Tony Wyss-Coray - US grants

DESCRIPTION (From Abstract): Cerebrovascular deposition of amyloid, or cerebral amyloid angiopathy (CAA), is a prime cause of normotensive intracerebral hemorrhages in the elderly. CAA is also a major neuropathological lesion in Alzheimer's disease (AD) and is accompanied by degenerating cells of the vascular wall. Because cerebrovascular amyloidosis has implications for the pathogenesis of Alzheimer's disease and for central nervous system (CNS) function in general, understanding its etiology is of great importance. Although it is known that single amino acid substitutions in several different proteins can cause rare autosomal dominant forms of CAA and that the apolipoprotein (apo) E e4 allele is a genetic risk factor for CAA, the cause of this disease in the majority of cases remains elusive. Through studies addressing the role of injury in neurodegenterative diseases, we have identified transforming growth factor (TGF)-ß1 as an inducer of cerebrovascular amyloidosis and as a potential pathogenic factor for CAA in human Alzheimer's disease cases. The cytokine TGF-ß1 is rapidly produced after all forms of CNS injury and may function as an organizer of the responses to brain injury. Overexpression of TGF-ß1 in astrocytes of transgenic mice caused cerebrovasular amyloid deposition and prominent perivascular astrocyte activation along with a degeneration of cortical capillaries reminiscent of Alzheimer's disease. Here we propose experiments to define the role of TGF-ß1 in cerebrovascular amyloidosis at the molecular level. We will determine whether chronic activation of astrocytes by TGF-ß1 is necessary and sufficient to cause cerebrovascular amyloidosis in vivo and whether this process is modulated by different human apoe isoforms. We will use transgenic mice that overexpress dominant-active or dominant-negative TGF-ß receptors in astrocytes or comparable levels of apoE3 or apoE4 in neurons. In addition, we will initiate studies to determine if chronic TGF-ß1 production and astrocytosis cause the capillary degeneration that precedes amyloid deposition and whether these processes can be modulated by apoE3 or apoE4. The proposed studies will allow us to better understand the etiology and pathogenesis of cerebrovascular amyloidosis in vivo and clarify the roles of TGF-ß1, CNS injury, and astrocyte activation in this process. Our findings will have implications for the pathogenesis of human CAA and Alzheimer's disease in general and will help to assess whether TGF-ß1 could be a future target of therapeutic interventions.

Stroke is a leading cause of death and a major source of disability and suffering. Alzheimer' s disease is the most frequent cause of dementia in the elderly, affecting an estimated four million people in the US alone. Apolipoprotein E has been identified as a modulator or a susceptibility gene in both these diseases. Of the three common Apo E isoforms, Apo E4 is associated with poor outcome after stroke, traumatic brain injury, intracerebral hemorrhages, and is a major risk factor for Alzheimer's disease and possibly vascular dementia. In contrast, Apo E3 and Apo E2 isoforms are protective and associated with lower risk for these diseases. Similar observations were made in Apo E transgenic mice. The long-term objective of this study is to elucidate the molecular mechanism by which Apo E exerts these isoform-specific effects in the brain and specifically how Apo E3 is neuroprotective. The preliminary results show that Apo E3 and to a lesser extent Apo E4 stimulates the synthesis of a neuroprotective protease inhibitor, plasminogen activator inhibitor 1 (PAI-1) in vitro and that PAI-1 protects mice against different forms of neurodegeneration most likely by inhibiting serine protease tissue plasminogen activator (tPA). TPA is probably the most abundant protease in the brain and it cause neurodegeneration in mice and possibly humans. Therefore, the investigators hypothesize that Apo E3 activates a signaling pathway that leads to the production of PAI-1, which then protects neurons against injury, whereas Apo E4 induces less PAI-1 and does not protect efficiently against neurodegeneration. The proposed studies are designed to assess this novel function of Apo E in brain injury and neuroprotection. In Specific Aim #1, how Apo E induces PAI-1 will be determined at the molecular level. In Aim #2, apo E induction of PAI-1 and its influence on neurotoxicity will be examined in cell cultures. In Aim #3, the neuroprotective effect of Apo E will be assessed in vivo. These results may help devise novel therapeutic strategies to mimic the beneficial Apo E3 effects or inhibit the detrimental Apo E4 effects in brain injury and neurodegeneration.

Acute or chronic neuronal injury can lead to cell death and degeneration in stroke or neurodegenerative diseases. A number of animal models have been developed to study these diseases but in most cases, animals have to be sacrificed to gain information on disease severity or to study pathogenic pathways. This is in contrast to many peripheral diseases where disease markers can be more easily measured in the blood or biopsies can be obtained without interfering dramatically with the disease. Therefore, new methods are necessary to study disease progression or cellular changes in the brain noninvasively. Bioluminescence imaging in living animals has recently been used to monitor and quantitate gene activity and disease progression in peripheral organs with great success. Although, this imaging modality lacks high resolution and cannot be used at present to localize signals with high accuracy, it is quantitative and can faithfully report gene activation if appropriate fusion gene constructs are used. Here we propose to use bioluminescence imaging in transgenic mice that harbor an injury-responsive Lucifer's reporter gene to assess neuronal injury or use mice that express luciferase at high levels in neurons to quantitate cell number. Strong preliminary data indicate that we can indeed collect photons that correlate with reporter gene activity from injured brains of living mice. We propose to use these mice, which carry a reporter gene responsive to the TGF-beta signaling pathway, to optimize non-invasive bioluminescence imaging of the brain and to try to quantitate neuronal injury non-invasively in two acute models of brain injury using bioluminescence imaging (Specific Aim 1}. We also propose to engineer related mice that express luciferase and YFP constitutively at high levels in defined groups of neurons to try to correlate bioluminescence signals in living mice with neuronal cell number (Specific Aim 2). If we are successful, our studies will demonstrate feasibility of using non-invasive bioluminescence imaging to measure gene activity in the brain and to use such measurements as indicators of pathological or physiological processes in individual mice. These, and other reporter mice could also be used to screen drugs for efficacy to interfere with or activate specific signaling pathways in living mice and to assess drug availability in the brain.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative disease that leads to progressive cognitive dysfunction. Current knowledge of the processes leading to Alzheimer's disease is still limited, and no effective treatments are available. Alzheimer's disease is characterized by loss of neurons and the abnormal accumulation of amyloid beta into amyloid plaques and hyperphosphorylated tau into neurofibrillary tangles. However, in the vast majority of AD cases there are no obvious alterations in the molecular pathways causing amyloid beta production or tau hyperphosphorylation and it has been difficult to assign them a causal role in sporadic Alzheimer's disease. It is perhaps more likely that other factors trigger these toxic pathways which subsequently lead to neurodegeneration. Age is the major known risk factor for Alzheimer's disease. It is associated with an increase in cellular injury and inflammation and it has been postulated that an age-related reduction in trophic support makes neurons vulnerable to injury and degeneration. TGF-beta1 is an injury response factor and has been implicated in AD pathogenesis. TGF-beta1 levels in AD brains are increased and correlate inversely with the amount of parenchymal amyloid beta deposition but positively with the amount of cerebrovascular amyloid betadeposition. This is consistent with findings in transgenic mice where TGF-beta1 produced by astrocytes reduces the accumulation of parenchymal amyloid beta while the remaining amyloid beta is found in blood vessels. TGF-beta1 has also potent neurotrophic/neuroprotective effects in numerous cell culture and in vivo models of brain injury and mice lacking TGF-beta1 show spontaneous neuronal cell death and neurodegeneration. Our most recent preliminary studies demonstrate that reduced TGF-beta signaling in neurons promotes AD-like disease in mice suggesting that neuronal TGF-beta signaling may be beneficial. Based on these findings we propose the hypothesis that aging and reduced trophic support to neurons promotes neurodegeneration and sporadic Alzheimer's disease and that neuronal TGF-beta signaling is a critical trophic signal for neurons to age successfully. To test our hypothesis we will determine how lack of neuronal TGF-beta signaling affects survival, APR processing, and tau phosphorylation in cell culture studies and we will use unbiased screens to identify the transcription factors and TGF-beta responsive genes mediating neuroprotection in neurons. We will block neuronal TGF-beta signaling in AD mouse models in vivo before or during active disease to accelerate disease, and we will specifically increase neuronal TGF-beta signaling before or during active disease to ameliorate disease. These in vivo studies will help us to assess the relevance and potential therapeutic implications of our hypothesis and will evaluate the TGF-bet1 signaling pathway as a potential target for the treatment of Alzheimer's disease.

Alzheimer's disease (AD)is a neurodegenerative disease that leads to progressive cognitive dysfunction. Current knowledge of the processes leading to AD is still limited, and no effective treatments are available. Because neurodegeneration in is associated with injury and an activation of innate immune responses in the brain, drugs that could mimic the beneficial aspects of this response are potential therapeutic candidates. The cytokine transforming growth factor (TGF)-(31 is an organizer of the brain's response to injury and has been shown to have neuroprotective effects in models of brain injury and degeneration. Recombinant TGF- (31 has been used to treat various forms of brain injury in vivo but delivery is not suitable for human use. Studies from our lab have demonstrated that TGF-(31 can reduce the overall accumulation of A3, a key factor in AD pathogenesis, in mouse models for AD and in cell culture. Numerous studies have also demonstrated that TGF-P1 is a potent neurotrophic factor although high-level chronic TGF-P1 production can also be detrimental. Recently, we reported that reduced TGF-|31 expression in vivo or in cultured neurons increases neurodegeneration. Additional preliminary studies presented here show that reducing TGF-p signaling in neurons of a mouse model for AD increases A(3accumulation and neurodegeneration and that the brain is a major site of TGF-p signaling in the mouse. We have started to search for small molecule chemical compounds that can activate the TGF-p signaling pathway. With reporter cell lines for the TGF-p signaling pathway we screened a diverse small molecule drug library containing and identified several compounds that are able to activate the reporter system in vitro and early tests indicate that at least one may be active in vivo. We propose to screen a larger chemical library for compounds that activate TGF-p signaling in vitro to test their activity in AD-relevant cell culture assays and mouse models and in a reporter mouse for the TGF-p signaling pathway. This combined screening approach should allow us to identify compounds that have activity in vivo to test the hypothesis that chemical activators of the TGF-p signaling pathway can be used to delay or reduce neurodegeneration and AD-like disease in mice. Together with our collaborators at the high-throughput screening facility at Stanford and at SRI International we expect to find such compounds that activate the TGF-p signaling pathway and test them in vivo in TGF-p reporter mice and in mouse models for AD. After the completion of the proposed studies we hope to be in a position to initiate lead optimization studies with non-profit or commercial collaborators. This would for the first time bring a novel neuroprotective and amyloid reducing drug based on the TGF-p signaling pathway towards testing in AD.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative disease that leads to progressive cognitive dysfunction. Current knowledge of the processes leading to Alzheimer's disease is still limited, and no effective treatments are available. Alzheimer's disease is characterized by loss of neurons and the abnormal accumulation of amyloid beta into amyloid plaques and hyperphosphorylated tau into neurofibrillary tangles. However, in the vast majority of AD cases there are no obvious alterations in the molecular pathways causing amyloid beta production or tau hyperphosphorylation and it has been difficult to assign them a causal role in sporadic Alzheimer's disease. It is perhaps more likely that other factors trigger these toxic pathways which subsequently lead to neurodegeneration. Age is the major known risk factor for Alzheimer's disease. It is associated with an increase in cellular injury and inflammation and it has been postulated that an age-related reduction in trophic support makes neurons vulnerable to injury and degeneration. TGF-beta1 is an injury response factor and has been implicated in AD pathogenesis. TGF-beta1 levels in AD brains are increased and correlate inversely with the amount of parenchymal amyloid beta deposition but positively with the amount of cerebrovascular amyloid betadeposition. This is consistent with findings in transgenic mice where TGF-beta1 produced by astrocytes reduces the accumulation of parenchymal amyloid beta while the remaining amyloid beta is found in blood vessels. TGF-beta1 has also potent neurotrophic/neuroprotective effects in numerous cell culture and in vivo models of brain injury and mice lacking TGF-beta1 show spontaneous neuronal cell death and neurodegeneration. Our most recent preliminary studies demonstrate that reduced TGF-beta signaling in neurons promotes AD-like disease in mice suggesting that neuronal TGF-beta signaling may be beneficial. Based on these findings we propose the hypothesis that aging and reduced trophic support to neurons promotes neurodegeneration and sporadic Alzheimer's disease and that neuronal TGF-beta signaling is a critical trophic signal for neurons to age successfully. To test our hypothesis we will determine how lack of neuronal TGF-beta signaling affects survival, APR processing, and tau phosphorylation in cell culture studies and we will use unbiased screens to identify the transcription factors and TGF-beta responsive genes mediating neuroprotection in neurons. We will block neuronal TGF-beta signaling in AD mouse models in vivo before or during active disease to accelerate disease, and we will specifically increase neuronal TGF-beta signaling before or during active disease to ameliorate disease. These in vivo studies will help us to assess the relevance and potential therapeutic implications of our hypothesis and will evaluate the TGF-bet1 signaling pathway as a potential target for the treatment of Alzheimer's disease.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative disorder that leads to progressive cognitive dysfunction. Current knowledge of the processes leading to AD is still limited, and no effective treatments are available. Because neurodegeneration is associated with injury and activation of innate immune responses in the brain, drugs that could mimic the beneficial aspects of this response are potential therapeutic candidates. The cytokine transforming growth factor (TGF)-¿1 is an organizer of the brain's response to injury and has been shown to have neuroprotective effects in models of brain injury and degeneration. Recombinant TGF-¿1 has been used to treat various forms of brain injury in vivo but delivery is not suitable for human use. Studies from our lab have demonstrated that TGF-¿1 can reduce the overall accumulation of A¿, a key factor in AD pathogenesis, in mouse models for AD and in cell culture. Numerous studies have also demonstrated that TGF-¿1 is a potent neurotrophic factor, although high-level chronic TGF-¿1 production can also be detrimental. Recently, we reported that reduced TGF-¿1 expression in vivo or in cultured neurons increases neurodegeneration. Additional studies show that reducing TGF-¿ signaling in neurons of a mouse model for AD increases A¿ accumulation and neurodegeneration and that TGF-¿ receptor expression is reduced in human AD brains. We have identified bioactive small molecule chemical compounds that can activate the TGF-¿ signaling pathway in hippocampal neurons of mice and that pass the blood-brain barrier. With reporter cell lines for the TGF-¿ signaling pathway we screened a diverse small molecule drug library and identified several compounds that are able to activate the reporter system in vitro and in TGF-¿ reporter mice in vivo. The compounds induce specific TGF-¿-responsive genes in cell culture consistent with Smad dependent activation of the TGF-¿ pathway. These chemicals share common properties from which we propose here to derive a lead compound within 5 years. This project includes structure activity relationship analysis of identified active compounds, medicinal chemistry, toxicology and pharmacology in a subcontract with SRI International. Compounds will be tested in neuroprotection and neurotoxicity assays in cell culture and in TGF-¿ reporter mice in vivo. The two most promising compounds will then be tested in an in vivo model of neurodegeneration and in a mouse model for AD. Part of the in vivo analysis on neurodegeneration will be done in collaboration with researchers at UCSD. At the end of our studies we propose to have for the first time a novel neuroprotective and amyloid reducing investigational new drug based on the TGF-¿ signaling pathway for testing in patients with AD.

1-(3-(dimethylamino)propyl)-4-((hydroxyimino)methyl)pyridinium, chloride; 3-Pyrrolidineacetic acid, 2-carboxy-4-(1-methylethenyl)-, (2S-(2alpha,3beta,4beta))-; 4-hydroxyiminomethyl-1-(3-N,N-dimethylaminopropyl)pyridinium chloride; Abnormal Assessment of Metabolism; Absorption; Acquired brain injury; Activation, Gene; Age; Agonist; Alzheimer; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer's; Alzheimer's Disease; Alzheimers Dementia; Alzheimers disease; Ammon Horn; Amyloid; Amyloid Substance; Animal growth regulators, transforming growth factors; Assay; Behavioral; Bioassay; Bioavailability; Biologic Assays; Biologic Availability; Biological Assay; Biological Availability; Bioluminescence; Blood - brain barrier anatomy; Blood-Brain Barrier; Bone-Derived Transforming Growth Factor; Brain; Brain Injuries; C 3-5 Converting Enzyme; C3 Proactivator; C3PA; CVFBb; Canine Species; Canis familiaris; Cell Communication and Signaling; Cell Line; Cell Line, Transformed; Cell Lines, Strains; Cell Signaling; CellLine; Cells; Characteristics; Chemicals; Chemistry, Pharmaceutical; Chronic; Clinical Trials; Clinical Trials, Unspecified; Cognitive Disturbance; Cognitive Impairment; Cognitive decline; Cognitive function abnormal; Collaborations; Common Rat Strains; Complement 3 Proactivator; Complement Factor B; Complement Factor B, Alternative Pathway; Complement Protein B; Complement Protein Factor B; Cornu Ammonis; Cultured Cells; Cytochrome P-450; Cytochrome P-450 Enzyme System; Cytochrome P450; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Development; Digenic Acid; Disturbance in cognition; Dogs; Dose; Drug Interactions; Drug Kinetics; Drugs; Drugs, Investigational; ELIG; Eligibility; Eligibility Determination; Encephalon; Encephalons; Evaluation; FDA; FLR; Factor B; Failure (biologic function); Food and Drug Administration; Food and Drug Administration (U.S.); Gene Activation; Genes; Genes, Reporter; Genetic Toxicology; Hemato-Encephalic Barrier; Hippocampus; Hippocampus (Brain); Human; Human, General; Hydrogen Oxide; Image; Immune response; Impaired cognition; In Vitro; Injury; International; Intracellular Communication and Signaling; Investigational Drugs; Investigational New Drug Application; Investigational New Drugs; Investigators; Kainic Acid; Knowledge; Lead; Libraries; Life; Liver; MT-bound tau; Mammals, Dogs; Mammals, Mice; Mammals, Rats; Man (Taxonomy); Man, Modern; Materials Testing; Maximal Tolerated Dose; Maximally Tolerated Dose; Maximum Tolerated Dose; Measures; Medication; Medicinal Chemistry; Metabolic; Metabolic Studies; Metabolism Studies; Mice; Mice, Transgenic; Milk Growth Factor; Modeling; Monitor; Murine; Mus; Nerve Cells; Nerve Degeneration; Nerve Unit; Nervous System, Brain; Neural Cell; Neurocyte; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Neuron Degeneration; Neurons; Oral; Overexpression; P450; Pathogenesis; Pathology; Pathway interactions; Patients; Pb element; Permeability; Pharmaceutic Chemistry; Pharmaceutic Preparations; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacokinetics; Pharmacology; Pharmacology and Toxicology; Physiologic Availability; Platelet Transforming Growth Factor; Primary Senile Degenerative Dementia; Process; Process of absorption; Production; Properdin Factor B; Property; Property, LOINC Axis 2; Protein Overexpression; Protocol Screening; Range; Rat; Rattus; Recombinant Transforming Growth Factor; Reporter; Reporter Genes; Reporting; Research Personnel; Researchers; Route; SCHED; Safety; Schedule; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Structure; Structure-Activity Relationship; System; System, LOINC Axis 4; TGF B; TGF-beta; TGFbeta; Testing; Therapeutic; Time; Toxic effect; Toxicities; Toxicogenetics; Toxicokinetics; Toxicology; Toxicology Genetics; Transformed Cell Line; Transforming Growth Factor beta; Transforming Growth Factors; Transgenic Mice; Transgenic Organisms; Tumor Growth Factors; USFDA; United States Food and Drug Administration; Water; absorption; analog; base; bioavailability of drug; biological signal transduction; body system, hepatic; brain damage; brain lesion (from injury); canine; chemical structure function; clinical investigation; cognitive dysfunction; cognitive loss; cognitively impaired; compound 30; conditioned fear; cultured cell line; cytokine; dementia of the Alzheimer type; design; designing; domestic dog; drug/agent; experiment; experimental research; experimental study; failure; fear conditioning; heavy metal Pb; heavy metal lead; hippocampal; host response; imaging; immunoresponse; improved; in vitro Assay; in vivo; in vivo Model; injury response; metabolic abnormality assessment; microtubule associated protein tau; microtubule bound tau; microtubule-associated protein tau; microtubule-bound tau; mouse model; mutant; neural degeneration; neurodegeneration; neurodegenerative illness; neurofibrillary degeneration; neurofibrillary lesion; neurofibrillary pathology; neuron cell death; neuron loss; neuron toxicity; neuronal; neuronal cell death; neuronal degeneration; neuronal loss; neuronal toxicity; neuroprotection; neurotoxicity; neurotrophic factor; neurotrophin; neutrophin; novel; organ system, hepatic; overexpress; pathway; pre-clinical; preclinical; prevent; preventing; primary degenerative dementia; receptor expression; research study; response; response to injury; scaffold; scaffolding; scale up; senile dementia of the Alzheimer type; small molecule; small molecule libraries; structure function relationship; tangle; tau; tau Proteins; tau factor; transgenic

DESCRIPTION (provided by applicant): Alzheimer's Disease (AD) is an age-related disorder that causes a dramatic loss of cognitive function and affects millions of elderly individuals worldwide. It is characterized pathologically by the presence of protein aggregates of beta amyloid (AB) and tau and a progressive neurodegeneration. There is exceedingly strong evidence that abnormal assemblies of AB are neurotoxic and have a key role in AD. Why AB accumulates or induces neurodegeneration is unclear. Following up on a report that linked Beclin 1, an essential protein involved in the early steps of autophagy, to neurodegeneration and cell death in the lurcher mouse we decided to explore the possibility that Beclin 1 and autophagy may have a role in AD. Autophagy is the major pathway involved in the degradation of long-lived proteins and organelles, cellular remodeling, and survival during nutrient starvation. It is unclear whether autophagy exerts a pathological or protective role in neurodegeneration and Alzheimer's Disease. We discovered that expression of Beclin 1 is reduced more than 50% in gray matter of the frontal cortex in postmortem brains from AD cases compared with age-matched cases of Lewy body variant of AD, Huntington's disease, Parkinson's disease, or nondemented controls. This decrease was not simply due to a loss of neurons since levels of the neuronal protein neuron specific enolase were not altered. We found that genetic reduction of Beclin 1 expression in beclin 1-/+ haploinsufficient mice results in less autophagy in primary neurons and is associated with neurodegeneration in 9-month-old mice. Beclin 1 deficiency in APP transgenic mice, a model for AD, results in increased accumulation of fragments of APP and AB in cells and in the extracellular space and was associated with increased inflammation. In addition, increased autophagy in cultured neuroblastoma cells reduces APP fragments while siRNA mediated reduction in Beclin 1 expression increases APP fragments and AB. Together, these studies provide strong evidence for a role of Beclin 1 and autophagy in AD pathogenesis and they open a new pathway to potentially target this disease. The goal of this application is to determine how Beclin 1 is regulated in neurons and in mouse brains, how it affects the production and turnover of AB and its precursors, and whether increased production of Beclin 1 may be protective and ameliorate neurodegeneration and AD-like disease in mice. We also expect to establish that Beclin 1 is a major modifier of AD pathogenesis and that increasing Beclin 1 levels reduces disease. If successful, our findings may provide new targets for the treatment of AD and neurodegeneration. PUBLIC HEALTH RELEVANCE: Alzheimer's Disease (AD) affects millions of people worldwide. It is characterized by the accumulation of proteins in the brain inside and outside of neurons. We discovered a novel mechanism that neurons use to normally rid themselves of old or damaged proteins to be defective in AD. If we inhibit this protein degradation process in AD mice they develop more disease. We propose to study this process to try to reduce disease in mice and possibly in AD.

DESCRIPTION (provided by applicant): Cognitive function in humans declines in essentially all domains starting around age 50-60, and neurodegeneration and dementia seem to be inevitable in all but a few who survive to very old age. Mice with a fraction of the human lifespan show similar cognitive deterioration indicating that specific biological processes rather than time alone are responsible for brain aging. While age-related cognitive dysfunction and dementia in humans are clearly distinct entities and affect different brain regions, the aging brain shows the telltale molecular and cellular changes that characterize most neurodegenerative diseases including synaptic loss, dysfunctional autophagy, increased inflammation, and protein aggregation. Remarkably, the aging brain remains plastic and exercise or dietary changes can increase cognitive function in humans and animals, with animal brains showing a reversal of some of the aforementioned biological changes associated with aging. Using heterochronic parabiosis we showed recently that blood-borne factors present in the systemic milieu can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing an old mouse to a young systemic environment or to plasma from young mice increased neurogenesis, synaptic plasticity, and improved contextual fear conditioning and spatial learning and memory. Preliminary proteomic studies show several proteins with stem cell activity increase in old rejuvenated mice supporting the notion that young blood may contain increased levels of beneficial factors with regenerative capacity. In this application we intend to test the hypothesis that blood-borne protein factors in young mice are sufficient to increase adult neurogenesis and regenerate the old brain, and that a focused proteomic screen will allow us to identify the most potent such factors. Our studies pursue the innovative concept that brain aging and cognitive dysfunction is at least in part under control of factors from the circulatory environment and that such factors are sufficient to rejuvenate the aging brain.

DESCRIPTION (provided by applicant): Aging in humans is associated with a progressive decline in cognitive function, the consequences of which are enormous for affected individuals. Any scientific advance that could delay or prevent age-related cognitive decline would have a profound impact at every level of society given the demographic changes that are occurring with an exponentially increasing percentage of elderly individuals and the percentage of those individuals who are affected by cognitive decline. The use of animal models has greatly accelerated the pace of research on factors that influence cognitive function and age-related changes. One of the most robust interventions that can enhance cognitive function is physical activity. This has been shown in organisms ranging from rodents to humans. Despite the importance and potency of this intervention, the mechanisms by which exercise enhances cognitive activity remain elusive. Here we propose to test the provocative hypothesis that there are factors secreted by muscle that promote neurogenesis and synaptic plasticity to maintain cognitive function and that these factors are increased by muscle activity during exercise (exercise factors). This hypothesis is firmly rooted in the expanding field of research on muscle as a secretory organ, participating in various endocrine networks that function to regulate physiological phenomena such as energy metabolism, angiogenesis, and bone formation. Within the context of regulation of cognitive function, we propose that a muscle-brain axis is an evolutionarily conserved endocrine pathway that links two primordial organ systems, with muscle-derived factors promoting maintenance of neuronal homeostasis. We will use both in vitro and in vivo approaches to explore this hypothesis in murine models of neurogenesis, neuronal function, and cognitive activity. Capitalizing on our expertise in plasma proteomics, we will characterize the muscle proteome from control muscle and muscle altered by exercise or aging. Both muscle and brain (hippocampus) will be tested for transcriptional and epigenetic changes induced by exercise, both to explore the mechanisms by which exercise modifies muscular and neuronal function and also to test for any molecular memory to explain any persistent effects of exercise on the brain. Direct tests of secretomes will be performed using parabiotic pairings and plasma injections, and candidate testing will include studies of neurogenesis in vitro and muscle-specific gene deletions in vivo. These multifaceted approaches will allow us to characterize the muscle-brain axis, to examine the molecular basis and regulation of that axis with exercise, and to understand the basis for the lasting effects of exercise on neuronal activity, each of which would provide an entirely new framework within which to understand the beneficial effects of physical activity on brain function and together offering a potentially revolutionary approach to the treatment of age-related cognitive decline.

DESCRIPTION (provided by applicant): Aging leads to a functional deterioration of multiple physiological systems, including those underlying sleep and wakefulness. Older individuals tend to have reduced sleep amounts and changes in sleep composition and distribution, decreased alertness, increased fragmentation, reduced nocturnal sleep and amplitude of delta EEG frequency. Sleep disruptions in the elderly also severely affect the health and well-being of their caregivers to the point that, in pathologies such as Alzheimer's disease, sleep fragmentation is the main cause of institutionalization. The underlying mechanisms of these changes in sleep architecture and efficiency during aging are unknown. In this proposal, we will use optogenetics to interrogate the role of specific neuronal cell types and circuits in the decline of sleep qualit and cognition during aging. Optogenetics is an ideal method to study sleep/wake mechanisms in rodents because pharmacological approaches far exceed the short time scale of sleep/wake cycles (in the order of minutes), and electrical stimulations cannot provide cellular specificity. n particular we propose to determine whether the ability of three neurotransmitters (Hcrt/orexin, norepinephrine and acetylcholine) to facilitate wakefulness is reduced in old mice. In aim 2, we will determine whether specific features of sleep quality driven by these three transmitters are relevant for the cognitive decline observed during aging. In a third aim we will test whether a young systemic environment is able to improve sleep composition and distribution in an old mouse using plasma transfer and parabiosis experiments. The data obtained from these experiments may lead to selective therapeutic interventions to improve the quality of life of the elderly and their caregivers.

Project Summary: Core D The overall goals of the Neuropathology and Biospecimen Core are to obtain ante- and postmortem biospecimens and tissues from ADRC participants in support of research on Alzheimer disease and related disorders, to provide state-of-the art neuropathologic diagnoses for center participants, and report Core data to the NACC. The special emphasis of our Core lies in the integration of classical neuropathology with systems biology and advanced clinical and imaging studies. This emphasis guided the selection of Dr. Tony Wyss- Coray (a neurobiologist with a background in neuroimmunity, proteomics, and Alzheimer disease biomarkers) as the Core Leader and Dr. Edward Plowey (a neuropathologist trained in cutting-edge diagnostic techniques and neurodegeneration research as a neuropathology fellow with the University of Pittsburgh ADRC) as the Core co-leader and director of the Stanford ADRC brain bank. The core also benefits from the experience and track record of a previously funded Alzheimer's Disease Core Center at Stanford University and the Palo Alto VA from 2000 to 2009 during which time 57 autopsy cases were collected and 43 submitted to NACC. Tissues from these cases will be made available for distribution under the new core with the assistance of Dr. Ahmad Salehi, director of the brain bank at the VA. As part of the proposed focus on neuro-immune interactions and novel genomics approaches, cerebrospinal fluid, plasma, blood, and skin biopsies will be collected for proteomic, genomic, and cellular measurements, which will be made available upon request as part of the resources provided by the Core. The Neuropathology and Biospecimen Core will closely collaborate with the Administrative, Clinical, Imaging, and Data Management and Biostatistics Cores to optimize the use of these biospecimens by investigators at Stanford, other Alzheimer's Disease Centers, and other qualified institutions.

? DESCRIPTION (provided by applicant): Age is the major risk factor for a majority of the leading causes of death in the US including heart disease, cancer, stroke, and dementia. With a rapidly aging population pushing the boundaries of what our health care system can absorb, there has been an unprecedented interest in aging and longevity from the public, funding bodies, and industry. I believe that based on dramatic technological advances in chemical biology and empirical findings advanced in part by our group, the time is right to ask some of the fundamental questions about aging: what controls organismal aging? Does it start in one tissue and spread to another? Or does aging start in one cell type, such as in stem cells or senescent cells, and spread to other cells and tissues? Or does it start simultaneously in all cells, driven y aging mitochondria, by failures in DNA or protein maintenance, or other mechanisms? The proposed research will try to address these questions and follows from our groundbreaking discovery that soluble, heat labile factors in blood plasma are sufficient to accelerate brain agin or regenerate and rejuvenate old brains. These and studies with heterochronic parabiosis by others provide the foundation for my proposal by demonstrating that soluble factors carry information about the biological age of an organism, that soluble factors can actively modulate aging, and that aging is malleable and possibly reversible. I will take advantage of the extraordinary developments in chemical biology, which allow for the orthogonal (i.e. independent, causing no interference) introduction of genetically encoded, non-canonical amino acids into living organisms and the subsequent detection of the proteins into which they are incorporated. This revolutionary concept is based on a rewired translation in which the genetic code is expanded resulting in the production of modified proteins, which can be chemically identified. We will generate transgenic mice using this technol

PROJECT SUMMARY/ABSTRACT Alzheimer's disease is a chronic neurodegenerative disease that causes progressive cognitive decline and has thus far proven incurable. With an aging population, this devastating disease will become more widespread in the near future, making the search for effective treatments a principle objective. Brain pathology is marked by amyloid plaques and neurofibrillary tangles, but it is unknown if these are causative, and numerous treatment strategies targeting amyloid have proven unsuccessful thus far. Cerebrovascular signaling has been shown to play an important role in aging and in neurodegenerative diseases including Alzheimer's. In particular, cerebrovascular TGF-? signaling undergoes a marked decline in these conditions, potentially exacerbating disease symptoms. Indeed, mice lacking TGF-? signaling specifically in endothelial cells have increased blood- brain barrier permeability and hemorrhage. Restoring deficient TGF-? signaling is beneficial and promotes amyloid clearance in mouse models of Alzheimer's disease while inhibiting signaling exacerbates pathology, but the mechanism and cell types responsible remain unclear. Brain endothelial cells are responsible for neurovascular homeostasis and mediating the response to vascular signals, and have been shown to be dysfunctional in Alzheimer's disease. This current study will characterize changes in brain endothelial cell TGF- ? signaling during aging and in mouse models of Alzheimer's disease, and determine the effects of these changes on blood-brain barrier permeability. Specifically, using genetic models to regulate TGF-? signaling in brain endothelial cells in mice, an orally bioavailable small molecule activator of TGF-? signaling in the brain, and novel bioorthogonal chemistry and imaging tools to assess blood-brain barrier function, this study aims to determine whether the loss of TGF-? signaling during aging and Alzheimer's disease impairs endothelial and neurovascular function, contributing to disease pathology, and whether reversing this loss in signaling has therapeutic potential. Ultimately, these studies pursue the innovative concept that cerebrovascular TGF-? signals play an important and overlooked role in Alzheimer's disease, and that restoring these signals can be exploited therapeutically.

Alzheimer?s disease (AD) is the most prevalent neurodegenerative disease in the world, characterized by a progressive loss of cognitive functions leading to dementia. Hallmark pathological features of AD are amyloid plaques, caused by abnormal accumulation of amyloid-? (A?), and neurofibrillary tangles consisting of hyperphosphorylated tau. However, it has been difficult to assign a causal role to these features in sporadic AD, and it is increasingly accepted that AD is a multifaceted disorder driven by aging and dysfunction in many cellular pathways. Recently neuroinflammation, in particular microglia dysfunction, has been shown to be an essential component in the development of functional deficits in AD. Remarkably, new reports suggest that in the aged and diseased brain different microglia subsets with unique transcriptional and functional signatures co-exist, which can be protective or detrimental for the brain. Consequently, it is of high importance to identify and target those microglia subsets that are dysfunctional and ?harmful? for the aging and diseased brain such as AD. We recently identified a novel, lipid-droplet containing microglia (LAM) subtype in the aging brain. Lipid droplets (LD), which are neutral lipid storing cytoplasmic organelles, are increasingly recognized as structural markers of inflammation. Yet surprisingly, LDs and their role in brain myeloid cells- especially in the context of AD pathogenesis- have been completely neglected so far. We discovered that LAM represent a dysfunctional, pro-inflammatory state in the aging brain and further identified genes that have been previously linked to neurodegeneration (SLC33A1, SNX17, PGRN) as genetic modulators of LD formation in microglia. These results suggest that LAM represent a detrimental microglia state in the aging brain and in AD that exerts a critical role in age-related neurodegeneration. This current study will characterize the molecular and cellular signature of LAM in aging and AD. Further, using microglia transplantation techniques, we will study whether LAM are the result of environmental determinants or whether LAM can instruct their environment. Lastly, using genetic models to regulate microglial lipid formation, we will determine molecular regulators and functions of LAM in aging and AD model mice. Ultimately, these studies pursue the innovative concept that lipid droplet containing microglia play an important and overlooked role in Alzheimer?s disease, and that targeting LAM may provide a promising approach for therapeutic intervention.

PROJECT SUMMARY Age is the main risk factor for Alzheimer's disease (AD), a neurodegenerative disorder rapidly increasing in both incidence and prevalence as the population becomes older. Unfortunately, AD is the only top ten cause of death with no effective treatments. Therefore, the development of disease-altering treatments for AD is an urgent and unmet need. Although the exact etiology of AD is unknown, microglia, the tissue-resident macrophages of the brain, have been implicated in disease pathogenesis based on the observation that genetic variants in several microglia-specific genes significantly alter disease risk. In the healthy brain, microglia maintain homeostasis through multiple modalities including phagocytic clearance of pathogens, apoptotic cells, and debris. In aging and AD brains, microglia are dystrophic, hypo-motile, and burdened with lysosomal deposits indicative of impaired homeostatic function. These findings suggest that the general decline in microglial function with age might underlie pathological neurodegeneration. However, the mechanisms of age-related microglial dysfunction are poorly understood. This proposal aims to elucidate the mechanisms of impaired microglial homeostasis in the aging brain and to uncover therapeutic strategies to reverse this impairment in AD. Our preliminary data suggest that CD22, a sialic-acid binding immunoglobulin-like lectin typically expressed on B-cells, inhibits phagocytosis in aged microglia and serves as a dominant regulator of microglial homeostasis. Aim 1 combines biochemical and genetic tools to identify upstream and downstream signaling partners that cooperate with CD22 to inhibit phagocytosis in microglia. Aim 2 will elucidate the role and regulation of microglial CD22 expression during development, aging, and AD. Aim 3 will define the neuronal response to restoration of microglial homeostasis upon CD22 blockade. Finally, Aim 4 will evaluate the therapeutic potential of blocking CD22 to ameliorate cognitive decline in mouse models of AD. These experiments will uncover a novel mechanism of microglial dysfunction during normal aging with direct translational implications for patients with AD.

1. SUMMARY (Biomarker Core) Biomarkers have enormous value for the detection, management, and treatment of disease, but also for the development of novel therapeutics. The utility of biomarkers is most evident in the management of cardiovascular disease and diabetes, but biomarkers, especially predictive, easily obtainable ones, are still largely absent with respect to neurodegenerative diseases. The best fluid biomarkers currently available for Alzheimer?s disease (AD) include; A?, tau, and neurofilament in CSF, a biofluid which is difficult to collect in healthy, at-risk populations or on a repeated basis. Other biomarkers for AD include imaging modalities which are often very expensive or have low sensitivity and specificity at the individual level. The members of this core have considerable experience in unbiased multi-omic screens and data analysis and, over the years, have published numerous studies towards developing new biomarkers for neurodegenerative and other diseases. Enabled by the current ADRC, the core leaders and collaborators have used biospecimens from Stanford ADRC participants and generated extensive preliminary data with unbiased deep immune phenotyping, proteomics, and transcriptomics of human CSF resident cells, and discovered novel Parkinson's disease (PD) biomarkers. Based on this expertise, the mission of this Biomarker Core is to facilitate the discovery of novel biomarkers for AD and PD, as well as new biology underlying the pathological processes that lead to dementia in line with the core mission of the NAPA. This will be achieved by pursuing the collection of genetic and molecular measurements from a broad source of tissues from ADRC participants; the processing and dissemination of this information in useable formats through web portals and other means (i.e., ?Deep Phenotyping Database?); the analysis and bioinformatics integration of the collected information with clinical and imaging data, as well as information from public databases; and the development and dissemination of new data analysis algorithms and pipelines.

PROJECT SUMMARY Aging is the single greatest cause of disease and death worldwide, and rejuvenating the body by targeting biological aging processes therefore holds potential to simultaneously prevent multiple chronic diseases like cancer, heart disease, dementia, and diabetes. While decades of research has unveiled common hallmarks of aging, like mitochondrial dysfunction, inflammation, and loss of proteostasis, therapies targeting these hallmarks have elicited only modest rejuvenation in animal models. This may be in part because most aging studies have focused on only one or a few organs or cell types, with little to no temporal resolution, limiting our ability to interpret how and when aging impacts these interconnected systems. Recently, however, we have attempted such a systematic characterization of aging. Using bulk RNA-sequencing (RNA-seq) and single-cell RNA- sequencing (scRNA-seq) on dozens of mouse organs and cell types across the lifespan (termed Tabula Muris Senis), we discovered global and specific aging signatures throughout the body. But it remains unknown how, or if, rejuvenation paradigms affect these global aging pathways, or rather instigate nascent biochemical programs. The rational design of new therapeutics is therefore challenging. One method of rejuvenation which has garnered beneficial effects across organ systems is heterochronic parabiosis, in which a young and old mouse share a common circulation. Phenotypes like cognition, muscle strength, and bone repair have all shown functional improvement through exposure to young blood. Parabiosis research has largely focused on age-related changes to circulating proteins, and several have been determined to mediate at least some of the observed effects. However, such individual factors have yet to achieve robust rejuvenation throughout the body, likely in part due to an incomplete understanding of the effects of parabiosis on disparate organs and cells. Using our newly created Tabula Muris Senis data to represent normal aging, we investigated scRNA-seq changes in 3 tissues following parabiosis: gonadal and mesenteric adipose tissues, which undergo age-related gene expression changes prior to other organs, and liver, as hepatocytes were one of the first cell types observed to benefit from exposure to a young circulatory system. Interestingly, individual cell types vary greatly in their response to parabiosis, with vascular endothelial cells from all 3 tissues showing prominent transcriptomic changes consistent with normal aging genes. It is our hope that by expanding this analysis to scRNA-seq of 9 tissues, and to bulk RNA-seq of 21 tissues, we can discover signatures that will serve as the basis for identifying small molecules capable of robust rejuvenation and healthspan extension. As surgically intensive parabiosis is confounded by cell trafficking and simultaneous exposure to young and aged circulating factors, we further propose to compare parabiosis signatures to those derived from young plasma transfer, thereby uncovering aspects of rejuvenation specifically sensitive to alterations in plasma factors. Similar to our earlier datasets, all data will be made publically available.