Howard J. Federoff - US grants

Synapse formation occurs principally during nervous system development. However, in response to injury new synapses can be formed both in the peripheral nervous system and to a more limited extent in the central nervous system (CNS) . In addition, some mature neurons within discreet CNS regions are able to undergo restructuring of their synapses in response to changes in their environment. This property of mature neuronsis often referred to as synaptic plasticity and may serve as the basis for memory formation and sexually dimorphic physiology. The mechanisms that regulate these diverse forms of synapse formation are probably different, but all involve the highly regulated expression of specialized neuronal gene products. One such neural gene product, termed GAP-43, has been implicated in these varied forms of synaptogenesis. Our program is devoted to elucidating the molecular mechanisms involved in the regulation of synapse formation. The approach is to define the mediators and mechanisms that regulate gene expression during synaptogenesis. We have initiated this work by characterizing the regulation of GAP-43 gene expression by nerve growth factor (NGF) and corticosteroid in cultured neurons and in pheochromocytoma cells (PC12). This application identifies three areas for further study: 1) The identification of the mechanisms responsible for NGF up-regulation and corticosteroid down-regulation of GAP-43 gene expression; 2) The examination of GAP-43 gene regulation by synapse formation with appropriate target cells; and 3) The isolation and characterization of other genes expressed during synaptogenesis.

Long term rehabilitation is often required for patients who have suffered traumatic and ischemic brain injury. The requirement for long-term rehabilitation is directly determined by the extent of permanent neuron loss. Since neuron loss occurs during a delayed as well as an acute phase, one approach to improving the outcome in such patients is to develop therapies that limit delayed neuron destruction. We propose the development of a herpes virus based gene therapy approach in which the expression of neuroprotective proteins in the peri-injury period will reduce the extent of neuron death and limit the loss of function of surviving neurons. Specifically, we will test the efficacy of gene transfer and expression of neurotrophic factors in an in vivo model of traumatic injury, and the gene for bcl2 in an organotypic hippocampal slice culture system and an in vivo model of ischemic injury. A role for neurotrophic factors in the maintenance of neuron function in experimental traumatic injury has been previously suggested. We hypothesize that delayed neuron loss following ischemic injury to the hippocampal formation to be a form of apoptosis. For this reason, we will transfer the gene for bcl2, because its expression has been shown to be neuroprotective in other apoptotic models.

DESCRIPTION (Adapted from applicant's abstract): The underlying basis for cognitive impairment in psychiatric and much neurologic disease is obscure. Abnormal synaptic networks arising from developmental and/or adult perturbations may be the pathophysiological substrate for cognitive impairment. At the level of individual cellular components of the network we can study molecules that are important for its establishment and maintenance. Moreover, with newer molecular genetic tools we can more precisely dissect the contribution of particular molecules to these processes. Nerve growth factor (NGF), the prototypic neurotrophin, has been well established in the peripheral nervous system, where its function as a target derived factor is essential for the development and survival of sympathetic and a subpopulation of sensory neurons. In the central nervous system (CNS), NGF is implicated in modulating cognition through its stimulatory effect on the septohippocampal cholinergic pathway, as suggested by pharmacological studies involving infusion of NGF or blocking antibodies. To explore the cellular basis for this cognitive effect in mouse and define the relationship between neurons synthesizing and those responding to NGF in the hippocampus, we have developed a somatic genetic mosaic approach. A binary gene system employing the bacteriophage P1 derived cre/loxP recombinant system provides a method to create somatic tissues in which some cells differ from their neighbors by a single gene's activation, producing a gain of NGF function. With our approach a germline transmitted NGF transgene is flanked by loxP elements (cis recombinant elements) that are activated to recombine by the somatic expression of the cre recombinase gene delivered by a viral vector. This affords temporal and spatial control of recombination and the ability to regulate the number of activated cells within a mosaic tissue. We have constructed multiple lines of transgenic mice harboring an NGF excision activation transgene (XAT) that produces a gain of function when activated by a herpes simplex amplicon vector that expresses cre recombinase (HSVcre). The studies proposed herein utilize the conditional NGF gain of function mice to investigate the role of NGF in septohippocampal cholinergic function. These studies will reveal much new information regarding the function of NGF in the adult nervous system. Moreover, continued refinement of the somatic mosaic approach will likely lead to its broad application to study the function of other genes in the intact CNS.

DESCRIPTION: Advances in gene transfer methods have created the opportunity for development of gene therapy for human neurological disease such as Parkinsons disease (PD). One goal is to slow dopaminergic neuron loss and the success of such neuroprotective gene therapy is contingent on the development of safe and effective gene transfer vectors that can express a therapeutic gene over long periods of time in specific neuronal populations. The plasmid based herpes simplex virus (HSV) "amplicon" vectors accommodate a large (9kb) typrosine hydroxylase (TH) promoter and provide highly selective and relatively long term (10 weeks) gene expression in dopamine (DA) neurons in the rat substantia nigra. But, HSV amplicon vector stocks also contain replication defective HSV helper virus which under some conditions and particularly at high multiplicities of infection produces cellular injury and death. The overall goals of this application are three fold: Aim 1 will compare several complementary new methods to reduce and perhaps eliminate helper virus related toxicity and determine whether they provide increased efficiency and stability of amplicon transgene gene expression in vitro (in cultured primary neurons and astrocytes measuring titers of amplicon and helper, number of lac positive cells and cytotoxicity) and Aim 2 will test their efficacy in vivo using the long tyrosine hydroxylase promotor - b lacZ reporter amplicon (delivering virus to the striatum and scoring bgal expression (mRNA and protein) in DA neurons (TH positive) in the striatum and SN at 1,6, and 16 weeks after infection at several MOIs.). Finally Aim 3 will determine whether the approach of decreasing cytotoxicity leads to a more effective gene therapy. The applicant will construct new amplicon vectors carrying three candidate neuroprotective genes thought to work by different pathways, GDNF, BDNF and bcl-2, use the least cytotoxic system to produce infectious particles, and evaluate them in FluoroGold/6-OHDA lesioned rats, a progressive injury model of PD, examining gene expression characteristics (RNAse protection to measure vector mRNA levels, ELISA and immunostaining to measure gene products), neuroprotection (quantitation of FluoroGold labeled neurons) and neurochemical changes (levels of DA and metabolites-DOPAC, HVA as a measure of both intra and inter-neuron DA turnover and effects on serotonergic neurons with 5-HT, 5-HIAA) at 1 and 4 months after injection of viral stock.

DESCRIPTION (Abstract of the application) The development of gene therapies for chronic diseases critically depends on the development of non-toxic vectors that are capable of achieving long term gene expression. All currently used vectors have a limited' capacity for sustained gene expression. In the relatively immune-privileged central nervous system (CNS) it is possible to study vector transcriptional silencing, one of the major mechanisms limiting prolonged gene expression. Our hypothesis is that the extent and duration of expression of a somatically transferred transcription unit is a function of chromatin organization. We propose that transcriptional silencing is a consequence of heterochromatin formation on the vector genome. We will directly test this hypothesis by analyzing gene expression and chromatin structure in the HSV amplicon vector. In addition, we will examine one of the predictions of the hypothesis: inclusion within amplicon vectors of cis elements will direct chromatin, organization and increase the likelihood for long term gene expression. Specifically, we will test whether a matrix attachment region (MAR) element placed in a HSV amplicon vector imparts resistance to heterochromatin formation and its consequence- transcriptional silencing. A glucocorticoid-responsive transcription unit will be used because it has a DNA-protein signature recognizable in biochemical studies to examine chromatin structure I on vector genomic DNA. This project focuses on HSV amplicon vectors, versatile plasmid-based vectors that do not contain any viral genes and which can be prepared free of helper virus. Achieving long term expression from HSV amplicon vectors requires an understanding of how to direct the organization of the transcription unit within the target cell. We believe that the characterization of chromatin structure as it pertains to expression from the HSV amplicon vector in the CNS will have implications for extending gene expression from other episomal vectors and in different tissues.

DESCRIPTION: (Adapted from the Investigator's Abstract) Environmental exposure to some neurotoxicants produce damage to the basal ganglia. Dopaminergic neurons of the substantia nigra are most vulnerable to the action of specific intracellular toxins. We hypothesize that genetic determinants render dopaminergic neurons susceptible to neurotoxicant damage thus producing the phenomenon of selective vulnerability. We will model such environmental-genetic interactions to determine whether modification of a single gene within a subpopulation of substantia nigra dopaminergic neurons increases their susceptibility to neurotoxicants. To test our hypothesis we will employ an approach we recently developed, somatic mosaic analysis in the mouse, to dysregulate the expression of the dopamine transporter (DAT) in dopaminergic neurons. Somatic mosaic analysis is a binary cre/loxP molecular genetic method utilizing a germline transmitted recombinational substrate containing a dormant transcription unit and somatic gene transfer of a herpes virus vector that expresses cre recombinase and "activates" the gene of interest. In this application we will construct the DAT XAT (Cat eXcision Activation Transgene) dormant transcription unit which is designed to direct transcription only in dopaminergic neurons of the DAT gene and co-transcribed reporter gene, beta-galactosidase, following cre recombinase "activation". DAT XAT activation efficiency will be tested in vitro, mice harboring DAT XAT generated and in vivo activation studied. Vulnerability to systemically administered neurotoxicants MPTP and paraquat will be examined in mice in which a subset of nigral dopaminergic neurons have increased DAT expression. Increased vulnerability will be manifest by increased cell death in neurons carrying the "activated" DAT XAT transgene. Recombination-mediated gene activation permanently changes the genetic constitution of that subset of dopaminergic neurons thus allowing us to study neuronal vulnerability during chronic low dose exposure to paraquat as a model of environmental exposure. Taken together, these studies will provide the opportunity to model environmental neurotoxicant-genetic interactions as a cause of basal ganglia damage and provide a paradigm to explore etiologic mechanisms underlying idiopathic Parkinson's disease.

DESCRIPTION (Adapted from applicant's abstract): The underlying basis for cognitive impairment in psychiatric and much neurologic disease is obscure. Abnormal synaptic networks arising from developmental and/or adult perturbations may be the pathophysiological substrate for cognitive impairment. At the level of individual cellular components of the network we can study molecules that are important for its establishment and maintenance. Moreover, with newer molecular genetic tools we can more precisely dissect the contribution of particular molecules to these processes. Nerve growth factor (NGF), the prototypic neurotrophin, has been well established in the peripheral nervous system, where its function as a target derived factor is essential for the development and survival of sympathetic and a subpopulation of sensory neurons. In the central nervous system (CNS), NGF is implicated in modulating cognition through its stimulatory effect on the septohippocampal cholinergic pathway, as suggested by pharmacological studies involving infusion of NGF or blocking antibodies. To explore the cellular basis for this cognitive effect in mouse and define the relationship between neurons synthesizing and those responding to NGF in the hippocampus, we have developed a somatic genetic mosaic approach. A binary gene system employing the bacteriophage P1 derived cre/loxP recombinant system provides a method to create somatic tissues in which some cells differ from their neighbors by a single gene's activation, producing a gain of NGF function. With our approach a germline transmitted NGF transgene is flanked by loxP elements (cis recombinant elements) that are activated to recombine by the somatic expression of the cre recombinase gene delivered by a viral vector. This affords temporal and spatial control of recombination and the ability to regulate the number of activated cells within a mosaic tissue. We have constructed multiple lines of transgenic mice harboring an NGF excision activation transgene (XAT) that produces a gain of function when activated by a herpes simplex amplicon vector that expresses cre recombinase (HSVcre). The studies proposed herein utilize the conditional NGF gain of function mice to investigate the role of NGF in septohippocampal cholinergic function. These studies will reveal much new information regarding the function of NGF in the adult nervous system. Moreover, continued refinement of the somatic mosaic approach will likely lead to its broad application to study the function of other genes in the intact CNS.

We must here a proposal for a Nathan Shock Center of Excellence in Basic Biology of Aging that would build on the long tradition of geriatrics and gerontology at the University of Rochester Medical Center. The proposed Center would have a four-fold purpose: (1) To enhance the quality of research in the basic biology of aging, (2) to facilitate coordination of research on aging, (3) to create a regional/national resource for molecular and cellular technologies and (4) to create an environment for faculty growth in aging research. These goals would be implemented by an Administrative Core coordinating an integrated set of others Cores. The Gene Expression Vector Core provide investigators in the biology of aging with the viral vectors to create transfected cell lines, primary cells or transduce organs in intact animals. Investigators could then use the DNA array technology of the Microarray Analysis Core to survey and quantitate gene expression as a function of age and experimental manipulation. Ascertaining which array data sets are free of bias or artifact requires independent validation. Validation of a subset of genes in the array data set could then be achieved by real time quantitative PCR using the Gene Expression Vector Core and/or by quantitative in situ hybridization at the cellular level using the Molecular and Cellular Imaging Core. The resulting extensive data sets could then be analyzed using multi-variate statistics and informatics analyses provided by the Bioinformatics and Biostatistics Core. These Core can, of course, be utilized as stand alone capabilities when warranted by experimental design area. Reagents and techniques developed by the Cores would be available nationally. The Research Development Core will play an important role in the reaction of an environment for faculty growth in aging research through recruitment of outstanding new faculty, programs supporting pilot projects, providing partial salary support to investigators in the biology of aging, mentoring faculty and by instituting seminars, lectures and retreats.

DESCRIPTION (ADAPTED FROM THE APPLICANT'S ABSTRACT): The Interdepartmental Program in Neuroscience (the Program) is a broad, multidisciplinary structure that spans resources across departments and centers throughout the University. The Program has undergone several evolutionary changes and most notably was structurally reorganized in 1997. Its new form is expanded and modernized to include additional departments and centers and has established a diverse set of governing and operating committees to insure long-term structure and flexibility required of a dynamic scientific and educational enterprise. The Program now encompasses the full spectrum of neurosciences, providing training opportunities in molecular, cellular, behavioral as well as systems. Particularly important to the vitality and future success of the Program is the large infusion of resources to recruit 15 - 18 new faculty in the neurosciences. These new faculty will further strengthen our goal to provide training opportunities across disciplines ranging from molecular to systems and behavioral neurosciences. A reorganizing principle was the development of a training environment where interdisciplinary approaches to problems could be pursued, thus allowing students to study a problem at multiple levels. Some structure for the approach was embodied in the upper level research themes; tracks which represent not boundaries to train within but represent the diverse levels of problem solving available to students. The program proposed in this application will be used exclusively for broad and basic support of students in their first two years of graduate study. Following commitment to a dissertation laboratory, students will be supported by either an investigator's individual grant or a specialized training grant. The continuity of training objectives established by the Program occurs throughout the student's tenure irrespective of the mechanism of his or her support and leads to a Ph.D. in Neuroscience.

The genetic programs that drive neural development establishes the anatomic substrate that endows the mammal with the capacity to respond to external environmentally available cues. The retention of adaptive plasticity in some adult brain regions, notably the hippocampus, underlies are our ability to learn and remember. It is presumed that such adaptive plasticity must be mediated by selective changes in gene and protein expression within neurons that comprise the anatomic substrate. To understand the temporal and ordered sequence of molecular events that result in adaptive plasticity we need novel tools that must be designed to work in concert. This application will develop and validate such methodology in a well-described neural system. This application will result in the creation of the following four interleaved and novel technologies that will be scalable and broadly applicable. These new technologies are: l) A method and reagents for producing single gene changes within an experimentally determined number of neural cells in the intact nervous system. 2) A combined single cell microdissection and mRNA amplification approach to profile all mRNAs from genetically altered cells or others of interest. 3) A bioorganic transistor to detect change in the levels of different proteins from genetically altered cells or others of interest. 4) Bioinformatics methods to extract genetic regulatory networks from mined profile data.

DESCRIPTION (provided by applicant): Gene transfer methods have created the opportunity for developing gene therapy for human neurological diseases such as Parkinson's Disease (PD). Since PD represents a group of clinically similar syndromes each triggered by a different mechanism we hypothesize the existence of a shared downstream pathophysiologic pathway. Our goal is to develop therapy for PD directed at a shared common node in the pathway. The elaboration of such neuroprotective gene therapy is contingent on the development of safe and efficacious gene transfer vectors that can express a therapeutic gene for a prolonged period in specific neuronal populations. Of the currently available vehicles for direct gene therapy only plasmid based herpes simplex virus (HSV) "amplicon" vectors have been demonstrated to both accommodate a large (9 kb) tyrosine hydroxylase (TH) promoter fragment and to provide highly selective gene expression in dopamine (DA) neurons in the substantia nigra. However, HSV amplicon vectors exhibit transgene silencing that is an impediment to one-time dosing for a chronic disease such as PD. Our data indicate that transgene silencing results from heterochromatin formation. One of the goals of this project is to subvert transgene silencing by altering the propensity of vector to form heterochromatin. In Specific Aim 1 we examine multiple different approaches to stimulate euchromatin formation, that chromatin state posited to support long term gene expression. A second issue pertinent to the development of PD gene therapy is to direct different therapeutic genes to each compartment of the diseased nigrostriatal pathway: dopamine neurons and target striatum. In Specific Aim 2 we will develop separate vectors which will afford direct expression of different gene products to each anatomical compartment. A third issue for successful PD gene therapy is evaluation in appropriate animal models of the disease. Specific Aim 3 will employ two animal models: Our novel a-synuclein mice which develop progressive nigrostriatal dysfunction, reduction of substantia nigra TH and hypokinetic activity; and our modified chronic MPTP model which produces striatal denervation, dopaminergic cell loss and a neurobehavorial syndrome. The proposed studies will yield optimized HSV vectors, provide a detailed understanding of their characteristics, and evaluate their effectiveness in mechanistically different models of PD.

The Biological Measurement Core (BMC) will provide PDGTSG-affiliated investigators with state-of-the-art molecular biological resources that facilitate the study of gene transfer effects at the cellular and molecular levels, and a comprehensive computer database that will promote the seamless exchange of data within the PDGTSG. The BMC will perform quantitative nucleic acid measurements using "real-time" PCR to assess vector genome retention, transgene expression, and vector-elicited immune marker expression. The "real-time" quantitative PCR technology has proved to be a reproducible and high throughput method for quantitation of specific nucleic acid sequences (RNA or DNA) from cells and tissues isolated from animals receiving the proposed gene-based treatments. In addition, inverse PCR will be performed by the BMC to assess the effects vector integration may impart on host chromosomal integrity. By taking advantage of the expertise and facilities extant at the University of Rochester, the BMC seeks to provide these technologies to PDGTSG investigators. The Core will also maintain the PDGTSG Database that will serve as the central repository for all collected data, including gene expression data, vector construct sequences, safety assessments, and images. The BMC will be housed on the first floor of the recently constructed Arthur Komberg Medical Research Building within the Center for Aging and Developmental Biology. The BMC will be directed by Dr. Howard J. Federoff, while day-to-day Core laboratory activities will be performed by a technical associate and database operations by a database manager.

[unreadable] DESCRIPTION (provided by applicant): A unifying characteristic of all transmissible spongiform encephalopathies (TSE) is the conversion of the normal cellular prion protein (PrPc) to an abnormal conformation, designated PrPsc. The conversion confers an increase in beta- sheet content and a decrease in sensitivity to protease K. Recent data in PrPsc challenged transgenic mice that constitutively express PrPc-specific antibodies and PrPc-specific antibody treated cell cultures chronically infected with Prpsc showed a failure to accumulate PrPsc and demonstrated an ability to clear chronic infection of PrPsc. This application proposes to positively affect the conversion of PrPc in C57BL/6 mice challenged with PrPsc by utilizing a herpes amplicon-based gene therapy strategy to express novel single-chain variable fragment (scFv) antibody coding sequences. Three aims are proposed. Isolation, subcloning, and in vitro expression of PrPc-specific scFv antibody coding sequences: A previously characterized combinatorial phage display library expressing human immunoglobin heavy and light chains variable regions will be used to identify phage clones capable of binding to recombinant mouse prion protein (rec MoPrP). Immunoglobulin coding sequences from identified clones and a murine Ig kappa secretion signal will be subcloned into HSV amplicon vectors. Efficiency of in vitro expression and secretion of functional PrPc-specific scFv antibodies will be assessed by real-time quantitative RT-PCR, ELISA, and Western blot analysis. Characterization of in vivo expression of PrPc-specific scFv antibodies in C57BL/6 mice: C57B1/6 mice will be administered HSV amplicons via intracerebral (IC) route. In vivo secreted expression levels, kinetics, and migration from IC administration site of PrPc-specific scFvs in vaccinated mice will be assessed via immunohistochemical (IHC) at 3, 7, 14, 30, 60, and 90 days post-injection time-points via detection of an scFv epitope tag. Deleterious effects of expressing PrPc-specific scFvs on behavior of C57BL/6 mice and signs of autoimmune disease will be examined. Assessment of vaccine efficacy in PrPsc-challenged mice: Efficacy of approach will be tested in C57BL/6 mice challenged with mouse-adapted PrPsc. Mice will be IC-challenged with PrPsc at the apex of PrPc-specific scFv expression. Efficacy will be assessed by end-point PrPsc levels in brains and spleens of treated, challenged mice. Gross motor coordination behavioral will also be evaluated in treated mice by rota-rod performance. [unreadable] [unreadable]

Parkinson's disease (PD) affects about 1 million people in North America. Medications, such as levodopa, and some surgical approaches are available for PD, but offer only symptomatic therapy. New information contribute to current optimism that gene therapy might correct the molecular disturbances of PD, alleviate the symptoms of the illness and/or in retarding disease progression. Setbacks in gene therapy for other diseases underscore the importance of a purposely deliberate and careful approach that demands substantial assurances of safety and potential efficacy in advance of human testing. It is this philosophy of conservatism that will characterize the activities of our group. A coordinated stepwise progression from basic research through exhaustive preclinical evaluation prior to clinical testing is required. A multicenter, multidisciplinary collaborative group (The PD Gene Therapy Study Group [PDGTSG]) has formed and seeks support for those activities that will lead to a large-scale clinical trial of gene therapy for patients with PD. The PDGTSG consists of three different components: Cores, Principal Projects, and Pipeline Projects. Core A. Administrative Core (PI: Dr. Federoff): Houses a Steering Committee, and Vector (Chair: Dr. Lowenstein), Human Subjects/Clinical Assessment (Chair: Dr. Kurlan), Bioethics (Chair: Ms. Greenlaw), Intellectual Property (Chair: Ms. Hunter) and Biostatistics Modules (Chair: Dr. Oakes). Provides for the coordination of budgeting, committee scheduling, reports, progress preparation, and interface with NINDS staff, the clinical, scientific and lay community. Core B. Biological Measurement Core (PI: Dr. Federoff: Functions in the application shared quantitative measurements. Houses the database and the bank of vector constructs used in all studies. Project I. "Enzymatic Gene Transfer in MPTP Monkeys" (PIs: Bankiewicz and Kordower) Will comprehensively evaluate two vector platforms (rHIV and rAAV), each transducing the identical AADC gene cassette in the standardized non-human primate model. Project II. "Trophic Gene Transfer in MPTP Monkeys" (PIs: Bankiewicz and Kordower) Will comprehensively evaluate two vector platforms (rHIV and rAAV), each transducing the identical regulated GDNF gene cassette in the standardized non-human primate model. PIPELINE PROJECTS 0PPs) Focus 1: 1reproved regulation of gene expression PP I. "Tet-Regulated Vectors for Parkinson's Disease" (PI: Bohn). PP II. "Engineering RNA Switches that Respond to Dopamine and its Analogs" (PI: Breaker). Focus 2: Development of new vector platforms for application in PD disease models. PP III. "High Capacity Gutless Adenovirus" (PI: Lowenstein). PP IV. "Development of Integrating HSV Amplicons for Parkinson's Disease" (PI: Bowers). Focus 3: Development of methods to effectively control and distribute lentiviral and AAV vectors throughout the non-human primate brain. PP V. "Enhanced Delivery Methods for Widespread Distribution of Lentiviral and AAV Vectors" (PI: Bankiewicz). PP VI. "Analysis of Vector and Transgene Product Transport and Distribution" (PI: Bankiewicz) Focus 4: Development of imaging methods to detectgene expression. PP VII. "Design, Construction and Evaluation of PET Imaging Gene Surrogate" (PI: Muhkerjee). Focus 5: Development of a hioethics approach to PD gene therapy. PP VIII. "Measuring and Explaining PD Patients' Participation Preferences Regarding Phase I Studies in Gene Transfer Therapy" (PI: Kim).

DESCRIPTION (provided by applicant): Alzheimer's Disease (AD) pathogenesis has been linked to the abnormal processing of amyloid precursor protein (APP) resulting in excessive extracellular accumulation of Abeta peptide within plaque. In a pathogenic state Abeta entry into plaque must exceed its rate of removal. Peripheral vaccination with aggregated Abeta elicits an immune response resulting in reduction of apparent central nervous system amyloid by a mechanism that is presumed to involve CNS entry of antibodies and in situ removal from plaque. Humoral immunity has been implicated in this protective response, as some believe peripherally derived Abeta-specific antibodies act by altering Abeta equilibrium between the CNS and periphery thus preventing CNS deposition. The contribution of each of these processes may be a function of the type of antibody and its antigenic recognition properties. We hypothesize that immunization with forms of Abeta which undergo progressively enhanced degrees of fibrillarization will elicit immune responses against toxic higher-order Abeta structural intermediates that, in turn, will engender differential protection against disease pathogenesis. This application proposes to use a potent herpes virus-based vaccine strategy to determine the role of Abeta fibril formation in elicitation of strong anti-Abeta immune responses and how such host responses disrupt disease processes. Three aims are proposed. Characterization of fibrillar and non-fibrillar Abeta peptide-expressing; amplicon-based vectors. Amplicons encoding the wild-type sequence for Abeta1-40 will be constructed along with vectors encoding mutated forms of Abeta shown previously to more readily form fibrillar structures. All vectors will be assessed in vitro for expression, antigen presentation, transgene product fibrillarization, and transgene product cytotoxicity profiles. Assessment of amplicon vectors in C57BL/6J mice. In vivo analyses of these vectors in C57BL/6J mice will be undertaken, including the profile of humoral and cellular immune responses as well as any untoward effects Abeta immunization may impart on behavior in normal mice. Assessment of immunization strategy in murine models of AD: Amplicon vectors will be administered to Sw C57BL/6J, SwDI C57BL/6J, and Tg2576 C57BL//6J mice to examine efficacy of the approach. Abeta burden, levels, and effects on CNS in these mice will be assessed using immunological, biochemical, and behavioral methods. Overall, these studies will elucidate the role of fibrillarization in the genesis of disease and establish the utility of amplicon-based vaccination for AD.

DESCRIPTION (provided by applicant): Late onset Alzheimer's disease (LOAD) is a chronic neurodegenerative disorder that typically manifests clinically in the elderly. Interestingly, a variety of postmortem evidence suggests that the pathological hallmarks of AD, and by inference the disease itself, begin to occur early in an individual's life. This has led to an emerging view of LOAD whereby a set of disparate mechanistic triggers over a life-time converge upon shared biochemical pathways to elicit a phenotypically similar clinical syndrome and neuropathological state. This convergent pathophysiological hypothesis asserts that specific downstream biochemical pathways mediate the synaptic loss, cellular injury, and death observed in LOAD. Furthermore, many of these pathophysiological changes will be manifest in peripheral systems, which share these signaling pathways. We hypothesize that the hematopoietic system shares many cellular signaling pathways with the nervous system and is affected by many of the same pathophysiological changes that characterize LOAD. Specifically, we propose that peripheral leukocytes are affected by LOAD pathogenic processes, which will be reflected m alterations in protein levels and functions. As such, these changes will serve as important biomarkers for LOAD diagnosis and progression and will provide valuable insights into its pathophysiology and potential therapeutics. Our Specific Aims will test the following hypotheses using both exploratory and directed proteomics approaches in two cohorts of well-characterized, age-matched subjects with and without LOAD. Aim 1; Exploratory proteomics approaches will identify unique protein profiles in peripheral leukocytes in LOAD subjects compared to non-LOAD subjects. Hypothesis: Quantitative protein profiling in leukocytes will identify profiles of proteins that will identify LOAD subjects from age- and gender-matched non-demented control, non-demented Parkinson's disease, and mild cognitive impairment (MCI) subjects. Aim 2: Directed proteomics approaches will validate these biomarkers and will classify subjects with LOAD from non-LOAD subjects. Hypothesis: Directed Proteomics methods (Western blots, immunoprecipitation Western blots, ELISAs, activity assays) will validate our biomarker set in the first cohort of subjects and will classify LOAD from non-demented control subjects in a second cohort. Taken together, these studies will identify a unique set of leukocyte proteins that will differentiate LOAD from non-LOAD subjects and will provide important information on the diagnosis, progression, pathophysiology, and potential therapies for LOAD.

[unreadable] DESCRIPTION (provided by applicant): Parkinson's disease (PD) is an age-related neurodegenerative disorder typified by selective loss of nigrostriatal dopaminergic neurons. The etiology of Parkinson's disease is currently unknown but hypothesized to involve both genetic vulnerability and environmental toxicants. Despite disparate initiating factors a shared biochemical pathway is posited to elicit a phenotypically similar clinical syndrome and neuropathological state. This convergent pathophysiological hypothesis asserts that specific downstream biochemical pathways mediate the synaptic loss, cellular injury, and death observed in Parkinson's disease. We posit that these pathophysiological changes will be manifest in peripheral compartments, which share these signaling pathways. Specifically, we propose that peripheral leukocytes will report the PD pathogenic process and will be reflected in alterations in protein levels and functions. As such, these changes will serve as important biomarkers for PD diagnosis and progression and will provide valuable insights into its pathophysiology and potential therapeutics. We propose to utilize exploratory approaches on human peripheral leukocytes within select clinical populations, coupled with directed methodologies for validation. In the proposed study we will use an exploratory proteomics approach to provisionally identify differential protein expression in leukocytes from patients with PD (non-demented), late onset Alzheimer's disease (LOAD), mild cognitive impairment (MCI) and from age-matched control (non-demented) subjects. We currently have leukocyte protein samples from a total of 12 subjects per group and all subjects have been well characterized with respect to clinical stage and neuropsychological testing. 2-dimensional gels have been completed for these samples. In this study we will use computer-based analysis (Progenesis software) to identify differentially expressed proteins. Furthermore, we propose to sample an additional 13 subjects/group for a total number of 25 subjects in each of the four groups. Following exploratory analyses, these profiles will be validated using standard proteomics methods in the initial cohort as well as in a separate cohort of PD and control subjects (15 subjects each). We currently have leukocyte samples from all 30 subjects in this second cohon and prepared to validate identified biomarkers. We hypothesize that these approaches will in aggregate identify profiles of proteins within peripheral leukocytes that will categorize PD subjects from non-PD subjects. We believe that these unique and innovative studies will provide a wealth of proleomics information that will translate to improved PD diagnosis and evaluation of therapeutic interventions. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Nectin-1 is a cell adhesion molecule localized the puncta adherentia junctions in synapses. Nectin-1 associates with other proteins, which collectively participate in the formation of neuronal synapses. We hypothesize that nectin-1-undergoes a regulated multi-step set of endoproteolytic cleavage events by several sheddases in an activity-dependent manner and that these events regulate synaptogenesis and contribute to synaptic plasticity. Our preliminary data indicate that nectin-1 undergoes ectodomain shedding by at least two sheddases that result in the production of two C-terminal fragments (CTFs). These CTFs are further cleaved intramembraneously by y-secretase and liberate the NE-ICD from the plasma membrane. The released NEICD translocates into the nucleus and where we postulate it induces gene expression. In Specific Aim 1, we will determine the secretase cleavage sites of nectin-1 by immunoaffinity purification, followed by Edman degradation sequencing. We will also investigate which genes are regulated by NE-ICD in hippocampal neurons by CodeLink Bioarrays. Then, using quantitative RT-PCR, ICC, and Western blotting we will confirm differentially expressed genes in neurons. The molecules that interact with NE-ICD will be identified by a yeast two-hybrid screen. Once interactors are identified, their biological function will be assayed by cellular and molecular approaches. In Specific Aim 2, we will investigate the biological role of BACE1 in nectin-1 processing. Initial experiments indicate that BACE1 associates with and participates in the shedding of nectin-1. We will investigate how disruption of nectin-1 shedding, through loss of BACE1 function, affects synapse formation and synaptic plasticity by ICC, Western blotting and-vesicle recycling assays. Our preliminary data indicate that two nectin-1 point mutations, T310A and Y311A, are refractory to BACE1 cleavage and can /ra".y-dominantly interfere with processing of endogenous nectin-1. We will examine how these point mutants affect the synapse formation and synaptic function by transduction of hippocampal neurons and in vivo adult hippocampus with recombinant adeno-associated viral vectors. We will quantitatively measure the changes in synaptic markers, synapse morphology, and size by ICC, live cell imaging and synaptic activity. Subsequently, we examine whether expression of trans-dominant nectin-1 mutants will affect hippocampal dependent learning

[unreadable] DESCRIPTION (provided by applicant): Late onset Alzheimer's disease (AD) and Parkinson's disease (PD) are chronic neurodegenerative disorders that affect nearly six million Americans and are estimated to cost the United States economy over $100 billion annually. Unfortunately, current therapies and treatments are only symptomatic and these diseases remain relentlessly progressive. Thus, our need to fully understand the pathogenesis of these diseases and to design molecular diagnostics and improved pharmacotherapies is vitally important to our nation and to our health care systems. Interestingly, while these diseases typically manifest clinically later in life, a variety of postmortem evidence suggests that the pathological hallmarks of both AD and PD, and by inference the disease itself, occur early in an individual's life. This has led to an emerging view of the disease whereby a set of disparate mechanistic triggers over a lifetime converge upon shared biochemical pathways to elicit a phenotypically similar clinical syndrome and neuropathological state. This convergent pathophysiological hypothesis asserts that specific downstream biochemical pathways mediate the synaptic loss, cellular injury, and death observed in either AD or PD. Furthermore, many of these pathophysiological changes will be manifest in peripheral systems, which share these signaling pathways. We hypothesize that the hematopoietic system shares many cellular signaling pathways with the nervous system and is affected by many of the same pathophysiological changes that characterize AD and PD. Specifically, we propose that peripheral monocytes/macrophages are affected by early pathogenic processes, as well as may contribute to disease pathology, which will be reflected in alterations in transcript and protein levels and functions. As such, these changes will serve as important antecedent biomarkers for disease diagnosis and progression and should provide valuable insights into disease pathophysiology and potential therapeutics. We propose to use two murine models of these diseases, the triple transgenic model of AD (3xTg-AD) and the human wild-type synuclein transgenic model of PD (hSYN-WT+/+), which will allow us to collect tissues over the time course of each "disease." In Specific Aim 1 we will collect peripheral monocytes/macrophages from these transgenic mouse strains and wild type control mice and interrogate gene expression using microarrays at three time points. We will identify transcriptomic profiles that will be specific for each disease model (e.g. AD vs. PD vs. control) and that will be more generalizable to common neurodegenerative processes. In Specific Aim 2 we will test the specificity and sensitivity of these profiles in peripheral monocytes/macrophages isolated from human AD, PD, and control subjects. We hypothesize that the transcriptomic signatures identified in these studies will be important to our understanding of disease diagnosis, pathogenesis, and therapy in AD and PD. With the aging of America's "baby boomers" the need to fully understand the pathogenesis of this disease and to design molecular diagnostics and improved pharmacotherapies is vitally important to our nation and our health care systems. As such, it is necessary to develop easily accessible, robust, specific, and sensitive biomarkers of early AD and PD, which would greatly facilitate the diagnosis and treatment of these diseases. [unreadable] [unreadable] [unreadable]

Stroke is a leading cause of death and disability in the United States, for which effective treatments remain elusive. Astrocyte survival is likely an important determinant of stroke volume and neuronal survival as loss of astrocyte viability would be predicted to augment stroke damage by glutamate release, collateral damage, and loss of their neuronal supportive functions. Thus, dissecting the molecular mechanisms by which astrocyte viability is altered during stroke would be an important advance. A principal mechanism by which astrocytes adapt to the hypoxic environment during stroke through the actions of hypoxia inducible factor 1 alpha (HIF-1a). HIF-1a, the master regulator of the cellular response to hypoxia, is a transcription factor stabilized and activated during hypoxia. HIF-1a markedly increases the expression of multiple genes including those that would be predicted to improve survival and ones that would have a pro-apoptotic function. The conditions that control the pro-survival or pro-apoptotic functions of HIF-1a remain undefined. Preliminary data suggests that HIF-1apro- apoptotic functions predominate in astrocytes during severe hypoxic stress enhancing cell death. The molecular mechanisms by which HIF-1cc induces cell death is unknown but may involve induction of pro-apoptotic members of the Bcl2 family Nip3, Nix, or Noxa. Furthermore, by associating with thepro- apoptotic transcription factor p53, HIF-1a may increase the pro-apoptotic functions of p53 during severe hypoxic stress. In contrast, pro-survival functions may be selectively induced during mild hypoxia. In fact, several reports suggest that HIF-1a plays a prominent role in the neuroprotection afforded by the mild hypoxic stimulus of hypoxic preconditioning (HPC). Furthermore, hypoxia mimetic (HM) compounds, which induce HIF-1a protein and transactivation of HIF-1a target genes, are neuroprotective when used as a preconditioning stimulus and are actively being pursued as stroke therapeutics. This proposal will evaluate the role of HIF-1a in mediating pro-survival or pro-death functions in astrocytes during stroke, following HPC, and with administration of HM compounds. We postulate that HIF-1a function in astrocytes contributes to astrocyte cell death during the severe hypoxia and ischemia present during stroke. In contrast, we predict that during the mild hypoxic stress of hypoxic preconditioning, HIF-1a function in astrocytes mediates improved astrocyte and neuronal viability. Using in vitro and in vivo methodology, we will examine these postulates.

DESCRIPTION (provided by applicant): This is an M01 application for a new General Clinical Research Center (GCRC) sponsored by Georgetown University and MedStar Health named the "Georgetown University-MedStar General Clinical Research Center." The Georgetown University Clinical Research Center was first established in 1995, and was funded as a new GCRC by the NIH in September, 1999 (M01-RR020359). Due to intervening financial difficulties and faculty attrition at Georgetown University, at the time of the first competitive renewal in 2004 a decision was made to allow Children's National Medical Center to become the sponsoring institution of the Georgetown University GCRC, with Georgetown University as a satellite. The competitive renewal application was reviewed in June, 2004 and was approved with a recommendation for administrative review of the Georgetown University satellite at the end of the second year. This M01 application is therefore for a "new" GCRC at Georgetown University as was required by the NCRR Advisory Council in the 2004 review of our original GCRC. We are proposing a change in our name from the previous "Georgetown University GCRC" to the "Georgetown University-MedStar GCRC" in recognition of our successful partnership with the MedStar Health System and the MedStar Research Institute. This application first summarizes the history and accomplishments of the Georgetown University GCRC since its inception in 1999;our strategic partnership with MedStar Health in 2000;and our success at stabilizing the research, educational and clinical missions of Georgetown University Medical Center since that time. We then propose a detailed plan to re-establish the Georgetown University-MedStar GCRC as the central component of a much larger integrated regional clinical research network throughout the MedStar Health System in the greater Baltimore-Washington DC area.

[unreadable] DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a chronic neurodegenerative disorder that typically manifests clinically in the elderly. Interestingly, a variety of postmortem evidence suggests that the pathological hallmarks of AD, and by inference the disease itself, begin to occur early in an individual's life. This has led to an emerging view of AD whereby a set of disparate mechanistic triggers over a life-time converge upon shared biochemical pathways to elicit a phenotypically similar clinical syndrome and neuropathological state. This convergent pathophysiological hypothesis asserts that specific downstream biochemical pathways mediate the synaptic loss, cellular injury, and death observed in AD. Furthermore, many of these pathophysiological changes will be manifest in peripheral systems, which share these signaling pathways. We hypothesize that the hematopoietic system shares many cellular signaling pathways with the nervous system and is affected by many of the same pathophysiological changes that characterize AD. Specifically, we propose that peripheral leukocytes are affected by AD pathogenic processes, which will be reflected in alterations in protein levels and functions. As such, these changes will serve as important biomarkers for AD diagnosis and progression and will provide valuable insights into its pathophysiology and potential therapeutics. We propose to identify and collect serial clinical measurements and biological samples from three cohorts: subjects at high risk for developing mild-cognitive impairment (MCI)/AD (>75 years old with a first degree relative diagnosed with AD); age-and gender-matched subjects at low risk; and newly diagnosed, drug-naive subjects with MCI/AD. In Specific Aim 1 we will undertake an extensive clinical and biomolecular examination of all high risk subjects that progress to a diagnosis of MCI/AD compared to an appropriately matched subset of low risk subjects without MCI/AD to discover and validate a potential biomarker profile of disease. In Specific Aim 2 we will test the specificity and sensitivity of this profile in a second subset of the low risk cohort without MCI/AD and early, drug-naive MCI/AD subjects. We hypothesize that the clinical-biomolecular profile identified in these studies will be important to our understanding of disease diagnosis, pathogenesis, and therapy in AD. With the aging of America's baby boomers the need to fully understand the pathogenesis of this disease and to design molecular diagnostics and improved pharmacotherapies is vitally important to our nation and our health care systems. As such, it is necessary to develop robust, specific, and sensitive biomarkers of early AD, which would greatly facilitate the diagnosis and treatment of this disease. PUBLIC HEALTH RELEVANCE: We hypothesize that the clinical-biomolecular profile identified in these studies will be important to our understanding of disease diagnosis, pathogenesis, and therapy in AD. With the aging of America's baby boomers the need to fully understand the pathogenesis of this disease and to design molecular diagnostics and improved pharmacotherapies is vitally important to our nation and our health care systems. As such, it is necessary to develop robust, specific, and sensitive biomarkers of early AD, which would greatly facilitate the diagnosis and treatment of this disease. [unreadable] [unreadable] [unreadable]

Parkinson's disease is a slowly progressive degenerative disorder with classic motor symptoms that include resting tremor, cogwheel rigidity and bradykinesia. The disease produces invariant loss of dopamine neurons in the substantia nigra (SN) with the hallmark pathological features of activated microglia and proteinaceous cytoplasmic inclusions called Lewy bodies in the remaining neurons. Epidemiological data supports that gene-environment interactions are responsible for the largest proportion of sporadic PO cases. One of the key issues in PO is the identification of the initiating triggering mechanism(s) and the locus of its injury. Efforts to elucidate th[unreadable]ls mechanism have been aided by the linkage between toxicant (e.g., MPTP, paraquat. rotenone) injury, oxidative stress, inherited defects in turnover of the presynaptic and Lewy Body constituent protein a-synuclein (SYN), and involvement of cytosolic dopamine. Using established animal models of wild-type (wtSYN+/+) and mutant SYN (dmSYN+/+) overexpression in OA producing cells we will address the hypothesis that an early effect of SYN overexpression is increased microglial activation and proinflammatory processes leading to presynaptic dysfunction. We further posit that wtSYN and dmSYN activate microglia differentially but both result in an increased quinone oxidant response. Three aims are proposed to test this hypothesis: Ai m 1. Characterization of microglial activation and presynaptic function in mice overexpressing wild-type (wtSYN+/+) or double-mutant SYN (dmSYN+/+); Aim 2. Characterization of the proinflammatory response to wtSYN and dmSYN: Aim 3. Examination of the role of quinone-mediated oxidative stress in microglial activation. We will utilize several unique transgenic mouse models including wtSYN+/+ that overexpress wIld-type SYN, dmSYN+/+ that overexpress mutant SYN, wtSYN+/+::AREhPLAP and dmSYN+/+::AREhPlAP which overexpress SYN in the background of a mouse capable of reporting quinone-mediated stress. These studies will produce clear and interpretable data concerning the role of microglia in the presynaptic injury elicited by SYN. Parkinson 's Disease (PO) is the second most common neurodegenerative disease and impacts both patients and their caregivers. The goal of this project is to evaluate the progressive changes in the brain during PD. We focus our work on both tissue culture and animal models of PO and ask what is the role of pro-inflammatory molecules and microglia in disease initiation and progression . Particularly we will study early changes in PD. The results of these studies may lead to the identIfication of potential therapies for PD.

DESCRIPTION (Provided by Applicant): Parkinson's disease (PD) is a neurodegenerative disorder characterized by death of substantia nigra (SN) dopamine neurons (DANs), dopamine deficiency within the striatum and a clinical movement disorder. Most PD is sporadic/idiopathic that may arise from gene-environmental interactions. Among familial forms, mutations in the LRRK2 are most common contributing to over 10% of autosomal dominant familial PD and 3.6% of sporadic cases. The most frequent mutation, G2019S, occurs within the kinase domain of this 2527 amino acid protein. Mutations within the GTPase domain (e.g., R1441C) have also been associated with disease in the human population. We have shown that viral vector delivery to the mouse nigrostriatal pathway of human LRRK2-G2019S, but not human wild-type LRRK2, results in marked dopaminergic neuron loss in the SN. Given the need to develop additional non-toxicant models that may possibly be more predictive in drug development, we plan to extend the murine observations to the non-human primate (NHP). Herein, we examine whether HSV amplicon-directed expression of G2019S or R1441C in the striatum of NHPs will establish a useful and novel PD model for drug discovery. Our approach involves: 1) Production and characterization of HSV amplicon LRRK2 mutant viral vectors. Wild-type LRRK2 (LRRK2WT), G2019S (LRRK2G2019S), LRRK2 kinase dead (LRRK2KD) and R1441C (Roc/GTPase domain;LRRK2R1441C) will be subcloned into HSV amplicon vectors that co-express enhanced green fluorescent protein (eGFP;HSVPrPuc/CMVegfp). These will then be packaged into helper virus-free HSV amplicon vectors, titred and further tested for expression. 2 and 3) Establishment, Validation and Characterization of a novel PD model for drug discovery. Convection-enhanced delivery of each amplicon vector unilaterally into the striatum of rhesus macaque monkeys, which will be followed longitudinally by 18fluoro-methyl tyrosine PET imaging and a Clinical Rating Scale (CRS) off and on levodopa and apomorphine. At euthanasia brains will be harvested for histological analysis of neuropathology, neurochemistry and gene expression. 4) Translational evaluation of a LRRK2 Kinase Inhibitor (KI). Screening of a library of KI compounds has yielded one molecule, GW5074, as a candidate with specificity against LRRK2-G2019S. An improved novel compound has been prepared and will be further evaluated. ADME will be undertaken to establish an active dose, schedule of administration and metabolites. The selected dose will be confirmed as neuroprotective in the HSV amplicon transduced mouse model using DAN number as a readout. SAR will be undertaken on the pharmacophore to derive an optimized molecule. Evidence of compound neuroprotection against LRRK2G2019S toxicity will enable NHP testing. The compound will be evaluated using PET imaging and clinical rating. Subjects will be euthanized and brains harvested for neuropathology, neurochemistry and gene expression. Other organs will be harvested for future pathological analyses. Blood will be collected for future drug and metabolite analyses. This translational effort is intended to be IND-enabling. Public Health Relevance: Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by death of substantia nigra dopamine neurons (DANs). There is a great need for novel non-toxicant models of PD that may possibly be more predictive in drug development. We propose to extend our previous murine observations to non-human primate models by overexpressing various forms of the most commonly mutated PD gene. This model will also be used to test novel therapeutic small molecule LRRK2 inhibitors.

DESCRIPTION (provided by applicant): The Division of Comparative Medicine (DCM) is the sole centralized animal resource core facility at Georgetown University. The mission of the facility is to provide optimal husbandry and veterinary care, specialized technical support and appropriate regulatory oversight for research studies involving laboratory animals. The 50,740 sq. foot facility actively supports 133 research investigators with 218 animal care and use protocols. The building is comprised of 2 wings - the "Grey" Side and the "Blue" Side, constructed in 1986 and 1995, respectively. Fifty-seven investigators house animals on the Grey Side and utilize research support spaces. In 2006, the University initiated a 3-phase, 5 year plan to replace the 23 year old mechanical systems on the Grey Side. Two NCRR G20 grant projects are currently underway to complete the first 2 planned phases including the replacement of non-operational humidifiers;aged exhaust fans, cage wash equipment and the building management system;and upgrade of the dedicated cage wash facility HV system. This proposal seeks to complete Phase 3 of the institutional long range plan by replacement of 3 air handling units (AHUs) and associated mechanical support equipment. "In-kind" equipment replacement is requested, with all work performed solely in mechanical spaces (4,072 sf). Redundancy of the existing AHUs will allow phased replacement with minimal disruption of ventilation and ongoing research studies. This project embraces sustainable design principles consistent with LEED criteria. American jobs (15-23 FTE) would be maintained during the 21 month project period. Ten additional full time DCM jobs would be maintained after renovation. Mechanical systems renovation on the Grey Side of the resource core facility is critical to maintain the animal research program at Georgetown University. Optimal ventilation and proper sanitation of equipment/facilities safeguard animal health and support the conduct of PHS and other extramural supported research. If these mechanical systems fail, the institution risks the loss of irreplaceable animal models, 57 funded research programs, AAALACi accreditation and USDA registration.