Deborah C. Mash - US grants

nicotinic receptors; Alzheimer's disease; aging;

The incidence of cocaine-related sudden deaths has reached epidemic proportions in many metropolitan areas around the United States. Widespread cocaine abuse is associated with significant neuropsychiatric and from 1978 - 1985 has revealed a number of important findings regarding the temporal trends and risk factors surrounding cocaine-related sudden death. The epidemiological findings indicate that in Dade County only 6 to 11% of the cases had significant underlying coronary artery disease or ventricular hypertrophy, suggesting that these risk factors may account for only a subgroup of the cocaine-related deaths. In addition, polydrug abuse does not appear to be a significant risk factor in Dade County. High dose cocaine toxicity is associated with seizures. However, the percentage of cocaine-related deaths with seizures has declined during the epidemic. This pattern is consistent with toxicology findings which demonstrate that median plasma concentration of cocaine associated with sudden death also appear to be declining annually. In contrast, an increased incidence of preterminal excited delirium and sudden death has been reported which closely follows the epidemic curve for cocaine-related deaths in Dade County. These observations suggest that the causes of cocaine-related death in decedents with comparatively low concentrations of blood cocaine may be different from the high dose toxicity group. The precise pathophysiological mechanisms leading to cocaine-related sudden death are not well defined and, at present, all known mechanisms fail to explain why cocaine abuse may have divergent effects on the heart. Our hypothesis is that cocaine abuse results in abnormal neuronal activity in limbic and brainstem centers comprising the neural substrates which mediate central autonomic functions. Abnormal neurochemical transmission in these pathways may, in addition to the direct cardiotoxic effects of cocaine, lead to the pathogenesis of cocaine-related sudden death. The present proposal seeks funds for systematic post mortem studies of the chemo- and pathoarchitecture in brains from victims of cocaine-related death. Using rigorous inclusion criteria for cocaine exposure, the cases selected for neuroanatomical studies will be classified into the following subgroups : high dose toxicity (seizures); low dose toxicity without significant underlying cardiac pathology; and cocaine-related death in subjects exhibiting preterminal excited delirium. We propose to characterize post mortem neurochemical alteration in brains from victims of cocaine-related sudden death using in vitro autoradiography on whole-brain sections. The distribution and status of norepinephrine and dopamine terminals will be visualized in whole-brain sections by receptor subtypes and sigma receptors will be correlated with limbic and medullary pathoarchitecture and whenever possible with the pattern of use and drug and metabolite levels in plasma and brains. The proposed anatomical studies may disclose the toxic effects of an neural sites for the action of cocaine in the human brain. These studies may provide a basis for pharmacologically targeting multiple, interactive dysfunctions in the brain in order to halt cocaine abuse in chronic users.

This proposal seeks funds from the small grant program (R03) for a comprehensive autoradiographic study of the alterations in neuroamine receptor subtypes in a primate model of alcoholism. We propose to conduct these studies in animals taken from an experimental colony of Vervet monkeys (Cercopithecus aethiops) that have been previously characterized for their descriptive behaviors and alcohol preference. The Vervet or African green monkeys, which are housed in stable social groups, are drawn from a large isolated and non-endangered Caribbean population on the island of St. Kitts, W.I. Interestingly, the feral population from which these animals came has lived for 300 years in an environment of sugar cane, burned cane and fermented cane. Population screening studies indicate that 17% of the Vervet monkeys will select alcohol over vehicle alone. The Vervet monkeys which spontaneously select alcohol and drink to intoxication have advantages for the proposed chemoanatomical studies and complement the more established animal models of alcoholism. We propose to characterize baseline neurochemical markers in alcohol-selecting and control Vervets. We will utilize in vitro autoradiography to quantify the regional density of dopaminergic and serotonergic receptor subtypes. Dopaminergic and serotonergic innervation densities will be visualized by labelling dopamine and serotonin transporters in slide-mounted brain sections. The baseline neurochemical status will be compared to altered neuroreceptor regulation during the induction and alcohol withdrawal periods. Adjacent sagittal brain sections will be processed for in vitro autoradiography to demonstrate potential alterations in the affinity .and number of dopaminergic, serotonergic and beta adrenergic subtypes during the intoxication/withdrawal phase. We will attempt to correlate the neurochemical findings with blood alcohol levels and with measures of amine-aldehyde adducts in urine and brain. The proposed preclinical studies in primates which voluntarily select alcohol are designed to provide a basis for identifying the interrelated neurochemical correlates of alcohol abuse in man.

The University of Miami School of Medicine has demonstrated excellence in performing clinical and basic research dealing with the health problems associated with drug abuse. To bring together multidisciplinary approaches to ongoing drug abuse-related research, the University of Miami School of Medicine has recently established the Comprehensive Drug Research Center (CDRC). The programs described in this application relate to the timely topics of the toxicological consequences of cocaine abuse, perinatal cocaine exposure, and drugs of abuse as possible co-factors for human immune deficiency (HIV) infection. The state-of-the-art Finnigan GC/MS /DS Ion Trap Witness System will significantly enhance the PHS-funded projects and provide new research directions. The proposed system is optimally configured and fully capable of meeting present, as well as, the future requirements of the major users in this application. This proposal discusses the specific application of the Finnigan MAT GC/MS/DS Witness System by PHS-funded investigators within the Departments of Neurology and Cellular and Molecular Pharmacology (Dr. Deborah Mash), the Departments of Epidemiology and Cancer and Oncology (Dr. Clyde McCoy), and the Department of Psychiatry (Dr. Bryan Page), the Departments of Psychiatry and Molecular Biology (Dr. Paul Shapshak), the Department of Psychology (Dr. Tiffany Fields) and the Division of Neonatology (Dr. Emmalee S. Bandstra). We propose to house the GC/MS at the toxicology laboratories at the Metro-Dade County Medical Examiner Department. The newly constructed facility is located immediately adjacent to the University of Miami School of Medicine/Jackson Memorial Hospital complex.

technology /technique development; substance abuse related disorder; male; mental disorders; human tissue; biomedical resource; bioengineering /biomedical engineering; Mammalia; biomedical equipment development; human subject; nervous system; female; behavioral /social science research tag;

technology /technique development; substance abuse related disorder; male; mental disorders; human tissue; biomedical resource; bioengineering /biomedical engineering; Mammalia; biomedical equipment development; human subject; nervous system; female; behavioral /social science research tag;

DESCRIPTION: (provided by applicant) This R21 application request support for the development of a rapid method for: gamma-hydroxybutyrate (GHB) detection and measurement in biological fluids. An increasing number of reports indicate that the abuse of GHB is widespread. GHB is regularly encountered in cases of intoxicated drivers and fatal GHB overdoses are reported throughout the country. In Dade County, Florida, a number of fatal intoxications have been documented, some of them due to GHB alone. There are also reports suggesting that GHB is often used to facilitate sexual assault. Particularly in suspected poisoning cases, the need for a rapid method to detect and quantify GHB is obvious, since the symptomatology may be similar to, but the management and treatment different from, that of other kinds of intoxications. The analytical methods currently available are mainly based on gas chromatography with flame ionization (GC-FID) or mass spectrometric (GC-MS) techniques that involve laborious extraction steps for sample preparation. These methods are very time-consuming and inexpedient for use in emergency settings. We propose to develop an enzymatic assay based on the conversion of GHB to succinic semialdehyde as a rapid and specific method for the detection and measurement of GHB in biological fluids. This reaction is catalysed by a group of highly selective GHB dehydrogenase enzymes that utilize either NADP+ or NAD+ as cofactors. The NADPH or NADR produced in stoichiometric quantity by the reaction can be measured spectrophotometrically. Our approach is based upon the same principles as the classic alcohol dehydrogenase method for analysis of ethanol with substitution of highly selective GHB dehydrogenase. The GHB dehydrogenase of Syrian hamster liver, which has been purified and well characterized, will be used to evaluate the accuracy and reliability of the rapid spectrophotometric method of analysis. The rapid and sensitive analytical approach based on 4-hydroxybutyrate catabolism and spectrophotometric detection of the reaction product will be evaluated by comparing the results with a highly specific GC-MS method, currently in use at the toxicology unit of the Miami-Dade County Medical Examiner Office under the direction of Dr. W. Lee Ream. A novel approach will be to extend the methods developed in hamster liver for use with GHB dehydrogenase of Clostridium kluyveri. In this approach, a plasmid vector containing genes that express stable levels of 4-hydroxybutyrate dehydrogenase is grown and expressed in Escherichia coli. The enzyme will be isolated from the culture medium and employed in a reagent system adaptable to automated chemistry analyzers of the types used in medical and drug screening laboratories. In addition, we plan to extend the enzymatic method to develop a rapid spot test based on the same principle. Proof-of-concept and inter-assay validity will be evaluated by comparing the results of both these methods with GC-MS. Both of these methods will be tested on GHB-spiked controls and residual blood and urine specimens obtained from the Miami-Dade County Medical Examiner Department and the Emergency Department of Jackson Memorial Hospital.

Beta-N-methylamino-L-alanine (BMAA) is a potent neurotoxic amino acid that produced by cyanobacteria and can be found in almost every habitat, from oceans to fresh water to bare rock to soil. The role of BMAA in neurotoxicity and neurodegeneration is unknown; however, dietary exposure to BMAA has lead to some of the highest rates of devastating neurodegenerative diseases in humans such as Amyotrophic Lateral Sclerosis/Parkinsonism dementia complex (ALS/PDC) and Alzheimer?s disease. This evidence leads scientists to believe that the food chain may be an important route of exposure for BMAA to the human populations

With funding from this Small Grant for Exploratory Research, researchers from the University Of Miami?s School Of Medicine will collaborate with scientists from the University of Hawaii and the Institute for Ethnomedicine to document the presence of BMAA in the coastal marine food chain in South Florida waters. Through multiple field sampling and analytical techniques, the principle investigators will evaluate the transfer of BMAA through the coastal marine food web, determining its presence and concentration, as well as its affects on the health of the human population.

The broader impacts of this research are directly related to global public health. With the worldwide spread of algal blooms caused by anthropogenic pollution, the risk of human exposure to this cyanobacterial neurotoxin becomes a significant public health concern. The results of this study will be valuable in the future prevention of neurodegenerative diseases and the establishment of policies regarding the prevention of future anthropogenic marine pollution.

[unreadable] DESCRIPTION (provided by applicant): Although PD is predominated by motor symptoms (tremor, rigidity and bradykinesia) during its initial stages, with disease progression the majority of patients develop additional non-motor manifestations like cognitive impairment, psychiatric complications and autonomic dysfunction. Among the most common are visual hallucinations seen in about 1/3 of PD patients. Visual hallucinations are usually related to L-dopa therapy, significantly influence quality of life and lead to increased mortality in PD. Although their clinical variables have been somewhat defined, their neurochemical basis remains unknown. Lack of such knowledge limits therapeutic efficacy in PD and may compromise the development and use of future therapies like stem cell transplantation and nerve growth factor gene transfer. This project focuses on the characterization of the neurochemical changes leading to visual hallucinations in PD. We propose that these symptoms result from a complex process that involves accumulation of the protein alpha-synuclein and dysregulation of DAergic pathways affecting the ventral tegmental area (VTA), to later extend to the ventral striatal (nucleus accumbens) and mesocorticolimbic regions (amygdala). To this end, we will evaluate the neurodegenerative process as well as key components of the DAergic system in these regions. We propose three specific objectives to address these issues: 1) To characterize the expression of alpha-synuclein in the substantia nigra, vental tegmental area and striatum using immunocytochemistry, immunoblotting, and in situ hybridization techniques in postmortem brain specimens of PD patients with and without visual halluinations as well as age-matched controls. 2) To map and visualize the loss of DAergic projections to the limbic target areas by determining the expression of dopamine transporter (DAT) binding and 3) To examine the regulation of D1, D2 and D3 DAergic receptors using autoradiographic binding techniques in limbic brain regions in PD. Our long term objective is to identify neurochemical mechanisms related to the development of drug-induced psychotic symptoms in PD. This Small Grant (R03) application from a new investigator is concentrated in obtaining key pilot data on the etiopathogenesis of visual hallucinations (VH) in Parkinson's disease. Recent publications from our group, our preliminary results, the brain tissue availability and the multidisciplinary team assembled provide strong support for the feasibility of this project. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Carbon-14 Birth Dating of Neurons in Addiction The dynamic turnover of neurons in the human brain is essentially unknown. Recent rodents and primates studies suggest that new neurons are not only generated throughout life, they integrate into the circuitry of the brain and actively participate in its functions throughout life. By combining biomedical approaches with recent developments in accelerator mass spectrometry (AMS), it is now possible to measure the incorporation of ambient 14C derived from above-ground nuclear bomb tests in neuronal DNA. We propose to use this novel approach to retrospectively establish the birth date of cells in adult human brain and to test for the effect of abused drugs and alcohol on cell turnover. This proposal is supported by our access to a large archived biorespository of well-characterized postmortem brain specimens from chronic substance abusers. Our hypothesis is that addiction is a condition that impairs the neural stem and progenitor cell pools in adult brain. A substantial literature describes the capacity of all addictive drugs to slow neurogenesis, but there are no studies, which have assessed the effect of abused drugs or alcohol on adult-generated neurons across the lifespan in humans. Chronic abuse of addictive drugs or alcohol may affect the stem/progenitor cell pool providing a link between dysfunctional neurogenesis and the pathobiology of addiction. We propose to map sites of neurogenesis in select regions of the adult brain to determine the effect of different classes of abused substances on cell turnover using the 14C birth dating method and AMS in parallel with immunologic detection of early fate and proliferation markers. Drug or alcohol-induced changes in hippocampal neurogenesis may be one of the underlying biological mechanisms responsible for the intractable cycle of compulsive use and failed abstinence. The proposed studies will contribute to an understanding of the neurobiology of drug and alcohol addiction, and may have important implications for the development of novel therapeutic approaches targeted to these cell populations.

Project Summary Multiple interacting genes and environmental factors determine the risk and trajectory of drug addiction. Expression of genes involved in the risk and consequences of addiction is influenced by environmental factors that in many cases are likely to leave persistent chemical modifications (epigenetic marks) in DNA and chromatin. We propose to comprehensively identify in archived postmortem human brain, changes in gene expression and linked epigenetic marks associated with chronic cocaine dependence. We will study two dopamine-dependent brain regions, the dorsal and ventral striatum. We will profile epigenetic marks in cocaine abusers with both short and long-term drug exposures. For analysis of the transcriptome, we will perform whole genome, strand- specific sequencing of mRNA transcripts (RNA-seq). For the DNA methylome, we will use MeDIP or equivalent technology to map the distribution of methyl CpG's at genome- wide and locus specific levels. The ultimate aim of this proposal is to discover which epigenetic changes in brain accompany the transition from cocaine abuse to chronic cocaine dependence. Discovery of cocaine-associated networks and pathways will provide clues to the etiology of cocaine addiction and will identify likely points of intervention in molecular and biochemical pathways that represent potential therapeutic targets.

Although many cells and neural circuits clearly contribute to opiate and other substance abuse disorder, the path to drug addiction travels through midbrain dopaminergic neurons. Though a rare cell type (it is estimated that a mere 1 of every 200,000 neurons in the human brain is of a dopaminergic phenotype), changes in dopaminergic neurotransmission are thought to play a role in various stages of addiction, from acute reward mechanisms and goal-directed actions, to the development of habitual behavior and increased salience of cues associated with drug use, as well as the anhedonia and dysphoria associated with drug withdrawal. Surprisingly little is actually known about persistent changes in gene expression that presumably underlie the dysfunction of dopamine systems in brain exposed to opiates and other drug of abuse. Our project is centered on three Specific Aims. In Aim #1,we will extract chromatin from immunotagged midbrain dopaminergic neuron nuclei collected by fluorescence-activated sorting from 150 controls and 150 cases diagnosed with opiate abuse and then profile, on a genome-wide scale, the transcriptome and open chromatin landscapes and promoter-enhancer loopings and other types of chromosomal conformations (the ?3D genome?) in cell type-specific manner. In Aim #2, we will apply integrative genomics approaches and leverage Aim #1 postmortem brain data with population-scale genotypes and phenotypes provided by the Million Veterans Project and the Psychiatric Genomics Consortium to build causal probabilistic networks and predict key drivers within the regulatory non-coding DNA space of the dopaminergic system. In Aim #3, we will validate addiction-relevant cis-regulatory sequences (from Aim #1, #2) with small RNA-guided epigenomic editing systems in cultured human dopaminergic neurons. Collectively, our midbrain dopaminergic neuron- focused project will fill critical voids in the field of human addiction research and human neurogenomics and embark, for the first time, on a deep epigenomic assessment of one of the key cell populations in reward and addiction circuitry.