Robert A. Colvin - US grants

Funding is requested under RFA NS/AG-91-03 to study the mechanisms of neuronal cell death in normal human aging and Alzheimer's disease. The cognitive decline associated with aging and the human neurodegenerative diseases (e.g. Alzheimer's disease, Parkinson's disease and Huntington's disease) is most certainly caused by neuronal dysfunction, eventual cell death, and loss of synapses (1). But, what are the causes of neural degeneration and death? The "calcium hypothesis of brain aging" (2) proposes that changes in the cellular mechanisms that act to modulate the concentration of free intracellular calcium ([Cai]) within the neuron contribute to the causative factors leading to neuronal dysfunction and degeneration. A similar hypothesis has also been proposed to explain the neurodegenerative processes occurring in Alzheimer's disease (AD). The "calcium hypothesis of AD" proposes that a loss of Cai homeostasis is the final common pathway to neuronal degeneration. Unfortunately, very little is known about the effects of human aging and AD on neuronal Ca2+ homeostatic processes. This proposal describes a focused study of the properties of neuronal plasma membrane Na+/Ca2+ countertransport in the aging rat brain, the aging human central nervous system (CNS) and in AD. The specific aims of this study are as follows: 1. Determine the effect of an animal model of aging, normal human aging and AD on several important molecular and kinetic properties of neuronal plasma membrane Na+/Ca2+ countertransport. a. the Km and Vmax for Ca2+ activation of Ca2+ transport b. the passive permeability of the plasma membrane to Ca2+ c. evidence for the presence of K+ sensitive and insensitive forms of the Na+/Ca2+ exchanger in rat brain and the human central nervous (CNS) d. the effect of lipophilic peptides (including betaA41-40, betaA425-35, and substance P analogs) on the Na+/Ca2+ exchanger e. molecular weight determination by SDS PAGE and western blot analysis using monoclonal antibodies (MAB) raised to peptide sequences of the dog heart exchanger f. the cellular location of the exchanger depicted by immunocytochemistry using the same MAB 2. Analyze the relationship between age of onset of AD and items 1.a-e above. 3. Using tissues from well documented familial AD, determine the effect on Km and Vmax for Ca2+ activation of Ca2+ transport and western blot analysis.

The long term goals of this research project are to understand the interplay of cellular processes contributing to neuronal and glial cell zinc ion homeostasis in the human central nervous system. In addition we will elucidate the underlying mechanisms of any changes in cellular zinc ion homeostasis that occur in neurodegenerative disorders such as Alzheimer's disease. This pilot grant application proposes experiments that will provide much needed new information on the mechanism of plasma membrane zinc ion influx and efflux in the human brain. Further, we will identify any changes in plasma membrane zinc transport that are associated with Alzheimer's disease pathology. These data will then form the basis of a subsequent RO1 application focused towards accomplishing our long term goals. Published studies from this laboratory have provided convincing evidence of a robust zinc transport activity associated with plasma membrane vesicles isolated from rat brain. The results point to a reversible transport of zinc which is highly influenced by pH. These results have led to the following working hypothesis, which forms the conceptual framework for the proposed research. A zinc ion transporter exists in the plasma membrane that mediates both influx and efflux pathways for zinc (i.e., the transporter is freely reversible). The transport mechanism may involve antiport of protons. This zinc ion transporter is present in human brain and its activity may be altered in neurodegenerative disorders such as Alzheimer's Disease. By performing a thorough kinetic analysis of zinc fluxes in human brain plasma membrane vesicles we will find the answers to the following questions, which test the validity of the above hypothesis. 1. Is the zinc transporter present in human brain? 2. What is the mechanism of zinc transport? 3. Do high affinity and low affinity zinc transporters exist? 4. Do major areas of human brain which contain high levels of zinc (e.g., hippocampus), have higher levels of zinc transport activity? 5. Is zinc transporter activity in human brain altered in Alzheimer's Disease?

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand cellular Zn2+ homeostasis in the brain. Perturbations of Zn2+ homeostasis have been implicated in selective neuronal death following transient brain ischemia and in the pathogenesis of Alzheimer's disease. Thus, a better understanding of Zn2+ homeostasis in the brain will aid in the clarification of the role of Zn2+ in ischemic brain injury and Alzheimer's disease. Yet, homeostasis of Zn2+ in the brain is poorly understood. In previous studies, we have characterized a robust mechanism of plasma membrane Zn2+ transport in cortical neurons that shows a striking dependence on pH. Several lines of experimental evidence suggest that pH plays a very fundamental role in the plasma membrane transport of Zn2+ and other transition elements. Unfortunately, the mechanism(s) underlying pH effects are still unclear. The next logical step to elucidating these mechanisms in neurons now hinges on the identification of the responsible protein(s). The compartmentalization of extrasynaptic Zn2+ in neurons is a newly emerging area of exploration. This laboratory has used zinquin (a Zn2+ specific fluorescent dye) to demonstrate the extrasynaptic compartmentalization of Zn2+ in neurons (see preliminary studies p. 21). The next logical step to understanding the significance of the extrasynaptic compartmentalization of Zn2+ in neurons is to identify the cytoplasmic organelles that sequester Zn2+. Building upon our previous studies and those of others, the research plan described in this grant application outlines the experiments that will take these next logical steps and advance our research effort toward accomplishing our long-term goal. The research plan is organized into two specific aims. Specific Aim 1: Mechanism(s) of plasma membrane Zn2+ transport. Which protein(s) is/are responsible for pH dependent plasma membrane Zn2+ transport in cortical neurons? Specific Aim 2: Extrasynaptic Zn2+ compartmentalization. What is the identity of Zn2+ containing compartments in the cell body and processes of cortical neurons?

DESCRIPTION (provided by applicant): The International Society of Zinc Biology (ISZB) was founded in 2007, as a nonprofit organization. Our fundamental goal is to bring together scientists from diverse fields with common interest in the structural, biochemical, genetic, physiological and medical aspects of zinc biology. Membership is open to individuals from any nation. Our Society is a consensus-based organization that strongly encourages exchange of information and ideas among zinc researchers. To that aim, the ISZB has organized and supported one of a kind biennial international conferences (ISZBC) dating back to 2008. Work at these meetings has shown that fundamental mechanisms of zinc biology are of critical relevance to understanding health and disease across a broad spectrum that includes but is not limited to cancer, immunology, neurology, as well as metabolic and endocrine disorders. Bringing these investigators together has contributed to substantial advances in these fields, and promoted new collaborations between fields. The number of attendees has steadily grown and now exceeds 140 with an intentional balance of graduate students, postdoctoral fellows, junior faculty, and established investigators. The 4th ISZBC is being held in Pacific Grove, California at Asilomar from September 14th-19th, 2014, and will be organized by a diverse and experienced team of investigators. Asilomar is ideally suited for a meeting of this size and has been chosen because it provides an exceptional environment for participant interaction and scientific exchange. Our conference is the premier and only scientific venue focused exclusively on the field of zinc biology. The ISZBC strongly supports the development of the next generation of researchers including those from underrepresented populations and highly values trainee development and diversity. The ISZBC; 1) encourages the exchange of unpublished data; 2) is highly interactive between young and established investigators to foster meaningful collaborations; and 3) promotes networking between investigators, trainees and their respective institutions from around the world. As a society we are driven by innovation and discovery. As a testament to this, we have a separate scientific program committee that has recruited the top senior and junior talent in established and emerging areas of zinc biology regardless of society affiliation. The 2014 conference will integrate junior faculty/trainee podium presentations into every scientific session. Five graduate student or postdoctoral fellows chosen from submitted abstracts are also invited to present orally, providing these trainees with an invaluable opportunity to showcase their research. We propose to select from abstract submissions ten U.S. trainees for registration waivers, focusing on underrepresented minorities. Importantly, this year's ISZBC will have an interactive forum focused on career development for trainee attendees. Special efforts have been made in general to encourage attendance by underrepresented minorities in science, including women. Thus, the ISZBC supports a variety of activities that are directly relevant to the scientific missions of the National Institutes of Healh and public health.