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Impact of enzyme replacement therapy on lysosomal function in Gaucher disease
Abstracts / Molecular Genetics and Metabolism 114 (2015) S11–S130
doi:10.1016/j.ymgme.2014.12.119
117 Impact of enzyme replacement therapy on lysosomal function in Gaucher disease Margarita Ivanovaa, Ryan Chengb, Chidima Martina, Ozlem Goker-Alpana, a Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA, USA, bUniversity of Virginia, Charlottesville, VA, USA
TE
DP
Lysosomes are primarily responsible for the degradation of proteins and organelles through a process called “autophagy”. Functional lysosomes are not only critical for the activation autophagy, but also for efficient degradation of their contents. Therefore, autophagy is logical target in lysosomal diseases (LSD). Gaucher disease (GD) is one of the most common LSD, caused by accumulation of glucocerebroside in the lysosomes due to deficient glucocerebrosidase (GC) activity. Autophagic abnormalities have previously been reported in cultured cells in GD. However, in vivo autophagic pathway has not been studied in patients with GD, and the effect of enzyme replacement therapy (ERT) on autophagy is unknown. Objective: to investigate the lysosomal function in patients with GD receiving ERT, and effects of rhGC on autophagy. To test the autophagy function and the effect of ERT on activation of autophagy, peripheral blood mononuclear cells (PBMCs) from patients with GD receiving ERT were analyzed with Cyto-ID autophagy assay, LysoTracker staining, and Western blots. PBMCs were obtained pre- and immediately post-ERT from 7 patients with GD1, 3 with GD3, and compared with 6 controls from healthy donors, and patients with other LSD. Isolated PBMCs were further treated with recombinant-human(rh)-GC. Autophagy inducer through inhibition of TORC1(Rapamycin), inhibitors (3-MA, Leupeptin, NH4Cl), and protease inhibitors (E64D, chloroquine-CQ) were used to study functional autophagy. We observed a decrease in the number of autophagic vacuoles in PBMCs from GD patients. Additionally, we noticed an accumulation of lysosomes. Although calpain inhibitor (E64D) and endosomal acidification inhibitor (CQ) increased autophagic vacuoles similar to controls, Rapamycin did not induce autophagosome formation. In vivo, ERT did not impact basal autophagy levels, nor number of lysosomes. However in vitro rh-GC improved autophagosome formation. In conclusion, there is dysfunction of mTOR pathway in patients with GD even with therapy, leading to impaired basal autophagy and lysosomal proliferation. The rhGC partially improves level of autophagic vacuole formation.
The mucopolysaccharidoses (MPS) are a group of heritable lysosomal disorders, characterized by the loss or reduction of a distinct lysosomal enzyme required for the degradation of specific glycosaminoglycans. Hematopoietic stem cell (HSC) transplant and enzyme replacement therapy, two clinically available forms of treatment, are able to correct the soft tissue aspects of MPS disease but have had a limited effect on the more complex skeletal and neurological symptoms. Stem cell therapy utilizing mesenchymal stem cells (MSC) has the potential to overcome these limitations due to their ability to differentiate into cells that are the major sites of MPS pathology. MSCs produce and secrete significantly higher levels of multiple MPS enzymes than HSCs in vitro and can be engineered to over-produce MPS enzymes using a lentiviral system. For example, MSCs secrete up to 5559 fold greater MPS I enzyme after lentiviral transduction, suggesting a greater potential to correct MPS pathology than HSCs. Lentiviral transduction was stable and persistent in vitro, and over-expression of MPS enzyme did not affect MSC differentiation down osteogenic, adipogenic, chondrogenic or neurogenic lineages. Systemically administered human MSCs distribute widely and cross the blood brain barrier. Significantly elevated brain α-liduronidase was observed two months post administration and was associated with functional improvements in neuromuscular strength, motor control, coordination and spatial learning. MSCs were found to limit astroglial activation and modulate brain inflammatory gene expression of Cd68 and Tnf. Vertebral body width also returned towards normal. However, no improvement in gag storage or α-liduronidase was observed in other tissues. Overall, MSCs have superior enzyme production and secretion capacity compared to HSCs and can modulate the host environment to limit inflammation, while promoting functional recovery and repair. However, further work is needed to elucidate if behavioral changes were due to a direct effect of MSC presence or indirectly through anti-inflammatory pathways.
OF
underexpressed: PGK1 and VIME. Fold changes between different groups were in the range 1.65–0.78. All proteins differentially expressed by iTRAQ were selected for verification by MRM. The following proteins were validated: FLNA and KPYM were underexpressed in SBD versus HC; in the case of MBD versus HC, all proteins differentially expressed by iTRAQ were validated and at last, VIME and PGK1 were validated as underexpressed proteins in SBD versus MBD. Further studies in larger cohorts are required for deeping into the role of these proteins as potential biomarkers for bone disease.
RO
S58
doi:10.1016/j.ymgme.2014.12.121
UN
CO
RR
EC
119 Patients' need to design a patient education program in Fabry disease (Filigrane)
doi:10.1016/j.ymgme.2014.12.120
118 α-l-Iduronidase transduced mesenchymal stem cells improve the behavioral deficits in mucopolysaccharidosis type I mice Matilda R. Jacksona,b, Ainslie L.K. Derrick Robertsa,b, Stan Gronthosa, Sharon Byersa,b, aUniversity of Adelaide, Adelaide, Australia, bSA Pathology, Adelaide, Australia
Roland Jaussauda, Laurence Lawb, Nathalie Triclin-Conseilc, AnneSophie Lapointed, Olivier Lidovee, aHôpital Robert Debré, CHU de Reims, Reims, France, bEdusanté, Paris, France, cAssociation des Patients de la Maladie de Fabry, APMF, Vendresse, France, dVaincre les Maladies Lysosomales, Massy, France, eHôpital Croix-Saint-Simon, Paris, France
Background/purpose: Fabry disease (FD) is an X-linked rare progressive multisystemic disorder. Renal failure, cardiomyopathy, and peripheral and central nervous system alterations are the main causes of morbidity and reduced life expectancy. A better understanding of patients' and physicians' perceptions and experience of FD will help to improve the management of the patients and aid the development of appropriate educational strategies. Aim: To identify perceptions, experience, educational needs and barriers to learning in FD patients at high risk of severe cardiovascular, neurologic and nephrologic complications. Methods design of study: A qualitative study involving patients who joined French patients associations and physicians involved in the care of FD patients. A physician focus group was conducted. Setting: 9 centers in Reims, Paris, Garches, Dijon, Bordeaux, Lille, Toulouse, Lyon, and Caen. Methods: A questionnaire on their expectations in terms of a patient education program (PEP) was sent to the members of the FD association (Association des Patients de la Maladie de Fabry, APMF) and Lysosomal association (Vaincre les maladies Lysosomales, VML)
doi:10.1016/j.ymgme.2014.12.119
117 Impact of enzyme replacement therapy on lysosomal function in Gaucher disease Margarita Ivanovaa, Ryan Chengb, Chidima Martina, Ozlem Goker-Alpana, a Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA, USA, bUniversity of Virginia, Charlottesville, VA, USA
TE
DP
Lysosomes are primarily responsible for the degradation of proteins and organelles through a process called “autophagy”. Functional lysosomes are not only critical for the activation autophagy, but also for efficient degradation of their contents. Therefore, autophagy is logical target in lysosomal diseases (LSD). Gaucher disease (GD) is one of the most common LSD, caused by accumulation of glucocerebroside in the lysosomes due to deficient glucocerebrosidase (GC) activity. Autophagic abnormalities have previously been reported in cultured cells in GD. However, in vivo autophagic pathway has not been studied in patients with GD, and the effect of enzyme replacement therapy (ERT) on autophagy is unknown. Objective: to investigate the lysosomal function in patients with GD receiving ERT, and effects of rhGC on autophagy. To test the autophagy function and the effect of ERT on activation of autophagy, peripheral blood mononuclear cells (PBMCs) from patients with GD receiving ERT were analyzed with Cyto-ID autophagy assay, LysoTracker staining, and Western blots. PBMCs were obtained pre- and immediately post-ERT from 7 patients with GD1, 3 with GD3, and compared with 6 controls from healthy donors, and patients with other LSD. Isolated PBMCs were further treated with recombinant-human(rh)-GC. Autophagy inducer through inhibition of TORC1(Rapamycin), inhibitors (3-MA, Leupeptin, NH4Cl), and protease inhibitors (E64D, chloroquine-CQ) were used to study functional autophagy. We observed a decrease in the number of autophagic vacuoles in PBMCs from GD patients. Additionally, we noticed an accumulation of lysosomes. Although calpain inhibitor (E64D) and endosomal acidification inhibitor (CQ) increased autophagic vacuoles similar to controls, Rapamycin did not induce autophagosome formation. In vivo, ERT did not impact basal autophagy levels, nor number of lysosomes. However in vitro rh-GC improved autophagosome formation. In conclusion, there is dysfunction of mTOR pathway in patients with GD even with therapy, leading to impaired basal autophagy and lysosomal proliferation. The rhGC partially improves level of autophagic vacuole formation.
The mucopolysaccharidoses (MPS) are a group of heritable lysosomal disorders, characterized by the loss or reduction of a distinct lysosomal enzyme required for the degradation of specific glycosaminoglycans. Hematopoietic stem cell (HSC) transplant and enzyme replacement therapy, two clinically available forms of treatment, are able to correct the soft tissue aspects of MPS disease but have had a limited effect on the more complex skeletal and neurological symptoms. Stem cell therapy utilizing mesenchymal stem cells (MSC) has the potential to overcome these limitations due to their ability to differentiate into cells that are the major sites of MPS pathology. MSCs produce and secrete significantly higher levels of multiple MPS enzymes than HSCs in vitro and can be engineered to over-produce MPS enzymes using a lentiviral system. For example, MSCs secrete up to 5559 fold greater MPS I enzyme after lentiviral transduction, suggesting a greater potential to correct MPS pathology than HSCs. Lentiviral transduction was stable and persistent in vitro, and over-expression of MPS enzyme did not affect MSC differentiation down osteogenic, adipogenic, chondrogenic or neurogenic lineages. Systemically administered human MSCs distribute widely and cross the blood brain barrier. Significantly elevated brain α-liduronidase was observed two months post administration and was associated with functional improvements in neuromuscular strength, motor control, coordination and spatial learning. MSCs were found to limit astroglial activation and modulate brain inflammatory gene expression of Cd68 and Tnf. Vertebral body width also returned towards normal. However, no improvement in gag storage or α-liduronidase was observed in other tissues. Overall, MSCs have superior enzyme production and secretion capacity compared to HSCs and can modulate the host environment to limit inflammation, while promoting functional recovery and repair. However, further work is needed to elucidate if behavioral changes were due to a direct effect of MSC presence or indirectly through anti-inflammatory pathways.
OF
underexpressed: PGK1 and VIME. Fold changes between different groups were in the range 1.65–0.78. All proteins differentially expressed by iTRAQ were selected for verification by MRM. The following proteins were validated: FLNA and KPYM were underexpressed in SBD versus HC; in the case of MBD versus HC, all proteins differentially expressed by iTRAQ were validated and at last, VIME and PGK1 were validated as underexpressed proteins in SBD versus MBD. Further studies in larger cohorts are required for deeping into the role of these proteins as potential biomarkers for bone disease.
RO
S58
doi:10.1016/j.ymgme.2014.12.121
UN
CO
RR
EC
119 Patients' need to design a patient education program in Fabry disease (Filigrane)
doi:10.1016/j.ymgme.2014.12.120
118 α-l-Iduronidase transduced mesenchymal stem cells improve the behavioral deficits in mucopolysaccharidosis type I mice Matilda R. Jacksona,b, Ainslie L.K. Derrick Robertsa,b, Stan Gronthosa, Sharon Byersa,b, aUniversity of Adelaide, Adelaide, Australia, bSA Pathology, Adelaide, Australia
Roland Jaussauda, Laurence Lawb, Nathalie Triclin-Conseilc, AnneSophie Lapointed, Olivier Lidovee, aHôpital Robert Debré, CHU de Reims, Reims, France, bEdusanté, Paris, France, cAssociation des Patients de la Maladie de Fabry, APMF, Vendresse, France, dVaincre les Maladies Lysosomales, Massy, France, eHôpital Croix-Saint-Simon, Paris, France
Background/purpose: Fabry disease (FD) is an X-linked rare progressive multisystemic disorder. Renal failure, cardiomyopathy, and peripheral and central nervous system alterations are the main causes of morbidity and reduced life expectancy. A better understanding of patients' and physicians' perceptions and experience of FD will help to improve the management of the patients and aid the development of appropriate educational strategies. Aim: To identify perceptions, experience, educational needs and barriers to learning in FD patients at high risk of severe cardiovascular, neurologic and nephrologic complications. Methods design of study: A qualitative study involving patients who joined French patients associations and physicians involved in the care of FD patients. A physician focus group was conducted. Setting: 9 centers in Reims, Paris, Garches, Dijon, Bordeaux, Lille, Toulouse, Lyon, and Caen. Methods: A questionnaire on their expectations in terms of a patient education program (PEP) was sent to the members of the FD association (Association des Patients de la Maladie de Fabry, APMF) and Lysosomal association (Vaincre les maladies Lysosomales, VML)