Elias K. Michaelis - US grants

We have observed that two neuronal plasma membrane activities, the glutamate binding sites/glutamate receptors and the Na+- Ca2+ exchange carriers are quite sensitive to the effects of ethanol in vitro and in vivo. Long-term administration of ethanol to experimental animals also led to an increase in the maximal glutamate binding and Na+ - Ca2+ exchange capacity of synaptic membranes. We believe that these changes represent important biochemical and physiological neuronal adaptations to the long- term effects of ethanol on brain neurons. We propose to examine the following issues: a) the cellular mechanisms that produce these neuronal changes, and b) the biochemical/physiological consequences of the effects of ethanol exposure on these two membrane-related activities. The specific studies that we plan to conduct are: a) To develop internally-labeled monoclonal antibodies and to use these monoclonal antibodies to quantify the GBP units in membrane fractions from several brain regions of controls, chronically ethanol-treated animals and ethanol-withdrawn animals; b) To determine the effect of ethanol on L-glutamate binding and glutamate-activated ion channels in hippocampal primary cell cultures, measure changes in the number of GBP units in cultured neurons, and measure the effects of ethanol treatment on the rate of synthesis and membrane transfer of GBP; c) To measure free (Ca2+) in synaptosomes and hippocampal neurons with Fura-2 and determine to what extent ethanol's effects are due to inhibition of the antiporter; d) To purify the Na+ - Ca2+ exchange carrier protein and raise polyclonal and monoclonal antibodies against the protein which will be used to determine whether chronic ethanol treatment produces quantitative changes in the carrier protein units detected by immunochemical procedures.

We have observed that two neuronal plasma membrane activities, the glutamate binding sites/glutamate receptors and the Na+- Ca2+ exchange carriers are quite sensitive to the effects of ethanol in vitro and in vivo. Long-term administration of ethanol to experimental animals also led to an increase in the maximal glutamate binding and Na+ - Ca2+ exchange capacity of synaptic membranes. We believe that these changes represent important biochemical and physiological neuronal adaptations to the long- term effects of ethanol on brain neurons. We propose to examine the following issues: a) the cellular mechanisms that produce these neuronal changes, and b) the biochemical/physiological consequences of the effects of ethanol exposure on these two membrane-related activities. The specific studies that we plan to conduct are: a) To develop internally-labeled monoclonal antibodies and to use these monoclonal antibodies to quantify the GBP units in membrane fractions from several brain regions of controls, chronically ethanol-treated animals and ethanol-withdrawn animals; b) To determine the effect of ethanol on L-glutamate binding and glutamate-activated ion channels in hippocampal primary cell cultures, measure changes in the number of GBP units in cultured neurons, and measure the effects of ethanol treatment on the rate of synthesis and membrane transfer of GBP; c) To measure free (Ca2+) in synaptosomes and hippocampal neurons with Fura-2 and determine to what extent ethanol's effects are due to inhibition of the antiporter; d) To purify the Na+ - Ca2+ exchange carrier protein and raise polyclonal and monoclonal antibodies against the protein which will be used to determine whether chronic ethanol treatment produces quantitative changes in the carrier protein units detected by immunochemical procedures.

biomedical equipment purchase;

health science research support; pharmacy;

The Kansas Center for Mental Retardation and Human Development includes settings at the University of Kansas in Lawrence, Kansas, at the University of Kansas Medical Center in Kansas City, Kansas and at Parsons State Hospital and Training Center, Parsons, Kansas, and bears on a broad range of biobehavioral problems. The center staff has developed research that calls for a combination of disciplines and experimental approaches. These sustained efforts have resulted in a significant number of published research studies and other valuable products in behalf of the retarded. The work of the three campus settings for the next five years has been organized under eleven themes: 1. Speech, Language and Communication (three campuses) 2. Neurobiological Mechanisms (KU and KUMC) 3. Experimental and Professional Training (three campuses) 4. Cognitive Development (three campuses) 5. Social and Environmental Intervention and Integration (three campuses) 6. Psychobiological Aspects of Audition (three campuses) 7. Family Center, Ethics and Policy (KU) 8. Impaired Fetal and Infant Development (KU and KUMC) 9. Human Genetics (KUMC) 10. Implantation and Placental Physiology (KUMC) 11. Reproductive Physiology and Neuroendocrinology (KUMC) The distribution of campuses and settings provide wide access to basic and applied problems and resources. The center has access to the problems of mentally retarded citizens, their families, their work and living environments, and their medical, educational and occupational support systems. These arangements provide for a scientific effort in both basic laboratory and applied training settings.

biomedical equipment purchase;

Aging in mammals is associated with progressive loss of muscle mass, bone demineralization, vascular calcification and progressive hypertension, sensory perception deficits, hypo functioning immune systems, and loss of tissue elasticity. The original program project proposal five years ago was based on the two hypotheses of cell aging, the free radical theory and the Ca2+ hypothesis of aging, that we believe explain many of the cell and molecular changes that occur with aging. Chronic imbalances between oxidant and anti-oxidant processes in cells cause oxidative stress during aging and progressive deterioration of macromolecular structures such as proteins, DNA, and lipids. Progressive deterioration of processes that control [Ca2+]i leads down a path of cellular deterioration and organ malfunction. One of the operating mechanisms for the loss of the capacity of cells to handle the rise in [Ca2}]i may be oxidant-induced protein and membrane damage, especially of proteins that regulate Ca2+ entry into cells, transport out of cells, transport into intracellular organelles, or release from such organelles. The research performed in our laboratories under the auspices of the program project has contributed substantial and detailed new information to support the two major hypotheses of aging. We have provided chemical evidence for the presence of post-translational modification of proteins brought about by ROS. These modifications occur in proteins that regulate intracellular Ca2+ and the function of Ca2+ as an intracellular messenger. The current program project renewal application expands upon the work already accomplished and attempts to define not only the chemical and biochemical modifications due to increasing oxidative stress during aging, but also the cellular and physiological consequences of such modifications. An additional focus of the proposed work is that of cellular mechanisms for protein repair or degradation following insults produced by oxidation. The areas of focus in the continuation of this program project are: a) characterization of the pathways of repair or degradation of calmodulin and the Ca-ATPases b) mechanisms of protein repair by methionine sulfoxide reductase c) age-dependent changes in NMDA receptor function, intracellular calcium channels, protein phosphatases and kinases, and the multi- catalytic proteases d) chemical sensitivity of tyrosine nitration, sulfhydryl group oxidation, and the chemical nature of protein carbonyl groups. To accomplish these goals we will continue to use the expertise of the protein and peptide analysis core. We have also established a new core in cell culture and molecular biology that will assist us in our investigations of changes in cell biology, cell physiology and molecular biology. It is our hope and expectation that the studied proposed in this program will provide us with new insights into basic biological and chemical processes associated with aging. And, that this program will also provide us with targets for potential therapeutic intervention to ameliorate the gradual deterioration in cell and organ functions that occur with aging.

The experimental work proposed is a continuation of our research efforts to characterize the effects o chronic alcohol intake on nerve cell function and, especially, on the adaptation made by neurons in the brain to prolonged exposure to ethanol. This proposal has two major themes, a characterization of the molecular events involved in alcohol-induced changes in the expression and function of brain neuronal glutamate/N- methyl-D-aspartate (NMDA) receptors and in the changes in the regulation of intracellular calcium (Ca2+) through altered expression and function of the Na+-Ca2+ exchange proteins (NCX). Ethanol interferes with the normal function of glutamate as an excitatory transmitter in brain and as a result of such acute interference with glutamate neurotransmission, an adaptation occurs in neurons that brings about the appearance of enhanced glutamate receptor activity. This adaptive response is of importance in the manifestation of post-ethanol withdrawal reactions, especially the appearance of seizures. Part of the adaptive response that brain neurons make to the chronic presence of ethanol is to increase the NCX activity by altering the expression of NCX proteins. This altered activity has a protective effect when the cells are confronted with disruption of Ca2+ homeostatic mechanisms. We have provided experimental evidence that NMDA receptors are not a single family of proteins and therefore the complex responses of neurons to ethanol are the result of a great variation of potential macromolecular targets with which ethanol interacts to produce its effects. In addition, we have shown that there are two forms of NCX proteins in brain neurons, therefore the neuronal adaptations may include changes in both types of NCX transporters. The proposed studies are designed to probe the specific changes that occur during chronic ethanol treatment in the expression of the two major forms of NMDA receptor and NCX transport proteins. These studies include the following: an analysis of the expression of the various subunits of the NMDA receptors and of the NCX proteins in brain following chronic ethanol treatment; an analysis of the roles that the NMDAR1 and GBP subunits of the two forms of the receptors play in the development of enhanced sensitivity of neurons chronically treated with ethanol to NMDA; a characterization of the role of two signal transduction pathways in the function of the NMDA receptors in neurons chronically exposed to ethanol; an analysis of the regulation of expression of NMDA receptors and of NCX proteins in cells exposed to ethanol in vivo and in brain in situ; an examination of the effects of ethanol on resting levels and the decay of stimulated elevations in intracellular [Ca2+] in control neurons and neurons exposed to ethanol for several days; and a comparison of the regulatory elements in the promoter regions of the genes for the NMDAR1 and GBP subunits of the two forms of NMDA receptors.

The experimental work proposed is a continuation of our research efforts to characterize the effects o chronic alcohol intake on nerve cell function and, especially, on the adaptation made by neurons in the brain to prolonged exposure to ethanol. This proposal has two major themes, a characterization of the molecular events involved in alcohol-induced changes in the expression and function of brain neuronal glutamate/N- methyl-D-aspartate (NMDA) receptors and in the changes in the regulation of intracellular calcium (Ca2+) through altered expression and function of the Na+-Ca2+ exchange proteins (NCX). Ethanol interferes with the normal function of glutamate as an excitatory transmitter in brain and as a result of such acute interference with glutamate neurotransmission, an adaptation occurs in neurons that brings about the appearance of enhanced glutamate receptor activity. This adaptive response is of importance in the manifestation of post-ethanol withdrawal reactions, especially the appearance of seizures. Part of the adaptive response that brain neurons make to the chronic presence of ethanol is to increase the NCX activity by altering the expression of NCX proteins. This altered activity has a protective effect when the cells are confronted with disruption of Ca2+ homeostatic mechanisms. We have provided experimental evidence that NMDA receptors are not a single family of proteins and therefore the complex responses of neurons to ethanol are the result of a great variation of potential macromolecular targets with which ethanol interacts to produce its effects. In addition, we have shown that there are two forms of NCX proteins in brain neurons, therefore the neuronal adaptations may include changes in both types of NCX transporters. The proposed studies are designed to probe the specific changes that occur during chronic ethanol treatment in the expression of the two major forms of NMDA receptor and NCX transport proteins. These studies include the following: an analysis of the expression of the various subunits of the NMDA receptors and of the NCX proteins in brain following chronic ethanol treatment; an analysis of the roles that the NMDAR1 and GBP subunits of the two forms of the receptors play in the development of enhanced sensitivity of neurons chronically treated with ethanol to NMDA; a characterization of the role of two signal transduction pathways in the function of the NMDA receptors in neurons chronically exposed to ethanol; an analysis of the regulation of expression of NMDA receptors and of NCX proteins in cells exposed to ethanol in vivo and in brain in situ; an examination of the effects of ethanol on resting levels and the decay of stimulated elevations in intracellular [Ca2+] in control neurons and neurons exposed to ethanol for several days; and a comparison of the regulatory elements in the promoter regions of the genes for the NMDAR1 and GBP subunits of the two forms of NMDA receptors.

A need to obtain, manipulate or display visual information is common to essentially all MRDDRC investigators. The objective of the Biobehavioral Imaging and Graphics Core (BIG) is to provide knowledgeable and skilled technical support staff and state-of-the-art equipment so that all MRDDRC investigators can acquire, analyze, and output the highest quality images in the most cost-effective manner. This primary objective is central to the mission of the MRDDRC, as a strong image acquisition and graphics core contributes significantly to the productivity of Center investigators, presents new opportunities for investigators and collaboration, and helps to highlight the quality of research conducted within the Center. To assure that this primary objective is attained, BIG activities are centered around three specific objectives that embrace 14 individual services. These are: (1) To provide full-service facilities and staff for preparing biological material for imaging. Services include Tissue Processing, Tissue Sectioning, and Biological Staining. (2) To provide state-of-the-art equipment and technical expertise for obtaining, processing, and analyzing images from biological materials and other sources. Services and equipment are directed toward: Image Acquisition from wide variety of microscopic and non-microscopic platforms; Image Correction to control for unwanted distortion introduced during the acquisition process; Image Correction to control for unwanted distortion introduced during the acquisition processing; Image Editing such as grouping and labeling images for publication; Image Analysis and Quantitation to obtain quantitative data from acquired images; 3-D Reconstruction for providing spatial representations of biological data; and where appropriate, Illustration and Animation. (3) To provide state-of-the-art equipment and technical expertise for rendering digital images into appropriate formats for presentation or display. Output formats include: Photogenic-Quality Prints generated from digital images for journal publications; High-Resolution Photographic Slices for oral presentations, and High Resolution Large Format Printing for poster presentations at scientific meetings; and HTML for Web-Based Publications.

DESCRIPTION (provided by applicant): Many of the faculty at the University of Kansas have maintained an excellent tradition of conducting basic biomedical research on the main campus in Lawrence and, through collaborations with clinical scientists at the Medical Center campus, have been involved in transitional research in areas such as mental retardation, aging, Alzheimer's Disease, neurological diseases, pregnancy and early development, and cancer. The University plans to construct two major, bi-campus, multidisciplinary research institutes, one in neuroscience and the other in cancer research. These institutes will combine the efforts of 50 neuroscientists and 30 cancer researchers. Both institutes have identified the development of experimental animal models for neuro-development, neurological diseases, and cancer biology as a key component in achieving an understanding of basic biological mechanisms for neurological diseases and cancer and for developing new therapeutic interventions. Transgenic and gene knockout approaches in mice are the main pathway to such animal model development. The focus of this proposal is on renovating space within the main animal care facility at the University of Kansas in Lawrence to be used to generate, house, and breed transgenic and gene knockout mice. Over the past two years a microinjection laboratory was established and personnel trained to generate transgenic mice. Limited rodent housing was designated for mouse housing and breeding rooms for transgenic animals and a small bio-behavioral laboratory configured to allow for sensitive measures of changes in behavioral and neurological function as a result of over-expression or disruption of various genes. However, these facilities are not optimal for the conduct of the experimental work planned or for the housing of major instrumentation that will be purchased in fiscal year 2005. They also are not centralized in a manner that would effectively support existing and future PHS-funded research. This proposal has as its main focus the renovation/restructuring of a space of approximately 5,600 gross square feet so that it will become a centralized transgenic/gene knockout facility that will serve the community of scientists of the whole University. A unique feature of this facility besides the laboratory for micro-injection, mouse housing/breeding and mouse genotyping, is the inclusion of a well designed space for bio-behavioral measurements and one for complete pathological examination of new transgenic/knockout mice generated.

3-mononitrotyrosine; 3-nitrotyrosine; ATP[{..}]protein-tyrosine O-phosphotransferase; Acquired brain injury; Actins; Active Oxygen; Affect; Age; Aged 65 and Over; Aging; Agonist; Ammon Horn; Anabolism; Animal Model; Animal Models and Related Studies; Axon; Behavioral; Body Tissues; Brain; Brain Injuries; Brain region; Caloric Restriction; Cell Communication and Signaling; Cell Function; Cell Process; Cell Signaling; Cell physiology; Cellular Function; Cellular Physiology; Cellular Process; Central Nervous System; Characteristics; Collaborations; Complex; Cornu Ammonis; D Aspartate; DNA; Dehydrogenases; Dendrites; Deoxyguanosine; Deoxyribonucleic Acid; Development; Diet; Drugs; EPH- and ELK-Related Tyrosine Kinase; EPH-and ELK-Related Kinase; EPHA8; Elderly; Elderly, over 65; Electromagnetic, Laser; Encephalon; Encephalons; Enzymes; EphA8 Protein; Ephrin Type-A Receptor 8; Ephrin Type-A Receptor 8 Precursor; Event; Exhibits; Extracellular Space; Flies; GFP; Gene Expression; Generalized Growth; Generations; Genes; Glutamate Dehydrogenase; Glutamate Receptor; Glutamate Translocase; Glutamate Transport Glycoprotein; Glutamate Transporter; Glutamates; Green Fluorescent Proteins; Growth; Guanosine, 2'-deoxy-; HEK3; Hippocampus; Hippocampus (Brain); Human; Human, General; Hyperactive behavior; Hyperactivity; Hyperactivity, Motor; Hyperkinesia; Hyperkinesis; Hyperkinetic Movements; Injury; Intercellular Space; Intermediary Metabolism; Intracellular Communication and Signaling; Kidney; Knock-out; Knockout; L-Glutamate; L-Glutamate[{..}]NAD+ oxidoreductase (deaminating); L-Proline; Lasers; Lead; Learning; Length of Life; Link; Liver; Longevity; METBL; Maintenance; Maintenances; Mammalia; Mammals; Mammals, General; Mammals, Mice; Mammals, Rodents; Man (Taxonomy); Man, Modern; Measures; Mediating; Medication; Membrane; Memory; Memory Deficit; Memory impairment; Metabolic; Metabolic Processes; Metabolic stress; Metabolism; Mice; Mice, Transgenic; Mitochondria; Modeling; Molecular; Morphology; Murine; Mus; Muscle, Skeletal; Muscle, Voluntary; N Methyl D aspartic Acid; N methyl D aspartate; N-Methyl-D-aspartate; N-Methylaspartate; NMDA; Nerve; Nerve Cells; Nerve Impulse Transmission; Nerve Transmission; Nerve Unit; Nervous; Nervous System Diseases; Nervous System Injuries; Nervous System Trauma; Nervous System damage; Nervous System, Brain; Nervous System, CNS; Neural Cell; Neuraxis; Neurites; Neurochemistry; Neurocyte; Neurologic; Neurologic Disorders; Neurologic Dysfunctions; Neurological; Neurological Damage; Neurological Disorders; Neurological Injury; Neurological trauma; Neuron-Specific Enolase; Neuronal Transmission; Neurons; Overexpression; Oxidative Stress; Oxidoreductase; Oxygen Radicals; PI Transfer Protein; PTK; Pathway interactions; Pb element; Peripheral; Pharmaceutic Preparations; Pharmaceutical Preparations; Phosphatidylinositol Exchange Protein; Phosphatidylinositol Transfer Protein; Phosphorylation; Population; Principal Investigator; Pro-Oxidants; Process; Production; Programs (PT); Programs [Publication Type]; Proline; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Protein Overexpression; Protein Phosphorylation; Protein Tyrosine Kinase; Protein Tyrosine Kinase EEK; Proteins; Pyramidal neuron; Radiation, Laser; Rate; Reactive Oxygen Species; Recovery; Recovery of Function; Reductases; Research; Resistance; Rodent; Rodentia; Rodentias; Science of neurochemistry; Senescence; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Protein; Skeletal Muscle Tissue; Skeletal muscle structure; Somatosensory Cortex; Stress; Structure; Subcellular Process; Testing; Therapeutic Intervention; Tissue Growth; Tissues; Toxic effect; Toxicities; Transgenes; Transgenic Mice; Transgenic Organisms; Trauma, Nervous System; Tyrosine Kinase; Tyrosine-Protein Kinase Receptor EEK; Tyrosine-Specific Protein Kinase; Tyrosylprotein Kinase; Urinary System, Kidney; Wild Type Mouse; advanced age; age dependent; age effect; age related; aged; aging brain; aging effect; analog; base; biological signal transduction; biosynthesis; body system, hepatic; brain damage; brain lesion (from injury); calorie restriction; cytotoxic; density; drug/agent; elders; extracellular; fly; frontal cortex; frontal lobe; functional recovery; gene product; geriatric; glutamic dehydrogenase; heavy metal Pb; heavy metal lead; hippocampal; hippocampal pyramidal neuron; hydroxyaryl protein kinase; innovate; innovation; innovative; intervention therapy; juvenile animal; late life; later life; life span; lifespan; mRNA Expression; membrane structure; mid life; mid-life; middle age; middle aged; midlife; mitochondrial; model organism; nervous system disorder; neurite growth; neurochemistry; neurological disease; neurological dysfunction; neuron cell death; neuron loss; neuronal; neuronal cell death; neuronal loss; neuronal survival; neurotransmission; nitrotyrosine; novel; older adult; older person; ontogeny; organ system, hepatic; overexpress; oxidation; pathway; programs; renal; resistant; senescent; senior citizen; somatosensory; somesthetic sensory cortex; transgenic; tyrosyl protein kinase; young animal

The Program Project on Reactive Oxygen Species and Aging will include three Projects and two scientific Cores, all located at the University of Kansas. The Principal Investigator will continue in his function as the Leader of Core A, the Administrative Core. The goals of Core A are: a) Provide administrative leadership and coordination of the research effort in the study of aging, b) Provide appropriate administrative support, c) Offer expert external review and consultation, d) Organize scientific exchanges in the form of symposia, e) Promote scientific interactions and create a cohesive research environment. And, f) Obtain support from the Institution as needed. The overarching goal is that the whole of this Program Project is greater than the sum of the individual Projects and Cores. Meeting these goals defines the purpose of the Administrative Core, which is: to provide for the overall direction and management of the Program; to make sure that the Project and Core staff remain cohesive and focused on common scientific goals; to provide opportunities for reviews, consultations, and exchanges with the scientific community; and, to represent the scientific interests of the investigators to the University and secure support for laboratory facilities as needed. The progress that Core A has made in providing for the successful, collaborative and forward-looking scientific efforts of the investigators in the Projects and Cores is attested by the productivity of the investigators, their commitment to the goals of the Project, their collaborations, and their efforts to introduce new technologies and new resources. The aims of Core A are to: 1) Provide administrative leadership and coordination of the overall research effort with the goal of producing high quality science in the study of aging; 2) Provide appropriate administrative, accounting and other management support; 3) Offer expert external review and consultation for optimal progress of the research; 4) Organize bi-annual symposia on Molecular Mechanisms of Aging, Oxidative Stress and Ca2+ Regulation; 5) Promote scientific interactions, exchange information and share data/resources; and 6) Obtain support from the Institution as needed for the expansion of the research capabilities of the Project.

ABSTRACT: MITOCHONDRIAL GENOMICS AND METABOLISM (MGM) CORE During the previous funding cycle the Mitochondrial Genomics and Metabolism (MGM) Core assisted investigators interested in the genetics and function of mitochondria in Alzheimer's disease and aging. Our mission was to provide investigators with tools to conduct genomic, transcriptomic, proteomic, and metabolomic analyses of mitochondria, and to relate such measures to mitochondrial bioenergetics in aging and AD. Our goals were to provide cybrid cell lines to investigators exploring the hypothesis that AD is a systemic disease that affects metabolism in mitochondria derived from platelets, to genotype APOE and TOMM40 for all ADC Clinical Cohort subjects, to perform mtDNA haplotype analyses and sequence mtDNA from Clinical Cohort subjects, and to advise and assist scientists studying mitochondrial genomics or metabolism in AD and aging. An additional goal was to generate unique datasets and provide those datasets to KU ADC and outside investigators. The MGM Core accomplished each of these goals. During the next period the MGM Core will provide state of the art tools to investigators that will allow them to study mitochondrial genetics and metabolism and develop possible biomarkers for AD. We will facilitate or provide: (1) New cybrid lines derived from platelet mitochondria from 15 amyloid positive and 15 amyloid negative cognitively normal (CN) individuals (CDR=0) that are also studied by the Clinical and Neuroimaging Cores, and new cybrid lines with modified APOE genotypes. These cybrids will help investigators explore differences between amyloid positive and amyloid negative CN adults, and explore the relationship between APOE isoforms and mitochondrial function. (2) Information on mtDNA mutations associated with AD by performing next generation sequencing (NGS) on the complete Clinical Cohort. We will document the validity of mutations through duplex mtDNA sequencing, and compare brain and blood mtDNA NGS to generate data that enables correlations of disease diagnoses or endophenotypic characteristics with mtDNA mutations or heteroplasmy. (3) Development of potential AD biomarkers by characterizing epigenomic modifications of DNA bases in AD, MCI, and CN, and information on post-translational modifications of mitochondrial proteins in AD, MCI, and CN subjects from the Clinical Cohort group. (4) Assistance in methodology development for the study of mitochondrial genomics and metabolism in AD and aging, and arrange seminars or workshops focused on mitochondrial genetics, metabolism, and neurodegeneration. Through its efforts the MGM Core will assist ADC and other investigators in making new discoveries. Our involvement in pioneering studies such as the deep sequencing of mtDNA, those planned in DNA epigenomics, and the quantification and characterization of post-translational modifications of mitochondrial proteins will lead to new AD-relevant discoveries and hypotheses.