Thomas H. McNeill - US grants

The clinical effectiveness of nitroimidazole hypoxic cell radiation sensitizers such as misonidazole (MISO) is reduced by the dose limitation imposed by neurotoxic side effects. Considerable recent effort has been made to develop more efficient or less toxic sensitizers to replace MISO for clinical use. Our studies have concentrated on the development of an animal model which integrates drug distribution, quantitative functional tests and histopathology in order to screen for nitroimidazole neurotoxicity in terms of predictive significance. Our data indicate that the mouse model is useful in giving a quantitative rank order for the neurotoxicity of the sensitizers tested which differ in relative toxicity by a factor of 1.5-2.0. Although differences in mouse strain sensitivity to sensitizers do occur and the route of administration may be important in determining relative neurotoxicity of similar sensitizers, a general finding has been that for sensitizers of equal electron affinity, less lipophilic compounds are less neurotoxic. We have also shown that inhibition of glycolysis is a potential mechanism for sensitizer neurotoxicity. The major general objectives of this renewal proposal are (1) to determine the relative neurotoxicity of several newly synthesized sensitizers (eg. Ro-03-8799, SR 2508, RSU-1087) which are candidates for the replacement of MISO in the clinic and (2) to better define the biochemical mechanism(s) of radiation sensitizer toxicity to normal tissue, with an emphasis on inhibition of glycolysis and the pentose phosphate shunt. We propose to use the techniques developed during out studies on the neurotoxicity of 2- and 5-nitroimidazoles in the mouse in order to select the least neurotoxic newly synthesized sensitizer to replace MISO for clinical use and thereby improved the potential therapeutic ratio. The studies proposed to investigate biochemical mechanism(s) of toxicity of nitroimidazoles will provide data on the cofactor and substrate changes produced by administration of such compounds in neuronal tissue in vivo in order to identify specific enzyme systems related to glycolysis for further investigation and to provide information on potential methods of protection. Overall, these investigations should provide information which will improve our understanding of the neurotoxicity of radiation sensitizers and which will guide our efforts to select the most effective replacement of MISO as an adjuvant agent in the clinical management of human solid tumors with radiation therapy.

These studies will examine the differential effect of aging as a contributing factor to drug-related changes in the morphological and biochemical parameters of neurotransmitter and neuropeptide neurons of the central (CNS) and autonomic nervous system (ANS) associated with anticancer agents. Drugs chosen for these studies are either currently being used in the clinic (WR-2721) or will enter clinical trials within the next year (BSO). An integrated methodological approach encompassing light microscopic immunocytochemistry and histochemistry, radioimmunoassay, radioenzymatic assays and HPLC will be used to identify changes in cholinergic, catecholaminergic and somatostatin systems. Tissues to be examined include the substantia nigra and striatum, locus coeruleus, cholinergic nuclei of the basal forebrain, superior cervical ganglia and adrenal gland. We will address two specific questions: first, does buthionine sulfoxine (BSO), an inhibitor of glutathione synthesis or the thiol-containing radiation protector, WR-2721 alter neurotransmitter or neuropeptide systems of the CNS or ANS; second, does advancing age provide a confounding variable in determining either acute or late-effect therapy- related drug toxicity in young (3, 6 months) and old (10, 20, 25, 30 months) mice? Our approach will be twofold. First, we will determine acute effects by comparing drug-related morphological and biochemical changes in neurotransmitter or neuropeptide neurons in young animals with similar data obtained in old mice. Second, we will determine late-effect toxicities by comparing morphological and biochemical changes in middle and old aged mice which have received treatments at earlier ages with age- matched, untreated controls. Data from these investigations will provide information fundamental to our understanding of the underlying mechanisms of the neurotoxicity of anticancer agents and will increase our awareness of potential age-related hazards associated with cancer therapy in man.

A major research goal of our laboratory is to understand how age-related changes in the striatal motor system may contribute to the on-set and progression of human neurodegenerative disorders, such as Parkinson's (PD) and Huntington's (HD) diseases. In particular, studies conducted in our laboratory over the last several years have used the C57BL/6N mouse as an animal model to define the temporal sequence of age-related morphological changes that occur in two areas of the brain critically involved in PD and HD, ie. the striatum (ST) and substantia nigra (SN). However, while these studies have provided solid examples of how age-related morphological changes in the brain are both brain region- and cell type-specific it is currently unclear how closely our findings in mice parallel normal aging in the SN and ST of man since detailed comparative studies between rodent and postmortem human brain are lacking. In addition, the issue of age-related DA cell loss is of particular importance in man, because the dogma that aging is associated with a progressive loss of DA neurons in the SN of man has been entrenched in the literature since the data was published in 1977 (78) and remains the single factor that ties together all the current theories regarding the etiopathology of PD (reviewed in 29,70). However, the data upon which this dogma is based is uncertain at best (3,44,76,78,91) and recent findings from our own laboratory as well as those of others suggest that DA cell loss is not characteristic of normal aging in either mice, non-human primates or man (see preliminary data). As part of the studies proposed we will test the hypothesis that aging is not associated with a progressive decline in DA neurons of the SN and compare our findings with our previous data obtained from mice and non-human primates. If our hypothesis is proven to be correct the data obtained will form the basis for reexamining all of the basic theories regarding the etiopathology of PD as well-as studies which examine the cellular basis for the death of DA neurons. In addition, data from our studies will provide information which is fundamental to establishing the extent to which rodents and non-human primates may serve as useful animal models of normal aging in man and further our understanding of how age-related changes in the SN and ST may contribute to the onset and progression of human neurodegenerative diseases, such as PD.

A basic knowledge of how we age is essential for our understanding of age-related impairments in brain function that lead to major personal and economic problems for older citizens. In addition, changes in the brain considered part of normal aging may contribute to the onset and progression of age-related neurodegenerative diseases of the central nervous system (CNS), the classic examples of which are Alzheimer's, Huntington's and Parkinson's diseases. However, while many of the neurodegenerative diseases of the CNS have a characteristic age of onset after midlife, it is unclear what role age-related changes in the morphological, biochemical and electrophysiological properties of CNS neurons play in the onset and progression of a disease process. The proposed program project targets this gap in our knowledge of the aging brain by testing hypotheses about mechanisms of age-related changes in brain function. We propose an integrated program of basic research to examine the cellular and molecular mechanisms involved in brain aging, with particular emphasis on the substantia nigra and striatum, structures especially vulnerable in Parkinson's and Huntington's diseases. The study of reactive synaptogenesis, neuron death and functional adaptability to aging and injury represent the main areas of focus on our renewal application. Furthermore, we propose to manipulate age-related changes in dopaminergic function using chronic dietary restriction or treatment with the dopamine agonist pergolide. These studies will investigate the cellular and molecular events associated with oxidative stress in aging, with an emphasis on GFAP expression and other glial responses that we have documented in response to neurodegeneration. In addition, we will analyze the expression of several recently discovered genes, including apoJ, SCG-10, BDNF, GDNF, which seem to play key roles in processes determining cell survival and plasticity. The proposed experiments will help to understand the cellular and molecular mechanisms that lead to cell death in some regions of the aged brain and will provide the basis for the development of future therapeutic strategies aimed at the treatment of age-related neurodegenerative diseases of the CNS.

16. Caloric Restriction and Aging: This study reflects a new area of research for the PI and will focus on the fundamental question of how changes in neurochemical pathways may act as possible mediators of the caloric restriction effect on brain aging. Specifically, we want to determine whether alteration of the cellular redox state of neurotransmitter neurons, that are known to modulate neurite outgrowth and synapse replacement after brain injury, may mediate the caloric restriction effect on neural plasticity in the aged brain. Dopamine neurons are the focus of our investigation because 1) synapse replacement in the striatum after a corticostriatal lesion is modulated by mesostriatal dopamine cells, and 2) dopamine neurons are particularly vulnerable to oxidative insult with age due to the generation of toxic free radicals during dopamine metabolism and the formation of dopamine quinones that can modify cellular proteins and damage dopamine neurons. Specifically, we will test the hypothesis that Iowerinq cellular levels of the antioxidant .qlutathione (GSH), as a strategy to alter the cellular redox state of mesostriatal dopamine neurons, leads to an increase in dopamine oxidation and the formation of protein cysteinyl-catechols that can damaqe dopamine neurons and disrupt dopamine modulation of neural plasticity after brain iniury. We will test our hypothesis through a combination of in vitro and in vivo experimental models that selectively express dox-inducible reductions in the level of GSH in dopamine neurons. Aim 1 will use PC12 cells with reduced levels of GSH to assess the effect of lowering cellular GSH in dopamine neurons on the regulation of dopamine autoxidation and the formation of protein cysteinyl-catechols after exposure to an elevation in the steady-state concentration of nitric oxide, a condition typically seen after brain injury. Aim 2 will characterize changes in the temporal schedule of the upregulation of candidate molecules known to participate in the regulation of neurite outgrowth and synaptogenesis (i.e., pCREB, BDNF, GAP-43, SCG-10) in the striatum after a unilateral corticostriatal lesion in transqenic mice with reduced levels of GSH in dopaminergic mesostriatal neurons. Data from these pilot studies will provide the basis for additional mechanistic studies that will identify which particular proteins undergo damage or lose their functional capacity under conditions of oxidative stress using mass spectrometry, as well as, future in vivo studies to determine if age-related protein modifications in dopamine neurons are prevented by caloric restriction.

stroke therapy; neural plasticity; rehabilitation; medical rehabilitation related tag; clinical research;

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Stroke is the leading cause of serious, long-term disability among American adults and places a tremendous burden on both the private 'and public health resources of the nation. Of all impairments that result from stroke, one of the most in need of effective rehabilitation studies is hemiparesis of the upper limb, which significantly impacts functional independence and health of stroke survivors. One approach that has shown promise in rehabilitation of upper limb disabilities is Constraint Induced Movement Therapy (CIMT), which emphasizes repetitive use of the impaired limb using task-specific training, while restricting movement of the better limb. However, the "best practice" strategy for the rehabilitation of upper limb paresis using CIMT is still unclear, and little is known about how critical factors such the focus of therapy (skilled learning vs. motor activity), the intensity and timing of therapy, patient motivation, initial impairment and the neural mechanisms that underlie the recovery process interact to impact the effectiveness of rehabilitation therapy. [unreadable] [unreadable] In order to make significant advances in the field of stroke rehabilitation we believe that a concerted interdisciplinary approach among the biological, engineering, computer, and clinical sciences will be needed to solve this complex problem. The studies proposed in this planning grant are designed to meet this challenge and will examine the synergy between neural plasticity and treatment strategies that promote the recovery of upper limb motor function after stroke-induced brain injury. Participants include faculty from the biological, behavioral, computational and engineering sciences with expertise in methods encompassing molecular and cell biology, behavioral neuroscience, bioinformatics, computational modeling, virtual environment technology, haptics, biostatistics and physical rehabilitation. The long-term goals of our study are: 1) to broaden our understanding of the key factors that modulate neuroplasticity and the recovery of function after brain injury, 2) build a foundation of interdisciplinary scientific knowledge that can be used in the development of innovative and more effective therapeutic interventions to enhance the health and independence of persons with post-stroke disabilities; and 3) provide an interdisciplinary training opportunity for (basic science and clinical) graduate and post-doctoral students to develop as independent research scientists equipped to work both within and across scientific disciplines. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The studies proposed use a multidisciplinary approach (i.e. anatomical, pharmacological, molecular and behavioral) to further our understanding of the cellular events that underlie neural plasticity in the CMS by investigating the roles that dopamine and BDNF play in the modulation of axonal sprouting and synapse replacement in the striatum. The rationale for the study is based on previous reports from our laboratory, as well as those of others, showing that: 1) axon terminals from contralateral corticostriatal neurons reinnervate denervated synaptic sites in the striatum after a unilateral cortex lesion; 2) synapse replacement involves the differential regulation of growth associated proteins and neurotrophins that are lesion-specific; and 3) depletion of striatal dopamine in combination with a unilateral cortex lesion results in an aberrant pattern of neurotrophin and growth associated protein gene expression and synapse replacement in the striatum that slows the recovery of motor function. The overarching hypotheses to be tested are: 1) that dopamine acts through specific subtypes of dopamine receptors to up-regulate candidate molecules known to participate in the regulation of neurite outgrowth and synaptogenesis after brain injury, and 2) that a key mediator in the compensatory response to a unilateral cortex lesion is the induction of BDNF in contralateral corticostriatal neurons. We will use the rat unilateral cortex lesion model and selective dopamine agonist and antagonist drug treatments to define the roles that specific subtypes of dopamine receptors play in the regulation of reactive synaptogenesis after brain injury. We will use intra-striatal infusions of the neurotrophin antagonist trkB-IgG to test the hypothesis that neurite outgrowth and synapse formation after the cortex lesion is mediated through BDNF and the trkB receptor. We will assess lesion- and treatment effect on: 1) upregulation of candidate molecules known to participate in the regulation of neurite outgrowth, synaptogenesis and cell survival after brain injury; and 2) synapse loss and replacement. Molecular and anatomical changes will be correlated with the recovery of sensorimotor function, as measured by the rotorod, forelimb-use asymmetry and forelimb placing tests. Data from our studies are fundamental for broadening our understanding of the capacity of the brain to compensate for injury, and central to the development of new treatment strategies that can translate the basic principles of neuroplasticity into effective clinical interventions. [unreadable] [unreadable]