William Klein, PhD - US grants

Long-range goals of this project relate to the basic mechanisms underlying the action of abused drugs. The immediate focus is on nicotine; dependence on nicotine by tens of millions of cigarette smokers, with its associated health hazards, is an extraordinarily widespread and costly form of drug abuse. The proposed work deals in new ways with the effect of nicotine on the structure and function of CNS neurons. Two hypotheses, suggested by recent observations, will be investigated: (1) By interfering with the physiological control of neuronal nicotinic ACh receptors, nicotine may significantly perturb receptor concentration or position; (2) By excessively stimulating receptor-mediated ion flux, nicotine may cause Ca++ activated proteolytic damage to the structure of neuronal synapses. The specific experiments proposed are designed to investigate the normal cell biology of neuronal nicotinic receptors in addition to testing the effects of nicotine. Cells from the chick retina will be used because of their high level of receptors as well as their convenience for biochemical, cellular and developmental experiments. Two novel approaches will be used in these experiments. First, methods have been developed for isolating highly arborized neurons in order to identify which cells have nicotinic receptors and to determine where on those cells receptors are localized. Second, methods have been developed for examining isolated neurons with the high voltage electron microscope; this technique permits three-dimensional ultrastructural analysis of selected subcellular regions with remarkable resolution, clarity and speed. Overall, the specific experiments posed should help identify which cells in the CNS are sensitive to nicotine, provide new insight into the assembly of neuronal postsynaptic cholinergic membranes, and establish how cholinergic drugs such as nicotine might perturb this ongoing process. Such perturbations could have lasting effects on neuron structure and function well after the drugs are withdrawn.

The long-range goal of this work is to understand how signal transduction is established and maintained at CNS cholinergic synapses; its focus is on the biochemistry, development and cellular regulation of muscarinic ACh receptors. These receptors mediate a large proportion of CNS cholinergic signaling, have been implicated in many behaviorally and clinically significant phenomena and show a broad spectrum of transductional and regulatory responses. At present, it appears most appropriate to isolate and begin characterizing receptor system proteins and to generate receptor specific antibodies for studies of muscarinic cell and developmental biology. The following three aims have therefore been chosen: AIM #1--To generate a library of muscarinic receptor-specific monoclonal antibodies. Partially purified (250-fold) bovine brain receptors will be used for antibody induction using a combination of methods already tested for their effectiveness. Binding assays and western blots will be used in testing antibody specificity. AIM #2--To purify and begin characterization of receptor molecules. Immunoaffinity chromatography will be added to tested separations based on subcellular fractionation, hydrophobicity, charge, molecular weight and specific glycosylation. 2D-Gel, sedimentation equilibrium and N-terminal analyses will be used in testing purity. The ligand specificity, number of subunits, number of binding sites, and overall amino acid composition of the purified molecules will be assessed. AIM #3--To localize receptors as a function of synaptic development, post-translational modification, and cholinergic stimulation. The monoclonal antibodies obtained above will be used to localize receptors on avian retina neurons at the subcellular and ultrastructural levels. A novel approach employing isolation of differentiated neurons and HVEM whole mount analysis will be used.

The proposed work concerns the growth cones of vertebrate CNS neurons, focusing on the developmental transition from growth cone to synaptic arbor. It is designed to learn more about signals that may regulate the activity of growth cones and to assess possible synaptogenic roles of their filopodia. The experimental strategy comprises continued use of video-enhanced contrast differential interference contrast microscopy (VEC-DICM) in tandem with the high voltage electron microscope (HVEM). Using this state-of-the-art microscopy, one first can record the real-time activity of living neurons at greater than 10,000X magnification, and then, after fixation, examine neurites, growth cones, filopodia, and synapses as whole mounts to obtain ultrastructural images that have remarkable perspective and detail. Circumventing time-lapse photography as well as traditional embedding and sectioning, this approach makes it possible to obtain new images of cellular activity and organization and to obtain these data quickly. The work will be done primarily with avian retina neurons in culture. Culture models for CNS differentiation provide ease of observation and experimentation. The retina, with its abundance of local circuit interactions and well-defined cell types, is well-suited to cell culture and is widely-used in studies of development and synaptogenesis. Four hypotheses provide a framework for the current experimental aims. While speculative, they are supported by some intriguing observations made during past project period: To test the hypothesis that formation of microtubule loops may terminate neurite extension; To test the hypothesis that neurohumoral agents may influence the activity and structure of vertebrate CNS growth cones; To test the hypothesis that filopodia may be structural precursors of synapses; To test the hypothesis that formation of cleft-spanning filaments may stabilize nascent synapses. An investigation of these four hypotheses should provide deeper understanding of how neurons become polarized for synaptic function as well as give insight into how synapses may be maintained, modified or regenerated later in life.

This award will support Prof. Rama Bansil and Prof. William Klein of the Center for Polymer Studies of Boston University in a research collaboration with Prof. A. Coniglio of the University of Naples. The objective of the collaboration is to seek a better understanding of how basic physical laws are altered on random materials and also of the physics of inhomogeneous media. In order to carry out this objective, the researchers propose to study 5 general topics: Topic I, the phenomena of multifractality in random materials, whereby an infinite hierarchy of exponents is sufficient to characterize virtually any system near its critical point. Topic II is concerned with applications of modern methods of statistical mechanics to physical processes on disordered media and to how a random material is fractured under sufficiently high stresses. In Topic III the researchers plan to address questions concerned with one specific class of random materials, linear and branched polymers. Topic IV is concerned with the relation between geometric clusters and thermal phase transitions, while Topic V will involve experiments using dynamic light scattering techniques to investigate the problem of probe diffusion in polymer gels. This work will continue a highly productive collaboration that has led to useful concepts and applications to problems like irreversible growth, resistor networks and many others.

9305575 Klein This is an experimental and theoretical study of phase separation, ordering, and crystallization kinetics in metal alloys. Research on kinetics of martensitic transformations emphasizes simultaneous ordering and shape change reactions occurring in copper/gold alloys. The research also includes time-resolved studies of quasimartensitic kinetics in which the martensitic reaction is slowed by heat diffusion. The crystallization kinetics near spinodals is a focus in the theoretical and simulation effort. Both homogeneous and heterogenous nucleation are considered. The concept of liquid forming a "clump" phase at a spinodal point is examined. Ordering is examined in FeAl and FeCo alloys. %%% This research expands our knowledge base on the kinetics associated with several phase transformations. Phase transformations play an essential role in the processing and properties of technologically important materials. ***

The long-range goal of this work concerns molecular mechanisms of cell death in Alzheimer's disease (AD). Its immediate focus is to study the molecular cell biology of amyloid precursor protein (APP). The cytoarchitectural nature of APP in situ, its functional role, and the factors influencing its expression are essentially unknown. The proposed aims address these gaps and, additionally, will help to establish a useful new model for experimental studies of AD neuropathogenesis. AIM #1- To characterize the structural biology of APP at cell and extracellular surfaces, focusing on tests of the hypothesis that APP may be a cell adhesion molecule. While the cellular function of APP is unknown, one major hypothesis based on sequence analysis is that this protein is a cell adhesion molecule. To test this hypothesis directly, and to characterize the structural biology of the APP molecule in situ, we recently have developed a whole mount EM-immunogold method for studying APP expression in cultured human nerve cell lines. Based on our pilot studies, we propose (1) To verify and extend characterization of the relationship between APP forms and adhesive microfibrils found at cell and extracellular surfaces, applying whole mount transmission electron microscopy-immunogold protocols to SHSY5Y human neuroblastoma cell cultures. (2) To evaluate the adhesive role of APP, using paradigms that test adhesion-linked functions such as neurite extension and growth cone motility (3) To provide initial characterization of a postulated APP receptor, using isolation protocols based on the adhesive properties of APP. AIM #2- To evaluate the influence of cellular stress factors on APP expression. The APP promoter is known to have multiple control elements, including a stress-related heat shock sequence. Experiments therefore have been designed to study APP expression in response to external influences that can stress nerve cells. Six factors incriminated directly or indirectly in the pathogenesis of AD will be studied (heat shock, Ca++ overload, glucose withdrawal, hypoxia, oxidant injury, and aluminum). The distribution of specific APP forms on cell and culture surfaces will be compared using imaging techniques; the levels of APP forms associated with cells and culture substratum will be measured by quantitative immunoblots. The data will provide essential tests of the hypothesis that cell stress causes significant changes or anomalies in APP expression.

9633385 Klein, Gould and Brower This is a new award which is funded jointly by the Office of Multidisciplinary Activities/MPS, and the Divisions of Materials Research, Mathematical Sciences and Advanced Scientific Computing. An integrated theoretical and computational investigation will be made of the structural properties of fragile glass-forming liquids and the relation of this structure to the mechanisms of relaxation. Glasses have become an increasingly important class of materials in several new technologies. They possess advantages such as light weight and ease of processing, but suffer from degradation through creep, fatigue, and embrittlement. To understand these mechanisms, it is necessary to understand the structure of glasses and how they relax. At present, experiments can provide information about relaxation processes in glasses, but do not yield much information about their structure or relation of this structure to the relaxation mechanisms. In contrast, computer simulations can provide information about structure, but only on relatively short time and length scales. Moreover, theoretical understanding of glasses is in a relatively crude state. In this research, a sequence of models will be studied beginning with a mean-field model of a glass-forming liquid and proceeding via various approximation methods to include non-mean-field effects. Several parallel computer architectures will be used to extend the size and the time scale of the systems simulated. The simulations will be based on an efficient message passing molecular dynamics program, and by more speculative methods based on Fourier acceleration algorithms and on cellular automata models. The results of the theoretical investigations and computer simulations, in conjunction with ongoing laboratory experiments at Boston University and other locations, will facilitate an understanding of the relation between the relaxation observed experimentally in deeply supercooled liqui ds and glasses and the existence of structures seen in computer simulations. Understanding of this relation will make it possible to use the structure of glasses to predict their temporal evolution. The research will be done in collaboration with colleagues at Boston University in the Departments of Physics and Electrical, Computer and Systems Engineering, and the Center for Computational Science. In addition, colleagues in the Physics Departments at Clark and Brandeis Universities, the Center for Computational Materials at NIST, and at Thinking Machines Corporation will participate. Graduate students will also participate through the NSF-sponsored Graduate Research Training Program at the Center for Computational Science at Boston University. Computer platforms include Boston University's 38-processor SGI Power Challenge array, a work station cluster and several CAM-8 machines. A workstation cluster at Clark University and the NIST Cray YMP will also be used. %%% This is a new award which is funded jointly by the Office of Multidisciplinary Activities/MPS, and the Divisions of Materials Research, Mathematical Sciences and Advanced Scientific Computing. The research combines aspects of NSF programs on Advanced Materials and Processing and on High Performance Computing and Communications. An integrated theoretical and computational investigation will be made of the structural properties of fragile glass-forming liquids and the relation of this structure to the mechanisms of relaxation. Glasses have become an increasingly important class of materials in several new technologies. They possess advantages such as light weight and ease of processing, but suffer from degradation through creep, fatigue, and embrittlement. To understand these mechanisms, it is necessary to understand the structure of glasses and how they relax. At present, experiments can provide information about relaxation processes in glasses, but do not yield much information about t heir structure or relation of this structure to the relaxation mechanisms. In contrast, computer simulations can provide information about structure, but only on relatively short time and length scales. Moreover, theoretical understanding of glasses is in a relatively crude state. The results of the theoretical investigations and computer simulations, in conjunction with ongoing laboratory experiments at Boston University and other locations, will facilitate an understanding of the relation between the relaxation observed experimentally in deeply supercooled liquids and glasses and the existence of structures seen in computer simulations. Understanding of this relation will make it possible to use the structure of glasses to predict their temporal evolution. The research will be done in collaboration with colleagues at Boston University in the Departments of Physics and Electrical, Computer and Systems Engineering, and the Center for Computational Science. In addition, colleagues in the Physics Departments at Clark and Brandeis Universities, the Center for Computational Materials at NIST, and at Thinking Machines Corporation will participate. Graduate students will also participate through the NSF-sponsored Graduate Research Training Program at the Center for Computational Science at Boston University. Computer platforms include Boston University's 38-processor SGI Power Challenge array, a work station cluster and several CAM-8 machines. A workstation cluster at Clark University and the NIST Cray YMP will also be used. ***

Alzheimer's disease (AD) is a progressive neurodegenerative dementia affliciting over 4 million people in the United States. There is no treatment and the disease inevitably causes derangement and death. AD has multiple etiologies, but in all cases a major pathological hallmark is the accumulation of amyloid beta protein (Abeta). Accumulated Abeta occurs in varius multimeric forms and in association with a variety of other molecules. Abeta also can accumulated in the absence of neurodengeneration of dementia, which suggests that its deposition is not a simple marker for extensive cell death. We propose to investigate the relation of Abeta to AD neuropathology in the context of the following working theory: When monomers of Abeta self-assemble into multimers, they form toxic ligand-like domains that alternatively can be exposed or cryptic, depending on the final multimeric structure. If the toxic domains cannot interact with neurons, the multimers are innocuous. If, on the other hand, the toxic domains do interact with neurons, themultimers are pathogenic and they play a role in the progressive neurodegeneration of Alzheimer's disease. The final multimeric structure is highly sensitive to conditions of assembly. Certain glial derived proteins assoicated with Alzheimer's pathology (such as ApoJ or butyrylcholinesterase) promote a particularly dangerous form of oligomeric Abeta that is both toxic and diffusible. These Abeta derived active ligands (ADALs) bind to nerve cell surfaces and corrupt vital neuronal signal transductionpathways, forcing nerve cell dysfunction and death. Preliminary data are presented that support each element of this working theory. Predictions of the theory will be tested using a series of characterized amyloid obtained from collaborations with Drs. Drafft and Van Eldik. Specific responses of nerve cell lines and brain slice cultures will be monitored using assay established here. Significant conclusions from these experiments will be tested for their relevance to neurodegenerationin Alzheimer's- afflicted brain tissue, in collaboration with Dr. Mesulam. AIM 1 Determine how Abeta neurotoxicity is altered by experimentally controlled variations in its multimeric state; from experiments with cloned cell lines, data will compare the cytotoxicity of a series of amyloid made by mixing synthetic Abeta with AD-related glial derived proteins. AIM2 Define a molecular signature for each particular amyloid and show whether common as well as unique elements exist in the response to neurodegenerative amyloids; data will compare cell line responses to the amyloids evaluated in AIM 1 via assays germane to mechanisms of neurodegeneration. AIM 3 Test the pathogenic relevance of cell line responses to Ab; data will show if responses to Abeta amyloids seen in AIMs 1 and 2 are reproduced in mammalian brain tissue, and will show if evidence of such changes may exist in tissue from Alzheimer's-afflicted patients. Data will provide salient tests of the modified amyloid cascade hypothesis. New insight is expected into specific pathways that make neurons vulnerable to toxic insults and into factors that modulate the toxicity of Abeta. Hypothetically, the proposed impact of Abeta amyloids on signal tranduction could lead to severe, end-stage dementia via nerve cell death; at earlier stages, corrupted signaling could lead to memory failure and other cognitive impairments via degeneration of cytoskeletal-dependent synaptic plasticity. If the amyloid hypothesis ultimately should prove valid, this project will provide important models for identifying drug target candidates and testing neuroprotective agents.

DESCRIPTION (From the Applicant's Abstract): This application concerns a new possibility for the role of Abeta in AD. It is designated to investigate the loss of synaptic plasticity and death of nerve cells caused by nonfibrillar Abeta oligomers. Oligomers accumulate in AD brain, and current best evidence indicates they contribute to disease progression, potentially accounting for the imperfect correlation with amyloid. Aims focus on three key properties of oligomers (referred to as "ADDLs," for Abeta derived diffusable ligands). (i) ADDLs inhibits LTP in under an hour, one of the fastest known responses to any form of Abeta. (ii) ADDLs kill hippocampal neurons by a mechanism blocked by germline knockout of Fyn, a protein tyrosine kinase coupled to NMDA receptors and tetanus-induced LTP. (iii) ADDLs bind to cell surface proteins that are trypsin-sensitive and cluster at punctate "hot spots." The goal is to understand the molecular basis for ADDL neurotoxicity. Two hypotheses will be evaluated. First, ADDLs may be small protein ligands that cause damage by disturbing Fyn signal transduction, a consequence mediated by specific toxin receptors. Alternatively, ADDLs may disrupt cell integrity with little or no specificity, generating a global breakdown of cell physiology that includes loss of LTP and culminates in cell death. Predictions of these hypotheses will be tested by the proposal's AIMs. Aim 1. LTP or neurotransmission-Is the synaptic impact of ADDLs specific for LTP, or is synaptic function broadly impaired. Aim 2. Types of plasticity-Do ADDLs affect multiple types of neuronal plasticity, or only tetanus induced LTP? Aim 3. Molecular impact-Does toxicity stem form ADDLs impact on Fyn, or do ADDLs attack at alternative and perhaps multiple sites? Aim 4. Cell surface reactions-Are ADDLs bound by specific "toxin receptors," or is ADDL activity not dependent on unique binding sites? Results from this application will give a new basis for understanding the actions of Abeta oligomers found in human brain. In a best-case outcome, findings would provide novel targets for therapies that ultimately could reverse and not just slow down AD memory impairment.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a progressive and untimely fetal dementia. Its cause is not yet known, but a correlation exists with synaptic pathology, which may be responsible for cognitive deterioration. We, and recently others, have proposed that synaptic pathology in AD is caused by synaptically active Abeta oligomers, which we have called ADDLs. The oligomer hypothesis is supported by compelling evidence from multiple laboratories. The evidence, however, comes almost exclusively from studies with model systems and investigator-generated oligomers. Need addressed by this proposal: A major concern is whether experimental evidence that favors the ADDL hypothesis is clinically relevant. This study therefore is designed to determine the level of occurrence and toxicology of ADDLs in human samples. AIM 1: Quantify ADDL occurrence in human subjects AD. We will determine the structural forms of ADDLs that are present, their distribution in situ, and their abundance in brain, CSF, and plasma. AIM 2: Evaluate the CNS toxicology of human ADDLs. We will determine where ADDL binding sites are found in relation to synapses in rat hippocampus culture models and test the prediction that binding of human ADDLs causes synaptic pathology. To characterize human ADDLs, we have developed sensitive ADDL antibodies and immunoassays. Proposed experiments will extend preliminary findings that indicate (i) ADDLs are present in human cortex and increase as much as 70-fold in AD (ii) ADDLs act extracellularly and bind to plasma membranes at particular post-synaptic terminals (iii) Synaptically-bound ADDLs alter synapse homeostasis, causing aberrant growth of pre- and post-synaptic terminals and the formation of ectopic dendritic spines. This study will provide the first clinically relevant evaluation of the ADDL hypothesis. In addition to testing a series of key predictions, it lays a foundation for two future goals: (1) To test the degree of correlation between dementia and ADDLs in a large group of subjects, including those with mild cognitive impairment (MCI) and early to middle-level AD; (2) To develop new approaches to Alzheimer's therapeutics and diagnostics using ADDLs as optimal targets.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a progressive and untimely fetal dementia. Its cause is not yet known, but a correlation exists with synaptic pathology, which may be responsible for cognitive deterioration. We, and recently others, have proposed that synaptic pathology in AD is caused by synaptically active Abeta oligomers, which we have called ADDLs. The oligomer hypothesis is supported by compelling evidence from multiple laboratories. The evidence, however, comes almost exclusively from studies with model systems and investigator-generated oligomers. Need addressed by this proposal: A major concern is whether experimental evidence that favors the ADDL hypothesis is clinically relevant. This study therefore is designed to determine the level of occurrence and toxicology of ADDLs in human samples. AIM 1: Quantify ADDL occurrence in human subjects AD. We will determine the structural forms of ADDLs that are present, their distribution in situ, and their abundance in brain, CSF, and plasma. AIM 2: Evaluate the CNS toxicology of human ADDLs. We will determine where ADDL binding sites are found in relation to synapses in rat hippocampus culture models and test the prediction that binding of human ADDLs causes synaptic pathology. To characterize human ADDLs, we have developed sensitive ADDL antibodies and immunoassays. Proposed experiments will extend preliminary findings that indicate (i) ADDLs are present in human cortex and increase as much as 70-fold in AD (ii) ADDLs act extracellularly and bind to plasma membranes at particular post-synaptic terminals (iii) Synaptically-bound ADDLs alter synapse homeostasis, causing aberrant growth of pre- and post-synaptic terminals and the formation of ectopic dendritic spines. This study will provide the first clinically relevant evaluation of the ADDL hypothesis. In addition to testing a series of key predictions, it lays a foundation for two future goals: (1) To test the degree of correlation between dementia and ADDLs in a large group of subjects, including those with mild cognitive impairment (MCI) and early to middle-level AD; (2) To develop new approaches to Alzheimer's therapeutics and diagnostics using ADDLs as optimal targets.

People often face ambiguous information about their risks, skills, personality attributes, and opinions. Patients may need to cope with seemingly conflicting advice about the value of optional protective behaviors (e.g., prostate cancer screening), employees may receive a range of feedback about their job performance, and students may need to integrate an array of potentially discrepant information in order to make reasoned academic and career decisions.

Research in the decision sciences suggests that people are often averse to the sorts of ambiguity described above. On the other hand, research in social psychology suggests that people try to use ambiguity to their advantage. They are more likely to believe they are superior to their peers on dimensions that can be defined many different ways (e.g., leadership ability) than on dimensions that are more unambiguously defined (e.g., punctuality). Moreover, they are more likely to see themselves as being superior relative to ambiguously defined referents (e.g., the "average" person) than relative to more unambiguously defined referents (e.g., a specific acquaintance). These separate lines of research suggest a need to better understand the role that ambiguity plays in self-judgment, reactions to personal feedback, and decision-making.

This study examines these issues. The project consists of three experiments. Experiment 1 will present chance-based games and skill-based games with probabilities of winning that vary in ambiguity. It is predicted that people will prefer the skill-based games even when the probabilities of winning the chance games are ambiguous, and particularly when their chances of winning are contingent on ambiguous performance. The design of the experiment is such that this sort of pattern would reflect ambiguity aversion despite perceptions of high competence, and would therefore be consistent with both the decision science and self-evaluation literatures. Experiments 2 and 3 will test the hypothesis that people prefer games linked to performance on tasks for which they have received ambiguous feedback. In order to separate the confounding effects of personal competence and controllability, all experiments will include measures of how participants believe others should behave, and one will vary whether performance has occurred in the future or in the past (where controllability is low because past performance cannot be altered).

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the role played by ADDLs (soluble AB oligomers) in the molecular etiology of Alzheimer's disease. We previously established that ADDLs are metastable neurotoxins that accumulate in Alzheimer's-affected brain. Though not yet proven, it is increasingly likely that this accumulation may cause AD's early memory loss. Recent findings from our laboratory suggest, moreover, that the neuronal impact of ADDLs could provide a unifying mechanism for major facets of AD pathology. We propose to investigate these important new links to AD pathology at the level of molecular mechanisms. When exposed to ADDLs, neurons manifest three major pathologies germane to AD: ROS generation, tau hyperphosphorylation and synapse degeneration. Our first aim is to determine how ADDLs initiate this neuronal damage and whether a common mechanism of initiation links all three pathologies. A strong clue to the mechanism is our finding that ADDLs attach to neurons as high-affinity synaptic ligands, a gain-of- function restricted to high-n oligomers. Our hypothesis is that ADDL attachment to synapses is the mechanistic starting point common to all AD neuronal pathology. Specific subaims include identifying the molecular nature of synaptic attachment and its role in initiating ADDL-induced pathologies. Our second aim is to determine the mechanism, triggered by ADDL binding that ultimately results in synaptic degeneration. This objective is of particular importance because of the correlation between synapse loss and Alzheimer's dementia. Synaptic aberrations induced by ADDLs include major down-regulation of NMDA receptors, appearance of immature synaptic spine morphology, and significant decrease in synaptic spine abundance. Preceding this damage is an excessive accumulation of Arc, a synaptic F-actin regulating protein whose proper transient expression is required for long-term memory formation. Our hypothesis is that excessive Arc accumulation, by disrupting F-actin, is an early instigating step in the mechanism responsible for molecular and structural deterioration of synaptic spines. This project addresses the need to comprehensively characterize AD-relevant pathological changes induced in neurons by ADDLs, to define the cellular and molecular mechanisms that underlie this pathology, and to identify the earliest events responsible for initiating pathogenic mechanisms. Anticipated results have the potential to provide a unifying mechanism for early AD pathology and memory loss. Successful completion of the project should identify new AD drug targets and provide mechanism-based assays for development of novel neuroprotective compounds useful for AD therapeutics.

DESCRIPTION (provided by applicant): This application concerns the pathological accumulation of Abeta oligomers (ADDLs) in brains and CSF of individuals at various stages of Alzheimer's disease (AD). This disease strikes 10 percent of individuals over 65, inflicting painful emotional burden to families and a cost to the country of more than $100 billion annually. Despite its serious impact, our ability to contend with AD suffers from imprecise diagnostics and inadequate therapeutics. The current project addresses these problems by investigating the clinical properties of ADDLs, which we identified as metastable neurotoxins that accumulate in AD brain and CSF. It now appears likely that ADDLs play a significant role in the neuronal dysfunction and damage responsible for AD's progressively catastrophic dementia (Health &Human Services 2004-2005 Progress Report on Alzheimer's Disease). To advance our understating of this pathogenic role, we propose to test the hypothesis that an attack on synapses by ADDLs initiates tau hyperphosphorylation, oxidative damage and synapse deterioration, thus providing a comprehensive mechanism for memory failure and the major facets of AD neuropathology. This hypothesis, which is strongly supported by findings from cell biology and neuropathology, will be investigated primarily using human ADDLs and CNS samples in order to directly establish clinical relevance. We also propose to investigate why AD-affected brain is unable to remove ADDLs, following up on newly reported neuropathological data suggesting a failure of glial clearance mechanisms. Building on recent results, our seven aims are to: (i) Validate that ADDLs in CSF/plasma provide a reliable AD biomarker;(ii) Verify that the earliest preclinical sign of AD is ADDL accumulation around individual neurons;(iii) Test the prediction that neurons surrounded by ADDLs develop AD pathology;(iv) Verify that perineuronal deposits of ADDLs are due to ADDL attachment to synapses;(v) Identify the pathogenically significant oligomeric species (putatively 12mers) and obtain new monoclonal antibodies that specifically target disease-relevant oligomers;(vi) Test the predicted role of synaptic membrane proteins in the mechanism by which ADDLS attack only particular neurons;and, (vii) Confirm the novel ADDL-related pathology discovered in astrocytes, testing its relevance to failed ADDL clearance mechanisms in disease onset and progression. Important technical innovations include ultrasensitive nanotechnology-based ADDL assays, monoclonal antibodies specific for ADDLs in solution, and a series of physicochemical methods capable of characterizing oligomer size in dilute aqueous solutions without the use of SDS or other harsh chemicals. Results are expected to provide important advances needed to validate and optimize ADDLs as major targets for Alzheimer's diagnostics and disease-modifying therapeutics. PUBLIC HEALTH RELEVANCE: This application concerns the role of Abeta oligomers (ADDLs) in Alzheimer's disease, focusing on human samples to establish direct clinical and pathological relevance. Results obtained with human ADDLs and tissue will help answer important questions central to ADDL involvement in pathogenesis and substantiate the targeting of ADDLs for future diagnostics and therapeutics.

People often react defensively when they are faced with personal feedback and messages that challenge positive beliefs about themselves. To illustrate, smokers endorse a variety of myths that allow them to rationalize their smoking behavior, and people receiving negative feedback about their performance use many strategies to reduce the credibility or personal relevance of the feedback. However, people are less likely to show such defensiveness when they have an opportunity to reflect on their personal values in advance, an act of self-affirmation. The purpose of this grant is to explore a set of physiological, cognitive, and motivational processes that can account for the beneficial effects of self-affirmation. The first two experiments examine effects of self-affirmation on adaptive physiological responses to threat such as reduced heart rate and cortisol response. Five additional experiments explore cognitive effects such as unconscious attention to threatening stimuli, the ability to extract the "gist" of a threatening message, the determination to allocate attention to threat when competing demands are present, and the tendency to generate and test more even-handed hypotheses (e.g., "perhaps I do eat too much fat"). Finally, two experiments look at motivational effects, particularly the extent to which self-affirmation makes people more receptive to opportunities they might have to enact behavioral change. In addition, these investigations look at whether self-affirmation closes the oft-documented gap between people's resolutions to change behavior and their actual behavior change.

Together, these experiments follow the sequence from self-affirmation to behavioral outcomes, helping to elucidate why self-affirmation reduces defensiveness. Thus, the findings will advance self-affirmation theory as well as related theories of the self. Moreover, the findings will help in the development of interventions that succeed in sidestepping defensive reactions in such wide-ranging domains as health, performance evaluation, education, and interpersonal relationships. Finally, the project features an international collaboration between investigators in the United States and United Kingdom, providing unique educational opportunities for the students involved with the project as well as taking advantage of the unique expertise in the United Kingdom on the interplay between social processes and health outcomes.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the major cause of dementia in the elderly, with a US incidence of 5.4 million and an annual cost of $183 billion. There is no effective treatment. This proposal is in response to an NIA request for R21 high risk/high reward proposals to generate new AD drug discovery strategies. It focuses on current knowledge indicating that significant neuron damage in AD can be attributed to the impact of toxic Ab oligomers. These small diffusible molecules are distinct from amyloid plaques and are thought to instigate AD memory loss through their ability to target synapses and disrupt synaptic plasticity. Current efforts to prevent AD largely focus on metabolic inhibitors that block accumulation of the Ab monomer and on passive vaccines that remove either the monomer or its toxic assemblies. These efforts have not as yet been successful. This proposal introduces an alternative strategy that focuses on the first step in the mechanism of oligomer toxicity. To elici damage, oligomers must first bind to cellular receptors. These receptors mediate association of oligomers with particular neurons and transduce binding into synaptotoxic responses. Although receptor identity would be valuable for elucidating the mechanism of toxicity, it is feasible even without this knowledge to use oligomer-receptor binding as a target for drug discovery. What is required is an assay suitable for large- scale high-throughput screening (HTS) of binding antagonists. We propose a novel strategy to achieve this goal using an approach that generates artificial nanoscale membranes (Nanodiscs). This is a well-established methodology that has been adapted here to provide unbiased and functional soluble preparations of synaptic plasma membrane proteins. Binding of oligomers to synaptic plasma membrane (SPM) Nanodiscs has been demonstrated and exhibits characteristics expected of ligand-receptor interaction. The binding reaction has been adapted to a homogeneous chemiluminescence assay well-suited to HTS for antagonists of oligomer binding. Unlike high content, cell-based assays, the biochemical assay for binding to soluble receptors has the bandwidth and precision required for the primary screening of very large libraries of compounds. Parallel investigations of these SPM-Nanodiscs, separate from this project, are expected to identify the receptor protein(s). Our Approach to drug discovery follows a screening tree in which hits from the primary assay using SPM-Nanodiscs are validated in cell-based assays for binding and toxicity, first with synthetic oligomers and then with brain-derived oligomers. The Nanodisc HTS and secondary screens will be optimized at first using a small library and then greatly expanded. Hit-to-lead resources o the Northwestern Center for Molecular Innovation and Drug Discovery will be implemented as needed. The Aim, expected to be achieved by the end of two years, is to establish a fully functioning strategy for HTS of the very large libraries now available under the auspices of NIH. Results ultimately have strong potential for discovering lead compounds that target an underexploited but significant aspect of AD pathogenesis. PUBLIC HEALTH RELEVANCE: This proposal is in response to an NIA request for R21 high risk/high reward proposals to generate new Alzheimer's disease (AD) drug discovery strategies. AD is the major cause of dementia in the elderly, with a US incidence of 5.4 million and an annual cost of $183 billion. There is no effective treatment. This project is expected to have strong potential to identify lead compounds that target binding of synaptotoxic Ab oligomers to their receptors, an underexploited but critical early-step in AD pathogenesis.

DESCRIPTION (provided by applicant): Progress in developing a treatment for Alzheimer's disease (AD) remains minimal, despite the disease affecting one in eight persons over 65 at a burden to the US economy of over $200 billion per year. A significant factor is the lack of effective methods for AD diagnosis and for quantifying the efficacy of investigational new drugs. Despite exciting progress with PET probes in imaging amyloid fibrils found in plaques, which have provided an important focus for AD studies over many years, plaques do not correlate well with cognitive loss, do not offer a timely indicator of disease onset, and are no longer regarded as the initial cause of AD neuron damage. This project addresses the need for a probe that targets molecules that appear earlier in AD pathology and that are responsible for initiating neuron damage. Our choice of target is the toxic A? oligomer (A?O), for which we have prepared a novel molecular MRI probe. Toxic A?Os are regarded by many researchers as instigating the initial stages of AD neuron damage. Evidence indicates A?Os begin to accumulate in early AD, perhaps the first indicator of AD pathology. Our immediate goal is to obtain proof of concept with the 5XFAD tg mouse that our MRI probe (NU4MNS) will track A?Os in vivo as predicted. NU4MNS comprises aqueous-stable magnetic nanostructures (MNS), which act as powerful MRI contrast agents that have been conjugated to our team's NU4 monoclonal antibody. The NU4 parent antibody selectively targets A?Os with high affinity and, when delivered intranasally, it readily targets hippocampal A?Os in a mouse AD model. The NU4MNS probe has shown outstanding potential for MRI diagnostics in preliminary studies. The probe retains full ability to selectively bind A?Os, and it can distinguish in vitro human AD brain samples from controls by MRI signal. The project will undertake three studies to extend our successful initial findings and advance the probe toward our long-range clinical goal of imaging A?Os in AD patients by MRI. (Study 1) Carry out prototype assays for A?O-dependent MRI signals using brain sections from the 5X FAD mouse model. (Study 2) Optimize intranasal delivery of probe to brain of AD and control mice. (Study 3) Obtain proof of concept that mice with AD pathology can be identified using the NU4MNS MRI probe, and that the probe can determine drug efficacy in lowering A?Os in treated animals. Our imaging goal builds on landmark breakthroughs from other teams that have made it possible to clinically image amyloid plaques in Alzheimer's patients. This project is expected to advance imaging resources by developing a new probe can detect neuron- damaging A?Os at early stages of mouse pathology and monitor toxin reduction by an experimental drug. Development of targetable nanostructures specific for neurotoxic A?Os has potential to provide an MR imaging modality for evaluating the disease-modifying efficacy of investigational new drugs (INDs) and ultimately deliver early-stage AD diagnosis and subsequent disease management.

Surprisingly, evolution has retained an amino acid sequence for the human A?42 peptide that favors formation of large homo-oligomers. It is widely regarded that these unusual molecules are pathogenic. These oligomers accumulate in human and animal model brain tissue in an Alzheimer's disease-dependent manner. They have been shown experimentally to impact the CNS in a manner that causes memory dysfunction and recapitulates AD neuropathology (e.g., tau hyperphosphorylation, neuroinflammation, synapse loss, selective nerve cell death). Pathological A?O buildup begins early in the disease, before plaques, but it is not evident in healthy, non-demented adults. In vitro, toxic A?Os readily assemble from nanomolar concentrations of synthetic human A?. The same oligomer-forming sequence is found in certain animal species (e.g., nonhuman primates, rabbit, chicken, but not rodents). It is peculiar that these species have retained the apparently dangerous capacity to form molecules that instigate the neural damage leading to AD dementia. The current project addresses the possibility that circumstances exist whereupon A?Os manifest a functional, physiological presence. In the long- run, determining such circumstances is expected to give a significantly new perspective into the triggers and mechanisms of toxic A?O buildup in AD. The working hypothesis tested is that A?Os have a transient, functional role during CNS development, a possibility strongly supported by preliminary findings. Western blots and immunohistochemistry show the presence of A?Os in the developing chick, whose A? sequence is the same as humans. A?Os in embryonic retina are selectively expressed by ganglion cells, which, somewhat remarkably, likewise show a presence of tau phosphorylated at the AD serine-396 site. Nothing is known about the function of the developmentally-regulated A?Os, but it is feasible that the developmental impact may resemble the pathological impact observed in adult brain. Looked at another way, when A?Os re-emerge pathologically in adults, they impact brain tissue in a manner re-capitulating developmental functions. Developmental functions of A?Os thus might comprise, e.g., stimulation of tau hyperphosphorylation, microgliosis, synaptic pruning or selective nerve cell death. The Aims test the hypothesis using developing chick retina for experimentation. Aim 1 ? Determine the onset, peak expression and disappearance pattern for particular A?O species relative to AD-like tau hyperphosphorylation and microglial differentiation during retina development. Aim 2 ? Determine the influence that transient A?Os exert on cellular development of retina, particularly with respect to microglia and tau hyperphosphorylation. Results are expected to establish that Alzheimer's-related A?Os serve a physiological role during development. Embryonic chick will be introduced as an exceptionally well-suited model for investigating the regulation of A?O expression and normal A?O function, including its relationship to tau and microglia. By achieving the first insights into normal A?O biology, it is expected that new and deeper insight into A?O pathobiology will be achieved.

This project concerns the etiological origins of sporadic Alzheimer's disease (AD). While plaques and tangles still define AD, it is thought that oligomeric forms of A? and tau play major roles in the pathogenic mechanism. What triggers the buildup of these toxins, however, and whether the etiological triggers affect toxin impact, remain unknown. The long-term goal of this project therefore is to understand the etiological origins of sporadic AD, with a strategic focus on the pathobiology of AD risk factors. Its central hypothesis is that risk factors have a common ability to stimulate pathogenic A?O buildup, but that each has its own signature regarding the nature and clinical outcome of this buildup. The important relationship between risk factors and A?O pathobiology has not been studied before in unbiased, non-transgenic models. The immediate focus is on two metabolic AD risk factors: hypercholesterolemia (HypC) and type 2 diabetes (T2D). Investigations will address three aims and will provide results that test the central hypothesis and a prediction of potential diagnostic value. AIM 1 ? Determine the pathobiology that makes HypC and T2D act as risk factors for sporadic AD. The working hypothesis is that the dysfunctional metabolic states of HypC and T2D are AD risk factors because they promote the buildup of pathogenic A?Os. AIM 2 ? Establish a mechanistic principle to explain why AD manifests as heterogeneous phenotypes. The working hypothesis is that although the risk factors HypC and T2D each promote buildup of pathogenic A?Os, there is an etiology-sensitive signature to that buildup that influences the cognitive outcome. AIM 3 ? Establish in vivo biomarkers that can diagnose the etiology-sensitive status of A?O neuropathology. The working hypothesis is that tandem imaging of brain A?Os and brain function will optimally diagnose AD in a manner that is etiology-sensitive. Rabbit will be used as a non-transgenic model. Pilot data show that substantial memory dysfunction is caused by diets associated with HypC and T2D. This dysfunction is linked to A?O buildup, and it manifests with an etiology-specific signature. New analytics will establish the impact of HypC and T2D on brain region-selective memory performance, functional and molecular MRI, AD-linked neuropathology, and buildup of distinct A?O species. The approach is the first rigorous investigation into the onset of A?O pathobiology in an animal model unbiased by transgene expression. The expected outcome is establishment of a molecular mechanism to connect risk factors HypC and T2D to sporadic AD. Results are expected, moreover, to establish a platform for future mechanistic studies of the now-burgeoning list of AD risk factors, illustrate the significance of etiology to AD diagnosis and precision medicine, and accelerate development of effective AD treatments and prevention strategies.

Abstract Distinct amyloid-beta (A?) conformers such as peptides, oligomers (A?Os), and fibrils have long been targets studied for the cause, diagnosis and treatment of Alzheimer?s disease (AD). Spatiotemporal spreading of A?Os is theorized to underly AD progression; however, because of significant polydispersity, no consensus has been reached into which A?O structural elements or size distribution leads to potent neurotoxicity. Indeed, reports suggest some A? species may play a protective role in the CNS, through a mechanism in which herpesviridae infection promotes A? amyloidosis. Evidence also suggests A? exists in diverse modified proteoforms or associate with cofactors (e.g., metals). The diversification of A? monomers may contribute to different rates of A? oligomerization, in a manner that results in distinct A?Os populations that trigger synaptic dysfunction. Our work suggests that chemical diversification of A? through post-translational modifications (PTMs) and non- covalent interactions (e.g., metals) leads to potentially hundreds of native monomeric A? proteoforms. We propose that the compositional makeup of these monomers varies in a manner that is associated with stages and brain regions during AD onset and progression, analogous to the stages established for plaques and tangles by Braak and Braak. A new native Top-down mass spectrometry (nTDMS) procedure pioneered by our team has provided us momentum to test this hypothesis by providing a sensitive measure of the native A? proteoforms that exist in A?Os of virtually any size. The assay reads the A? PTM-status and characterizes bound co-factors, including metals, in a single detection event. Aim 1 will describe the spatial pattern of native A? proteoforms in demented patients and animal models relative to controls. Data mining will describe signatures of A? related by covalent PTMs or non-covalent interactions, correlating the signatures to pathological co-variables. Aim 2 will utilize data mining to define proteoform signatures that associate with cellular phenotypes (e.g., synapse binding and neuroinflammation). Aim 3 will describe the temporal variability of A? proteoforms relative to distinct neuropathological features in animal models. Partnering with neuroscientists, in Aim 4 we will create a Proteinopathy Proteoform Knowledgebase that aggregates proteoform data in a manner that links subsets of proteoforms to disease relevant phenotypes (e.g., A? pathologies) or other clinical data. Overall, our work will provide fundamental insights on spatiotemporal signaling leading to dementia, and will inform many A? research tracks, including hypothesis testing in relation to in vivo targeting of A? imaging probes or diagnostic or therapeutic antibodies.