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Don J Mahuran - One of the best experts on this subject based on the ideXlab platform.
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systemic gene transfer of a hexosaminidase variant using an scaav9 47 vector corrects GM2 gangliosidosis in sandhoff mice
Human Gene Therapy, 2016Co-Authors: Karlaina J L Osmon, Don J Mahuran, Patrick Thompson, Steven J. Gray, Brian L. Mark, Evan Woodley, Subha Karumuthilmelethil, John G Keimel, Jagdeep S. WaliaAbstract:GM2 gangliosidosis is a group of neurodegenerative diseases caused by β-hexosaminidase A (HexA) enzyme deficiency. There is currently no cure. HexA is composed of two similar, nonidentical subunits, α and β, which must interact with the GM2 activator protein (GM2AP), a substrate-specific cofactor, to hydrolyze GM2 Ganglioside. Mutations in either subunit or the activator can result in the accumulation of GM2 Ganglioside within neurons throughout the central nervous system. The resulting neuronal cell death induces the primary symptoms of the disease: motor impairment, seizures, and sensory impairments. This study assesses the long-term effects of gene transfer in a Sandhoff (β-subunit knockout) mouse model. The study utilized a modified human β-hexosaminidase α-subunit (μ-subunit) that contains critical sequences from the β-subunit that enables formation of a stable homodimer (HexM) and interaction with GM2AP to hydrolyze GM2 Ganglioside. We investigated a self-complementary adeno-associated viral (scAAV)...
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Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 Ganglioside in vivo.
Molecular therapy. Methods & clinical development, 2016Co-Authors: Michael B. Tropak, Sayuri Yonekawa, Subha Karumuthil-melethil, Patrick Thompson, Warren W. Wakarchuk, Steven J. Gray, Jagdeep S. Walia, Brian L. Mark, Don J MahuranAbstract:Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 Ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 Ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 Ganglioside levels.
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Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2 Gangliosidosis in Sandhoff Mice.
Human gene therapy, 2016Co-Authors: Karlaina J L Osmon, Don J Mahuran, Subha Karumuthil-melethil, Patrick Thompson, Steven J. Gray, Brian L. Mark, Evan Woodley, John G Keimel, Katalina Ong, Jagdeep S. WaliaAbstract:GM2 gangliosidosis is a group of neurodegenerative diseases caused by β-hexosaminidase A (HexA) enzyme deficiency. There is currently no cure. HexA is composed of two similar, nonidentical subunits, α and β, which must interact with the GM2 activator protein (GM2AP), a substrate-specific cofactor, to hydrolyze GM2 Ganglioside. Mutations in either subunit or the activator can result in the accumulation of GM2 Ganglioside within neurons throughout the central nervous system. The resulting neuronal cell death induces the primary symptoms of the disease: motor impairment, seizures, and sensory impairments. This study assesses the long-term effects of gene transfer in a Sandhoff (β-subunit knockout) mouse model. The study utilized a modified human β-hexosaminidase α-subunit (μ-subunit) that contains critical sequences from the β-subunit that enables formation of a stable homodimer (HexM) and interaction with GM2AP to hydrolyze GM2 Ganglioside. We investigated a self-complementary adeno-associated viral (scAAV) vector expressing HexM, through intravenous injections of the neonatal mice. We monitored one cohort for 8 weeks and another cohort long-term for survival benefit, behavioral, biochemical, and molecular analyses. Untreated Sandhoff disease (SD) control mice reached a humane endpoint at approximately 15 weeks, whereas treated mice had a median survival age of 40 weeks, an approximate 2.5-fold survival advantage. On behavioral tests, the treated mice outperformed their knockout age-matched controls and perform similarly to the heterozygous controls. Through the enzymatic and GM2 Ganglioside analyses, we observed a significant decrease in the GM2 Ganglioside level, even though the enzyme levels were not significantly increased. Molecular analyses revealed a global distribution of the vector between brain and spinal cord regions. In conclusion, the neonatal delivery of a novel viral vector expressing the human HexM enzyme is effective in ameliorating the SD mouse phenotype for long-term. Our data could have implications not only for treatment of SD but also for Tay-Sachs disease (α-subunit deficiency) and similar brain disorders.
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In cellulo examination of a beta-alpha hybrid construct of beta-hexosaminidase A subunits, reported to interact with the GM2 activator protein and hydrolyze GM2 Ganglioside.
PloS one, 2013Co-Authors: Incilay Sinici, Sayuri Yonekawa, Warren W. Wakarchuk, Steven J. Gray, Brian L. Mark, Ilona Tkachyova, R. Jude Samulski, Don J MahuranAbstract:The hydrolysis in lysosomes of GM2 Ganglioside to GM3 Ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 Ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 Ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 Ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 Ganglioside.
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A sensitive fluorescence-based assay for monitoring GM2 Ganglioside hydrolysis in live patient cells and their lysates
Glycobiology, 2009Co-Authors: Michael B. Tropak, Scott W Bukovac, Sayuri Yonekawa, Warren W. Wakarchuk, Brigitte Rigat, Don J MahuranAbstract:Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is anattractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are alsoinhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Herewe demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 Ganglioside, like natural GM2 Ganglioside, is specifically carried out by the β-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant β-hexosaminidase A and substrate-hydrolysis as compared to mock treated cells.
Jagdeep S. Walia - One of the best experts on this subject based on the ideXlab platform.
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systemic gene transfer of a hexosaminidase variant using an scaav9 47 vector corrects GM2 gangliosidosis in sandhoff mice
Human Gene Therapy, 2016Co-Authors: Karlaina J L Osmon, Don J Mahuran, Patrick Thompson, Steven J. Gray, Brian L. Mark, Evan Woodley, Subha Karumuthilmelethil, John G Keimel, Jagdeep S. WaliaAbstract:GM2 gangliosidosis is a group of neurodegenerative diseases caused by β-hexosaminidase A (HexA) enzyme deficiency. There is currently no cure. HexA is composed of two similar, nonidentical subunits, α and β, which must interact with the GM2 activator protein (GM2AP), a substrate-specific cofactor, to hydrolyze GM2 Ganglioside. Mutations in either subunit or the activator can result in the accumulation of GM2 Ganglioside within neurons throughout the central nervous system. The resulting neuronal cell death induces the primary symptoms of the disease: motor impairment, seizures, and sensory impairments. This study assesses the long-term effects of gene transfer in a Sandhoff (β-subunit knockout) mouse model. The study utilized a modified human β-hexosaminidase α-subunit (μ-subunit) that contains critical sequences from the β-subunit that enables formation of a stable homodimer (HexM) and interaction with GM2AP to hydrolyze GM2 Ganglioside. We investigated a self-complementary adeno-associated viral (scAAV)...
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Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 Ganglioside in vivo.
Molecular therapy. Methods & clinical development, 2016Co-Authors: Michael B. Tropak, Sayuri Yonekawa, Subha Karumuthil-melethil, Patrick Thompson, Warren W. Wakarchuk, Steven J. Gray, Jagdeep S. Walia, Brian L. Mark, Don J MahuranAbstract:Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 Ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 Ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 Ganglioside levels.
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Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2 Gangliosidosis in Sandhoff Mice.
Human gene therapy, 2016Co-Authors: Karlaina J L Osmon, Don J Mahuran, Subha Karumuthil-melethil, Patrick Thompson, Steven J. Gray, Brian L. Mark, Evan Woodley, John G Keimel, Katalina Ong, Jagdeep S. WaliaAbstract:GM2 gangliosidosis is a group of neurodegenerative diseases caused by β-hexosaminidase A (HexA) enzyme deficiency. There is currently no cure. HexA is composed of two similar, nonidentical subunits, α and β, which must interact with the GM2 activator protein (GM2AP), a substrate-specific cofactor, to hydrolyze GM2 Ganglioside. Mutations in either subunit or the activator can result in the accumulation of GM2 Ganglioside within neurons throughout the central nervous system. The resulting neuronal cell death induces the primary symptoms of the disease: motor impairment, seizures, and sensory impairments. This study assesses the long-term effects of gene transfer in a Sandhoff (β-subunit knockout) mouse model. The study utilized a modified human β-hexosaminidase α-subunit (μ-subunit) that contains critical sequences from the β-subunit that enables formation of a stable homodimer (HexM) and interaction with GM2AP to hydrolyze GM2 Ganglioside. We investigated a self-complementary adeno-associated viral (scAAV) vector expressing HexM, through intravenous injections of the neonatal mice. We monitored one cohort for 8 weeks and another cohort long-term for survival benefit, behavioral, biochemical, and molecular analyses. Untreated Sandhoff disease (SD) control mice reached a humane endpoint at approximately 15 weeks, whereas treated mice had a median survival age of 40 weeks, an approximate 2.5-fold survival advantage. On behavioral tests, the treated mice outperformed their knockout age-matched controls and perform similarly to the heterozygous controls. Through the enzymatic and GM2 Ganglioside analyses, we observed a significant decrease in the GM2 Ganglioside level, even though the enzyme levels were not significantly increased. Molecular analyses revealed a global distribution of the vector between brain and spinal cord regions. In conclusion, the neonatal delivery of a novel viral vector expressing the human HexM enzyme is effective in ameliorating the SD mouse phenotype for long-term. Our data could have implications not only for treatment of SD but also for Tay-Sachs disease (α-subunit deficiency) and similar brain disorders.
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long term correction of sandhoff disease following intravenous delivery of raav9 to mouse neonates
Molecular Therapy, 2015Co-Authors: Jagdeep S. Walia, Naderah Altaleb, Alexander Bello, Christa Kruck, Matthew C Lafave, Gaurav K Varshney, Shawn M Burgess, Biswajit Chowdhury, David J HurlbutAbstract:GM2 gangliosidoses are severe neurodegenerative disorders resulting from a deficiency in β-hexosaminidase A activity and lacking effective therapies. Using a Sandhoff disease (SD) mouse model (Hexb−/−) of the GM2 gangliosidoses, we tested the potential of systemically delivered adeno-associated virus 9 (AAV9) expressing Hexb cDNA to correct the neurological phenotype. Neonatal or adult SD and normal mice were intravenously injected with AAV9-HexB or –LacZ and monitored for serum β-hexosaminidase activity, motor function, and survival. Brain GM2 Ganglioside, β-hexosaminidase activity, and inflammation were assessed at experimental week 43, or an earlier humane end point. SD mice injected with AAV9-LacZ died by 17 weeks of age, whereas all neonatal AAV9-HexB–treated SD mice survived until 43 weeks (P < 0.0001) with only three exhibiting neurological dysfunction. SD mice treated as adults with AAV9-HexB died between 17 and 35 weeks. Neonatal SD-HexB–treated mice had a significant increase in brain β-hexosaminidase activity, and a reduction in GM2 Ganglioside storage and neuroinflammation compared to adult SD-HexB– and SD-LacZ–treated groups. However, at 43 weeks, 8 of 10 neonatal-HexB injected control and SD mice exhibited liver or lung tumors. This study demonstrates the potential for long-term correction of SD and other GM2 gangliosidoses through early rAAV9 based systemic gene therapy.
Kohji Itoh - One of the best experts on this subject based on the ideXlab platform.
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Enhancement by Uridine Diphosphate of Macrophage Inflammatory Protein-1 Alpha Production in Microglia Derived from Sandhoff Disease Model Mice
JIMD reports, 2015Co-Authors: Eri Kawashita, Daisuke Tsuji, Yosuke Kanno, Kaho Tsuchida, Kohji ItohAbstract:Sandhoff disease (SD) is a lysosomal β-hexosaminidase (Hex) deficiency involving excessive accumulation of undegraded substrates, including GM2 Ganglioside, and progressive neurodegeneration. Macrophage inflammatory protein-1α (MIP-1α) is a crucial factor for microglia-mediated neuroinflammation in the onset or progression of SD. However, the transmitter-mediated production of MIP-1α in SD is still poorly understood.
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The total chemical synthesis of the monoglycosylated GM2 Ganglioside activator using a novel cysteine surrogate
Chemical communications (Cambridge England), 2015Co-Authors: Kohei Sato, Daisuke Tsuji, Kohji Itoh, Keisuke Kitakaze, Takahiro Nakamura, Naoto Naruse, Keisuke Aihara, Akira Shigenaga, Tsubasa Inokuma, Akira OtakaAbstract:We describe a novel peptide ligation/desulfurization strategy using a β-mercapto-N-glycosylated asparagine derivative. The newly developed procedure was successfully applied to the total chemical synthesis of the GM2 Ganglioside activator protein bearing a monosaccharide on the native glycosylation site.
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Lyso-GM2 Ganglioside: A Possible Biomarker of Tay-Sachs Disease and Sandhoff Disease
PloS one, 2011Co-Authors: Takashi Kodama, Daisuke Tsuji, Kohji Itoh, Tadayasu Togawa, Takahiro Tsukimura, Ikuo Kawashima, Kazuhiko Matsuoka, Keisuke Kitakaze, Yo-ichi Ishida, Minoru SuzukiAbstract:To find a new biomarker of Tay-Sachs disease and Sandhoff disease. The lyso-GM2 Ganglioside (lyso-GM2) levels in the brain and plasma in Sandhoff mice were measured by means of high performance liquid chromatography and the effect of a modified hexosaminidase (Hex) B exhibiting Hex A-like activity was examined. Then, the lyso-GM2 concentrations in human plasma samples were determined. The lyso-GM2 levels in the brain and plasma in Sandhoff mice were apparently increased compared with those in wild-type mice, and they decreased on intracerebroventricular administration of the modified Hex B. The lyso-GM2 levels in plasma of patients with Tay-Sachs disease and Sandhoff disease were increased, and the increase in lyso-GM2 was associated with a decrease in Hex A activity. Lyso-GM2 is expected to be a potential biomarker of Tay-Sachs disease and Sandhoff disease.
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therapeutic potential of intracerebroventricular replacement of modified human β hexosaminidase b for GM2 gangliosidosis
Molecular Therapy, 2011Co-Authors: Kazuhiko Matsuoka, Daisuke Tsuji, Keisuke Kitakaze, Hitoshi Sakuraba, Tomomi Tamura, Yukie Dohzono, Kazuki Ohno, Seiji Saito, Kohji ItohAbstract:To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 Ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 Ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 Ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.
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Therapeutic evaluation of GM2 gangliosidoses by ELISA using anti-GM2 Ganglioside antibodies.
Clinica chimica acta; international journal of clinical chemistry, 2006Co-Authors: Daisuke Tsuji, Kazuhiko Matsuoka, Hitoshi Sakuraba, Yukari Higashine, Kohji ItohAbstract:GM2 gangliosidoses, including Tay-Sachs disease, Sandhoff disease and the AB variant, comprise deficiencies of beta-hexosaminidase isozymes and GM2 Ganglioside activator protein associated with accumulation of GM2 Ganglioside (GM2) in lysosomes and neurosomatic clinical manifestations. A simple assay system for intracellular quantification of GM2 is required to evaluate the therapeutic effects on GM2-gangliosidoses. We newly established a cell-ELISA system involving anti-GM2 monoclonal antibodies for measuring GM2 storage in fibroblasts from Tay-Sachs and Sandhoff disease patients. We succeeded in detecting the corrective effect of enzyme replacement on elimination of GM2 in the cells with this ELISA system. This simple and sensitive system should be useful as additional diagnosis tool as well as therapeutic evaluation of GM2 gangliosidoses.
C. Emiliani - One of the best experts on this subject based on the ideXlab platform.
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Occurrence of an anomalous endocytic compartment in fibroblasts from Sandhoff disease patients
Molecular and Cellular Biochemistry, 2010Co-Authors: Brunella Tancini, Alessandro Magini, Loredana Latterini, Lorena Urbanelli, Virginia Ciccarone, Fausto Elisei, C. EmilianiAbstract:Sandhoff disease (SD) is a lysosomal storage disorder due to mutations in the gene encoding for the β-subunit of β-hexosaminidase, that result in β-hexosaminidase A (αβ) and β-hexosaminidase B (ββ) deficiency. This leads to the storage of GM2 Ganglioside in endosomes and lysosomes, which ends in a progressive neurodegeneration. Currently, very little is known about the biochemical pathways leading from GM2 Ganglioside accumulation to pathogenesis. Defects in transport and sorting by the endosomal–lysosomal system have been described for several lysosomal storage disorders. Here, we have investigated the endosomal–lysosomal compartment in fibroblasts from SD patients and observed that both late endosomes and lysosomes, but not early endosomes, have a higher density in comparison with normal fibroblasts. Moreover, Sandhoff fibroblasts have an intracellular distribution of terminal endocytic organelles that differs from the characteristic perinuclear punctate pattern observed in normal fibroblasts and endocytic vesicles also appear larger. These findings reveal the occurrence of an alteration in the terminal endocytic organelles of Sandhoff fibroblasts, suggesting an involvement of this compartment in the disruption of cell metabolic and signalling pathways and in the onset of the pathological state.
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Occurrence of an anomalous endocytic compartment in fibroblasts from Sandhoff disease patients.
Molecular and cellular biochemistry, 2009Co-Authors: Brunella Tancini, Alessandro Magini, Loredana Latterini, Lorena Urbanelli, Virginia Ciccarone, Fausto Elisei, C. EmilianiAbstract:Sandhoff disease (SD) is a lysosomal storage disorder due to mutations in the gene encoding for the beta-subunit of beta-hexosaminidase, that result in beta-hexosaminidase A (alphabeta) and beta-hexosaminidase B (betabeta) deficiency. This leads to the storage of GM2 Ganglioside in endosomes and lysosomes, which ends in a progressive neurodegeneration. Currently, very little is known about the biochemical pathways leading from GM2 Ganglioside accumulation to pathogenesis. Defects in transport and sorting by the endosomal-lysosomal system have been described for several lysosomal storage disorders. Here, we have investigated the endosomal-lysosomal compartment in fibroblasts from SD patients and observed that both late endosomes and lysosomes, but not early endosomes, have a higher density in comparison with normal fibroblasts. Moreover, Sandhoff fibroblasts have an intracellular distribution of terminal endocytic organelles that differs from the characteristic perinuclear punctate pattern observed in normal fibroblasts and endocytic vesicles also appear larger. These findings reveal the occurrence of an alteration in the terminal endocytic organelles of Sandhoff fibroblasts, suggesting an involvement of this compartment in the disruption of cell metabolic and signalling pathways and in the onset of the pathological state.
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Restoration of the GM2 Ganglioside Metabolism in Bone Marrow–Derived Stromal Cells from Tay-Sachs Disease Animal Model
Neurochemical Research, 2002Co-Authors: S. Martino, C. Cavalieri, C. Emiliani, D. Dolcetta, M. G. Cusella De Angelis, V. Chigorno, G. M. Severini, K. Sandhoff, C. Bordignon, S. SonninoAbstract:The therapeutic potential of bone marrow–derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 Ganglioside storage. With this aim, we produced bone marrow–derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the α-subunit of the lysosomal enzyme β-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow–derived stromal cells have β-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, β-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 Ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.
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Restoration of the GM2 Ganglioside metabolism in bone marrow-derived stromal cells from Tay-Sachs disease animal model.
Neurochemical Research, 2002Co-Authors: S. Martino, C. Cavalieri, C. Emiliani, D. Dolcetta, V. Chigorno, G. M. Severini, K. Sandhoff, C. Bordignon, M. G. Cusella De Angelis, Sandro SonninoAbstract:The therapeutic potential of bone marrow–derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 Ganglioside storage. With this aim, we produced bone marrow–derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the α-subunit of the lysosomal enzyme β-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow–derived stromal cells have β-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, β-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 Ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.
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Restoration of the GM2 Ganglioside metabolism in bone marrow-derived stromal cells from Tay-Sachs disease animal model.
Neurochemical research, 2002Co-Authors: S. Martino, C. Cavalieri, C. Emiliani, D. Dolcetta, M. G. Cusella De Angelis, V. Chigorno, G. M. Severini, K. Sandhoff, C. Bordignon, S. SonninoAbstract:The therapeutic potential of bone marrow-derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 Ganglioside storage. With this aim, we produced bone marrow-derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the alpha-subunit of the lysosomal enzyme beta-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow-derived stromal cells have beta-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, beta-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 Ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.
Steven U Walkley - One of the best experts on this subject based on the ideXlab platform.
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Growth of Ectopic Dendrites on Cortical Pyramidal Neurons in Neuronal Storage Diseases Correlates with Abnormal Accumulation of GM2 Ganglioside
Journal of neurochemistry, 2008Co-Authors: Donald A. Siegel, Steven U WalkleyAbstract:: Ganglioside analysis and quantitative Golgi studies of the cerebral cortex of cats with Ganglioside and nonGanglioside lysosomal storage diseases reveal a correlation between the amount of accumulated GM2 Ganglioside and the extent of ectopic dendrite growth on cortical pyramidal neurons. This correlation was not observed with any of the other Gangliosides assayed for, including GM1 Ganglioside. These results suggest a specific role for GM2 Ganglioside in the initiation of ectopic neurites on pyramidal cells in vivo and are consistent with the developing hypothesis that different Gangliosides have specific roles in different cell types dependent upon the receptor or other effector molecules with which they may interact.
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Gangliosides as Modulators of Dendritogenesis in Normal and Storage Disease-affected Pyramidal Neurons
Cerebral cortex (New York N.Y. : 1991), 2000Co-Authors: Steven U Walkley, Mark Zervas, Samson WisemanAbstract:Pyramidal cells initiate the formation of dendritic arbors in a prolific burst of neurite outgrowth during early cortical development. Although morphologically mature pyramidal neurons do not normally sprout additional primary dendrites, the discovery of ectopic dendritogenesis in neuronal storage diseases has revealed that these cells do retain this ability under appropriate stimulation. The capacity for renewal of dendritogenesis has been found to exhibit a species gradient with human > cat, dog, sheep > mouse. A consistent metabolic feature of ectopic dendrite-bearing pyramidal neurons is a heightened intracellular expression of GM2 Ganglioside. Elevated expression of this same glycosphingolipid has also been found to correlate with normal dendritogenesis. Immature neurons in developing cat and ferret cortex exhibit high levels of GM2 Ganglioside immunoreactivity coincident with normal dendritic sprouting and a similar relationship has now been shown for human cortical development. Ultrastructural studies of all three species revealed GM2 localized to vesicles in a manner consistent with Golgi synthesis and exocytic trafficking to the somatic-dendritic plasmalemma. We propose that GM2 Ganglioside functions in glycosphingolipid-enriched microdomains (lipid rafts) in the plasmalemma to promote dendritic initiation through modulation of specific membrane proteins and/or their associated second messenger cascades.
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GM2 Ganglioside as a Regulator of Pyramidal Neuron Dendritogenesisa
Annals of the New York Academy of Sciences, 1998Co-Authors: Steven U Walkley, Donald A. Siegel, Kostantin Dobrenis, Mark ZervasAbstract:ABSTRACT: One of the most profound events in the life of a neuron in the mammalian CNS is the development of a characteristic dendritic tree, yet little is understood about events controlling this process. Pyramidal neurons of the cerebral cortex are known to undergo a single explosive burst of dendritic sprouting immediately after completing migration to the cortical mantle, and following maturation there is no evidence that new, primary dendrites are initiated. Yet in one group of rare genetic diseases-Tay-Sachs disease and related neuronal storage disorders-cortical pyramidal neurons undergo a second period of dendritogenesis. New dendritic membrane is generated principally at the axon hillock and in time is covered with normal-appearing spines and synapses. In our studies of normal brain development and storage diseases we consistently find one feature in common in cortical pyramidal neurons undergoing active dendritogenesis: They exhibit dramatically increased expression of GM2 Ganglioside localized to cytoplasmic vacuoles within neuronal perikarya and proximal dendrites. There is also evidence that the increase in GM2 precedes dendritic spouting, and that after dendritic maturation is complete (in normal brain) the GM2 levels in neurons become substantially reduced. These findings are consistent with GM2 Ganglioside playing a pivotal role in the regulation of dendritogenesis in cortical pyramidal neurons.
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ELEVATED GM2 Ganglioside IS ASSOCIATED WITH DENDRITIC PROLIFERATION IN NORMAL DEVELOPING NEOCORTEX
Developmental Brain Research, 1996Co-Authors: Linda A. Goodman, Steven U WalkleyAbstract:Abstract Mature pyramidal neurons of cerebral cortex in several neuronal storage disease elaborate dendrites. These dendrites appear specifically on pyramidal neurons containing elevated GM2 Ganglioside and a variety of studies support the hypothesis that this Ganglioside is responsible for inducing the new dendrite growth. To determine whether a similar association between GM2 Ganglioside and dendrite growth occurs in normal neurons, we used an antibody to localize GM2 in developing cat neocortex. Our results show that GM2 Ganglioside is elevated in normal cortical neurons during the period when dendritogenesis is occurring, but is greatly diminished in these cells after dendritic differentiation is complete. Elevations of GM2 occur in deep neurons earlier than in superficial ones, a sequence that corresponds closely to the inside-first, outside-last progression of cortical neuron differentiation. Ultrastructurally, GM2 immunoreactivity is found sequestered in vesicles with a distribution that coincides with sites of Ganglioside synthesis and transport. The close association between elevated GM2 Ganglioside and dendrite growth in cortical pyramidal neurons during normal development, coupled with a similar correlation between GM2 and ectopic dendritogenesis in neuronal storage disease, support the view that this specific Ganglioside plays a pivotal role in regulating dendritogenesis.
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GM2 Ganglioside and pyramidal neuron dendritogenesis
Neurochemical Research, 1995Co-Authors: Steven U Walkley, Donald A. Siegel, Kostantin DobrenisAbstract:GM2 Ganglioside, although scarce in normal adult brain, is the predominant Ganglioside accumulating in several types of lysosomal disorders, most notably Tay-Sachs disease. Pyramidal neurons of cerebral cortex in Tay-Sachs, as well as many other types of neuronal storage disorders, are known to exhibit a phenomenon believed unique to storage disorders: growth of ectopic dendrites. Recent studies have shown that a common metabolic abnormality shared by storage diseases with ectopic dendrite growth is the abnormal accumulation of GM2 Ganglioside. The correlation between increased levels of GM2 and the presence of ectopic dendrites has been found in both Ganglioside and nonGanglioside storage disorders, the latter including sphingomyelin-cholesterol lipidosis, mucopolysaccharidosis, and α-mannosidosis. Quantitative HPTLC analysis has shown that increases in GM2 occur in proportion to the incidence of ectopic dendrite growth, whereas, other Gangliosides, including GM1, lack similar increases. Immunocytochemical studies of all nonGanglioside storage diseases which exhibit ectopic dendritogenesis have revealed heightened GM2 Ganglioside-immunoreactivity in the cortical pyramidal cell population, whereas neurons in normal adult brain exhibit little or no staining for this Ganglioside. Further, studies examining disease development have consistently shown that accumulation of GM2 Ganglioside precedes growth of ectopic dendrites, indicating that it is not simply occurring secondary to new membrane production. These findings have prompted an examination for a similar relationship between GM2 Ganglioside and dendritogenesis in cortical neurons of normal developing brain. Results show that GM2 Ganglioside-immunoreactivity is consistently elevated in immature neurons during the period when they are undergoing active dendritic initiation, but this staining diminishes dramatically as the dendritic tress of these cells mature. Collectively, these studies on diseased and normal brain offer compelling evidence that GM2 Ganglioside plays a pivotal role in the regulation of dendritogenesis in cortical pyramidal neurons.