Eliezer Masliah, MD, National Autonomous University of Mexico 1983 - US grants

The cognitive alterations observed in Alzheimer's disease (AD) might be the result of an extensive loss of cortico-cortico afferent combined with an aberrant sprouting in the areas of synapse loss. We hypothesize that in those areas the dystrophic neurites scattered in the neuropil and in the plaques originate in the abnormal axons and presynaptic terminals, as the result of the aberrant sprouting where amyloid precursor protein (APP) and growth associated protein (GAP-43) may play a role. In order to understand the subcellular basic and mechanisms of response to synapse loss and pathology in AD and its relationship to the neuritic plaque formation, this project will utilize two approaches. One is to perform 3-D reconstructions of synapses, scattered dystrophic neurites and neuritic plaques in AD cortex and the other is to develop an experimental model of denervation with widespread dystrophic changes. The 3-D reconstructions will be performed from serial semithin sections obtained from AD frontal cortex biopsies processed with a Silicon Graphics Iris 4D/210VGX workstation. Reconstructions will also be performed from optical sections obtained from the laser confocal imaging of double immunolabeled sections of the AD frontal cortex. The sections will be labeled with fluorescent tagged antibodies that recognize presynaptic terminals and dystrophic neurites with anti-synaptophysin and tau respectively. We also will analyze the role of APP and GAP-43 in the sprouting response in the AD cortex by comparing the patterns of APP-immunoreactive synapses and dystrophic neurites to those of GAP-43 and synaptophysin in double immunolabeled sections. The quantitative information derived from these studies and 3-D images of the AD neuropil and neuritic plaques are expected to provide important information about the relationship between the pathological synapses in the neuropil and in the plaques with the dystrophic neurites. The other approach that we propose is to develop an animal model of neocortical dystrophic changes. For this study, rats will receive unilateral aspirative lesion of the fronto-parietal cortex followed by the injection of a neurotoxin that will alter the process of plasticity. The rationale is that AD pathogenesis may be a multi-step process in which several sequential hits are necessary to damage the repair mechanisms in the cortex. In the first step, there is a widespread synapse loss in cortex, followed by a neurotoxic event that eventually will lead to an ineffective and abnormal repair process in the denervated regions. The main neurotoxin we will test is the phorbol 12-myristate 13-acetate (PMA). The rationale behind using PMA in our model of denervation, is that it will interfere with reinnervation, promote an aberrant sprouting response similar to the one observed in AD, affect PKC and GAP-43, and promote the expression of amyloid genes in rat. We expect that this lesion and neurotoxin combination will reproduce, in the rodent model, the widespread dystrophic alterations observed in AD and provide information about the pathogenesis of the synaptic alterations observed in AD.

Recently studies have suggested that amyloid precursor protein (APP) plays a major role in synaptic plasticity. In Alzheimer's disease (AD), APP is abnormally accumulated in aberrant sprouting neurites in the plaque. Furthermore, APP is localized in the synaptic apparatus and, depending on the dosage, it promotes neuritic outgrowth, synaptogenesis and neuronal survival in vitro and in vivo. Therefore, we hypothesize that a mutation in the APP gene (or a related gene) represents a predisposing condition which, in combination with adjuvant factors (eg. ischemia, aging, trauma), triggers the development of the disease. Ischemia, aging and trauma are conditions known to be associated with synaptic loss. If APP is involved in mechanisms of synaptic repair and plasticity, altered APP might lead to an abnormal reparative response and accentuation of synaptic damage. For the present project, we propose to analyze the patterns of synaptogenesis and sprouting in the brains of transgenic (tg) mice into which the human APP gene has been introduced, and that later in life received a nervous system injury (ischemia, denervation). In order to analyze the effect of these conditions on the synaptic organization of the brain, sections double-immunolabeled for APP/synaptophysin/GAP-43 will be studied with the aid of laser scanning confocal microscopy and computer aided image analysis. The following lines of mice will be analyzed at different ages: a) tg with neuron specific enolase (NSE) promoter-APP751 construct (with no mutation), b) tg with NSE-APP751 construct (with Hardy's mutation in 717), c) tg with NSE-APP695 (no mutation), d) tg with NSE-APP695(with G->A mutation), and e) corresponding non-transgenic (non-tg) controls. Groups of tg and non-tg animals will receive either entorhinal cortex lesion, global ischemia, or kindling. The information derived form this study will provide important data as to the role of APP in plasticity and could help to understand the mechanisms of synaptic pathology in AD.

At the inception of the HNRC, the concept of HIV encephalitis had not been fully defined. Over the past 5 years it has become clear that HIV infection of the CNS is not an all-or-nothing phenomenon, rather there is a spectrum of severity of HIV infection within the CNS; but to define these changes requires sensitive quantitated histology that goes beyond traditional gross and light microscopic analysis, as well as regional estimation of viral burden. The central objectives of the Neuropathology Core are to delineate HIV and CMV related brain changes in a comprehensive fashion, and to relate anatomic findings and regional estimates of viral burden to antemortem neurobehavioral and imaging information. To achieve this, the Co-Pls, Drs. Wiley and masliah, will collaborate in the following studies; Dr. Masliah (Co-Pl of the UCSD site) will quantify neurologic damage on all deceased HNRC participants. The techniques include quantitative assessment of gliosis, morphometric quantitation of neuronal numbers in mid-frontal, superior temporal and inferior parietal cortex, laser confocal immunocytochemistry quantitation of synaptic density, and Golgi quantitation of synaptic spines in mid-frontal cortex. Dr. Wiley (Co-Pl of Pittsburgh site), will assess HIV CNS burden by performing semi-quantitative immunocytochemistry for HIV gp41 on cortical gray, white and deep gray matter of all autopsy cases, and quantitative PCR and HIV p24 antigen capture assays on CSF and CNS homogenates from the same regions. Together the quantitation of viral burden and CNS damage will provide the foundation for comparisons with neurobehavioral and neuroradiologic measurements of CNS disease.

male; female; microscopy; nervous system; model design /development; human subject; biomedical resource; Mammalia; aging;

DESCRIPTION (Adapted from applicant's abstract) : Synaptic loss and neurodegeneration in the neocortex and limbic system in patients with Alzheimer's disease (AD) results in characteristic cognitive alterations. Therefore, to better understand the pathophysiology of AD it is necessary to understand the cellular and molecular mechanisms involved in synaptic damage. During the course of funding for this project the applicants have shown that abnormal functioning of amyloid precursor protein (APP) at the synaptic site might be involved in mechanisms of neurodegeneration in AD. For the renewal period, they hypothesize that abnormal APP functioning might result in deficient functioning of glutamate transporters (GT) which in turn results in increased levels of glutamate at the synaptic cleft and exitotoxicity/neurogeneration. Furthermore, they hypothesize that low density lipoprotein receptor-related protein (LRP) plays an important role in mediating the effects of APP on the GT. Therefore, the main objectives of this revised version of the project are to 1) characterize in vivo the specifity of interactions between APP and GT; 2) assess the in vivo role of putative receptors of APP in mediating the effects of APP on GTs; 3) determine the subcellular mechanisms mediating the effects of APP on GT; 4) determine if altered GT activity, promoted by APP, plays a role in neuroprotection and neurodegeneration. To achieve these aims, levels of mRNA and protein expression and activity (Kd, Bmax) for neuronal and glial GTs will be assessed in the nervous system of challenged (kainic acid injection) and naive NSE-APP transgenic, APP- and LRP-deficient mice by RNAse protection assay, immunochemistry, and competitive and saturated uptake binding assays with D-and L- aspartate. Neuronal and synaptic integrity will be evaluated by immunoquantification of synaptophysin (SYN, synaptic marker) and microtubule associated protein 2 (MAP2, dendritic marker). Additional studies of the role of APP effect on GTs will be performed in a human neuronal cell line (HNT) after treatment with APP, LRP blockers, protein kinase C (PKC) inhibitors and stimulators, and calcium channel blockers. They hypothesize that APP binding to LRP triggers, in addition to the internalization of the ligand, the activation of PKC, which in turn results in GT activity regulation. In this context, they propose to study the role of APP and LRP interactions in regulating the levels of excitotoxins at the synaptic site and to determine how dysfunction of this process might result in neurodegeneration. These studies will help to better understand the mechanisms of synaptic pathology in AD and to develop potential neuroprotective agents that will operate by enhancing the GT activity.

neuropathology; AIDS /HIV neuropathy; neurotoxicology; cerebral degeneration; methamphetamine; drug abuse; apoptosis; HIV infections; brain mapping; clinical research; human subject;

NACP, the precursor of non-A component of Alzheimer's disease (AD) amyloid (alpha-synuclein) is a synaptic molecule that accumulates in AD plaques. Recent studies have shown that a mutation in NACP is associated with familial Parkinson disease and that Lewy bodies are immunoreactive with antibodies against this molecule. In this context, the central hypothesis of this project is that abnormal accumulation/compartmentalization of NACP is involved in the process of neurodegeneration in Lewy body disease (LBD). The main objective of this proposal is to better understand the mechanisms through which abnormal accumulation of NACP leads to neurodegeneration. For this purpose, we propose the following Specific Aims: 1) To determine the relationship between abnormal NACP/alpha- synuclein accumulation and neurodegeneration in the brains of patients with LBD. We hypothesize that in LBD abnormal accumulation of NACP/alpha- synuclein will result in neurodegeneration of cells within the mesolimbic, mesocortical and striatonigral systems. For this purpose, we propose to determine the relationship between NACP/alpha-synuclein levels in synapses, neurons and neurites and cell counts, synapse density and apoptosis in postmortem brains (frontal, temporal, hippocampus, basal ganglial, cingulate and mesencephalon) from patients with LBD. 2) To develop in vivo models to investigate mechanisms which NACP/alpha- synuclein promotes neurodegeneration. We hypothesize that abnormal NACP/alpha-synuclein accumulation resulting over-expression of NACP/alpha-synuclein will result in synaptic damage and neuronal cell death in transgenic (tg) mice. Furthermore, we postulate that mutant NACP/alpha-synuclein might accelerate this process. For this purpose we propose to investigate the patterns of neurodegeneration in the brains of young and old tg mice over-expressing mutant and wildtype human NACP/alpha-synuclein under the control of the platelet-derived growth factor (PDGF) promoter. 3) To determine if risk factors associated with AD increase susceptibility to NACP/alpha-synuclein-induced neurodegeneration in tg mice. We hypothesize that known genetic risk factors for AD such as the presence of apolipoprotein E4 allele (ApoEepsilon4) and amyloid precursor protein (APP) mutations will enhance NACP/alpha-synuclein tg mice will be crossbred with apoE-deficient (knockout), ApoEepsilon3 or E4 tg mice, and with amyloid precursor protein (APP) wildtype and mutant tg MICE. Taken together these studies will help to better delineate the molecular and cellular mechanisms involved in neurodegeneration in LBD. The models and paradigms developed will also help to identify potential targets that will prevent neuronal cell injury in neurodegenerative disorders.

The new title of this revised application: "beta- synuclein as a treatment for Lewy body disease" reflects the modified and sharper focus on our scientific approach in response to the reviewers' major concern that synuclein might be more relevant for understanding Lewy body disease (LBD). We now propose a new concept where beta-synuclein, a naturally occurring anti-aggregation molecule and non-amyloidogenic homologue of alpha-synuclein, might prevent the neurotoxic effects of alpha- synuclein and could be a suitable target for the development of an alternative treatment for LBD. In this context, we propose the following Specific Aims: 1) To characterize the mechanisms by which beta-synuclein blocks alpha-synuclein aggregation. We hypothesize that beta-synuclein may interact with alpha-synuclein through specific beta-synuclein domains, leading to inhibition of alpha-synuclein aggregation. To test this hypothesis, we will study the effects of mutant recombinant beta-synucleins and synthetic beta-synuclein derived peptides on alpha-synuclein aggregation, using immunoblotting analysis, Congo red/Thioflavine-S staining, and electron microscopy. Additional studies of synuclein binding will be performed by immunoblotting with His-tagged alpha- and beta-synuclein 2) To determine if the anti-aggregation effect of beta-synuclein is protective in neuronal cell lines expressing alpha-synuclein. We hypothesize that beta-synuclein may be neuroprotective by blocking alpha- synuclein aggregation. To test this hypothesis, alpha-synuclein- overexpressing GT1-7 and B103 neuronal cells will be co- transfected with beta-synuclein GT1-7 cells will be evaluated for cell viability, mitochondrial function, oxidative stress conditions, GnRH secretion, altered mitochondria morphology and inclusion body formation. B103 cells will be evaluated by analysis of neurite formation and cell adhesion. Immunoblotting experiments will be performed with cells co-transfected with c- myc-tagged beta-synuclein to assess binding to alpha-synuclein. To determine potential novel treatments, alpha-synuclein- overexpressing GT1-7 and B103 cells will be treated with beta- synuclein-expressing recombinant adeno-associated viral vector (rAAV). 3) To determine if beta-synuclein blocks alpha-synuclein aggregation and neurodegeneration in in vivo model systems of LBD. We hypothesize that beta-synuclein may inhibit alpha- synuclein aggregation in vivo. To test this hypothesis, we will cross alpha-synuclein tg mice with either beta- synuclein tg or knockout mice where murine alpha- or beta-synuclein gene is deleted. Mice will undergo detailed behavioral, neurochemical and neuropathological examination to determine if beta-synuclein expression affects the functional and structural alterations promoted by alpha-synuclein and might act as a basis for treatment of LBD. To assess the potential for treatment development, alpha-synuclein tg mice will be treated with a beta- synuclein-expressing rAAV. In summary, we will utilize a multi-system approach (a cell-free, cell culture and tg mouse systems) to ascertain the anti- aggregation potential of beta-synuclein as a therapeutic target for development of novel treatments for LBD.

NEUROBIOLOGY CORE PROJECT SUMMARY/ABSTRACT: The overall objective of the Neurobiology Core (NB) is to provide the NeuroAIDS community with a set of neurobiological resources that will enhance the analysis and discovery of the mechanisms of neurodegeneration associated with prolonged survival with HIV infection, from a comprehensive and dynamic perspective. The NB Core has been re-organized into In Vivo and In Vitro Units to better serve the current and future needs of the neuroAIDS field. The main resource objectives will be to: (a) provide a set of innovative state-of-the-art neuropathological and neurobiological in vivo and in vitro resources to support studies of the mechanisms of viral persistence/eradication, influence of microbiome in neuro-inflammation and role of aging in neurodegeneration with HIV infection, (b) encourage and facilitate collaborative work addressing these and other scientific themes of the Center, and (c) provide user training and consultation to new investigators. In addition, the NB Core Scientific objectives will include: (1) To provide support for studies of HIV infection in the CNS that address viral persistence and eradication, including molecular studies of chromatin modifiers, epigenetic markers and markers of viral production and cycling; (2) To provide in vitro and in vivo neuropathological resources and assays in support of the Microbiome theme, that could include studies of patterns of neuro-inflammation, neuro-vascular unit injury and gut pathology in patients with HAND; (3) To provide quantitative analysis of novel sets of HIV related neuropathologies in support of the theme on Aging. Examples of work on the Aging theme could include neuropathological and biochemical studies of: amyloid-? protein (A?) deposits including cerebral amyloid angiopathy; Tau and ?-synuclein accumulation; markers of autophagy and lysosomal activation; markers of mitochondrial biogenesis and mitophagy; brain immunophilin response; and human primary neuro-glial, brain endothelial and vascular smooth muscle cell cultures. And finally, (4) we will continue supporting clinico-pathological studies which investigate the relationship between new markers of neurodegeneration and HAND. Understanding the neuropathological basis for viral persistence and neural injury in the context of aging and the microbiome as contributing factors, will elucidate mechanisms through which HIV leads to HAND and inform new treatments for this disabling condition.

DESCRIPTION (provided by applicant): The neurocognitive alterations in HIV disease are associated with neurodegeneration of selective neuronal populations. Survival of these populations are dependent on specific trophic factors, such as fibroblast growth factor (FGF). In this context, it is possible that dysregulation of patterns of FGF expression in individuals with AIDS might render neurons more vulnerable to degeneration under the combined challenge of excitotoxins and viral products. Thus, the main objective of this proposal is to investigate mechanisms of FGF1 neuroprotection in HIV disease. The central hypothesis is that neuroprotective effects of FGF 1 against HIV products (e.g.: Tat and gp 120) are mediated in the short term via regulation of glycogen synthase kinase 3 13 (GSK313), a modulator of cell death pathways (53) and in the long term by regulating the expression of chemokine receptors (e.g.: CXCR4) that mediate HIV toxicity. The following Specific Aims are proposed: 1) To determine if patterns of FGF1 expression in the brains of AIDS patients are associated with neuroprotection vs. neurodegeneration. For this purpose levels of FGFI, FGF receptor 1 (FGFRI), GSK313, 13-catenin and CXCR4 will be determined in AIDS brains subdivided into four groups based on the presence or absence of neurodegeneration and HIVE. 2) To determine if FGF1 is neuroprotective in vivo against HIV-products. In order to test this hypothesis in vivo, transgenic (tg) mice overexpressing FGF1 will be generated and crossbred with gpl20 or Tat tg mice. Additional experiments will be performed where FGF1 tg mice will receive intracerebral injections of gpl20 or Tat. Levels of neurodegeneration and GSK313, g-catenin and CXCR4 will be compared among mice. 3) To determine the mechanisms responsible for FGF1 neuroprotective effects against HIV neurotoxicity. For this purpose, expression of GSK313, 13- catenin and CXCR4 will be deternained in human fetal primary neurons in the presence or absence of FGF1 and gpl2 and Tat. Neurodegeneration will be assesed by lactase dehydrogenase (LDH), MTT, DNA fragmentation and caspase activation assays. The involvement of the intracellular signaling pathways will be further evaluated via inhibition or activation of FGFR1 or GSK313. These studies will be performed primarily at the Institute of Psychiatry in London. Clarification of the mechanisms by which FGF1 protects neurons against HIV will provide a potential target for the development of new treatment directed at ameliorating the neuronal damage to in AIDS patients with cognitive impairment.

Alzheimer's disease (AD) is the most commonly found neurodegenerative disorder in the elderly population. Although the incidence of this devastating disorder continues to increase thus creating a serious public health problem, to date this disorder is neither curable nor preventable. For the current project, we propose a new approach for this modeling and treatment of AD based on the use of lentiviral vectors expressing proteins that enhance or decrease accumulation of products of amyloid precursor protein (APP) metabolism and tau. We postulate that injection of lentiviral vectors expressing mutant presenilin 1 (PS1) or tau into hAPP tg mice will help develop more comprehensive models of AD, while lentiviruses expressing amyloid degrading enzymes as neprilysin (Nep) or insulin degrading enzyme (IDE) could be used as targets for the development of an alternative treatment for AD. In this context, we propose the following Specific Aims: 1. To determine the potential use of recombinant lentiviral vectors expressing mutant PS1 or APP in developing tg models of amyloid deposition in the brain. The hypothesis is that lentiviral vectors expressing pro-amyloidogenic vectors will accelerate plaque formation in a time- and region-specific manner. 2. To determine the potential therapeutic use of recombinant lentiviral vectors expressing Nep or IDE to reduce amyloid in the brain of tg models. The hypothesis is that injection of lentiviral vectors expressing Nep or IDE will reduce the amyloid burden in the brains of hAPP tg mice and that this will result in improved functional performance and reduced neurodegeneration. 3. To determine the potential use of recombinant lentiviral vectors expressing mutant tau in developing new tg models of AD. The hypothesis is that injection of lentiviral vectors expressing mutant tau into the brains of hAPP tg mice will result in formation of neurofibrillary tangles (NFTs) in a time- and region-specific manner. For all these aims, tg mice will receive lentiviral injections either in the frontal cortex, hippocampus, or cerebellum. Mice will undergo detailed neurochemical and neuropathological analysis to assess the regional specificity and potential for formation of amyloid and NFTs by the lentiviral constructs. Taken together these studies will help develop new treatments for AD based on the use of anti-amyloidogenic viral vectors.

Abstract not provided.

This is a revised application for Project by Masliah that has been extensively modified to address the reviewers' concerns by: 1) expanding our analysis to include stereology and quantitative real time PCR (qRT-PCR), 2) broadening our neuropathological and in vitro studies of astroglial cells, and 3) incorporating new preliminary data in support of the more innovative concept that interferons (IFNs) produced by HIV and HCV-infected cells might, in conjunction with METH, enhance astroglial responses that might promote neurodegeneration and block the effects of neurotrophic factors such as fibroblast growth factors (FGFs) on calbindin (CB) and dopaminergic neurons. Specifically, we hypothesize that loss of CB-IR in the neocortex and hippocampus will be associated with cognitive deficits, while loss of markers in the nigrostiatal system will correlate with motor and cognitive deficits. For the renewal and in collaboration with other components of the PPG our aims are: 1) To elucidate patterns of regional neuronal damage and astrogliosis in the brains of METH+, HIV+ cases and their relationship with neuropathological markers of HIVE and levels of IFN expression and trophic factors selective for cortical interneurons and NS cells. 2} To determine the relationship between regional patterns of neuronal injury and astrogliosis in the brains of METH-using HIV infected cases who vary in HCV status, specific cerebrospinal fluid (CSF) biomarkers and behavioral alterations. 3) To investigate the synergistic neurotoxic effects of METH and HIV in vitro via alterations in astroglial IFN-alpha and IFI gene expression and neuronal FGF/SHH signaling. Dissection of the molecular mechanisms will be performed in neuronal cell lines, primary neuronal cultures, and human fetal brain aggregates exposed to HIV proteins in the presence or absence of METH and FGF2, or FGFS and SHH. These studies will help advance understanding of the cellular and molecular mechanisms of cognitive and motor alterations in HIV infected METH users and in developing novel treatment strategies for these complex disorders.

bioimaging /biomedical imaging; technology /technique development

Lewy body disease (LBD) is a group of disorders characterized by alpha-synuclein (alpha-syn) accumulation and parkinsonism. During the previous period, the objective was to understand the mechanisms by which (3- synuclein ((3-syn)¿a close homologue to alpha-syn¿blocks a-syn aggregation and might have a role in the treatment of degenerative disorders such as Alzheimer's disease (AD) and LBD. Both p-syn and antibodies against alpha-syn target alpha-syn aggregates for clearance probably via autophagy, a process of degradation and recycling of cellular constituents. Alterations in autophagy might play a role in the pathogenesis of AD and LBD, and might represent a target for treatment development. The objectives of this renewal application are: i) to gain new knowledge as to the involvement of the autophagy pathways in the mechanisms of neurodegeneration in LBD, ii) to develop new experimental therapies for LBD by targeting the autophagy pathways and iii) to better understand the involvement of the autophagy pathways in the mechanisms of asyn clearance mediated by immunotherapy. We propose the following aims: AIM 1. Characterize in vivo the contribution of selected molecular components of the autophagy pathway to the pathogenesis of LBD. AIM 2. Investigate in in vivo models of alpha-synucleinopathy the therapeutic and neuroprotective effects of activators of the autophagy pathway. AIM 3. Better understand the cellular mechanisms involved in the clearance of toxic alpha-syn aggregates via specific antibodies. AIM 4. Determine in immunized animals, the contribution of autophagy to the molecular mechanisms involved in alpha-syn clearance. alpha-Syn transgenic mice will be crossed with mice either deficient in or transgenic for components of the autophagy pathway (e.g. Beclinl, LAMP2, mTor). Mice will be treated with stimulators of autophagy (rapamycin, immunotherapy) and analyzed behaviorally, biochemically and neuropathologically. Studies will be complemented with primary neuronal cultures treated with lentiviral vectors and analyzed for markers of autophagy. Better understanding the autophagic pathways involved in alpha-syn clearance is of central importance toward elucidating the pathogenesis of LBD and developing new treatments for these conditions. Thus, enhancing autophagy and lysosomal degradation of alpha-syn may represent a promising therapeutical strategy for the treatment not only for LBD but also for AD.

[unreadable] DESCRIPTION (provided by applicant): The HIV epidemic continues to be one of the most serious public health problems in the US. The number of treated subjects with chronic HIV infection is increasing and with it the prevalence of HIV-associated cognitive & neurological impairment. In these patients cognitive impairment is associated with damage to the synapto-dendritic complex of mature neurons and defects in neurogenesis in adult hippocampus. Therefore, development of alternative treatments to reduce neuronal damage and rescue the alterations in neuronal stem cells (NSCs) in patients with NeuroAIDS might be of significant importance in managing the cognitive disturbances. During the previous period of funding for this grant we discovered that in patients with HIVE, neurodegeneration is associated with alterations in fibroblast growth factors (FGF) & that FGFs might protect neurons from the toxic effects of HIV proteins by inactivating the glycogen synthase kinase-3 (GSK3) signaling pathway. These studies have moved towards pilot clinical trials with lithium. More recently, we found that FGF might also be neuroprotective by regulating other intracellular cascades that are affected in patients with HIVE, such as the cyclin dependent kinase-5 (CDK5) signaling pathway. In this context, the main objectives are to: i) investigate the role of the CDK5 pathway in the mechanisms of synapto-dendritic damage and defective neurogenesis in HIVE, and ii) to test the potential neuroprotective &therapeutic effects of CDK5 inhibitors in models of HIV neurotoxicity. The central hypothesis is that HIV-derived proteins via calcium-mediated calpain activation promote the aberrant cleavage of p35 into p25 with concomitant CDK5 hyperactivation. CDK5-mediated abnormal phosphorylation of synaptic and cytoskeletal substrates might lead to neurodegeneration by interfering with synaptic plasticity and neurogenesis. To test thiswe propose: AIM 1. To investigate the role of alterations in CDK5 signaling pathway in the molecular mechanisms of synapto-dendritic damage triggered by HIV proteins. AIM 2. To determine the involvement of the CDK5 signaling pathway in the mechanisms of defective neurogenesis triggered by HIV proteins. AIM 3. To study if pharmacological inhibitors of CDK5 ameliorate synaptic pathology and alterationsin neurogenesis in in vivo models of HIV protein-mediated neurotoxicity. For this purpose, in vitro & in vivo p35-CDK5 transgenic models exposed to HIV proteins will be used. At the end of the treatments mice will be tested in water maze & neuropathologically. End points will include analysis of neurodegeneration, synaptic structure, CDK5 activity & phosphorylation state of CDK5-related substrates. Neurogenesis will be evaluated by BrdU incorporation, OCX, & PCNA expression in the hippocampus. These studies will be complemented by testing the neuroprotective effects of roscovitine andrelated analogs in primary neuronal cultures and NSCs in the presence of HIV proteins (gp120, tat, vpr), or in HIV infected mixed neuronal/microglial cultures.Together these studies will help to elucidate the role of the CDK5 pathway in neurodegeneration in patients with HIVE and help at developing new neuroprotective treatments. [unreadable] [unreadable] [unreadable]

Parkinson's disease (PD) and related Lewy body diseases are associated with the abnormal intraneuronal accumulation of alpha-synuclein. Mutations that enhance the propensity of alpha-synuclein to aggregate cause early onset familial PD. Notably, the majority of patients with Alzheimer's disease (AD) also have alpha-synuclein immunoreactive Lewy bodies and a substantial proportion of them develop a form of parkinsonism that defies conventional therapeutic approaches. This suggests that factors involved in the pathogenesis of AD might promote the development of particularly recalcitrant forms of PD. We have shown that amyloid beta peptides (Abeta), which play a central role in AD pathogenesis, promote the intracellular accumulation of alpha-synuclein and accelerate alpha-synuclein-dependent motor deficits in alpha-synuclein/amyloid precursor protein transgenic mice, an animal model that mimics aspects of Lewy body disease. However, the mechanisms underlying these effects remain unknown. The main objectives of this proposal are to elucidate these mechanisms and to determine whether blocking Abeta effects might prevent or ameliorate PD and other Lewy body diseases. In Aim 1 we will determine whether the increase in neuronal alpha-synuclein accumulation and in alpha-synuclein-dependent deficits in a transgenic mouse model of Lewy body disease depends on the ratio of the two predominant Abeta species (Abeta1-42/Abeta1-40). For this purpose, alpha-synuclein transgenic mice will be crossed with wildtype or mutant human amyloid precursor protein (hAPP) transgenic mice and detailed biochemical, neuropathological and behavioral analysis will be performed. These experiments will be complemented with in vitro studies in alpha-synuclein-transfected cell fines treated with Abeta1-42 or Abeta1-40 or a mixture of both. In Aim 2 we will determine whether the increase in neuronal alpha-synuclein accumulation and in alpha-synuclein-dependent deficits in a transgenic mouse model of Lewy body disease depends on the uptake of secreted Abeta via the LDL receptor-related protein (LRP). For this purpose, mice expressing both alpha-synuclein and wildtype or mutant hAPP will be crossed with receptor associated protein-deficient mice, which have reduced LRP expression. These experiments will be complemented with in vitro studies in alpha-synuclein-transfected cell lines. In Aim 3 we will determine whether Abeta-dependent alpha-synuclein aggregation and alpha-synuclein-dependent neuronal deficits can be reduced by antioxidants. For this purpose, alpha-synuclein/hAPP mice will be crossed with superoxide dismutase 1 or 2 transgenic mice. These experiments will be complemented with in vitro studies in synuclein-transfected cell lines treated with antioxidants. These experiments will shed light on the overlap between AD and PD and on the role of hAPP/Abeta in the pathogenesis of PD and other Lewy body diseases.

[unreadable] DESCRIPTION (provided by applicant): Lewy body disease (LBD) is a group of disorders characterized by alpha-synuclein (a-syn) accumulation and Parkinsonism. During the previous period, the objective was to understand the mechanisms by which alpha- synuclein (a-syn) a close homologue to a-syn blocks a-syn aggregation and might have a role in the treatment of degenerative disorders such as Alzheimer's disease (AD) and LBD. Both p-syn and antibodies against a-syn target a-syn aggregates for clearance probably via autophagy, a process of degradation and recycling of cellular constituents. Alterations in autophagy might play a role in the pathogenesis of AD and LBD, and might represent a target for treatment development. The objectives of this renewal application are: i) to gain new knowledge as to the involvement of the autophagy pathways in the mechanisms of neurodegeneration in LBD, ii) to develop new experimental therapies for LBD by targeting the autophagy pathways and iii) to better understand the involvement of the autophagy pathways in the mechanisms of a- syn clearance mediated by immunotherapy. We propose the following Aims: AIM 1. Characterize in vivo the contribution of selected molecular components of the autophagy pathway to the pathogenesis of LBD. AIM 2. Investigate in in vivo models of alpha-synucleinopathy the therapeutic and neuroprotective effects of activators of the autophagy pathway. AIM 3. Better understand the cellular mechanisms involved in the clearance of toxic a-syn aggregates via specific antibodies. AIM 4. Determine in immunized animals, the contribution of autophagy to the molecular mechanisms involved in a-syn clearance. a-Syn transgenic mice will be crossed with mice either deficient in or transgenic for components of the autophagy pathway (e.g. Beclinl, LAMP2, mTor). Mice will be treated with stimulators of autophagy (rapamycin, immunotherapy) and analyzed behaviorally, biochemically and neuropathologically. Studies will be complemented with primary neuronal cultures treated with lentiviral vectors and analyzed for markers of autophagy. Better understanding the autophagic pathways involved in a-syn clearance is of central importance toward elucidating the pathogenesis of LBD and developing new treatments for these conditions. Thus, enhancing autophagy and lysosomal degradation of a-syn may represent a promising therapeutic strategy for the treatment not only for LBD but also for Alzheimer's disease. [unreadable] [unreadable] [unreadable]

The mission of the Neuropathology and Animal Behavior Core will be to provide the projects of the NIEHS Center with well-characterized autopsy brain material from human PD cases as well as with transgenic (tg) animal models expressing a-synuclein (a-syn), DJ-1, Parkin and PINK1 for neuropathological, molecular, biochemical and behavioral studies. The Core will provide support with human tissues from PD patients and animal models of PD to Projects 1, 2, and 3; support behavioral studies in Project 3; and help with survival/apoptosis/necrosis analysis of human embryonic stem cell (hESC)-derived developing neurons for Project 4. The Core has developed state of the art techniques for neuropathological analysis of neurodegeneration, biochemical analysis of misfolded protein accumulation, and behavioral analysis of motor and cognitive alterations. Moreover, we have developed unique animal models of PD in which to test and screen new molecular pathways and small molecules. Please note that a human clinical component, as defined in the NIEHS Center RFA, is contained within this Core.

Ablation; Accounting; Affect; Aging Process; Aging-Related Process; Amentia; Ammon Horn; Amyloid; Amyloid A4 Protein Precursor; Amyloid Protein Precursor; Amyloid Substance; Amyloid beta-Protein Precursor; Animal Model; Animal Models and Related Studies; Antigens, Leukemia, Common Acute Lymphoblastic; Apopain; Area; Atriopeptidase; Autonomic Dysfunction; Autophagocytosis; Biochemical; Blood Coagulation Factor IV; Brain; Brain Stem; Brainstem; C-terminal; CALLA Antigen; CASP-3; CASP3; CD10 Antigens; CPP-32; CPP32; CPP32 protein; CPP32B; CPP32beta; Ca++ element; Ca2+-Activated Protease; Calcium; Calcium Channel; Calcium Channel Antagonist Receptor; Calcium-Activated Neutral Protease; Calcium-Activated Neutral Proteinase; Calcium-Activated Protease; Calcium-Dependent Neutral Protease; Calcium-Dependent Neutral Proteinase; Calpain; Caspase 3, Apoptosis-Related Cysteine Protease; Cell Line; Cell Lines, Strains; Cell-Death Protease; CellLine; Cells; Cleaved cell; Coagulation Factor IV; Cognitive; Collaborations; Common ALL Antigen; Complex; Cornu Ammonis; Corpus Striatum; Corpus striatum structure; Cysteine Protease CPP32; Cytoskeletal Proteins; DA Neuron; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Dementia; Dementia with Lewy Bodies; Dementia, Lewy Body; Desminase; Diffuse Lewy Body Disease; Disease; Disorder; Dopamine neuron; Dyskinesia Syndromes; Encephalon; Encephalons; Endopeptidase-24.11; Endopeptidases; Enkephalin Dipeptidyl Carboxypeptidase; Enkephalinase; Enkephalinase-24.11; Environment; Esteroproteases; Experimental Models; Experimental Models, Other; Extravasation; Factor IV; Funding; GRIK1 gene product; GRM5; Generations; Genetic; Glutamate Receptor; Hippocampus; Hippocampus (Brain); Human; Human, General; ICE-like protease; Impairment; Injury; Intervention; Intervention Strategies; Ion Channels, Calcium; Kidney-Brush-Border Neutral Proteinase; Label; Lead; Leakage; Length; Lentiviral Vector; Lentivirus Vector; Lewy Body Disease; Lewy Body Disease, Cortical; Lewy Body Type Senile Dementia; Limbic System; MGLUR5; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Membrane; Membrane Metallo-Endopeptidase; Membrane Metalloendopeptidase; Metabotropic Glutamate Receptors; Mice; Mice, Transgenic; Modeling; Models, Experimental; Movement Disorder Syndromes; Movement Disorders; Murine; Mus; Neocortex; Neprilysin; Nerve Cells; Nerve Degeneration; Nerve Unit; Nervous System, Brain; Neural Cell; Neurocyte; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Neuron Degeneration; Neurons; Neutral Endopeptidase; Neutral Endopeptidase 24.11; Oxidative Stress; PARP Cleavage Protease; PDGF; Papain-Like Cysteine Protease; Parkin; Parkinsonian; Parkinsonian Condition; Parkinsonian Diseases; Parkinsonian Disorders; Parkinsonian Syndrome; Parkinsonism; Pathogenesis; Pathway interactions; Patients; Pattern; Pb element; Peptidases; Peptide Hydrolases; Peptide Peptidohydrolases; Plant Embryos; Plant Roots; Platelet-Derived Growth Factor; Population; Predisposition; Principal Investigator; Production; Programs (PT); Programs [Publication Type]; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Proteases; Proteinases; Proteins; Proteolytic Enzyme; Proteolytic Enzymes; RNA, Small Interfering; Rate; Receptors, Calcium Channel Blocker; Receptors, Metabotropic Glutamate; Role; SCA-1; SREBP Cleavage Activity 1; Seeds; Signal Pathway; Small Interfering RNA; Spillage; Striate Body; Striatum; Susceptibility; Synapses; Synaptic; System; System, LOINC Axis 4; Testing; Thermolysin-Like Metalloendopeptidase; Toxic effect; Toxicities; Transgenic Mice; Transgenic Organisms; VDCC; Viral Vector; Voltage-Dependent Calcium Channels; Vulnerable Populations; YGG-Forming Enzyme; Yama; Yama protein; Zygotes, Plant; amyloid precursor protein; autophagy; calcium flux; calcium mobilization; caspase; caspase-3; cleaved; cultured cell line; cystein protease; cystein proteinase; cysteine endopeptidase; cysteine protease P32; disease/disorder; dopaminergic neuron; experiment; experimental research; experimental study; familial Alzheimer disease; familial Alzheimer disease (FAD); gene product; heavy metal Pb; heavy metal lead; hippocampal; homotypical cortex; immunocytochemistry; in vivo; inhibitor; inhibitor/antagonist; interventional strategy; isocortex; kidney brush border neutral peptidase; membrane structure; metabotropic glutamate receptor 5; model organism; mutant; neopallium; neural degeneration; neurodegeneration; neurodegenerative illness; neuronal; neuronal degeneration; neurotoxic; novel; oncogene protein pp60(v-src) (137-157); parkin gene/protein; pathway; peptide A; prevent; preventing; programs; protease; proteinase; pyridine; release of sequestered calcium ion into cytoplasm; research study; root; seed; siRNA; social role; striatal; synergism; synuclein; transgenic

DESCRIPTION (provided by applicant): Lewy body disease (LBD) is a group of disorders characterized by alpha-synuclein (a-syn) accumulation and Parkinsonism. During the previous period, the objective was to understand the mechanisms by which alpha- synuclein (a-syn) a close homologue to a-syn blocks a-syn aggregation and might have a role in the treatment of degenerative disorders such as Alzheimer's disease (AD) and LBD. Both p-syn and antibodies against a-syn target a-syn aggregates for clearance probably via autophagy, a process of degradation and recycling of cellular constituents. Alterations in autophagy might play a role in the pathogenesis of AD and LBD, and might represent a target for treatment development. The objectives of this renewal application are: i) to gain new knowledge as to the involvement of the autophagy pathways in the mechanisms of neurodegeneration in LBD, ii) to develop new experimental therapies for LBD by targeting the autophagy pathways and iii) to better understand the involvement of the autophagy pathways in the mechanisms of a- syn clearance mediated by immunotherapy. We propose the following Aims: AIM 1. Characterize in vivo the contribution of selected molecular components of the autophagy pathway to the pathogenesis of LBD. AIM 2. Investigate in in vivo models of alpha-synucleinopathy the therapeutic and neuroprotective effects of activators of the autophagy pathway. AIM 3. Better understand the cellular mechanisms involved in the clearance of toxic a-syn aggregates via specific antibodies. AIM 4. Determine in immunized animals, the contribution of autophagy to the molecular mechanisms involved in a-syn clearance. a-Syn transgenic mice will be crossed with mice either deficient in or transgenic for components of the autophagy pathway (e.g. Beclinl, LAMP2, mTor). Mice will be treated with stimulators of autophagy (rapamycin, immunotherapy) and analyzed behaviorally, biochemically and neuropathologically. Studies will be complemented with primary neuronal cultures treated with lentiviral vectors and analyzed for markers of autophagy. Better understanding the autophagic pathways involved in a-syn clearance is of central importance toward elucidating the pathogenesis of LBD and developing new treatments for these conditions. Thus, enhancing autophagy and lysosomal degradation of a-syn may represent a promising therapeutic strategy for the treatment not only for LBD but also for Alzheimer's disease.

The overall objective of the Neurobiology Core is to provide the NeuroAIDS community with a set of neuropathological methods and in vivo neurobiological resources that will enhance the analysis and discovery of the mechanisms of neurodegeneration associated with prolonged survival with HIV infection, from a comprehensive and dynamic perspective. During the previous period of funding we developed novel neuropathogy-based techniques to asses neurogenesis and neurodegeneration, and supported over 35 funded NeuroAIDS investigators. For the renewal we will provide: 1) an array of techniques to facilitate quantitative analysis of neuronal injury that will facilitate studies on HIV-mediated neuropathogenesis; 2) technical assistance and consultation on state-of-the-art neuropathological approaches; 3) support for preliminary studies that utilize neuropathology data; 4) mentorship for students and junior faculty in neurobiology and neuropathology; 5) Collaboration with the Coordinating Core to disseminate information on HlV-related neuropathology and 6) technical support and facilitation of international collaborations. In anticipation of current and future needs derived from such investigations, we will expand our studies to include new markers of neurodegeneration (e.g. autophagy) and detection of novel sets of HIV related neuropathology that have emerged as a result of aging (AB, TAU, a-synuclein, lysosomal markers) and other co-morbidities (HCV, methamphetamine). We will also assist investigators in the neuroAIDS field with the better characterization of the patterns of white matter damage, neuro-inflammation and blood brain barrier alterations in HIV patients, and we will provide support for studies of transcriptional and epigenetic dysregulation resulting from acute and chronic HIV infection in the CNS. Our ability to provide scientific and technical support for such studies will be strengthened by our ongoing collaborations with the other Center Cores. Collaborative capacity building in resource limited settings will help research into possibly altered HIV neuropathogenesis related to viral and host differences in international settings.

For the past four years, the Neuropathology Core at the Neurosciences and Pathology Departments at UCSD has been operating under the auspices of the NIH Blueprint Neurosciences Core Grant (Stuart Lipton, PI), and is primarily focused at better understanding the mechanisms of synaptic pathology and at developing new experimental therapies for Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Neurodevelopmental abnormalities, HIV associated dementia (HAD), and other neurodegenerative conditions. This Core Laboratory is headed by Dr. Eliezer Masliah, who has collaborated with a number of neuroscientists on the La Jolla Torrey Pines Mesa and elsewhere for many years, but these facilities had not been previously available to all neuroscientists as a Core until the NIH Blueprint Neuroscience Core Grant was funded. Since the Blueprint Core Grant program is being discontinued by NIH, this NINDS P30 core funding is now requested to continue this core for La Jolla Neuroscientists who are NINDS investigators. Dr. Masliah will continue serve as Director of the Neuropathology Core, which will continue to be located at UCSD. This Core will foster further collaborations and enhance research into neurodegenerative disorders in the San Diego area by NINDS scientists.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The cognitive impairment in patients with Alzheimer's disease is closely associated with synaptic loss in the neocortex and limbic system. Although the neurotoxic effects of aggregated amyloid-beta oligomers in Alzheimer's disease have been studied extensively in experimental models, less is known about the characteristics of these aggregates across the spectrum of Alzheimer's disease. In this study, postmortem frontal cortex samples from controls and patients with Alzheimer's disease were fractionated and analyzed for levels of oligomers and synaptic proteins. We found that the levels of oligomers correlated with the severity of cognitive impairment (blessed information-memory-concentration score and mini-mental state examination) and with the loss of synaptic markers. Reduced levels of the synaptic vesicle protein, vesicle-associated membrane protein-2, and the postsynaptic protein, postsynaptic density-95, correlated with the levels of oligomers in the various fractions analyzed. The strongest associations were found with amyloid-beta dimers and pentamers. Co-immunoprecipitation and double-labeling experiments supported the possibility that amyloid-beta and postsynaptic density-95 interact at synaptic sites. Similarly, in transgenic mice expressing high levels of neuronal amyloid precursor protein, amyloid-beta co-immunoprecipitated with postsynaptic density-95. This was accompanied by a decrease in the levels of the postsynaptic proteins Shank1 and Shank3 in patients with Alzheimer's disease and in the brains of amyloid precursor protein transgenic mice. In conclusion, this study suggests that the presence of a subpopulation of amyloid-beta oligomers in the brains of patients with Alzheimer's disease might be related to alterations in selected synaptic proteins and cognitive impairment.

DESCRIPTION (provided by applicant): Currently over 30 million people live with HIV worldwide. In the US, the aging population represents one of the fastest growing groups with HIV. The Center for Disease Control estimates that by the year 2015, half of all Americans living with HIV will be over the age of 50. The mechanisms of neurodegeneration in aged individuals are not completely understood, however HIV activates apoptotic pathways, dysregulates calcium homeostasis and promotes oxidative stress. Moreover, recent studies have shown that HIV proteins might interfere with clearance pathways such as autophagy, a pathway necessary for protein quality control and elimination of defective older intracellular organelles. Deficits i autophagy have been described in Alzheimer's Disease (AD), Parkinson's Disease (PD) and other aging-related disorders, similarly, neurodegeneration has been linked to defects in autophagy in patients with HIV. We have recently shown that abnormal functioning of the autophagy pathway is associated with progressive accumulation of Amyloid-beta (A¿), ¿-synuclein (¿-syn) and Tau in the CNS of aged HIV human cases and in transgenic (tg) mice expressing HIV-gp120 protein (GFAP-gp120 tg). In this context our hypothesis is that HIV proteins such as Nef might interfere with autophagy by interacting with components of the autophagocytic pathway such as Beclin1. In aged patients with chronic HIV infection, this might result in reduced clearance of neurotoxic proteins and neurodegeneration. The main objectives of this proposal will be to a) better understand the mechanisms through which HIV proteins interfere with autophagy leading to protein accumulation and neurotoxicity, and b) to determine whether activation of the autophagy pathway is neuroprotective in preclinical models of HIV neurotoxicity and aging. For this purpose we propose the following Aims: Aim 1: To analyze interactions between HIV proteins and components of the autophagy pathway in brains of aged patients with chronic HIV infection. Aim 2: To investigate the role of HIV proteins in the cellular mechanisms of autophagy dysfunction and neurotoxicity. Aim 3. To determine whether autophagy activation in vivo ameliorates neurodegenerative and behavioral deficits in aged transgenic rodent models of HIV neurotoxicity. This project has the potential to further elucidate the role of autophagy as key downstream mediator of HIV-protein neurotoxicity during aging, which could lead to the development of new therapies and models of HIV-associated neurodegeneration and neuroprotection. Since alterations in autophagy are also present in AD and PD, this project may have broader applications for therapeutic advancements in other age-related neurodegenerative disorders.

Multiple system atrophy (MSA) is a progressive, degenerative neurological disorder characterized by parkinsonism, ataxia & dysautonomia. The cardinal pathological feature of MSA is the presence of glial cytoplasmic inclusions composed of alpha-synuclein (SYN) in oligodendrocytes. Recent studies suggest that abnormal SYN accumulation in neurons & glia leads to cellular dysfunction & neurodegeneration. During the previous funding period we developed in vitro & in vivo models of MSA showing that mitochondrial damage & hyperphosphorylated SYN aggregate generation may contribute to the pathogensis of MSA. However, mechanisms by which these pathways promote oligodendrogial dysfunction & neurodegeneration are unclear. In this renewal we will investigate the role of mitochondrial dysfunction in SYN phosphorylation & toxicity. Our central hypothesis is that oxidative stress due to mitochondrial dysfuntion may promote G-protein coupled receptor kinase (GRK) activation & toxic SYN phosphorylation. The main objective is to investigate neurodegeneration in MSA-like SYN transgenic (tg) models to determine if reducing SYN accumulation represents a therapeutic strategy for MSA. Aim 1. In order to determine the role of hyperphosphorylated SYN accumulation in oligodendrocytes in the mechanisms of neurotoxicity, we will analyze SYN accumulation & neurodegeneration in myelin basic protein (MBP)-SYN tg mice expressing wild-type (wt) human SYN or a nonphosphorylatable SYN mutant (S129A). MBP-SYNwt tg mice will be crossed with GRK2- or GRKS-deficient mice & MBP-SYNwt tg mice & MBP-SYN(S129A) tg mice will receive intra-cerebral infections with lentivirus expressing GRK2 or GRK5 under a oligodendroglial specific promoter (MBP). Aim 2. In order to determine the role of mitochondrial dysfunction & oxidative stress on GRK activation & SYN phosphorylation, MBP-SYN (wt and S129A) mice & oligodendroglial cells will be challenged with 3- nitropropionic acid. Aim 3. In order to determine if neuronal impairments in MSA models can be ameliorated by reducing SYN aggregation or inhibiting GRKs, MBP-SYN wt tg mice will be treated with rifampicin or GRK blockers. Behavioral performance, neurodegeneration, SYN oligomerization & phosphorylation & GRK activity will be assessed.

Lewy body disease (LBD) is a group of disorders characterized by alpha-synuclein (alpha-syn) accumulation and parkinsonism. During the previous period, the objective was to understand the mechanisms by which (3- synuclein ((3-syn)¿a close homologue to alpha-syn¿blocks a-syn aggregation and might have a role in the treatment of degenerative disorders such as Alzheimer's disease (AD) and LBD. Both p-syn and antibodies against alpha-syn target alpha-syn aggregates for clearance probably via autophagy, a process of degradation and recycling of cellular constituents. Alterations in autophagy might play a role in the pathogenesis of AD and LBD, and might represent a target for treatment development. The objectives of this renewal application are: i) to gain new knowledge as to the involvement of the autophagy pathways in the mechanisms of neurodegeneration in LBD, ii) to develop new experimental therapies for LBD by targeting the autophagy pathways and iii) to better understand the involvement of the autophagy pathways in the mechanisms of asyn clearance mediated by immunotherapy. We propose the following aims: AIM 1. Characterize in vivo the contribution of selected molecular components of the autophagy pathway to the pathogenesis of LBD. AIM 2. Investigate in in vivo models of alpha-synucleinopathy the therapeutic and neuroprotective effects of activators of the autophagy pathway. AIM 3. Better understand the cellular mechanisms involved in the clearance of toxic alpha-syn aggregates via specific antibodies. AIM 4. Determine in immunized animals, the contribution of autophagy to the molecular mechanisms involved in alpha-syn clearance. alpha-Syn transgenic mice will be crossed with mice either deficient in or transgenic for components of the autophagy pathway (e.g. Beclinl, LAMP2, mTor). Mice will be treated with stimulators of autophagy (rapamycin, immunotherapy) and analyzed behaviorally, biochemically and neuropathologically. Studies will be complemented with primary neuronal cultures treated with lentiviral vectors and analyzed for markers of autophagy. Better understanding the autophagic pathways involved in alpha-syn clearance is of central importance toward elucidating the pathogenesis of LBD and developing new treatments for these conditions. Thus, enhancing autophagy and lysosomal degradation of alpha-syn may represent a promising therapeutical strategy for the treatment not only for LBD but also for AD.

CORE D: NEUROPATHOLOGY - ABSTRACT The UCSD-ADRC Neuropathology Core has been instrumental in providing support for establishing the accuracy of clinical diagnosis of Alzheimer's Disease (AD) and dementia with Lewy bodies (DLB), delineating structural and clinico-pathological correlates of dementia in AD, identifying new neuropathological entities causing dementia, provide tissues to investigators and helping to better understand the mechanisms of synaptic degeneration in AD. For the renewal the Aims of the Neuropathology Core will be to: 1) perform rapid autopsies and procure brains from the ADRC participants, using a standardized protocol; 2) perform standardized neuropathological diagnoses and immunocytochemical analysis of demented and normal aged (control) patients clinically evaluated by the UCSD ADRC following the new NIA criteria; 3) maintain a state of the art brain repository to provide the ADRC projects and other investigators with well characterized including early AD and MCI cases; 4) perform immunochemical analysis relevant to neurodegeneration and synapse loss in MCI and early AD cases and 5) foster the utilization of the ADRC Neuropathology tissue repository for new research and inter- center collaborations. Approximately 50 to 60 cases and over 20 tissue requests will be processed a year. The neuropathological results will be submitted to the National Alzheimer's Coordinating Committee (NACC) in compliance with NIA requirements. As part of the mission of the Core we will also continue to support extensive collaborations with national and international investigators and train fellows, residents, graduate and undergraduate students in neuropathology and microscopy techniques. Support ADRC Projects including providing post-mortem confirmation and analysis of SORL-1 for Project 1, FTD tissues and analysis of C9ORF for Project 2, and providing brain tissues from control, MCI, AD for Project 3. With the ADRC Outreach, Recruitment and Education (ORE) Core organize meetings to encourage the use of the neuropathology core. New and junior investigators will be encouraged to conduct research with the postmortem tissues. Continue to provide tissues to local and national investigators and for multi-center initiatives such as the Genome-Wide Analysis Studies organized by NIA.

? DESCRIPTION (provided by applicant): Synucleonopathies are a group of neurodegenerative disorders that affect over 1.5 million people in the US. Multiple system atrophy (MSA) is a fatal, rapidly progressive synucleonopathy characterized by parkinsonism and oligodendroglial accumulation of ?-synuclein (?-syn). While considerable effort has been devoted at understanding the pathogenesis of Parkinson's Disease, less is known about MSA and the mechanisms through which ?-syn accumulates in oligodendroglial cells, resulting in neurodegenerative pathology, is not completely clear. One possibility is that autophagy failure could lead to ?-syn propagation from neurons to oligodendroglial cells. We recently found that in MSA microRNA (miR-101, miR-30a, miR183, miR-96) that regulate autophagy are affected. The HYPOTHESIS is that miRNA dysregulation in MSA might down-regulate autophagy, which in turn results in defective ?-syn clearance with the consequent propagation from neurons to glia. The OBJECTIVES will be to better understand the mechanisms through which alterations in autophagy- related miRNAs are involved in the pathogenesis of MSA and to evaluate the potential value of modulating miRNA's as a novel therapeutical approach for MSA. For this purpose we will utilize a combined strategy including studies in unique mixed cell cultures in chambers, transgenic mouse models of MSA and brain tissues from MSA patients from multiple sites. The AIMS are: ONE. To investigate in mixed neuron- oligodendroglial cell cultures the mechanisms through which alterations in miRNAs might lead to ?-syn propagation into glial cells. TWO. To determine in transgenic models of MSA if modulating miRNAs that regulate autophagy ameliorate the ?-syn pathology and spreading. THREE. To analyze the regional relationship between alterations in specific miRNAs and autophagy targets in MSA. These goals are in agreement with the NINDS 2014 PD Basic Research recommendations. Finding a link between miRNA dysregulation, autophagy deficits, and ?-syn spreading will shed light on pathogenesis of MSA, and will open the door for the study of these interactions in other neurodegenerative disorders. These studies could also lead to the development of novel therapeutical strategies for MSA.