FUNDING PERIOD: APRIL 1, 2008 - MARCH 31, 2011

Paul J. Lombroso, M.D.
Yale University
New Haven, CT
The Role of STEP in Alzheimer's Disease
$400,000

Alzheimer's disease is a devastating disease with few effective treatments. Understanding the molecular basis of this disease should lead the way to new therapies. We are testing the hypothesis that a protein called STEP disrupts communication between neurons in Alzheimer's disease. STEP is a brain-specific protein that regulates the activity of several proteins required for the stabilization of memories. One of these proteins is the NMDA glutamate receptor. This receptor complex normally moves from intracellular pools to the neuronal surface where it can receive neurotransmitter signals required for the formation of long-term memories. The trafficking to and from membranes is a tightly regulated process, and STEP participates in this process. We recently discovered that STEP is inappropriately activated by beta amyloid. Moreover, active STEP removes NMDA receptors from neuronal surfaces. Inappropriate activation of STEP leads to loss of glutamate receptors and subsequent disruption to memory formation. We predict that reducing STEP activity will reduce the loss of NMDA receptors from their active sites. We will test this hypothesis by using an animal model of Alzheimer's disease with reduced levels of STEP proteins. We predict these mice will be 'rescued' and have restored cognitive abilities. These findings will have potential therapeutic implications.

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Linda J. Van Eldik, Ph.D.
Northwestern University
Evanston, IL
Attenuating proinflammatory cytokine up-regulation as an AD therapeutic strategy
$400,000

New Alzheimer's disease (AD) drugs that alter disease progression are urgently needed. The long term goal of our research is to develop safe and effective drugs for AD by targeting the overproduction of inflammatory molecules from glial cells, the cells in the brain that produce detrimental inflammation responses. Glial cells normally cooperate with the nerve cells to keep the brain operating smoothly. When an injury or change in the brain occurs, the glial cells mount a beneficial inflammation response to fight off the insult and restore the brain to its proper functioning. While a controlled inflammatory response is an important element in protecting the brain, this beneficial process sometimes gets out of balance and the inflammation becomes too strong or does not shut off on schedule. In AD, glial cells are over-activated and produce detrimental inflammatory molecules called proinflammatory cytokines that can contribute to nerve cell death and accelerate the progression of the disease. This raises the logical question of whether drugs can be developed to selectively target cytokine up-regulation in glia, with the hope that such drugs would slow down or perhaps even prevent disease progression.

In our Center for Drug Discovery and Chemical Biology, we are using a drug discovery platform that integrates what we call 'smart chemistry' with 'smart biology' to develop new small molecule compounds that selectively suppress the overproduction of proinflammatory cytokines in the brain. We have developed two exciting new drug-like compounds, named Minozac and Minokine, that are safe in animals, readily enter the brain, and are able to be taken by mouth. We have found that these compounds suppress the glial cytokine production back towards normal levels, which prevents subsequent nerve cell damage and learning deficits in an animal model of early stage AD. Our proposed project will extend these findings to test Minozac and Minokine for effectiveness in a more severely affected AD transgenic mouse model, in order to ask whether the compounds are effective not only at preventing or suppressing cytokine-driven neurologic damage in early stages, but are also effective under conditions of increasing disease severity. Successful completion of this project will provide immediate impetus for further development of these compounds into future, potentially disease-modifying drugs for AD.

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Alison M. Goate, M.D.
Washington University
St. Louis, MO
Identification of functional alleles that influence cerebrospinal fluid levels of Aβ and risk for Alzheimer's disease
$400,000

My proposal has been designed to identify novel genes and pathways that influence a person's risk for developing Alzheimer's disease (AD). I am taking a unique approach to this question by using cerebrospinal fluid protein levels that are known to correlate with whether someone has AD or not in a genetic study. This approach provides a specific biological mechanism for the effect of the genetic variation on AD, making subsequent experiments to identify the function of the genetic variation simple and straightforward. I will first follow-up my previous findings in independent samples to prioritize genes for further study. Second, I will study the high priority genes in depth, identifying the variation responsible for the effects on AD. Third, I will characterize the biological mechanism by which the variation causes these effects. The successful identification and characterization of genetic variation that affects risk for AD will help us learn about why people develop AD, thus providing new targets for AD drug treatment.

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Hui Zheng, Ph.D.
Baylor College of Medicine
Houston, TX
Role of APP in synaptogenesis
$400,000

Beta-amyloid plaque is the hallmark of Alzheimer's disease (AD). The plaque is formed from the amyloid precursor protein (APP). Genetic mutations in APP lead to familial AD. Thus, APP's role is essential in AD pathogenesis. In addition to the plaque pathology, synapse dysfunction is believed to directly contribute to dementia. However, the link between APP and synaptic activity is poorly understood. Because it is difficult to know what happens in human brains during the disease progression, my laboratory uses mice as a model system to learn the human disease. Specifically, we are looking to understand the function of APP, and what goes wrong in familial cases that lead to AD using mice that either delete APP or express the mutant form of APP. We found that APP directly regulate synapse formation and we want to know how it works and whether the amyloid affect this activity. Our studies will allow understanding how APP functions in synaptic activity and what happens when APP is mutated, and what can we do to enhance the neuronal activity. The latter has the potential to discover novel therapeutic targets for AD.

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Eric A. Schon, Ph.D.
Columbia University
New York, NY
(Embargoed for release until after JUNE 1)
$150,000

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Paul H. Axelsen, M.D.
University of Pennsylvania
Philadelphia, PA
Oxidative stress and amyloidogenesis
$150,000

Oxidative stress and amyloid fibril formation are consistent major themes among processes thought to be involved in the pathogenesis of Alzheimer's disease. However, a mechanistic link between these two processes has not been defined. The research being proposed will probe human brain tissues and animal models of Alzheimer's disease for evidence of a specific chemical mechanism that will be amenable to therapeutic intervention.

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Kathryn J. Moore, Ph.D.
Massachusetts General Hospital
Boston, MA
Innate immune signaling in Alzheimer's disease pathogenesis
$265,000

Microglia are the primary immune cells of the brain and in Alzheimer's disease (AD) these cells accumulate at sites of ß-amyloid (Aβ) deposits, including senile plaques. Microglial interactions with Aβ incite a chronic inflammatory response that leads to neuronal degeneration, increased Aβ deposition and disease progression. The microglial receptors and signaling pathways triggered by Aβ that promote this chronic inflammation remain a matter of speculation. Our long-term goals are to identify the mechanisms of microglial activation by Aβ and the impact of these pathways on disease. We hypothesize that the Toll-like receptors (TLR), an evolutionarily ancient family of microbial recognition receptors, initiate and maintain the microglial inflammatory response to Aβ. This hypothesis is based on preliminary findings that targeting of members of this signaling pathway block microglial inflammatory responses to Aβ. We propose to define the TLRs and co-receptors responsible for initiating this signalling, their impact on microglial inflammatory responses and the implications for disease. Understanding the mechanism(s) of microglial interactions with Aβ and identifying the receptors involved in these interactions will provide valuable insight into the role of these cells in the pathogenesis of AD and potentially identify therapeutic targets in AD.

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James K. Foskett, Ph.D.
University of Pennsylvania
Philadelphia, PA
IP3R-Presenilin Interaction: Calcium Dysregulation in AD
$400,000

Alzheimer's disease (AD) is a common form of dementia involving slowly developing degeneration of neurons in the brain. The causes of AD are still not clear, but mutations in some proteins that result in early-onset cases of the disease provide clues. One of these proteins, presenilin, causes the amount of calcium in cells to be abnormally regulated. Because calcium regulates many brain functions, this abnormality may be a key part of the disease. We have discovered a mechanism whereby mutant forms of presenilin that cause AD alter the function of an important protein that regulates calcium signals in cells. Calcium in cells is precisely regulated, because it is toxic if its concentration is too high. Chronic abnormal calcium regulation as a result of mutations in presenilin may therefore cause cellular toxicity that leads to cell death. We plan to study how presenilin interacts with this important calcium signaling protein to alter its function, and how altered calcium signaling in turn affects cell functions. These studies should provide new insights into the molecular mechanisms of AD and into the development of novel targets for therapeutic interventions.

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Huaxi Xu, Ph.D.
Burnham Institute for Medical Research
La Jolla, CA
Roles of a novel gene FG01 in inhibiting Alzheimer's amyloid and tau pathologies and GSK3 activity
$400,000

Too much of ß-amyloid (Aβ) protein in the brain is believed to initiate the pathological cascade culminating in Alzheimer's disease (AD) - the most common form of dementia. Aâ is derived from the proteolytic cleavage of â-amyloid precursor protein by β- and β-secretase activities. Inhibiting either secretase is a major goal in AD therapeutics. The β-secretase, which determines the generation of the toxic form of Aβ, can be regulated by many factors or genetic pathways. Because ã-secretase can also cleave a variety of membrane proteins, which is important for normal physiological activities, significant unwanted effects may appear if we would develop drugs to inhibit the β-secretase. In this proposal, we utilized a technology named Random Homozygous Knockout to identify genes that can inhibit the β-secretase activity and hence reduce brain Aβ levels. Through years of intensive research, we have found a novel gene, FG01, that can significantly reduce Aβ without affecting the action of β-secretase toward other important proteins when it is expressed in mouse and human neuron-like cells. We will continue to characterize the functions of this gene to understand its mechanism of action which may eventually be applied to develop therapeutic interventions specifically inhibiting Aβ without unwanted side-effects.

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Tae-Wan Kim, Ph.D.
Columbia University
New York, NY
Modulation of BACE1 by a Novel Sorting Nexin
$265,000

Aberrant trafficking of Alzheimer’s disease (AD)-associated molecules, such as beta-amyloid precursor protein (APP) and beta-site APP-cleaving enzyme 1 (BACE1), has been extensively implicated in the neuropathogenesis of AD. BACE1 mediates the first of two cleavage events of APP to yield amyloid beta-peptide (A-beta). Recent studies suggest that aberrant regulation of molecular components of the endosome and trans-Golgi network (TGN) may contribute to the enhanced A-beta levels associated with AD. We discovered that a novel sorting nexin, a member of the family of trafficking proteins that bind phospholipids, binds BACE1 and regulates the beta-site cleavage of APP. Our proposed studies will investigate the mechanism underlying the regulation of BACE1 trafficking and beta-amyloid generation in neurons by this novel sorting nexin. Gaining insight into these cellular mechanisms will lead to development of novel therapeutic approaches for preventing or treating AD.

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FUNDING PERIOD: APRIL 1, 2008 – MARCH 31, 2010

Youssef Z. Wadghiri, Ph.D.
New York University School of Medicine
New York, NY
Improving Delivery And Labeling Efficiency Of MRI Probes For Alzheimer's Plaques
$150,000

Our group has undertaken the task to develop safe MRI molecular probes to visualize amyloid β (Aβ) plaques, one of the earliest pathological hallmark of Alzheimer's disease that can only be confirmed by post-mortem examination of the brain. The early diagnosis of this slow neurodegenerative disease that leads to dementia and death, would be critical when new and future therapeutic approaches may be most effective in preventing the irreversible neuronal damage. Transgenic mice that develop Aβ plaques similar to AD patients are now being used to test experimental approaches for amyloid clearance. We were the first to demonstrate that magnetically labeled peptides could be used to detect Aβ plaques in the brain of living mice with MRI. The goal of this proposal is to further improve the means of delivery of our MRI probes as well as their magnetic labeling efficiency. We wish to implement a minimally invasive injection approach while maintaining the shortest injection route to the brain by the use of an ultrasound-guided intra-cardiac injection. We also hypothesize that the pre-opening of the blood-brain-barrier prior to the infusion of our construct and the use of paramagnetic agent with stronger contrast effect will increase the plaque population visualized. Furthermore, increasing the relatively short plasma half-life of our constructs (currently few minutes) will increase their recirculation and diffusion throughout the brain thereby improving their delivery across the entire brain. Our aims are to visualize the AD plaques with a higher specificity and sensitivity by refining the characteristics of the label while assessing the safety and effectiveness of the approach in mouse models of AD. In essence, the work proposed will serve as the basis for establishing a safer and more efficient animal protocol that could be utilized routinely to better understand the role of amyloidoisis as well as to test current and new anti-amyloid therapies before their examining in humans.

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Ruben Vidal, Ph.D.
Indiana University School of Medicine
Indianapolis, IN
Double transgenic model of familial Danish dementia with plaques and tangles
$100,000

Alzheimer disease (AD) afflicts more than 4 million Americans at a cost to the economy in excess of $100 billion. The psychological and emotional burden to patients and care-givers is incalculable. Given the absence of disease-modifying therapeutics and accurate diagnostics, there is a need to better comprehend AD's pathogenic mechanism. We have developed an animal model for a similar neurodegenerative disease named familial Danish dementia (FDD). FDD shares many clinical and pathological similarities with AD, including deposition of amyloid (only different from the one found in AD in primary sequence) and neurofibrillary tangle formation (similar to the one found in AD). We will perform biochemical, neuropathological and behavioral studies in the animal model that should provide the basis for the identification of the cascade of events that lead to FDD. We believe that the study of this model may provide insights into the role of amyloid and neurofibrillary tangle not only in FDD but also in AD.

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Mathias Lösche, Ph.D.
Carnegie Mellon University
Pittsburgh, PA
Lipid Bilayer Reorganization by Amyloid-&beta Oligomers
$149,940

How do misfolded peptide aggregates interact with lipid membranes? More specifically, how do amyloid-Beta (ABeta) oligomers affect neuronal cell membranes? -- A progressively larger body of evidence has recently accrued that the toxic form of ABeta -- a 40 or 42 amino acids long peptidic cleavage product of the amyloid precursor protein APP which has long been implicated to play a crucial role in the etiology of Alzheimer's Disease (AD) -- is a soluble oligomeric aggregation state of the peptide, and that such ABeta oligomers interact strongly with cell membranes. Using novel synthetic membrane models, we have developed suitable experimental tools to address the questions posed above in structural, functional and dynamic terms on the molecular level. In this ADR Pilot Grant, we propose to study the response of such synthetic membranes to ABeta oligomers as a function of membrane composition. We are particularly interested in bilayer compositions that mimic the lipid inventory of neuronal cell membranes to investigate whether ABeta oligomers interact with such membranes strongly.

The proposed research may have implications for a molecular-scale understanding of the damage that ABeta oligomers afflict on neuronal membranes and may in the long run also help to devise synthetic strategies for the early detection of ABeta oligomers in patient samples.

The specific aims of this research are to investigate the interaction of ABeta oligomers with synthetic lipid membranes of well-defined compositions and to correlate the structural, functional and dynamic response of such membranes to ABeta with the aggregation state of the peptide.

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Anne M. Cataldo, Ph.D.
McLean Hospital
Belmont, MA
A Novel Treatment Strategy for Neurorepair in Alzheimer's Disease
$150,000

Neurodegenerative disorders such as Alzheimer's Disease (AD) have devastating effects on patients and society; they are difficult to predict, diagnose, and treat and weigh heavily on families and the healthcare system. Alzheimer's Disease affects 5 million Americans, 7 in 10 of whom are cared for by family members. While medications can ameliorate the symptoms of this disorder, none reverse the death of nerve cells. For these reasons, we are looking at novel avenues--namely stem cells--for brain cell regeneration. Unlike many studies using embryonic stem cells, this treatment will rely on stem cells derived from the bone marrow of adults, called marrow-derived adult progenitor cells (MAPCs) that could provide a valuable alternative to the use of embryonic stem cells. Our studies thus far show that new and selective nerve cell growth can be encouraged from MAPCs, and importantly that these cells have the ability to generate the same types of nerve lost in AD. Unlike embryonic stem cells, whose use has generated a number of ethical concerns, MAPCs are autologous, meaning these cells can be obtained from the marrow of the same individual with AD. Patients thus serve as their own donors, eliminating the occurrence of a graft-host rejection -- an immune response that often occurs with the transplantation of tissue obtained from one individual to another. MAPCs also can be delivered to brain through intravenously injection, which we have observed in our initial animal studies. In addition, MAPCs are capable of migrating to those areas in brain with the most severe cell loss. The proposed studies will employ a novel therapeutic approach which focuses on the potential use of MAPCs to carry an important cholinergic cell growth factor that is reduced in AD, sAPPAlpha, into the brains of an animal model with AD-like cholinergic cell loss and its ability when combined with other nerve cell growth factors to retard nerve cell loss and promote new nerve cell development. The promise of stem cell therapies doesn't stop at neurodegenerative diseases but may impact other diseases of brain. If we can develop bone marrow cells into the brain cells lost in AD, maybe we can transform them into other subpopulations of nerve cells that may be lost or compromised in a wide range of disorders of brain which will have important implications for clinical care.

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Luciano Domenici, M.D., Ph.D.
National Council of Research (C.N.R.)
Pisa, Italy
RAGE, MAP kinases and Abeta induced synaptic dysfunction
$148,000

Alzheimer's disease (AD) is one of the main form of progressive neurodegenerative disorders leading to cognitive impairment with dementia. Typical neurophatological feature of AD is the presence of beta amyloid (Aβ) deposits with the form of plaques in different brain areas. Recent studies have higlighted the apparent discrepancy between neuropathological findings and cognitive impairment such as memory loss in AD. In the present project we raise the hypothesis that impairment of memory and neuronal dysfunction during an early phase of AD is due to oligomeric Aβ, i.e. a step before the formation of plaques. In particular, for the first time, we aim to demonstrate that oligomeric Aβ induces a synaptic dysfunction in entorhinal cortex one of the brain areas affected in AD. The project will use a combination of electrophysiological and molecular approaches to show the mechanisms underlying oligomeric Aβ induced neuronal dysfunction including the identification of receptors mediating Aβ dysfunction.

The outcome of these studies will be important to clarify the role of Aβ and its receptors in different temporal phases of cortical neurodegenerative processes in AD. Deciphering new molecular aspects in AD pathology will be essential to define new therapeutical strategies leading to halt the progress of disease.

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Francine Grodstein, Sc.D.
Harvard University
Boston, MA
Mid-life Telomere Length and Cognitive Decline in Later Life
$100,000

Alzheimer’s disease develops over many years. Thus, preventing Alzheimer’s disease may require interventions beginning at younger ages. However, to provide interventions only to those who most need them, we will have to find ways to identify at younger ages, those who would be most likely to get Alzheimer’s disease at older ages.

We hypothesize that a novel marker of aging and bodily stress may also indicate the likelihood of developing memory problems in older persons. Much research would be needed before this marker could be used in clinical practice, and virtually no research has been done to date. Thus, our proposal to generate early data on a new marker that might predict memory problems in older people, would be critical to interest in supporting future widespread research on this marker.

Overall, the success of our proposed pilot study could significantly change the field of research on Alzheimer’s disease prevention by indicating ways of predicting dementia risk at middle-age.

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Randall J. Bateman, M.D.
Washington University
St. Louis, MO
Human CNS-Apolipoprotein E Isoform Production and Clearance
$150,000

Alzheimer's disease is the most common cause of dementia, and the most feared disease of Americans over the age of 65. ApoE is the strongest genetic risk factor for Alzheimer's disease, and is a potential target for disease-modifying therapies. The ApoE4 allele, which occurs in 15 percent of the population, increases one's risk of developing Alzheimer's Disease by 3-fold, while two copies of ApoE4 increase this risk by 12-fold. Whereas the E2 allele, occurring in 7 percent of the population, decreases one's risk of developing Alzeimer's disease. The hypothesis being tested is the three ApoE isoforms (E2, E3 and E4) have different rates of synthesis and clearance. We will measure the metabolism of ApoE from the cerebrospinal fluid of young, non-demented participants. This study will provide an important link regarding how ApoE4 may be involved in the pathophysiology of Alzheimer's disease, and may lead to improved therapeutics, which target the major genetic risk factor of Alzheimer's disease, ApoE.

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Gal Bitan, Ph.D.
University of California, Los Angeles
Los Angeles, CA
{Embargoed for release}
$100,000

Mark E. Girvin, Ph.D.
Albert Einstein College of Medicine of Yeshiva University
Bronx, NY
Structural Determinants for Bri2 Inhibition of ABeta Production
$150,000

The main protein that leads to Alzheimer's disease (called A-beta) is only a small portion of a larger protein that has to be cut into smaller pieces to be toxic. One way to avoid building up the levels of the toxic piece, is to prevent its parent protein from being cut. Several researchers are working on inhibitors of that cutting enzyme. But there's a problem -- that enzyme is needed to cut other important proteins in the cell into their active form. Two groups recently found a new protein called BRI2 that only binds to the parent protein, and protects it from getting cut. One group has isolated the smallest piece of this BRI2 protein that does the job, and would like to modify it to use it as a drug to prevent build-up of the toxic A-beta, but they don't know what makes it bind tightly and specifically to the A-beta parent protein. So, we are going to use a technique called Nuclear Magnetic Resonance (NMR) to get a detailed picture of that piece of the BRI2 protein alone (in Aim 1), and when it is bound to the A-beta parent protein (in Aim 2 and 3). This will tell the drug designers what parts of the protein need to be kept (or improved) for good binding to the target, and what parts can be changed to make the molecule a better drug. Hopefully this will lead to a drug that keeps down the levels of the toxic A-beta protein, but doesn't have any side effects.

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Terrone L. Rosenberry, Ph.D.
Mayo Clinic Jacksonville
Jacksonville, FL
Detection of cross-linked amyloid-beta oligomers in Alzheimer's disease by mass spectrometry
$150,000

A hallmark of Alzheimer's disease (AD) is the presence of deposits called amyloid plaques in the brain. These plaques are mostly composed of a peptide called amyloid-beta. Recent research indicates that soluble clusters of Abeta, called oligomers, are perhaps the primary cause of AD. We are trying to determine the chemical structure of these oligomers and, in particular, whether the structure contains a feature called a cross-link. This is a challenging goal, as the amount of these oligomers in the brain is too low for conventional chemical analyses. To our knowledge, no other researchers have tried to address this question in a technically rigorous manner. Our project could have significant therapeutic benefits. Diagnostic tests could be designed to specifically quantify cross-linked oligomers in cerebrospinal fluid and correlate their levels with cognitive deficits in elderly individuals. Other tests of the neurotoxicity of these oligomers could be conducted by chemical synthesis of their precise structures and evaluation their toxicity in cellular and intact animal assays. Such synthetic peptides could even be investigated as possible vaccines. However, before we can determine whether cross-linked oligomers are enriched in individuals with AD, we must first develop the analytical techniques required for sensitive oligomer detection. This developmental effort is best directed at synthetic Abeta oligomers that are prepared and chemically cross-linked in our laboratory. Our grant proposal has three aims. In Aim 1 we will prepare stable oligomers of Abeta by chemical cross-linking in vitro and isolate individual oligomers. In Aim 2 we will evaluate and then establish procedures to detect covalently cross-linked Abeta oligomers and identify sites of cross-linking by two mass spectrometry techniques. In Aim 3 we will apply our optimized techniques to determine the structure and amounts of Abeta oligomers that can be isolated from AD brain tissue.

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Cynthia A. Massaad, Ph.D.
Baylor College of Medicine
Houston, TX
The Role of Mitochondrial Superoxide in Alzheimer's Pathology
$100,000

Alzheimer's disease (AD) is a neurodegenerative disease characterized by deposition of amyloid plaques leading to dementia and memory loss. Oxidative stress is also associated with the pathology of the disease and it is believed that amyloid beta and oxidative stress are linked; it is not known however which comes first. Our project aims at understanding the role of oxidative stress in AD via using 2 animal models: an AD model and a model that overexpresses an antioxidant enzyme. We are in a unique position not only to study the biochemical and behavioral effects, but also the in vivo physiological effects of antioxidants on AD symptoms. We specifically propose to (1) measure the increase of various amyloid species in AD mice at several age points and test whether this increase is alleviated by an increased antioxidant protection. (2) measure the integrity of axonal transport (indicative of nerve cell integrity) in AD model mice by in vivo imaging with MRI, and test the effect of antioxidant protection using the same technique. (3) Measure blood flow in the brain of AD model mice and test whether increased antioxidant protection will be beneficial. This aim will be achieved using perfusion imaging by MRI. The results of such study are extremely important to identify novel targets for the design and use of pharmacological antioxidant agents for the treatment of AD.

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Marguerite Prior, Ph.D.
The Cleveland Clinic Foundation
Cleveland, OH
Inhibition of RTN3 aggregation as a novel therapeutic target to reduce cognitive failure in AD
$100,000

Alzheimer's disease (AD) is the most common cause of dementia in the elderly affecting over 5 million Americans. Unfortunately despite extensive research there is no effective treatment for this disease up to today. Hence, every approach that will help to alleviate cognitive dysfunction in AD patients deserves through investigation. A protein called reticulon 3 (RTN3) has recently been found to be accumulated in the brains of AD patients in a distinct population of damaged neurons that cannot be seen in non-demented brains. This accumulation of RTN3 in AD brains leads to the formation of RTN3 aggregation and the extent of aggregation correlates with the cognitive decline as revealed in our animal study. We hypothesize that inhibition of RTN3 aggregation will block the formation of neuronal dystrophy and reduce amyloid deposition, offering a new therapeutic target in AD. A biochemical assay has been developed in our laboratory to monitor the accumulation of RTN3 in cells and this assay has identified a compound that can effectively inhibit the accumulation of RTN3. Our overall aim is to determine the potential therapeutic application of the inhibitor in reducing cognitive failure in AD animal models. Inhibition of the formation of these RTN3 damaged neurons and improved cognitive performance in animal models may lead to human clinical trials. In specific aim 1 we will further characterize how the compound inhibits accumulation of RTN3 by using structure activity studies. In specific aim 2 we will take advantage of our ability to detect biochemically tractable accumulated RTN3 to study the in vivo effect of this inhibitor of RTN3 accumulation on the formation of RTN3 damaged neurons using mice that express higher levels of RTN3, produce RTN3 accumulation and RTN3 damaged neurons and mice, which produce age related neuritic plaques containing the toxic amyloid peptide.

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Carmen E. Westerberg, Ph.D.
Northwestern University
Evanston, IL
Connections Between Memory Dysfunction and Sleep in Alzheimer's Disease
$100,000

Our studies of Alzheimer's disease (AD) are aimed at elucidating relationships between memory, sleep, and brain dysfunction. We explore a possible link between poor memory and poor sleep in AD, with possible implications for disease detection and treatment.

In early stages of AD, people experience problems in only one type of memory, declarative memory (memory for episodes and facts). Declarative memory provides each of us with a vast but imperfect storehouse of information. However, declarative memories do not persist in the brain in a static state. Rather, restructuring is required as new information is added. Over time, some declarative memories change into a resilient state such that they might last a lifetime, whereas others are lost. Declarative memories reach an enduring state by undergoing consolidation. Sleep processing may provide the vehicle whereby consolidation can move forward. We recently postulated that memory change during sleep constitutes a fundamental characteristic of declarative memories. This is a relatively new idea, but supporting research findings are rapidly appearing. An alliance has formed between the traditionally separate fields of memory research and sleep research. We propose to take another step forward in that direction.

Why do Alzheimer's patients so often experience difficulties with declarative memory? To address this question, we will examine sleep and memory in patients at the earliest stages of AD and in healthy individuals of the same age. Detailed analysis of a variety of physiological signals will be used to detect abnormal sleep patterns and to examine relationships between neural signals during sleep and subsequent memory abilities.

Our proposal also involves a potential treatment based on the findings of our co-workers in Germany. In their young subjects, electrical stimulation at the scalp significantly enhanced slow-wave sleep and memory storage. This DC stimulation was harmless, and so mild that it was not even perceived. We will implement the same procedures in our study. Any memory improvement could be beneficial for patients whose memory capabilities are beginning to deteriorate, and major improvements are conceivable. Our investigations will determine whether such methods could eventually be useful for improving quality of life in patients. We begin by studying patients with a preclinical form of AD, but plan to develop a more extensive research program in this area that will include patients with a diagnosis of probable AD.

When we are awake we have the opportunity to learn, acquire knowledge, relate new information to what we already know, and creatively put information together in novel ways. In this research we consider the fascinating possibility that when we are asleep, memory storage may progress similarly, and that a failure in memory processing during sleep may exacerbate memory deficits in AD.

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Daniel Zwilling, Ph.D.
J. David Gladstone Institutes
San Francisco, CA
The Role of the Kynurenine Pathway in Microglia in Alzheimer's Disease
$100,000

Microglia are specialized brain cells that support the health and function of neurons. In response to brain injuries, these cells produce a large number of molecules that participate in inflammation and wound repair. While acute glial responses may help prevent neuronal damage, prolonged or aberrant activation of these cells may contribute to neurological disease. I am using genetic and pharmacological strategies to assess whether glial cells and their products contribute to cerebral amyloidosis and neurodegeneration in Alzheimer's disease. Researchers found that a certain microglial enzyme that is active in the metabolism of tryptophane (an amino acid) might be responsible for the production of neurotoxic substances. In AD patients this enzyme might be more active than usual. However, the inactivation of this enzyme by drugs might help to prevent the progression of AD pathology. The proposed experiments will increase our understanding of the role of microglia in Alzheimer's disease and may also provide information critical for the preclinical development of drugs for Alzheimer's disease.

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Ping He, M.D., Ph.D.
Sun Health Research Institute
Sun City, AZ
Regulatory Mechanism of BACE by Inflammatory Factors
$100,000

A major protein component of amyloid plaques in Alzheimer's disease (AD) is amyloid-beta-peptide (Abeta). To produce Abeta, the amyloid precursor protein (APP) must be cleaved by beta-secretase, which produces C99 fragments and releases soluble APPbeta(sAPPBeta). C99 is then cleaved by a second enzyme, gamma-secretase, leaving us with Abeta. Higher levels of both BACE activity and protein expression were recently discovered in the brains of those with sporadic AD. Patients with Mild Cognitive Impairment (MCI), are characterized as individuals with an increased risk of developing AD in comparison to healthy controls and AD patients. We have recently reported that both the level and activity of BACE were increased in the cerebral spinal fluid (CSF) of MCI patients, which indicates that such an elevation may be an early sign of the onset of AD. Meanwhile, the mechanisms of 'upstream' genotypic events that lead to increased BACE levels still remain unclear. Because our preliminary Northern blot analysis indicated an increase of BACE mRNA in AD brains, translational regulation of BACE will be one of our potential mechanisms to answer that question. However, whether BACE mRNA could increase during the MCI stage remains unknown. On the other hand, our published studies have demonstrated cytokine involvement in AD-related insults and our preliminary data demonstrate that the 5'-uncoding region (5'-UTR) contains NF-KappaB responsive elements. As these inflammatory events are implicated during the progress of AD pathogenesis, we hypothesize that BACE level and activity, in addition to transcriptional activity may potentially increase during the MCI stage mediated by cytokines, resulting in Abeta accumulation, and later onset of AD. Therefore, we will examine BACE transcriptional levels in MCI brains and observe whether BACE transcriptional activity is regulated by TNFalpha response signal pathway.

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Sergio Casas-Tinto, Ph.D.
University of Texas Medical Branch
Galveston, TX
XBP1, a novel suppressor for Amyloid-beta neurotoxicity
$100,000

Alzheimer's disease (AD) is a terminal age-associated dementia characterized by learning and memory deficits, loss of brain neurons, and eventually death. The accumulation of abnormal amyloid beta (Aβ) peptide is the primary neurotoxic event in the AD brain, however the molecular mechanisms mediating Aβ toxicity are largely unknown. To shed light on this issue, I searched for genes that suppress Aβ neurotoxicity in a fly model of Alzheimer's disease. To do this, I used the fly eye as experimental system because accumulation of Aβ causes progressive degeneration of photoreceptor neurons. Thus, by crossing flies producing Aβ in the eye with 5,132 stocks of flies that activate different genes, I was able to search the fruit fly genome for mutations that specifically protect against Aâ neurotoxicity. This led me to the identification of a small group of genes with remarkable suppressor activity. The goal of this project is to characterize the ability of one of these suppressors, known as XBP1, to protect against Aβ-induced neurodegeneration in the fly brain and also in human cultured neurons. Our results may provide new information into the mechanisms of AD pathogenesis and will be relevant for the design of novel therapies for this devastating disorder.

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Mary N. Haan, Dr.P.H., M.P.H.
The Regents of the University of Michigan
Ann Arbor, MI
A Pilot Study of the Association of Plasma Beta Amyloid 40/42 and Risk of Alzheimer's disease in Mexican Americans
$60,000

Most of my research focuses on chronic neurodegenerative disease in aging populations, especially on Alzheimer's disease and dementia. As an epidemiologist, I am primarily interested in risk factors that increase disease risk and on identifying ways to prevent the development of disease. My research is trying to find environmental factors or biomarkers that predict which people are more likely to get Alzheimer's Disease. Evidence is mounting that exposures leading to Alzheimers occur in early or midlife. Research studies are important to find biomarkers that can predict an increased risk of Alzheimer's Disease (AD). It is likely that prevention will be more effective in early stages of disease development. I also think that studying diverse populations can better help to identify both environmental and genetic risk factors that cause disease. Beta amyloid 40 (AB40) and 42(AB42) are potential biomarkers that could be used in screening for early AD and neurodegenerative processes. Before we can do that, we need to know if this will work in a variety of populations. This research proposes to test plasma samples taken from elderly people of Mexican ancestry for these two biomarkers: AB40 and AB42. These tests were previously done in studies of people of European ancestry and showed that a higher ratio of 42 to 40 gave a significantly increased risk of developing of Alzheimer's Disease.
The aims of this pilot project will be:

1. To describe plasma amyloid B42 and B40 in Mexican ancestry individuals, including overall levels, correlations with inflammatory markers (i.e. C-reactive protein, IL-6 and receptors, TNF-alpha and receptors), lipids, insulin, insulin degrading enzyme (IDE), body fat and other metabolic markers.
2. To estimate the risk of Alzheimer's disease and CIND associated with AB40, AB42 and the ratio of AB42/AB40.
3. To evaluate whether the effects of AB40 and AB42 on AD or CIND are modified by presence of the 'e4' allele of ApoE lipoprotein.
4. To evaluate the influence of key biomarkers on the association between AB42 or 40 or the 42/40 ratio and AD and CIND as intermediate pathways or modifiers.
   

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FUNDING PERIOD: APRIL 1, 2007 – MARCH 31, 2010

George R. Jackson, M.D., Ph.D.
University of California, Los Angeles
Los Angeles, CA
Project: Validation and Characterization of Tau Modifiers In Vivo
$400,000

Neurofibrillary tangles containing tau are a hallmark of Alzheimer’s disease. However, little is known about ways to protect brain cells from degeneration caused by tau. We looked at gene expression in several parts of the brain of mice expressing human tau including a mutation that causes FTD. This was done in combination with testing in the simple fruit fly. We identified several novel modifiers of tau neurotoxicity including the highly conserved protein, puromycin-sensitive aminopeptidase (PSA). Here, we propose further studies of the role of PSA in tauopathy, and plan to validate other genes identified as putative protective or susceptibility genes in the transgenic human tau P301L mouse model. These will involve crossing fruit flies that express tau with other lines that have greater and/or lesser expression of the genes identified in the mouse. Genes that succeed in changing tau toxicity can easily be identified under the microscope by examining the size of the fly eye. Validation and characterization of mechanisms of action of tau modifiers using the fly model will provide a first step toward identifying those modifiers which are most promising as therapeutic targets for AD; these may then be further studied in cell culture and mice.

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David E. Kang, Ph.D.
University of California, San Diego
LaJolla, CA
Project: Novel Domain of LRP Cytoplasmic Tail in APP Processing
$400,000

Alzheimer's disease (AD) is a progressive and irreversible disease of the brain leading to deterioration of mental function and eventual morbidity and death. The major defining characteristic of AD brains is the excessive accumulation of amyloid plaques, composed of a sticky protein called amyloid b (Ab). Ab is toxic to nerve cells, and this may explain the progressive degeneration seen in AD brains. Ab is formed when “molecular scissors” cut APP into 2 places, resulting in the release of Ab. This is a normal process that also occurs in healthy individuals. However, for reasons we do not understand at present, Ab is either excessively produced or not removed fast enough in AD patients. One obvious way to block Ab formation is to inactivate the “molecular scissors”. However, these proteins also have other important functions, such that blocking the “molecular scissors” can have undesirable side effects. An alternative and perhaps additive design might be to block APP transport inside nerve cells so that it does not reach the sites where the “molecular scissors” reside and are most active. In our studies, we found that a protein called LRP normally promotes Ab generation by directing the transport of APP inside cells to compartments where the “molecular scissors” are most active. More than 80% of Ab production is dependent on the presence of LRP. Remarkably, we found that a very small region of LRP (LRP-C37) by itself is sufficient to mimic LRP in robustly increasing Ab generation. In addition, we identified new proteins that physically interact with the C37 domain and modify the cuts made in APP. At present, how LRP-C37 by itself or in the context of the full protein increases Ab production is not known. Furthermore, it is not known how the two new proteins we identified alter APP processing and Ab generation. We hypothesize that the LRP-C37 domain plays a critical role in transporting LRP and APP to compartments where Ab is normally generated. In this application, we propose to characterize the mechanistic basis of the LRP-C37 domain in LRP and APP transport inside cells and Ab generation. In addition, we will determine the role of the two new LRP-C37 interacting proteins in these processes. These studies are expected to form the basis of designing a novel therapeutic approach to block Ab generation.

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James Lah, M.D., Ph.D.
Emory University
Atlanta, GA
Project: Targeted Discovery of LR11/sorLA-Based AD Therapeutics
$300,000

Because of the strong association between aging and Alzheimer's disease, it is becoming an increasingly important public health concern as life expectancies increase and the number of affected individuals grows. Despite rapid growth in our scientific understanding of Alzheimer’s disease, current treatments are relatively ineffective.

In earlier studies, we discovered a novel association between a receptor called LR11 (also known as SorLA) and Alzheimer's disease. LR11 levels were consistently reduced in the brains of patients with AD compared to normal individuals. Additional research findings indicate that changes in LR11 may occur early in the disease process and that LR11 may play an important role in regulating the levels of amyloid-beta peptide, which is believed to play a central role in causing Alzheimer's disease. The evidence emerging from our study of LR11 suggests that it represents an important new therapeutic target for Alzheimer’s disease. We hypothesize that LR11 will be a good target for discovery of candidate compounds that can modulate amyloid-beta through interactions with LR11.

We propose to initiate a search for LR11-interacting compounds, which will produce leads in the development of new therapeutic agents. To accomplish this, we will combine our scientific expertise with the capabilities of the NIH-funded Emory Molecular Libraries Screening Center to develop tools to identify and evaluate candidate compounds that interact with LR11. In Aim 1, we will develop high throughput assays to screen a library of small compounds for those capable of interacting with specific portions of LR11. In Aim 2, we will develop cell-based assays to identify relevant LR11-interacting compounds.  In Aim 3, we will test the ability of LR11-interacting molecules to modulate amyloid-beta levels to refine the list to the most promising compounds and expedite future efforts to develop LR11-based therapeutic agents.

The long-term goal of these studies is to exploit our understanding of LR11 to develop new treatments that can slow or prevent the development of Alzheimer's disease.

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Bruce Lamb, Ph.D.
The Cleveland Clinic Foundation
Cleveland, OH
Project: Microglia, CX3CR1 and Alzheimer's Disease Pathogenesis
$400,000

Alzheimer's disease (AD), the most common dementing disorder of late life, is now the fourth major cause of death in the developed world.  A definitive diagnosis of AD requires examination of brain tissue for the presence of distinctive AD pathological alterations including filamentous inclusions (termed neurofibrillary tangles) and extracellular deposits of the ß-amyloid peptide (Aß, termed senile plaques).  In addition, while there is considerable data that suggests there is a marked activation of the immune system within the AD brain, there is little evidence that altered inflammation plays a direct role in the observed neurodegeneration.  It was recently demonstrated that alterations in inflammation within the brain, through genetically engineered  mutations in the chemokine receptor, CX3CR1, can directly result in increased neuronal cell loss in three different mouse models of neurodegeneration.  The focus of the current proposal is to determine the role of CX3CR1 plays in activation of the immune system, neuronal cell death and Aß deposition in two different mouse models of AD as well as to gain insight into the mechanisms involved.  The long-term goal of this project is to gain insight into the role inflammation play in AD and thus provide potential new avenues of therapeutic intervention.

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Anton Roebroek, Ph.D.
KU Leuven
Leuven, Belgium
Project: The Dual Role of LRP1 in Generation and Clearance of AB
$240,000

Alzheimer’s Disease (AD) is caused by the increasing appearance of abnormal structures in the brain of AD patients, named senile plaques. These plaques consist mainly out of aggregates of a small peptide, Ab, which arises from cleavage from a larger protein. Ab is also present in the brain of healthy people, but for some reason the amount of Ab in the brain goes strongly up and it is deposited in the senile plaques in AD patients. It seems that the normal balance between production and breakdown of Ab, in scientific terms Ab metabolism, is disturbed. Recent scientific investigations revealed that a receptor, LRP1, present on the outside of the cells in the brain might be involved at the same time in both production and breakdown of Ab. Lately, the applicant of the research project generated mice and cells with a modified LRP1, which can be used to clarify the double role of LRP1 in Ab metabolism. It is the strong believe of the applicant that this scientific research will result in essential additional knowledge on the role of LRP1 in the balance of production and breakdown of Ab. This could eventually contribute to new approaches for a treatment for AD.

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Michael R. Sierks, Ph.D.
Arizona State University
Tempe, AZ
Project: Morphology Specific Antibodies to beta-amyloid
$150,000

Alzheimer's Disease (AD) is characterized by the presence of neuritic plaques and neurofibrillary tangles. The role of Aß in AD is still controversial, an issue that has been complicated greatly by the multiple lengths and morphologies of Aß.  A wealth of literature suggests the various lengths and morphologies have different effects on neuron viability and memory.  In order to reliably assess the roles of Aß and anti-Aß vaccination strategies in AD, highly specific and very well-defined reagents such as single chain antibody binding variable domains (scFvs) that target individual Aß forms and morphologies are needed.  Using a novel technology combining antibody diversity and microscopic imaging techniques, scFvs against specific Aß morphologies can be isolated.  The isolated scFvs can be affinity matured to have extremely high specificity for the target ligands.  The hypothesis of this proposal is that highly specific and well defined scFvs to specific oligomeric morphologies of Aß can be isolated by useful therapeutics for treating AD.  The long term goal is to use the pool of morphology specific scFvs to probe the roles of the various Aß morphologies in AD and to test the value of these scFvs as potential diagnostic and therapeutic agents. 

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Paul M. Salvaterra, Ph.D.
Beckman Research Inst. of the City of Hope
Duarte, CA
Project: AB and Neurodegeneration
$400,000

Chronic and progressive dying neurons are the major feature of Alzheimer's disease that can best explain the clinical symptoms.  More than 20 years of intensive research has implicated two toxic peptides generated from a larger normal protein as playing an important role in the disease process.  It is not known, however, if these peptides are a cause of the disease or just an effect of some other problem in brain cells.  Our work is designed to investigate the role of these peptides, both alone and in combination, as direct causes of chronic brain cell death.  We will accomplish this by using a simplified genetically controlled model organism.  We also believe that our preliminary observations indicate a new cellular pathway that may be responsible for Alzheimer's related cell death.  We will thus try and prove this hypothesis using genetic and drug based experimental strategies in our model system.  If this new pathway is indeed responsible for cell death in Alzheimer's disease our work could lead to the identification of new treatment and prevention strategies.

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Jashvant D. Unadkat, Ph.D.
University of Washington
Seattle, WA
Project: P-Glycoprotein and Alzheimer's Disease
$398,176

P-glycoprotein (P-gp) is an export pump that is highly active at the barrier that separates brain tissue from blood (called the blood brain barrier or BBB). P-gp can transport amyloid-β (Aβ), a compound that accumulates in the brain of people with Alzheimer’s disease (AD). We believe that reduced activity of P-gp at the BBB results in accumulation of Aβ in the brain of patients with AD. In the proposed study we will compare P-gp activity at the BBB in patients with AD and in age-matched volunteers without AD, using a method called positron emission tomography (PET). The proposed studies are particularly relevant to Alzheimer’s disease for a number of reasons. For the first time, our studies will test the idea that P-gp activity at the BBB is compromised in AD. If it is, we will test in future studies if compounds that are known to increase P-gp activity in the human intestine, such as rifampin or St. John’s Wort, can increase P-gp activity at the BBB. If both or one of these compounds do increase P-gp activity at the BBB, they can be tested for their effectiveness in stopping progression of Alzheimer’s disease.

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CENTENNIAL AWARDS
FUNDING PERIOD: APRIL 1, 2007 – MARCH 31, 2009

Bradley T. Hyman, M.D., Ph.D.
Massachusetts General Hospital
Charlestown, MA
Project: Role of apoE in Neurodegeneration
$1,000,000

Dr. Hyman and his collaborators are studying different forms of a protein that is associated with Alzheimer’s disease. One form of the protein dramatically increases the risk of developing the disease, while another form protects against it.  By studying the different forms of this protein, Dr. Hyman’s team hopes to identify features of its molecules that might be used as targets of future medicines.  The close collaboration among Dr. Hyman and his co-investigators at Stanford University and Washington University is an excellent example of the scientific synergy key to new discoveries.

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Donald Weaver, M.D., Ph.D.
Dalhousie University
Halifax, Nova Scotia, Canada
Project: Development, Optimization and Comprehensive Biological Evaluation of Compounds Shown to Inhibit Aggregation of AB, Tau and  α-synuclein
$1,000,000

Currently, there are no drugs that stop or reverse the progression of Alzheimer’s disease. The drugs that are available treat the symptoms but not the cause of this disease. Dr. Weaver and his collaborators are investigating small molecules that can disrupt the protein buildup that damages the brains of Alzheimer’s patients.  By disrupting these proteins, they hope to stop their aggregation and in turn, prevent brain cell death.  The ultimate goal of Dr. Weaver’s research is to discover new and useful drug treatments for Alzheimer’s disease.

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FUNDING PERIOD: APRIL 1, 2007 – MARCH 31, 2009

Barbara Calabrese, Ph.D.
The Scripps Research Institute
LaJolla, CA
Project: Rapid Effects of Soluble AB on Synaptic Structure
$100,000

Barbara Calabrese, Ph.D. of the Scripps Research Institute, proposes studying in rat hippocampal neurons how the amyloid beta (Aβ) peptide, a key trigger of Alzheimer’s disease pathology, induces early changes at synapses - the specialized connections between neurons that are essential for learning and memory. Research suggests that in early stages of Alzheimer’s disease cognitive disruption reflects significant loss in the numbers and/or function of synapses. The soluble forms of Aβ have been found to induce memory impairments in animal models. However, it is still not understood how soluble Aβ alters the structure and function of synapses, especially in early stages of the disease. Dr. Calabrese hypothesizes that exposure of neurons to low levels of soluble Aβ results in definable changes in the numbers, shape, and stability of synapses. Pharmacological and gene transfer manipulations of synaptic Aβ targets will be used in combination with high resolution live cell imaging to test this hypothesis. These studies may provide novel insights to treat the earliest stages of AD, during which intervention is likely to be most effective. The hope is that by understanding soluble. Aβ-induced synaptic destabilization we can improve upon therapies that prevent synapse loss or restore synapse function.

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Jungsu Kim, Ph.D.
Washington University
St. Louis, MO
Project: New Method to Assess apoE and Abeta Metabolism
$100,000

Alzheimer's disease (AD) is the most common cause of dementia. Mutations in specific genes (APP, PSEN1, and PSEN2) cause rare forms of familial AD.  While these mutations have been very useful, >99% of AD (late-onset) does not appear to be due to these mutations. Defects in clearance of Abeta from brain could underline many cases of sporadic AD. There is only one proven genetic risk factor for both early and late-onset AD, one's APOE genotype. ApoE4 is associated with an increased risk and apoE2 is associated with a decreased risk for AD. A large amount of evidence suggests that apoE is likely to influence risk for AD by acting as a molecular chaperone for Abeta and influencing Abeta fibrillogenesis and clearance. The hypothesis being tested is that different human apoE isoforms and lipidation states of apoE alters apoE and Abeta clearance in the CNS. We further hypothesize that the perturbation in regulation of apoE metabolism will then influence Abeta metabolism and will alter both the time course and amount of Abeta depostion in brain. Results from these experiments may provide insights into normal apoE metabolism in the CNS as well as clarify why APOE isofom genotype influences risk for AD.

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Thomas L. Kukar, Ph.D.
Mayo Clinic Jacksonville
Jacksonville, FL
Project: Anti-Amyloid Effects of Truncated Abeta Peptides
100,000 over 2 years

Understanding the factors responsible for Alzheimer's disease (AD) is critical for the development of therapeutic strategies for this debilitating neurodegenerative disease. Intriguingly, epidemiological studies suggest that chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) protects from the development of AD. We have shown that certain NSAIDs selectively lower production of the 42 amino acid form of the amyloid beta peptide (Aβ42). Based on evidence that the accumulation of Aβ42 in the brain leads to AD, it has been hypothesized that this unique property may contribute to the protective effect of some NSAIDs. These compounds not only selective lower Aβ42 but also increase the levels of shorter Aβ peptides such as Aβ34, 37, and 38. Studies have shown that Aβ42 is much more prone to form amyloid than shorter fragments. Based on these results, we hypothesized that the elevations in shorter Aβ peptides induced by some NSAIDs further enhances their anti-amyloidogenic effect. Experiments will directly investigate the effects of smaller Aβ peptides on Aβ aggregation and fibril formation in vitro. Most importantly a unique technology to rapidly and specifically increase the levels of these short Aβ peptides in the brains of mice will be used to see if they protect from Aβ plaque formation.

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Kun Ping Lu, Ph.D., M.D.
Beth Israel Deaconess Medical Center
Boston, MA
Role of the Pin1 and Presenilin-1 Interaction In Vivo
$150,000

Alzheimer’s disease is the most common form of dementia and its pathological hallmarks are tangles made of a protein called tau and plaques comprising small peptides called Aβ peptides generated from its precursor protein called APP.  We have recently identified a new enzyme, Pin1 that regulates the structure and function of certain proteins such as tau and APP.  Moreover, Pin1 is pivotal for protecting against tangle formation, Aβ accumulation and neurodegeneration.  Notably, Pin1 is inhibited by various mechanisms in Alzheimer’s patients. These results suggest that Pin1 deregulation is an important factor in Alzheimer’s development, although its molecular targets and mechanisms are not fully elucidated.  Our hypothesis in this proposal is that Pin1 might also regulate the function of presenilin 1, an essential component of the enzyme responsible for Aβ production, and that this regulation might be disrupted by some Alzheimer’s mutations in presenilin 1.  To test this hypothesis, we will determine whether Pin1 regulates presenilin 1 structure in a test tube, whether manipulating Pin1 function affects PS1 activity in cell culture and animal models, and whether this Pin1-dependent regulation is disrupted by presenilin 1 mutations. These studies would provide new insight into Alzheimer’s development and might have important therapeutic implication.

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Enrico Malito, Ph.D.
The University of Chicago
Chicago, IL
Project: Insulin Degrading Enzyme and Control of Amyloid B levels
$100,000

Accumulation in the brain of a specific protein in the form of insoluble plaques is a critical event in Alzheimer’s disease pathology. The imbalance between production of this protein and its degradation is the critical event responsible for its accumulation. Investigating the defects of enzymes that degrade this protein is an essential effort for a better understanding and for the treatment of Alzheimer’s disease. Insulin degrading enzyme (IDE) is among the enzymes responsible for the degradation of the peptide that then accumulates in the brain in an insoluble form, and IDE deficiency is correlated with a significant net increase in accumulation of insoluble plaques in the brain. Consequently, IDE represents a new target for the development of drugs for the treatment of Alzheimer’s disease. Our structural studies of IDE allowed us to understand the basic features of this important enzyme. Starting from these observations we can now modify IDE in order to obtain a decrease of accumulated insoluble plaques. We propose to further investigate the molecular mechanism by which IDE exerts its function, with the final aim of finding a hyperactive form of IDE able to more effectively degrade the protein responsible for the onset of Alzheimer’s disease. These studies will provide valuable insights into the design of IDE-based therapeutics against Alzheimer’s disease.

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Michael P. McDonald, Ph.D.
Vanderbilt University Medical Center
Nashville, TN
Project: Targeting GD3S to Reduce Plaque and Improve Memory
$150,000

Alzheimer's disease is characterized by the accumulation of plaques in the brain, widespread neurodegeneration, and cognitive decline.  We have shown that by eliminating an enzyme called GD3S we are able to reduce plaque formation, block cell death, and prevent memory deficits in a mouse model of Alzheimer's disease.  This suggests that blocking GD3S may useful in treating Alzheimer's disease.  However, in these mice the mutation that blocked GD3S was made before birth, and the enzyme plays an important role in many processes that are important for normal brain development.  Thus it's important to test the therapy in adult mice, after they've gone through normal brain development.  This is analogous to what a genetic therapy will be like for Alzheimer's patients, i.e., the treatment begins in adulthood.  A new technique, called siRNA, allows us to simply and efficiently suppress the expression of the GD3S gene in live mice.  We propose to use siRNA to suppress GD3S expression in a mouse model of Alzheimer's disease.  Consistent with our previous results, we expect that this novel genetic therapy will significantly reduce plaque formation and completely block the memory deficits normally exhibited by these mice.

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Robert Alan Nichols, Ph.D.
Drexel University College of Medicine
Philadelphia, PA
Project: Beta Amyloid Regulation of Presynaptic Nicotinic Current
$145,049

Alzheimer’s disease is a relentlessly progressive degenerative disorder for which there is currently only limited treatment. Early in the disease memory deficits occur, followed later by disruption of many facets of thinking and even language. Eventually, patients can no longer recognize anyone, including family members, and cannot care for themselves. Thus, it would appear that the disease results from a number of brain functions going awry at different times over its course, involving multiple pathological entities. One entity in particular is a small, sticky peptide known as beta amyloid, which accumulates in the brains of aging adults and eventually forms large, dense deposits (otherwise known as plaques). What free beta amyloid is doing in the brain in the first place is completely unknown, but its accumulation into deposits is one of the first signs in the course of the disease. It is largely produced from nerve endings where they make contacts with and signal to other nerve cells. Normally, this signaling from nerve endings to nerve cells is the primary way that communication occurs within the brain and we are hypothesizing that the free beta amyloid, accumulated at pathological levels, disrupts this basic communication. At present, one particular hope is to identify individuals at risk for heavy accumulation of the beta amyloid, using brain imaging techniques, and then to remove the free beta amyloid. In animal models where beta amyloid is artificially expressed in the brain, early removal of accumulating free beta amyloid prevents the memory deficits that result from the presence of beta amyloid; however, removal from the brain has required vaccination against beta amyloid and to date that has been problematic, though still remains an important and hopeful avenue. Another approach is to disrupt beta amyloid’s pathological actions, potentially preserving its normal function. In order to address this idea, it is essential to understand beta amyloid’s normal function. This project is aimed at understanding its physiological role. Using a defined nerve cell system in culture, we can express target protein receptors for beta amyloid and then examine the functional consequence via several techniques. We can also introduce mutations into the receptors or make deletions to define precisely the molecular components targeted by beta amyloid. Finally, we can explore derivatives of beta amyloid as potential means to disrupt beta amyloid’s action

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Eric Norstrom, Ph.D.
University of Chicago
Chicago, IL
Project: In Vivo Identification of APP-Interacting Proteins
$99,431

Alzheimer's disease is an incurable neurodegenerative disease characterized by the accumulation of amyloid plaques - deposits of protein in the brain whose main constituent is the Ab peptide, which is itself derived from the metabolism of a larger protein called APP.  Reducing the Ab load in the brain is a major goal of Alzheimer’s research, and to accomplish this, many strategies aim to inhibit the metabolism of APP.  Thus, understanding which proteins APP interacts with is important because 1) this aids in the design of small molecule drugs, and 2) if APP metabolism is to be inhibited, an understanding of its natural function is critical.  Although many studies have investigated the metabolism of APP in cultured cells, confirmation of these results in animal studies has not yet been achieved.  Thus, we aim to generate a transgenic mouse that expresses APP containing a peptide tag.  By using this tagged APP as”bait" in the living animal, we can subsequently purify it and those proteins with which it is bound.  Analyzing the purified material and comparing it to a protein database will confirm binding partners identified by cell culture studies and identify new binding partners with new specific targets for drug therapy.

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Troy Townsend Rohn, Ph.D.
Boise State University
Boise, ID
Project: Caspase-cleavage of Tau in Alzheimer's Disease
$131,140

Introduction: Recent studies have suggested that proteolytic cleavage of tau by caspases may be an important event linking beta-amyloid with neurofibrillary tangles in Alzheimer's disease (AD).  These studies suggest that caspase activation may play an important role in driving AD disease pathology and simply do not represent end-stage events associated with this disease.

Hypothesis: Direct, functional evidence for the involvement of caspases in driving AD pathology is currently lacking.  The current proposal will test directly the role of caspases in AD by blocking caspase activation in an AD transgenic mouse model and examining whether such inhibition prevents the pathology associated with these animals.

• Aim 1: Crossing 3xTg-AD mice with Bcl-2 OE Tg mice will prevent the caspase cleavage of tau.
• Aim 2: Mice resulting from crossing 3xTg-AD mice with Bcl-2 OE Tg mice will exhibit fewer tangle alterations.

Long-term goals: Direct evidence indicating a causative role for caspases in AD may stimulate the development of caspase inhibitors for their potential in treating this disease. In addition, results from this pilot study will provide the necessary feasibility and data for the development of a more comprehensive proposal examining all pathological aspects of these novel AD mice.

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Alejandro F. Schinder, Ph.D.
Instituto Leloir
Buenos Aires, Argentina
Project: Impaired Synaptogenesis as an Early Event in AD
$150,000

The complexity of the human brain can be easily conceived if we think about 1011 neurons connected by 1015 synapses. Those connections are highly dynamic. Synapses are continuously formed and eliminated in a manner that depends on the activity of brain circuits. Activity-dependent remodeling of neuronal networks is essential for higher brain functions.  Its impairment has been associated with mental retardation syndromes and may also play an important role in neurodegenerative disorders.

Alzheimer’s disease (AD) has been associated with amyloid plaques, neurofibrillary tangles and neuronal death, which were thought to be the cause of cognitive decline. Recently, animal models of AD have taught us that amyloid-beta (Ab) peptides can impair synaptic transmission in the absence of plaques or tangles, but the specific effects and sites of action of Ab remain unknown. Does Ab impair neuronal communication and/or synapse formation and elimination?

Our goal is to study the effects of Ab on synapse formation and function in the hippocampus of adult mice in vivo. We are using a novel strategy based on the fact that new neurons are continuously generated in the adult hippocampus and go through an intense period of synapse formation. We manipulate the genetic information of those adult-born neurons to increase their levels of Ab and analyze their development and maturation using electrophysiology and microscopy.  Addressing these questions will contribute to the better understanding of the early changes underlying cognitive impairment in AD and other neurodegenerative diseases.

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Vijay Sharma, Ph.D.
Washington University
St. Louis, MO
Project: Imaging Pgp-Mediated Transport in Alzheimer's Disease
$150,000

Alzheimer’s Disease (AD) patients demonstrate loss of neurons in regions of the brain responsible for learning and memory (hippocampus) and the presence of distinct protein aggregates, commonly known as amyloid plaques. Emerging new models for occurrence of the disease indicate that pathways in disease progression are likely mediated by transporters prevalent in the brain. Among these transporters, P-glycoprotein (Pgp) known to block penetration of numerous drugs or cytotoxins into brain may likely be involved in buildup of amyloid plaques within the brains of AD patients. We hypothesize that natural function involving Pgp mediating efflux of amyloid plaques out of the CNS may likely be compromised in diseased patients compared with the normal ones and the process initiates prior to appearance of symptoms for the disease. Thus, ultrasensitive-diagnostic agents capable of evaluating that novel risk factor in terms of individual variations in Pgp transport would likely assist in patient stratification and guide therapeutic choices. Herein, we propose to evaluate the potential of lead Pgp-targeted agent to act as noninvasive probe to detect those defects in brains of mouse models via PET imaging. Additionally, our strategy is amenable to kit formulation with potential for widespread deployment of a test for managing AD.

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Jane M. Sullivan, Ph.D.
University of Washington School of Medicine
Seattle, WA
Project: Role of Presenilin in Synaptic Transmission
$149,950

The earliest manifestations of Alzheimer’s disease are deficiencies in cognitive function, specifically problems with memory.  These earliest symptoms of the disease are most likely caused by abnormal synaptic transmission.  As the disease progresses and dementia becomes more severe, neurons will die, but the earlier changes, those that are hypothesized to be caused by more subtle effects on the way that synapses operate before neurons die, have not been well studied.  This is because these changes are occurring in patients that are still alive, and they cannot be investigated with existing techniques.  In order to understand what changes are taking place at synapses before neurons die, a model system must be used.  This model system must replicate the changes that are thought to be taking place in the brains of patients with early Alzheimer’s disease.  This grant proposal describes a series of pilot studies that will develop such a model system using brain cells from mice.  Identifying specific changes in synaptic function produced by mutant presenilin, a protein strongly implicated in the inherited form of Alzheimer’s disease, will provide molecular targets for novel therapies to improve cognitive function and delay further neurodegeneration in patients with early Alzheimer’s disease

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Nan Wang, Ph.D.
Columbia University
New York, NY
Project: Role of ABCG1 and ABCG4 in Abeta Generation in Brain
$150,000

Recent studies suggest that cholesterol balance in the brain may affect development of Alzheimer's disease. Increased cholesterol is associated with increased risk of Alzheimer's disease while decreased cholesterol appears to reduce it. Recently, we have identified two membrane transporters that are involved in cholesterol transport in cells. These transporters are highly expressed in the brain. Now we have evidence suggesting that increased activity of these transporters enhances generation of Abeta, the molecule aberrant elevation and deposition of which is considered to be the major cause of Alzheimer's disease. Importantly, deficiency of the transporters appears to decrease Abeta production in the brain. ABCG4, one of two transporters, is mainly expressed in the brain and studies with mouse models of ABCG4 deficiency suggest that ABCG4 deficiency does not affect animal development and no impaired physiological functions have been identified in these mice. Therefore, ABCG4 may represent a novel drug target for treatment of Alzheimer's disease.

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Gregory J. Zipfel, M.D.
Washington University
St. Louis, MO
Project: Immunotherapy for Cerebral Amyloid Angiopathy
$150,000

Cerebral amyloid angiopathy (CAA) involves deposition of a protein called amyloid-β (Aβ) into brain blood vessels.  It is almost universally found in patients with Alzheimer's Disease (AD).  CAA can lead to stroke and dementia, likely by causing blood vessel dysfunction and lowering blood flow to the brain.  We hypothesize that treating CAA with an anti-Aβ antibody (an antibody directed against the Aβ protein) will improve blood vessel function and blood flow to the brain.  If true, this would prove that Aβ deposits are the reason why CAA leads to blood vessel dysfunction and reduced brain blood flow.  It would also mean that these antibodies may represent a new treatment for CAA and AD. The specific aims of this project are as follows:

• To determine whether “preventive” anti-Aβ antibodies prevent or substantially reduce blood vessel dysfunction and reductions in brain blood flow caused by CAA.
• To determine whether “therapeutic” anti-Aβ therapy decreases the severity of CAA, and if so, will such therapy reduce blood vessel dysfunction and reductions in brain blood flow caused by CAA.

The long-term goals of this research are to determine how CAA causes stroke and dementia and to discover effective treatments for CAA and AD. 

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FUNDING PERIOD: APRIL 1, 2007 – MARCH 31, 2008

Gary E. Landreth, Ph.D.
Case Western Reserve University
Cleveland, OH
Project: PPARgamma as a Therapeutic Target in Alzheimer's Disease
$400,000

Alzheimer's disease is characterized by the deposition of b amyloid (Ab) within the brain and there is good biological evidence that that therapies that reduce Ab deposition and enhance its clearance from the brain will be beneficial. This application investigates the ability of a new class of drug that activate a transcription factor, termed peroxisome proliferator-activated receptor gamma (PPARg). Importantly, drugs that activate PPARg are already in clinical use and have been shown to result in enhanced learning and memory in AD patients. The central problem is that we do not know how the drugs work to elicit the behavioral improvement and this application is focused on establishing the mechanism of drug action. We show that treatment of animal models of AD with PPARg stimulators lead to lower levels of plaque deposition. Moreover, we show that this is likely due to the ability of the drug to stimulate Ab degradation, reducing brain levels of amyloid.  We think that PPARg activation works to stimulate the production of factors that allow cellular proteases to cut the amyloid peptides into pieces too small to be deposited. This research provides an explanation for why the present generation of drug that PPARg are beneficial and will inform the development of the next generation of drugs that target this receptor.

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FUNDING PERIOD: APRIL 1, 2006 – MARCH 31, 2008
GRANTS IN EXTENSION

William Klein, BSc, Ph.D.
Northwestern University
Evanston, IL
Project: Synaptic Attack by ADDLs: A Mechanism for Memory Loss
$300,000

Memory formation begins at synapses, and it appears likely that destruction of memory formation is due to synapse failure.  This proposal investigates the molecular cause of this failure.  It focuses on a new neurotoxin, called ADDLs, that was discovered and characterized by this group over the past several years. Dr. Klein previously showed that ADDLs accumulate in AD brains, so it is important to know how they act. His most recent work established that ADDLs attack synapses, a fact that gives very strong support to the idea that AD is a synapse failure. He now wants to discover what type of structural and molecular damage ADDLs cause to synapses after they bind. The preliminary evidence strongly indicates that ADDLs rapidly change the geometry of synapses into a shape often seen in mental retardation. Evidence also indicates that the neurotransmitter receptor molecules required for information storage are removed from synapses. Dr. Klein plans to verify and extend these preliminary results by new experiments using state-of-the-art experimental models to study synapse biology and the impact of ADDLs. The experiments in the long run will determine how the ADDLs attack on synapses could result in the catastrophic memory failure suffered in early Alzheimer's disease.

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Ricardo Miledi, M.D.
University of California, Irvine
Irvine, CA
Project: Glutamate and GABA Receptors in the AD Brain
$250,918

All the functions of the brain, sensations, memory etc. depend on the transmission of signals across the myriads of synapses that interconnect the billions of neurons of the brain. In the synaptic process neurotransmitter substances, released from one neuron, act on receptor proteins embedded in the membrane of neighboring neurons.  Alzheimer’s is a synaptic disease accompanied by neuronal loss. Nevertheless, very little is known about the neurotransmitter receptors of the Alzheimer’s brain. Dr. Miledi will study the structure and function of these receptors, using a method that he developed to micro-transplant receptors from the human brain to frog oocytes. Membranes, isolated from brains frozen post-mortem, are injected into oocytes. These membranes, carrying the original receptors from the Alzheimer’s brain, and still embedded in their original membrane, fuse with the oocyte membrane. Remarkably, the receptors are still functional and can be subjected to detailed structural and functional analyses. Receptors from Alzheimer’s brains will be compared with those from non-Alzheimer’s brains, focusing on the receptors to GABA and Glutamate: the main inhibitory and excitatory neurotransmitters in the human brain. The effects of Amyloid beta will also be studied. All this will help determine the cause of Alzheimer’s disease and help to develop new treatments.

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Sheue-Houy Tyan , Ph.D.
University of California, San Diego
La Jolla, CA
Project: WLDs Protein and Aβ-induced Synaptic Dysfunction
$100,000

Dysfunction at synapses in the brain may underlie many of the deficits present in Alzheimer's disease.  Factors that influence the health of synapses are thus vital areas of research focus.  The proposed research will be focused on a protein which has shown promise as a protective factor in other models of neurodegeneration, but which has not been examined in Alzheimer's Disease models.  The objective is to elucidate whether this protein (known as WLDs) is functionally useful as a protective factor in an Alzheimer's disease model.  Given the protective effect that the WLDs protein has had in other models, the working hypothesis is that the protein will attenuate synaptic dysfunction Alzheimer's disease models.  Dr. Schroeder will first determine the effects of the WLDs protein on synaptic transmission and learning deficits known to exist in Alzheimer's disease models.  In the second part of this proposal, he will examine the effect of the WLDs protein on known structural and synaptic protein expression changes in Alzheimer's disease models.  In the long-term, he hopes that the potential protective effects of the WLDs protein will serve to clarify the mechanism of synaptic dysfunction in Alzheimer's disease as well as lead to therapeutic options for treating synaptic dysfunction.

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Guilian Xu, Ph.D.
University of Florida
Gainesville, FL
Project: The Role of LRP in Amyloid Deposition in APP/PS1 Mice
$100,000

There is substantial evidence to suggest that the deposition of beta-amyloid (small fragments of a protein called the amyloid precursor protein) triggers a cascade of events that ultimately causes the symptoms of Alzheimer’s disease. The life-long accumulation of amyloid peptide in the brain is determined by the rate of its generation versus clearance. A large number of studies have provided evidence that a protein called the low-density lipoprotein receptor-related protein (LRP) may play a pivotal role in regulating the production or clearance of amyloid peptides. This study will use transgenic mouse models to study the roles of LRP in modulating amyloid pathology. Dr. Xu will use a genetic system, called Cre/lox, to eliminate LRP expression in specific types of cells in the forebrains of mice and then assess how this manipulation has altered amyloid deposition. It has been suggested that the binding of LRP to proteins involved in amyloid peptide production and clearance is the mechanism of action. If so, then it may be possible to identify drugs that modulate LRP binding to these proteins and thus influence its role in the formation of amyloid pathology.

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Riqiang Yan, Ph.D.
Cleveland Clinic Foundation
Cleveland, OH
Project: The Role of NgR2 in Pathogenesis of Alzheimer’s Disease
$300,000

The etiology of Alzheimer’s disease (AD) is still unclear, and many factors appear to affect AD pathogenesis.   Hence, it is imperative that every approach be considered in the effort to stem this disease.   Neuritic plaques (or called senile plaques) and neurofibrillary tangles are two well known pathological features in patients’ brains.  Neuritic plaques that are predominantly seen in brains of Alzheimer's patients are due to the presence of amyloid depositions surrounded by dystrophic neuritis, reactive astrocytes and activated microglia.  In this proposal, Dr. Yan proposes to study a molecule called Nogo Receptor 2 (NgR2) and its role in the formation of neuritic plaques.  The information gained from this study will be used for developing therapeutic agents to prevent and/or treat Alzheimer's patients.

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