Greenwood Genetic Center Inc

PATHOGENIC MECHANISMS OF LYSOSOMAL DISEASE

CLONING GENES THAT CAUSE MENTAL RETARDATION

IDENTIFICATION OF NOVEL X-LINKED INTELLECTUAL DISABILITY GENES

PATHOGENIC MECHANISMS OF LYSOSOMAL DISEASE - PROJECT SUMMARY MAINTAINING NORMAL ACTIVITY OF LYSOSOMES IS ESSENTIAL FOR HUMAN HEALTH, AS EVIDENCED BY THE MANY RARE DISORDERS CAUSED BY DEFECTS IN LYSOSOMAL BIOGENESIS AND FUNCTION. HOW LYSOSOMAL DYSFUNCTION LEADS TO THE TISSUE-SPECIFIC PHENOTYPES ASSOCIATED WITH LYSOSOMAL STORAGE DISORDERS (LSDS) IS POORLY UNDERSTOOD. OUR LONG- TERM GOAL IS TO DEFINE THESE PATHOGENIC MECHANISMS, AND USE THIS INFORMATION TO IDENTIFY NEW THERAPEUTIC STRATEGIES. OUR RECENT EFFORTS HAVE FOCUSED ON THE INVESTIGATION OF SECRETED PROTEASES AS KEY INITIATORS OF DISEASE IN MUCOLIPIDOSIS II (MLII). IN MLII, THE ENZYME (GLCNAC-1-PHOSPHOTRANSFERASE) THAT SYNTHESIZES THE CARBOHYDRATE-BASED TAG NEEDED FOR RECEPTOR-MEDIATED LYSOSOMAL TARGETING IS MISSING. THIS CAUSES CATHEPSIN PROTEASES TO BE SECRETED OUTSIDE THE CELL WHERE THEY BECOME ACTIVATED. USING POWERFUL ZEBRAFISH TOOLS, WE ESTABLISHED THAT THESE SECRETED CATHEPSIN PROTEASES ALTER THE KEY SIGNALING EVENTS THAT CONTROL NORMAL CARTILAGE AND CARDIAC DEVELOPMENT. THE CURRENT PROPOSAL WILL EXTEND THESE STUDIES AND TEST THE CENTRAL HYPOTHESIS THAT CATHEPSIN-MEDIATED MECHANISMS ARE PATHOGENIC DRIVERS ACROSS MULTIPLE LSDS WITH DIFFERENT ETIOLOGIES. THIS EFFORT IS PREMISED ON OBSERVATIONS THAT DEMONSTRATE: I) CATHEPSIN ACTIVITY IS ALTERED IN OTHER LSDS INCLUDING SIALIDOSIS (NEU1) AND MPSIVA (GALNS), II) INCREASED LYSOSOMAL EXOCYTOSIS IS ASSOCIATED WITH ALTERED CATHEPSIN ACTIVITY, AND III) EXTRACELLULAR CATHEPSIN K (CTSK) ACTIVITY IS MODULATED BY SPECIFIC GLYCOSAMINOGLYCANS (GAGS). WE WILL USE A UNIQUE AND INNOVATIVE SET OF TOOLS IN THE ZEBRAFISH SYSTEM TO INVESTIGATE THE PROTEASE- MEDIATED PATHOGENESIS IN CARTILAGE, WITH THE GOAL OF IDENTIFYING NOVEL DISEASE MECHANISMS FOR THESE LSDS. WE BELIEVE THIS WILL POINT TO NEW MODES OF TREATMENT FOR DISORDERS LIKE MLII AND SIALIDOSIS (WHERE NO APPROVED THERAPIES EXIST), AND FOR MPSIVA (WHERE ENZYME REPLACEMENT HAS LIMITED EFFICACY IN CARTILAGE AND BONE). UNDERSTANDING HOW EXTRACELLULAR CATHEPSIN ACTIVITY IMPACTS DIFFERENT TISSUES IS ALSO CRUCIAL SINCE THERE ARE A GROWING NUMBER OF PROPOSED THERAPIES AIMED AT INCREASING LYSOSOMAL EXOCYTOSIS AS A MEANS OF RESOLVING LYSOSOMAL STORAGE. SUCH THERAPIES MAY POSITIVELY IMPACT CERTAIN TISSUES BUT RELEASE OF LYSOSOMAL ENZYMES IN OTHER TISSUES MAY HAVE UNEXPECTED AND DETRIMENTAL CONSEQUENCES. TO ADDRESS THE CENTRAL HYPOTHESIS, WE WILL PROFILE CATHEPSIN ACTIVITY AND TGFSS RELATED GROWTH FACTOR SIGNALING IN SIALIDOSIS AND MPSIVA ZEBRAFISH (AIM 1) AND ADDRESS WHETHER CHANGES IN THE ABUNDANCE OR TYPE OF GAGS PRESENT CAN TUNE THIS PROTEASE-DEPENDENT PATHOGENIC CASCADE (AIM 2). NEXT, WE WILL DEFINE THE EXTENT TO WHICH LYSOSOMAL EXOCYTOSIS DRIVES CARTILAGE PATHOLOGY IN THESE DISORDERS AND ASK IF MODULATING EXOCYTOSIS IMPROVES OR EXACERBATES PHENOTYPES (AIM 3). WE WILL ALSO INVESTIGATE THE NEURONAL PATHOGENESIS ASSOCIATED WITH NUS1 (NOGOB RECEPTOR; NGBR) DEFICIENCY, A NEWLY CHARACTERIZED DISORDER WITH LYSOSOMAL DYSFUNCTION AND CHOLESTEROL STORAGE. HERE WE WILL ASK IF THE NEURONAL PHENOTYPES STEM FROM LOSS OF NGBR OR NIEMANN-PICK TYPE C2 (NPC2) IN SPECIFIC CELL TYPES, AND WHETHER CHOLESTEROL ACCUMULATION IMPAIRS MYELINATION AND INCREASES LYSOSOMAL EXOCYTOSIS. (AIM 4).

PROMOTING GENETIC LITERACY IN STUDENTS AND TEACHERS: THE EFFECTIVENESS OF NON-CLA

PATHOGENIC MECHANISMS OF CONGENITAL DISORDERS OF GLYCOSYLATION - THE CONGENITAL DISORDERS OF GLYCOSYLATION (CDG) ARE A GROWING GROUP OF RARE INHERITED DISEASES CAUSED BY MUTATIONS IN GENES INVOLVED IN PROTEIN AND LIPID GLYCOSYLATION. OUR UNDERSTANDING OF THE MECHANISMS DRIVING CDG PATHOGENESIS REMAINS LIMITED, GREATLY IMPEDING DEVELOPMENT OF NEW THERAPIES. TO OVERCOME THIS BARRIER, OUR GROUP DEVELOPED AND CHARACTERIZED A ZEBRAFISH MODEL FOR THE MOST COMMON CDG, PMM2-CDG. PMM2- CDG RESULTS FROM MUTATIONS IN PHOSPHOMANNOMUTASE 2 (PMM2), WHICH ENCODES AN ENZYME THAT CONVERTS MANNOSE-6-PHOSPHATE (M6P) TO MANNOSE-1-PHOSPHATE (M1P). DEFECTS IN PMM2 LIMIT PRODUCTION OF LIPID-LINKED N-GLYCOSYLATION PRECURSORS, IMPAIRING PROTEIN GLYCOSYLATION AND CAUSING NUMEROUS CLINICAL MANIFESTATIONS. THE CONNECTION BETWEEN INDIVIDUAL MISGLYCOSYLATED PROTEINS AND DISEASE PHENOTYPES, HOWEVER, IS POORLY UNDERSTOOD. USING THE PMM2-CDG ZEBRAFISH MODEL (PMM2M/M), WE IDENTIFIED TWO CLASSES OF ENZYMES, THE PROTEIN PROCONVERTASES AND MATRIX METALLOPROTEINASES (MMPS), AS CANDIDATE DRIVERS OF PATHOLOGY. ANALYSES OF CARTILAGE DEFECTS IN PMM2 MUTANT ZEBRAFISH REVEALED A BLOCK IN EARLY CHONDROCYTE DEVELOPMENT THAT IS ASSOCIATED WITH DEFECTIVE PROCESSING OF THE CELL ADHESION MOLECULE N-CADHERIN, AND ALTERED ACTIVITY OF BOTH MMPS AND PROCONVERTASES THAT PROCESS N-CADHERIN. WE WILL TEST THE HYPOTHESIS THAT ALTERED GLYCOSYLATION FUNCTIONALLY IMPAIRS ONE OR MORE OF THESE ENZYMES, INITIATING A CASCADE OF ABERRANT PROCESSING THAT PREVENTS N- CADHERIN CLEAVAGE AND DISRUPTS CHONDROGENESIS. PARALLEL EFFORTS IDENTIFIED MULTIPLE METABOLITES THAT ARE ALTERED IN PMM2M/M EMBRYOS, INCLUDING ELEVATED LEVELS OF THE POLYOL SORBITOL. SORBITOL IS INCREASED IN PMM2-CDG PATIENTS AND ITS LEVEL CORRELATES WITH DISEASE SEVERITY. TREATMENT WITH EPALRESTAT, A DRUG UNDER EVALUATION FOR PMM2-CDG, REDUCED SORBITOL LEVELS AND PARTIALLY RESTORED CARTILAGE DEVELOPMENT IN PMM2M/M EMBRYOS. LIKEWISE, INHIBITING PROCONVERTASE ACTIVITY RESTORED SOME OF THE CARTILAGE PHENOTYPES, BUT FAILED TO ALLEVIATE THE PRONOUNCED CELLULAR VACUOLATION IN PMM2M/M CARTILAGE. THESE FINDINGS INDICATE THAT MULTIPLE PATHOGENIC MECHANISMS – ONE RELATED TO ALTERED PROTEASE FUNCTION AND N-CADHERIN PROCESSING, ANOTHER TO SORBITOL-DRIVEN CELLULAR STRESS – CONTRIBUTE TO PMM2-CDG DISEASE PATHOGENESIS. THIS GRANT WILL LEVERAGE A POWERFUL SUITE OF NOVEL ZEBRAFISH TOOLS TO UNRAVEL PMM2-CDG PATHOGENESIS AT THE MOLECULAR LEVEL, WITH THE LONG-TERM GOAL OF BROADLY DEFINING HOW DEFECTS IN CDG GENES CAUSE DISEASE AND USING THIS INFORMATION TO IDENTIFY THERAPIES. THE STUDIES IN AIM 1 WILL INVESTIGATE THE MECHANISMS LINKING ALTERED ACTIVITY OF PROCONVERTASES AND MMPS TO ABERRANT N-CADHERIN PROCESSING, ADDRESSING HOW PROTEIN-SPECIFIC MISGLYCOSYLATION DRIVES THESE PHENOTYPES. IN AIM 2, MULTIPLE APPROACHES WILL BE USED TO MODULATE ENZYMES INVOLVED IN SUGAR METABOLISM AND POLYOL PRODUCTION TO DEFINE THEIR ROLE IN PMM2-CDG CARTILAGE PATHOGENESIS. AIM 3 TAKES ADVANTAGE OF NEW ZEBRAFISH MUTANTS IN THE OLIGOSACCHARYLTRANSFERASE (OST) COMPLEX TO STUDY THE RELEVANCE OF THESE MECHANISMS IN CDG THAT DISRUPT OTHER STEPS WITHIN THE N-GLYCOSYLATION PATHWAY.

NOVEL METABOLIC BIOMARKER FOR AUTISM SPECTRUM DISORDER

APNEA INDEX AS AN OUTCOME MEASURE OF IGF-1 TREATMENT IN RETT SYNDROME

STRATEGIC INITIATIVE

BIRTH DEFECTS CONFERENCE SUPPORT FOR 2010 DAVID W. SMITH WORKSHOP ON MALFORMATION

TOPIC AREA NUMBER: NCBDDD-300.1 BIRTH DEFECTS CONFERENCE SUPPORT FOR 2009 DAVID W

BIRTH DEFECT CONF SUPPORT FOR 2008 DAVID W. SMITH WORKSHOP ON MALFORMATIONS & MOR

THIRD INTERNATIONAL SCIENTIFIC WORKSHOP ON GLYCOPROTEINOSES