Cheng-Xin Gong - US grants

The long term objective of this proposal is to understand the mechanism of neurofibrillary degeneration in Alzheimer disease (AD). The specific objective of this pilot project is to study the role of neuroleptics, chlorpromazine and trifluoperazine, in the abnormal hyperphosphorylation of tau and AD-like neurofibrillary protein pathology in rat brain. The specific aims of this proposal are: (1) study the activities of protein phosphatases (PP), PP-1 PP-2A, and PP-2B, in rat brains after chronic infusion with chlorpromazine and trifluoperazine which are also PP-2B inhibitors. Infusion will be carried out by using Alzet osomtic minipumps which will continually deliver drugs to lateral ventricle of rat brain for 2 to 6 months. Another PP-2B inhibitor cyclosporin A and PP-2A/PP-1 inhibitors, okadaic acid and calyculin A will also be infused individually and in combination with the neuroleptics to study the effect by inhibition of all the three protein phosphatases. The phosphatase activities of rat brain extracts will be assayed in vitro using (32P]phosphorylase (for PP-1 and PP2A) and [32P]phosphorylase kinase (for PP-2B) as substrates; (2) study the histopathological changes in treated and control rat brains from Specific Aim #1. Formalin (10%) fixed sections of the rat brain will be prepared for Congo red staining and immunocytochemistry. Phosphorylation-dependent tau antibodies Tau-1, AT8, PHF-1, 12E8, M4, 102c and R21657 will be used to examine the accumulation of hyperphosphorylated tau. Any neurofibrillary changes observed by light microscope will be examined by electron microscopy; (3) quantitate the phosphorylation level of tau in rat brains from Specific Aim #1. Western blots of rat brain homogenates developed with [125I]-labelled secondary antibody and quantitated by Phosphorimager will be carried out for this purpose. Tau antibodies as described above will be employed as primary antibodies to detect the phosphorylation of tau at the specific site(s) recognized by each antibody. These studies will help reveal whether in rat brain the inhibition of protein phosphatase activity by neuroleptics and other phosphatase inhibitors induces Alzheimer-like abnormal hyperphosphorylation of tau and consequent neurofibrillary degeneration.

The long term objective of this proposal is to understand the mechanism of neurofibrillary degeneration in Alzheimer's disease (AD). The specific aims of this project are to reveal the nature and extent of glycosylation of microtubule associated protein tau during various stages of Alzheimer neurofibrillary degeneration, and to understand the role of tau glycosylation in the conversion of tau into tangles of paired helical filaments (PHF) in AD brain. Towards these aims it is proposed: (1) to identify the saccharides and the glycosylation sites of various species of tau representing different stages of neurofibrillary degeneration. Normal tau from normal human brains, and AD non- hyperphosphorylated tau, soluble AD abnormally phosphorylated tau, readily detergent soluble PHF-tau and sparingly detergent soluble PHF-tau from AD brains will be bulk isolated. The presence and the quantity of saccharides conjugated to these proteins will be determined by lectin binding techniques and gas chromatography-Mass spectrometry, respectively. The types of glycosidic linkages will be identified by their sensitivity to selective glycosidases. The glycoylation sites will be mapped by generation and isolation of glycopeptides, and their amino acid sequencing and matrix-assisted laser desorption/ionization-Mass spectrometry. (2) To study the interactions between abnormal glycosylation and abnormal hyperphosphorylation of tau and the role of tau glycosylation in the AD pathogenesis. The effects of one of the two tau posttranslational modifications (glycosylation and phosphorylation) on each other will be investigated by comparing the kinetics of the modification with and without the prior presence of the other modification. The sensitivity of glycosylated tau to proteolysis and its tendency to polymerize into filaments will be studied before and after removal of glycans from the proteins. Finally, the structure of PHF tangles will be examined by electron microscopy after the tangles are deglycosylated. These studies will elucidate the abnormal tau glycosylation and its impact on the formation of PHF tangles and, therefore, should provide a new inside view of the mechanism of Alzheimer neurofibrillary degeneration.

[unreadable] DESCRIPTION (provided by applicant): Abnormal hyperphosphorylation and aggregation of tau protein in the brain are critical to neurodegeneration of Alzheimer Disease (AD). Glucose uptake/metabolism is impaired in AD brain, which is believed to cause neurodegeneration. However, how this impairment contributes to neurodegeneration is unknown. The specific goal of this project is to reveal the nature and functional role of tau O-GlcNAcylation [a unique type of O-glycosylation by which beta-N-acetylglucosamine (GlcNAc) is linked to serine or threonine residues of proteins] and to uncover the mechanism by which impaired brain glucose uptake/metabolism of AD contributes to neurodegeneration. The long-term objective of this proposal is to understand the mechanism of neurodegeneration in AD and, based on this knowledge, to develop strategies to prevent and treat the disease. Hence, the Specific Aims are: (1) Map the O-GlcNAcylation sites of tau and determine the change of tau O-GlcNAcylation in AD brain. The underlying cause of the change in tau O-GlcNAcylation will also be studied by comparing brain level of UDP-GlcNAc and activities of O-GlcNAc transferase and O-GlcNAcase between AD and controls. (2) Study the interactions between O-GlcNAcylation and phosphorylation of tau both in vitro and in differentiated PC12 cells. The functional role of tau O-GlcNAcylation will be studied by measuring its microtubule-binding and -assembly activities and examining cell morphology and organelle transport upon alteration of tau O-GlcNAcylation. (3) Investigate the molecular mechanism by which impaired brain glucose uptake/metabolism contributes to neurodegeneration of Alzheimer's disease. Two animal models of impaired brain glucose uptake/metabolism-fasted mice and mice after intracerebroventricular injection of cytochalasin B-will be used to study its effects on tau O-GlcNAcylation and phosphorylation. The exact role of O-GlcNAcylation in tau hyperphosphorylation and neurodegeneration induced by low glucose uptake/metabolism will also be elucidated in the mouse brains. These studies will reveal the nature and functional role of tau O-GlcNAcylation and its dysregulation in AD brain and uncover the mechanism by which impaired brain glucose uptake/metabolism of AD contributes to neurodegeneration. Completion of these studies will provide new insight into the mechanism of neurodegeneration of AD and help develop novel strategies to prevent and treat Alzheimer's disease and probably other neurodegenerative disorders. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Abnormal hyperphosphorylation and aggregation of tau protein in the brain are critical to neurodegeneration of Alzheimer Disease (AD). Glucose uptake/metabolism is impaired in AD brain, which is believed to cause neurodegeneration. However, how this impairment contributes to neurodegeneration is unknown. The specific goal of this project is to reveal the nature and functional role of tau O-GlcNAcylation [a unique type of O-glycosylation by which beta-N-acetylglucosamine (GlcNAc) is linked to serine or threonine residues of proteins] and to uncover the mechanism by which impaired brain glucose uptake/metabolism of AD contributes to neurodegeneration. The long-term objective of this proposal is to understand the mechanism of neurodegeneration in AD and, based on this knowledge, to develop strategies to prevent and treat the disease. Hence, the Specific Aims are: (1) Map the O-GlcNAcylation sites of tau and determine the change of tau O-GlcNAcylation in AD brain. The underlying cause of the change in tau O-GlcNAcylation will also be studied by comparing brain level of UDP-GlcNAc and activities of O-GlcNAc transferase and O-GlcNAcase between AD and controls. (2) Study the interactions between O-GlcNAcylation and phosphorylation of tau both in vitro and in differentiated PC12 cells. The functional role of tau O-GlcNAcylation will be studied by measuring its microtubule-binding and -assembly activities and examining cell morphology and organelle transport upon alteration of tau O-GlcNAcylation. (3) Investigate the molecular mechanism by which impaired brain glucose uptake/metabolism contributes to neurodegeneration of Alzheimer's disease. Two animal models of impaired brain glucose uptake/metabolism-fasted mice and mice after intracerebroventricular injection of cytochalasin B-will be used to study its effects on tau O-GlcNAcylation and phosphorylation. The exact role of O-GlcNAcylation in tau hyperphosphorylation and neurodegeneration induced by low glucose uptake/metabolism will also be elucidated in the mouse brains. These studies will reveal the nature and functional role of tau O-GlcNAcylation and its dysregulation in AD brain and uncover the mechanism by which impaired brain glucose uptake/metabolism of AD contributes to neurodegeneration. Completion of these studies will provide new insight into the mechanism of neurodegeneration of AD and help develop novel strategies to prevent and treat Alzheimer's disease and probably other neurodegenerative disorders. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Alzheimer's disease (AD) manifests both by the development of extracellular amyloid deposits (plaques) and intracellular aggregates known as neurofibrillary tangles (NFTs). The plaques result from accumulation of the insoluble peptide amyloid b (A2) that is proteolytically released from the amyloid b precursor protein (APP). The NFTs arise from an abnormally hyperphosphorylated form of the cytoskeletal protein tau that assembles into aggregated, paired helical filaments (PHFs). Accumulating evidence suggests that a synergistic interplay between Ab and hyperphosphorylated tau results in the full development of AD symptoms. It has emerged over the past number of years that the addition of O-linked N-acetylglucosamine units (O-GlcNAc) on intracellular proteins can regulate their activities and stabilities. Recently, one mechanism for regulating tau phosphorylation has been shown to involve addition of O-GlcNAc units. Tau O-GlcNAc levels in human AD brains are markedly lower than in normal brains and hyperphosphorylated tau bears little O- GlcNAc. These results, and others, demonstrate that tau O-GlcNAc levels and tau hyperphosphorylation are linked in a reciprocal manner. Maintenance of an appropriate balance between these two modifications may be critical to avoid the development of the pathogenic tau species that are associated with disease progression in AD. These observations suggest that blocking the removal of O-GlcNAc from tau will prevent hyperphosphorylation and, in turn, arrest the formation of tau aggregates. APP has also been shown to be O-GlcNAc modified, and preliminary reports indicate that O-GlcNAc levels affect APP processing. Accordingly, there is clear potential for pharmacological enhancement of O-GlcNAc levels of tau and, to a lesser extent, APP as a method to prevent the pathological processes of tau hyperphosphorylation and plaque formation. We have recently devised several potent and selective small-molecule inhibitors of O-GlcNAcase, the enzyme responsible for removal of O-GlcNAc from proteins. We have also demonstrated that oral dosing with several of these inhibitors dramatically lowers tau phosphorylation levels in rats. We aim to establish proof-of- principle that pharmacologic blockade of O-GlcNAcase prevents tau hyperphosphorylation and thereby blocks the development of AD-like symptoms. This experiment will be carried out in vivo using an animal model of AD. Transgenic TAPP mice (containing mutant forms of human tau and APP) will be dosed orally with inhibitor for 36 weeks and the effects of treatment will be assessed using Western blot analyses, immunocytochemistry, and histology. Motor skills and cognitive performance will be assessed using behavioral tests from week 12 onward. Mice will be sacrificed at weeks 14, 24, and 36, and tested for levels of tau phosphorylation, NFT formation, and Ab deposition. Results for the dosed groups will be compared to those for control groups, receiving vehicle alone, to assess the effects of inhibitor on disease progression. PUBLIC HEALTH RELEVANCE: The defining features of Alzheimer's disease are the formation of pathogenic forms of tau protein and deposition of amyloid peptide in the brain. The proposed work will test an inhibitor of an enzyme involved in the regulation of these proteins in transgenic mice; mice that develop symptoms similar to Alzheimer's disease. If the mice receiving inhibitor show delayed disease progression, this will validate this enzyme as a drug target and may lead to a new and more effective class of drugs for treatment of Alzheimer's disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Alzheimer disease (AD) is an age-related progressive neurodegenerative disease. As a result of increases in the size of the aged population worldwide, AD has become an important social, economical and medical burden, affecting ~27 million individuals worldwide and 4-5 million individuals in the U.S. alone. Because the molecular mechanism of AD is not yet understood, there is no cure for the disease so far. The long-term objective of this application is to understand the molecular mechanism of the neurodegeneration in AD and, based on this knowledge, to develop strategies to prevent and treat the disease. The specific goal of this international research collaboration FIRCA grant (R03) is to foster international research collaboration between the applicant's laboratory in the U.S. and the foreign collaborator's laboratory in China. Because abnormal phosphorylation (a modification of protein by phosphate) and accumulation of neurofilaments (the major cytoskeletal proteins of the neuron) in the brain may contribute to degeneration of neurons in AD, the specific focus of the proposed studies is on understanding the role of O-GlcNAcylation (a modification of protein by sugar) in the phosphorylation and function of neurofilaments, which are abnormal in the brains of individuals with AD. The Specific Aims of this project are to (1) study the functional impact of O-GlcNAcylation on filament assembly and on axonal transport of neurofilaments and (2) study the vulnerability of various brain regions to NF hyperphosphorylation induced by decreased O-GlcNAcylation in the brain. This project will not only elucidate the biological relevance of neurofilament O-GlcNAcylation and its functional roles, thus providing the fundamental understanding of the role of dysregulation of neurofilaments in neurodegeneration of AD, but also foster the ongoing international research collaboration between the U.S. and China. With ~6 million individuals with AD, China has a huge demand for adequate research on the disease. The foreign collaborator of this FIRCA application is Dr. Yanqiu Deng, an Associate Professor of Pathophysiology at Tianjin Medical University, Tianjin, China. The proposed studies will be carried out at both the applicant's and the foreign collaborator's laboratories. Studies proposed in this application will complement, extend and enhance the significance of the applicant's parent grant (NIH R01 AG027429, 03/01/06-02/28/11) that targets the mechanism of the involvement of O-GlcNAcylation of tau protein in the neurodegeneration of AD. PUBLIC HEALTH RELEVANCE: Alzheimer disease is an age-related progressive neurodegenerative disease and is characterized by impaired memory, thinking, and behavior, leading to dementia and death. The objective of this project is to enhance international research collaboration on studies targeting the molecular mechanism of neurodegeneration of Alzheimer disease and, on the basis of this knowledge, to develop strategies to prevent and treat the disease.