Ted M. Dawson - US grants

In primary neuronal cultures, N-methyl-D-aspartate neurotoxicity is mediated in part by nitric oxide (NO). The mechanisms involved in NO neurotoxicity, as well as the source of NO is not known. Accordingly, experiments will be performed and designed to further elucidate the role of NO in neurotoxicity. Conditions for the selective removal or inactivation of NO synthase (NADPH diaphorase) neurons will be developed. this will be followed by a detailed analysis of excitatory amino acid neurotoxicity in these cultures. It will be determined whether an inducible NO synthase is formed after excitatory amino acid administration, and whether it plays a role in NMDA neurotoxicity. NO synthase (NADPH diaphorase) neurons are known to be relatively resistant to NMDA neurotoxicity. Experiments will be performed to determine whether NO synthase is involved in this protection. Conditions for transient expression of NO synthase in neurons will be developed and excitatory amino acid neurotoxicity will be studied in detail. In addition, the role that antioxidant enzymes, such as copper/zinc-superoxide dismutase, manganese- superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase play in neurotoxicity and in neuroprotection will be studied. The role of the superoxide anion in mediating NO cell death will be investigated by exploring NMDA neurotoxicity, as well as exogenously applied NO in the presence of various inhibitors of superoxide dismutase and liberators of the superoxide anion. Furthermore, cell lines over and under-expressing manganese-superoxide dismutase will be developed. Employing these cell lines, studies on exogenously applied NO and its subsequent toxicity will be investigated. Finally, the functional consequences of phosphorylation or dephosphorylation of NO synthase will be investigated in primary neuronal cultures after excitatory amino acid administration. In addition, determination of the subtype(s) of glutamate receptor responsible for NO synthase activation will be identified by co-transfection studies.

DESCRIPTION: (adapted from Applicant's Abstract) In primary neuronal cultures, N-methyl-D-aspartate (NMDA) neurotoxicity, and in vitro model of cerebral ischemia, is mediated, in part, by nitric oxide (NO). Despite numerous studies implicating NO in glutamate mediated neurotoxicity, there is evidence that NO may play no role in NMDA neurotoxicity and may in fact be neuroprotective. NO may be neuroprotective by nitrosylating and inactivating the NMDA receptor. NO s double edge sword may be related to it s redox state, with the NO radical being toxic and the nitrosonium ion being neuroprotective. In order to better understand the role of NO in a variety of physiologic processes in which NO has been implicated to regulate including glutamate neurotoxicity, the applicants have generated mice carrying a selective mutation in the neuronal NOS gene by targeted deletion in embryonic stem cells. Utilizing the NOS knockout mice and their wild type controls, a series of experiments are proposed to clarify the potential mechanisms and involvement of NO in neurotoxicity and to identify alternative pathways of toxicity. Neuronal damage due to cerebral ischemia may occur through excess NO production. Combined oxygen-glucose deprivation in neuronal cultures as well as excitatory amino acid administration will be used as in vitro model of cerebral ischemia. The susceptibility to neuronal injury will be evaluated in neuronal NOS knockouts and compared to wild type controls. Experiments will be performed to determine whether targeted deletion of neuronal NOS leads to additional changes that might account for the resistance to glutamate mediated toxicity. The distribution and density of glutamate receptors will be evaluated. 45Ca2+ accumulation will be examined in response to excitatory amino acids in neuronal NOS knockouts and compared to wild type controls. NADPH diaphorase or NOS neurons are resistant to NMDA type neurotoxicity and are highly susceptible to kainate and quisqualate neurotoxicity. NOS and NADPH diaphorase neuronal colocalize with somatostatin and NPY. The NOS knockouts possess normal NPY and somatostatin neurons. As such the susceptibility of these neurons to NMDA, kainate and quisqualate will be examined in NOS knockouts versus wild type controls to determine whether the susceptibility of these neurons is altered in NOS knockouts. Additionally, the distribution of glutamate receptors will be examined on NOS neurons to evaluate whether the differential expression of glutamate receptors accounts for differential susceptibility to toxicity. Other potential pathways of neurotoxicity exist as NOS inhibitors are only partially protective. The neuronal NOS knockouts provide a unique opportunity to examine other potential pathways of glutamate neurotoxicity without the influence of NO. As such, glutamate neurotoxicity and oxygen-glucose deprivation in neuronal cultures will be examined in the neuronal NOS knockouts and compared to wild type controls after the administration of a variety of inhibitors of other potential pathways of neurotoxicity. These agents will include scavengers of the superoxide anion, and free radicals. In addition, the effects of phospholipase A2 inhibitors, cyclooxygenase inhibitors, and lipoxygenase inhibitors will be examined.

neurotoxicology; nerve injury; nitric oxide synthase; nitric oxide; enzyme induction /repression; genetic transcription; oxidative stress; neuroprotectants; ischemia; hypoglycemia; genetic promoter element; growth factor; neurotrophic factors; cytotoxicity; hypoxia; neurotoxins; tissue /cell culture; laboratory mouse;

The overall goals of this proposal are to understand the role of alpha- synuclein in the pathogenesis and pathology of Parkinson's disease (PD) and to define the molecular mechanisms of dopaminergic neuronal injury in animal models of PD. The program represents a multi-disciplinary, mechanistic approach involving interactive, productive investigators with complementary areas of expertise who have long been committed to the studies of neurodegenerative diseases. Their aim will be to integrate the activities of various disciplines than if each project were pursued individually. The program has two themes. First, the biology and pathobiology of alpha- synuclein, implicated in familial PD, will be defined in human brain from PD with and without cognitive impairment and age matched controls. This will be complemented by studying the cell biology of alpha-synuclein in in vivo and in vitro model systems. Transgenic animal models of familial PD will be produced by generating animals over-expressing the A53T and A30P mutations of alpha-synuclein. Second, molecular, transgenic, neuropathologic, cell biologic and neurobehavioral approaches are proposed to examine the mechanism of neuronal injury and neuroprotection in the MPTP model of PD. We will determine the neurotoxic mechanisms associated with nitric oxide signaling and superoxide anion formation and scavenging and the role of poly (ADP ribose) polymerase and potential derangements in the biology of alpha-synuclein in response to MPTP. Also, identification of differentially expressed genes following the MPTP neurotoxicity paradigm will be characterized in the striatum and substantia nigra dopamine containing cells. We believe that our multi-disciplinary approach has the capacity to produce unique information concerning mechanisms of neuronal injury and neuroprotection in animal models of Parkinson's Disease and to lead to better understanding of the function and the role of alpha-synuclein in normal and pathophysiologic processes related to PD. The program consists of four projects: 1) Cognitive Impairment in Parkinson's Disease: The Role of alpha-Synuclein 2) Molecular Mechanism's of MPTP Neurotoxicity; 3) alpha-Synuclein and Interacting Proteins: In Vitro Studies; 4) Biology of alpha-Synuclein in Mouse Models of PD; and is supported by four core facilities: A) Administration and Training; B) Transgenic and Neurobehavior; c) Neuropathology and D) Clinical.

The overall goal of this project is to understand the role of poly(ADP- ribose)polymerase in the pathogenesis and pathology of neuronal injury following excitotoxicity and ischemia-reperfusion injury. The Viral Gene Transfer Core C, has a central role in the Program Project grant and it will interact closely and support the activities of all three projects. The goals of the Viral Gene Transfer Core C are: 1. To provide adenovirus or sindbus virus vectors for expression of poly(ADP-ribose) polymerase (PARP), PARP mutants, and reporter genes to Projects 1, 2, and 3 for infection of neurons in vitro and in vivo. 2. To make new mutations to the various functional domains of PARP and generate adenovirus or sindbus virus vectors of the new mutations and to make viruses containing poly (ADP-ribose) glycohydrolase (PARG) for use by Projects 1, 2 and 3. The staff of the Viral Gene Transfer Core C have experience in generating and amplifying viral vectors, both adenovirus and sindbus virus. The Viral Gene Transfer Core C will be a shared resource of this Program Project grant and will play a central role in our investigations into the actions of PARP in neuronal injury.

Stroke is the third leading cause of death and disability in the United States. Understanding the molecular mechanisms that lead to neuronal injury and cell death may lead to novel therapeutics to treat this devastating disorder. A variety of studies indices in models of ischemia indicate the involvement of oxygen free radicals, oxidative stress and DNA damage in the pathogenesis of stroke. Preliminary studies indicate a potential pivotal role for Poly (ADP-ribose) polymerase (PARP) in the pathogenesis of stroke. PARP is a ubiquitous and unique protein which is activated by DNA strand nicks and breaks which can be induced by neuronal injury. Activation of PARP may lead to the loss of cellular NAD+ and ATP, resulting in acute cell injury and death. These events may be crucial in both short- and longer-term deleterious effects of stroke. However, it is unclear whether this pathway (PARP activation and NAD+ loss) caused by ischemia is solely responsible for cell death, or whether other pathways such as caspase activation, which involves cleavage of PARP and other substrates may contribute to neuronal injury. Accordingly, this project is designed to explore the molecular mechanisms and consequences of PARP activation and cleavage following excitotoxic neuronal injury, as well as, the molecular consequences of poly PARP activation and cleavage following excitotoxic neuronal injury, as well as, the molecular consequences of poly (ADP-ribosyl)ation of target proteins. In Specific Aim #1 we will determine the role of PARP activation and PARP mediated cell death following excitotoxic (NMDA versus kainate/AMPA) and mitochondrial toxin mediated PARP mediated cell death following excitotoxic injury. In Specific Aim #3 we will examine the role of nitric oxide, superoxide generation and peroxynitrite formation in PARP activation and PARP mediated cell death following excitotoxic (NMDA) mediated neuronal injury. Clarifying and understanding the molecular consequences of PARP activation and caspase cleavage and the role reactive oxidants in this process following excitotoxic neuronal injury may yield important insight into the function of PARP that may identify novel targets for therapy aimed at preserving neurologic function following neuronal injury and stroke.

DESCRIPTION (provided by applicant) Parkinson's Disease is a chronic progressive neurologic disorder that affects over half a million men and women in the U.S. alone. Underlying the clinical symptoms of Parkinson's Disease is the degeneration of the neuromelanin-containing neurons predominantly located in the pars compacta of the substantia nigra. Although the pathology underlying the disease is clear, it is still unknown why these substantia nigra pars compacta neurons die. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), can selectively damage neurons in the nigrostriatal dopaminergic pathway and produce Parkinsonism in humans, non-human primates, and mice has provided a valuable model for investigating mechanisms of selective sensitivity of nigral neurons. MPTP mediates this toxicity through inhibition of complex I in the mitochondria. Excitotoxic and oxidant injury are important mediators in MPTP neurotoxicity. We propose a novel and unique pathway that integrates these classic signals with evolving caspase-independent mitochondrial coordinated pathways of cell death. Apoptosis inducing factor (AIF) is a novel cell death effector protein. It is normally confined to mitochondria but translocates to the nucleus following toxic insults. AIF causes chromatin condensation in isolated nuclei and large-scale fragmentation of DNA. The morphology of AIF mediated cell death in HeLa cells is reminiscent of excitotoxic morphology in neurons that is induced by NMDA receptor activation, NO release and PARP activation. MPTP is a toxin that induces dopaminergic cell loss and results in experimental animal models of Parkinson's disease. We propose that AIF might be a link between excitotoxicity and known proteins that mediate apoptosis. This proposal is designed to assess the feasibility of this novel avenue of investigation and develop data in support of this innovative hypothesis of neuronal cell death. We propose that excitotoxicity is a unique form of cell death that involves molecules associated with classic necrosis or apoptosis but these mediators are activated in a unique program that ultimately results in excitotoxic death. This hypothesis would be a paradigm shift in the conceptualization of neuronal cell death. Furthermore, understanding the regulation of AIF and its role in excitotoxicity might provide clues to preventing its release from the mitochondria thus providing neurons a window of opportunity to restore NAD levels and survive toxic insults.

[unreadable] DESCRIPTION (provided by applicant): The overall goals of this proposal are to serve as one of the clinical centers in the Parkinson's Disease (PD) Clinical Neuroprotection Trial and to participate in the large, collaborative, randomized, double-blind trial testing neuroprotective agents in patients with early PD. Our program represents a multidisciplinary, interactive and collaborative group of investigators and clinicians who have long been committed to the studies and care of patients with Parkinson's disease and other related neurodegenerative diseases. Our aim will be to recruit and follow PD patients, including women and minority patients, early in their clinical course per established protocol and rates established by the PD Neuroprotection Clinical Trial.PD is the second most common neurodegenerative disorder, and it affects nearly I million Americans. Although available medical therapies are effective for the treatment of PD early in its course, there are no proven agents that prevent or slow the progression of the neurodegeneration of PD. Tremendous insight into the mechanisms of neurodegeneration of PD has created an opportunity to begin to rationally study potential neuroprotective agents in the treatment of this disorder. As part of the Parkinson's disease research agenda for the NIH published in April 2000, there was a call for the initiation of randomized, controlled clinical trials to test potential neuroprotectants in PD. We propose to participate as one of the clinical centers in this large collaborative trial testing neuroprotective agents in patients with early PD. To address the goals of the trial, we will (1) recruit and follow PD patients, including women and minority patients early in their clinical course per established protocol and rates established by the PD neuroprotection clinical trial; (2) operate a clinical center to a) enroll patients, b) monitor and examine patients per protocols established by the coordination center, c) record, manage, maintain and process clinical trial data, and provide the data to the coordination and statistical centers, and d) ensure safety of subjects and confidentiality of data; (3) adhere to common protocols and cooperate with other centers and the NINDS in the conduct of both pilot studies and the main efficacy studies; and (4) maintain an administrative structure that allows for close collaboration with the selected coordination and statistical centers, other clinical centers, NINDS scientific program personnel, the NIH oversight committee, referring physicians, and lay organizations and support groups.

Stroke is the third leading cause of death and disability in the United States. Understanding the molecular mechanisms that lead to neuronal injury and cell death may lead to novel therapeutics to treat this devastating disorder. A variety of indices in models of ischemia indicate the involvement of oxygen free radicals, oxidative stress and DNA damage and a potential pivotal role for Poly (ADP-ribose) polymerase-1 (PARP-1) in the pathogenesis of stroke. PARP-1 is a ubiquitous and unique protein which is activated by DNA strand nicks and breaks, which can be induced by neuronal injury. Activation of PARP-1 may lead to the loss of cellular NAD+ and ATP, and the mitochondrial release and translocation of apoptosis inducing factor (AIF) resulting in acute cell injury and death. These events may be crucial in both short- and longer-term deleterious effects of stroke. However, it is unclear how PARP-1 activation leads to AIF release. Is it the NAD+ and ATP loss that accompanies PARP-1 activation that leads to AIF release and cell death or does poly(ADP-ribosyl)ation signal the release of AIF and induce the cell death cascade? Accordingly, this project is designed to explore the molecular mechanisms and consequences of PARP-1 activation following excitotoxic neuronal injury, as well as, the molecular consequences of poly (ADP-ribosyl)ation. In Specific Aim #1 we will evaluate the mechanism by which PARP-1 activation leads to release of AIF from the mitochondria and its translocation to the nucleus following cellular injury. In Specific Aim #2 we will identify PAR polymer binding proteins and investigate their role in cell death induced by PARP-1 activation. In Specific Aim #3 we will determine the role of poly (ADP-ribose) glycohydrolase (PARG) in PARP-1 mediated cell death following reactive oxygen species induced cellular injury and excitotoxic mediated neuronal injury through RNAi approaches and in PARG knockouts. And in Specific Aim #4 we will evaluate the role of overexpression of PARG in PARP-1 mediated cell death following reactive oxygen species induced cellular injury and excitotoxic mediated neuronal. Clarifying and understanding the molecular consequence of PARP-1 activation and the role poly(ADP-ribosyl)ation in the release of AIF and cell death following excitotoxic neuronal injury may yield important insight into the function of PARP-1, poly(ADP-ribosyl)ation, PARG and AIF that may identify novel targets for therapy aimed at preserving neurologic function following excitotoxic neuronal injury and stroke.

DESCRIPTION (provided by applicant): The overall goals of this proposal are to understand the role of alpha-synuclein, parkin, DJ-1 and synphilin-1 in the pathogenesis and pathology of Parkinson's disease (PD) and to define the molecular mechanisms of neuronal injury in animal models of PD. The program represents a multi-disciplinary, mechanistic approach involving interactive, productive investigators with complementary areas of expertise who have long been committed to the studies of neurodegenerative diseases. Their aim will be to integrate the activities of various disciplines such that the interrelationships will result in a greater scientific contributions and achievements if each project were pursued individually. The program has one major theme: To understand the role of familial associated genes alpha-synuclein, parkin and DJ-1 in the pathogenesis of Parkinson's disease and related disorders. The role of alpha-synuclein, parkin, DJ-1 and synphilin- 1 in PD pathogenesis will be investigated using molecular, transgenic, neuropathologic, cell biologic and neurobehavioral approaches to examine the mechanism of neuronal dysfunction and injury clue to alterations in these gene products. The mechanism of neuronal loss in Parkin knockout mice and alpha-synuclein A53T transgenic mice will be characterized. We will determine whether parkin interacts with alpha-synuclein and further explore the relation between and parkin, alpha-synuclein and synphilin-1. We will explore alpha-synuclein processing and modifications and the relationship of synphilin-1 to alpha-synuclein. Furthermore, we will investigate the potential function of DJ-1 and it role in PD Pathogenesis. We believe that our multi-disciplinary approach has the capacity to produce unique information concerning the mechanisms of neurodegeneration in genetic animal models of Parkinson's disease and the related synucleinopathies and to lead to better understanding of the function and the role of alpha-synuclein, parkin, DJ-1 and synphilin-1 in normal and pathophysiologic processes related to PD. The program consists of four projects: 1) Mouse Models of Parkin Biology and Pathobiology 2) PD Cell Models: Alpha-synuclein and Interacting Proteins; 3) Mechanisms of Neurodegeneration in Human Alpha-synuclein Transgenic Mice; 4) The Role of DJ-1 in Parkinson's Disease and four cores A) Administration and Training; B) Transgenic and Neurobehavior; C) Neuropathology and D) Clinical.

Mutations in the parkin gene are the main genetic cause of autosomal recessive Parkinson's disease (PD) and mutations in parkin also play a major role in familial PD. Preliminary studies indicate a potential pivotal role for parkin in the ubiquitin proteasomal pathway (UPP) by functioning as an ubiquitin E3 ligase. Most disease causing mutations of parkin are thought to be loss of function mutations that ultimately lead to the absence of ubiquitination and the subsequent failure of UPP-mediated degradation of parkin substrates. Thus, the abnormal accumulation of parkin substrates is thought to play a role in the demise of substantia nigra dopaminergic neurons in patients with parkin mutations. A number of putative parkin substrates have been identified, but their importance in the pathogenesis of PD due to parkin mutations is not known. We propose to characterize parkin knockout mice to formally test the hypothesis that the absence of parkin function is the cause of PD due to parkin mutations. Furthermore, biochemical and proteomic characterization of the parkin knockout mice may shed light on the substrates that are important in the pathogenesis of PD due to parkin mutations. Accordingly experiments are proposed to further characterize the role of parldn and it's substrates in the pathogenesis of PD. In Specific Aim #1 we will characterize parkin knockout mice. In Specific Aim #2 we will evaluate the sensitivity of parkin knockouts to environmental toxins. In Specific Aim #3 we will evaluate the interaction of parkin with the alpha-synuclein interacting protein, synphilin-1 and determine whether parkin mediates K48 or K63 ubiquitin linkages. In Specific Aim #4 we will determine whether parkin interacts with alpha-synuclein by evaluating of the effect of crossing parkin knockout mice with A53T mutant alpha-synuclein transgenic mice and further evaluate the interaction of parkin with the alpha-synuclein interacting protein, synphilin-1. In Specific Aim #5 we will identify and characterize parkin interacting proteins and identify compensatory changes in parkin knockout mice. Development and characterization of parkin knockout, understanding the relationship of parkin, alpha-synuclein and synphilin- 1 in the pathogenesis of PD may provide insight into the molecular mechanisms by which these gene products induce neuronal damage and may provide novel therapeutics and targets to prevent the toxic effects of these familial associated genes in the degenerative process of PD.

DESCRIPTION (provided by applicant): Mutations in the DJ-1 gene are a rare genetic cause of autosomal recessive Parkinson's disease (PD). The DJ-1 protein is either absent or appears to be functionally inactive in the families in which mutation have been identified. Thus, mutations in the DJ-1 gene probably cause PD through a loss of function. It is difficult at this juncture to fully appreciate how mutations in the DJ-1 gene cause PD, as its function is largely unknown. DJ-1 was identified as a hydroperoxide-responsive protein that becomes more acidic following oxidative stress suggesting that it may function as an antioxidant protein. Furthermore, DJ-1 is sumoylated through binding to the SUMO-1 ligase, PIAS, suggesting that it might be involved in the regulation of transcription. Other putative functions of DJ-1 have been raised, but how a loss of function of DJ-1 leads to loss of DA neurons and PD awaits further study. We propose to generate and characterize DJ-1 knockout mice to formally test the hypothesis that the absence of DJ-1 function is the cause of PD due to DJ-1 mutations. Accordingly experiments are proposed to further characterize the role of DJ-1 in the pathogenesis of PD. In Specific Aim #1 we will develop and characterize DJ-1 knockout mice. In Specific Aim #2 we will evaluate the sensitivity of DJ-1 knockouts to environmental toxins including MPTP-induced dopaminergic cell death. In Specific Aim #3 we will determine whether DJ-1 interacts with parkin by evaluating the effect of crossing DJ-1 knockout mice with parkin knockout mice. Development and characterization of DJ-1 knockouts, understanding the relationship of DJ-1 and parkin in the pathogenesis of PD may provide insight into the molecular mechanisms by which these gene products induce neuronal damage and may provide novel therapeutics and targets to prevent the toxic effects of these familial associated genes in the degenerative process of PD.

Mutations in parkin are largely associated with autosomal recessive juvenile parkinsonism (AR-JP). The underlying mechanism of pathogenesis in parkin-associated Parkinson's disease (PD) is thought to be due to the loss of parkin's E3 ubiquitin ligase activity leading to accumulation of parkin substrates due to failure of the ubiquitin proteasome system. A large number of possible parkin substrates have been identified, yet their role in the pathogenesis of PD due to parkin mutations have yet to be clarified. Moreover, the post-translational modifications that potentially regulate parkin's function are not known. We propose to generate and characterize parkin knockout mice to test the role of proteasome dysfunction in PD. Furthermore, we propose to characterize and identify parkin substrates, and identify potential post-translational modifications of parkin that regulate its function, thus providing important new information about the role of UPS dysfunction in PD. To accomplish these goals we propose the following specific aims. In Specific Aim #1 we will generate and characterize parkin knockout mice. In Specific Aim #2 we will evaluate the sensitivity of parkin knockouts to proteasome inhibitors. In Specific Aim #3 we will characterize the Role of Nitrosative Stress on Parkin Function. In Specific Aim #4 we will identify additional and potentially authentic parkin substrates in parkin knockout mice in response to proteasome inhibition. Identification and characterization of parkin substrates and regulatory mechanisms of parkin function may provide novel therapeutics and targets to prevent the toxic effects of this familial associated gene in the degenerative process of PD.

The Transgenic and Neurobehavior Core B Facility has three major aims: 1. To generate and maintain breeding colonies oftransgenic and gene-targeted mouse lines and to provide cohorts of transgenic and gene-targeted mice for studies proposed in Projects #1 to #4 of the Center grant. 2. To establish C57B16 congenic lines of the transgenic mice used throughout the study. 3. To establish methods to examine motor and clinical deficits in mouse models of Parkinson's Disease. The transgenic and neurobehavior core will be a shared resource of the Parkinson's Disease Research Center and will play a central role in our investigations in the pathogenesis of Parkinson's Disease.

DESCRIPTION (provided by applicant): Mutations in the PINK1 gene are a rare genetic cause of autosomal recessive Parkinson's disease (PD). The PINK1 protein is either absent or appears to be functionally inactive in the families in which the mutations have been identified. Thus, mutations in the PINK1 gene probably cause PD through a loss of function. It is difficult at this juncture to fully appreciate how mutations in the PINK1 gene cause PD, as its function is largely unknown. PINK1 was identified as a mitochondrial enriched protein kinase. Loss of function of PINK1 increases cellular susceptibility to oxidative stress. How a loss of function of P1NK1 leads to loss of DA neurons and PD awaits further study. We propose to generate and characterize PINK1 knockout mice to formally test the hypothesis that the absence of PINK1 function is the cause of PD due to PINK1 mutations. Accordingly experiments are proposed to further characterize the role of PINK1 in the pathogenesis of PD. In Specific Aim #1 we will develop and characterize PINK1 knockout mice. In Specific Aim #2 we will we will evaluate the sensitivity of PINK1 knockouts to environmental toxins including MPTP-induced dopaminergic cell death. In Specific Aim #3 we will determine whether PINK1, Parkin and/or DJ-1 participate in a common pathogenic pathway by crossing PINK1 knockouts with Parkin and DJ-1 knockouts. Development and characterization of PINK1 knockouts, understanding the relationship of PINK1 and mitochondrial function in the pathogenesis of PD may provide insight into the molecular mechanisms by which these gene products induce neuronal damage and may provide novel therapeutics and targets to prevent the toxic effects of this familial associated gene in the degenerative process of PD.

[unreadable] DESCRIPTION (provided by applicant): Mutations in the LRRK2/Dardarin gene are a common cause of autosomal dominant Parkinson's disease (PD). Several dominantly inherited missense mutations have been identified in a number of families that exhibit a broad spectrum of neuropathological features, including deposition of alpha-synuclein and tau proteins. LRRK2 (leucine-rich repeat kinase 2) encodes a large, multifunctional protein. It belongs to the ROCO protein family and includes a protein kinase domain of the MAPKKK class and several other major functional domains including Ras/GTPase and WD40 domains. It is difficult at this juncture to fully appreciate how mutations in the LRRK2 gene cause PD, as its function is largely unknown. We propose to generate and characterize LRRK2 knockout mice to gain a better understanding of LRRK2 function and to determine whether the absence of LRRK2 function is the cause of PD due to LRRK2 mutations. We plan to utilize the recent technological advances in the generation of inhibitor sensitizing mutations to introduce a mutation into the LRRK2 kinase domain that will allow the specific inhibition of the LRRK2 signaling cascade. Introduction of the drug in drinking water or by injection will permit the specific and reversible inhibition of LRRK2 kinase activity at any time during development or aging. Such an innovative approach provides an extremely valuable method to dissect the role of LRRK2 signaling in PD. Moreover, it will enable a molecular dissection of the role of LRRK2 kinase activity in maintaining a functional nigrostriatal dopamine system during development, early postnatal development and within the mature and diseased brain. Our targeting strategy will also enable us to create a null mutation in LRRK2 as well as tissue specific deletion of LRRK2 using the Cre-Lox system. Accordingly experiments are proposed to further characterize the role of LRRK2 in the pathogenesis of PD. In Specific Aim #1 we will develop and characterize a reversible and temporally inducible LRRK2 knockout mouse using a novel method of reversible and temporally specific interruption of kinase activity in cells and animals. In Specific Aim #2 we will we will evaluate the sensitivity of LRRK2 gene targeted mice to environmental toxins including MPTP-induced dopaminergic cell death. In Specific Aim #3 we will determine whether LRRK.2, a-synuclein and/or tau participate in a common pathogenic pathway by crossing LRRK2 gene-targeted mice with a-synuclein and tau transgenic mice. Development and characterization of a reversible and temporally inducible LRRK2 knockout mouse, will increase our understanding of the relationship of LRRK2 kinase activity in the pathogenesis of PD. Moreover, it may provide insight into the molecular mechanisms by which mutations in this gene induce neuronal damage and may provide novel therapeutics targets to prevent the toxic effects of this familial associated gene in the degenerative process of PD. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The overall goals of this proposal are to serve as one of the clinical centers in the Parkinson's Disease (PD) Clinical Neuroprotection Trial and to participate in the large, collaborative, randomized, double-blind trial testing neuroprotective agents in patients with early PD. Our program represents a multidisciplinary, interactive and collaborative group of investigators and clinicians who have long been committed to the studies and care of patients with Parkinson's disease and other related neurodegenerative diseases. Our aim will be to recruit and follow PD patients, including women and minority patients, early in their clinical course per established protocol and rates established by the PD Neuroprotection Clinical Trial.PD is the second most common neurodegenerative disorder, and it affects nearly I million Americans. Although available medical therapies are effective for the treatment of PD early in its course, there are no proven agents that prevent or slow the progression of the neurodegeneration of PD. Tremendous insight into the mechanisms of neurodegeneration of PD has created an opportunity to begin to rationally study potential neuroprotective agents in the treatment of this disorder. As part of the Parkinson's disease research agenda for the NIH published in April 2000, there was a call for the initiation of randomized, controlled clinical trials to test potential neuroprotectants in PD. We propose to participate as one of the clinical centers in this large collaborative trial testing neuroprotective agents in patients with early PD. To address the goals of the trial, we will (1) recruit and follow PD patients, including women and minority patients early in their clinical course per established protocol and rates established by the PD neuroprotection clinical trial; (2) operate a clinical center to a) enroll patients, b) monitor and examine patients per protocols established by the coordination center, c) record, manage, maintain and process clinical trial data, and provide the data to the coordination and statistical centers, and d) ensure safety of subjects and confidentiality of data; (3) adhere to common protocols and cooperate with other centers and the NINDS in the conduct of both pilot studies and the main efficacy studies; and (4) maintain an administrative structure that allows for close collaboration with the selected coordination and statistical centers, other clinical centers, NINDS scientific program personnel, the NIH oversight committee, referring physicians, and lay organizations and support groups.

The extent and cost of drug addiction and neuronal injury is staggering. In the National Drug Threat Assessment 2006 published in January 2006 the estimated economic cost of drug abuse to the United States was $180.9 billion. Of this, $97.7 billion was due to non-alcohol related drug abuse. In 1995 the White House Office of National Drug Control Policy estimated that Americans spent $57.3 billion on drugs of abuse. In 1999, about 14.8 million Americans were current users of illicit drugs with an estimated 3.5 million that were dependent on illicit drugs. The economic burden due to illicit drug use is due to both social issues as well as medical problems associated with drug addiction including brain injury and stroke. To address the problem of brain and nervous system disorders investigators have focused attention on describing cell injury mechanisms. However, discovery of cell survival strategies could have profound impact on the treatment of brain injury and loss of function and is an area that has not yet been rigorously investigated. Neuronal preconditioning is a phenomenon in which tissue is severely stressed but allowed to recover. Subsequently, the tissue is remarkably resistant to further toxic events. Preconditioning is dependent on new gene transcription and protein expression. In the last grant we developed a novel screen and identified 31 putative neuroprotective genes and over a thousand late response activity dependent genes. Several neuroprotective genes were known. We characterized several unknown or novel genes. We focused our attention on an unknown gene we named Iduna and a poorly studied transcription factor NF1-A. Expression of both Iduna and NF1-A are regulated by a NMDA and nitric oxide (NO) dependent signaling pathway and expression of either gene provides neuroprotection. We have also identified a novel non-coding RNA regulated by NO that is critically important for neuronal survival. This non-coding RNA we have termed NO inducible gene 17 (NOIG17). We believe NOIG17 may code for a microRNA cassette. Recently microRNA's have been discovered to play a critical role in neuroplasticity, but to date there are no known neuroprotective microRNA's. This proposal will focus on understanding NO signaling resulting in neuroprotection. The long range goal of this work will be to identify targets that can be developed in the future to ease the burden of individuals suffering brain injury due to drug abuse.

The overall goals of this proposal are to understand the role of a-synuclein, parkin, LRRK2 and the relationship with oxidative stress in the pathogenesis and pathology of Parkinson's disease (PD) and to define the molecular mechanisms of neuronal injury in animal models of PD. The program represents a multi-disciplinary, mechanistic approach involving interactive, productive investigators with complementary areas of expertise who have long been committed to the studies of neurodegenerative diseases. Our aim will be to integrate the activities of various disciplines such that the interrelationships will result in greater scientific contributions and achievements if each project were pursued individually. The program has one major theme: To understand the role of familial associated genes a-synuclein, parkin and LRRK2 in the pathogenesis of Parkinson's disease and related disorders. The role of a-synuclein, parkin, LRRK2 and oxidative stress in PD pathogenesis will be investigated using molecular, transgenic, neuropathologic, cell biologic, and neurobehavioral approaches to examine the mechanism of neuronal dysfunction and injury due to alterations in these gene products. We believe that our multi-disciplinary approach has the capacity to produce unique information concerning the mechanisms of neurodegeneration in genetic animal models of Parkinson's disease and the related synucleinopathies and lead to better understanding of the function and the role of a-synuclein, parkin and LRRK2 in normal and pathophysiologic processes related to PD. The program consists of three projects: 1) Biology of Parkin and its Role in Parkinson's Disease; 2) Mechanisms of Neurodegeneration in Human alpha-Synuclein Transgenic Mice; 3) LRRK2 Biology in Parkinson's disease and four cores A) Administration and training, B) Bioenergetics, C) Transgenic and Neurobehavior and D) Clinical.

Project 1: Biology of Parkin and Its Role in Parkinson's Disease Mutations in the parkin gene play a prominent role in Parkinson's disease (PD) as mutations in parkin are the main genetic cause of autosomal recessive PD and mutations in parkin also appear to play a role in familial PD. Parkin plays a pivotal role in the ubiquitin proteasomal pathway (UPP) by functioning as an ubiquitin E3 ligase. Most disease causing mutations of parkin are thought to be loss of function mutations that ultimately lead to the absence of ubiquitination and the subsequent failure of UPP-mediated degradation of parkin substrates. Thus, the abnormal accumulation of parkin substrates could play a role in the demise of substantia nigra dopaminergic neurons in patients with parkin mutations. Moreover, inactivation of parkin through dopaminergic and oxidative and nitrosative stress may play a role in sporadic PD. The stress activated non-receptor tyrosine kinase c-Abl phosphorylates and inactivates parkin and may play a critical role in sporadic PD by inactivating parkin. We propose to characterize the role of c-Abl mediated inactivation of parkin and its relationship to oxidative and nitrosative stress in sporadic PD as well as the role of parkin substrates in the pathogenesis of PD. Understanding the function and role of c-Abl and oxidative/nitrosative stress mediated inactivation of parkin may provide novel therapeutics targets to prevent the toxic effects of parkin deficiency in the degenerative process of PD. RELEVANCE (See instructions): Parkinson Disease (PD) is common neurodegenerative disease with no proven neuroprotective or neurorestorative therapy. Understanding the molecular mechanisms by which parkin inactivation leads to PD may provide novel therapeutic opportunities to maintain parkin in a catalytically active neuroprotective state.

Core Summary: Administrative and Training Core A Facility has four major aims: 1. To provide a centralized administrative facility for the entire Center grant. 2. To provide scientific leadership to the entire Research Center. 3. To provide biostatistical and data management support to all projects and investigators participating in the Center grant. 4. To facilitate and coordinate the training of new investigators in Parkinson's disease and Parkinson's syndromes. The Administrative and Training Core will be a shared resource of the Parkinson's Disease Research Center and will play a central role in our investigations in the pathogenesis of Parkinson's Disease.    

DESCRIPTION (provided by applicant): Stroke and other neurodegenerative disorders are a leading cause of death, disability and loss of quality of life. The importance of glutamate neurotoxicity in cerebral ischemia and neurodegenerative diseases is well documented. Both in vitro and in vivo administration of glutamate and its analogs effectively kill neurons via excitotoxic mechanisms. Poly(ADP-ribose) polymerase-1 (PARP-1) is pivotal in glutamate neurotoxicity and cerebral infarction. Prior studies indicate that NO, or peroxynitrite plays a prominent role in glutamate excitotoxicity and cerebral infarction. Amongst other responses NO, or peroxynitrite, can activate PARP-1, which leads to cell death through the formation of complex and branched poly(ADP-ribose) (PAR) polymer. Recently, apoptosis inducing factor (AIF) has been identified as key mediator of neurotoxicity triggered by glutamate, reactive oxygen species, DNA damage and PAR polymer. AIF resides in the mitochondria in normal healthy cells, but moves to the nucleus following a lethal stimulus in a PARP-1 dependent manner. Blocking AIF from entering the nucleus can spare cells from death. The proteins and the mechanisms that are involved in facilitating the release of AIF from the mitochondria following PARP-1 activation are not known. Accordingly, experiments are proposed to elucidate the underlying molecular mechanisms accounting for AIF release following PARP-1 activation and to investigate the role of PAR polymer in excitotoxic and stroke induced neuronal injury. We hope that understanding the mechanisms of PAR polymer induced cell death, the activation of AIF and the identification of additional components of the PAR/AIF signaling complex will lead to new methods to terminate the toxic actions of PAR polymer and AIF and offer innovative therapeutic approaches to treat neurodegenerative diseases and stroke.

DESCRIPTION (provided by applicant): We are seeking to improve the diagnosis and treatment of patients with Parkinson disease (PD). Five years of support will allow us to characterize a cohort of patients throughout the PD disease spectrum and matched healthy controls in a systematic and generalizable manner, and then obtain their blood and cerebrospinal fluid (CSF) to identify biomarkers. We will assess whether specific posttranslational modifications to c-Abl, a-synuclein, parkin and parkin substrates (AIMP2, FBP-1 and PARIS) are potential biomarkers. Our clinical and cognitive testing and our biofluid ascertainment methods will follow guidelines from the RFA-NS-1211, the Michael J. Fox Parkinson's Progression Markers Initiative (PPMI), and the consensus opinion of the Udall Centers regarding cognitive testing. The clinical characterization will include extensive motor testing as well as assessments of many of the non-motor facets of PD, including cognition, sleep, and smell. Many of our study participants will have agreed to autopsy through their participation in the Johns Hopkins Medicine Morris K. Udall Center of Parkinson Disease Research of Excellence, allowing for confirmation of their clinical diagnosis and further investigation of the biomarkers that we identify. The blood will be obtained every 6 months at the time of clinical characterization and the CSF will be obtained yearly. Success of the clinical characterization will allow for a cohort of well-characterized individuals with blood and CSF that others and we may correlate with markers in their blood and CSF. Multiple reaction monitoring mass spectrometric assays using a Perfinity workstation inline to a Thermo Vantage triple quadrupole mass spectrometer with on-column trypsin digestion will be used to identify specific posttranslational modifications (PTMs) to proteins integral to PD pathogenesis to differentiate individuals with PD from healthy controls and if these PTMs and proteins integral to PD follow the clinical progression of PD. Success of the biomarker testing will determine if these peptides are diagnostic or progression markers for PD.

? DESCRIPTION (provided by applicant): Parkinson's disease (PD) is relentlessly progressive and despite effective symptomatic therapy in the early stages of the illness most patients have substantial morbidity and disability. There is an urgent need to understand the etiology and pathogenesis of PD so that more effective symptomatic therapies and ultimately preventive therapies can be developed. Our hypothesis is that a-synuclein, parkin and LRRK2 contribute to a complex signaling network that results in the pathogenesis of Parkinson's Disease and related disorders. The center will approach this hypothesis in a multidisciplinary manner. The center represents a multi-disciplinary, mechanistic approach involving interactive, productive investigators with complementary areas of expertise who have long been committed to studies of neurodegenerative diseases. Our center will integrate diverse advanced technologies within the center to accelerate discovery. It will work collaboratively within the Udall Center Network to accelerate discovery of disease, new therapies and disease and progression biomarkers. Through the integration of the activities of the various disciplines, the interrelationships will result in a greater scientific contribution than could be achieved if each project or center investigation were pursued individually. The Johns Hopkins Udall Center will serve as a local and national resource for PD research including broad sharing of data and clinical and biospecimen resources and fostering community outreach activities. We will provide and coordinate training of young investigators in the pathogenesis and study of Parkinson's disease and Parkinson's syndromes. The Johns Hopkins Udall Center is focused on understanding the interrelationship between parkin (Project 1), the most common cause of autosomal recessive PD, ?-synuclein (Project 2), which is causal in sporadic PD and LRRK2 (Project 4), the most common cause of autosomal dominant PD and to use our discoveries to develop disease and progression markers (Project 3) and ultimately new therapies. The insights into how these three genes interact in the pathogenesis of PD and the discovery of biomarkers would not be possible without the Udall Center structure as it enables the evaluation of these major causes of PD and the ability to evaluate findings in each arm of PD to elucidate common pathways if they exist as these may provide the best opportunities for comprehensive new therapies and biomarkers. We believe that our multi-disciplinary approach has the capacity to produce unique information concerning the mechanisms of neuronal injury in animal models of PD that will lead to a better understanding of the function and the role of ?-synuclein, parkin, and LRRK2 in normal and pathophysiologic processes related to PD.

PROJECT SUMMARY - PROJECT 1: BIOLOGY OF PARKIN AND ITS ROLE IN PARKINSON'S DISEASE Parkinson's disease (PD) is a complex neurodegenerative disorder that is both sporadic and familial. Mutations in parkin are the most common cause of autosomal recessive PD. In sporadic PD dopaminergic, oxidative and nitrosative stress as well as c-Abl phosphorylation result in inhibition of parkin. Thus, loss of parkin function is elemental to both familial and sporadic PD. Parkin is an E3 ligase, this loss of function leads to accumulation of the substrates, AIMP2 and PARIS. We have found that AIMP2 expression leads to age dependent DA neurodegeneration due to parthanatos. And PARIS expression may lead to loss of mitochondrial quality control that promotes neurodegeneration. Our hypothesis is that parkin inactivation in sporadic PD by nitrosative/oxidative stress, and c-Abl activation leads to phosphorylation of parkin on Y143 (pY143 parkin) and inactivation followed by the accumulation of parkin substrates, loss of mitochondrial quality control and toxicity. In parallel, ?-synuclein is phosphorylated on Y39 (pY39 ?-synuclein) resulting in aggregation and subsequent toxicity. Since aggregated ?-synuclein can lead to mitochondrial dysfunction it creates a feed forward cycle. Aim 1: One of the unifying features of PD is mitochondrial dysfunction. This aim will explore the inter- relationship of PARIS and mitochondrial dysfunction caused by mutations in parkin. We have shown that PARIS is an important pathophysiologic substrate of parkin in PD that transcriptionally represses PGC-1? a major transcriptional co-activator that regulates mitochondrial biogenesis and mitochondrial oxidant stress responses. Aim 2: Inactivation of parkin results in accumulation of both AIMP2 and PARIS. Expression of either AIMP2 or PARIS is sufficient to promote age dependent DA neurodegeneration. The sequence of events activated by PARIS and AIMP2 will be explored to determine if and how these two proteins interact to initiate the cell death program, parthanatos. Aim 3: We observe pY143 parkin and elevated AIMP2 and PARIS in A53T ?-synuclein transgenic mice that raises the question of whether parkin inactivation, PARIS and AIMP2 upregulation and PARP1 activation play a role in ?-synuclein induced neurodegeneration? This possibility will be explored with the ?-synuclein preformed fibrils (PFFs) model of PD. Aim 4: State-of-the-art technology including deep sequencing and SILAM (stable isotope labeling by amino acids in mammals) will be deployed to identify genes and proteins that are regulated by adult conditional knockout of parkin and their relationship to PARIS induction with the goal of identify nodal points in the signal cascade of neurodegeneration that can provide new targets for the treatment of PD.

DESCRIPTION (provided by applicant): Stroke and other neurodegenerative disorders are a leading cause of death, disability and loss of quality of life. The importance of glutamate neurotoxicity in cerebral ischemia and neurodegenerative diseases is well documented. Both in vitro and in vivo administration of glutamate and its analogs effectively kill neurons via excitotoxic mechanisms. Poly(ADP-ribose) polymerase-1 (PARP-1) is pivotal in glutamate neurotoxicity and cerebral infarction. Prior studies indicate that NO, or peroxynitrite plays a prominent role in glutamate excitotoxicity and cerebral infarction. Amongst other responses NO, or peroxynitrite, can activate PARP-1, which leads to cell death through the formation of complex and branched poly(ADP-ribose) (PAR) polymer. Recently, apoptosis inducing factor (AIF) has been identified as key mediator of neurotoxicity triggered by glutamate, reactive oxygen species, DNA damage and PAR polymer. AIF resides in the mitochondria in normal healthy cells, but moves to the nucleus following a lethal stimulus in a PARP-1 dependent manner. Blocking AIF from entering the nucleus can spare cells from death. This form of cell death has recently been designated parthanatos to distinguish it from other types of cell death such as apoptosis, necrosis or autophagic death. Interference with each step of the parthanatic cascade has been shown to be neuroprotective in a variety of models. Once AIF enters the nucleus, large scale DNA fragmentation (chromatinolysis) occurs through poorly characterized mechanisms, which is likely to be the execution step in parthanatic cell death. Accordingly, experiments are proposed to identify and characterize the parthanatos AIF associated endonuclease-1 (PAAN-1) and to investigate the role of PAAN-1 in excitotoxic and stroke induced neuronal injury. The identification of AIF interactors and understanding the mechanisms of PAAN-1 in neuronal injury will lead to new methods to terminate the toxic actions of NO, PAR and AIF and offer innovative therapeutic approaches to treat neurodegenerative diseases and stroke.

? DESCRIPTION (provided by applicant): Parkinson's disease (PD) is relentlessly progressive and despite effective symptomatic therapy in the early stages of the illness most patients have substantial morbidity and disability. There is an urgent need to understand the etiology and pathogenesis of PD so that more effective symptomatic therapies and ultimately preventive therapies can be developed. Our hypothesis is that a-synuclein, parkin and LRRK2 contribute to a complex signaling network that results in the pathogenesis of Parkinson's Disease and related disorders. The center will approach this hypothesis in a multidisciplinary manner. The center represents a multi-disciplinary, mechanistic approach involving interactive, productive investigators with complementary areas of expertise who have long been committed to studies of neurodegenerative diseases. Our center will integrate diverse advanced technologies within the center to accelerate discovery. It will work collaboratively within the Udall Center Network to accelerate discovery of disease, new therapies and disease and progression biomarkers. Through the integration of the activities of the various disciplines, the interrelationships will result in a greater scientific contribution than could be achieved if each project or center investigation were pursued individually. The Johns Hopkins Udall Center will serve as a local and national resource for PD research including broad sharing of data and clinical and biospecimen resources and fostering community outreach activities. We will provide and coordinate training of young investigators in the pathogenesis and study of Parkinson's disease and Parkinson's syndromes. The Johns Hopkins Udall Center is focused on understanding the interrelationship between parkin (Project 1), the most common cause of autosomal recessive PD, ?-synuclein (Project 2), which is causal in sporadic PD and LRRK2 (Project 4), the most common cause of autosomal dominant PD and to use our discoveries to develop disease and progression markers (Project 3) and ultimately new therapies. The insights into how these three genes interact in the pathogenesis of PD and the discovery of biomarkers would not be possible without the Udall Center structure as it enables the evaluation of these major causes of PD and the ability to evaluate findings in each arm of PD to elucidate common pathways if they exist as these may provide the best opportunities for comprehensive new therapies and biomarkers. We believe that our multi-disciplinary approach has the capacity to produce unique information concerning the mechanisms of neuronal injury in animal models of PD that will lead to a better understanding of the function and the role of ?-synuclein, parkin, and LRRK2 in normal and pathophysiologic processes related to PD.

ABSTRACT Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder after Alzheimer's disease. Although PD is associated with Lewy body formation in the substantia nigra and other regions of the brain, the pathologic and metabolic alterations occurring during the onset and progression of PD have not been clearly defined. Despite a critical need for a reliable diagnostic marker for PD, there is currently no such biomarker that can be used accurately in clinical practice for establishing a definitive diagnosis of PD. The difficulty of identifying reliable biomarkers can be attributed to the variability of clinical samples, low abundance of proteins that are involved in PD pathogenesis and the lack of reproducibility in validating biomarker candidates. To overcome these limitations, we propose use of a large cerebrospinal fluid (CSF) cohort with greater statistical power for true discovery and deep proteome analysis to discover PD biomarkers that are involved in PD pathogenesis, but are present at low abundance. In addition, multiplexed sample analysis by isobaric tandem mass tagging (TMT) with a common reference for data normalization will ensure robust analytical precision of quantitative proteomic data for discovery from a larger set of samples. Moreover, additional proteomic analysis of substantia nigra will be used to select those biomarkers that show differential expression in CSF as well as substantia nigra. These discovery platforms will use a bioinformatics approach to select the most plausible candidates for targeted validation studies followed by an intensive validation of the discovered biomarker candidates. To achieve these goals, we propose three aims: Specific Aim 1: To discover proteins that are differentially expressed in patients with Parkinson's disease. We plan to carry out a quantitative proteomic analysis of CSF and substantia nigra samples from patients with PD and from controls by employing TMT-based multiplexing technology. With this approach, we expect to obtain a more comprehensive coverage of a larger number of proteins quantified across the analyzed samples. Specific Aim 2: To prioritize candidates based on an integrative analysis of alterations in CSF and substantia nigra. By integrating the expression changes in CSF and substantia nigra with a network approach that takes advantage of the known biological pathways that have been described in PD, our approach should be able to select reliable PD biomarker candidates for validation by targeted PRM experiments. Specific Aim 3: To validate candidate protein biomarkers in a larger cohort using targeted parallel reaction monitoring (PRM) mass spectrometry using CSF samples from a PD cohort at Johns Hopkins. Biomarkers that are selected by selection algorithms based on these PRM experiments will finally be confirmed with blinded PDBP CSF samples. Through the approaches outlined above, we expect to discover and validate reliable PD biomarkers in a reproducible fashion.

? DESCRIPTION (provided by applicant): Parkinson's disease (PD) is relentlessly progressive and despite effective symptomatic therapy in the early stages of the illness most patients have substantial morbidity and disability. There is an urgent need to understand the etiology and pathogenesis of PD so that more effective symptomatic therapies and ultimately preventive therapies can be developed. Our hypothesis is that a-synuclein, parkin and LRRK2 contribute to a complex signaling network that results in the pathogenesis of Parkinson's Disease and related disorders. The center will approach this hypothesis in a multidisciplinary manner. The center represents a multi-disciplinary, mechanistic approach involving interactive, productive investigators with complementary areas of expertise who have long been committed to studies of neurodegenerative diseases. Our center will integrate diverse advanced technologies within the center to accelerate discovery. It will work collaboratively within the Udall Center Network to accelerate discovery of disease, new therapies and disease and progression biomarkers. Through the integration of the activities of the various disciplines, the interrelationships will result in a greater scientific contribution than could be achieved if each project or center investigation were pursued individually. The Johns Hopkins Udall Center will serve as a local and national resource for PD research including broad sharing of data and clinical and biospecimen resources and fostering community outreach activities. We will provide and coordinate training of young investigators in the pathogenesis and study of Parkinson's disease and Parkinson's syndromes. The Johns Hopkins Udall Center is focused on understanding the interrelationship between parkin (Project 1), the most common cause of autosomal recessive PD, ?-synuclein (Project 2), which is causal in sporadic PD and LRRK2 (Project 4), the most common cause of autosomal dominant PD and to use our discoveries to develop disease and progression markers (Project 3) and ultimately new therapies. The insights into how these three genes interact in the pathogenesis of PD and the discovery of biomarkers would not be possible without the Udall Center structure as it enables the evaluation of these major causes of PD and the ability to evaluate findings in each arm of PD to elucidate common pathways if they exist as these may provide the best opportunities for comprehensive new therapies and biomarkers. We believe that our multi-disciplinary approach has the capacity to produce unique information concerning the mechanisms of neuronal injury in animal models of PD that will lead to a better understanding of the function and the role of ?-synuclein, parkin, and LRRK2 in normal and pathophysiologic processes related to PD.

The synucleinopathies are collection of disorders characterized by accumulation of aggregated ?-synuclein and include dementia with Lewy bodies (DLB), Parkinson's disease (PD), Parkinson's disease with dementia (PDD), and multisystem atrophy (MSA). These synucleinopathies show different distributions of ?-synuclein pathology but all show pathology in the substantia nigra dopamine neurons. Together, DLB and PDD are estimated to make up ~25% of all degenerative dementias worldwide. Based on post-mortem brain studies, Heiko Braak and colleagues hypothesized that ?-synuclein pathology spreads from cell to cell in a retrograde (dendrite to axon) pattern from the brain stem anteriorly. Subsequent studies have observed ?-synuclein aggregates in PDD and DLB gastrointestinal (GI) tract. Coupled with observations that vagotomy is significantly protective against PD suggests that the origin of ?-synuclein spreading pathology may be in the GI tract. We have modeled this process using injection of ?-synuclein pre-formed fibrils (PFFs) into the pylorus and duodenum of mice. We observe a spread of ?-synuclein pathology in a pattern reminiscent of that described by Braak. Using this model, we propose a series of studies to identify neural circuits that accumulate ?-synuclein aggregates in GI-inject PFF mice and determine how this accumulation affect the synaptic function and connectivity within the circuit. In this application, we propose to (1) molecularly and anatomically identify input neurons to subtantia nigra dopamine neuron using recombinant rabies virus based RNAseq method, (2) determine the timecourse and extent of toxic ?-synuclein spread in these input neurons, and (3) determine the consequence of ?-synuclein spread on the function and connectivity of neurons innervating the nigral dopamine neurons. At the completion of these experiments, we will have improved our understanding how ?-synuclein disrupts neural circuits involved in cognitive processes, the underlying cause of dementia in DLB and PDD.

ABSTRACT Progressive supranuclear palsy (PSP) is a devastating atypical parkinsonian disorder that currently lacks meaningful symptomatic therapies, reduces lifespan and greatly impairs daily function and quality of life. It is often difficult to distinguish from Parkinson disease (PD) clinically, which is crucial for appropriate and timely management, prognosis and clinical trial enrollment. Despite a critical need for a reliable diagnostic marker for parkinsonian disorders, there is currently no biomarker that can be used in routine clinical practice to distinguish between PSP and PD. The purpose of this project is to discover cerebrospinal fluid (CSF) biomarkers that reliably distinguish between PSP, PD and healthy individuals. The difficulty of identifying reliable biomarkers can be attributed to the variability of clinical samples, low abundance of proteins that are involved in the pathogenesis of PSP and PD, and the lack of reproducibility in validating biomarker candidates. To overcome these limitations, we propose use of a large CSF cohort with greater statistical power for true discovery, and deep proteome analysis to reveal PSP biomarkers that are involved in PSP pathogenesis, but are present at low abundance. In addition, multiplexed sample analysis by isobaric tandem mass tagging (TMT) with a common reference for data normalization will ensure robust analytical precision of quantitative proteomic data for discovery from a larger set of samples. Moreover, additional proteomic analysis of brain tissue will be used to select those biomarkers that show differential expression in CSF as well as the globus pallidus, a representative brain region used to pathologically define PSP. These discovery platforms will utilize a bioinformatics approach to select the most plausible candidates for targeted validation studies followed by an intensive validation of the discovered biomarker candidates. To achieve these goals, we propose four aims: Specific Aim 1: To prospectively collect CSF on patients with clinically well-characterized PSP. Specific Aim 2: To discover proteins that are differentially expressed in patients with PSP compared to controls and PD. We plan to carry out a quantitative proteomic analysis of CSF and globus pallidus samples from patients with PSP, PD and from controls by employing TMT-based multiplexing technology. With this approach, we expect to obtain a more comprehensive coverage of a larger number of proteins quantified across the analyzed samples. Specific Aim 3: To prioritize PSP biomarker candidates based on an integrative analysis of alterations in CSF and globus pallidus. By integrating the expression changes in CSF and brain tissue with a network approach that takes advantage of the known biological pathways that have been described in PSP, our proposal will be able to select reliable PSP biomarker candidates for validation by targeted PRM experiments. Specific Aim 4: To validate candidate protein biomarkers in a larger cohort using targeted parallel reaction monitoring (PRM) mass spectrometry using CSF samples from a PSP cohort at Johns Hopkins University, the University of Pennsylvania, UCSF and PDBP. Biomarkers that are selected by algorithms based on these PRM experiments will finally be confirmed using blinded PDBP CSF samples from PSP and will be compared to CSF samples from PD. Through the approaches outlined above, we expect to discover and validate reliable PSP biomarkers that are distinguishable from PD in a reproducible manner.

Transneuronal Propagation of Pathologic ?-Synuclein from the Gut to the Brain Models Parkinson?s disease. Sangjune Kim1,2, Seung-Hwan Kwon1,2, Seung Pil Yun1,2, Tae-In Kam1,2, Nikhil Panicker1,2, Senthilkumar S. Karuppagounder1,2, Saebom Lee1,2, Jun Hee Lee1,2,10, Wonjoong Richard Kim1,2, Minjee Kook1,2, Catherine A. Foss3, Chentian Shen3,11, Subhash Kulkarni4, Pankaj J. Pasricha4, Gabsang Lee1,2,5, Martin G. Pomper3, Valina L. Dawson1,2,5,6,8, Ted M. Dawson1,2,5,7,8,9,#, and Han Seok Ko1,2,8,9,# 1Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, 2Department of Neurology, 3The Russell H. Morgan Department of Radiology and Radiological Science, 4Center for Neurogastroenterology, Department of Medicine, 5Solomon H. Snyder Department of Neuroscience, 6Department of Physiology, 7Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA 8Adrienne Helis Malvin Medical Research Foundation, 9Diana Helis Henry Medical Research Foundation, New Orleans, LA 70130, USA Present Address: 10Department of Pharmacology and Toxicology, University of Alabama at Birmingham School of Medicine, Birmingham, AL 35294, USA 11Department of Nuclear Medicine, Shanghai Jiao Tong University Affiliated Sixth People?s Hospital, Shanghai 200233, China #Correspondence: tdawson@jhmi.edu (T.M.D.), hko3@jhmi.edu (H.S.K.) Analysis of human pathology led to the Braak hypothesis that ?-synuclein (?-syn) pathology spreads from gut to brain, via the vagus nerve. Here, we test Braak?s hypothesis by assessing ?-synucleinopathy in the brain in a novel gut-to-brain ?-syn transmission mouse model, where pathological ?-syn preformed fibrils (PFF) were injected into the duodenal and pyloric muscularis layer. Spread of pathologic ?-syn in brain, as assessed by phosphorylation of serine 129 of ?-syn, was observed first in the dorsal motor nucleus, then in caudal portions of the hindbrain including the locus coeruleus, and much later in basolateral amygdala, dorsal raphe nucleus, and the substantia nigra pars compacta. Moreover, loss of dopaminergic neurons, motor and non-motor symptoms were observed in a similar temporal manner. Truncal vagotomy and ?-syn deficiency prevented the gut-to-brain spread of ?- synucleinopathy and associated neurodegeneration and behavioral deficits. This study implicates the Braak hypothesis in the etiology of idiopathic PD.

PROJECT SUMMARY There is emerging evidence that SARS-CoV2 or COVID19 gains entry into human brain cells leading to a sequela of neurologic symptoms. There is concern that SARS-CoV2 may lead to neurotoxicity and that neuronal death in the regions of the brain that control respiration and cardiac function may be a contributing factor to the acute loss of cardio respiratory function and death. SARS-CoV2 could gain access to the brain by several routes including through the nose, or neurons innervating infected lung tissue or through the cells lining blood capillaries in the brain. COVID-19 patients lose their sense of smell or taste often before the onset of respiratory symptoms and a patient presented with Guillain-Barré syndrome before testing positive for SARS- CoV2. It appears that at least 36% of COVID19 patients had neurologic manifestations including headache, nausea, loss of consciousness, strokes, confusion, encephalitis, meningitis and seizures. These clinical observations strongly indicate a role for SARS-CoV2 in the death of neurons and importantly the brain may be one of the first tissues infected and affected. The actions of SARS-CoV2 on the different cells in the brain, as well as the infectivity, tropism, and replication in brain cells is not yet known. In this application we propose to evaluate: (1) The tropism and replication of SARS-CoV2 in human microglia, astrocytes, neurons, and determine relative susceptibility? (2) The mechanisms of cellular injury and evaluate potential protective approaches. (3) Determine the transcriptional responses to SARS CoV2 infection in human neurons, astrocytes, and microglia at the single cell level to gain new insight into the differential response of brain cells to SARS CoV2 to better understand the neural deficits the virus causes.