- Email: [email protected]
28. Alpha-synuclein aggregation in Gaucher patients and carriers with synucleinopathies
S14
Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41
the effectiveness of intrathecal ERT as a treatment for another neurological symptom of MPS I, cognitive decline. This study is a 24-month open-label prospective randomized study in 16 MPS I patients age 6 or older who have documented evidence of cognitive decline. The study will test the safety and efficacy of intrathecal recombinant human a-L-iduronidase (rhIDU) to reduce or stabilize cognitive decline by assessing the subjects at baseline with neuropsychological, clinical, radiological, and biochemical evaluations and then monitoring the change in these parameters during a regimen of first monthly, then quarterly intrathecal treatments with rhIDU. The study will randomize subjects to a treatment and a control group for 12 months, and then all subjects will receive treatment on a 12-month open-label continuation. If shown to be effective, intrathecal ERT would be the only treatment for cognitive decline in patients who do not qualify for and/or are unable to have hematopoietic stem cell transplantation. doi:10.1016/j.ymgme.2009.10.044
28. Alpha-synuclein aggregation in Gaucher patients and carriers with synucleinopathies Jae Choia, Barbara Stubblefielda, Ozlem Goker-Alpana, Mark Cooksonb, Nahid Tayebia, Ellen Sidranskya, aSectionon Molecular Neurogenetics, Medical Genetics Branch, NHGRI, National Institutes of Health, Bethesda, MD, USA, bCell Biology and Gene Expression Unit, Laboratory of Neurogenetics, NIA, National Institutes of Health, Bethesda, MD, USA Recent findings demonstrate an increased frequency of mutations in glucocerebrosidase (GBA), the enzyme deficient in the lysosomal storage disorder, Gaucher disease (GD), among patients with synucleinopathies. Some patients have been observed to develop both GD and parkinsonism. Neuropathology revealed Lewy bodies and synuclein-positive inclusions in the vulnerable regions. In this study, samples from cerebral cortex from subjects with different synucleinopathies and GBA mutations were extracted. These include samples from patients with Lewy body dementia, Lewy body variant Alzheimers disease and normal controls with and without GBA mutations. There were ten subjects with GBA mutations, four of them carrying homozygous mutations. The samples were homogenized and fractionated into total (PBSsoluble), soluble (SDS-soluble) and insoluble (Urea-soluble) fractions. Upon examination, most patients with GD with synucleinopathies were shown to exhibit aggregation of oligomeric forms of alpha-synuclein in the insoluble fraction, while others showed monomeric form of alpha-synuclein in the same fraction. Those with extensive Lewy body pathologies tended to have more of the oligomeric forms of alphasynuclein. These studies indicate that patients with synucleinopathies carrying GBA mutations show biochemical characteristics typical of Lewy body disorders. doi:10.1016/j.ymgme.2009.10.045
29. Genetic and pharmacological chaperone modulation of brain GCase activity affects synuclein accumulation in mice Sean Clarka, Ying Sunb, You-Hai Xub, Lee Pellegrinoa, Yi Luna, Michelle Frascellaa, Richie Khannaa, Gregory Grabowskib, Brandon Wustmana, Ken Valenzanoa, David Lockharta, aAmicus Therapeutics, Cranbury, USA, bDivision of Human Genetics, Cincinnati Childrens Hospital Medical Center, Cincinnati, USA Pharmacological chaperones are orally available small molecules that bind and stabilize a target protein, and increase cellular trafficking, levels and activity. Parkinsons disease (PD) and Dementia with Lewy Bodies (DLB) share both the accumulation of – synuclein in the brain and a genetic risk factor: mutations in Gba which encodes the lysosomal enzyme acid b-glucocerebrosidase (GCase). While Gba mutations occur at a frequency of 0.4–6.2% in the general population, Gba carriers are found at a frequency of 2.3–31.3% in idiopathic PD, and up to 23% in DLB. In support of a biochemical basis for this genetic association, a Gba mutant mouse with reduced GCase activity was found to accumulate both glucosylceramide (the substrate of GCase) and endogenous synuclein in the brain. We tested the hypothesis that an increase in GCase activity could prevent synuclein accumulation in mice that express normal levels of endogenous, wild-type GCase but that overexpress wild-type human synuclein. We used the pharmacological chaperone AT 2101 to increase wild-type GCase enzyme levels in the brain. Oral administration of AT 2101 for 12 weeks to mice overexpressing human synuclein prevented the accumulation of synuclein in the hippocampus that otherwise occurs in untreated animals. In addition, we have investigated the effects of pharmacological chaperones in mice that express even higher levels of human synuclein (the Thy-1-hSNCA model), and that have motor deficits as well as synuclein deposits even at an early age. doi:10.1016/j.ymgme.2009.10.046
30. Open-label Phase I/II clinical trial of pyrimethamine for the treatment of chronic GM2 gangliosidosis Joe Clarkea, Edwin Kolodnyb, Don Mahurana, Maria Fullerc, Michael Tropaka, Jack Keimeld, Swati Satheb, Sophia Pesotchinskye, Brigette Rigata, aResearch Institute, Hospital for Sick Children, Toronto, Canada, bNew York University Medical Center, New York, USA, cWomens and Childrens Hospital, Adelaide, Australia, dUniversity of Minnesota, Minneapolis, MN, USA, eLSVP International, Inc., Los Altos, USA Background: Chronic GM2 gangliosidosis (CGM2G) is a lysosomal storage disease caused by deficiency of hexosaminidase A (Hex A) due to mutations in either the HEXA or HEXB genes. The disease is often the result of misfolding and premature degradation of the mutant polypeptide. In cellulo studies showed that mutant enzyme activity was enhanced by exposure to pyrimethamine (PYR), a competitive inhibitor of Hex A. Aim: This is a Phase I/II clinical trial to evaluate the safety and efficacy of PYR administered to adults with CGM2G. Methodology: It is an open-label, two-center, dose escalation study, with plans to enroll a total of 20 patients. In addition to safety data, information on efficacy is collected by measurement of plasma PYR levels, leukocyte and plasma Hex A activities using MUGS as substrate, and leukocyte and plasma GM2 ganglioside levels. Patients start on 25 mg PYR increasing every 4 weeks to a final dosage of 100 mg per day. All patients receive folinic acid 5 mg daily throughout the study. Results: A total of 10 patients have been enrolled (7 male, 3 female), ages 22– 50 yrs. The drug is well-tolerated. Preliminary analyses show significant enhancement of Hex A activities in leukocytes. Some patients exhibited subjective improvement in speech and muscle power. Conclusion: The clinical trial is progressing without significant adverse events and with some preliminary evidence that PYR treatment results in enhanced Hex A activity in leukocytes of patients with CGM2G. doi:10.1016/j.ymgme.2009.10.047
31. TRPML1 downregulation is associated with changes in lysosomal enzyme levels Grace Colletti, Mark Miedel, Yi Wang, Ora A. Weisz, Kirill Kiselyov, University of Pittsburgh, Pittsburgh, USA Mucolipidosis Type IV (MLIV) is an autosomal recessive, neurodegenerative lysosomal storage disorder that results from inactivating mutations in the gene MCOLN1 that encodes the lysosomal ion channel Mucolipin-1 (TRPML1). TRPML1s function in the lysosome is undefined; however there is evidence supporting two models for TRPML1 function. The ‘‘trafficking’’ model suggests that TRPML1 plays a role in the trafficking or fusion of vesicles along the endocytic pathway. The ‘‘metabolic’’ model argues that TRPML1 plays a role in regulating the ionic environment of the lysosome. Recent data have shown that TRPML1 may regulate lysosomal pH as well as iron uptake, supporting the latter model [Miedel, 2008. Journal of Experimental Medicine; Dong, 2008. Nature]. TRPML1-mediated changes in lysosomal homeostasis could lead to either reversible or chronic inactivation of lysosomal enzymes. To test this possibility, we have used proteomic approaches to identify changes in lysosomal enzyme levels or processing upon RNAi mediated TRPML1 loss. Lysosome-enriched subcellular fractions and whole cell extracts were subjected to 2-D DIGE and western blot analyses. These studies revealed changes in the proteins Annexin 2A, Cathepsin B, and Lysosomal acid lipase (LAL). Annexin 2A and Cathepsin B have dramatically increased levels upon TRPML1 loss, while LAL levels are decreased. These data provide insight into the early pathology of MLIV and suggest a novel role for TRPML1 in regulating lysosomal function. doi:10.1016/j.ymgme.2009.10.048
32. Distinct features of disease phenotypes in two genetic models of NCL Susan Cotmana, Yi Caoa, Sunita Biswasa, Pavlina Wolfa, Ashish Masseyb, Ana Maria Cuervob, Marcy MacDonalda, Jong-Min Leea, aMassachusetts General Hospital, Boston, MA, USA, bAlbert Einstein College of Medicine, Bronx, NY, USA CLN3 and CLN6 are novel genes mutated in juvenile and late-infantile Neuronal Ceroid Lipofuscinosis (NCL), respectively. Research suggests that CLN3 and CLN6 proteins reside in late endosomes and lysosomes and endoplasmic reticulum, respectively, and that their loss of function leads to NCL disease with strongly overlapping features including lysosomal accumulation of subunit c of the mitochondrial F0 ATP synthase. Here, we sought to establish precise genetic models of these two forms of NCL for genotype–phenotype studies aimed at testing the hypothesis
Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41
the effectiveness of intrathecal ERT as a treatment for another neurological symptom of MPS I, cognitive decline. This study is a 24-month open-label prospective randomized study in 16 MPS I patients age 6 or older who have documented evidence of cognitive decline. The study will test the safety and efficacy of intrathecal recombinant human a-L-iduronidase (rhIDU) to reduce or stabilize cognitive decline by assessing the subjects at baseline with neuropsychological, clinical, radiological, and biochemical evaluations and then monitoring the change in these parameters during a regimen of first monthly, then quarterly intrathecal treatments with rhIDU. The study will randomize subjects to a treatment and a control group for 12 months, and then all subjects will receive treatment on a 12-month open-label continuation. If shown to be effective, intrathecal ERT would be the only treatment for cognitive decline in patients who do not qualify for and/or are unable to have hematopoietic stem cell transplantation. doi:10.1016/j.ymgme.2009.10.044
28. Alpha-synuclein aggregation in Gaucher patients and carriers with synucleinopathies Jae Choia, Barbara Stubblefielda, Ozlem Goker-Alpana, Mark Cooksonb, Nahid Tayebia, Ellen Sidranskya, aSectionon Molecular Neurogenetics, Medical Genetics Branch, NHGRI, National Institutes of Health, Bethesda, MD, USA, bCell Biology and Gene Expression Unit, Laboratory of Neurogenetics, NIA, National Institutes of Health, Bethesda, MD, USA Recent findings demonstrate an increased frequency of mutations in glucocerebrosidase (GBA), the enzyme deficient in the lysosomal storage disorder, Gaucher disease (GD), among patients with synucleinopathies. Some patients have been observed to develop both GD and parkinsonism. Neuropathology revealed Lewy bodies and synuclein-positive inclusions in the vulnerable regions. In this study, samples from cerebral cortex from subjects with different synucleinopathies and GBA mutations were extracted. These include samples from patients with Lewy body dementia, Lewy body variant Alzheimers disease and normal controls with and without GBA mutations. There were ten subjects with GBA mutations, four of them carrying homozygous mutations. The samples were homogenized and fractionated into total (PBSsoluble), soluble (SDS-soluble) and insoluble (Urea-soluble) fractions. Upon examination, most patients with GD with synucleinopathies were shown to exhibit aggregation of oligomeric forms of alpha-synuclein in the insoluble fraction, while others showed monomeric form of alpha-synuclein in the same fraction. Those with extensive Lewy body pathologies tended to have more of the oligomeric forms of alphasynuclein. These studies indicate that patients with synucleinopathies carrying GBA mutations show biochemical characteristics typical of Lewy body disorders. doi:10.1016/j.ymgme.2009.10.045
29. Genetic and pharmacological chaperone modulation of brain GCase activity affects synuclein accumulation in mice Sean Clarka, Ying Sunb, You-Hai Xub, Lee Pellegrinoa, Yi Luna, Michelle Frascellaa, Richie Khannaa, Gregory Grabowskib, Brandon Wustmana, Ken Valenzanoa, David Lockharta, aAmicus Therapeutics, Cranbury, USA, bDivision of Human Genetics, Cincinnati Childrens Hospital Medical Center, Cincinnati, USA Pharmacological chaperones are orally available small molecules that bind and stabilize a target protein, and increase cellular trafficking, levels and activity. Parkinsons disease (PD) and Dementia with Lewy Bodies (DLB) share both the accumulation of – synuclein in the brain and a genetic risk factor: mutations in Gba which encodes the lysosomal enzyme acid b-glucocerebrosidase (GCase). While Gba mutations occur at a frequency of 0.4–6.2% in the general population, Gba carriers are found at a frequency of 2.3–31.3% in idiopathic PD, and up to 23% in DLB. In support of a biochemical basis for this genetic association, a Gba mutant mouse with reduced GCase activity was found to accumulate both glucosylceramide (the substrate of GCase) and endogenous synuclein in the brain. We tested the hypothesis that an increase in GCase activity could prevent synuclein accumulation in mice that express normal levels of endogenous, wild-type GCase but that overexpress wild-type human synuclein. We used the pharmacological chaperone AT 2101 to increase wild-type GCase enzyme levels in the brain. Oral administration of AT 2101 for 12 weeks to mice overexpressing human synuclein prevented the accumulation of synuclein in the hippocampus that otherwise occurs in untreated animals. In addition, we have investigated the effects of pharmacological chaperones in mice that express even higher levels of human synuclein (the Thy-1-hSNCA model), and that have motor deficits as well as synuclein deposits even at an early age. doi:10.1016/j.ymgme.2009.10.046
30. Open-label Phase I/II clinical trial of pyrimethamine for the treatment of chronic GM2 gangliosidosis Joe Clarkea, Edwin Kolodnyb, Don Mahurana, Maria Fullerc, Michael Tropaka, Jack Keimeld, Swati Satheb, Sophia Pesotchinskye, Brigette Rigata, aResearch Institute, Hospital for Sick Children, Toronto, Canada, bNew York University Medical Center, New York, USA, cWomens and Childrens Hospital, Adelaide, Australia, dUniversity of Minnesota, Minneapolis, MN, USA, eLSVP International, Inc., Los Altos, USA Background: Chronic GM2 gangliosidosis (CGM2G) is a lysosomal storage disease caused by deficiency of hexosaminidase A (Hex A) due to mutations in either the HEXA or HEXB genes. The disease is often the result of misfolding and premature degradation of the mutant polypeptide. In cellulo studies showed that mutant enzyme activity was enhanced by exposure to pyrimethamine (PYR), a competitive inhibitor of Hex A. Aim: This is a Phase I/II clinical trial to evaluate the safety and efficacy of PYR administered to adults with CGM2G. Methodology: It is an open-label, two-center, dose escalation study, with plans to enroll a total of 20 patients. In addition to safety data, information on efficacy is collected by measurement of plasma PYR levels, leukocyte and plasma Hex A activities using MUGS as substrate, and leukocyte and plasma GM2 ganglioside levels. Patients start on 25 mg PYR increasing every 4 weeks to a final dosage of 100 mg per day. All patients receive folinic acid 5 mg daily throughout the study. Results: A total of 10 patients have been enrolled (7 male, 3 female), ages 22– 50 yrs. The drug is well-tolerated. Preliminary analyses show significant enhancement of Hex A activities in leukocytes. Some patients exhibited subjective improvement in speech and muscle power. Conclusion: The clinical trial is progressing without significant adverse events and with some preliminary evidence that PYR treatment results in enhanced Hex A activity in leukocytes of patients with CGM2G. doi:10.1016/j.ymgme.2009.10.047
31. TRPML1 downregulation is associated with changes in lysosomal enzyme levels Grace Colletti, Mark Miedel, Yi Wang, Ora A. Weisz, Kirill Kiselyov, University of Pittsburgh, Pittsburgh, USA Mucolipidosis Type IV (MLIV) is an autosomal recessive, neurodegenerative lysosomal storage disorder that results from inactivating mutations in the gene MCOLN1 that encodes the lysosomal ion channel Mucolipin-1 (TRPML1). TRPML1s function in the lysosome is undefined; however there is evidence supporting two models for TRPML1 function. The ‘‘trafficking’’ model suggests that TRPML1 plays a role in the trafficking or fusion of vesicles along the endocytic pathway. The ‘‘metabolic’’ model argues that TRPML1 plays a role in regulating the ionic environment of the lysosome. Recent data have shown that TRPML1 may regulate lysosomal pH as well as iron uptake, supporting the latter model [Miedel, 2008. Journal of Experimental Medicine; Dong, 2008. Nature]. TRPML1-mediated changes in lysosomal homeostasis could lead to either reversible or chronic inactivation of lysosomal enzymes. To test this possibility, we have used proteomic approaches to identify changes in lysosomal enzyme levels or processing upon RNAi mediated TRPML1 loss. Lysosome-enriched subcellular fractions and whole cell extracts were subjected to 2-D DIGE and western blot analyses. These studies revealed changes in the proteins Annexin 2A, Cathepsin B, and Lysosomal acid lipase (LAL). Annexin 2A and Cathepsin B have dramatically increased levels upon TRPML1 loss, while LAL levels are decreased. These data provide insight into the early pathology of MLIV and suggest a novel role for TRPML1 in regulating lysosomal function. doi:10.1016/j.ymgme.2009.10.048
32. Distinct features of disease phenotypes in two genetic models of NCL Susan Cotmana, Yi Caoa, Sunita Biswasa, Pavlina Wolfa, Ashish Masseyb, Ana Maria Cuervob, Marcy MacDonalda, Jong-Min Leea, aMassachusetts General Hospital, Boston, MA, USA, bAlbert Einstein College of Medicine, Bronx, NY, USA CLN3 and CLN6 are novel genes mutated in juvenile and late-infantile Neuronal Ceroid Lipofuscinosis (NCL), respectively. Research suggests that CLN3 and CLN6 proteins reside in late endosomes and lysosomes and endoplasmic reticulum, respectively, and that their loss of function leads to NCL disease with strongly overlapping features including lysosomal accumulation of subunit c of the mitochondrial F0 ATP synthase. Here, we sought to establish precise genetic models of these two forms of NCL for genotype–phenotype studies aimed at testing the hypothesis