Kathleen Maguire-Zeiss - US grants

[unreadable] DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of nigrostriatal dopaminergic neurons. The etiology of sporadic Parkinson's disease remains unknown although epidemiologic studies implicate to gene environment interaction. Progress in Parkinson's disease modeling in rodents has been achieved by administration of specific toxicants and through construction of transgenic mice harboring human a-synuclein. The pathogenic linkage between toxicant and a-synuclein appears to lie in their unique capacities to produce oxidative injury. Less well investigated is role of glial-neuronal interactions promoting oxidative damage and death of ventral midbrain dopamine neurons. We hypothesize that neuronal overexpression of \vildtype human a-synuclein triggers ROS that are, in part, defended by local glial anti-oxidant responses. Over time this defense mechanism fails resulting in pathologic a-synuclein misfolding, presynaptic dopamine neuron injury and ultimately cell death. To test each facet of this hypothesis, we engineered a compound transgenic mouse to specifically overexpress human wild type a-synuclein in dopaminergic cells (SYN+/+) on the background of glial depleted glutathione peroxidase 1 (GPX-/-) and an antioxidant promoter-reporter. This compound transgenic animal (SYN+/+::GPX-/-::AREhPLAP) affords the study of and cellular locus of oxidative injury wrought by a-synuclein and the impact of impaired glial anti-oxidant capacity on dopaminergic neuron function and viability. Three Aims have been proposed. Aim 1. The evolution of oxidant injury in human wild type a-synuclein homozygous mice (SYN+/+::AREhPLAP). Aim 2. Synergistic injury in SYN+/+::GPX-/-::AREhPLAP mice: a model of accelerated dopaminergic neuron compromise. Aim 3 In which cell types does restoration ofGpx-1 mitigate environmental toxicant injury in SYN+/+::GPX-/-::AREhPLAP mice. These studies will produce clear and interpretable data concerning the role of glia anti-oxidant defense in oxidative injury elicited by a-synuclein alone and in combination with a known dopaminergic toxicant. The mechanistic information derived may enable new glial-oriented therapeutic initiatives. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Classical microglial activation may contribute to neuronal injury occurring with HIV associated neurological disorders (HAND). Emerging evidence suggests that matrix metalloproteinases (MMPs) could play a critical role in stimulating such activation. For example, inhibition of MMP activity blocks microglial activation in response to stimuli including lipopolysaccharide. In addition, minocycline, a potent inhibitor of MMP expression and activity, abrogates microglial activation occurring with osmotic demyelination as well as with simian immunodeficiency viral (SIV) infection. The mechanisms by which MMPs might activate microglia are not completely understood. Of interest, however, is the potential for MMPs to generate ligands for integrins that are highly expressed on microglia. Integrin dependent signaling can in turn stimulate changes associated with an activated phenotype. That this mechanism could be important is underlined by data showing that inhibition of microglial integrin expression, or function, blocks microglial phagocytosis and migration. In addition, recent studies have shown that select integrin antagonists can substantially reduce microglial activation and associated neurotoxicity in more than one disease model. In previous publications we have shown that HIV proteins can increase MMP release from brain derived cells, and that MMP levels are increased in spinal fluid samples from patients with HAND. In the present application, we hypothesize that these MMPs generate specific cell adhesion molecule (CAM) fragments that will in turn engage microglial integrins. Our focus on CAM fragments is based on several considerations. CAMs are easily accessible by virtue of their proximity to the cell surface, an area where MMP activity may be concentrated. We and others have shown that MMPs stimulate ectodomain shedding of these molecules, and elevated levels of soluble forms can be detected in spinal fluid samples from patients with brain inflammation. Moreover, we have recently published data showing that the shed domain of at least one CAM can interact with a microglial integrin that has been well linked to an activated phenotype. In the present R21 proposal we plan to identify microglial integrin-binding ligands that may be increased in association with HIV, to investigate the hypothesis that these ligands stimulate classical, pro-inflammatory microglial activation, and to determine whether this activation is of sufficient magnitude to be inimical to vulnerable neurons. We will focus on CAMs that are widely expressed in the CNS, including the immunoglobulin (Ig) domain containing intercellular cell adhesion molecules (ICAMs) and synaptic CAMs (synCAMs), as well as the cadherins. We will also focus on integrins that mediate microglial activation in other disease models, such as LFA-1 and Mac- 1. This dual PI grant relies on expertise related to MMPs (KC), as well as microglia and dopaminergic neurons (KMZ). The possibility that MMPs contribute to microglial activation in the setting of HIV is yet untested and clinically relevant, in that it would allow MMP inhibitor to be used in an attempt to reduce such activation. Further study of the receptors that underlie MMP dependent effects is also clinically relevant. At least one soluble CAM can interact with LFA-1, a microglial integrin for which a clinically tolerable antagonist has been developed.

DESCRIPTION (provided by applicant): Project Summary/Abstract This is an application for the competitive renewal of an Institutional Training Grant in Neural Injury and Plasticity (NIP). We request support for 4 advanced predoctoral students who will be trained in research on neural injury and plasticity by faculty participating in the Center for Neural Injury and Recovery (CNIR) at Georgetown University. The purpose of this training program is to prepare scientists to investigate fundamental mechanisms of neural injury, by trauma, stroke or neurodegenerative processes and to understand basic mechanisms of neural plasticity that may be functionally beneficial to the repair processes after neural injury. Our goal is to train researchers who will b capable of and committed to the basic science component of developing novel and effective treatment strategies to reduce the functional impairments that result from neural injury. An experienced and well-funded group of 27 faculty members with a wide range of research interests and expertise in neural injury and plasticity will participate in the Ph.D. training program. Students will enter graduate school through the Georgetown University Interdisciplinary Program in Neuroscience (IPN). In the first two years they will take course work and rotate through the laboratories of potential mentors. Those interested in the NIP Program will take one or more specifically relevant courses, begin to participate in the NIP Journal Club and also obtain a clinical experience with an outstanding group of clinician-scientists, to provide basic scientists with an appreciation of the experiences of patients and clinicians dealing with neural injury and plasticity. In the second year, students will formally apply to the NIP Training Program with the outline of a thesis research proposal approved by a potential mentor or co-mentors from the NIP Training Faculty. As NIP Trainees they will participate in the weekly NIP Journal Club as well as educational programs of the CNIR and plan to report results from their research in yearly student seminars, presentations at national meetings and as publications in peer reviewed journals. With respect to public health, this program will create a cadre of neuroscientists trained for research and/or management of research programs, through which new and more effective treatments for acute and degenerative disorders of the nervous system can be treated. PUBLIC HEALTH RELEVANCE: Project Narrative This research training program is focused on the development of advanced predoctoral students in the area of neural injury and responses to injury. With respect to public health, this program will create a cadre of neuroscientists traind for research and/or management of research programs, through which new and more effective treatments for acute and degenerative disorders of the nervous system can be treated.

DESCRIPTION (provided by applicant): Protease activated receptor-1 (PAR-1) is a G protein coupled receptor (GPCR) that is highly expressed on neurons and microglia. Levels of PAR-1 activators, including plasmin and matrix metalloproteinases (MMPs), are substantially increased in HIV associated neurological disorders (HAND) and one study has shown that levels of PAR-1 are increased as well. Though not yet studied in the context of HAND, PAR-1 antagonists can prevent microglial activation and neurotoxicity in animal models of Parkinson's disease and cerebral ischemia. Recent studies have shown that select GPCRs can associate with ?-arrestins to activate the kinase glycogen synthase kinase-3??(GSK-3?) through a novel, non-canonical signaling pathway. Importantly, GSK- 3?? inhibitors have been shown to reduce neuronal injury in response to HAND relevant stimuli. In addition, increased GSK-3?? activity has been implicated in HAND relevant pathology including microglial activation, neurotoxicity, and long term depression of synaptic transmission. In the present dual PI R01 proposal, we hypothesize that excess activation of PAR-1 stimulates non- canonical GPCR dependent signaling pathways to measurably contribute to HAND relevant microglial activation and neuronal injury. Our plan will be to test underlying mechanisms with in vitro studies (Aims 1 and 2) and the relative in vivo importance of this pathway to phenotypic changes observed in mouse models (Aim 3). Preliminary data in support of our hypothesis will include evidence for GSK-3?? activation and cognitive impairment in mice that overexpress a potent PAR-1 agonist. Preliminary data will also show evidence for PAR-1 dependent, non-canonical signaling, in CNS derived cells isolated from these animals. Innovation comes from the study of a relatively unexplored receptor as related to HAND, the study of a novel signaling pathway for this receptor, and the use of a unique mouse model. Innovation also comes from techniques that include recordings of neuronal activity via multielectrode arrays, and small animal magnetic resonance spectroscopy. The overall goal of our proposal is to identify targets for adjunct therapeutics. If PAR-1 activation can stimulate HAND relevant pathology through increased GSK-3?? activity, novel drugs being developed to more specifically inhibit the activity of this kinase could be considered for treatment of this condition. Moreover, newly developed orally available PAR-1 antagonists that are now in clinical trials for coronary artery disease might be considered for treatment of the same.

Project Summary/Abstract This is an application for the competitive renewal of an Institutional Training Grant in Neural Injury and Plasticity (NIP). We request support for 4 advanced predoctoral students who will be trained in research on neural injury and plasticity by faculty participating in the Center for Neural Injury and Recovery (CNIR) at Georgetown University. The purpose of this training program is to prepare scientists to investigate fundamental mechanisms of neural injury, by trauma, stroke or neurodegenerative processes and to understand basic mechanisms of neural plasticity that may be functionally beneficial to the repair processes after neural injury. Our goal is to train researchers who will be capable of and committed to the basic science component of developing novel and effective treatment strategies to reduce the functional impairments that result from neural injury. An experienced and well-funded group of 20 faculty members with a wide range of research interests and expertise in neural injury and plasticity will participate in the Ph.D. training program. Most students will enter graduate school through the Georgetown University Interdisciplinary Program in Neuroscience (IPN). In the first two years they will take course work and rotate through the laboratories of potential mentors. Those interested in the NIP Program will take one or more specifically relevant courses and begin to participate in the NIP Journal Club. In the second year, students will formally apply to the NIP Training Program with the outline of a thesis research proposal approved by a potential mentor or co-mentors from the NIP Training Faculty. As NIP Trainees they will participate in NIP Specific Training which includes: clinical experience with an outstanding group of clinician-scientists, to provide basic scientists with an appreciation of the experiences of patients and clinicians dealing with neural injury and plasticity; the weekly NIP Journal Club and extended integrative reasoning and statistical literacy training; professional development training (i.e., Elevator pitches, grant writing and presentation skills, effective communication skills) as well as monthly meetings focused on in-depth discussions of disorders specific to neural injury and plasticity. Trainees will report results from their research in yearly student seminars, presentations at national meetings, and as publications in peer-reviewed journals. With respect to public health, this program will create a cadre of neuroscientists trained for research and/or management of research programs, through which new and more effective treatments for acute and degenerative disorders of the nervous system can develop.

Due to strong excitatory input, reliable GABA release and fast firing, parvalbumin expressing (PV) neurons are thought to represent critical pacemakers for synchronous network events. PV neurons also represent the predominant GABAergic neuronal population that is enveloped by the perineuronal net (PNN), a lattice like extracellular matrix that is thought to localize glutamatergic input. Disruption of the PNN has been linked to reductions in PV excitability. Importantly, deficits in PV excitability influence synchronous network events critical to both attention and long-term memory consolidation. In support of this, recent studies have demonstrated that reduced glutamatergic input to hippocampal PV cells, through knockout of PV selective glutamate receptors or a reduction in presynaptic glutamatergic innervation, is linked to increases in sharp wave ripple (SWR) density and deficits in long term memory consolidation. PNN processing occurs through the actions of specific proteases. While metalloproteinases of the ?a disintegrin and metalloproteinase with thrombospondin motifs? (ADAMTS) and secreted matrix metalloproteinase (MMP) family members can cleave specific PNN components, the latter may be particularly important in the background of human immunodeficiency virus (HIV) infection. Soluble MMPs are expressed by neurons and microglia and known to digest PNN components including aggrecan and brevican. In addition, while ADAMTS protein expression is not detected in astrocytes in a simian immunodeficiency virus (SIV) model, PNN degrading MMPs are highly expressed by astrocytes, the most numerous cell type in the brain. Moreover, in murine models of brain injury, selective MMP inhibition reduces PNN remodeling. It has previously been demonstrated that human HIV encoded Tat protein can increase the expression and/or cellular release of MMP-9, a potent modulator of PNN processing. Tat protein is detectable in the cerebrospinal fluid of individuals receiving combination anti-retroviral treatment (cART). In new preliminary data included herein, we show that Tat significantly increases release of MMP-13 from astrocytes. Moreover, we see active forms of MMP-13 in brain tissue lysates from virologically suppressed HIV-infected individuals. In preliminary studies, MMP-13 can efficiently cleaves PNN components. Published work has linked MMP-13 expression to HIV infection, and also shown reduced PNN integrity in the background HIV associated cognitive dysfunction (HAND). Importantly, however, causes and potentially critical neurophysiological consequences of PNN disruption in the setting of HAND have not been well examined. In the present application, we plan to test the hypothesis that HIV relevant stimuli including Tat can stimulate MMP-dependent PNN processing in vitro and in vivo, with consequent effects on hippocampal PV activity, neuronal population dynamics and memory consolidation. !