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Ellen Sidransky - One of the best experts on this subject based on the ideXlab platform.
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Glucocerebrosidase and its relevance to Parkinson disease
Molecular neurodegeneration, 2019Co-Authors: Cindy E. Mckinney, Pankaj Sharma, Ellen SidranskyAbstract:Mutations in GBA1, the gene encoding the lysosomal enzyme Glucocerebrosidase, are among the most common known genetic risk factors for the development of Parkinson disease and related synucleinopathies. A great deal is known about GBA1, as mutations in GBA1 are causal for the rare autosomal storage disorder Gaucher disease. Over the past decades, significant progress has been made in understanding the genetics and cell biology of Glucocerebrosidase. A least 495 different mutations, found throughout the 11 exons of the gene are reported, including both common and rare variants. Mutations in GBA1 may lead to degradation of the protein, disruptions in lysosomal targeting and diminished performance of the enzyme in the lysosome. Gaucher disease is phenotypically diverse and has both neuronopathic and non-neuronopathic forms. Both patients with Gaucher disease and heterozygous carriers are at increased risk of developing Parkinson disease and Dementia with Lewy Bodies, although our understanding of the mechanism for this association remains incomplete. There appears to be an inverse relationship between Glucocerebrosidase and α-synuclein levels, and even patients with sporadic Parkinson disease have decreased Glucocerebrosidase. Glucocerebrosidase may interact with α-synuclein to maintain basic cellular functions, or impaired Glucocerebrosidase could contribute to Parkinson pathogenesis by disrupting lysosomal homeostasis, enhancing endoplasmic reticulum stress or contributing to mitochondrial impairment. However, the majority of patients with GBA1 mutations never develop parkinsonism, so clearly other risk factors play a role. Treatments for Gaucher disease have been developed that increase visceral Glucocerebrosidase levels and decrease lipid storage, although they have yet to properly address the neurological defects associated with impaired Glucocerebrosidase. Mouse and induced pluripotent stem cell derived models have improved our understanding of Glucocerebrosidase function and the consequences of its deficiency. These models have been used to test novel therapies including chaperone proteins, histone deacetylase inhibitors, and gene therapy approaches that enhance Glucocerebrosidase levels and could prove efficacious in the treatment of forms of parkinsonism. Consequently, this rare monogenic disorder, Gaucher disease, provides unique insights directly applicable to our understanding and treatment of Parkinson disease, a common and complex neurodegenerative disorder.
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Validation of anti-Glucocerebrosidase antibodies for western blot analysis on protein lysates of murine and human cells
The Biochemical journal, 2019Co-Authors: Brad A. Davidson, Ellen Sidransky, Matthew Nguyen, Taylor Lindstrom, Richard Grey, Robert Burnett, Elma Aflaki, Wendy WestbroekAbstract:Gaucher disease is a rare lysosomal storage disorder caused by mutations in the GBA1 gene, encoding the lysosome-resident Glucocerebrosidase enzyme involved in the hydrolysis of glucosylceramide. The discovery of an association between mutations in GBA1 and the development of synucleinopathies, including Parkinson disease, has directed attention to Glucocerebrosidase as a potential therapeutic target for different synucleinopathies. These findings initiated an exponential growth in research and publications regarding the Glucocerebrosidase enzyme. The use of various commercial and custom-made Glucocerebrosidase antibodies has been reported, but standardized in-depth validation is still not available for many of these antibodies. This work details the evaluation of several previously reported Glucocerebrosidase antibodies for western blot analysis, tested on protein lysates of murine gba +/+ and gba -/ - immortalized neurons and primary human wild type and type 2 Gaucher disease fibroblasts.
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Identification of miRNAs that modulate Glucocerebrosidase activity in Gaucher disease cells
RNA biology, 2014Co-Authors: Marina Siebert, Arash Velayati, Wendy Westbroek, Yu-chi Chen, Nima Moaven, Maria Luiza Saraiva-pereira, Scott E. Martin, Ellen SidranskyAbstract:Gaucher disease is an autosomal recessive disorder caused by deficiency of the enzyme Glucocerebrosidase. Although it is a monogenic disease, there is vast phenotypic heterogeneity, even among patients with the same genotype. MicroRNAs (miRNAs) are small non-coding RNAs involved in many biological processes and diseases. To determine whether miRNAs can affect Glucocerebrosidase activity, we performed a screen of 875 different miRNA mimics. The screen was performed using Gaucher fibroblasts, and Glucocerebrosidase activity was used as the initial outcome parameter. We found several miRNAs that either up- or down-regulated Glucocerebrosidase activity. In follow-up assays, we confirmed that one specific miRNA (miR-127–5p) down-regulated both Glucocerebrosidase activity and protein levels by down-regulation of LIMP-2, the receptor involved in proper trafficking of Glucocerebrosidase from the endoplasmic reticulum to the lysosome. A conditioned media assay demonstrated that cells treated with this miRNA secret...
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Glucocerebrosidase is shaking up the synucleinopathies
Brain, 2014Co-Authors: Marina Siebert, Ellen Sidransky, Wendy WestbroekAbstract:The lysosomal enzyme Glucocerebrosidase, encoded by the Glucocerebrosidase gene, is involved in the breakdown of glucocerebroside into glucose and ceramide. Lysosomal build-up of the substrate glucocerebroside occurs in cells of the reticulo-endothelial system in patients with Gaucher disease, a rare lysosomal storage disorder caused by the recessively inherited deficiency of Glucocerebrosidase. Gaucher disease has a broad clinical phenotypic spectrum, divided into non-neuronopathic and neuronopathic forms. Like many monogenic diseases, the correlation between clinical manifestations and molecular genotype is not straightforward. There is now a well-established clinical association between mutations in the Glucocerebrosidase gene and the development of more prevalent multifactorial disorders including Parkinson's disease and other synucleinopathies. In this review we discuss recent studies advancing our understanding of the cellular relationship between Glucocerebrosidase and α-synuclein, the potential impact of established and emerging therapeutics for Gaucher disease for the treatment of the synucleinopathies, and the role of lysosomal pathways in the pathogenesis of these neurodegenerative disorders.
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Reduced Glucocerebrosidase is associated with increased α-synuclein in sporadic Parkinson's disease.
Brain, 2014Co-Authors: Karen E. Murphy, Nahid Tayebi, Ellen Sidransky, Amanda M Gysbers, Sarah K. Abbott, Woojin S. Kim, Anthony Cooper, Brett Garner, Glenda M. HallidayAbstract:Heterozygous mutations in GBA1, the gene encoding lysosomal Glucocerebrosidase, are the most frequent known genetic risk factor for Parkinson’s disease. Reduced Glucocerebrosidase and α-synuclein accumulation are directly related in cell models of Parkinson’s disease. We investigated relationships between Parkinson’s disease-specific Glucocerebrosidase deficits, Glucocerebrosidase-related pathways, and α-synuclein levels in brain tissue from subjects with sporadic Parkinson’s disease without GBA1 mutations. Brain regions with and without a Parkinson’s disease-related increase in α-synuclein levels were assessed in autopsy samples from subjects with sporadic Parkinson’s disease (n = 19) and age- and post-mortem delay-matched controls (n = 10). Levels of Glucocerebrosidase, α-synuclein and related lysosomal and autophagic proteins were assessed by western blotting. Glucocerebrosidase enzyme activity was measured using a fluorimetric assay, and Glucocerebrosidase and α-synuclein messenger RNA expression determined by quantitative polymerase chain reaction. Related sphingolipids were analysed by mass spectrometry. Multivariate statistical analyses were performed to identify differences between disease groups and regions, with non-parametric correlations used to identify relationships between variables. Glucocerebrosidase protein levels and enzyme activity were selectively reduced in the early stages of Parkinson’s disease in regions with increased α-synuclein levels although limited inclusion formation, whereas GBA1 messenger RNA expression was non-selectively reduced in Parkinson’s disease. The selective loss of lysosomal Glucocerebrosidase was directly related to reduced lysosomal chaperone-mediated autophagy, increased α-synuclein and decreased ceramide. Glucocerebrosidase deficits in sporadic Parkinson’s disease are related to the abnormal accumulation of α-synuclein and are associated with substantial alterations in lysosomal chaperone-mediated autophagy pathways and lipid metabolism. Our data suggest that the early selective Parkinson’s disease changes are likely a result of the redistribution of cellular membrane proteins leading to a chronic reduction in lysosome function in brain regions vulnerable to Parkinson’s disease pathology.
Anthony H V Schapira - One of the best experts on this subject based on the ideXlab platform.
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functional assessment of Glucocerebrosidase modulator efficacy in primary patient derived macrophages is essential for drug development and patient stratification
Haematologica, 2020Co-Authors: Natalie J Welsh, Christina Gewinner, Kavita Mistry, Mumta Koglin, Juniebel Cooke, Matt Butler, Ben Powney, Malcolm Roberts, James Staddon, Anthony H V SchapiraAbstract:Gaucher disease (GD) is a lysosomal storage disorder caused by mutations in the Glucocerebrosidase 1 ( GBA1 ) gene encoding the lysosomal enzyme Glucocerebrosidase. Patients with type 1 GD present with accumulation of glucosylceramide in macrophages leading to a range of systemic manifestations,
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Glucocerebrosidase mutations and synucleinopathies toward a model of precision medicine
Movement Disorders, 2019Co-Authors: Fabio Blandini, Anthony H V Schapira, Roberto Cilia, Silvia Cerri, Gianni Pezzoli, Stephen Mullin, Jose L LanciegoAbstract:Glucocerebrosidase is a lysosomal enzyme. The characterization of a direct link between mutations in the gene coding for Glucocerebrosidase (GBA1) with the development of Parkinson's disease and dementia with Lewy bodies has heightened interest in this enzyme. Although the mechanisms through which Glucocerebrosidase regulates the homeostasis of α-synuclein remains poorly understood, the identification of reduced Glucocerebrosidase activity in the brains of patients with PD and dementia with Lewy bodies has paved the way for the development of novel therapeutic strategies directed at enhancing Glucocerebrosidase activity and reducing α-synuclein burden, thereby slowing down or even preventing neuronal death. Here we reviewed the current literature relating to the mechanisms underlying the cross talk between Glucocerebrosidase and α-synuclein, the GBA1 mutation-associated clinical phenotypes, and ongoing therapeutic approaches targeting Glucocerebrosidase. © 2018 International Parkinson and Movement Disorder Society.
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Glucocerebrosidase and Parkinson Disease: Molecular, Clinical, and Therapeutic Implications:
The Neuroscientist : a review journal bringing neurobiology neurology and psychiatry, 2018Co-Authors: Roberta Balestrino, Anthony H V SchapiraAbstract:Parkinson disease (PD) is a complex neurodegenerative disease characterised by multiple motor and non-motor symptoms. In the last 20 years, more than 20 genes have been identified as causes of parkinsonism. Following the observation of higher risk of PD in patients affected by Gaucher disease, a lysosomal disorder caused by mutations in the Glucocerebrosidase (GBA) gene, it was discovered that mutations in this gene constitute the single largest risk factor for development of idiopathic PD. Patients with PD and GBA mutations are clinically indistinguishable from patients with idiopathic PD, although some characteristics emerge depending on the specific mutation, such as slightly earlier onset. The molecular mechanisms which lead to this increased PD risk in GBA mutation carriers are multiple and not yet fully elucidated, they include alpha-synuclein aggregation, lysosomal-autophagy dysfunction and endoplasmic reticulum stress. Moreover, dysfunction of Glucocerebrosidase has also been demonstrated in non-GBA PD, suggesting its interaction with other pathogenic mechanisms. Therefore, GBA enzyme function represents an interesting pharmacological target for PD. Cell and animal models suggest that increasing GBA enzyme activity can reduce alpha-synuclein levels. Clinical trials of ambroxol, a Glucocerebrosidase chaperone, are currently ongoing in PD and PD dementia, as is a trial of substrate reduction therapy. The aim of this review is to summarise the main features of GBA-PD and discuss the implications of Glucocerebrosidase modulation on PD pathogenesis.
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Ambroxol effects in Glucocerebrosidase and α-synuclein transgenic mice.
Annals of neurology, 2016Co-Authors: Anna Migdalska-richards, Liam Daly, Erwan Bezard, Anthony H V SchapiraAbstract:Objective. Gaucher disease is caused by mutations in the Glucocerebrosidase 1 gene that result in deficiency of the lysosomal enzyme Glucocerebrosidase. Both homozygous and heterozygous Glucocerebrosidase 1 mutations confer an increased risk for developing Parkinson disease. Current estimates indicate that 10 to 25% of Parkinson patients carry Glucocerebrosidase 1 mutations. Ambroxol is a small molecule chaperone that has been shown to increase Glucocerebrosidase activity in vitro. This study investigated the effect of ambroxol treatment on Glucocerebrosidase activity and on α-synuclein and phosphorylated α-synuclein protein levels in mice. Methods. Mice were treated with ambroxol for 12 days. After the treatment, Glucocerebrosidase activity was measured in the mouse brain lysates. The brain lysates were also analyzed for α-synuclein and phosphorylated α-synuclein protein levels. Results. Ambroxol treatment resulted in increased brain Glucocerebrosidase activity in (1) wild-type mice, (2) transgenic mice expressing the heterozygous L444P mutation in the murine Glucocerebrosidase 1 gene, and (3) transgenic mice overexpressing human α-synuclein. Furthermore, in the mice overexpressing human α-synuclein, ambroxol treatment decreased both α-synuclein and phosphorylated α-synuclein protein levels. Interpretation. Our work supports the proposition that ambroxol should be further investigated as a potential novel disease-modifying therapy for treatment of Parkinson disease and neuronopathic Gaucher disease to increase Glucocerebrosidase activity and decrease α-synuclein and phosphorylated α-synuclein protein levels.
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Ambroxol effects in Glucocerebrosidase and α-synuclein transgenic mice.
Annals of Neurology, 2016Co-Authors: Anna Migdalska-richards, Liam Daly, Erwan Bezard, Anthony H V SchapiraAbstract:Gaucher disease is caused by mutations in the Glucocerebrosidase 1 gene that result in deficiency of the lysosomal enzyme Glucocerebrosidase. Both homozygous and heterozygous Glucocerebrosidase 1 mutations confer an increased risk for developing Parkinson disease. Current estimates indicate that 10 to 25% of Parkinson patients carry Glucocerebrosidase 1 mutations. Ambroxol is a small molecule chaperone that has been shown to increase Glucocerebrosidase activity in vitro. This study investigated the effect of ambroxol treatment on Glucocerebrosidase activity and on α-synuclein and phosphorylated α-synuclein protein levels in mice.
Paul Saftig - One of the best experts on this subject based on the ideXlab platform.
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Parkinson's disease: acid‐Glucocerebrosidase activity and alpha‐synuclein clearance
Journal of Neurochemistry, 2016Co-Authors: Judith Blanz, Paul SaftigAbstract:The role of mutations in the gene GBA1 encoding the lysosomal hydrolase β-Glucocerebrosidase for the development of synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, was only very recently uncovered. The knowledge obtained from the study of carriers or patients suffering from Gaucher disease (a common lysosomal storage disorder because of GBA1 mutations) is of particular importance for understanding the role of the enzyme and its catabolic pathway in the development of synucleinopathies. Decreased activity of β-Glucocerebrosidase leads to lysosomal dysfunction and the accumulation of its substrate glucosylceramide and related lipid derivatives. Glucosylceramide is suggested to stabilize toxic oligomeric forms of α-synuclein that negatively influence the activity of β-Glucocerebrosidase and to partially block export of newly synthesized β-Glucocerebrosidase from the endoplasmic reticulum to late endocytic compartments, amplifying the pathological effects of α-synuclein and ultimately resulting in neuronal cell death. This pathogenic molecular feedback loop and most likely other factors (such as impaired endoplasmic reticulum-associated degradation, activation of the unfolded protein response and dysregulation of calcium homeostasis induced by misfolded GC mutants) are involved in shifting the cellular homeostasis from monomeric α-synuclein towards oligomeric neurotoxic and aggregated forms, which contribute to Parkinson's disease progression. From a therapeutic point of view, strategies aiming to increase either the expression, stability or delivery of the β-Glucocerebrosidase to lysosomes are likely to decrease the α-synuclein burden and may be useful for an in depth evaluation at the organismal level. Lysosomes are critical for protein and lipid homeostasis. Recent research revealed that dysfunction of this organelle contributes to the development of neurodegenerative diseases such as Parkinson's disease (PD). Mutations in the lysosomal hydrolase β-Glucocerebrosidase (GBA1) are a major risk factor for the development of PD and the molecular events linked to the reduced activity of GBA1 and the pathological accumulation of lipids and α-synuclein are just at the beginning to be understood. New therapeutic concepts in regards to how to increase the expression, stability, or delivery of β-Glucocerebrosidase to lysosomes are currently developed. This article is part of a special issue on Parkinson disease.
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Parkinson's disease: acid-Glucocerebrosidase activity and alpha-synuclein clearance.
Journal of neurochemistry, 2016Co-Authors: Judith Blanz, Paul SaftigAbstract:The role of mutations in the gene GBA1 encoding the lysosomal hydrolase β-Glucocerebrosidase for the development of synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, was only very recently uncovered. The knowledge obtained from the study of carriers or patients suffering from Gaucher disease (a common lysosomal storage disorder because of GBA1 mutations) is of particular importance for understanding the role of the enzyme and its catabolic pathway in the development of synucleinopathies. Decreased activity of β-Glucocerebrosidase leads to lysosomal dysfunction and the accumulation of its substrate glucosylceramide and related lipid derivatives. Glucosylceramide is suggested to stabilize toxic oligomeric forms of α-synuclein that negatively influence the activity of β-Glucocerebrosidase and to partially block export of newly synthesized β-Glucocerebrosidase from the endoplasmic reticulum to late endocytic compartments, amplifying the pathological effects of α-synuclein and ultimately resulting in neuronal cell death. This pathogenic molecular feedback loop and most likely other factors (such as impaired endoplasmic reticulum-associated degradation, activation of the unfolded protein response and dysregulation of calcium homeostasis induced by misfolded GC mutants) are involved in shifting the cellular homeostasis from monomeric α-synuclein towards oligomeric neurotoxic and aggregated forms, which contribute to Parkinson's disease progression. From a therapeutic point of view, strategies aiming to increase either the expression, stability or delivery of the β-Glucocerebrosidase to lysosomes are likely to decrease the α-synuclein burden and may be useful for an in depth evaluation at the organismal level. Lysosomes are critical for protein and lipid homeostasis. Recent research revealed that dysfunction of this organelle contributes to the development of neurodegenerative diseases such as Parkinson's disease (PD). Mutations in the lysosomal hydrolase β-Glucocerebrosidase (GBA1) are a major risk factor for the development of PD and the molecular events linked to the reduced activity of GBA1 and the pathological accumulation of lipids and α-synuclein are just at the beginning to be understood. New therapeutic concepts in regards to how to increase the expression, stability, or delivery of β-Glucocerebrosidase to lysosomes are currently developed. This article is part of a special issue on Parkinson disease.
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limp 2 is a receptor for lysosomal mannose 6 phosphate independent targeting of β Glucocerebrosidase
Cell, 2007Co-Authors: David Reczek, Michael Schwake, Jenny Schroder, Heather Hughes, Judith Blanz, William Brondyk, Scott M Van Patten, Tim Edmunds, Paul SaftigAbstract:Summary β-Glucocerebrosidase, the enzyme defective in Gaucher disease, is targeted to the lysosome independently of the mannose-6-phosphate receptor. Affinity-chromatography experiments revealed that the lysosomal integral membrane protein LIMP-2 is a specific binding partner of β-Glucocerebrosidase. This interaction involves a coiled-coil domain within the lumenal domain. β-Glucocerebrosidase activity and protein levels were severely decreased in LIMP-2-deficient mouse tissues. Analysis of fibroblasts and macrophages isolated from these mice indicated that the majority of β-Glucocerebrosidase was secreted. Missorting of β-Glucocerebrosidase was also evident in vivo, as protein and activity levels were significantly higher in sera from LIMP-2-deficient mice compared to wild-type. Reconstitution of LIMP-2 in LIMP-2-deficient fibroblasts led to a rescue of β-Glucocerebrosidase levels and distribution. LIMP-2 expression also led to lysosomal transport of a β-Glucocerebrosidase endoplasmic reticulum retention mutant. These data support a role for LIMP-2 as the mannose-6-phosphate-independent trafficking receptor for β-Glucocerebrosidase.
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limp 2 is a receptor for lysosomal mannose 6 phosphate independent targeting of β Glucocerebrosidase
Cell, 2007Co-Authors: David Reczek, Michael Schwake, Jenny Schroder, Heather Hughes, Judith Blanz, William Brondyk, Tim Edmunds, Xiaoying Jin, Scott M Van Patten, Paul SaftigAbstract:beta-Glucocerebrosidase, the enzyme defective in Gaucher disease, is targeted to the lysosome independently of the mannose-6-phosphate receptor. Affinity-chromatography experiments revealed that the lysosomal integral membrane protein LIMP-2 is a specific binding partner of beta-Glucocerebrosidase. This interaction involves a coiled-coil domain within the lumenal domain. beta-Glucocerebrosidase activity and protein levels were severely decreased in LIMP-2-deficient mouse tissues. Analysis of fibroblasts and macrophages isolated from these mice indicated that the majority of beta-Glucocerebrosidase was secreted. Missorting of beta-Glucocerebrosidase was also evident in vivo, as protein and activity levels were significantly higher in sera from LIMP-2-deficient mice compared to wild-type. Reconstitution of LIMP-2 in LIMP-2-deficient fibroblasts led to a rescue of beta-Glucocerebrosidase levels and distribution. LIMP-2 expression also led to lysosomal transport of a beta-Glucocerebrosidase endoplasmic reticulum retention mutant. These data support a role for LIMP-2 as the mannose-6-phosphate-independent trafficking receptor for beta-Glucocerebrosidase.
Anna Migdalska-richards - One of the best experts on this subject based on the ideXlab platform.
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Ambroxol effects in Glucocerebrosidase and α-synuclein transgenic mice.
Annals of neurology, 2016Co-Authors: Anna Migdalska-richards, Liam Daly, Erwan Bezard, Anthony H V SchapiraAbstract:Objective. Gaucher disease is caused by mutations in the Glucocerebrosidase 1 gene that result in deficiency of the lysosomal enzyme Glucocerebrosidase. Both homozygous and heterozygous Glucocerebrosidase 1 mutations confer an increased risk for developing Parkinson disease. Current estimates indicate that 10 to 25% of Parkinson patients carry Glucocerebrosidase 1 mutations. Ambroxol is a small molecule chaperone that has been shown to increase Glucocerebrosidase activity in vitro. This study investigated the effect of ambroxol treatment on Glucocerebrosidase activity and on α-synuclein and phosphorylated α-synuclein protein levels in mice. Methods. Mice were treated with ambroxol for 12 days. After the treatment, Glucocerebrosidase activity was measured in the mouse brain lysates. The brain lysates were also analyzed for α-synuclein and phosphorylated α-synuclein protein levels. Results. Ambroxol treatment resulted in increased brain Glucocerebrosidase activity in (1) wild-type mice, (2) transgenic mice expressing the heterozygous L444P mutation in the murine Glucocerebrosidase 1 gene, and (3) transgenic mice overexpressing human α-synuclein. Furthermore, in the mice overexpressing human α-synuclein, ambroxol treatment decreased both α-synuclein and phosphorylated α-synuclein protein levels. Interpretation. Our work supports the proposition that ambroxol should be further investigated as a potential novel disease-modifying therapy for treatment of Parkinson disease and neuronopathic Gaucher disease to increase Glucocerebrosidase activity and decrease α-synuclein and phosphorylated α-synuclein protein levels.
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Ambroxol effects in Glucocerebrosidase and α-synuclein transgenic mice.
Annals of Neurology, 2016Co-Authors: Anna Migdalska-richards, Liam Daly, Erwan Bezard, Anthony H V SchapiraAbstract:Gaucher disease is caused by mutations in the Glucocerebrosidase 1 gene that result in deficiency of the lysosomal enzyme Glucocerebrosidase. Both homozygous and heterozygous Glucocerebrosidase 1 mutations confer an increased risk for developing Parkinson disease. Current estimates indicate that 10 to 25% of Parkinson patients carry Glucocerebrosidase 1 mutations. Ambroxol is a small molecule chaperone that has been shown to increase Glucocerebrosidase activity in vitro. This study investigated the effect of ambroxol treatment on Glucocerebrosidase activity and on α-synuclein and phosphorylated α-synuclein protein levels in mice.
Wendy Westbroek - One of the best experts on this subject based on the ideXlab platform.
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Validation of anti-Glucocerebrosidase antibodies for western blot analysis on protein lysates of murine and human cells
The Biochemical journal, 2019Co-Authors: Brad A. Davidson, Ellen Sidransky, Matthew Nguyen, Taylor Lindstrom, Richard Grey, Robert Burnett, Elma Aflaki, Wendy WestbroekAbstract:Gaucher disease is a rare lysosomal storage disorder caused by mutations in the GBA1 gene, encoding the lysosome-resident Glucocerebrosidase enzyme involved in the hydrolysis of glucosylceramide. The discovery of an association between mutations in GBA1 and the development of synucleinopathies, including Parkinson disease, has directed attention to Glucocerebrosidase as a potential therapeutic target for different synucleinopathies. These findings initiated an exponential growth in research and publications regarding the Glucocerebrosidase enzyme. The use of various commercial and custom-made Glucocerebrosidase antibodies has been reported, but standardized in-depth validation is still not available for many of these antibodies. This work details the evaluation of several previously reported Glucocerebrosidase antibodies for western blot analysis, tested on protein lysates of murine gba +/+ and gba -/ - immortalized neurons and primary human wild type and type 2 Gaucher disease fibroblasts.
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a new Glucocerebrosidase chaperone reduces α synuclein and glycolipid levels in ipsc derived dopaminergic neurons from patients with gaucher disease and parkinsonism
The Journal of Neuroscience, 2016Co-Authors: Elma Aflaki, Barbara K. Stubblefield, Wendy Westbroek, Nima Moaven, Wei Zheng, Daniel K Borger, Steven A Rogers, Samarjit Patnaik, Frank J Schoenen, Patricia SullivanAbstract:Among the known genetic risk factors for Parkinson disease, mutations in GBA1 , the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how Glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased Glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of Glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored Glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of Glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal Glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory Glucocerebrosidase chaperone, which successfully restored Glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning Glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.
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Identification of miRNAs that modulate Glucocerebrosidase activity in Gaucher disease cells
RNA biology, 2014Co-Authors: Marina Siebert, Arash Velayati, Wendy Westbroek, Yu-chi Chen, Nima Moaven, Maria Luiza Saraiva-pereira, Scott E. Martin, Ellen SidranskyAbstract:Gaucher disease is an autosomal recessive disorder caused by deficiency of the enzyme Glucocerebrosidase. Although it is a monogenic disease, there is vast phenotypic heterogeneity, even among patients with the same genotype. MicroRNAs (miRNAs) are small non-coding RNAs involved in many biological processes and diseases. To determine whether miRNAs can affect Glucocerebrosidase activity, we performed a screen of 875 different miRNA mimics. The screen was performed using Gaucher fibroblasts, and Glucocerebrosidase activity was used as the initial outcome parameter. We found several miRNAs that either up- or down-regulated Glucocerebrosidase activity. In follow-up assays, we confirmed that one specific miRNA (miR-127–5p) down-regulated both Glucocerebrosidase activity and protein levels by down-regulation of LIMP-2, the receptor involved in proper trafficking of Glucocerebrosidase from the endoplasmic reticulum to the lysosome. A conditioned media assay demonstrated that cells treated with this miRNA secret...
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Glucocerebrosidase is shaking up the synucleinopathies
Brain, 2014Co-Authors: Marina Siebert, Ellen Sidransky, Wendy WestbroekAbstract:The lysosomal enzyme Glucocerebrosidase, encoded by the Glucocerebrosidase gene, is involved in the breakdown of glucocerebroside into glucose and ceramide. Lysosomal build-up of the substrate glucocerebroside occurs in cells of the reticulo-endothelial system in patients with Gaucher disease, a rare lysosomal storage disorder caused by the recessively inherited deficiency of Glucocerebrosidase. Gaucher disease has a broad clinical phenotypic spectrum, divided into non-neuronopathic and neuronopathic forms. Like many monogenic diseases, the correlation between clinical manifestations and molecular genotype is not straightforward. There is now a well-established clinical association between mutations in the Glucocerebrosidase gene and the development of more prevalent multifactorial disorders including Parkinson's disease and other synucleinopathies. In this review we discuss recent studies advancing our understanding of the cellular relationship between Glucocerebrosidase and α-synuclein, the potential impact of established and emerging therapeutics for Gaucher disease for the treatment of the synucleinopathies, and the role of lysosomal pathways in the pathogenesis of these neurodegenerative disorders.