Khalid Iqbal, PhD - US grants

The long term objective of this proposal is to learn the etiology and pathogenesis of neurofibrillary tangle of paired helical filaments (PHF) which is the most striking lesion in dementias of the Alzheimer type and in several conditions with mental retardation. The concentration of this lesion correlates directly to the degree of dementia. The studies proposed in this application are designed to determine the biochemical origin of the PHF. We have developed a method for isolation of highly purified preparations of PHF. We will purify PHF from autopsied Alzheimer brains to near homogeneity, identify its protein subunit/s, purifiy each of these proteins and in comparison with purified normal neurofibrous proteins characterize each PHF protein a) chemically by peptide maps, amino-terminal and carboxy-terminal analysis and amino acid composition, and b) immunochemically and immunocytochemically by Ouchterlony double diffusion test, immunobinding by 125I Protein A method on strips of polyacrylamide slab gels, and immunolabeling with and without immunoabsorption at light microscopic level by immunofluorescence and peroxidase-antiperoxidase (PAP) methods, and electron microscopic level by PAP and immunoperoxidase methods. We will also purify PHF from cases of Guam Parkinsonism dementia complex and Down syndrome (adult cases) and isolate PHF proteins and characterize these in comparison with PHF proteins from cases of Alzheimer disease, and study the immunocytochemical reaction of antisera to PHF proteins from Alzheimer brains with a) neurofibrillary changes of PHF in other human conditions such as normal aging, senile dementia, Guam Parkinsonism dementia, dementia pugilistica, Down syndrome, Hallerworden-Spatz disease and subacute sclerosing panencephalitis, b) neurofibrillary accumulation of 15 nm fibers in human diseases such as Steele-Richardson-Olszewski syndrome and c) neurofibrillary accumulation of 10 nm filaments in human disorders such as sporadic motor neuron disease, vincristine neuropathy and infantile neuroaxonal dystrophy, and in experimental animal conditions such as colchicine-induced neurofibrillary changes in rabbits.

There has been a tremendous increase in the rate of excellent publications on Alzheimer's disease and related disorders. The First International Conference on Alzheimer's disease and Related Disorders attracted over 350 scientists from around the world. There is a strong need to have an open and international forum on a biennial basis for discussion of the latest findings. Since the First International Conference in 1988, considerable progress has been made in differential diagnosis, biological markers, molecular pathology and genetics. The specific aims of the grant application include publicizing the meeting, soliciting and reviewing abstracts, encouraging talented junior U.S. investigators and ensuring participation of distinguished U.S. researchers. Toronto, Canada has been selected as a site for several reasons. This is intended to be an international conference and the first conference was in Las Vegas. A leading Alzheimer disease researcher in Toronto is a collaborator of the P.I. Toronto is in close proximity to many U.S. investigators. The cost of holding the meeting at a high quality facility is much lower than New York City. Finally, the Alzheimer Disease Society of Canada is eager to become the primary non-governmental sponsor of the meeting. The application requests funds to pay for the travel of junior U.S. investigators; they will be selected on a competitive basis. Funds are also requested to pay for the travel of invited speakers from the U.S. Finally, funds are sought to pay for a half-time secretary and general administrative expenses to help organize the conference and get out the proceedings. If this application is funded, it will make a major contribution to the success of the Second International Conference on Alzheimer's Disease and Related Disorders.

The long term objective of tis proposal is to learn the etiology and the pathogenesis of Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT) which constitutes one of the major public health problems in modern society. In the United States alone presently over three million senior citizens are affected and these numbers will keep increasing at a frightening rate unless the disease is understood and prevented. Several lines of evidence suggest that the state of phosphorylation/ dephosphorylation of neuronal proteins including the microtubule associated proteins tau might be affected in AD/SDAT and that the Alzheimer paired helical filaments (PHF) might contain abnormally phosphorylated tau. The specific aims of this project are 1) a thorough investigation of the ability of Alzheimer brain tau and PHF, with or without prior in vitro dephosphorylation, to stimulate in vitro assembly of microtubules from bovine tubulin-determined by turbidimetric measurements, negative stain electron microscopy and SDS-PAGE; 2) identification of the protein kinase/s responsible for the phosphorylation of tau and PHF in AD/SDAT brain and measurements of this kinase activity in AD/SDAT and in age-matched unaffected brains-- determined by autoradiography and Western blots of SDS-PAGE; and 3) study the effect of phosphorylation on the self assembly of tau into filaments -- determined by negative stain electron microscopy, turbidimetric measurements and SDS-PAGE. The studies will test whether the abnormal phosphorylation of tau might be the cause of the microtubule assembly defect in AD/SDAT brain and whether dephosphorylation of tau/PHF can reverse this defect in vitro. Identification of the protein kinase/s responsible for the phosphorylation of tau in AD/SDAT brain and determination of the conditions for the self assembly of tau into filaments and their depolymerization might be critical to devise a rational approach in correcting this defect. These studies will help eluciate how alterations of the normal cytoskeleton might contribute to neurofibrillary pathology and functional deficits in AD/SDAT brain.

The long term objective of this project is to learn the etiology and the pathogenesis of Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT) which constitutes one of the most important public health problems in our country since over two million people are affected. The specific aim of this proposal is to determine the origin of paired helical filaments (PHF) which are the common structural element of the Alzheimer neurofibrillary tangles and the neuritic (senile) plaques, the two most characteristic brain lesions of AD/SDAT and in a much less concentration the normal aged humans. During the current grant period we have demonstrated that PHF and microtubule associated protein tau polypeptides comigrate on SDS-PAGE, and immunochemically crossreact with each other, tau is abnormally phosphorylated in PHF, by ELISA tau is a major component of PHF and both tau and PHF polypeptides are amino-terminally blocked. We now propose to determine the amino acid sequences of PHF and tau polypeptides by cleaving them into peptides with CNBr, trypsin and chymotrypsin, study their peptide maps, isolate the peptides by HPLC, sequence them by automated gas phase sequencing, obtain synthetic peptides corresponding to the sequences determined, raise antibodies to the synthetic peptides, confirm their localization to PHF immunocytochemically and by determining their crossreactivities with PHF polypeptides by Western blots and study their in vitro polymerization into fibrils. The techniques involved in this study are biochemical, immunological and morphological. Identification and characterization of the PHF polypeptide/s will be a major step towards learning the origin of PHF and may ultimately lead to an understanding of the disease mechanism. Immunochemical data strongly suggest that tau is a major protein of PHF. Amino acid sequence of tau and PHF polypeptides will demonstrate the presence and the amount of tau and any non-tau polypeptide/s in PHF. The in vitro polymerization of PHF/tau peptides might reveal the identity of the crucial amino acid sequence responsible for the formation of PHF.

Alzheimer Disease has become one of the major areas of research in neuroscience. There has been a tremendous increase in the rate of excellent publications on Alzheimer's disease and related disorders. The First International Conference on Alzheimer's Disease and Related Disorders held in Las Vegas in 1988 attracted over 350 scientists from around the world. The Second International Conference on Alzheimer's Disease held in Toronto, Canada in July, 1990 was attended by approximately 750 scientists, representing practically every Alzheimer's disease research group. Over 400 papers were presented at the Second Conference. This more than doubling the size of the meeting, from the First to the Second Conference, has clearly demonstrated the need to have an open and international forum on a biennial basis for discussion of the latest findings. Approximately 1,000 scientists are expected to attend and present over 500 abstracts at the Third Conference in Padova, Italy in 1992. Since the last Conference in 1990, considerable progress has been made in differential diagnosis, biological markers, molecular pathology and genetics. The specific aims of the grant application include publicizing The Third Conference, soliciting and reviewing abstracts, encouraging talented junior U.S. investigators and ensuring participation of distinguished U.S. researchers. Padova, Italy has been selected as a site for the Third Conference for several reasons. This is intended to be an international conference. The First Conference was held in the United States (Las Vegas), and The Second Conference was in Canada (Toronto). For the Third Conference, Europe (Padova, Italy) was selected to encourage greater participation of our European colleagues. Finally, the Fidia Research Laboratories is eager to become the primary non-governmental sponsor of the meeting. The application requests funds to pay for the travel of junior U.S. investigators; they will be selected on a competitive basis. Funds are also requested to pay for the travel of invited speakers from the U.S. Finally, funds are sought to pay for a part-time secretary and general administrative expenses needed to help organize the conference and issue the proceedings. If this application is funded, it will make a major contribution to the success of the Third International Conference on Alzheimer's Disease and Related Disorders.

The long term objective of this project is to learn the etiology and the pathogenesis of Alzheimer's disease/senile dementia of the Alzheimer's type (AD) which constitutes one of the major public health problems in our country since over two million people are affected. Increasing evidence suggests that microtubule-associated protein tau is abnormally phosphorylated in AD brain and is a major component of the Alzheimer paired helical filaments (PHF). Our working hypothesis is (1) that the protein phosphorylation-dephosphorylation system is defective in AD brain leading to abnormally phosphorylated tau, and (2) that this abnormal phosphorylation is at least partly due to a deficit in the phosphoprotein phosphatase system. Towards this hypothesis we propose to: (1) determine in AD and control brains the levels of activities of phosphoprotein phosphatases using in vitro phosphorylated phosphorylase kinase and light chain of myosin as exogenous substrates, and in vitro phosphorylated normal tau and AD brain abnormally phosphorylated tau as endogenous substrates; (2) isolate phosphoprotein phosphatases from AD and control brains and determine their enzyme kinetics towards standard exogenous substrates, phosphorylase kinase and light chain of myosin and towards PHF, unpolymerized ,abnormally phosphorylated tau, normal tau and in vitro phosphorylated tau; (3) generate rabbit antibodies to isolated phosphatases, and determine immunocytochemical distribution, and immunoassay the levels of each phosphatase in various areas of AD and control brains; (4) study stimulation of microtubule assembly from tubulin with PHF-tau, unpolymerized abnormal tau, and normal tau, before and after dephosphorylation with different phosphatases from Specific Aim #2. The enzyme activities in Specific Aim #1 will be assayed radiometrically towards in vitro phosphorylated [32P] substrates. The phosphatases indicated from Specific Aim #1 will be isolated by tissue fractionation followed by salting or ethanol precipitation, liquid and affinity chromatographies. Immunocytochemical distribution of phosphatases (Aim #3) will be studied by light microscopy. Microtubule assembly in Specific Aim #4 will be determined both by turbidimetric measurements and by negative stain electron microscopy. Studies proposed in this application are on the identification of the protein phosphatase/s responsible for the abnormal phosphorylation in Alzheimer disease brain which information is critical to devise a rational approach in correcting this defect.

DESCRIPTION: Our long term objective is to understand the etiology and the pathogenesis of Alzheimer disease (AD) and to find a rational therapeutic treatment of the disease. The specific aims of this proposal are: (1) to sort the protein kinases into those that do and those that do not make the tau functionally inactive by phosphorylation at one or more of the same abnormal sites as in the AD abnormally phosphorylated tau both proline-directed protein kinases (PDPKs) MAP kinase, GSK-3, and P34 cdc2 and non-PDPKs protein kinase-A1 protein kinase-C, casein kinase (CK)-11 CK-2, CaM kinase-II, and Ser 262 kinase will be employed. Fetal and adult human recombinant taus, normal human tau and AD abnormally phosphorylated tall (AD P-tau)-dephosphorylated with different protein phosphatases will be phosphorylated, Kinetics - of phosphorylation at the abnormal sites determined by Western blots and radioimmunoslot blot assays using antibodies to various abnormal phosphorylation sites of AD P-tau, and the stoichiometry of phosphorylation will be determined. The effect of phosphorylation on the biological activity of will be determined by its binding to tubulin and normal tall, and by assaying its ability to promote assembly of bovine tubulin into microtubules measured by turbidity at 350 nm and by negative stain-electron microscopy (2) Determine the role of protein-protein interaction in the phosphorylation tau by- comparing the phosphorylation of free tau with tau in microtubules, and tau bound to AD P-tau. Both the stoichiometry of phosphorylation and the kinetics of phosphorylation at each site will -be determined a- above. (3) Study the role of site-site interaction in the abnormal phosphorylation of tau. The effect of phosphorylation of tau by one kinase on the kinetics of phosphorylation by a second kinase will be investigated. The site specific phosphorylation at the abnormal sites will be examined by employing antibodies to the phosphor or the non-phosphor state of the epitomes. The tall conformation found to be the best substrate for the abnormal hyperphosphorylation from the Specific Aim #1 will be employed a the substrate for the site-site interaction studies. These studies will reveal the identification of protein kinases that make tall functionally inactive, and whether the abnormal phosphorylation of tau is a result of a specific conformational state of the substrate protein, protein-protein interaction and/or site- sit interaction. Identification of the protein kinase/s responsible far the abnormal phosphorylation in AD brain is critical to devise a rational approach in correcting this defect.

neurofibrillary tangles; neural degeneration; Alzheimer's disease; enzyme activity; phosphoprotein phosphatase; myosin light chain kinase; tau proteins; guanine nucleotide binding protein; cell population study; phosphorylation; brain cell; microtubules; molecular site; phosphorylase kinase; microtubule associated protein; tubulin; animal tissue; western blottings; human tissue; postmortem; immunocytochemistry; immunologic assay /test; sedimentation; antibody; electron microscopy;

Alzheimer Disease has become one of the major areas of research in neuroscience. There has been a tremendous increase in the rate of excellent publications on Alzheimer's disease and related disorders. The Biennial International Conferences on Alzheimer's Disease and Related Disorders which started in 1988 attracting over 350 scientists from around the world. The First International Conference on Alzheimer s Disease was held in Las Vegas in July, 1988, the Second conference in Toronto, Canada in July, 1990, attended by approximately 750 scientists, the Third conference in Padova, Italy, 1992 attended by 1,000 scientists, the Fourth conference in Minneapolis in 1994 attended by 1,220 scientists, and the Fifth conference in 1996 is Osaka, Japan attended by over 1,600 scientists. Since the last Conference in 1996, important information continues to be made regarding the etiology, clinical course, differential diagnosis, epidemiology and risk factors, histopathological course, genetics, molecular genetics, model systems, and therapeutics, as well as related neurodegeneration. The specific aims of the grant application include publicizing The Sixth Conference, soliciting and reviewing abstracts, encouraging talented junior U.S. investigators (graduate student, post doctoral fellows and faculty members up the rank of assistant professor). Amsterdam, The Netherlands has been selected as a site for the Sixth Conference for several reasons. This is intended to be an international conference. The First and Fourth conferences were held in the United States (Las Vegas and Minneapolis), The Second Conference was in Canada (Toronto), the Third in Padova, Italy, and the Fifth in Osaka, Japan. For the Sixth Conference, scientists in Europe are producing a large volume of high quality dementia research and the time has come to increase their interaction and collaboration with their colleagues in the United States. Additionally, Professor Dick Swaab is not only respected by his international academic scientific colleagues, but by the researchers and senior managers of the European Pharmaceutical companies, which will enable the local organizers to underwrite and subsidize many of the conference expenses. The application requests funds to pay for the travel of junior U.S. investigators; they will be selected on a competitive basis. If this application is funded, it will make a major contribution to the success of the Sixth International Conference on Alzheimer's Disease and Related Disorders.

The long term objective of this project is to learn the etiology and the pathogenesis of Alzheimer's disease/senile dementia of the Alzheimer's type (AD) which constitutes one of the major public health problems in our country since over two million people are affected. Increasing evidence suggests that microtubule-associated protein tau is abnormally phosphorylated in AD brain and is a major component of the Alzheimer paired helical filaments (PHF). Our working hypothesis is (1) that the protein phosphorylation-dephosphorylation system is defective in AD brain leading to abnormally phosphorylated tau, and (2) that this abnormal phosphorylation is at least partly due to a deficit in the phosphoprotein phosphatase system. Towards this hypothesis we propose to: (1) determine in AD and control brains the levels of activities of phosphoprotein phosphatases using in vitro phosphorylated phosphorylase kinase and light chain of myosin as exogenous substrates, and in vitro phosphorylated normal tau and AD brain abnormally phosphorylated tau as endogenous substrates; (2) isolate phosphoprotein phosphatases from AD and control brains and determine their enzyme kinetics towards standard exogenous substrates, phosphorylase kinase and light chain of myosin and towards PHF, unpolymerized ,abnormally phosphorylated tau, normal tau and in vitro phosphorylated tau; (3) generate rabbit antibodies to isolated phosphatases, and determine immunocytochemical distribution, and immunoassay the levels of each phosphatase in various areas of AD and control brains; (4) study stimulation of microtubule assembly from tubulin with PHF-tau, unpolymerized abnormal tau, and normal tau, before and after dephosphorylation with different phosphatases from Specific Aim #2. The enzyme activities in Specific Aim #1 will be assayed radiometrically towards in vitro phosphorylated [32P] substrates. The phosphatases indicated from Specific Aim #1 will be isolated by tissue fractionation followed by salting or ethanol precipitation, liquid and affinity chromatographies. Immunocytochemical distribution of phosphatases (Aim #3) will be studied by light microscopy. Microtubule assembly in Specific Aim #4 will be determined both by turbidimetric measurements and by negative stain electron microscopy. Studies proposed in this application are on the identification of the protein phosphatase/s responsible for the abnormal phosphorylation in Alzheimer disease brain which information is critical to devise a rational approach in correcting this defect.

The long term objective of this proposal is to understand the molecular mechanism of neurofibrillary degeneration and then based on this knowledge develop therapeutic treatment for Alzheimer disease (AD) and related disorders. Our working hypothesis is (1) that the protein phosphorylation/dephosphorylation is imbalanced in AD brains, leading to abnormal hyperphosphorylation of tau and some other neuronal proteins; (2) that this defect is in part due to a deficit in the phosphoprotein phosphatase (PP) -2A and -1 which leads to the breakdown of microtubules and consequent impairment of axoplasmic flow and retrograde neuronal degeneration; and (3) that inhibition of neurofibrillary degeneration will arrest the progression of AD. Towards this hypothesis we propose to: (1) study the structures and the activities of the two physiological inhibitors of PP-2A, called I1PP2A and I2PP2A from human brain by generation of rabbit affinity purified antibodies to synthetic peptides corresponding to the unique regions of these proteins, isolation of these proteins from brain by column chromatographies and preparative SDS-PAGE, amino acid sequencing of I2PP2A which very much differs in its molecular mass from that in kidney, cloning of these phosphatase inhibitors from a human brain cDNA library, generation and purification of the recombinant brain inhibitors and inhibition of the phosphatase activities by these inhibitors; (2) study the regulation of the activities of PP-2A and PP-1 in AD and age-matched control brains by assaying the levels (by 125I-immuno-dot-blots) and the activities (by radiometric enzyme assays) of I1PP2A, I2PP2A and DARPP-32 in nuclear and cytoplasmic compartments of various areas of these brains, and by immunocytochemical distribution and cellular localization of the inhibitors in various histopathologically affected and unaffected areas of AD and corresponding areas of control brains; and (3) study the effect of the downregulation of PP-2A and PP-1 activities in the phosphorylation of tau and cellular degeneration by generating stably transfected neural progenitor cells isolated and propagated from hippocampus that overexpress the phosphatase inhibitors in a regulatable manner; these studies will include the effect of the overexpression of each phosphatase inhibitor on viability (MTT assay) and morphology of the cells (phase contrast and immunofluorescence), phosphorylation of tau at specific sites with site specific phosphorylation dependent antibodies (125I Western blots), ability of the resulting phosphorylated tau to promote/inhibit microtubule assembly by light scattering assay and sequestration of normal MAPS by binding and sedimentation assays. These studies will help elucidate the role of PP-2A- and PP-1- inhibitors in the abnormal hyperphosphorylation of tau and the neurodegeneration that occurs in AD.

[unreadable] DESCRIPTION (provided by applicant): The overall objective of this proposal is to investigate the nature of different signaling pathways involved in the etiopathogenesis of neurofibrillary degeneration of abnormally hyperphosphorylated tau, a hallmark brain lesion of Alzheimer disease (AD), Down syndrome, frontotemporal dementia, and other tauopathies, and employ this information to identify and diagnose the different subgroups of Alzheimer's disease. We postulate that more than one disease mechanism and signaling pathway are involved in producing AD pathology, and that various subgroups of this disease can be identified based on CSF levels of proteins associated with plaques and neurofibrillary tangles and of taus abnormally phosphorylated at various specific sites. To test this hypothesis we propose (1) to develop and validate ultrasensitive bienzyme-recycle ELISAs for various abnormal phosphorylation sites of tau. (2) To determine CSF levels of A[unreadable], ubiquitin and total tau, and tau phosphorylated at various specific sites using the assays developed in Aim #1 in AD and control cases, and identify subgroups of AD based on these data by cluster analysis. APOE genotype frequencies and clinical profiles of each cluster, including symptoms such as depression, hallucinations, hypokinesia, and rigidity, will be analyzed. The % sensitivity and % specificity of each phosphorylation site at appropriate cut-off points will be determined to evaluate its diagnostic potential. (3) To study the relationship of levels of soluble and aggregated A[unreadable]1, 2, ubiquitin and various phosphotaus between CSF and brain in Alzheimer's disease. Levels of soluble and aggregated A[unreadable]2, ubiquitin and various phosphotaus will be assayed by ELISA and radioimmuno-dot-blots in the frozen autopsied brains of AD cases from which lumbar CSFs are available. The levels of these markers in the brain will be correlated to the histopathological staging of the disease, and to the CSF levels of these markers. These studies will help (i) identify subgroups of AD based on CSF markers, (ii) provide a lead on the nature of signaling pathways involved in various subgroups, (iii) reveal the diagnostic potential of CSF levels of tau phosphorylated at different specific sites and (iv) identify the relationship of the CSF levels of A[unreadable], ubiquitin and tau to these markers in the brain and to the various histopathological stages of Alzheimer's disease. Better classification of AD at the molecular level and identification of biomarkers that represent the underlying disease process of various subtypes of the disease, in the long term, will lead to improved diagnosis and better defined treatment opportunities for AD and other tauopathies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Alzheimer disease (AD) is the most common cause of dementia in the elderly, which accounts for over five million cases in the United States, six million in the P.R. China, and over 20 million cases worldwide. Abnormal hyperphosphorylation of the microtubule associated protein tau and formation of b-amyloid (Ab) in the brain are the two hallmark pathological processes in AD. Although the mechanisms underlying tau hyperphosphorylation and Ab overproduction have been extensively studied, there is currently no effective cure for this disease. The therapeutic clinical trials aimed at eliminating Ab alone have been disappointing, to date. Therefore, new target(s) to inhibit simultaneously abnormal hyperphosphorylation of tau and Ab overproduction warrant investigation. Based on our previous studies which showed a selective decrease in brain protein phosphatase 2A (PP2A) in AD brain and the involvement of the endogenous protein inhibitors, I1PP2A and I2PP2A, of this enzyme in the etiopathogenesis of AD, our long-term objective is to develop an effective treatment for AD neurodegeneration based on this disease mechanism. The specific objective of this three-year Fogarty International Research Collaboration Award (FIRCA) is to study whether knockdown of inhibitor-2 (I2PP2A) is a valuable target to inhibit tau/Ab pathologies and to rescue the memory deficit in a well-defined triple transgenic mouse model of AD (3xTgAD). Towards this goal, we propose the following two specific aims: (1) To study whether knockdown of I2PP2A can inhibit abnormal hyperphosphorylation of tau/neurofibrillary degeneration and b-amyloidosis in 3xTgAD mice; and (2) To study whether knockdown of I2PP2A can inhibit neurodegeneration and associated cognitive impairment in these animals. These studies will help validate a rational therapeutic target for drug development of AD, and will also foster international research collaboration between the applicant's laboratory in the United States and the Foreign Collaborator's laboratory in China. This research will be done primarily in Huazhong University of Science and Technology, Wuhan, P.R. China, in collaboration with Jian-Zhi Wang, as an extension of NIH Grant No. R01 AG019158, 5/1/2007 to 4/30/2012. The objective of this FIRCA application is to extend and expand the research programs of both the United States laboratory and the Foreign Collaborator's laboratory that will help elucidate whether inhibition of I2PP2A can restore PP2A activity and rescue AD-type histopathology and cognition, and thus to provide an effective therapeutic target for AD drug development. Validation of a disease-based rational therapeutic target that can lead to the development of one or more effective therapeutic drugs for AD and related disorders is highly relevant and a high priority both for the United States and for the P.R. China.