GRANTS AWARDED IN FISCAL YEAR 2001 - 2002
FUNDING PERIOD: APRIL 1, 2002 - MARCH 31, 2003

Thomas Fritsch, Ph.D.
Case Western Reserve University
Cleveland, Ohio
Project: Cognitive Performance in Adolescence, APOE and AD

Researchers have suggested that there is a relationship between cognitive performance early in life and the development of Alzheimer's disease (AD) later in life, but few studies have directly tested this relationship. Also, there have been no investigations to examine whether the genetic factor, apolipoprotein E4 (apoE4), modifies the possible relationship. There are two main goals of this investigation by Dr. Fritsch. First, he will use information from archived school records and yearbooks to study relationships between cognitive performance, grades and participation in school activities in adolescence and the development of AD in adulthood. Second, he will collect genetic material in order to study the possible modifying role of apoE. These studies should lead to understanding the early roots of AD and help evaluate whether AD is a developmental process or a long-term chronic disorder.

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GRANTS AWARDED IN FISCAL YEAR 2000 - 2001
FUNDING PERIOD: APRIL 1, 2001 - MARCH 31, 2003

Steven L. Gonias, M.D., Ph.D.
The University of Virginia
Charlottesville, VA
Project: Amyloid beta Peptide/a₂-Macroglobulin Interactions

Recent studies suggest that if the clearance of amyloid beta from the extracellular spaces of the brain is enhanced (through a vaccine), the progression of Alzheimer's disease (AD) may be stopped or reversed. A protein called a₂-Macroglobulin (a₂M) is a naturally occurring protein present in human blood and in the spaces between cells. This large protein contains a number of distinct binding sites for other, smaller proteins, including a binding site for growth factors near the center of the a₂M structure. By binding growth factors, a₂M may harm cells and increase cell death when excessive amyloid beta is present. Dr. Gonias has evidence suggesting that there is a distinct region in the structure of a₂M that binds Alpha-beta. His goal is to further define the Alpha-beta binding site in a₂M, with the long-range goal of identifying a₂M miniproteins to mediate Alpha-beta catabolism and counteract the toxic activity of Alpha-beta. This study is a continuation of a project launched in 1999.

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Joanna L. Jankowsky, Ph.D.
The Johns Hopkins University
Baltimore, MD
Project: Environmental Enrichment of APP Transgenic Mice SRC

Several studies have suggested that environmental factors can influence the development of AD. However, detailed historical information is not available for most of the population, making it difficult to discern the relationship between specific environmental factors and AD. Dr. Jankowski is using a simple animal model that has some of the pathological features of AD to examine the extent to which environmental surroundings can influence the evolution of the disease. In this case mice that carry either the mutant APP gene alone or mutant APP and PS1 together are being examined in a controlled setting, to try to determine the extent to which environmental stimuli can influence the evolution of brain pathology. Previous studies by several laboratories have shown that "enriching" the mouse habitat with toys and playmates can lead to changes in the brains of normal rodents that are indicative of increased neurologic activity. Dr. Jankowsky hypothesizes that housing experimental mice with AD in enriched environments will lead to increased neurologic activity and will allow her to examine the role of brain activity in the evolution of AD. In addition, further experiments are planned to examine the role of an enriched environment on amyloid beta production, plaque formation, synapse formation, and learning and memory. These experiments can provide insight into the potential for human environmental factors such as social interaction, mental activity, and exercise to influence the onset and outcome of AD. Dr. Jankowski is one of the first ADR researchers to receive a Pilot Program Award, an award for researchers whose work shows special promise.

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Mary F. Knauer, Ph.D.
University of California
Irvine, CA
Project: LRP Internalizes Fxla Complexed to APP, PN1: Effect on Amyloid beta

LDL Receptor Related Protein (LRP) is a receptor on the cell surface that takes in the amyloid precursor protein (APP) so it can be broken down. Determining the role of LRP in this process is key to understanding how amyloid beta deposits are formed. Dr. Knauer has proposed that the metabolic process causing protein deposits to form occurs inside of cells, but is triggered by molecules outside of the cell called proteases. These proteases modify the amyloid protein that causes AD and also cause it to rapidly enter cells where it can set off a process that results in damaging deposits accumulating outside of cells. To test this hypothesis, Dr. Knauer is working to identify an inhibitor of these proteases that her laboratory recently discovered and determine to what extent it can contribute to, or reduce, the formation of deposits or "senile plaques." She will test the effectiveness of protease inhibitors on limiting the metabolic pathway that gives rise to deposits in order to search for the exact point of entry in this pathway. If this process can be elucidated, drugs may be developed to block the entry of amyloid protein into cells and inhibit the metabolic process that causes deposits to form.

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Dora M. Kovacs, Ph.D.
Massachusetts General Hospital
Boston, MA
Project: Effect of Cholesteryl-esters on PS1 and on Amyloid beta Ratios

High cholesterol levels have long been associated with atherosclerosis. Recent studies show that high cholesterol levels are also linked to the increased appearance of the senile plaques and neurofibrillary tangles characteristic of Alzheimer's disease. One possible explanation is that cholesterol inside the cellular membranes may somehow regulate the enzymes (secretases) that generate the amyloid beta peptide found in senile plaques. The cholesterol content in cellular membranes is maintained by the action of an enzyme called ACAT, which moves excess cholesterol from the membrane into the cell's cytoplasm in the form of insoluble lipid droplets. In earlier work, Dr. Kovacs found that decreased lipid droplets led to a decrease in the production of amyloid beta, whereas abnormally high levels of lipid droplets (even with normal membrane cholesterol levels) led to an increase in amyloid beta production. Dr. Kovacs suggests that the mechanism for this may involve the stabilization of presenilins, Alzheimer-associated proteins responsible for the production of amyloid beta. Dr. Kovacs is now exploring the mechanism by which presenilin activity and the production of amyloid beta may be regulated by lipid droplets. Understanding this mechanism could provide the basis for developing ACAT inhibitors that would lower amyloid beta generation and slow or prevent AD-related neuropathology.

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H.P.H. Kremer, M.D., Ph.D.
University Center
Nijmegen, The Netherlands
Project: Does Indomethacin Retard Disease Progression in AD?

Many studies have suggested that inflammatory mechanisms could play an important role in the deterioration of the brain in AD. Recent epidemiological studies in patients with arthritis found that the incidence of AD was lower than expected, possibly due to the long-term use of anti-inflammatory drugs. Small clinical studies have provided evidence to support this idea. Dr. Kremer is conducting tests to determine whether the anti-inflammatory drug indomethacin will slow the progression of mild or moderate AD. He is evaluating the effect of indomethacin on the decline of memory, behavior and functional status of patients with mild to moderate AD. Dr. Kremer will also evaluate the safety of long-term indomethacin treatment in patients with AD. If indomethacin is successful in treating early to moderate Alzheimer's disease, it would provide a cheap and effective medication for treating patients even in some of the poorest countries in the world.

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Ki-Young Lee, Ph.D.
University of Calgary
Calgary, Alberta, Canada
Project: CDK5 Activation and Tau Phosphorylation

Cyclin-dependent kinase 5 (Cdk5) is a small enzyme that has been associated mostly with the activity of neurons. Cdk5 contributes to the major kinase activity in neurons and has been implicated in altering the state of neuron-specific Tau proteins by making them "hyperphosphorylated." Hyperphosphorylated Tau is the major component of paired helical filaments (PHFs) that constitute larger structures (such as NFTs) in AD. Although other enzymes can alter the state of Tau, Cdk5 is a major candidate responsible for Tau hyperphosphorylation. For its activity, Cdk5 requires either a protein called p35nck5a, or a related protein called p39nck5ai, or their shorter forms, p25nck5a and p3Onck5ai, respectively. In a recent study, it was shown that Cdk5 activation by p25nck5a can be enhanced by a neuronal cell extract, suggesting the existence of a Cdk5-activating kinase, which has been named Cdk5-AK. Dr. Lee is exploring the hypothesis that Cdk5 is regulated primarily by its activators, nck5a and nck5ai, and an upstream Cdk5-AK. He also believes that abnormal regulation of Cdk5 contributes to hyperphosphorylation of Tau and neuronal cell death in AD. In this study, Dr. Lee hopes to (1) identify and characterize a Cdk5-AK and (2) to characterize the various Cdk5/activator complexes and their ability to phosphorylate Tau. If successful, these studies will further our understanding of how Cdk5 activity is regulated in the brain and how the enzyme may participate in the development of AD.

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Claus U. Pietrzik, Ph.D.
University of California
San Diego, CA
Project: LRP and Amyloid beta Generation: Role of Cytoplasmic Domain

Recently, several laboratories, including Dr. Pietrzik's, have suggested that a molecule called LRP contributes to the cause of Alzheimer's disease. LRP is a member of the family of lipid receptors. Dr. Dudley Strickland's laboratory (under an AHAF grant) reported earlier this year that LRP influences how much amyloid beta is made by cells, and proposed that APP and LRP associate outside of cells to influence amyloid beta production. Dr. Pietrzick's preliminary studies suggest that LRP influences APP and amyloid beta generation intracellularly. He is now testing the hypothesis that the cytoplasmic domains (intracellular parts) of APP and LRP are very important for these two molecules. He will measure the amount of amyloid beta made by cells that have been engineered to produce only shorter intracellular APP and LRP molecules. He is also studying other ways that LRP influences APP to give a more complete picture of how LRP is responsible for amyloid beta production. It is hoped that this work will lead to fundamental knowledge about the causes of AD and further the development of drugs to reduce amyloid beta production. Dr. Pietrzik is also the recipient of an ADR Pilot Program Award.

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Michael R. Sierks, Ph.D.
University of Arizona
Tempe, AZ
Project: Bispecific Proteolytic Antibodies for Treating Alzheimer's Disease

It is not yet certain that a vaccine to immunize patients with amyloid beta will be successful in fighting Alzheimer's disease. One reason is that not everyone who is immunized may produce an immune response. A potential new approach is called passive immunization. In this process, antibodies that bind to amyloid beta would be directly injected into AD patients. Dr. Sierks proposes the additional refinement of introducing an antibody that is genetically engineered to provide two different functions. The first function of the antibody is to bind to amyloid beta. The second function will be to break amyloid beta into two soluble fragments that can be cleared from the brain. Dr. Sierks' first step in producing such antibodies will be to identify cloned gene sequences of antibodies that will bind to certain regions of the amyloid beta protein. The next step will be to identify antibody sequences that break down the amyloid beta, and to introduce mutations that increase the activity and targeting of the antibody so that it breaks down amyloid beta but not other proteins. Finally, the antibody sequences will be combined into one molecule that can both target and cleave amyloid beta. If this project is successful, the new antibody can be further refined so it can cross the blood-brain barrier, setting the stage for preclinical testing.

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Gopal Thinakaran, Ph.D.
University of Chicago
Chicago, IL
Project: PS1-Nicastrin Interaction and Role in Trafficking

It is well known that presenilin proteins (PS-1 and PS-2) and other proteins interact to generate amyloid beta protein. Recently, researchers at the University of Toronto (with whom Dr. Thinakaran is collaborating) identified one of the proteins, Nicastrin, that interacts with presenilins. A better understanding of the association between presenilins and Nicastrin will shed more light on the mechanisms involved in amyloid beta production, and perhaps lead to new therapies for AD aimed at reducing amyloid beta deposits in the brain. Some studies also indicate that presenilins may be involved in assisting APP (the precursor for amyloid beta) in reaching appropriate destinations within the cell. Dr. Thinakaran is examining whether presenilins direct other proteins to their destinations. He will explore this issue by studying two proteins that resemble APP and others that have structures different from that of APP. It is hoped that these latter studies will help scientists understand what presenilins normally do. The information gained from studies like this will be critical to determining whether presenilins can be considered as appropriate targets for the treatment of AD.

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Donald J. Tipper, Ph.D.
University of Massachusetts
Boston, MA
Project: Prion Protein Mutations, Effects on ER Translocation

Similarities shared by prion diseases and Alzheimer's suggest common pathogenic mechanisms. Both prion diseases and AD result in the accumulation of amyloid proteins in the brain, and this accumulation indicates defects in the folding or processing of these proteins. The fact that mutations in the prion gene cause inherited forms of these diseases reinforces the connection. Dr. Tipper believes that gaining insight into these pathogenic mechanisms is vital to the design of effective treatments. Both the normal form of the prion protein (PrP) and the precursor of Alzheimer's amyloid beta are transmembrane (TM) proteins, meaning that they span the membranes that separate cells or cellular organelles. TM proteins must contain the information that allows them to reach the correct cellular location and orientation to the membrane. Recent evidence suggests that prion pathology results from a defect that causes the prion protein to be inserted backward in the membrane. Dr. Tipper is testing the proposed correlation between the improper insertion of prion proteins and pathogenicity in a yeast organism. He plans to extend these results to mammalian cells. If the correlation is confirmed, the next step would be to investigate the resulting processes that lead to cellular damage and the loss of brain function in AD.

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Ronald B. Wetzel, Ph.D.
University of Tennessee Medical Center
Memphis, TN
Project: Amyloid-specific Monoclonal Antibodies

Amyloid fibrils are intermediate structures formed during the assembly of the polymeric amyloid fibrils and are central to the mechanism of AD, yet we know very little about their structure. Dr. Wetzel is using two monoclonal antibodies with the ability to bind to amyloid fibrils in order to study their structure. There are antibodies that bind to amyloid fibrils through highly specialized surfaces that fit together with the fibrils like a hand in a glove. Dr. Wetzel is using a number of methods to learn more about the structures of these antibodies, since they will indirectly provide an understanding of fibril structure. In other words, by knowing what the glove looks like, Dr. Wetzel can build a model for the hand that fits into that glove. These antibodies will also be used to study different biochemical steps in the formation of larger aggregates of amyloid from smaller fibril subunits. This research could help to demonstrate the importance of particular forms of amyloid in AD, and lead to the development of a tool by which these important forms can be detected and measured in patients. This could then play an important role in the search for a vaccine against Alzheimer's.

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Philip C. Wong, Ph.D.
The Johns Hopkins University
Baltimore, MD
Project: Bace2 Transgenic Models and beta-Amyloid Modulation

Amyloid beta peptides are created by the activities of beta- and gamma-secretases, two enzymes that are required to cut amyloid precursor protein (APP). Two beta-secretases called BACE1 and BACE2 may be involved in the generation of the toxic amyloid beta proteins. Dr. Wong has bred mice that lack the BACE1 enzyme. He has found that neurons from the mice lacking BACE1 no longer produce amyloid beta peptides, and this suggests that BACEl is the principal enzyme required to generate amyloid beta proteins. Studies from other laboratories also suggest that BACE2 may serve to limit the production of amyloid beta. To test this hypothesis, Dr. Wong is attempting to create mice that produce high amounts of normal and/or mutant BACE2 to determine if these genetic alterations curtail amyloid beta production in ways that are consistent with the hypothesis. BACE2 mice will also be bred with "AD" mice to determine if the accumulation of amyloid beta is curtailed by the production of high amounts of BACE2. If it is, this knowledge could pave the way for scientists to design and test drugs that increase the level of BACE2 activity in humans with AD.

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