impact. It will increase understanding of the genetic underpinning of the enlarged brain humans have. In doing so, it will identify those encoded proteins and DNA regulatory elements that evolved in more recent human ancestry under the force of positive selection. This information could be of great value in the field of mental health such as in the search for new therapeutic agents for mental health practitioners.