Serge Przedborski, Md - US grants

Parkinson's disease (PD) is a neurodegenerative disorder associated with dopamine (DA) cell loss in the substantia nigra (SN). Recent evidence supports the involvement of a toxin in the cause of PD. Moreover, impairments in detoxifying mechanisms identified in PD patients suggest that predisposition to the disease may be related to the inability to detoxify the putative causal agent. The applicant is a clinically trained neurologist with a special interest in PD. The focus of this proposal is the study of possible causal and predisposing mechanisms for PD and has been designed to explore two observations made by the applicant. First, transgenic mice with increased superoxide dismutase (SOD) activity are resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin which damages SN DA cells as seen in PD. Further studies are needed to investigate the role of SOD, a key enzyme in cellular defenses against superoxide radicals, in the MPTP model and in PD. Second, there is an inverse relationship between the number of SN-DA cells an MPTP sensitivity in two different mouse strains. Further investigations are required to explore the basis of this relationship. Specific Aim (SA) I will examine in selected brain regions whether strain- and age-dependent MPTP sensitivity in mice are related to differences in activity of SOD and/or in number of neurons expressing SOD protein and mRNA. To further understand the mechanisms involved in the resistance of SOD-transgenic mice to mPTP, SA II will examine the effects of 1-methyl-4-phenylpyridinium (MPP+), the toxic metabolite of mPTP, and of other specific toxins on cultured DA cells from SOD-transgenic and nontransgenic mice. In SA III, activity of SOD and number of neurons expressing SOD protein and mRNA will be examined in post-mortem PD brains. Also examined will be SOD activity in blood samples from PD patients. Mutations for one SOD isoenzyme have recently been associated with familial amyotrophic lateral sclerosis (ALS), which suggests that, like PD, ALS may be caused by free radicals. Thus, post-mortem and blood sample studies for SOD will also be performed in ALS. In SA IV, the relationship between the number of SN DA neurons and MPTP sensitivity will be examined in different mouse strains in which the number of nigral neurons will be experimentally diminished by a neonatal striatal quinolinic acid lesion. SA V will examine the developmental basis for differences in nigral neuron number among different mouse strains as they relate to number of precursor cells or to degree of developmental cell death. The studies outlined in this proposal will provide further insight into the possible role of free radicals in the pathogenesis of PD and the role played by determinants of nigral neuron number.

DESCRIPTION (Adapted from Applicant's Abstract): The PI is a clinically trained neurologist specializing in Parkinson's disease (PD). The PI has been studying the molecular mechanism of I -methyl-4-phenyl- 1,2,3,6- tetrahydropyridine (MPTP), a toxin that damages substantia nigra (SN) dopamine (DA) neurons as seen in PD. The PI's work has provided compelling evidence that both superoxide radicals and nitric oxide (NO) are implicated in MPTP's toxicity. Both species are modestly reactive, but can combine to produce the highly reactive tissue-damaging peroxynitrite. To elucidate the source of N0 that participates in MPTP's toxicity, Specific Aim (SA)-I will compare the effects of MPTP on SN DA neurons of mutant mice deficient in neuronal, inducible, or endothelial NO synthase (NOS), the three isoforms of NOS. To demonstrate the production of peroxynitrite following MPTP administration, SA-II will quantify striatal and midbrain levels of nitrotyrosine, a stable fingerprint of peroxynitrite's deleterious effects on proteins, at different time-points and doses of MPTP. The requirement for superoxide, NO, and MPTP's active metabolite, 1- methyl-4-phenylpyridinium in protein nitrotyrosine formation, as well as its specificity for DA neurons will also be examined. To define the cell groups and organelles preferentially tyrosine-nitrated, SA-III will ascertain the cellular and subcellular distribution of nitrotyrosine immunoreactivity in the mouse midbrain after MPTP administration. To examine the potential biological consequences of protein tyrosine nitration, SA-IV will assess whether candidate proteins, tyrosine hydroxylase and manganese superoxide dismutase, are nitrated after MPTP administration. The catalytic activity of these enzymes and the search for other proteins that are tyrosine-nitrated after MPTP administration will also be undertaken. This proposal contains a comprehensive set of experiments, which should provide important insights into the role of NO in MPTP toxicity. It should also shed light on the mechanism(s) of neurodegeneration in PD.

This proposal is submitted to pursue our exploration of the pathogenesis of Parkinson's disease (PD). Pertinent to this goal, first, we have found, that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin that kills dopaminergic (DA) neurons in the substantia nigra pars compacta (Snpc) as seen in PD, stimulates the production of hypochlorous acid, a highly reactive species that may contribute to MPTP's deleterious effects. To acquire a better understanding of hypochlorous acid cytotoxic role in the MPTP model, Specific Aim (SA)-1 will quantify brain levels of chlorotyrosine and nitrotyrosine, the two main fingerprints of hypochlorous acid-induced protein oxidative attack, at different time points and doses of MPTP. Since myeloperoxidase (MPO) is the only mammalian enzyme which produced hypochlorous acid, SA-II, will analyze the response of MPO mRNA and protein expression at different time points and doses of MPTP, and will assess the effects of MPTP on the Snpc DA neurons of knockout mice deficient in MPO. Second, we have demonstrated that MPTP can oxidatively damage proteins. However, MPTP can compromise other vital cellular elements, such as DNA, by a similar damage proteins. However, MPTP can compromise other vital cellular elements, such as DNA, by a similar process. However, MPTP can compromise other vital cellular elements, such as DNA, by a similar process. TO demonstrate whether DNA is an intracellular target of MPTP-induced oxidative damage and to explore the consequences of such alteration, SA-III will study the occurrence of DNA strand breakage and of poly(ADP-ribose) polymerase (PARP) activation in the Snpc after MPTP administration. Third, we have found that transgenic mice expressing mutant copper/zinc superoxide dismutase (mSOD1) not only exhibit a dramatic loss of spinal cord motor neurons, but also of Snpc DA neurons. This raises the unique possibility that mSOD1 produces a model of progressive adult-onset degeneration of Snpc DA neurons. To further investigate this model, SA-IV will characterize the neuropathology of mSOD1-mediated Snpc DA neuron sensitivity to MPTP. This proposal contains a comprehensive set of experiments, which would provide insight into the role of hypochlorous acid and its synthesizing enzyme MPO, as well as into DNA damage and PARP activation in the MPTP model. It should also generate significant information on the mSOD1 model of Snpc neurodegeneration. Collectively, the proposed studies should shed light on the mechanisms of neurodegeneration in PD.

DESCRIPTION (from abstract): This proposal is submitted to pursue our exploration of the pathogenesis of Parkinson's disease (PD). Pertinent to this goal, we have found that synuclein expression is increased in the mouse midbrain after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, a toxin that damages the substantia nigra pars compacta (SNpc) as seen in Parkinson's disease. Mutations in the alpha synuclein gene are associated with the development of a familial form of PD> However, to date, the link between synuclein and SNpc neurodegeneration is unknown. To acquire a better understanding about this relationship, Specific Aim I will assess the time course of synuclein mRNA and protein expression alterations in selected brain regions following MPTP regimens that induce SNpc neuronal apoptotic or necrotic death. To determine whether increased expression of synuclein is specific to MPTP-induced neuronal death, Specific Aim II will explore the topographical and temporal expression of synuclein in another model of SNpc degeneration such as that produced by the mutant copper/zinc superoxide dismutase (SOD1) G93A enzyme. Changes in synuclein's secondary structure due to mutation or tyrosine nitration may underlie its toxicity. To assess this possibility, Specific Aim III will determine: (I) the magnitude and location of tyrosine nitration of synuclein following exposure of PC-12 cells to peroxynitrite and to MPTP's active metabolite, 1-methyl-4-phenylpyridinium (MPP+); (ii) the time course of synuclein tyrosine nitration in MPTP-treated mice; and (iii) the requirement for superoxide and nitric oxide (NO) in synuclein nitration by administering Altered pre-synaptic protein synuclein may impair synaptic machinery including MPTP metabolism. Thus, to elucidate whether modulation of synuclein expression can affect susceptibility to MPTP, Specific Aim IV will correlate the severity of MPP+-induced toxicity with the level of synuclein expression in PC-12 cell in which contains a comprehensive set of experiments which should shed light on the role of synuclein in experimental models of SNpc degeneration and possibly in Parkinson's disease.

This proposal is submitted to pursue our investigation of the pathogenesis of amyotrophic lateral sclerosis (ALS) using transgenic mice expressing the glycine-93 yields arginine mutant copper/zinc superoxide dismutase (SOD1G93A). Pertinent to this goal, first, we have shown that inducible nitric oxide synthase (iNOS) is upregulated in glial cells in the spinal cords of affected transgenic SOD1G93A mice. To elucidate the role of iNOS in this model of ALS, Specific Aim (SA)-I will determine the effect of iNOS inhibition or ablation on SOD1G93A-mediated neurodegeneration. Second, we have evidence that the production of the highly-reactive tissue damaging species hypochlorous acid is increased in the spinal cords of affected transgenic SOD1G93A mice. To acquire a better understanding the cytotoxic role of hypochlorous acid in this model of ALS, SA-II will quantify spinal cord levels of chlorotyrosine and nitrotyrosine, the two main fingerprints of hypochlorous acid-induced protein oxidative attack, at different disease stages, in different lines of transgenic mice that express either mutant or wild-type SOD1 and, in transgenic SOD1G93A mice after ablation of neuronal NOS (nNOS), iNOS, or myeloperoxidase (MOP), which is the only mammalian enzyme which produced hypochlorous acid. To explore further the role of MPO. SA-III will (1) define spinal cord expression of MPO mRNA and protein, as in SA-II, at different disease stages and transgenic lines; and (2) assess the effect of MPO ablation on SOD1G93A-mediated neurodegeneration. Third, we have observed that cyclooxygenase-2 (Cox-2), a key enzyme in the synthesis of the pro-inflammatory prostaglandin PGE2, is also markedly increased in the spinal cord of affected transgenic SOD1G93A mice. To demonstrate whether Cox-2 upregulation plays a role in SOD1G93A-mediated neurodegeneration, SA-IV will (1) characterize spinal cord Cox-2 mRNA and protein expression, and PGE2 content, as in SA-II, at different disease stages, transgenic lines, and in transgenic SOD1G93A mice after ablation of iNOS; and (2) assess the effects of Cox-2 inhibition or ablation on SOD1G93A-mediated neurodegeneration. This proposal contains a comprehensive set of experiments, which should provide insights into the role of iNOS, hypochlorous acid and its synthesizing enzyme MPO, as well as into Cox-2 in transgenic SOD1G93A mice. It should also shed light onto the mechanisms of neurodegeneration in ALS.

This proposal focuses on the role of inflammation in the pathogenesis of and therapeutic options for amyotrophic lateral sclerosis (ALS) using transgenic mice expressing the glycine-93--> arginine mutant copper/zinc superoxide dismutase (SOD1G93A). Pertinent to this goal, first, we have shown that content of pro-inflammatory prostaglandin PGE2 is increased in spinal cords of affected transgenic SOD1 G93A mice. To elucidate the role of PGE2-synthesizing enzyme, cyclooxygenase-1 (Cox-1) in this model of ALS, Specific Aim (SA)-I will determine the effect of Cox-1 inhibition and ablation on mutant SOD1-mediated neurodegeneration. Second, we have evidence that the reactive oxygen species-producing microglial enzyme NADPH-oxidase is activated in spinal cords of affected transgenic SOD1G93A mice. To acquire a better understanding of the cytotoxic role of NADPH-oxidase in this model of ALS, SA-II will (1) define spinal cord expression of NADPH-oxidase subunits, at different disease stages in different lines of transgenic mice that express either mutant or wild-type SOD1; and (2) assess the effect of NADPH-oxidase deficiency on mutant SOD1- mediated neurodegeneration. Third, in the spinal cords from affected transgenic SOD1G93A mice, we have observed a robust activation of microglial cells, which may lead to the release of cytotoxic factors. To elucidate whether microglial activation is instrumental in mutant SOD1-mediated neurodegeneration, SA-III will assess the effects of the inhibition of microglial activation on spinal cord inflammatory response and neurodegeneration in transgenic mutant SOD1 mice. Fourth, vaccination of transgenic mutant SOD1 mice with the myelin basic protein-related peptide copolymer-1 (Cop-1) allows accumulation of activated T-cells in the diseased spinal cord which produce anti-inflammatory cytokines such as IL-4 and IL-10 and several neurotrophins. To examine whether such an immunization strategy can be beneficial in transgenic mutant SOD1 mice, SA-IV, will (1) determine the effect of Cop-1 vaccination on disease symptomatology, (2) assess the consequences of Cop-1 vaccination on spinal cord inflammatory response and neuropathology, (3) confirm the role of Cop-1 specific T-cells by adoptive transfer and their accumulation in both the central and peripheral nervous systems, and (4) demonstrate spinal cord production and cellular origin of IL-4 and IL-10 and neurotrophins, at different disease stages.

DESCRIPTION (provided by applicant): In this project, we elect to explore the role of gene/environment interaction by looking at environment not as the repository of noxious factors, but as the vector of complex inanimate and social stimulations. Our hypothesis is that an enriched environment incorporating social interactions, learning, and exercise modulates the effects of genetic risk factors and pathogenic mutations on the natural history of ALS. To test this hypothesis, we will use transgenic mice expressing the glycine-93 --> arginine mutant copper/zinc superoxide dismutase (SOD1G93A), a recognized model of inherited ALS, and well-validated conditions of enriched environment, which have been shown to be beneficial to a variety of pathological situations affecting the adult nervous system. Specific Aim (SA)-I will evaluate the effect of such an enriched environment on the clinical expression of the disease in transgenic SOD1G93A mice. We will determine whether transgenic SOD1G93A mice exposed to an enriched environment become symptomatic later and survive longer than their counterparts exposed to a standard environment. SA-II will elucidate the anatomical correlates of the effects provided by this enriched environment in transgenic SOD1G93A mice. Here, the status of the lower motor neuron pathway and the magnitude of the glial reaction will be compared among transgenic SOD1G93A mice exposed to different environmental conditions. SA-III will investigate the molecular basis of the effects provided by this enriched environment in transgenic SOD1G93A mice. As a first step toward this goal, protein expression profiles in spinal cords from transgenic SOD1G93A mice exposed to the different environmental conditions will be compared using proteomic approaches. This development/exploratory project offers an high-risk/high-yield set of studies which may demonstrate, in an original way, how environment may impact on the occurrence and the progression of ALS.

This proposal is submitted to pursue the exploration of the pathogenesis of Parkinson's disease (PD). Pertinent to this goal, we have found that cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin E2 (PGE2) synthesis, is upregulated in nigrostriatal dopamine (DA)-containing neurons in post-mortem PD tissues and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. We have also shown that both ablation and inhibition of COX-2 protect against MPTP neurotoxicity via a non-inflammatory process. Given this, we now wish to examine first whether the deleterious effects of COX-2 depend upon cytosolic phospholipase A2 (cPLA2), as this enzyme produces COX-2's main substrate. Specific Aim (SA)-I will thus define the temporal and anatomical relationship between cPLA2 and COX-2 in MPTP mice and in PD tissues. A comparison of MPTP neurotoxicity between cPLA2 deficient mice treated with and without a COX-2 inhibitor will also be done. Second, it is also known that DA stimulates PGE2 production, which suggests that this neurotransmitter could exacerbate COX-2's effects on nigrostdatal DA neurons. SA-II will thus assess the effects of DA on cPLA2 and COX-2-activities and on neuronal death in primary ventral midbrain cultures exposed to MPTP's toxic metabolite. In addition, MPTP neurotoxicity will be compared between wild-type and knockout COX-2 mice pre-treated with drugs that increase or decrease brain DA. Third, PGE2 produced by injured nigrostriatal DA neurons may activate PGE2-EP3 receptors that are present on many neurons. To ascertain whether such a mechanism is operative here, SA-III will define EP3 receptor distribution in MPTP mice and in PD tissues; because EP3 receptors can be expressed on both plasma and nuclear membranes, EP3 subcellular distribution in MPTP mice will also be studied. Since activation of nuclear EP3 alters intranuclear calcium uptake and c-los transcription, occurrence of these two events will be determined in nigrostriatal DA neurons after MPTP injection. Evaluation of MPTP neurotoxicity in EP3-deficient mice or in COX-2-deficient mice treated with an EP3 agonist will complete SA-III investigations. Together, these studies should provide a deeper understanding of how COX-2 may contribute to the pathogenesis of PD and to the selective nature of the neurodegenerative process in this disease.

This Neuroscience Core will provide general laboratory services. It will be directed by Drs. Serge Przedborski and David Sulzer. Dr. Przedborski will chair the monthly facility meeting in which all issues related to proper care, maintenance, and availability of Core facilities are discussed. The day-to-day management is the responsibility of Dr. Vernice Jackson-Lewis. The services to be provided include maintenance and repair of all common laboratory equipment used by the Principal Investigators, and the provision of part-time dishwashing services. This Core will also provide common supplies utilized by all laboratories, including wet and dry ice, and common laboratory gases.

DESCRIPTION (provided by applicant): This proposal is designed to pursue our exploration of the pathogenesis of Parkinson's disease (PD). Pertinent to this goal, it has been found that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin that damages the dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc) as seen in PD, can kill these neurons by apoptosis. We have demonstrated that MPTP-induced SNpc DA neuron apoptosis is Bax-dependent and involves the recruitment of the mitochondrial apoptotic pathway. This molecular cascade cannot be recapitulated by MPTP in isolated brain mitochondria, supporting the notion that MPTP triggers the apoptotic demise of SNpc DA neurons by an indirect mitochondrial mechanism in which Bax is pivotal. Accordingly, Specific Aim (SA)-I will determine how Bax is upregulated after MPTP by studying the role of the tumor suppressor p53, one of the rare identified molecules known to regulate Bax expression. Since activation of p53 results, in turn, from DNA damage, we will assess the relationship between DNA damage, p53 activation and Bax upregulation after MPTP intoxication, as well as the dependence of this molecular cascade on MPTP-induced oxidative stress. In SA-II, we will determine how Bax is activated after MPTP by assessing the contribution of the JNK/c-Jun pathway on Bax oligomerization and mitochondrial insertion, which constitute the cornerstone of Bax pro-apoptotic effect. The specific JNK isoform responsible for this effect will be determined, as well as the role of the "BH3-only" molecule Bim. In SA-IIi, we will assess the occurrence of the caspase-independent apoptotic pathway mediated by apoptosis inducing factor (AIF), and its regulation by Bax, in the MPTP mouse model. Accordingly, we will determine the time-course of AIF mitochondrial release and nuclear translocation after MPTP intoxication in mice with and without ablation/inhibition of caspases, and its dependency on Bax and PARP activations. We will also test, in isolated brain mitochondria, whether AIF release directly results from the inhibitory effect of MPTP on mitochondrial respiration. Finally, the actual role of AIF in MPTP-induced SNpc DA neurodegeneration will be demonstrated in mutant mice with 80% reduction in AIF expression with and without caspase inhibition. Collectively, the proposed studies should shed light on the molecular mechanisms of neurodegeneration in PD and help to identify new molecular targets for therapeutic intervention.

This proposal is submitted to pursue the exploration of the pathogenesis of Parkinson's disease (PD). Pertinent to this goal, we have found that cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin E2 (PGE2) synthesis, is upregulated in nigrostriatal dopamine (DA)-containing neurons in post-mortem PD tissues and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. We have also shown that both ablation and inhibition of COX-2 protect against MPTP neurotoxicity via a non-inflammatory process. Given this, we now wish to examine first whether the deleterious effects of COX-2 depend upon cytosolic phospholipase A2 (cPLA2), as this enzyme produces COX-2's main substrate. Specific Aim (SA)-I will thus define the temporal and anatomical relationship between cPLA2 and COX-2 in MPTP mice and in PD tissues. A comparison of MPTP neurotoxicity between cPLA2 deficient mice treated with and without a COX-2 inhibitor will also be done. Second, it is also known that DA stimulates PGE2 production, which suggests that this neurotransmitter could exacerbate COX-2's effects on nigrostdatal DA neurons. SA-II will thus assess the effects of DA on cPLA2 and COX-2-activities and on neuronal death in primary ventral midbrain cultures exposed to MPTP's toxic metabolite. In addition, MPTP neurotoxicity will be compared between wild-type and knockout COX-2 mice pre-treated with drugs that increase or decrease brain DA. Third, PGE2 produced by injured nigrostriatal DA neurons may activate PGE2-EP3 receptors that are present on many neurons. To ascertain whether such a mechanism is operative here, SA-III will define EP3 receptor distribution in MPTP mice and in PD tissues; because EP3 receptors can be expressed on both plasma and nuclear membranes, EP3 subcellular distribution in MPTP mice will also be studied. Since activation of nuclear EP3 alters intranuclear calcium uptake and c-los transcription, occurrence of these two events will be determined in nigrostriatal DA neurons after MPTP injection. Evaluation of MPTP neurotoxicity in EP3-deficient mice or in COX-2-deficient mice treated with an EP3 agonist will complete SA-III investigations. Together, these studies should provide a deeper understanding of how COX-2 may contribute to the pathogenesis of PD and to the selective nature of the neurodegenerative process in this disease.

DESCRIPTION (provided by applicant): This proposal seeks to pursue our investigations on the pathogenesis of amyotrophic lateral sclerosis (ALS) by searching for the earliest cellular perturbations provoked by mutant superoxide dismutase-1 (SOD1), a known cause of this fatal disease. This project rests on the premise that mutant SOD1 induces responses in genes and proteins expressed by motor neurons even before the emergence of the clinical phenotype which are key to understanding the basis of motor neuron degenerative process and to developing effective neuroprotective strategies. We have shown that wild-type primary or embryonic stem cell-derived spinal motor neurons can be killed by astrocytes expressing mutant SOD1, a neurotoxic effect that can be recapitulated with medium pre-conditioned by mutant, but not wild-type astrocytes (Nagai et al., Nat. Neurosci. 2007). Given these results, we hypothesize that our in vitro cell system should afford a unique tool to identify those initial mRNA and protein changes that arise in wild-type motor neurons in response to a mutant SOD1. Accordingly, in Specific Aim (SA)-I, we will determine motor neuron response to mutant astrocyte conditioned medium by gene array using highly purified primary spinal cord and embryonic stem cell-derived motor neurons cultured on laminin, and then, at a defined moment, exposed to either mutant or wild-type astrocyte conditioned medium. At selected time points, motor neurons cultured in mutant or wild-type astrocyte conditioned medium will be harvested, and total mRNA will be isolated and quantitatively compared by gene expression profiling. Results will be confirmed by real-time PCR. In SA-II, we will define motor neuron response to mutant astrocyte conditioned medium by proteomics using the same neuronal cultures and conditional media as in SA-I. At selected time points, motor neurons, cultured as above, will be harvested and proteins will be isolated and quantitatively compared by differential fluorescence 2-D gel electrophoresis proteome profiling. Results will be confirmed by Western blot. This R21 project offers a high-risk/high-yield set of studies, which may identify, thanks to complementary approaches, molecular pathways and key mediators of motor neuron degeneration in the mutant SOD1 model. The generated information may be of critical importance for the development of effective neuroprotective therapies for the familial form of ALS linked to mutant SOD1 and perhaps for its sporadic form as well. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is an incurable fatal paralytic disorder of uncertain cause. We have found in a dish that specific inflammatory cells isolated from animals modeling this disease produce factors capable of killing the nerve cells responsible for ALS paralysis. In this project, we propose to use this unique disease model to hunt for the earliest pathological changes taking place in nerve cells in response to the insult mediated by neighboring inflammatory cells. We expect that these findings will open the door to the development of new therapeutic strategies aimed at protecting nerve cells against ALS even before they become significantly damaged by the disease process.

DESCRIPTION (provided by applicant): Mutations in superoxide dismutase-1 (SOD1) cause familial amyotrophic lateral sclerosis (ALS). Studies in chimeric or conditional mutant mice indicate that non- neuronal cells play an important role in ALS-related neurodegeneration. We and others find that mutant SOD1 astrocytes can kill motor neurons. However, mutant astrocytes do not kill spinal interneurons, which are also spared in the spinal cord of ALS patients. Here, we seek to investigate further the selective toxic effects of mutant SOD1 astrocytes on motor neurons. We find that this motor neuron toxicity involves both JNK and Bax. Thus, to further define the signaling pathways leading to motor neuron death triggered by mutant astrocytes, Specific Aim (SA)-I will: elucidate the MAPK core signaling module leading to JNK/Bax recruitment within motor neurons;characterize the functional link between JNK and Bax;and test the involvement of 14-3-3, p53 and Bim in the putative JNK/Bax interplay. The ability to generate large numbers of ES cell-derived motor neurons should facilitate the the proposed biochemical analyses. We find that the toxic activity is a polypeptide of ~5-15 kDa in size. Thus, to identify the toxic factor released by SOD1 mutant astrocytes, SA-II will: search for candidate toxic factors using both genomics and proteomics to compare wild- type and mutant astrocytes, and their conditioned media. Each candidate will be validated for toxicity and links to the JNK/Bax pathway. The role of non- neuronal cells in ALS remains unknown. Thus, to determine whether mutant astrocytes can affect the vitality of motor neurons in vivo, SA-III will: characterize the survival and development of mutant astrocytes grafted into the lumbar cord of wild-type rats of different ages;assess the number and morphology of neighboring wild-type motor neurons and inflammatory cells in response to mutant astrocyte grafts;and monitor the impact of mutant astrocyte grafts on motor function, axonal transport, and axonal dying back. This application contains a comprehensive set of experiments which should provide valuable insights into the cellular and molecular underpinning of mutant SOD1-mediated motor neuron degeneration. Information generated in this project should therefore have implications for the treatment and prevention of ALS. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is an incurable fatal paralytic disease in which inflammation is an increasingly recognized contributor to the disease process. We have found in a dish that specific inflammatory cells produce factors capable of killing the nerve cells responsible for ALS paralysis. Herein, we propose to;search for this toxic factor;to demonstrate by which mechanism it kills cells;and to demonstrate whether a similar situation occurs in an animal model of ALS.

The purpose of this clinical core is to collect and store skin fibroblasts, from patients with sporadic and familial Parkinson's disease (PD) and from healthy age-matched controls, destined for future genetic, epigenetic, and stem cell reprogramming investigations. Serge Przedborski will direct this effort, including selection of the potential subjects and supervising collection of relevant clinical information. Carol Moskowitz will obtain informed consent, perform the skin biopsy and draw blood for genotyping. We anticipate performing approximately 180 skin biopsies per year based on our average number of PD patients seen at our outpatient clinic (2,922 patients/yr of whom 422 are new patients) and on Carol Moskowitz's prior recruitment rate for other projects involving skin biopsy. Samples will be processed in Serge Przedborski's laboratory according to standard protocols and stored in a long-term liquid nitrogen tank. Lorraine Clark will genotype all blood samples for known PD mutations and associated SNPs in the following genes: alpha-Synuclein, Parkin, DJ-1, PINK1, LRRK2, UCHL1, ATP13A2, GIGYF2, GBA, MAPI and APOE. Aliquots of cell lines will be made available to both inside and outside investigators. To request aliquots, a Web-based formal request procedure similar to the New York Brain Bank at Columbia University will be developed and posted on the PD-DOC website. In case of high demand for limited samples, preference will be given to the members of the Udall Center network. RELEVANCE (Seeinstructions): Skin cells may harbor important information regarding the cause and mechanism of Parkinson's disease. By collecting such samples from thoroughly characterized patients and making these samples available to the research community, this Core provides a service important to public health and scientific advancement.

DESCRIPTION (provided by applicant): Mutations in superoxide dismutase-1 (SOD1) cause familial amyotrophic lateral sclerosis (ALS). Studies in chimeric or conditional mutant mice indicate that non- neuronal cells play an important role in ALS-related neurodegeneration. We have found that culture medium conditioned with mutant SOD1 astrocytes kills wild- type embryonic stem cell-derived motor neurons (ES-MNs) in vitro. This 2-year translational R21 aims at identifying neuroprotective agents for the treatment of ALS. We have succeeded in miniaturizing our ES-MNs cell model of ALS and have secured all of the necessary expertise, equipment, and chemical libraries to embark on a high-throughput screening (HTS) effort. We will begin by completing the optimization and validation of our assay. We will then perform our HTS in four steps. First, to fine-tune the assay parameters, we will screen a small collection of 2,000 biologically active compounds for their ability to enhance ES-MN survival in the presence of SOD1 astrocyte-conditioned medium. Second, a collection of ~80,000 compounds will then be screened one-by-one at a single concentration (10 5M) and with one parameter (ES-MN survival). Third, hits will be validated by: (a) repeat testing in multiple replicates;(b) retesting after resynthesis/repurchase;and (c) performing dose-response curves spanning the concentrations used in the initial screening. Fourth, structurally-related compounds from our collection will be tested in parallel with validated hits to confirm the bioactivity of each class of compounds, and to select the most potent. Leads will be defined as compounds with a reproducible neuroprotective activity, a potency of EC50 d 5 5M and a maximum effect e 75% of that of the positive control. In agreement with the intent of this program announcement, our proposal offers a set of investigations geared toward developing an assay for the screening of therapeutics. We anticipate that, by the end of this project, our entire library of small molecules will have been tested and that ~100 hits will have been selected, leading directly into a therapy development project for ALS. The latter will consist of preclinical studies based on in vitro secondary screens, a set of in vivo toxicity and pharmacokinetic investigations, and testing of the lead candidates in transgenic mice expressing mutant SOD1. Public Health Relevance: Amyotrophic lateral sclerosis (ALS) is a common fatal paralytic disorder affecting adults with no effective treatment. We have recently identified and validated a means of reproducing ALS in a laboratory dish - a breakthrough which has led to the development of functional cell models of this disease. By using these new cell models in conjunction with high- throughput technologies, we will screen large numbers of small molecules in a brief period of time with the ultimate goal of discovering compounds with neuroprotective activities against ALS.

DESCRIPTION (provided by applicant): This R21 exploratory/developmental project in translational research aims to test the small-molecule necrostatin-1 (Nec-1) as a potential neuroprotective agent for the treatment of amyotrophic lateral sclerosis (ALS), an incurable fatal paralytic disorder. Mutations in superoxide dismutase-1 (SOD1) cause familial ALS and studies in chimeric or conditional mutant mice indicate that non-neuronal cells play an important role in mutant SOD1-related neurodegeneration. We and others find that wild-type primary spinal motor neurons (MNs) are selectively killed by mutant SOD1-expressing astrocytes or their conditioned medium by programmed cell death. Now, we show that Nec-1, an allosteric inhibitor of the kinase receptor interacting protein-1 (RIP1), protects against this mutant astrocyte-induced MN death. Thus, to define the cellular target of Nec- 1, specific aim (SA)-1 will determine whether the protection afforded by Nec-1 in our MN/astrocyte co-culture model system of ALS is linked to an inhibition of RIP1 within MNs and/or within astrocytes. Whether Nec-1 protects MN cell bodies as potently as their nerve processes will also be examined. We also find, by in silico modeling, that Nec-1 is predicted to cross the blood-brain barrier raising the possibility that Nec-1 may be usable in vivo as a therapeutic agent for ALS. Thus, in our initial steps toward testing this possibility, SA-2 will assess Nec-1 and its inactive structural analogue Nec-1ia solubility and stability in suitable vehicles for chronic in vivo use, and their central nervous system penetration, pharmacokinetics, routes of administration and tolerability in wild- type mice. Then, based on the optimal conditions of Nec-1 administration defined in SA-2, SA-3 will determine the neuroprotective potency of Nec-1 in the transgenic mutant SOD1 mouse model of ALS using a comprehensive set of behavioral and morphological investigations. We anticipate that, by the end of this project, the preclinical suitability and effectiveness of Nec-1 will have been evaluated, which may lead into a therapeutic development project for ALS. The latter will include preclinical regulatory studies as part of the prerequisites for future clinical trials. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is an incurable fatal paralytic disease in which inflammation is an increasingly recognized contributor to the disease process. We have found in a dish that a small molecule called necrostatin blocks the deleterious effects of specific inflammatory cells on the nerve cells responsible for ALS paralysis. Herein, we propose to determine the suitability of using this small molecule is a living organism and to demonstrate whether it is protective in an experimental model of ALS.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a uniformly fatal neurodegenerative disease for which there is still no effective treatment. One explanation for this is that very few therapeutic targets - molecular events in the disease pathway whose inhibition confers benefit - have been identified, hindering rational translational approaches. One means of discovering novel targets is high-throughput in vitro screening of chemical libraries to identify compounds that can ameliorate disease-related phenotypes. Enzymes inhibited by such compounds constitute candidate therapeutic targets. However, to validate them for further development, it is necessary to demonstrate that their inhibition delays disease onset or progression in vivo. Among the cellular events that occur early in the ALS disease process is the physical die-back of motor axons from motor endplates, leading directly to muscle paralysis that spreads progressively throughout the body. Early muscle denervation is observed in mutant SOD1 rodent models as well as in patients with either sporadic or familial forms of the disease. Preventing axonal degeneration, or stimulating regrowth, would be predicted to delay disease onset or progression. However, since the underlying mechanisms remain unclear it has not been possible to test this therapeutic hypothesis directly. We recently screened a library of ~50,000 compounds to identify agents that enhance motor axon regeneration in an inhibitory context in vitro. The strongest hits were the statins, which enhanced axonal growth by up to 5- fold at concentrations 100-fold lower than with benchmarking compounds. Statin effects on axonal growth depend entirely on inhibition of their known target enzyme HMG-CoA reductase (HMGCR; 3-hydroxy-3-methyl- glutaryl-CoA reductase), which is the rate-limiting step for cholesterol synthesis and protein prenylation pathways. Our data identify HMGCR as a novel candidate therapeutic target in ALS. In this two-year project, we propose to validate HMGCR as a target in vivo using two potent ligands, cerivastatin and simvastatin, as probes. Our experiments will use compartmentalized motor neuron cultures to determine whether HMGCR inhibition needs to occur in the cell body or in the axon terminal. We will then establish protocol for statin administration in vivo that lead to significant HMGCR inhibition in motor neurons in the spinal cord. Finally, we will determine whether statin administration can delay muscle denervation in the mutant SOD1 mouse model of ALS. Overall, our experiments should allow us to determine whether HMGCR and the pathways downstream of it are valid therapeutic targets in ALS. They should also provide proof of principle for the idea that enhancing motor axon growth can delay functional denervation of muscle. Lastly, our data should stimulate further research into the specific processes modulated by statins in motor neurons, and thereby help identify more selective targets - such as protein prenylation - for future drug development.

? DESCRIPTION (provided by applicant): We have published (Re et al. Neuron, 2014) that wild-type spinal motor neurons (MNs) are selectively killed by both mouse familial mutant SOD1 (mSOD1)-expressing astrocytes and human sporadic ALS astrocytes-or their conditioned medium (CM)-through a caspase-independent mechanism. In this study, we also show that necrostatin-1 (Nec-1), an allosteric inhibitor of the kinase function of the receptor interacting protein-1 (RIP1), which is an obligatory mediator of necroptosis, affords full protection against MN death in our in vitro models. Consistent with the RIP1 finding, our pilot data demonstrate that silencing RIP3 and inhibiting mixed lineage kinase domain-like (MLKL), two other known determinants of necroptosis, also provide MN protection. These findings offer the first experimental evidence that necroptosis regulates neuronal death in a model of chronic human neurological disorder. Yet, remarkably, aside from RIP1, RIP3, and MLKL, the molecular network of necroptosis, especially in neurons, remains elusive. Thus, as an initial phase toward unraveling the neuronal molecular network of necroptosis, we propose a 2 year scope of work consisting of two sequential steps: First, to assign the complex gene expression changes that occur during necroptosis to the actions of a limited number of regulatory genes, we will use: (i) our MN interactome (a cell type-specific regulatory network); and (ii) RNAseq data obtained from purified mouse embryonic stem cell-derived MNs exposed to CM made with either mSOD1-astrocytes (toxic condition) or wild-type SOD1 astrocytes (non-toxic control condition). So as to differentiate between general changes in MNs induced by astrocytes and those involved in necroptosis, each experiment will be performed in the presence or the absence of the antagonist Nec-1. Candidate master regulators (MRs; i.e. sets of transcription factor and signaling pathway genes causally associated with the phenotype) of necroptosis will be identified using the MARINa algorithm. Second, to validate the role of the candidate MRs-identified in Step 1-in the MN death phenotype, each will be targeted using shRNA strategies as shown in our pilot data. This validation will be done in both our in vitro mouse and human models by monitoring both MN survival and neurite length. Also to be performed in Step 2 are multiplex qRT-PCR experiments on MN samples, in each a MR will be individually knocked-down. The goal here is to examine whether any of the identified MRs regulate the expression of the others. As future studies, we propose: (1) to use the information generated herein to develop a genomic signature and new reagents/tools to study necroptosis in both post-mortem mouse models of and patients with neurodegenerative disorders such as, but not limited to ALS; and, (2) to assess the role of the most promising MRs in the expression of the disease phenotype in transgenic mouse model of ALS.

DESCRIPTION (provided by applicant): Despite multiple clinical trials, there is still no effective therapy for the adult-onset neurodegenerative disease ALS (amyotrophic lateral sclerosis). One major reason for this is that, aside from the genes that are causal in familial ALS, no therapeutic targets have been validated. Examples of targets would be enzymes that play a critical role in disease progression and whose inhibition retards disease onset or slows progression. Strikingly, even in late-stage patients with amyotrophic lateral sclerosis (ALS), eye movement and continence are preserved, reflecting the near-complete resistance of motor neurons in oculomotor and Onuf's nuclei to the disease process. If it were possible to confer even a fraction of this resistance upon the normally vulnerable spinal motor neurons, there would be significant therapeutic benefit. Understanding the mechanisms of resistance therefore provides a method for defining new targets. In preliminary studies, we identified novel genes expressed in ALS-susceptible but not in ALS-resistant motor neurons, or vice versa, using laser-capture microdissection and microarray analysis. One of these is MMP-9 (matrix metalloproteinase-9), an extracellular enzyme which is absent from resistant oculomotor and Onuf's nuclei. We showed that its expression in different motor neuron subsets is tightly correlated with their vulnerability. Strikingly, we find that inactivation of the mmp9 gene in ALS model mice - whose normal lifespan is ~6 months - leads to a >3-month delay in muscle denervation and a 24% increase in survival. Significant benefit was observed even in mice that were heterozygotes for mmp9. MMP-9 is therefore a strong candidate as a potential therapeutic target in ALS. The overall goal of the proposed project is to understand the cellular and molecular mechanisms through which MMP-9 triggers motor neuron degeneration and to provide initial evaluation of potential therapeutic strategies to block this. The proposal is structured around three main questions. First, we will determine the molecular mechanism(s) through which MMP-9 triggers motor neuron degeneration, focusing on candidate pathways involving the Fas receptor and glutamate excitotoxicity. Second, we will investigate the cellular site of action of MMP-9, using different routes of administration of viral vectors expressing mmp9 shRNA. Third, we will ask whether inhibition of the enzymatic activity of MMP-9 is sufficient to confer benefit, or whether is known non-enzymatic modes of action are also implicated. Overall, the results should provide novel insights into the mechanisms of motor neuron degeneration in ALS and important preclinical indications as to the potential of MMP-9 as a therapeutic target for future development.

? DESCRIPTION (provided by applicant): Rapid advances in human genetics and in genome editing are creating unprecedented scientific opportunities to develop animal models of different human diseases. These opportunities, however, are already severely straining the animal facilities at Columbia University Medical Center (CUMC). This application reflects the deep commitment of the CUMC to improving the quality of animal care and welfare necessary to meet or exceed PHS Policy on the Care and Use of Laboratory Animals. Because of the expected concentration of efforts in neurological disorders, this project, which focuses on neurobiology of disease research and its translation to advance human health, will upgrade and equip a 1,365 square foot area within the existing CUMC animal facility as part of an effort to establish a high-throughput rodent behavior analysis core. This improved facility will be comprised of barrier-space for dedicated rodent housing, animal preparation for and performance of live animal surgery and viral vector injections, and behavioral testing. Specifically, the proposal seeks Federal funds for the purchase of three single and four double HEPA-filtered ventilated cage racks, four HEPA-filtered ventilated changing and transfer stations, and one biosafety cabinet. Associated construction costs related to the installation and connection of the ventilated racks and the biosafety cabinet will also be sought. The facility we establish here will allow CUMC investigators to seize the opportunities created by advances in genetics and genome engineering, while still providing for the very highest standards of animal care and welfare and human safety. In keeping with this, the requested equipment will allow us to fulfill three Specific Aims: (1) to maximize the dedicated rodent housing capacity; (2) to minimize the effects of environmental changes on animal behaviors and to minimize the risk of harmful human exposure to rodent allergens and breaks in colony biosecurity during cage changes and experimental manipulation of animals; and, (3) to allow the sterile performance of survival surgery and safe injection of viral vectors into animals to achieve optimal quality research. This proposal represents CUMC's critical and substantial effort to meet the present and anticipated needs of its neurobiology of disease community by providing a new and fully-functional barrier behavioral testing facility for rodents (primarily mice). Recognizing the importance of this effort the Dean's office, the Columbia Translational Neuroscience Initiative, and the Institute of Genomic Medicine have come together to collectively provide all of the necessary support to create the facility described above beyond the resource that this grant would provide. This institutional commitment will include: (1) integrating the costs of renovation/construction of the new facility into the ongoing capital project of CUMC's Institute of Comparative Medicine aimed at upgrading its animal care facilities and (2) covering the purchase of all of the behavior testin apparatii.

This R21 is submitted to pursue our investigations of the pathogenesis of Parkinson?s disease (PD) by exploring the role of extracellular alpha-synuclein (?-syn) in the neurodegenerative process of PD. While ?-syn is natively an intracellular protein, it can be recovered in the extracellular space due to, at least two, non- mutually exclusive mechanisms, namely atypical secretion and leakage from, healthy and damaged neurons, respectively. Once in the extracellular space, ?-syn is subject to oligomerization and/or modification (nitration or oxidization) that can trigger a microglial-derived inflammatory response, and, according to growing number of studies, a cell-to-cell transmission in both in vitro and in vivo models. We have compelling data showing that ?-syn is ingested by microglia via a receptor-mediated process, but the ?-syn species, which have been linked to PD (e.g. nitrated, oligomerized, and mutated) are less efficiently cleared than those which has not been linked to PD (e.g. native monomeric ?-syn). We thus hypothesize that the lesser the ingestion of extracellular ?-syn by microglia, the greater the microglial-derived inflammatory response and the cell- to-cell transmission and the ensuing neurodegeneration. The objective of this R21 is to characterize, in- depth, the relationship between ingestion, inflammation and cell-to-cell transmission triggered by extracellular ?-syn species both in vitro and in vivo. To achieve these stated goals, two specific aims (S.A.) are proposed. In S.A.1, in vitro investigations will: (i) complete the characterization of microglia receptor-mediated ingestion of selected ?-syn species; (ii) examine how different species of ?-syn elicit inflammation as measured by the production of selected inflammatory mediators; and (iii) assess the effects of Fc-mediated ingestion of the various forms of ?-syn and test subclasses of antibodies directed against selected forms of ?-syn to promote their microglial ingestion. How these strategies alter the ?-syn-related microglial production of inflammatory mediators will also be studied. All of these proposed experiments will be conducted in cultures of immortalized neonatal microglial cells using our novel assays to evaluate ?-syn ingestion and a panel of biochemical assays to quantify inflammatory markers in response to PD-linked and non-linked ?-syn species. In S.A.2, in vivo investigations will: (i) determine the effects of the deficiency in CD11b or SR-B2 ? two microglial receptors that we have identified as key determinants of ?-syn ingestion ? on brain inflammation and neuropathology in response to endogenous overexpression of wild-type or mutant A53T ?-syn; and (ii) examine the effects of the deficiency in CD11b or SR-B2 on brain inflammation, neuropathology, and cell-to-cell transmission in response to the injection of exogenous ?-syn. All of these proposed experiments will be conducted in transgenic mice expressing wild-type or mutant A53T ?-syn crossed with knockout CD11b or SR-B2 mice which will or will not be injected with ?-syn recombinant. This project is deemed significant and novel as it proposes an original pathogenic scenario, uses new methods, and may pave the way to new therapeutic avenues for PD.

Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disease characterized by neuromuscular junction (NMJ) denervation that precedes spinal motor neuron (MN) death and muscle weakness. We hypothesize that preventing denervation and stimulating reinnervation of NMJs will thwart muscle dysfunction and weakness in ALS, hence improving the patient's quality of life and, likely extending survival. Herein, we seek to demonstrate that protein prenylation, which was reported to operate as an endogenous brake on axonal growth, is a key determinant of ALS-related motor axon pathology. In support of this goal, our pilot work shows that dually silencing the prenylation enzymes, farnesyl transferase and geranylgeranyl transferase type-I, or uniquely silencing geranylgeranyl transferase type-II, mitigates NMJ denervation in the transgenic (Tg) mouse expressing mutant SOD1 (mSOD1). The rationale for this project is that, once it is known which prenylated proteins are essential for ALS-related motor axon pathology and which prenyl transferases catalyze their prenylation, new and innovative strategies can be devised for the treatment of ALS. Thus, the following three specific aims are proposed. In AIM 1, we will identify the prenyl transferase involved in motor axon pathology by silencing these enzymes individually or in combination in Tg mSOD1 mice and then, we will compare, at different time points, the number of lumbar and phrenic MNs and the NMJ innervation of ambulatory and respiratory muscles that are critical to the quality of life and lifespan, respectively. We will also demonstrate the generic nature of protein prenylation in ALS-related motor axon pathology by assessing the most effective silencing identified above in a non-SOD1 model of ALS. In AIM 2, we will ascertain the specificity of protein prenylation for motor axon pathology by monitoring behavioral, electrophysiological and anatomical parameters in Tg mSOD1 mice deficient in the pro-cell death gene Bax with and without prenylation inhibition. Since Bax deletion abrogates spinal MN death but not motor axon pathology in these mice, Tg mSOD1/Bax?/? animals will enable us to determine whether: (i) motor axon pathology and MN death are governed by distinct molecular programs and (ii) inhibition of both prenylation and Bax not only delays the onset of motor deficit but also extends lifespan. In AIM 3, we will elucidate the specific prenylated proteins that contribute to motor axon pathology by generating the MN prenylated proteome and then, use this information to perform a loss-of- function screening in an in vitro model of ALS-like axon pathology. Lastly, those silenced MN prenylated proteins that mitigate the axon phenotype in vitro will be validated in Tg mSOD1 mice using the same tests as in AIM 2. In light of the above, we expect that the successful completion of the proposed work will identify the prenylation pathway and its targets that contribute to motor axon pathology in ALS. These findings will have an important positive impact in that they will provide opportunities for preventive and therapeutic interventions and, fundamentally, advance our mechanistic understanding of ALS and related disorders.

SUMMARY This application is for an Administrative Supplement to the parent grant R01NS107442: Mechanisms of axon pathology in ALS, submitted in response to NOT-AG-18-039. Alzheimer?s disease and related dementias (AD/ADRD) are the most prevalent neurodegenerative disorders affecting more than 47 million people worldwide and conveying an ever growing societal and economic burden. AD alone, is estimated to affect 5.7 million Americans in 2018. However, the pathogenic mechanisms underlying the development AD/ADRD remain elusive. Interestingly, emerging evidence indicates that prenylation ? the same post-translational lipid modification of proteins under investigation in our parent R01 ? may also play an important role in the pathogenesis of AD. Prenylation reactions are catalyzed by prenyl transferases that attach isoprenoids, either farnesyl or geranylgeranyl pyrophosphate, to proteins with a characteristic C-terminal motif. The recognition that prenylation can modify the structure and function of many important proteins involved in human diseases has invigorated the interest about its involvement in diseases of the nervous system, and, of particular relevance to this project, in the importance of several targets of prenylation in neuronal loss and synapse plasticity. During the development of our parent project we found that farnesyl transferase and geranylgeranyl transferase-I inhibition by silencing of their common subunit FNTA is sufficient to attenuate NMJ denervation in ALS mice. Based on these results, we hypothesize that viral mediated knockdown of FNTA will inhibit protein prenylation and ameliorate the phenotype of the widely used mouse model of tauopathies. The experimental protocol will encompass different time points because the intent is: (a) to begin the study when cognitive impairment and neuronal loss are still not evident, which is at 4 months; (b) to monitor, longitudinally, the effect of inhibiting protein prenylation on the progression of tau pathology, neuronal loss, and cognitive impairment; and (c) to assess efficacy of intervention at different stages of the degenerative process. Thus, we predict that assessing cognitive impairment bi-monthly from month 4 to month 10 will enable us to compare, with adequate resolution, the beneficial effect of silencing FNTA in rTg4510 mice. The sensitivity of these functional endpoints to experimental modulation of tau pathology will be tested in parallel via the administration of doxycycline from 4 months of age in a subgroup of rTg4510 mice. The premise is that doxycycline will arrest the progression of tau burden and atrophy and thereby contribute to maintaining behavioral capacity at baseline levels. If successful this project could link prenylation and AD/ADRD pathogenesis and have far reaching consequence for the development of new therapeutic strategies.

Parkinson's disease (PD), PD with dementia (PDD) and dementia with Lewy bodies (DLB) are the the most common synucleinopathies. Although there is no cure for these diseases, passive immunization against ?- synuclein (?-syn) improves both the behavioral deficits and the neuropathology in mouse models of synucleinopathy and clinical trials testing the safety of monoclonal anti-?-syn antibodies in healthy volunteers and PD patients are ongoing. Despite the enthusiasm for ?-syn immunization, it is known that antibodies do no readily cross the blood brain barrier (BBB) and target engagement of ?-syn in the CNS may only be partial after the systemic antibody injection methods used in both rodent and human studies. Our hypothesis is that optimal neuroprotection may require higher anti-?-syn antibody accumulation in the brain. To overcome the challenge to immunization strategies for PD, PDD and DLB caused by the BBB, we will use low acoustic energy transcranial focused ultrasound stimulation (FUS), which opens the BBB reproducibly, selectively and safely, thereby enhancing antibody permeation to the CNS. Furthermore, FUS, in its own right, may mitigate ?-syn pathology. The rationale for the proposed research is that, once the anti-neurodegenerative effects of FUS alone and in combination of passive immunization in a mouse model of synucleinopathy are established, new and innovative strategies can be devised for the treatment of PD, PDD and DLB. So, the following two specific aims are proposed. In AIM 1, we will demonstrate the effects of FUS on behavior and neuropathology in a well-validated mouse model of synucleinopathy that will be injected with monoclonal anti-?-syn antibodies. Since mounting evidence indicates that microglia play a critical role in neurodegeneration and that FUS may modulate microglial function, in AIM 2, we will characterize the effects of FUS on the microglial response of a passively immunized mouse model of synucleinopathy. To achieve this stated goal, we will use state-of-the art single cell RNA sequencing technology to profile unbiasedly microglia from the brain of this mouse model of synucleinopathy that was subjected to FUS and/or immunization. In situ validation of the identified microglial subpopulations will also be performed in AIM 2. Successful completion of the proposed work is expected to overcome current limitations of using immunization for CNS disorders and will provide unique insights into the phenotypes of microglia in response to the disease process, FUS and/or immunization. These findings will have an important positive impact in that they will provide opportunities for therapeutic interventions and, fundamentally, advance our mechanistic understanding of PD and related disorders using ultrasound both for its potential direct effect on neurodegeneration and as an indirect immunotherapy enhancer through its action on the BBB.

Neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB) have no cure, but all three share signs of ?-synuclein (?-syn) pathology and of neuroinflammation. Although mounting evidence supports the notion that the immune cell response to neurodegeneration plays an active role in the pathogenesis of the above disorders, anti-inflammatory therapies have failed, thus far, to provide any beneficial effects in neurodegenerative diseases. We hypothesize that since immune cells exhibit a phenotypic heterogeneity, effective immune response-modifying therapy for neurodegenerative diseases requires the targeting of specific components of neuroinflammation rather than the broad inhibition of its signaling. The rationale for this project is that, once the regional and temporal genomic signatures of immune cells both inside and outside of the central nervous system (CNS) of animal models of synucleinopathies are known, meaningful biomarkers can be identified and innovative therapeutic strategies can be devised for PDD, DLB and even perhaps AD. Thus, the following two aims are proposed. To define the heterogeneity of the immune cell response to ?-syn pathology within the CNS, in AIM 1, we will define brain immune cell subpopulations in transgenic (Tg) mThy1-?-syn mouse line 61 (a recognized model of synucleinopathies) by droplet-based single-cell RNA-sequencing (scRNASeq) using freshly extracted immune cells from different brain regions at time points ranging from 2 (no/minimal neuroinflammation) to 14 months (overt neuroinflammation); non-Tg littermates will be used as controls. Using computational techniques adapted to low-depth single-cell RNA-seq data, we will identify transcriptomic subtypes of immune cells from the different mouse brain regions; this includes estimates of cluster robustness, characterization of cell type-specific genes, and identification of marker genes. In AIM1, we will also validate the single-cell data by probing for sets of newly- defined and existing marker genes in situ. Since ?-syn pathology also takes place outside of the CNS, in AIM 2, we will define the phenotypic diversity of the systemic immune cell response to ?-syn pathology in blood, spleen and lymph nodes from Tg Line 61 (and non-Tg littermates) at the same selected time points as in AIM 1, and will be subjected to the same scRNA-seq procedure and cell type analyses. We will then develop novel bi- compartmental models to relate changes in CNS and peripheral immune system cells, with the goal of identifying putative interactions between these two sets of cells in synucleinopathies. Successful completion of the proposed investigations will establish an atlas of immune cell phenotype heterogeneity in synucleinopathies and shed light into the cross talk between immune cells localized inside and outside of the CNS in response to ?-syn pathology. These findings will have an important positive impact on this area of research in that they will provide opportunities for biomarker identification and for therapeutic interventions and will advance our mechanistic understanding of PDD and DLB as well as possibly AD.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron (MN) disease that is associated with features of neuroinflammation. Although mounting evidence supports the notion that neuroinflammation may play an active role in ALS pathogenesis, anti-inflammatory therapies have to provide either no or minimal disease-modifying effect in ALS. Herein, we hypothesize that since immune cells exhibit a phenotypic heterogeneity, effective immune response-modifying therapy for ALS requires the targeting of specific components of neuroinflammation rather than broadly inhibiting its signaling. The rationale for this research is that, once the genomic signatures of immune cells in the central nervous system (CNS) and the peripheral nervous system (PNS) of ALS are known, meaningful biomarkers can be identified and innovative therapeutic strategies can be devised. Thus, the following three aims are proposed. To define the heterogeneity of the immune cell response within the CNS, in AIM 1, we will perform single-cell RNA-sequencing (scRNASeq) using freshly extracted immune cells from spinal cord (ALS susceptible region) and hippocampus (ALS resistant region) of patients with ALS as well as of the extensively used and validated transgenic (Tg) mutant SOD1 (mutSOD1) mouse model of ALS, from pre- symptomatic to end-stage paralysis. We will then utilize this large-scale multivariable dataset computationally to construct an integrated CNS immune cell response signature associated with MN degeneration. Since motor axon degeneration is a critical feature of ALS pathology and takes place outside of the CNS, in AIM 2, we will perform a phenotypical analysis of peripheral nerve infiltrating adaptive and innate immune cells by scRNASeq in sciatic nerves from both ALS patients and Tg mutSOD1 mice using the same analytic pipeline as in AIM 1. We will then computationally construct a bi-compartmental model that integrates the CNS and PNS immune cell information to generate a neuroinflammatory signature of ALS. Lastly, since subregions of the spinal cord degenerate unevenly in ALS, we will use the integrated, neuroinflammatory signatures generates in AIM 1 & 2 to: (i) interrogate an existing, comprehensive spatial transcriptome database from both ALS patients and Tg mutSOD1 mice, and by immunofluorescence and confocal microscopy (ii) localize the different spinal cord immune cell subpopulations in the respective tissue. Successful completion of the proposed investigations will establish heterogeneity of the immune cell phenotype in ALS in both the PNS and CNS in response to neurodegeneration. These findings will have an important positive impact in that they will provide opportunities for novel pathogenic hypothesis, for identification of biomarkers and for therapeutic interventions in ALS and related disorders.