Phillip A. Cole, M.D., Ph.D.
The Rockefeller University
Inhibition of N-acetyltransferase

Dr. Cole’s research is devoted to evaluating the effects of melatonin on mammalian physiology. Melatonin is a hormone thought to be important in the regulation of circadian rhythm. It is taken as a medication by some travelers to prevent jet lag. Melatonin has even been speculated to be useful in preventing aging. A particular enzyme called serotonin N-acetyltransferase regulates melatonin production in animals and people. Dr. Cole is attempting to develop specific agents which block this enzyme and will therefore inhibit melatonin production. He will then evaluate the effects of such inhibitors on behavior and circadian function in animals. Such inhibitors will also be tested to see how they effect the aging process in animals

Todd Golde, M.D., Ph.D.
Mayo Clinic, Jacksonville
Presenilins and g-secretase Cleavage of the Amyloid ß Protein Precursor

Dr. Golde compares Alzheimer's disease (AD) to atherosclerosis. Both are diseases associated with aging, both have strong genetic components, and both are diseases of deposition. Atherosclerosis involves deposition of cholesterol within blood vessels, which damages the cardiovascular system. Alzheimer's disease involves excessive deposition of a protein called the amyloid ß protein (Aß) in the brain. Like many who study AD, Dr. Golde believes that lowering Aß levels may delay or prevent AD akin to the way in which lowering cholesterol levels reduces the risk of developing atherosclerotic disease.

The focus of Dr. Golde's work is to find new ways to slow or prevent the deposition of the Aß. As part of this effort, Dr. Golde and colleagues are using novel genetic systems and more traditional approaches to better understand the ways in which the Aß is generated. Because Aß is initially made as a larger precursor protein, it must be "cut out" from its precursor by other proteins, generically referred to as proteases, that act as "molecular scissors". In fact, for Aß generation to take place two sequential proteolytic cleavages must occur. While the proteases responsible for the first cleavage, known as the ß-secretase cleavage, have recently been cloned, the proteases responsible for the second cleavage referred to as the g-secretase cleavage remain unidentified. Significantly, other proteins called presenilins (PSs) identified by studies of genetic forms of AD, have been reported to be the elusive g-secretases. Over the past few months our laboratory has generated data that shows PSs cannot be g-secretases, but somehow regulate the g-secretase cleavage. In the future, using knowledge gained from these studies we hope to identify the protease that make the g-secretase cleavages.

Although PSs are not the g-secretase, impairing PS function can decrease Aß production. Thus, PSs, like y-secretase and the enzymes responsible for ß-secretase cleavage, are therapeutic targets in AD as interfering with the function of each will decrease Aß production. However, because PSs have been shown to be important proteins that regulate many cellular functions, it is not currently known whether targeting PS will turn out to be a viable therapeutic strategy for the treatment or prevention of AD. Over the next year, using funds provided by the Ellison Medical Foundation, we will directly test whether PS are a therapeutic target by decreasing PS expression in an animal model of AD. To accomplish this we will treat the mice with peptide nucleic acids (PNA) that are designed to specifically lower PS expression. By monitoring the effects of PNA treatment on Aß production and deposition, as well as determining whether the treatment is well tolerated, we should gain significant insights into the feasibility of targeting PS for the treatment or prevention of AD. Moreover, if successful these studies will have broad reaching implications, as they would be the first demonstration that PNAs targeting a specific gene can influence the development of disease in an animal model.

Francine Grodstein, Sc.D.
Brigham and Women's Hospital Harvard Medical School
Estrogen, Antioxidants, and Cognitive Function in Women

In the US, 5% of women over age 60, and 28% over 85 may have dementia. While advances have been made recently to delay Alzheimer's disease progression, little population-based research is aimed at studying the earliest stages of cognitive decline in healthy elderly, a stage which might be most susceptible to intervention. Experimental studies suggest that brain tissue readily undergoes oxidative damage, and oxygen free radicals are believed to be involved in aging of the brain; thus, antioxidant supplementation might reduce the risk of dementia. In addition, estrogen appears to influence the cholinergic system, which regulates memory learning and other cognitive functions, suggesting that female hormones may protect against dementia. In this project, Dr. Grodstein will capitalize on a large epidemiologic data set with over 20 years of prospective data to study risk factors for early cognitive impairment; specifically, she will examine how dietary antioxidants and postmenopausal hormone therapy may influence cognitive decline in women free of major illness at baseline. In particular, she will examine the effect of duration and dose of these agents.

Bruce Hay, Ph.D.
California Institute of Technology
Apoptosis

Apoptosis is a genetically regulated form of cell death which is critical for the normal development and adult function of multicellular organisms. Inappropriate activation of apoptosis also contributes to the neuronal cell loss associated with acute brain injuries such as stroke, and in age-related neurodegenerative diseases such as Alzheimer’s disease. Proteases play multiple roles in the regulation of cell death in the brain by acting as cellular executioners that bring about cell death, and by processing proteins that regulate the development of Alzheimer’s disease. These proteases and regulators of their activity are thus important potential therapeutic targets. Dr. Hay has developed a reporter system that allows him to follow the activity of proteases that cleave specific target sites in living cells. He intends to use this technology to identify regulators of cell death, and to identify proteases that process proteins implicated in Alzheimer’s disease.

Elly Nedivi, Ph.D
Massachusetts Institute of Technology
CPG15, A Novel Growth Promoting Molecule Involved in Synaptic Plasticity

The capacity of the central nervous system (CNS) to modify connectivity properties as a result of activity is termed plasticity. Plasticity is not only a prominent feature of CNS development, but in the adult brain underlies learning and memory processes and adaptive reorganization of primary sensory maps. In the cases of both developmental and adult plasticity, there is evidence that synaptic modifications that are brought about by correlated activity may result from activation of gene expression. Dr. Nedivi has previously isolated a large number of candidate plasticity-related genes (CPGs), many of them novel. Dr. Nedivi now intends to use CPGs as molecular tools to dissect mechanisms of activity-dependent plasticity during development and in the adult.

Hong Yan, Ph.D.
Brigham and Women's Hospital, Harvard Medical School
Identification of Proteins Functionally Redundant to the Werner's Syndrome Gene Product

Werner syndrome (WS) is a rare genetic disorder associated with premature aging and increased risks for cancer. The gene underlining WS has been cloned and found to encode a member of the RecQ DNA helicase family. In addition to the drastic mutations found in WS patients, there are also mutations with unknown consequences in the general population. These natural variants (polymorphism) may contribute to the differential rate of aging in the population. Understanding the physiological function of WRN may therefore shed light on not only pathological aging but also natural aging.

We have purified a protein (FFA-1) based on its activity to induce the assembly of DNA replication foci in frog (Xenopus laevis) egg extracts. Sequence analysis indicates that FFA-1 is the homologue of human Werner syndrome gene (WRN), suggesting that WRN may be involved in DNA replication. However, WRN is not an essential gene. We hypothesize that another RecQ helicase family member, Bloom syndrome gene (BLM), may be functionally redundant to WRN.

To test this hypothesis, we have cloned the Xenopus homologue of BLM. We have been able to efficiently express xBLM as fusions to various affinity tags in E. coli. We have demonstrated that the His-xBLM fusion protein possesses DNA helicase activity. The GST-xBLM fusion protein has been used as antigen for antibody production. The antibody has been purified on an affinity column and is currently being used for immunofluorescence staining and immunodepletion studies on xBLM. Preliminary results suggest that xBLM and FFA-1 may indeed participate in the same DNA transaction pathway.