Michael J. Forster - US grants

biomedical equipment resource; biomedical equipment purchase;

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

The National Institute on Drug Abuse (NIDA), Medications Development Division (MDD) has established a Cocaine Treatment Discovery Program (CTDP) to identify potential treatments for the medical management of cocaine dependence. This program involves testing compounds through a decision-based screening scheme which consists of a number of in vitro and in vivo assays and which is designed to identify potential treatment agents which either substitute for cocaine or antagonize the effects of cocaine. The purpose of this contract is to preclinically evaluate the effects of 100 test compounds on the subjective effects of cocaine; as assessed by the rat drug discrimination procedure. Depending upon results obtained from the locomotor activity test, which is conducted under a separate contract, the test compound will be evaluated for the ability to substitute for, or antagonize, the discriminative effects of cocaine. The compounds to be tested under this contract will be selected and, for the most part, supplied by NIDA. Compounds will be supplied under an identification code number with appropriate information including physical properties, solubilities, and recommended starting doses, if available. CTDP data generated from this contract on proprietary compounds will be maintained in confidence by NIDA unless permission to release the data is granted to NIDA by the compound submitter. Compound submitters are free to disclose CTDP data generated on their compounds at any time.

This represents a competitive renewal of NIDA contract N01DA29305, entitled "Assessment of Potential Cocaine Treatment Medications in Rodents". The objectives of this research and development contract is to determine the effects of compounds in the rodent locomotor activity and drug discrimination procedures as a means of evaluating their potential efficacy as cocaine treatment medications. These tests have been selected by NIDA Medication Development Division's (MDD) Cocaine Treatment Discovery Program (CTDP) because they are believed to represent model systems that may be predictive of clinical efficacy of pharmacotherapies for cocaine dependence. The data generated by this contract will be utilized by the NIDA/MDD/CTDP in the selection of compounds for additional preclinical evaluation and subsequent development as pharmacotherapies for cocaine dependence. Briefly, the Contractor will be screening compounds in the mouse locomotor activity and rat drug discrimination test. We respect to the locomotor activity procedure, the effect of the compound, administered alone and in combination with cocaine, on locomotor activity in mice will be assessed. Depending on results obtained from the locomotor activity test, the compound will either be evaluated for the ability to antagonize the discriminative (subjective) effects of cocaine or to substitute for the discriminative (subjective) effects of cocaine. The anticipated end results of this contract will be written reports detailing study findings.

DESCRIPTION (provided by applicant): The endogenous antioxidants vitamin E, vitamin C, and coenzyme Q (CoQ) are thought to have significant interactions in the maintenance of cellular redox state and in cellular protection form oxidative insult. The proposed project will determine the extent to which these compounds can interact to ameliorate or prevent functional brain aging in mice, when supplemented in two- and three-way combinations. In one experiment (Aim1), supplementation with the antioxidant combinations will be initiated in late life, at age when brain dysfunction is already present. A battery of behavioral tests will be used to estimate the ability of the antioxidant supplementation regimens to reverse age-related losses of cognitive functions (associative learning, working memory, and spatial learning) as well as losses of sensory and psychomotor functions (auditory and somatosensory responsiveness, reaction time, coordination, balance, muscle strength). The same battery of behavioral tests will be used to determine whether or not the antioxidant supplementation regimens can prevent functional losses if supplementation is initiated prior to development of age-related brain dysfunction (Aim2). To determine whether or not beneficial effects of the antioxidative regimens depend on their ability to reduce oxidative stress/damage (Aim 3), brains from the mice tested in the first two aims will be dissected into different regions for determining: (i) amounts of oxidative damage to proteins or lipids (protein carbonyls, thiobarbituric reactive substances), (ii) shifts in glutathione redox state and amounts of aminothiols, protein sulfhydryl and mixed disulfides and (iii) levels of CoQ, vitamin E and vitamin C. The aminothiols status and albumin-associated carbonyl content of plasma will be determined at different times during treatments (Aim 4) to determine whether or not plasma markers of oxidative stress/damage are useful predictors of the effects of antioxidant supplementation on cognitive/psychomotor performance. These studies will provide specific information about the nature of antioxidant regimens most likely to be beneficial against brain aging and will identify the ages at which benefits should be expected. Moreover, they will improve understanding of the neurological consequences of antioxidant supplementation that are most critical to the beneficial effects and may identify clinically useful biological markers predictive of successful treatment.

DESCRIPTION (provided by applicant): Carisoprodol (Soma(R)) is a centrally-acting skeletal muscle relaxant frequently prescribed for the treatment of acute musculoskeletal conditions. In recent years, there has been increasing concern regarding carisoprodol's potential as a drug of abuse. In 1991, carisoprodol was ranked 54th among 234 drugs with abuse potential. Only eight years later, the Drug Abuse Warning Network identified carisoprodol as the 14th most abused drug, ranking higher than oxycodone, methadone, and LSD. In support of this, recent reports have substantiated the dangers involved in carisoprodol abuse, including severe withdrawal leading to seizures and death. However, its mechanism of action remains unclear. Until recently, it was widely accepted that the sedative effects of carisoprodol were predominantly due to its metabolite, meprobamate. However, preliminary in vivo and in vitro studies conducted in our laboratory demonstrate that carisoprodol itself is active, and its actions appear to be mediated via novel modulation of the GABAA receptor (GABAAR), the predominant inhibitory neurotransmitter receptor in mammalian brain. This may underlie the capacity of carisoprodol to enhance the sedative effects of CNS depressants, contributing to its potential for abuse. Indeed, concerns about the rise in abuse of carisoprodol have led the Drug Enforcement Agency to contact the National Institute on Drug Abuse for assistance in determining the abuse liability of carisoprodol. Based on our preliminary findings, we hypothesize that carisoprodol itself causes depressive CNS effects, and that these actions are mediated via a novel interaction with the GABAA receptor. The overall goal of the proposed study is to identify the mechanisms that mediate carisoprodol abuse, at both the behavioral and molecular level. We will utilize both in vivo (drug discrimination and observation of intoxication, tolerance and withdrawal, and pharmacokinetic analysis) and in vitro (assessment of carisoprodol on specific configurations of recombinant GABAA receptors) approaches to address the following specific aims: 1) To characterize the discriminative stimulus effects of carisoprodol;2) To investigate whether carisoprodol produces effects on its own in vivo or requires being metabolized to meprobamate;3) To assess the extent of carisoprodol dependence and severity of withdrawal;4) To identify GABAA receptor subunits critical for allosteric modulatory and direct gating effects of carisoprodol;and 5) To identify GABAA receptor subunit domains that confer carisoprodol sensitivity. Given the present and potential dangers posed by carisoprodol abuse, it is of crucial importance to determine the mechanism of action of this drug. Our proposed studies will substantially increase our understanding of the mechanism of action of carisoprodol as a therapeutic agent and as a drug of abuse. This knowledge may provide insight into treating carisoprodol dependence, and withdrawal. PUBLIC HEALTH RELEVANCE: The muscle relaxant Carisoprodol (Soma(R)) is being abused increasingly at an alarming rate;withdrawal from overuse of this drug can cause seizures and even death. There is an urgent need to develop new drugs that can treat these symptoms, as well as new drugs that are less likely to be abused. The proposed studies will provide the fundamental information needed to address both of these current unmet medical needs.

The ability of reactive oxygen species (ROS) to produce dysfunctional macromolecules is thought to play a significant role in brain aging and in neurodegenerative diseases. However, it is also well established that ROS serve as important signaling molecules involved in cellular processes that underlie different cognitive functions. In the current application, it is hypothesized that age-dependent increases in ROS may cause age- associated cognitive or motor impairment by (i) promoting deleterious accumulation of oxidative damage to specific mitochondrial proteins, (ii)causing a decrease in efficiency of ROS-mediated signaling and/or(iii) initiating compensatory shifts in signaling mechanisms underlying brain processes. Under Aim 1 of this application, we will identify proteins in mitochondria from different brain regions that are associated with age- related increases in carbonyls, in aged mice with and without cognitive or motor impairments. The functional significance of these impairment-associated oxidized proteins will be addressed further under Aim 2, in which it is proposed to determine the correlation between impaired bioenergetic activity of oxidized proteins and age-related behavioral dysfunction. Aim 3 of this application will assess whether or not the age-related increase in unregulated ROS contributes to impairment of synaptic plasticity and memory function, and assess whether or not this contribution is age-dependent. Aim 4 of the application is to determine if behavioral impairments associatedwith ROS can be reversed by experimental interventions that produce reductive shifts in the redox state of glutathione, or promote recovery of bioenergetic functions of specific oxidized proteins. The Aim 4 studies will also address whether or not the ability of interventions to reverse age-related impairments is age-dependent. The results of these studies should refute or validate the idea that oxidative damage to specific mitochondrial proteins is a factor in age-related decrements in cognitive or psychomotor performance, and may identify specific molecules that should be targeted by anti-aging interventions. These studies will also address the hypothesis that increases in unregulated ROS directly impair cognition during aging and promote relatively non-reversible derangement of underlying signaling processes. Finally, these studies will identify periods of senescence for which antioxidative treatment of cognitive decline is most likely to be successful.

PROJECT SUMMARY (See instructions): The over-arching aim of Core B is to provide to each Project, access to tissue and results relevant to the functional status of ovariectomized rats maintained in two experiments conducted in a standardized setting. In phases I and 11, five treatment regimens [vehicle, estrogen, diarylpropionitrile (DPN), progesterone, or estrogen + progesterone], implemented after 3 different post-ovariectomy delays, will be evaluated for their ability to reverse the functional deficits associated with OVX in young adult (4-month old) and after one delay in reproductive senescent (10 month old) rats. In a subsequent experiment (phase 111), the neuroprotective efficacy of the same treatment regimens will be assessed in the transient middle cerebral artery occlusion (tMCAO) model of stroke. The assessment of functional outcomes at the different post-ovariectomy delays will provide the critical data that allows the individual projects to relate changes in the functions of estrogen receptors, progesterone receptors and intracellular calcium channels, to specific periods of sensitivity or refractoriness to hormone receptor-targeted interventions. Core B will provide standardized implementation of the experimental variables; outcome assessments, environment, and animal husbandry needed for the project experiments, and provide for highly efficient leveraging of resources used by all projects, including staff and animals. To this end. Core B will procure, identify and monitor rats used in the experiments, perform all surgical interventions (ovariectomy, implantation of Silastic pellets and tMCAO), and perform comprehensive analyses of the functional status of the rats in the context of the different treatment regimens. Core B will in addition provide non-behaviorally characterized rats that will have been ovariectomized and exposed to the 5 treatment to Project 2 for synaptoneurosomal fraction preparation, and will maintain availability of neonatal brain tissue for the generation of primary neuronal cultures.

DESCRIPTION (provided by applicant): New and recurrent stroke is the third leading cause of death and the leading cause of long-term disability in the United States, yet no effective endogenous targets have been defined to prevent or attenuate stroke-induced brain injury. We have discovered that mitochondrial dihydrolipoamide dehydrogenase (DLDH) could be a target for chemical preconditioning against stroke injury. The objective of this application is thus to evaluate the neuroprotective efficacy of DLDH chemical preconditioning and delineate its underlying mechanisms. Our preliminary studies show that when rats were fed a 4-week diet supplemented with 5-methoxyindole-2- carboxylic acid (MICA), a specific and competitive DLDH inhibitor, brain infarction volume decreased by c. 60% after transient middle cerebral artery occlusion (tMCAO, 1 hr ischemia and 24 hr reperfusion). This result indicates that chronic DLDH inhibition by MICA affords robust cerebroprotection against stroke. Further studies of MICA-treated rats in the absence of stroke indicate that DLDH activity was lower than in control rats whilst NAD(P)H: quinone oxidoreductase-1 (NQO1) activity increased significantly in the MICA-treated rats. NQO1 is an inducible enzyme and its expression is activated by binding of the nuclear transcription factor E2-related factor 2 (Nrf2) to the antioxidant response element (ARE). Our preliminary studies also indicate that 4 weeks' MICA dietary administration did not affect food intake, body weight gain, blood glucose concentration, or mitochondrial respiratory complexes. These preliminary results support our central hypothesis that dietary inhibition of DLDH induces persistent cerebroprotection, affording enhanced recovery of cognitive and locomotor function after ischemic stroke, via activation of the Nrf2-ARE signaling cascade. The rationale for the proposed investigation is that identifying nontoxic, blood brain barrier-permeable chemical agents that afford brain protection from stroke, and defining the protective mechanisms, will foster development and clinical implementation of such agents to minimize death and disability in human victims of stroke. We plan to test our central hypothesis and, thereby, accomplish the objective of this application by addressing the following three Specific Aims: (1) To define the extent to which Nrf2 nuclear localization and NQO1 expression increase following MICA administration and tMCAO, (2) To measure the extent to which dietary preconditioning can produce persistent neuroprotection from stroke, and (3) To evaluate the effects of MICA diet and experimental stroke on neurobehavioral function and hippocampal synaptic plasticity measured as CA1 long-term potentiation (LTP). It is expected that the successfully completed study will provide novel strategies using DLDH as a target for stroke therapeutics.

Summary With the ?graying of America? we are faced with the need to address the ever increasing number of individuals in our society who have age-associated nervous system diseases and conditions. To address this problem, we need multidisciplinary approaches to facilitate the discovery of the mechanisms, treatments and prevention of these diseases. Active, integrated research-based training of pre-doctoral students is a key to re-supplying the research personnel needed to address these biomedical health care issues in a sustainable manner. Herein we propose to continue a successful pre-doctoral program at the University of North Texas Health Science Center (UNTHSC) that focuses on the neurobiology of aging. During this funding cycle we supported 33 fellows, of whom 22 completed their training and their Ph.D. The trainees were very productive and published 46 peer-reviewed papers to date; we also trained 7 (of 33 = 21.2%) minority students. Importantly, we increased our retention/graduation rate from 84.6% in the prior 5-year funding period to 100% in the most recent funding cycle. The proposed training program will enhance an already strong and successful training program in the neurobiology of aging. The continuation of our predoctoral training program is proposed, as our Institute for Healthy Aging has matured through the addition of faculty and continued funding of program project grants, such as that led by the PI, Dr. Meharvan Singh. Such growth has facilitated more basic, translational and clinical research into the causes, treatment and prevention of brain aging and Alzheimer's Disease at the UNTHSC. Collectively, these new initiatives create a stimulating environment for the training of predoctoral students in the neurobiology of aging. Unique and innovative features of the training program for the next period of funding include: (1) Several experiential learning opportunities to include the Geriatrics Skills Lab and the Translational Aging & Alzheimer's Disease Research Program; (2) our continued emphasis on diversity training, that resulted in greater than 20% minority participation in the training program; 3) Career counseling and interview skill development program, 4) techniques in neuroscience program; 5) our institutional support which will expand those students who will have access to some or all of this training program; 6) our focus on the development of research excellence and leadership among our trainees; and 7) our Mentoring-the-Mentor Program which trains junior faculty in mentoring trainees under the guidance of a senior faculty. Collectively, these attributes of our Neurobiology of Aging Training Program has produced an outstanding program of training that we wish to continue.