Roland D. Ciaranello - US grants

In this application we propose to investigate the genetic control on dopamine receptors and the way in which genetic variations influence behavioral responses to psychopharmacologic agents. Dopamine receptors are measured in the olfactory tubercle and corpus striatum using 3H-ADTN, a dopaminergic agonist, and 3H-spiroperidol, a dopamine receptor antagonist, as radioligands. Among eleven inbred mouse strains, variations in the number of dopamine receptors up to two-fold are observed. In contrast, no differences in radioligand affinity are seen. ADTN labels different sites than those labelled by spiroperidol. Spiroperidol binds to both serotonin and dopamine sites in the olfactory tubercle, but binds primarily to dopaminergic sites in the striatum. The number of ADTN or spiroperidol binding sites in the striatum does not correlate with the number of radioligand binding sites in the olfactory tubercle, suggesting that genetic controls on dopamine receptors operate independently in the olfactory tubercle and striatum. If this is true, the finding is of considerable potential clinical importance. The studies proposed in this application seek to further characterize ADTN and spiroperidol binding sites in both the olfactory tubercle and striatum. This is to differentiate serotonin from mouse strains to determine the number of genes involved in transmission of each ligand binding sites, the existence of structural gene variants, whether dopamine receptors in one region are under independent genetic control from receptors in the other region, and the mechanisms of gene control over dopamine receptors. The genetic data will then be used in psychopharmacology studies with amphetamine and haloperidol. These studies will determine if genetic differences in behaviors induced by administration of these dopaminergically-active drugs are associated with inheritance of dopamine receptors.

Elevated platelet serotonin in some autistic and mentally retarded children has been a finding repeatedly reported in the literature. The mechanisms underlying this finding and the relationship between peripheral hyperserotonemia and central serotonin metabolism are poorly understood. The possible efficacy of fenfluramine, reported to lower platelet serotonin and improve autistic functioning, makes it particularly timely for a definitive investigation of the mechanisms of platelet hyperserotonemia. A carefully diagnosed group of hyperserotonemic autistic children (HSA) will be compared to age, sex and SES-matched groups of normal serotonemic autistic children (NSA), hyperserotonemic mentally retarded children (HSMR) and normal children. The pathways of platelet serotonin production, storage and clearance will be examined systematically in the children and in their first degree relatives. A subgroup of HSA and NSA children will undergo CSF studies to determine the relationship of central to peripheral serotonin metabolism. Loci of metabolic defects in individual children, their family members, and in the clinical diagnostic groups will be determined. If multiple deficits are present in the same child or clinical group, a profile of metabolic dysfunction will be established. Fenfluramine, as part of multi-center collaborative project, will be administered to a selected population of children with autism. The studies will address the biochemical mechanisms responsible for hyperserotonemia, its familial or non-familial nature, the clinical features distinguishing hyperserotonemic autistic from normoserotonemic autistic children, the relationship of peripheral to central serotonin metabolism and the effects of lowering platelet serotonin on clinical symptoms of autism.

Our current program consists of both basic and clinical components. Our basic work consists of studies on the regulation of synthesis and degradation of neurotransmitter enzymes; regulation of the production and turnover of S-adenosylmethionine and related enzymes; genetic determination of dopamine and serotonin receptors; solubilization, isolation and purification of a high-affinity [3H]serotonin binding site; molecular biology studies cloning genes for catecholamin enzymes and neurotransmitter receptors; and neurochemical studies in canine narcolepsy. Our clinical studies are related to the major psychiatric disorders of childhood, specifically infantile autism, childhood schizophrenia, and depression. We are conducting three collaborative projects with autistic, schizophrenic and retarded children. These are studies determining biochemical mechanisms of hyperserotonemia in autistic and retarded children; studies on opiate peptides and pain responses in autistic children, and a large multicenter trial of fenfluramine administration to autistic children. In addition, we conduct collaborative biochemical studies with the Stanford Mental Health Clinical Research Center exploring neurotransmitter levels and neurotransmitter receptor concentration in unipolar, bipolar and schizophrenic patients. We intend to continue our work in these project areas. Emphasis will be placed on the molecular biology studies, the continued purification of a high-affinity serotonin receptor, and the descriptive genetics of dopamine and serotonin receptors. In our studies in autistic children, emphasis will be placed on elaborating biochemical mechanisms of hyperserotonemia.

This describes our work in the program on Neurochemistry and Childhood Psychopathology. Progress in the five component areas is described, along with a brief summary of our plans for the coming year. The program is currently in its first year; funding began in March, 1985. The component sections of the program include: Core Facilities, Biochemical Regulation; Neurotransmitter Receptors; Narcolepsy and Clinical Neurochemistry.

This proposal seeks to study several aspects of the regulation of important neurotransmitter enzymes, their reaction constituents, and the receptors which they stimulate and which in turn initiate critical cellular events. The application is a competing renewal to support our ongoing work in these areas. The main thrust of the work is regulation of neurotransmitter enzymes and receptors from a cellular, metabolic and genetic standpoint. The work will be carried out in brain and in adrenal both in vivo and in vitro. The projects to be conducted are: (A) regulation of phenylethanolamine N-methyltransferase (PNMT) turnover by glucocorticoids, by S-adenosylmethionine and by splanchnic neuronal neurotransmitters; (B) regulation of S-adenosylmethionine (SAM) levels by glucocorticoids and other factors; (C) regulation of hydroxyindole O-methyltransferase (HIOMT) turnover by glucocorticoids; (D) regulation of dopamine beta-hydroxylase (DBH) proteolysis in vivo by ascorbic acid; (E) isolation and characterization of the proteases which degrade PNMT and DBH in vivo; (F) genetic regulation of DBH turnover; (G) genetics of PNMT responsiveness in mice; (H) genetics of serotonin, serotonin enzymes and serotonin receptors in inbred mice and (1) deveopment of monoclonal antibodies to PNMT, DBH, HIOMT and the enzymes of S-adenosylmethionine metabolism.

This program consists of an administrative and scientific core serving four investigative areas: (1) Biochemical Regulation; (2) Biochemistry of Neurotransmitter Receptors; (3) Neurochemical Studies in Canine Narcolepsy and (4) Clinical Neurochemical and Genetic Studies in infantile autism. The central theme of the program is that pervasive developmental disorders in children reflect central nervous system dysfunction with defined biochemical and metabolic consequences. The investigative scope of the program ranges from basic work in gene regulation, protein structure and neuropharmacology to diagnostic and therapeutic strategies in infantile autism. Each component of the program interdigitate with the other project areas while retaining a separate base of scientific inquiry. The biochemical Regulation section focuses on the regulation of the catecholamine biosynthetic enzymes, dopamine s-hydroxylase and phenylethanolamine N-methyltransferase, from genomic through posttranslational events. Studies on the structure, subunit composition and amino acid sequence of these enzymes are ongoing as is work cloning cDNA probes encoding their structure. These probes and specific antibodies will be used to investigate molecular events underlying enzyme regulation. The Receptor section studies the molecular characteristics of 5HTIb and 5HTIc receptors, is solubilizing and puriFying these proteins and developing molecular probes to study their regulation. Developmental studies examining the brain serotonergic- noradrenergic system will be a coordinated effort of the Regulation and Receptor sections. The Narcolepsy section studies neurochemical deficits in the cholinergic, dopaminergic and noradrenergic systems implicated in canine narcolepsy, a genetic disorder also occurring in children. Work on the receptors and enzymes involved in these systems is underway, as are intergroup efforts studying their ontogeny and genetic regulation. The Clinical section has accumulated a large database of children with autism and related pervasive developmental disorders, and has developed methods of subtyping them into four, more distinct, homogeneous clinical groups. Work is underway to define discrete genetic, prenatal and biochemical markers associated with these subtypes. Finally, a new project area will investigate genetic linkage in multiplex families with autistic probands, using a series of cDNA probes to identify restriction fragment polymorphisms.

This program consists of an administrative and scientific core serving four investigative areas: (1) Biochemical Regulation; (2) Biochemistry of Neurotransmitter Receptors; (3) Neurochemical Studies in Canine Narcolepsy and (4) Clinical Neurochemical and Genetic Studies in infantile autism. The central theme of the program is that pervasive developmental disorders in children reflect central nervous system dysfunction with defined biochemical and metabolic consequences. The investigative scope of the program ranges from basic work in gene regulation, protein structure and neuropharmacology to diagnostic and therapeutic strategies in infantile autism. Each component of the program interdigitate with the other project areas while retaining a separate base of scientific inquiry. The biochemical Regulation section focuses on the regulation of the catecholamine biosynthetic enzymes, dopamine s-hydroxylase and phenylethanolamine N-methyltransferase, from genomic through posttranslational events. Studies on the structure, subunit composition and amino acid sequence of these enzymes are ongoing as is work cloning cDNA probes encoding their structure. These probes and specific antibodies will be used to investigate molecular events underlying enzyme regulation. The Receptor section studies the molecular characteristics of 5HTIb and 5HTIc receptors, is solubilizing and puriFying these proteins and developing molecular probes to study their regulation. Developmental studies examining the brain serotonergic- noradrenergic system will be a coordinated effort of the Regulation and Receptor sections. The Narcolepsy section studies neurochemical deficits in the cholinergic, dopaminergic and noradrenergic systems implicated in canine narcolepsy, a genetic disorder also occurring in children. Work on the receptors and enzymes involved in these systems is underway, as are intergroup efforts studying their ontogeny and genetic regulation. The Clinical section has accumulated a large database of children with autism and related pervasive developmental disorders, and has developed methods of subtyping them into four, more distinct, homogeneous clinical groups. Work is underway to define discrete genetic, prenatal and biochemical markers associated with these subtypes. Finally, a new project area will investigate genetic linkage in multiplex families with autistic probands, using a series of cDNA probes to identify restriction fragment polymorphisms.

This is a competing renewal application for continuing support of our research program in Molecular Neurobiology and Developmental Disorders. The program consists of an administrative and scientific core serving five major investigative areas. Each area has as its central theme the application of molecular neurobiology to an understanding of developmental processes and their regulation. Studies in the Regulation of Serotonin and beta-adrenergic Receptors section examine the regulation of serotonin and beta-adrenergic receptor mRNA and G-protein coupling during development, as well as the regulation of these receptor mRNAs by antidepressants and drugs which alter neurotransmission. The work seeks to understand those factors which initiate and maintain receptor gene expression. The section on Enzyme Regulation focuses on hormonal and neuronal regulation of phenylethanolamine N-methyltransferase (PNMT) gene expression during development. These studies examine differences in prenatal and postnatal mechanisms regulating enzyme expression. The section on Developmental Neurobiology of Canine Narcolepsy uses an animal model of narcolepsy, an autosomal recessive disorder which afflicts young adolescents and children, to examine defects in cholinergic and alpha(1)-adrenergic receptors. The research strategy utilizes the differential maturation of the relevant neurotransmitter systems to help localize the specific neurochemical lesion(s). In the section on Characterization of Developmentally Regulated Genes, we are devising methods of isolating and characterizing genes which are expressed during discrete developmental epochs. We hope to identify those genes which regulate the architectural development of the cerebral cortex. Finally, the section on Molecular and Linkage Genetics of Infantile Autism describes work seeking to find a gene marker for infantile autism in multiple-incidence families.

This is a revised application for an ADAMHA Research Scientist Award. It describes in part my work as principal investigator and program director for the research program in Molecular Neurobiology and Developmental Disorders. The program consists of an administrative and scientific core serving five major investigative areas. Each area has as its central theme the application of molecular neurobiology to an understanding of developmental processes and their regulation. Studies in the Regulation of Serotonin and beta-adrenergic Receptors section (Roland D. Ciaranello, M.D., Project Director) examine the regulation of serotonin and beta- adrenergic receptor mRNA and G-protein coupling during development, as well as the regulation of these receptor mRNAs by antidepressants and drugs which alter neurotransmission. The work seeks to understand those factors which initiate and maintain receptor gene expression. The section on Enzyme Regulation (Dona L. Wong, Ph.D., Project Director) focuses on hormonal and neuronal regulation of phenylethanolamine N-methyltransferase (PNMT) gene expression during development. These studies examine differences in prenatal and postnatal mechanisms regulating enzyme expression. The section on Developmental Neurobiology of Canine Narcolepsy (William C. Dement, M.D., Ph.D., Project Director) uses an animal model of narcolepsy, an autosomal recessive disorder which afflicts young adolescents and children, to examine defects in cholinergic and alpha-1-adrenergic receptors. The research strategy utilizes the differential maturation of the relevant neurotransmitter systems to help localize the specific neurochemical lesion(s). in the section on Characterization of Developmentally Regulated Genes (John L.R. Rubenstein, M.D., Ph.D., and Roland D. Ciaranello, M.D, Project Codirectors), we are devising methods of isolating and characterizing genes which are expressed during discrete developmental epoches. We hope to identify those genes which regulate the architectural development of the cerebral cortex. Finally, the section on Molecular and Linkage Genetics of Infantile Autism (Project F, Luigi Cavalli-Sforza, M.D., Project Director) describes work seeking to find a gene marker for infantile autism in multiple-incidence families. This Research Scientist Award application describes in detail my work in the sections on receptor regulation and autism genetics.