Peter Koulen - US grants

This application responds to research objective 14 "Functional Senescence" of PAR-02-049. Neurons of the central nervous system tightly regulate their intracellular free Ca 2+concentration. Small changes in the cytosolic Ca 2+ concentration and different patterns of Ca 2+transients are used to mediate important functional processes. In neurons both entry of extracellular Ca 2+ and release of Ca 2+ from intracellular stores are directly coupled to the activation of glutamate receptors and mediate physiological and pathophysiological processes. For the development of early-onset inherited AIzheimer's Disease (AD) and age-related cognitive impairment, numerous reports indicate that changes in the intracellular Ca 2+ concentration promote pathogenesis. The present application will test the hypothesis that the interaction of a group of intracellular Ca 2+ channels, ryanodine receptors, with presenilin-1 leads to a modulation of ryanodine receptor channel function. We further hypothesize that this interaction is mediated through specific binding sites that can be affected by presenilin-1 mutations, but can also be used as the target of potential pharmacological interventions aiming at modifying the presenilin-1 - ryanodine receptor interaction. Experiments will use a combination of single channel electrophysiology and spectrofluorimetric Ca 2+release assays. The specific aims of this proposal are to determine the modulation of biophysical and pharmacological characteristics of ryanodine receptors by presenilin-1 and to measure changes of ryanodine receptor activity after binding to mutated presenilin-1 as found in AD. These studies will allow the determination of mechanisms that influence intracellular Ca 2+ concentrations in neurons and that may play a crucial role in the development of the pathophysiology of Alzheimer's Disease and age-related cognitive impairment. Thus, potential new targets for treating those devastating conditions affecting the aging population may be identified.

DESCRIPTION (provided by applicant): Intracellular free Ca2+ concentrations determine several important functions in retinal neurons including neuronal development and differentiation, synaptic signaling, neurotransmitter release, gene expression and neurodegeneration. The application's broad, long-term objectives are to understand the contribution of intracellular Ca2+ channels (ICCs) to the function of retinal neurons and to identify the regulation of these proteins as potential targets for the treatment of neurodegeneration in the retina. The central hypothesis of the present application is that ICCs determine (a) physiological and pathophysiological processes in retinal neurons, and (b) functional properties of retinal neurons through their differential distribution and their interaction with regulatory factors. The specific aims for testing this hypothesis in neurons of the mammalian retina are: 1) to determine the subcellular localization of ICCs and of their signaling partners in the retina; 2) to analyze the biophysical and pharmacological characteristics of ICCs and their regulatory interactions with other proteins in the retina; 3) to identify and measure the contribution of ICCs to intracellular Ca2+ signaling of retinal neurons. In Aim 1, experiments will identify the structure and functional organization of intracellular Ca2+ signaling mechanisms with respect to glutamatergic synapses in the retina. ICCs and functionally associated proteins will be localized using immunocytochemistry, confocal laser scanning and electron microscopy. In Aim 2, the mechanisms of action and regulation of ICCs will be determined with single channel electrophysiology. The contribution of ICCs to intracellular Ca2+ signaling in glutamatergic retinal neurons will be measured by experiments in Aim 3 using imaging of intracellular Ca2+ concentrations, and confocal laser scanning microscopy. Both Aim 2 and 3 will lay the groundwork for a possible pharmacological modulation of ICCs and the control of intracellular Ca2+ concentration that are essential for the development of treatments of neurodegenerative processes in the retina such as glaucoma, ischemia and photoreceptor degeneration.

DESCRIPTION (provided by applicant): The intracellular free Ca 2+ concentration determines several important functions in lacrimal acinar cells including exocrine activity and exocytosis, development and differentiation, intracellular signaling and gene expression. The application's objectives are to gather preliminary data to develop a research basis for a subsequent application through the R01 mechanism and to explore the feasibility of the proposed research concept. The scientific goals are to understand the contribution of intracellular Ca 2+ channels (ICCs) to the function of lacrimal acinar cells and to identify the regulation of these proteins as potential targets for the treatment of ocular manifestations of rheumatic and skin diseases affecting lacrimal acinar cell function. The central hypothesis of the present application is that ICCs determine (a) physiological and pathophysiological processes in lacrimal acinar cells, and (b) functional properties of lacrimal acinar cells through their differential distribution and their interaction with regulatory factors. The specific aims for testing this hypothesis in mammalian lacrimal acinar cells are: 1) to determine the subcellular localization of ICCs and of their signaling partners in lacrimal acinar cells using immunocytochemistry, confocal laser scanning and electron microscopy; 2) to analyze the biophysical and pharmacological characteristics of ICCs and their regulation by the redox state in lacrimal acinar cells using single channel electrophysiology; 3) to identify and measure the contribution of oxidative stress to ICC mediated intracellular Ca 2+ signaling in lacrimal acinar cells with optical imaging of intracellular calcium concentrations. ICCs and functionally associated proteins will be localized using immunocytochemistry, confocal laser scanning and electron microscopy. The mechanisms of action and regulation of ICCs will be determined with single channel electrophysiology. The contribution of ICCs to intracellular Ca 2+ signaling in lacrimal acinar cells will be measured using imaging of intracellular Ca 2+ concentrations, and confocal laser scanning microscopy. The preliminary data that will be obtained from the specific aims will lay the groundwork for a possible pharmacological control of intracellular Ca 2+ concentration that are essential for the development of treatments of ocular manifestations of rheumatic diseases affecting lacrimal acinar cell function.

The purpose of the Neuroimaging Core (Core C) is to provide access to equipment and technical expertise for confocai microscopy, epi-fluorescence microscopy, and image processing and analysis. The range of experiments that will take advantage of this core include immunofluorescence, imaging of GFP and other organelle- and protein-specific dyes, time-lapse confocal microscopy, calcium imaging, and three-dimensional reconstruction and deconvolution of cells and tissue relevant to identify properties of intracellular calcium signaling mechanisms. Core C will take advantage of the University of North Texas Health Science Center's existing equipment in order to provide these services. Equipment in the core will include a digital imaging workstation with fluorescence microscope (Olympus IX70), fast CCD camera (ORCA ER C4742) and excitation wavelength switcher that provides wavelengths for dye excitation and uncaging (Sutter DG-4), a luminescence spectrofluorometer (AMINCO-Bowman Series 2), a Zeiss LSM 410 confocal microscope, a Bio-Rad Radiance 2100 confocal microscope and two computer workstations for off-line image processing and data analysis. Core C will also provide a Research Support Specialist who is experienced in fluorescence, deconvolution, confocal and multi-photon microscopy. This individual will assist members of each project with all experiments requiring the use of this core facility.

N-Acylethanolamines (NAEs) are potent, bioactive lipid signaling substances with diverse roles in mammalian physiology. We propose to investigate plant-derived NAEs functioning both as potential alternatives and supplements to currently existing neuroprotecting treatments for neurological disorders including Alzheimer's disease. Our preliminary data indicate that NAEs regulate the function of intracellular calcium channels allowing modulation of intracellular calcium signaling. NAEs are naturally occurring compounds, and therefore there are in place metabolic and clearance processes for these compounds lessening the likelihood of NAE-associated toxicities. The central hypothesis of this application is that NAEs exert protective effects on neurons. The mechanisms of neuroprotection mediated by NAEs will be analyzed and evaluated at the molecular level, in neuronal cell lines, primary neuronal cultures and in vivo as models of neurotoxic insults and neurodegeneration. In particular, the effect of NAEs will be evaluated for their ability to prevent cell death and elicit neuroprotection-related signaling pathways. The specific aims are: 1) Analyze the modulation of biophysical and pharmacological characteristics of intracellular calcium channels by NAEs; 2) to identify and measure the contribution of NAE mediated responses to intracellular Ca 2+ signaling of neurons; 3) to determine the neuroprotective effects of NAEs in neuronal cell lines as models of neurotoxic insults and neurodegeneration; 4) to identify the neuroprotective signal transduction pathways elicited by NAEs in primary neuronal cultures of the hippocampus as models of neurotoxic insults and neurodegeneration.; 5) to determine the effects of NAEs on ischemic damage in an animal model of stroke. Experiments will use a combination of biochemistry, electrophysiology, optical imaging of intracellular Ca 2+ concentrations, analyses of changes in intracellular signaling and neuroprotection assays. The overall goal of this study is to provide the necessary foundation for the development of novel alternative or supplemental treatments for neurodegeneration in Alzheimer's disease.

[unreadable] DESCRIPTION (provided by applicant): The proposal requests funding for the acquisition of a shared-use confocal laser-scanning microscope, the Zeiss LSM 510 system. This bioanalytical system will serve an increasing community of NIH-funded neuroscience researchers at the University of North Texas Health Science Center (UNTHSC) that have recently moved to the newly constructed Center for BioHealth (CBH building), a new off-site location that is part of a campus expansion of the UNTHSC. The capabilities of the one current confocal microscope at UNTHSC (located in the RES building) are insufficient due to the strong increase in the number of faculty and students using confocal microscopy for their research. In addition and more importantly, the new location across campus does not allow the majority of NIH-funded investigators of confocal microscopy to effectively use this technology, because their cell and tissue based assays and model systems do not tolerate transport or exposure to conditions associated with transport to a remotely located confocal microscopy facility. There is currently no confocal microscope at the off-site location at UNTHSC, in CBH, nor in a directly adjacent building that would allow researchers to perform this specialized data acquisition. The requested instrument will be housed in the microscopy core facility in CBH504, centrally located to allow all users direct access not only from within the CBH building but also for all major users adjacent to their respective laboratories. The Zeiss LSM 510 system, a state-of-the-art instrument, has a proven track record as a well-supported and established multi-user instrument. It will meet the current increased and future needs for advanced imaging, as well as modernize UNTHSC's teaching technology. Current research needs for a confocal microscope in CBH include: 1) measuring subcellular changes in signaling molecules in live cells; 2) optical sectioning of freshly isolated cells and tissue at a resolution that cannot be achieved with conventional fluorescence microscopy; 3) determining the distribution of low-abundance proteins over time while minimally affecting cell physiology; 4) elucidating mechanisms protein expression and trafficking in genetically modified cells; 5) conducting analyses of structure-activity relationships of novel neuroprotective agents in vitro and in vivo. The current application will primarily serve eight major groups around principal investigators that are extramurally funded by peer-reviewed grants from different NIH institutes. [unreadable] [unreadable] [unreadable]

N-Acylethanolamines (NAEs) are potent, bioactive lipid signaling substances with diverse roles in mammalian physiology. We propose to investigate plant-derived NAEs functioning both as potential alternatives and supplements to currently existing neuroprotecting treatments for neurological disorders including Alzheimer's disease. Our preliminary data indicate that NAEs regulate the function of intracellular calcium channels allowing modulation of intracellular calcium signaling. NAEs are naturally occurring compounds, and therefore there are in place metabolic and clearance processes for these compounds lessening the likelihood of NAE-associated toxicities. The central hypothesis of this application is that NAEs exert protective effects on neurons. The mechanisms of neuroprotection mediated by NAEs will be analyzed and evaluated at the molecular level, in neuronal cell lines, primary neuronal cultures and in vivo as models of neurotoxic insults and neurodegeneration. In particular, the effect of NAEs will be evaluated for their ability to prevent cell death and elicit neuroprotection-related signaling pathways. The specific aims are: 1) Analyze the modulation of biophysical and pharmacological characteristics of intracellular calcium channels by NAEs; 2) to identify and measure the contribution of NAE mediated responses to intracellular Ca 2+ signaling of neurons; 3) to determine the neuroprotective effects of NAEs in neuronal cell lines as models of neurotoxic insults and neurodegeneration; 4) to identify the neuroprotective signal transduction pathways elicited by NAEs in primary neuronal cultures of the hippocampus as models of neurotoxic insults and neurodegeneration.; 5) to determine the effects of NAEs on ischemic damage in an animal model of stroke. Experiments will use a combination of biochemistry, electrophysiology, optical imaging of intracellular Ca 2+ concentrations, analyses of changes in intracellular signaling and neuroprotection assays. The overall goal of this study is to provide the necessary foundation for the development of novel alternative or supplemental treatments for neurodegeneration in Alzheimer's disease.

Recent studies suggest that the steroid hormones estrogen and progesterone, their metabolic products, and chemical derivatives mediate protection against cellular damage and death. In a variety of organs and cell types, this has been documented for both acute insults and degenerative diseases. Among the protective mechanisms that are triggered by these steroid hormones is the re-establishment of the cytosolic free calcium ion homeostasis. This important gatekeeper of cellular decisions to progress towards differentiation, mitosis or apoptosis is critically dependent on the activity of intracellular calcium channels (ICC), inositol 1, 4, 5-trisphosphate receptor (IP3R) and ryanodine receptors (RyR). Intracellular calcium signaling mediated by these channels can be specifically altered by acute and chronic application of estrogen and/or progesterone. The present application will test the hypothesis that steroid hormones regulate intracellular calcium signaling through ICC that are important for neuronal function and viability. In particular, the effect of estrogen, progesterone, and related compounds, will be evaluated for their ability to induce posttranslational modifications of ICC and thereby elicit neuroprotection-related signaling pathways by controlling the cytosolic free calcium ion homeostasis. This is of high significance due to the fact that the non-genomic effects of estrogen and progesterone, which include intracellular calcium signaling, have the potential to provide the necessary information to design physiologically and clinically relevant cytoprotection strategies relevant for age-related disorders affecting the nervous systems and neurodegenerative diseases including Alzheimer's disease (AD). The overall goal of the study is to identify novel signaling pathways that are part of nongenomic actions of estrogen and progesterone in the nervous system. This identification of novel therapeutic targets will subsequently enable us to develop new strategies in cytoprotection for pathophysiological processes affecting neurons during aging and AD. AD is affecting the health and quality of life of an increasing number of individuals. In addition, changes in hormone levels in the aging population contribute as risk factors to AD and other age-related diseases. The present proposal addresses these pressing health issues that are also the focus of agency-wide NIH / NIA activities. Results from the proposed study will enable researchers to generate more effective drugs for AD and related diseases and clinicians to utilize more effective therapeutic approaches in hormone replacement therapy and in age related diseases including AD.

The intracellular free Ca2+ concentration of CMS neurons is highly regulated. Small changes in the cytosolic Ca2+ concentration and different patterns of Ca2+ transients are used by CMS neurons to mediate important functional and developmental processes. Increases in the cytosolic Ca2+ concentration can arise from entry of extracellular Ca2+ through ion channels in the plasma membrane or via the release of Ca2+ from intracellular stores. Both entry of extracellular Ca2+ and release of Ca2* from intracellular stores are directly coupled to neuronal function. For the development of acute and chronic degenerative diseases reducing the viability and function of CNS neurons several studies indicate that both changes in intracellular second messenger concentration and pathological increases in the intracellular Ca2+ concentration promote pathogenesis. The present application will test the two-pronged hypothesis that Ca2+ signaling of CNS neurons is functionally regulated by associated proteins of intracellular Ca2+ channels and that control of their expression and function represents a novel target for CNS neuroprotection. The proposed experiments designed to test this hypothesis will investigate the functional mechanism underlying this interaction under experimentally induced disease conditions in models of acute and chronic degenerative CNS diseases. The specific aims of this proposal are to determine a) changes in the expression and localization, b) function and c) modulation of these proteins based on therapeutic intervention studies in pre-clinical models of AD and age-related cognitive impairment. The overall goal of the study is to identify a novel mechanism of neuroprotection and determine its potential as a strategy for neuroprotective therapies targeting the aging brain and age-related neurodegenerative diseases such as Alzheimer's disease. This therapy approach will have the potential to be both preventative and therapeutic in nature and to complement existing treatment designs and rationales. Thus, potential new targets for treating those devastating conditions affecting the aging population may be identified.

DESCRIPTION (provided by applicant): The proposal requests funding for the acquisition of a shared-use confocal laser-scanning microscope system, a LSM 710 configuration #4 3ch 405, Ar, 561, 633 ZEN. The Zeiss LSM 710 Confocal Laser Scanning Microscope System with three spectral detection channels / PMTs, and a combination of six laser lines - 405, 458, 514, 488, 561 &633 nm as excitation sources allowing the simultaneous observation of fluorescent probes in distinct spectral ranges had been specifically developed as a state-of-the-art, highly sophisticated laser scanning instrument to image fixed biological specimens as wells as living cells and tissues utilizing state-of-the-art fluorescent probes for multiple standard confocal imaging application. This bioanalytical system will serve an increasing community of NIH-funded researchers at the University of Missouri (UMKC) School of Medicine. The system will meet the current increased and future needs for advanced imaging, as well as modernize the School of Medicine's teaching technology. Current research needs for a confocal microscope include: 1) measuring subcellular changes in signaling molecules in live cells;2) optical sectioning of freshly isolated cells and tissue at a resolution that cannot be achieved with conventional fluorescence microscopy;3) determining the distribution of low-abundance proteins over time while minimally affecting cell physiology;4) elucidating mechanisms protein expression and trafficking in genetically modified cells;5) conducting analyses of structure-activity relationships of novel agents in vitro and in vivo. The current application will primarily serve research groups around principal investigators that are extramurally funded by peer-reviewed grants from different NIH institutes. NIH-funded researchers at the School of Medicine in need of this instrumentation conduct research on diseases that affect significant and increasing portions of the U.S. population including minorities affected by disparities in health care delivery. The proposed equipment will accelerate research progress and enhance the quality and breadth of research results determining causes, mechanisms of action and subsequently contributing to discovering potential treatment strategies and improving health care.

DESCRIPTION (provided by applicant): Degeneration or damage of the optic nerve and the retina due to optic neuritis is a leading cause of visual loss and blindness in the United States and worldwide associated with multiple sclerosis and autoimmune damage to the CNS. The proposed multidisciplinary research project will focus on the development and characterization of a novel pharmacological intervention strategy that combines drugs to control structural and functional degeneration in autoimmune optic neuritis. Suppression of CNS inflammation, prevention of loss and damage of myelinated axons, and stimulation of regeneration and remyelination of damaged axons are the primary goals of the study. To this end, preclinical testing of the new therapeutic strategy will be performed in established models of human autoimmune optic neuritis. These experiments will determine efficacy of treatment in terminating and/or preventing autoimmune optic neuritis associated neuronal loss and preservation of visual function, and to generate data to support feasibility for and move positive findings to phase 1 or 2 clinical trials. Specifically, we will test the hypothesis that the proposed treatment strategy cn target and remedy specific phenotypes that include combinations of separate pathologies encountered during distinct stages of optic neuritis and multiple sclerosis, leading to improvement of visual impairment and functional deficits associated with the disease. The determination of neuronal viability and the acquired knowledge on associated therapeutic parameters will indicate the potential of the method to remedy autoimmune optic neuritis as the overall goal of the project. This therapy approach for autoimmune optic neuritis focuses on suppression of CNS autoimmunoreactivity, neuroprotection, axon regeneration and remyelination via different mechanisms. It has the potential to be both preventative and therapeutic and to complement existing treatment designs and rationales addressing other aspects of autoimmune optic neuritis treatment. PUBLIC HEALTH RELEVANCE: Multiple sclerosis affects approximately 2.5 million people worldwide and approximately 400,000 people in the United States. In multiple sclerosis, degeneration or damage of the optic nerve, the nerve that connects the eye to the brain and thereby makes vision possible, is a leading cause of loss of quality of life and productivity in th United States and worldwide. The project proposes the determination of the identity and function of novel targets for combination drug treatment that controls disease progression. As degeneration of the optic nerve affect significant and increasing portions of the U.S. population including minorities affected by disparities in health care delivery, determining causes, mechanisms of action and subsequently potential treatment strategies will contribute to improving health care, health and physical performance.

PROJECT SUMMARY (See instructions): In the previous funding period. Project 3 discovered that the steroid hormones estrogen (E2) and progesterone (P4) elicit a protective mechanism in CNS neurons representing a critical component of the reestablishment of the cytosolic free calcium ion homeostasis resulting in profound neuroprotection. Specifically, E2 and P4 as part of this mechanism are capable of changing the activity of intracellular calcium channels non-genomically through distinct kinase signaling pathways that induce specific post-translational modifications. However, a critical impediment to the full development of the neuroprotective potential of these hormones as pharmacotherapies is their limited therapeutic window and systemic side-effects. In particular, the use of steroid hormones to combat disease processes affecting neurons during aging and Alzheimer's disease (AD) is limited to a short time period after menopause in female individuals as shown by a number of seminal studies. In addition, usefulness is also extremely limited throughout the lifespan due to carcinogenic and estrogenic side-effects in male and postmenopausal female individuals. The present competitive renewal focuses on this mechanism and that improved neuronal viability is generated by steroid hormones controlling the state of specific PTMs of ICCs. Specifically, steroid hormone signaling induces these modifications and steroid hormone binding proteins functionally associated with ICCs modulate this activity. In particular, the time course and sustainability of neuroprotective PTMs will be measured for clinically relevant therapeutic windows; alternative therapeutic strategies will be determined to induce the same PTMs of ICC and thereby elicit neuroprotection. This strategy can bypass potential carcinogenic and estrogenic side-effects. This is of high significance because such effects of E2 and P4 on calcium signaling, data generated in the previous funding period, are potential clinically relevant cytoprotection strategies for age-related disorders affecting the nervous systems and neurodegenerative diseases including AD. When generated independently in clinically relevant penods, i.e. beyond the therapeutic window of steroid hormones themselves, such effects represent novel therapeutic approaches with high clinical relevance. The overall goal of the study is to identify the druggability of non-genomic actions of E2 and P4 in the CNS and of steroid hormone controlled calcium signaling proteins. This innovative strategy has a significant potential to expand the narrow therapeutic window for steroid mediated neuroprotection into an age range that is clinically relevant to achieve neuroprotection against processes underlying CNS aging and AD.

DESCRIPTION (provided by applicant): Degeneration or damage of the optic nerve and the retina due to optic neuritis is a leading cause of visual loss and blindness in the United States and worldwide associated with multiple sclerosis and autoimmune damage to the CNS. The proposed multidisciplinary research project will focus on the development and characterization of a novel pharmacological intervention strategy that combines drugs to control structural and functional degeneration in autoimmune optic neuritis. Suppression of CNS inflammation, prevention of loss and damage of myelinated axons, and stimulation of regeneration and remyelination of damaged axons are the primary goals of the study. To this end, preclinical testing of the new therapeutic strategy will be performed in established models of human autoimmune optic neuritis. These experiments will determine efficacy of treatment in terminating and/or preventing autoimmune optic neuritis associated neuronal loss and preservation of visual function, and to generate data to support feasibility for and move positive findings to phase 1 or 2 clinical trials. Specifically, we will test the hypothesis that the proposed treatment strategy cn target and remedy specific phenotypes that include combinations of separate pathologies encountered during distinct stages of optic neuritis and multiple sclerosis, leading to improvement of visual impairment and functional deficits associated with the disease. The determination of neuronal viability and the acquired knowledge on associated therapeutic parameters will indicate the potential of the method to remedy autoimmune optic neuritis as the overall goal of the project. This therapy approach for autoimmune optic neuritis focuses on suppression of CNS autoimmunoreactivity, neuroprotection, axon regeneration and remyelination via different mechanisms. It has the potential to be both preventative and therapeutic and to complement existing treatment designs and rationales addressing other aspects of autoimmune optic neuritis treatment.

PROJECT SUMMARY / ABSTRACT The present administrative supplement application responds to NOT-AG-20-034, ?Notice of Special Interest: Alzheimer's-focused administrative supplements for NIH grants that are not focused on Alzheimer's disease?. Under the parent grant for the present administrative supplement, R01 grant 1R01EY031248-01, entitled ?Novel mechanism controlling calcium signaling to treat and prevent neurodegeneration in early stage glaucoma?, we are targeting a novel signaling pathway for the development of a novel pharmacological intervention to control degeneration of nerve cells in the retina and optic nerve due to glaucoma, a major cause of visual loss and blindness in the United States and worldwide. Specifically, we plan to determine mechanisms of action and to measure preservation of neuronal viability and function in model systems of glaucoma. The proposed research of the parent award will allow us to generate preclinical data needed for the development of novel neuroprotectants to complement existing therapies targeting intraocular pressure. The research proposed under the present Alzheimer's disease (AD)-focused administrative supplement application will allow us to build on these novel preclinical data sets and thereby to develop a novel therapeutic strategy for the protection of neurons of the central nervous system (CNS) affected by AD. Specifically, we will test the hypothesis that similar to neurons of the retina and optic nerve affected by glaucoma, the lack of cellular defense mechanisms resulting from AD pathology reducing viability and function of CNS neurons can be attenuated by the novel therapeutic strategy under development in the parent award. To this end, a novel intervention approach will be developed that can be exploited to devise novel treatments that can be delivered to CNS neurons affected by AD protecting them from oxidative stress mediated damage and loss of function. Three-dimensional (3D) bio-printed human neural cell constructs will be used as in vitro models for AD and for drug discovery in AD and related dementias employing experimental strategies aligned with the parent award. The proposed experiments will determine the potential of the targeted novel therapeutic strategy for preventative and neuroprotective therapies in AD. The innovative strategy has the potential to generate a first-in-class pharmacotherapy approach for AD. The strategy's potentially high impact lies in its capacity to be both preventative and therapeutic in nature and to complement current and future treatment designs and rationales.

PROJECT SUMMARY/ABSTRACT Degeneration or acute damage of retinal pigment epithelial (RPE) and nerve cells in the retina due to Age-related Macular Degeneration (AMD) is a major cause of visual loss and blindness in the United States and worldwide. The proposed multidisciplinary research project will focus on the characterization of a novel signaling pathway for and the development of a novel pharmacological intervention to control degeneration of RPE cells and neurons in AMD. To this end, preclinical testing of the new therapeutic strategy will be performed, along with ocular biotransformation, transport and distribution studies, in established models of human AMD. These experiments will determine efficacy of treatment in terminating and/or preventing AMD associated neuronal loss and preservation of visual function, and to generate data to support feasibility for and move positive findings to phase 1 and 2 clinical trials. Specifically, we will test the two-pronged hypothesis that treatment based on the ocular targeting of a novel chemical antioxidant strategy protects RPE cells and neurons from apoptosis by topical delivery in established models of human AMD and, therefore, leads to prevention or improvement of visual impairment and functional deficits associated with AMD. The determination of neuronal viability and the acquired knowledge on associated biopharmaceutical and pharmacological parameters will indicate the potential of the method to remedy AMD as the overall goal of the project. This novel approach for therapy development in AMD focuses on complementary and alternative cellular protection and neuroprotection. The innovative strategy has the potential to generate a first-in-class pharmacotherapy approach for dry AMD using a topical rather than a systemic or invasive route of drug delivery. The strategy?s potentially high impact lies in its capacity to be both preventative and therapeutic in nature and to complement existing treatment designs and rationales addressing other aspects of AMD treatment such as those targeting neovascularization.