Charles Mactutus, Ph.D. - US grants

The recent establishment of a technically simple, economical, and practical techniques for chronic IV studies in which a vascular port is implanted prior to mating, offers a model for pregnant rats which reasonably mimics the rapidly peaking pharmacokinetics profile of cocaine abused via inhalation or IV administration. The proposed program specifically addresses the major question: Is maternal cocaine abuse during pregnancy a neurobehavioral teratogen when administered via a clinically-relevant route? The specific aims of the program are: 1) To determine the biodistribution of cocaine in the pregnant dam/fetus following intravenous administration. Using an IV vascular access port in unanesthetized pregnant rats we will determine both plasma and brain levels in dam and fetus of cocaine and its major metabolite, benzoylecgonine. Both time-response and dose-response functions will be determined. These studies are of fundamental importance in establishing the magnitude and duration of fetal cocaine exposure following maternal IV administration. 2) To establish the nature and extent of neurobehavioral alterations which occur in the offspring consequent to intravenous maternal cocaine exposure during pregnancy. Using unanesthetized IV-catheterized pregnant rats we will conduct ontogenetic and longitudinal analyses of the neurobehavioral effects of maternal cocaine exposure on the offspring with the inclusion of nutritional controls and surrogate fostering. These studies will identify dose-response functions and critical exposure periods. The dissociation of specific cognitive deficits from sensory or motor impairments will also be provided by the use of both multiple tasks and multiple dependent measures within each task. 3) To establish the nature and extent of the structural and functional alterations in the central noradrenergic system which occur following intravenous maternal cocaine exposure during pregnancy. Quantitative neuroanatomical measurements, quantitative protein analysis (Western blotting), synthetic enzyme activity, and in situ hybridization will be utilized to assess the structural and functional integrity of the central noradrenergic system. As a putative target of maternally administered cocaine, we hypothesize that alterations in the central noradrenergic system may be the neurobiological mechanism underlying the persistent and selective alterations observed in behavior, as particularly evident in "attentionally sensitive" neurobehavioral paradigms. The goals of the proposed program are to program are to provide a characterization of the neurobehavioral effects of maternal cocaine abuse during pregnancy, in an animal model based upon the clinically-relevant IV route of administration, and to characterize a neurobiological mechanism potentially underlying the persistent and selective neurobehavioral alterations.

Active maternal smoking during pregnancy is associated with adverse health effects for the infant at birth and throughout childhood. Only recently, however, has it been appreciated that involuntary passive cigarette smoke exposure of the pregnant woman (via household or work environmental exposures) may also be associated with adverse effects on fetal health and development. The present proposal is unique in representing the first programmatic attempt, in an animal model, to investigate the long-term consequences of passive maternal smoking during pregnancy on the conceptus. Using reference cigarettes with established smoke constituents to create clinically relevant conditions of sidestream smoke, as well as biological monitoring of smoke exposures, this proposal offers the following hypothesis: Passive maternal smoking during pregnancy has adverse effects on the neurobehavioral development of the fetus (as manifest in cognitive dysfunction) and these effects are caused by structural and functional disruption of the hippocampus. The specific aims of the program are: 1) To establish the nature and extent of cognitive dysfunction which occurs in the offspring following passive maternal cigarette smoking during pregnancy. Cognitive processes in the rodent will be evaluated via complex spatial and non-spatial tasks known to be sensitive indicators of hippocampal pathology. These studies will identify dose-response functions, critical exposure periods, and dissociations of specific cognitive deficits from sensory or motor impairments. 2) To establish the nature and extent of the structural alterations in the hippocampus which occur following passive maternal smoking during pregnancy. Quantitative neuroanatomical measurements, synthetic enzyme activity, and receptor autoradiography will be utilized to determine the structural integrity of the hippocampus. The hippocampus mediates spatial information processing in rodents and is also known to be highly susceptible to irreversible cytotoxic damage by moderate hypoxic episodes of varied origin. 3) To determine the relationship between the integrity of the hippocampus and the cognitive dysfunction using pharmacological probes. The functional integrity of two extrinsic neural pathways that project to the hippocampus (cholinergic & noradrenergic) and one intrinsic transmitter system (GABAergic) will be probed using selected pharmacological agonists and antagonists with the aim of predictively modifying the rats' behavior in spatial learning tasks. The goals of the current proposal are to quantitatively define the relationship between passive maternal smoke exposure during pregnancy and cognitive development of the offspring, and to identify characterize the neurobiological mechanism(s) underlying this cognitive deficit.

The data generated in the initial project period provide convergence across a variety of disciplines that indicate prenatal cocaine is a neurobehavioral teratogen when administered by a clinically relevant route (IV) and at clinically relevant peak arterial plasma levels, and moreover, that the central noradrenergic system is a very important target for these effects. Thus, we can now refine our original hypothesis to propose a mechanism for the effects of prenatal cocaine. The proposed program continuation maintains the hypothesis: Maternal cocaine abuse during a restricted portion of pregnancy causes Long-term and selective alterations in 1) "attentionally sensitive" neurobehavioral paradigms and 2) the structure and function of the central noradrenergic system: both of which are attributable to early noradrenergic cell dysfunction/loss in the locus coeruleus. The specific aims of the program are: 1) To establish the critical exposure period for the neurobehavioral alterations which occur in the offspring consequent to intravenous maternal cocaine exposure during pregnancy. Using ontogenetic and longitudinal analyses, the proposed studies will replicate and extend our prior studies by specifically identifying the critical exposure period(s). We will use noradrenergically mediated and/or attentionally sensitive tasks to provide an assessment of both ascending and descending noradrenergic projections of the locus coeruleus. Multiple dependent measures within each task will provide the dissociation of specific neural and cognitive deficits from sensory or motor impairments. 2) To establish the critical exposure period for the structural and functional alterations in the central noradrenergic system alterations which occur in the offspring consequent to IV maternal cocaine exposure during pregnancy. Quantitative neuroanatomical measurements (unbiased cell-counting/optical dissector technique), immunocytochemistry, synthetic enzyme activity, and in situ hybridization will be utilized to fundamentally establish whether the observed alterations in the central noradrenergic system, presumably reflecting compensatory processes in the developing CNS, occur in response to noradrenergic cell loss or, alternatively, to a less permanent cellular dysfunction. The goal of the proposed program is to determine whether the early cell dysfunction/loss in the locus coeruleus may provide a potential underlying basis for the long-term and selective alterations in attentionally sensitive neurobehavioral paradigms consequent to prenatal cocaine exposure.

[unreadable] DESCRIPTION (provided by applicant): Vertical transmission (Mother to child) is the primary mode of HIV-1 infection among young children worldwide, resulting in at least 1700 new infections each day or more than one pediatric infection every minute (UNAIDS/WHO, 1999; 2001). Viral products released by HIV-infected cells cause widespread metabolic derangement as evident in neuronal dysfunction, disruption of neuro-glial relationships, and immune dysregulation, all of which ultimately cause cerebral dysfunction and precipitate the development of HIV dementia. It has been well established that specific proteins encoded by the HIV genome, e.g., Tat and gp120, 1) are neurotoxic, 2) are elevated in brain tissue of patients with HIV dementia, and 3) when presented to the brain in vivo, both proteins cause histological changes that are consistent with those seen in patients with HIV dementia. These data suggest that clinically relevant studies of HIV neuropathogenesis may now be performed in a rodent model. One notable advantage of directly studying the virotoxins is that neuropathogenesis may be studied independent of the virus per se and the complications of secondary infections. The proposed program specifically addresses the major question: What are the neurodevelopmental effects of the HIV proteins Tat and gp120 and how are these effects mediated? Our hypothesis is that: Neonatal exposure to the HIV proteins, Tat and gp120, will produce specific neurodevelopmental disruptions in cognitive processes and that these disruptions will be evident in central catecholamine systems and attributable to interference of key developmental processes. Using a virotoxin exposure model (HIV proteins Tat/gp120) our programmatic studies will determine the adverse effects of these proteins on the developing central nervous system of perinatal animals. Several very important pieces of information will be provided. First, we will identify dose-response functions for the adverse neurobehavioral and neuroanatomical effects of perinatal HIV protein neurotoxicity. Second, we will identify the potential critical periods responsible for the adverse neurobehavioral and neuroanatomical effects of perinatal HIV protein neurotoxicity. Third, these deleterious effects will be identified independent from those of secondary infections, polydrug and/or nutritional interactions that characterize the clinical HIV/AIDS populations. Thus, this project will: A) contribute to our understanding of Tat/gp120 in a clinically relevant rodent model, B) further provide a comprehensive examination of potential catecholamine targets responsible for early developmental disorders independent of secondary opportunistic infections, and C) provide the foundation for an examination of these very same potential mechanisms for HIV protein neurotoxicity in humans with the use of fetal autopsy tissue. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The additive effects of HIV infection and methamphetamine (METH) abuse on cognitive function represent a serious medical problem. Experimental evidence indicates that neurotoxic viral proteins, particularly HIV-1 Tat and gp120, can cooperate with METH and promote neurodegeneration. However, mechanisms of such cooperation are elusive. Our prior research and preliminary studies indicate that the molecular mechanism of combined METH/Tat and/or METH/gp120 toxicity may involve concurrent adverse effects of these neurotoxins on key components of dopaminergic and glutamatergic transmission systems. This application will test the hypothesis that pathways of direct METH and HIV-1 protein-mediated neurotoxicity congregate to disrupt normal physical interactions between D1 and NMDA receptors. Thus, neurons, which co- express NMDA receptor complexes (NMDAR) and dopamine (DA) D1 receptors may be selectively sensitive to the injury concurrently incited by individually non-toxic doses of METH and HIV-1 proteins. This hypothesis will be tested in vitro using the primary rodent and human neural cell culture models of Tat/gp120/METH neurotoxicity. Faltered functional D1R/NMDAR interactions, which are often referred as the engine of cognition, may critically influence the development of persistent memory deficits in HIV-positive methamphetamine abusers. The broad goal of our studies is to elucidate the molecular base of additive deleterious cognitive effects of METH and HIV infection. Results of the project will have an impact on the preclinical research of effective protective strategies to improve the health and well-being of METH-dependent individuals living with HIV/AIDS. PUBLIC HEALTH RELEVANCE: The additive effects of HIV infection and methamphetamine (METH) abuse on cognitive function represent a serious medical problem. The present research proposal will investigate the role of D1R/NMDAR interactions in the overlapping pathway of METH/HIV-1 protein neurotoxicity. The broad goal of the research is to elucidate the molecular basis of METH/HIV-induced cognitive deficits.

DESCRIPTION (provided by applicant): Although combination antiretroviral therapy (CART) has been highly successful in reducing mother-to-child- transmission (MTCT) in the U.S., globally, MTCT is presently responsible for >1000 new HIV-1 infections each day or more than one new pediatric infection every two minutes. The dopamine (DA) system is a clinically relevant target as evidenced by recent imaging, neurocognitive, and post-mortem examinations of the HIV-1 infected patients. Using a prospective longitudinal design, in this competing renewal we will explore the hypothesis that the development and progression of neurocognitive dysfunction associated with HIV-1, is consequent of, and attributable to, pathology of the DA system, a system highly sensitive to inflammatory processes. Longitudinal studies, while challenging in non-rodent species, are critical for systematically addressing pediatric HIV-1/AIDS, and are also fundamental to our understanding of chronic HIV-1 associated neurological disorders (HAND). The specific aims are: 1) To determine the development and progression of neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV-1 protein exposure. Developmental milestones and an array of tasks directed at the behaviors expressing the prominent components of neurocognitive dysfunction that have been clinically identified will be assessed during the rodent preweaning period, adolescence, adulthood and middle age. 2) To determine the long-term alterations in the major DA receptor subtypes and DA transporter as a candidate neurochemical mechanism for the neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV- 1 protein exposure. Quantitative autoradiography will be used to assess the expression of DA receptors (D1, D2, and D3) and DA transporters (DAT) in the nigrostriatal and mesocorticolimbic pathways of the DA system during adolescence, adulthood and middle age. 3) To determine the integrity of DAT function in the development and progression of neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV-1 protein exposure. DAT function will be studied in a transgenic rat model of chronic HIV-1 protein exposure using in vivo NNF/microdialysis. 4) To determine whether the neurocognitive dysfunction consequent to chronic, low-level inflammation and HIV-1 protein exposure may be treated with currently used agonists or antagonists, or novel agents, targeted to the identified DA system dysfunction. These experiments will establish the functional role of the neurobiological changes in the DA receptors and DAT as a neurochemical mechanism contributing to, if not mediating, the neurocognitive impairments consequent to chronic expression of the HIV-1 transgene. The program goal is to advance the field with a translational model of the core components of cognition relevant to pediatric HIV-1/AIDS as well as to HAND, and more importantly, to identify (normal rats) and validate (the better characterized HIV-1 transgenic rat) novel neurotherapeutics to tune the cognition domains afflicted by HIV-1.

? DESCRIPTION (provided by applicant): Despite the widely acknowledged success of combination antiretroviral therapy (cART) in the incidence of HIV- 1 associated dementia (HAD), HIV-1-associated neurocognitive disorders (HAND) continue to afflict up to 50% of patients on cART. Neurological complications of HIV infection are the biggest challenge facing HIV researchers, and there is a critical need to develop treatment approaches for HAND. Using the HIV-1 transgenic (Tg) rat model in a prospective longitudinal design, we will explore the hypothesis that the progression of neurocognitive dysfunctions associated with HIV-1 are consequent to central nervous system (CNS) synaptodendritic pathology, neuroinflammation and oxidative stress, and that the progressive neurocognitive losses will be slowed or halted with a restorative therapeutic approach administered early in the course of transgene expression. The specific aims are: 1) To establish the progression of neurocognitive decline in the HIV-1 Tg rat on measures of attention and executive function; as distinct from potential changes in sensory, activity, and motivational confounds. The progression of neurocognitive dysfunction will be assessed with periodic tests from pre-adolescence through advanced age, given the exponential growth of 65+ year old HIV+ patients. Both males and females will be examined. 2) To establish the progression of CNS synaptodendritic pathology, neuroinflammation and oxidative stress, in the HIV-1 Tg rat. Investigations will focus on quantifying dendritic branching and spine parameter alterations with respect to the prefrontal cortex and medium spiny neurons of the nucleus accumbens. 3) We will protect (and restore) neurocognitive function and synaptodendritic complexity with S-equol, a metabolite produced via the gut microbiome following ingestion of soy isoflavone daidzein. Having established and replicated proof- of-concept (preliminary studies), we will assess the in vivo efficacy of S-equol to delay the progression to neurocognitive decline; synaptodendritic complexity, neuroinflammation and oxidative stress will be assessed in different time-sequential cohorts as the basis of the preserved neurocognitive function. Initially we will establish the optimal 2-month therapeutic window. Subsequently, we will establish the functional role of alterations in synaptodendritic complexity, neuroinflammation and oxidative stress as neurobiological mechanisms contributing to, if not mediating, the neurocognitive impairments consequent to chronic expression of the HIV-1 transgene. The program goal is to advance the field by establishing 1) the progression of neurocognitive decline in a translationally relevant model of HAND, 2) the efficacy of a regenerative approach targeted at synaptodendritic complexity in protecting (slowing or halting) the neurocognitive decline in the core cognitive components of attention and executive function, and 3) proof-of- principle for the microbiota-gut-brain axis as an innovative therapeutic approach for HAND.

Project Summary: In this competing renewal application, we will critically test a gene excision cure approach followed by neurorestoration therapy as a strategy to remove the HIV-1 provirus from the brain and promote neurocognitive recovery. Furthermore, efficacy of cure/restoration may be tailored for both males and females, as well as for those with a history of cocaine abuse. The hypothesis is that gene excision and estrogen/soy isoflavone therapy will restore neurocognitive function and synaptic complexity from the progressive decline characteristic of HIV-1 associated neurocognitive disorders (HAND). Thus, estrogenic compounds may serve as neurorestorative agents for the combined effects of cocaine/HIV proteins on producing synaptic impairments in the nucleus accumbens and prefrontal cortex, critical brain regions involved in the cognitive dysfunction associated with HAND. The specific aims are: 1) To establish efficacious neurorestoration of HIV-1/ cocaine-induced synaptopathy. The proposed in vitro experiments will establish neurorestoration at the synaptic level following both provirus activation and HIV-1 gene excision strategies. Novel compounds will be examined for neurorestoration of HIV-1 protein induced synaptopathy following cure strategies, with and without cocaine co-exposures, in sex-specified cell cultures. 2) To establish recovery from executive function deficits in HIV-1 transgenic animals, and the role of biological sex, with gene excision/ neurorestorative therapies. The ability to restore the neurocognitive/pathological consequences of HIV-1 proteins will be determined using assessment of executive function and synaptic plasticity/dendritic spines. Investigations will focus on quantifying spine density changes with respect to regulators of nucleus accumbens medium spiny neurons and prefrontal cortical neuronal plasticity. 3) To establish neurocognitive recovery in HIV-1 Tg animals, with deficits exacerbated by a history of cocaine abuse, with gene excision/ neurorestoration therapies. A critical in vivo test of gene excision/neurorestoration therapies will be provided with HIV-1 Tg animals that have an acquired history of cocaine self-administration; cocaine may accelerate the progression of HAND in HIV-1 infected patients by disrupting frontal cortex-nucleus accumbens circuitry. The robustness and generality of neurorestoration will subsequently be tested with self-administration of other abused substances (e.g. methamphetamine and methylphenidate). We are in a unique position to establish the trajectory of neurocognitive decline and synaptodendritic loss consequent to chronic HIV-1 protein/provirus expression and critically test a gene excision/regenerative-based therapeutic approach to protect and/or restore synaptodendritic complexity and neurocognitive function.

Project Summary: Apathy is a common motivational alteration in HIV-1+ individuals, affecting between 30-60% of the population, despite antiretroviral therapy. The high prevalence of apathy in HIV-1+ individuals is one of the biggest challenges facing HIV clinicians and researchers alike; there is a critical need to develop treatment approaches for HAND. Using the HIV-1 transgenic (Tg) rat model in a prospective longitudinal design, we will test the hypothesis that the development and progression of dysregulation of motivational processes associated with HIV-1 are consequent to synaptodendritic pathology, neuroinflammation and oxidative stress of the dopamine (DA) system in the prefrontal cortex-basal ganglia axis, and that the trajectory of motivational dysregulation will be altered with a therapeutic approach targeted to the mesocorticolimbic DA system. The specific aims are: 1) To establish the development and progression of motivational dysregulation in the HIV-1 Tg rat as measured with drug, food, and natural reinforcers, as distinct from potential changes in sensory and activity confounds. The progression of neurocognitive dysfunction will be assessed with periodic tests from pre-adolescence through advanced age, given the exponential growth of 65+ year old HIV+ patients. Both males and females will be examined. 2) To establish the progression of CNS synaptodendritic pathology in the prefrontal cortex (PFC) and medium spiny neurons (MSN) of the nucleus accumbens of the HIV-1 Tg rat with a focus on quantifying dendritic branching and spine parameter alterations. Specifically, synaptodendritic complexity, neuroinflammation, and oxidative stress will be assessed in different cohorts as the basis of the dysregulation of motivational processes. 3) To establish the treatment of the dysregulation of motivational processes consequent to chronic, low-level inflammation and HIV-1 protein exposure with currently used agonists or antagonists, or novel agents, targeted to the identified DA system dysfunction. With currently used agonists or antagonists, or novel agents, targeted to the identified DA system dysfunction we will mechanistically test the functional role of the neurobiological changes in the DA receptors and dopamine transporter (DAT) as a neurochemical mechanism contributing to, if not mediating, the motivational dysregulation consequent to chronic expression of the HIV-1 transgene. Initially we will establish the optimal therapeutic window. These studies will establish the functional role of alterations in synapto- dendritic complexity, neuroinflammation and oxidative stress as neurobiological mechanisms contributing to, if not mediating, the dysregulation of motivational systems consequent to chronic expression of the HIV-1 transgene. The program goal of our longitudinal studies is to establish our ability to predict motivational dysregulation as a function of chronic expression of the HIV-1 transgene, to provide a greater mechanistic understanding of the interrelationships of HIV-1-induced apathy and DA system dysfunction, and to improve the therapeutic options relevant to HIV-1+ individuals and improve their quality of life.