1985 — 2015 |
Dawson, Glyn |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Glycosphingolipid Metabolism and Mental Retardation
The broad, long-term objectives are to understand the role of glycolipids (glycoshingolipids (GSL) and inositolphosphoglycerides) in normal nervous function and the pathogenic processes involving them which lead to mental retardation. To achieve this goal we will use cultured cells which express either neural-specific properties (neurons, neurotumor hybrid cell lines, oligodendrocytes), or inherited metabolic defects (fibroblasts), in conjunction with metabolic studies, enzyme assays, specific antibodies, and cDNA transfection of specific proteins. The two major Specific Aims are: 1) To understand how receptors are coupled to the activation of phospholipase C (PLC) and the hydrolysis of phosphoinositides, and how this system is regulated by protein kinase A, protein kinase C, glycolipids and derived free sphingosine bases. a) We will focus on low molecular weight GTP-binding proteins such as rap1b, their turnover, phosphorylation, membrane association and coupling to receptors and PLC. We will study three cell lines in which receptor-PLC coupling seems to be regulated differently, namely NCB-20, WEHI-3 and human oligodendroglioma cells. b) We will determine how sphingosines activity, and how they could physiologically regulate both PK-C and PLC activity, and how their re- palmitoylation could be related to the acylation/deacylation of bioactive proteins such as GAP-43. 2) To elucidate the role of glycosphingolipids in the mechanism of certain types of neural cell injury. a) We will study hypoxia in neonatal rat oligodendrocytes since hypoxia initially restricts O2 for 2-hydroxy fatty acid GSL synthesis and depletes ATP levels sufficiently to disrupt inter- organellar translocation and phosphorylation of key myelin proteins. b) Since high titers of anti-GM1, GM2, and GD1b antibodies in humans appear to selectively destroy motor neuron-muscle synaptic contacts and cause motor neuron disease, we will study the role of gangliosides in regulating Ca2+, second messengers and synaptogenesis. c) We will determine the point mutations in the beta-Hex gene in patients with partial deficiency of the GM2-ganglioside degrading enzyme N-acetyl-beta-D-glucosaminidase (beta-Hex) in order to understand more about beta-Hex, how enzymes degrade GSL, and how better remedial therapy might be designed.
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1985 — 1994 |
Dawson, Glyn |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Opiate/Opioid Peptide Action
The objective of the project is to understand the mechanism of action of opiate and opioid peptides at the biochemical and physiological level by making use of neurotumor clonal cell lines which express different types of opiate receptors. The goals for the coming year are to continue studies on the cellular changes which result from acute or chronic exposure to opiates, specifically focussing on adenylate cyclase, protein kinase and glycosyltransferase linkage, and changes in ion fluxes and membrane potential. For these studies we will use cell lines N4TG1 and NG108-15 which express only DELTA (enkephalin) receptors, NCB-20, which express two BENZOMORPHAN (KAPPA AND SIGMA) receptors in addition to DELTA, and TCX-17 (which may express MU (MORPHINE) receptors in addition to DELTA receptors.
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1985 — 1986 |
Dawson, Glyn |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Mental Retardation Research Center
This program-project consists of studies aimed at the elucidation of certain aspects of mental retardation. Investigations will be concerned with the mechanisms of biosynthesis and control of biosynthesis of proteoglycans. Studies of the mechanism of differentiation will continue, using a tissue culture system which permits the change of chick limb bud mesenchyme to cartilage. It is hoped to study the changes in the cell during differentiation which lead to the synthesis of large amounts of cartilage-specific products, namely type II collagen, chondroitin sulfate proteolgycan, and link proteins. The genes for these proteins are being cloned in order to develop probes for measurement of changes in the genome or expression of appropriate mRNA. Clonal antibodies have been developed for the specific identification of core protein of chondroitin sulfate proteoglycans. A number of human heritable diseases involve defects in degradation of glycosaminoglycans, glycosphingolipids, and glycoproteins. Studies are continuing to elucidate the enzyme defects of these diseases as well as certain animal models. It is hoped that these may serve as bases for improved methods of diagnosis and perhaps point to future methods of treatment. Cultured cells of neural origin are being used to study the mechanism of synthesis of glycoproteins and glycolipids and the possible effects thereon of opiates, enkephalins and related compounds. The fine structure of the human cortex in normal and mentally retarded individuals is being investigated as well as dendritic development in a tissue culture system.
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1986 — 1988 |
Dawson, Glyn |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Glycolipid Metabolism and Motor Neuron Disease
Amyotrophic lateral sclerosis (ALS) is a devastating, progrssive degenerative disease of motor neurons (typically manifesting itself in middle age), which results in muscle wasting, paralysis and death. Since very little is known about the cause or precise pathogenesis of ALS, we propose to test the hypothesis that the disease can result from abnormal glycosphingolipid (GSL) (ganglioside) metabolism. We will structurally characterize the 3 major abnormal gangliosides already detected in all 9 ALS spinal cords thus far examined, but absent from non-ALS spinal cords, and will extend the search to ALS muscle and motor neuron-rich regions of the brain, including both gangliosides and neutral hexosamine-containing glycolipids. Structural analysis will require a combination of thin-layer chromatography, specific glycosidases, GLS-mass-spectrometry and specific monoclonal antibodies. Comparison will be made with GSL from unaffected brain regions. Fibroblasts from patients with various types of GM2-gangliosidosis, including a 25 year-old patient with a previously undescribed specific deficiency of N-acetyl-B-D-glucosaminidase B (Hex B) and typical ALS-like symptoms will be assayed for ability to degrade these gangliosides. Polyclonal antibodies to Hex B Alpha- or Beta-chains will be used to determine if Hex B is undersynthesized in these cells and if Heb B or a novel isoenzyme predominates in motor neurons. Finally, all 100 or more ALS patients seen at the University of Chicago ech year will be tested for Hex A and B levels and isoenzyme patterns. Our long-term objective is to show that abnormal accumulation of certain hexosamine-containing gangliosides in ALS motor neurons results in the type of neuronal degeneration seen in lysosomal storage diseases or a failure in motor neuron sprouting, which leads to progressive loss of neuromotor function. Such a finding would form the basis for ALS therapy.
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1991 — 1993 |
Dawson, Glyn |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Lipase and Cathespin Abnormalities in Batten Disease
The long-term objective of this proposal is to understand the biochemical basis of the three major forms (infantile, late infantile, and juvenile) of neuronal ceroid lipofuscinosis (NCL) or Batten disease. This will permit precise diagnosis of NCL, detection of heterozygotes, and facilitate new forms of therapy for this devastating neurodegenerative disease which causes retinal degeneration and loss of all neurofunctions in 3000 children each year. We will test a hypothesis that a deficiency of lysosomal phospholipase A1 (PLA1) can lead to the biochemical and pathological changes observed in Batten disease. To achieve this, we will first purify the PLA1 from human tissue and determine the conditions for optimum activity and substrate specificity. We will determine PLA1 activity in tissue homogenates prepared from patients with Batten disease to verify our initial data which suggests a marked deficiency. We will assay activity in immortalized lymphocytes from obligate NCL carriers to verify that PLA1 is the primary defect by showing 50% of normal activity in these carriers. We will then prepare polyclonal antibody to PLA1 and use it to study synthesis, processing, and subcellular localization of PLA1 in normal versus mutant cells. Because of the importance of animal models in devising therapy for storage diseases, we will attempt to confirm the PLA1 deficiency in English Setters with NCL, and use tissue from the obligate carriers to devise a reliable carrier test. Finally, in order to understand the pathogenesis of NCL, we will investigate how a lysosomal PLA1 deficiency can give rise to a "functional protease deficiency", involving primarily central system neurons and pigmentary epithelial cells of the retina. We will attempt to recreate the disease in vitro with the proposed pathogenic agent 4-hydroxynonenal, study two patients with apparent primary cathepsin H deficiency and clinical symptoms resembling NCL, and prepare [125I]labeled rhodopsin, and the putative major storage peptide in NCL (the C-subunit of mitochondrial ATP synthase) for use in in vitro assays with NCL tissue.
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1994 — 1996 |
Dawson, Glyn |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Lipase and Cathepsin Abnoralities in Batten Disease
This is a renewal of an application whose broad, long-term objectives are to understand the genetic and biochemical basis of Batten disease (Neuronal Ceroid Liofuscinosis; NCL). Particular emphasis will be placed on understanding the late-infantile (NCL2) and juvenile (NCL3) forms of the disease since these are common forms of inherited blindness, seizures and dementia in the U.S. We will test the hypothesis that NCL results from the inability to transport and degrade a normal mitochondrial protein (the DCCD-binding, proteolipid subunit c of mitochondrial ATP synthase (c- peptide for short). The lysosomal storage of this peptide, predominantly in neurons, retinal pigmentary epithelial cells and pancreas, is then hypothesized to give rise to clinical symptoms common to many lysosomal storage diseases. We will purify the c-peptide from NCL tissue, label it with [14C]DCCD or [125I], identify it by specific polyclonal antibody, check for any abnormal post-translational modifications such as trimethyl- lysine residues, and follow its catabolism after loading of normal and NCL fibroblasts and lymphoblastoid cell lines. This will only give indirect evidence so we have obtained cDNA for sheep c-peptide by PCR, spliced into a plasmid vector which contains a cytomegalovirus promoter etc. and stably overexpressed the gene in neuroblastoma cell line F-11. These and related lines are a model for the CNS. We will also transfect both normal and NCL lymphoblastoid cell lines. We will attempt to create autofluorescent pigment storage and then show that this also results in dolichol sugar accumulation, lysosomal phospholipase A1 inhibition.and other biochemical characteristics of NCL. We will determine if the ubiquitin pathway is normally involved in c-peptide disposal, look for a c-peptide transport protein by c-peptide affinity chromatography of brain extracts and look for c-peptide in lipoprotein complexes by their flotation densities in the range of 1.063-1.21g/ml on cesium chloride gradients in the ultracentrifuge. Any mutation will be assayed in the large number of cell lines available from patients, obligate heterozygotes (50% of normal activity) and a reliable diagnostic test will be devised. Understanding the defect in NCL will clearly open up a, whole new area of cell biology dealing with the transport and disposal of hydrophobic peptides. Finally, the, ability to detect carriers will have tremendous value in combatting this devastating neurodegenerative disorder of children.
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1999 — 2008 |
Dawson, Glyn |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Pathogenesis of Batten Disease
This project is aimed at understanding the biochemical pathology of Batten disease (a devastating mental retardation syndrome), using lymphoblastoid cell lines and fibroblast cultures from patients and neural tissues cultures similar to those used in the other projects. In the infantile form (CLN1) we will focus on the substrate specificity on the defective palmitoyl: protein thioesterase, the storage material and the source of this storage material from neural membrane turnover. In the late infantile form (CLN2) we will focus on the substrate specificity of the defective pepstatin-insensitive carboxyl peptidase, the storage material and its source, and in the juvenile form (CLN3) we will focus on the possible role of the protein gene product in facilitating proteolysis in the lysosome.
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2003 |
Dawson, Glyn |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Conference--Neuronal Ceroid Lipofuscinosis
DESCRIPTION (provided by applicant): Conference Support is sought for the Ninth International Congress on the Ceroid Lipofuscinoses to be held in Chicago, Illinois from April 10, 2003 to April 13, 2003. Neuronal Ceroid Lipofuscinoses (Batten Disease) are a group of inherited neurodegenerative disorders characterized by blindness, recurrent seizures and progressive, severe mental retardation. They are the most common neurodegenerative diseases of children and their pathological hallmark is the accumulation of auto fluorescent pigment, primarily in neurons. In the last 7 years, mutations in at least 8 different genes have been shown to result in a form of Batten disease. Two of these, palmitoyl; protein thioesterase (PPT1) and tripeptidylpeptidase (TPP 1) are mutated in CLN1 and CLN2 respectively and behave as lysosomal hydrolases. The genes involved in CLN3, 5, 6 and 8 code for membrane proteins of unknown function. An animal model of NC1 results from mutations in cathepsin D and the working hypothesis is that all these genes code for proteins involved in intracellular trafficking of neuronal proteins destined for catabolism. The development of transgenic animal models of the various forms of NCL has led to exciting new insights and vehicles for therapeutic endeavors. The Conference will focus on human genetics, diagnostics, mechanisms of intracellular trafficking and how this information can be used to devise therapeutic strategies. Particular emphasis will be put on drug, gene and stem-cell based therapies. The importance of the Conference is that it uniquely brings together senior scientists and junior investigators from all parts of the globe to foster exchange of research data and to stimulate future collaborative efforts aimed at eliminating this group of devastating diseases.
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2005 |
Dawson, Glyn |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Tenth International Congress On Ceroid Lipofuscinoses
DESCRIPTION (provided by applicant): Conference support is sought for the Tenth International Congress on Ceroid Lipofuscinoses (Battens disease) to be held at the Hanasaari Congress Center, Helsinki, Finland from June 8th to 12th, 2005. We anticipate 130 participants from all over the world and support is requested for some of the US participants. Matching funds are being solicited from the EU. The Congress center has capacity to accommodate 92 persons and the rest of the participants can be accommodated at nearby hotels. It makes an ideal congress venue due to the fact that the participants are gathered together but on the other hand it is readily accessible from Helsinki. The Conference center is fully equipped for handicapped access. This is essential both for the participation of scientists and for the ability of parents and their wheelchair bound children to attend and participate in the meeting. The Battens group of childhood neurodegenerative disorders involve at least 10 different genes and collectively are the most common inherited neurodegenerative diseases of children. They are characterized by vision loss, seizures and mental retardation. All are believed to involve impaired lysosomal protein degradation although only two of the genes involved, palmitoyl;protein thioesterase and tripeptidylpeptidase, have been identified as enzymes. The proteins coded by CLN3, 5,6 and 8 are of unknown function and one focus of this meeting will be to use proteomics, in combination with biochemistry and cell biology, to try to determine this function. Other potentially exciting areas to be covered will include the use of cord blood-derived stem cells, gene therapy, nutritional therapy, drug therapy and the use of transgenic animal models to develop effective treatments. The previous 9 Congresses have brought together physicians, basic researchers and parents and have provided a forum for young researchers, especially females, to present their work orally and participate in lively discussions. Helsinki was the site of the 6th Congress and the practice has been to alternate the Conference between Europe and the USA, resulting in the establishment of international collaborative research and facilitating better understanding of this devastating disease.
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2011 — 2015 |
Dawson, Glyn |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Proj 4: Chemical Chaperone Therapy of Batten Disease
PROJECT SUMMARY (See instructions): Pharmacological chaperones (eg: AcGDap(Palm)VKIKK)can be internalized by cells and re-fold misfolded proteins to an active configuration, but need to cross the blood brain barrier, enter neurons and escape endosomes. We have shown that the palmitoylated peptide motif is uniquely able to allow drugs to escape endosomes and that such chaperones can reactivate misfolded proteins such as palmitoyhprotein thioesterase (PPTl). We now propose to design sequences to permit the chaperones to cross the blood brain barrier by either attaching a fluorophore and target motif (eg: rabies virus glycoprotein coat peptide (RVG)) or coating on 6-1 Onm quantum dots. We will test short sequences of proline and histidine, with a glutamine spacer to attach to the surface of 635 nm red QDs capped with the 4-thiol PEG ligand. We will test this in cultured postnatal neurons, and lymphoblasts from patients with defined point mutations in PPTl origin, and then use either the E3 embryonic chick spinal cord injection system, in collaboration with Project I, or the rat hippocampal slice system through collaboration with Project II. The three model systems represent embryonic brain, neonatal brain and postnatal brain and the collaborations will allow us to better assess the toxicity (if any) of these drugs, their efficacy and their ultimate cellular distribution by adding our chaperones into their experimental systems. We will also test the idea that the palmitoylated peptide motif works by specifically localizing to lipid rafts, microdomains in membranes which are greatly enriched in cholesterol and sphingolipids and appear to be used to assemble signaling complexes. Chaperones can only treat 20-50% of the mutations so for the remaining 50-80% of INCL patients we propose that hydrophobic thiols such as thiocholesterol could chemically facilitate hydrolysis of the storage material itself. Finally we will extend our approach to the most common form of Batten disease caused by mutations in the CLN2 gene (tripepfidyl-peptidase-1). Compound heterozygote patients with milder disease should benefit from chaperone therapy based on borate complexes of the inhibitor (AAFX) delivered to the CNS with our unique peptide sequences either directly or coated on quantum dots.
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