Lester I. Binder - US grants

tubulin; cell population study; dendrites; neurons; myelination; brain; microtubules; axon; binding proteins; protein structure; neurogenesis; caudate nucleus; Alzheimer's disease; cellular pathology; protein biosynthesis; tissue /cell culture; density gradient ultracentrifugation; enzyme linked immunosorbent assay; cow; laboratory mouse; computer data analysis; stoichiometry; histochemistry /cytochemistry; monoclonal antibody; radioimmunoassay; gel electrophoresis; affinity chromatography;

The microtubule-associated proteins (MAPs), tau and MAP1B are highly regulated during normal neuronal development. Evidence is accumulating suggesting the re-emergence of fetal characteristics in vulnerable neurons involved in Alzheimer's Disease (AD) pathology. Both fetal and adult forms of tau are known to be integral components of the paired helical filament (PHF) and preliminary evidence indicates that AD-affected neurons express high levels of MAP1B. Therefore, we propose to determine the relative amounts of both of these proteins in brain regions and to determine the fetal/adult tau ratios in AD and control brains. Unlike other studies, we will attempt to control for neuronal death by also measuring the amount of neuronal tubulin using a specific monoclonal antibody and expressing the data as ratios of MAP/neuronal tubulin. In order to determine other proteins that may be associated with tau in the PHF or in other aspects of AD pathology, non-tubulin tau interacting proteins (TIPs) will be identified and quantified in AD and control brain. TIPs of demonstrated importance to ADD will be purified and characterized. Microtubule binding studies will be performed using AD and normal soluble tau as well as A68 to ascertain whether the microtubule binding capabilities of these different preparations are altered in the disease state. In addition, existing and novel monoclonal antibodies will be used to measure the amounts of tau in cerebral spinal fluids (CSFs) obtained from AD and control patients to ascertain whether tau can serve as a diagnostic marker for AD. Successful completion of these experiments will aid in establishing the degree to which tau and MAP1B are altered in either their expression, form or interactions in AD pathology.

This project focuses on the formation of the fibrillar pathology in AD and on its chief component, tau protein. We hypothesize that progressive modification and polymerization of tau proteins into straight and paired helical filaments (PHFs) result from conformational changes that can be identified and quantified using specific monoclonal antibodies. Conformation-selective antibodies, most of which were produced in the laboratory of Peter Davies at Albert Einstein College of medicine, can be used to determine th evolution of cytoskeletal abnormalities as a function of AD progression in vulnerable brain regions, and to correlate these with structural features of tau. Using these antibodies, the stability of the PHF- or AD-selective tau conformations can be assessed and quantified through affinity measurements against recombinant monomeric tau, synthetic polymeric tau, and authentic PHF tau. We propose that early changes in tau phosphorylation and /or conformation lead to the formation of fibrillar pathology in AD, and that these changes are stabilized and can be observed very early in the disease process using early marker monoclonal antibodies prior to the onset of PHF formation. This hypothesis will be tested as follows; 1. We will model the structural features of tau in vitro that underlie binding of PHF-selective antibodies in situ. This will be accomplished through oligonucleotide-directed mutagenesis and affinity measurements. Both phosphorylation-dependent and conformation-dependent antibodies will be characterized. This information will suggest a progression for tau conformation-dependent antibodies will be characterized. This information will suggest a progression for tau conformational changes associated with the formation of the fibrillar pathology; 2. We propose to select novel monoclonal conformation-sensitive tau antibodies for analysis and use in Aims 1 and 3, respectively; 3. We will probe for early conformational changes in tau protein that occur during disease progression using quantitative immunohisto- and cytochemistry at the light and E.M. level; and, 4. We propose to confirm that the staining documented in situ is due to conformationally altered tau by using two site capture ELISAs in vulnerable brain regions in aged control populations, mild, moderate and severe AD. These studies will be performed in conjunction with projects to correlate the formation of the fibrillar pathology with galanin hyperinervation of the basal forebrain cholinergic neurons and expression of specific NGF receptors. Data will be further correlated with structural atrophy as measured by MRI and behavioral symptomatology as well as electrophysiological measures of function.

tau proteins; protein biosynthesis; pathologic process; Alzheimer's disease; recombinant proteins; synthetic protein; protein structure function; intermolecular interaction; lipids; polymerization; microtubules; chemical kinetics; paired helical filament; phosphoproteins; animal tissue; peptide chemical synthesis; electron microscopy; human tissue; molecular cloning;

mental disorders; nervous system; biomedical resource; Invertebrata; aging;

This Americas Program award supports a regional workshop entitled "Cell and Molecular Aspects of the Birth, Life and Death of the Nervous System." The workshop is organized by Dr. Lester I. Binder, Northwestern University, and Dr. Ricardo Maccioni, University of Chile, and Dr. Alfredo Caceres, University of Cordoba, and will be held October 4-7, 2000 in Pucon, Chile. The general topic is cellular and molecular neuroscience with a specific focus on the cytoskeleton and its role in neural polarity, endocytosis and vesicular trafficking, neurite extension, vesicular transport, signalling events, and neurodegeneration. Scientists from the United States, Latin America, and Europe who are members of the American Society for Cell Biology, the AAAS, and of the Ibero-American Molecular Biology organization (IMBO) will be among the attendees. Graduate students and postdoctoral fellows will constitute a significant number of meeting participants and will present their work in poster sessions. Given the variety of topics and the strengths of the speakers, the workshop is expected to enhance U.S.-Latin American collaboration.

DESCRIPTION (provided by applicant): The Neurofibrillary Tangles (NFTs) are found mainly in highly vulnerable long projection neurons in the Alzheimer's disease (AD) brain. The cholinergic neurons of the cholinergic basal forebrain (CBF) are exquisitely prone to NFT formation, and a progression of cellular changes is associated with tangle formation. However, the molecular events that underlie the formation of these lesions by the microtubule-associated tau protein remain unknown. Studies on the in vitro assembly of tau protomers into filaments strongly suggest that tau's transition from the soluble to the fibrillar form can be driven, in part, by phosphorylation and by C-terminal truncation accomplished in part by the action of caspases. Additionally, isoforms of the casein kinase 1 (CK1) phosphokinase family that deposit in granulovacuolar degeneration bodies (GVDs) are upregulated 10-30 fold in end stage AD. We propose to determine the order of appearance of these tau alterations in correlation with NFT formation in the cholinergic basal forebrain (CBF) long projection neurons. We hypothesize that the formation of the fibrillar pathologies is induced by a definable sequence of molecular events that directly impact tau "s assembly competency through phosphorylation and truncation. We will test this hypothesis by accomplishing the following specific aims: 1. Using antibodies against specific tau phosphopeptides, we propose to determine whether CBF neurons exhibit a progression of site-specific phosphorylation events that correlates with the transition from non-cognitive impairment (NCI), to mild cognitive impairment (MCI), early, and end-stage Alzheimer's disease (AD); 2. We propose to determine the progression of C-terminal tau truncation using well-characterized antibodies to D421 (the caspase site) and E 391 (another truncation site known to occur in AD); 3. Using antibodies to CKIalpha, CK1delta, and CKIepsilon, we will assay for the appearance of GVD bodies in CBF neurons during the progression from NCI-->MCI-->AD; and, 4. Using gene array technology, we propose to determine the relative quantities of CK1 message and the amounts of caspase message in individual CBF neurons from patients with the aforementioned clinical diagnoses.

DESCRIPTION (provided by applicant): Tau undergoes a discrete set of shape changes during filament formation in Alzheimer's disease (AD). Concurrent with these changes are phosphorylation events throughout the molecule and cleavage at a caspase site (D421) that elevates the rate of tau filament formation in vitro. N-terminal truncation events that apparently change the shape of the tau molecule have also been discovered; the timing of their appearance during the course of tau filament formation and maturation in situ is important in understanding the role of modified tau, tau polymers, and tau toxicity in AD neurodegeneration. We hypothesize that such modifications at the carboxy and amino terminal regions of tau will correlate well with the cognitive transition from the non-cognitive impairment (NCI) to mild cognitive impairment (MCI) to AD. We will test this hypothesis in brain regions vulnerable in early AD as follows: 1. We will perform quantitative immunohistochemical analyses using markers of the C-terminus of tau in situ employing brain sections obtained as part of the Religious Orders Study (ROS). Specifically, we will order the events that precede and succeed cleavage of the D421 caspase site using existing monoclonal antibodies to correlate the relationship between C-terminal phosphorylation events and caspase cleavage with individual tests of memory function; 2. Using EM localization and immunochemical studies, we will determine which PHF/SF populations bind to Tau-C3; 3. Using standard protein chemistry coupled with mass spectrometry, we propose to identify N-terminal truncation and adjacent phosphorylation sites in soluble tau and SDS-insoluble PHF-tau. We will then produce antibodies specific for tau cleaved at the most abundant sites; 4. Novel and existing antibodies will be used to stain tissue sections taken from the entorhinal cortices and hippocampi of brains collected by the ROS to determine which of the amino truncations in the tau molecule correlate best with the transition from NCI->MCI->AD; and, 5. NFT staging will be correlated with galanin hyperinnervation and gene expression in Project 3 and with entorhinal cortex and hippocampal volumes in cases from Project 1.

DESCRIPTION (provided by applicant): Tau undergoes a discrete set of shape changes during filament formation in Alzheimer's disease (AD). Concurrent with these changes are phosphorylation events throughout the molecule and cleavage at a caspase site (D421) that elevates the rate of tau filament formation in vitro. N-terminal truncation events that apparently change the shape of the tau molecule have also been discovered;the timing of their appearance during the course of tau filament formation and maturation in situ is important in understanding the role of modified tau, tau polymers, and tau toxicity in AD neurodegeneration. We hypothesize that such modifications at the carboxy and amino terminal regions of tau will correlate well with the cognitive transition from the non-cognitive impairment (NCI) to mild cognitive impairment (MCI) to AD. We will test this hypothesis in brain regions vulnerable in early AD as follows: 1. We will perform quantitative immunohistochemical analyses using markers of the C-terminus of tau in situ employing brain sections obtained as part of the Religious Orders Study (ROS). Specifically, we will order the events that precede and succeed cleavage of the D421 caspase site using existing monoclonal antibodies to correlate the relationship between C-terminal phosphorylation events and caspase cleavage with individual tests of memory function;2. Using EM localization and immunochemical studies, we will determine which PHF/SF populations bind to Tau-C3;3. Using standard protein chemistry coupled with mass spectrometry, we propose to identify N-terminal truncation and adjacent phosphorylation sites in soluble tau and SDS-insoluble PHF-tau. We will then produce antibodies specific for tau cleaved at the most abundant sites;4. Novel and existing antibodies will be used to stain tissue sections taken from the entorhinal cortices and hippocampi of brains collected by the ROS to determine which of the amino truncations in the tau molecule correlate best with the transition from NCI->MCI->AD;and, 5. NFT staging will be correlated with galanin hyperinnervation and gene expression in Project 3 and with entorhinal cortex and hippocampal volumes in cases from Project 1.

ATP-protein phosphotransferase; Acetylcholine Agents; Address; Affect; Aged 65 and Over; Alzheimer; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer's; Alzheimer's Disease; Alzheimers Dementia; Alzheimers disease; Aminopeptidase; Antibodies; Antigenic Determinants; Appearance; Area; Assay; Binding; Binding (Molecular Function); Binding Determinants; Bioassay; Biologic Assays; Biological Assay; C-terminal; Ca2+-Activated Protease; Calcium-Activated Neutral Protease; Calcium-Activated Neutral Proteinase; Calcium-Activated Protease; Calcium-Dependent Neutral Protease; Calcium-Dependent Neutral Proteinase; Calpain; Casein Kinase 1; Cholinergic Agents; Cholinergic Drugs; Cholinergic Receptors; Cholinergics; Cholinoceptive Sites; Cholinoceptors; Cognitive Disturbance; Cognitive Impairment; Cognitive decline; Cognitive function abnormal; Data; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Desminase; Diagnosis, clinical; Disturbance in cognition; Dysfunction; Elderly; Elderly, over 65; Endopeptidases; Enzyme Gene; Enzymes; Epitopes; Event; Evolution; Exhibits; Exopeptidase; Exoproteases; Expression Profiling; Expression Signature; Functional disorder; Gene Expression; Gene Products, RNA; Ich-1 protein; Impaired cognition; Individual; Isoforms; Laboratories; Lead; MT-bound tau; Measures; Medial; Memory Loss; Micro-tubule; Microtubules; Modeling; Molecular; Molecular Configuration; Molecular Conformation; Molecular Fingerprinting; Molecular Interaction; Molecular Profiling; Molecular Stereochemistry; Nedd-2 protein; Neocortex; Nerve Cells; Nerve Growth Factor Receptors; Nerve Unit; Neural Cell; Neurobiology; Neurocyte; Neurofibrillary Tangles; Neuronal Dysfunction; Neurons; Neurotrophic Factor Receptor; PP2A; PP2A Subunit B Prime; PPP2R4; PR53; PTPA; Papain-Like Cysteine Protease; Pathologic; Pathologic Processes; Pathological Processes; Pathology; Pb element; Peptide Peptidohydrolases; Phosphoprotein Phosphatase; Phosphoprotein Phosphatase-2C; Phosphoprotein Phosphohydrolase; Phosphorylation; Phosphotyrosyl Phosphatase Activator; Physiopathology; Primary Senile Degenerative Dementia; Principal Investigator; Procedures; Process; Programs (PT); Programs [Publication Type]; Protein Isoforms; Protein Kinase; Protein Kinase CK1; Protein Kinase CKI; Protein Phosphatase 2A Regulatory Subunit B Prime; Protein Phosphatase 2A Regulatory Subunit PR53; Protein Phosphatase C; Protein Phosphatase-1; Protein Phosphatase-2A; Protein Phosphorylation; Protein phosphatase; Proteins; Proteolytic Enzyme; Purpose; Pyramidal Cells; Pyramidal neuron; RNA; RNA, Non-Polyadenylated; Receptors, ACh; Receptors, Acetylcholine; Receptors, NGF; Receptors, Nerve Growth Factor; Receptors, Neurotrophin; Ribonucleic Acid; Series; Site; Staging; Structure; Syndrome; System; System, LOINC Axis 4; Temporal Lobe; Testing; Work; abnormally aggregated tau protein; advanced age; aggregation of microtubule associated protein tau; aggregation of microtubule-associated protein tau; basal forebrain; basal forebrain cholinergic neurons; casein kinase I; caspase-2; cholinergic; clinical Diagnosis; cognitive dysfunction; cognitive function; cognitive loss; cognitively impaired; conformation; conformational state; dementia of the Alzheimer type; elders; filamentous tau inclusion; gene product; geriatric; glycogen synthase a kinase; heavy metal Pb; heavy metal lead; hippocampal pyramidal neuron; homotypical cortex; hydroxyalkyl protein kinase; in vitro Model; insight; isocortex; late life; later life; microtubule associated protein tau; microtubule associated protein tau aggregation; microtubule associated protein tau deposit; microtubule bound tau; microtubule-associated protein tau; microtubule-associated protein tau aggregation; microtubule-associated protein tau deposit; microtubule-bound tau; mild cognitive disorder; mild cognitive impairment; mild neurocognitive disorder; molecuar profile; molecular signature; neopallium; neurobiological; neurofibrillary degeneration; neurofibrillary lesion; neurofibrillary pathology; neurofibrillary tangle formation; neuronal; older adult; older person; paired helical filament; paired helical filament of tau; pathophysiology; phosphorylase b kinase kinase; primary degenerative dementia; programs; protease; protein expression; proteinase; puromycin-sensitive aminopeptidase; receptor expression; self-aggregate tau; senile dementia of the Alzheimer type; senior citizen; tangle; tangle formation; tau; tau PHF; tau PHF formation; tau Proteins; tau accumulation; tau aggregate; tau aggregation; tau conformation; tau factor; tau fibrillization; tau filament; tau filament assembly; tau function; tau neurofibrillary tangle; tau oligomer; tau paired helical filament; tau paired helical filament formation; tau polymerization; tau self-aggregate; tau-tau interaction; temporal cortex; temporal lobe/cortex

DESCRIPTION (provided by applicant): Tau proteins normally stabilize microtubules in neuronal processes in the brain. During the course of Alzheimer's disease and certain other neurodegenerative diseases, these proteins dissociate from the microtubule and begin to aggregate within cell bodies of neurons and glial cells in specific disease-related regions of the brain. This process is accompanied by an inflammatory response, one result of which is the nitration and oxidative crosslinking of certain proteins. We have discovered that tau gets nitrated predominantly at specific tyrosine residues in AD and related frontotemporal dementias;moreover, tau also can be crosslinked by dityrosine bonds. We propose to use biochemical modeling studies to determine the role of nitration in tau filament formation and to produce antibodies that only recognize specific nitrated residues and dityrosine crosslinked areas on tau for use in following this process in the brains of patients that died with AD or another tauopathy. Specifically, we will: 1. Study the affect of nitration on the assembly of each of the tau isoforms;2. Study the inhibition of assembly of normal tau by tau nitrated at specific sites;3. Use immunohistochemistry and laser scanning confocal microscopy with our specific antibodies to different nitrated sites on tau to assess the timing and role of nitrative events during the course of AD and other tauopathies. We will also produce monoclonal antibodies to additional nitrated sites on tau and assess them in diseased brains as well;and, 4. Manufacture monoclonal antibodies to dityrosine cross-linked tau in order to determine when this event occurs during the course of AD and other tauopathies. We hypothesize that site-specific tau nitration and dityrosine cross-linking events represent key elements in the formation and stabilization of the fibrillar pathologies in AD and other tauopathies. Successful completion of these studies will contribute greatly to our understanding of the role of inflammation in AD and other tauopathies. PUBLIC HEALTH RELEVANCE: In maladies such as Alzheimer's disease and related dementias, proteins known as tau aggregate within the cell bodies of neurons and certain glial cells. This aggregation is accompanied by an inflammatory process that causes these proteins to be altered on amino acids called tyrosines. We propose to determine the function of these alterations in disease and to determine when and where they occur.

DESCRIPTION (provided by applicant): Tau undergoes a discrete set of shape changes during filament formation in Alzheimer's disease (AD). Concurrent with these changes are phosphorylation events throughout the molecule and cleavage at a caspase site (D421) that elevates the rate of tau filament formation in vitro. N-terminal truncation events that apparently change the shape of the tau molecule have also been discovered; the timing of their appearance during the course of tau filament formation and maturation in situ is important in understanding the role of modified tau, tau polymers, and tau toxicity in AD neurodegeneration. We hypothesize that such modifications at the carboxy and amino terminal regions of tau will correlate well with the cognitive transition from the non-cognitive impairment (NCI) to mild cognitive impairment (MCI) to AD. We will test this hypothesis in brain regions vulnerable in early AD as follows: 1. We will perform quantitative immunohistochemical analyses using markers of the C-terminus of tau in situ employing brain sections obtained as part of the Religious Orders Study (ROS). Specifically, we will order the events that precede and succeed cleavage of the D421 caspase site using existing monoclonal antibodies to correlate the relationship between C-terminal phosphorylation events and caspase cleavage with individual tests of memory function; 2. Using EM localization and immunochemical studies, we will determine which PHF/SF populations bind to Tau-C3; 3. Using standard protein chemistry coupled with mass spectrometry, we propose to identify N-terminal truncation and adjacent phosphorylation sites in soluble tau and SDS-insoluble PHF-tau. We will then produce antibodies specific for tau cleaved at the most abundant sites; 4. Novel and existing antibodies will be used to stain tissue sections taken from the entorhinal cortices and hippocampi of brains collected by the ROS to determine which of the amino truncations in the tau molecule correlate best with the transition from NCI->MCI->AD; and, 5. NFT staging will be correlated with galanin hyperinnervation and gene expression in Project 3 and with entorhinal cortex and hippocampal volumes in cases from Project 1.