Laura L. Dugan - US grants

Free radicals have long been postulated to contribute to neuronal injury in acute central nervous system injury, including stroke and trauma, and in the more chronic neurodegenerative diseases, including Parkinsons disease, multi-infarct dementia, and Alzheimers disease. These conditions are fast becoming the most pressing health care issues for the coming decades. Globally, 15% of the population over age 65 is reported as having some form of dementia, and current estimates in the U.S. place the number of affected individuals at well over 4 million, a number which may double or triple in the next 20 years (NIA Special Report, 1991; Odenheimer, 1989). In addition, cerebral infarction (stroke) is a leading cause of neurologic disability in the elderly. Yet, to date, there is no effective therapy to prevent or treat a majority of these neurologic disease states. The goal of the proposed project is to characterize free radical processes initiated by glutamate receptor overactivation (excitotoxicity), and to assess the contribution of free radicals to the death of neurons excitotoxicity. Glutamate is the major excitatory neurotransmitter in the brain, and is felt to play a major role in learning and memory. However, both free radical injury and glutamate receptor-mediated excitotoxicity are believed to contribute to the death of neurons in acute and chronic neurologic diseases, including stroke and head trauma, Parkinsons Disease, Huntingtons Disease, and possibly Alzheimers dementia. Production of free radicals, in particular hydroxyl radical, may be one key mechanism by which excitotoxicity results in irreversible neuronal damage. The hypothesis we will test is whether glutamate receptor activation can initiate hydroxyl radical production in neurons, and whether hydroxyl radical in turn is a component of glutamate neurotoxicity. This hypothesis will be tested using state-of-the-art methods for detecting free radicals, such as Electron Spin Resonance and High Performance Liquid Chromatography, to measure hydroxyl radical production during excitotoxicity in mouse brain cell cultures, together with pharmacologic and molecular biology approaches to altering free radical production and clearance. Clarifying the sources of free radicals and the events that trigger their production may assist in the development of rational treatment in neurologic diseases, including stroke and many forms of neurodegenerative diseases.

Recent experimental evidence supports a role for AMPA/KA-type glutamate receptors in mediating ischemic neuronal death, such as occurs in stroke. Free radicals have also been implicated in the injury process that leads to ischemic cell death. The main objective of this proposal is to determine the involvement of free radical formation in AMPA/KA receptor- mediated neuronal death in oxygen-glucose deprivation in vitro and ischemic brain injury in vivo. The central hypothesis to be tested is that generation of oxygen free radicals contributes to AMPA/KA receptor- mediated neuronal death in oxygen-glucose deprivation in vitro and ischemia/reperfusion in vivo. Three specific aims and related research plans are directed at testing this hypothesis. The involvement of free radicals in AMPA/KA receptor-mediated cell death will be assessed in oxygen-glucose deprivation in vitro using mouse brain cortical cultures, and in two in vivo ischemia models (rat global ischemia and focal ischemia). In most experiments, NMDA receptors will be blocked to 'unmask' the contribution of AMPA/KA receptors. Production of free radicals will be measured using state-of-the-art methods for detecting free radicals, such as Electron Spin Resonance (ESR), and salicylate trapping/HPLC for detection of hydroxyl radical. Trapping agents (spin- traps for ESR or salicylate) will be delivered to the cells by microdialysis (in vivo) or by direct application to cells (in vitro). Protection by anti-oxidants/radical scavengers against the AMPA/KA receptor-mediated component of oxygen-glucose deprivation injury in vitro and ischemic injury in vivo will also be examined. Clarifying the sources of free radicals and the events that trigger their production may assist in the development of improved therapy for CNS ischemia.

Water soluble derivatives of C60 have been found to act as broadly effective neuroprotective agents in neuronal cell culture models. These compounds also delayed both motor deterioration and death in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). Investigation of these malonic acid C60 derivatives continues. Mass spectrometry provides a means of characterizing these compounds and determining the structure of oxidation products of these derivatives.

DESCRIPTION There is substantial evidence that oxidative injury plays a role in many types of cytotoxic insults, and that the role of free radical damage may be especially prominent in neurological disease states, such as stroke, trauma and neurodegenerative disorders such as Parkinson's disease. Recently, the applicant reported that water-soluble derivatives of C60 were highly effective neuroprotective agents capable of rescuing cortical neurons from a broad range of insults, including excitotoxicity induced by NMDA or AMPA, apoptosis produced by growth factor withdrawal or application of A-Beta (1-42), and neuronal death following oxygen-glucose deprivation. In addition, intraperitoneal administration of these compounds to a mouse model of familial ALS delayed both the onset of motor deterioration and death. Electron paramagnetic resonance spectroscopic studies confirmed that these water-soluble malonic acid derivatives of C60 retain the potent free radical scavenging capabilities of native C60 with the ability to eliminate both hydroxyl and superoxide radicals. Based on these observations, the applicants believe that neuroprotection provided by these antioxidants reflects their ability to scavenge not only hydroxyl radical but superoxide radical (O2-) at concentrations in the micromolar range. This application proposes to test the specific hypothesis that superoxide radical scavenging by C60 derivatives is a critical determinant of neuroprotective efficacy by C60 derivatives. They plan to study mechanisms of neuroprotection by generating C60 derivatives substituted with similar functional groups, but possessing differing degrees of O2- scavenging ability. The rank-order potency of derivatives will be compared for both neuroprotection and superoxide scavenging to see if these properties correlate. In addition, using Sod2mlucsf mice, which have a specific impairment in the ability to eliminate mitochondrial O2-, they will test the protective efficacy of these agents on neurons cultured from these mice, and in neonatal mice. The broader goals of this proposal are: 1) to develop these molecules as tools to study the role of O2- as both signaling molecule and neurotoxin, 2) to continue exploration of the contribution of mitochondrial O2- to neuronal cell death, 3) and to begin to define structure-function relationships for these promising compounds as a necessary step towards pre-clinical trials.

Carboxyfullerene synthesis involves two reactions addition of esters of malonic acid to give mixtures of adducts which are separated by chromatography and (2) conversion of esters to acids. We have prepared esters using dimethyl, diethyl and di-t-butyl malonates and confirmed that the product distribution is highly dependent on the size of the R group, yield of dimethyl C3> diethyl C3 >> di-t-butyl C3. The mass spectra (FAB+) confirmed the number of additions but the spectra were nearly identical for all isomers. Conversion of C3 esters (methyl or ethyl) to acid gave mixtures of acids which all retained the C3 symmetry. The three major components were identified as hexa acid and two isomeric penta acids. Penta esters were found in the unhydrolyzed portion of an incomplete reaction mixture, suggesting that the carboxyl was lost prior to hydrolysis. A variety of mass spectrometric techniques are used to characterize products and byproducts.

DESCRIPTION (Adapted from the abstract provided by the applicant): Age is the single greatest risk factor for most neurodegenerative disorders, even those that are genetically based. This delayed onset is believed to reflect an interaction between the risk factors for a neurodegenerative disease, and the aging process itself. Oxidative damage to mitochondrial DNA accumulates in brain of older individuals in many species, including man. This observation has led to the speculation that oxidative injury to mitochondria causes loss of mitochondrial metabolic reserve during aging, and that this contributes to the age-dependent onset of neurodegenerative processes. One class of proteins uniquely situated to contribute to, or modify, these age-dependent changes in mitochondrial function are the mitochondrial uncoupling proteins (UCPs). Mitochondrial uncoupling proteins are specifically designed to impair the efficiency of energy production by mitochondria to produce heat. Outside the nervous system, UCPs regulate body weight, temperature, and the response to starvation. Recently, however, we and others have shown that these proteins also regulate mitochondrial free radical production. Three UCPs (UCP2, 4, and 5) are expressed in brain, where their function(s) is essentially unknown. Our laboratory has been studying UCP5, and has determined that it is a neuronal protein with high expression in the forebrain of both mouse and man. We also found that over-expression of UCP5 in neurons decreased mitochondrial free radical production, a potentially beneficial effect, but decreased the efficiency of mitochondrial function and enhanced the vulnerability of neurons to injury and subsequent degeneration. We hypothesize that UCP5 in brain may be a two-edged sword which trades lower mitochondrial free radical production for greater mitochondrial metabolic inefficiency. We propose to determine whether expression and/or activity of UCP5 is altered in brain during aging. We will also determine whether this results in 1) constitutively higher levels of free radical production by mitochondria in older brain, and 2) increased vulnerability of brain to metabolic stress when UCP5 expression is induced. We will first identify factors, such as hormones or caloric restriction, which regulate expression and activity of UCP5. We will then use biochemical and fluorescence imaging techniques to evaluate mitochondrial function and free radical formation. Initial experiments will be performed in cultured neurons with modified levels of UCP5 or after treatment with agents to modify UCP5 levels or activity. We will then look at how altering UCP5 expression/activity impacts mitochondrial function and free radical production in brain of old mice. For many of these experiments, we will use Thy1-YFP mice, which exhibit neuronal expression of a fluorescent protein, to allow imaging of neuronal mitochondria in brain slices. Finally, we will generate mice deficient in UCP5 to study the normal function of UCP5 in brain and to elucidate how absence of UCP5 affects mitochondrial function and radical production in brain during aging, with the eventual goal of determining whether UCP5 contributes to the age-dependence of neurodegenerative disorders.

DESCRIPTION (provided by applicant): There is currently a need for improved spin probes to help with the diagnosis of and fundamental research into diseases mediated by reactive oxygen species (ROS). This is especially true for the study of age related diseases, since oxidative damage accumulates during the aging process, and many age related disorders such as Parkinson's and Alzheimer's disease are characterized by damage from excess ROS. Spin probes allow these unstable oxygen species to be detected and identified using various magnetic resonance techniques, such as electron paramagnetic resonance (EPR). While certain cancers in animals (Mikuni et al., 2004) and tissues, such as an isolated rat heart (Zweier et al., 1998), have been successfully imaged using EPR imaging, (EPRI), with the current generation of spin probes it is not possible to detect the generation of ROS in age related disorders. The development of new spin probes that allow in vivo detection of ROS produced by Parkinson's and other ROS diseases would represent a significant advance for diagnosing these conditions and for guiding their treatment. To overcome limitations of currently available spin probes, we propose to investigate spin probes based upon single paramagnetic nitrogen atoms encapsulated in C60 fullerenes, N@C60. In this species, the nitrogen is pinned at the center of the symmetric fullerene cage where its unpaired spins are completely protected from reaction with external species. Isolation from the outside environment in the fullerene cage endows N@C60 with one of the narrowest known EPR line widths, (Morton et al., 2006), giving it detection efficiency 100 to 1000 times better than the current spin probes. In addition, interactions with ROS occurring on the surface N@C60 will produce measurable shifts in the spectrum without direct reaction with the probe. These combined features make N@C60 a potentially ideal spin probe. Given the potential of this class of compounds as spin probes and the number of applications that would benefit from such compounds, the overall goal of this project is to synthesize a water-soluble, bioavailable N@C60 derivative, N@C3, and characterize its ability to measure molecular oxygen and biologically important ROS including superoxide using magnetic resonance techniques in vivo. The specific aims of this project are: 1) show that our N@C60 derivative has an EPR signal that is suitable for use as a spin probe for both oximetry and ROS detection, 2) compare N@C3 with currently available spin probes for both oximetry and detection of superoxide and other ROS in aqueous and lipid environments, in cells, and in isolated mitochondria, and 3) Use the technique of Overhauser-enhanced MRI to study the ability of the spin probe to a) enhance spatial resolution of the MRI image, b) substantially improve oxygen mapping by MRI, and c) detect and map ROS in vivo. The proposed studies are the first steps in developing endohedral fullerene-based compounds as novel spin probes, and may open up new avenues for the diagnosis and treatment of diseases ranging from cancer to Alzheimer's. There is growing evidence that reactive oxygen species (ROS) may contribute to the development of many human diseases, including cancer, diabetes, Alzheimer's dementia and Parkinson's disease, but there are currently no techniques which allow ROS (or free radicals) to be measured in patients or in intact animal models of human disease. This project is designed to develop a novel class of "spin probes", molecular agents which are able to interact with ROS to produce a signal which can be detected using various magnetic resonance imaging techniques, such as magnetic resonance imaging (MRI), to assist in early diagnosis and treatment of a broad range of human diseases.

DESCRIPTION (provided by applicant): Increasing evidence indicates that adverse health outcomes in older adults are strongly associated with the development of a state of chronic, mild inflammation. In humans, circulating markers of inflammation, including the inflammatory cytokine interleukin-6 (IL-6), are associated with, or predict, enhanced risk of frailty, loss of muscle mass and strength, disability, and early mortality, as well as acute and chronic cognitive decline, development of Alzheimer's disease, and drug- and stress-induced delirium. IL-6 activates a host of inflammatory actions through classical Jak/STAT pathway, but we recently found that IL-6, acting through non-cannonical activation of the transcription factor, NFkB, induces neuronal expression and activation of NADPH oxidase (Nox2), a multimeric enzyme complex first described as the respiratory burst oxidase in neutrophils. Nox2 is designed to produce large amounts of reactive oxygen species (ROS), primarily superoxide anion. We have further shown that Nox2 is induced and activated in neurons in the aging brain, and that Nox2 is in fact the major source of neuronal and synaptic superoxide production in aged mice. Inflammatory induction of Nox2 led to a superoxide-dependent loss of subsets of inhibitory interneurons in hippocampus, cortex, and amygdala. Loss of these neurons is observed in rodents, dogs, and primates, and has been proposed to underlie cognitive deficits across cognitive domains. We will test the hypothesis that age-related increase in IL-6, potentially mediated by the inflammatory cytokine TNFa or loss of the anti-inflammatory cytokine IL-10, induces neuronal Nox2 expression and that Nox2, in turn, results in persistent deficits in inhibitory neural circuits required for learning, attention, and memory encoding. Aged wild-type mice, and aged mice with modifications (either genetic or pharmacologic) which modify IL-6 expression, signaling, or specific downstream targets, will be used to test our hypothesis. A variety of techniques, including live animal fluorescence imaging of Nox2 activity, EPR, confocal imaging, immuno-fluorescence, electrophysiology and behavioral testing of brain region-specific function (e.g. spatial learning, a hippocampal CA3-dependent process) will be used to test each link in our hypothesized sequence. Finally, a series of interventional studies with drugs and immunotherapies which modulate IL-6, or the proposed sequence of events, will be carried out to determine whether age-related cognitive deficits in the mice are ameliorated, and to further test our hypothetical sequence. Importance to Human Health: Changes in cognitive function are an important health concern for older adults, and for society. The link between inflammatory pathway activation and aging in the brain remains to be fully defined. The studies proposed here are designed to explore one pathway which may link inflammatory pathways and age-related cognitive deficits, with the potential to provide additional insights into important and possibly reversible biological and neural circuitry changes in the aging brain.

According to the Centers for Disease Control (CDC), 5 million Americans are living with Alzheimer's disease (AD), and another 2 million with Parkinson's disease (PD) and other late-onset neurodegenerative conditions (LO-NDs), yet to date, disease-modifying treatments for these diseases have remained elusive. While aging is the single greatest risk factor for LO-NDs, genome wide association studies (GWAS) have identified genes involved in regulation of lysosomes (vesicular organelles responsible for degrading and recycling damaged cellular material), and activation of inflammation and innate immunity, as LO-ND risk genes. However, to date, it is unclear how these two processes may interact in aging brain to promote the development of LO-NDs. Our data suggests that lysosomes may progressively fail during normal aging ? even in the absence of a specific disease process or gene ? and further suggest that low-grade induction of the innate immune enzyme, NADPH oxidase (Nox), by IL-6, may specifically result in lysosome failure in the aging brain. Exciting new data, below, shows that lysosomal failure in aging brain can be rescued by inhibition of Nox. Therefore based on recent literature, and our published and new observations, we propose that age-related lysosome failure is due to IL-6-mediated activation of NADPH oxidase, and propose mechanistic studies to test each component of this hypothesis. The premise behind this application, that inflammation in aging brain causes progressive failure of lysosomes, is supported by existing literature, our published studies, and new preliminary data, but has not been systematically studied. Aim 1 will test the hypothesis that lysosome failure during aging is due to inflammatory activation of Nox, and the corollary that Nox induction is mediated by Il-6 and STAT3 signaling. Our data showing that lysosomal function can be rescued by a Nox inhibitor in aging animals supports the first step in this hypothesis, and is to our knowledge the first to show that Nox inhibition can improve brain lysosome function. Unique mouse models, including PV-tdTomato, IL-6-/- and inducible neuronal Stat3-/- mice with high-resolution confocal microscopy will be used. Aim 2 will determine mechanistically how inflammation produces lysosome failure and impaired degradation of cargo in aging brain. Induced pluripotent stem cell (iPSC)-derived human neurons (iPSC-huNs), and aged mice treated with Nox-modifying agents will determine mechanisms impairing lysosome function. Aim 3 tests the hypotheses that extruded lysosomal cargo contains active proteases which then damage nearby cells including neurons, and that extruded lysosomal material (cargo) is pro-inflammatory. Imaging of cathepsin activity, neuronal injury and markers of microglial and astrocyte activation will be employed. IMPACT: Age-related diseases of the nervous system are an ever-increasing health care issue, but no disease-modifying treatments have emerged for these diseases. This proposal will advance our understanding of how inflammation impairs the ability of lysosomes to clear and recycle damaged cellular material to provide new treatment targets for neurodegenerative disorders.