Patricia García-Sanz1,2, Lorena Orgaz1,2, Guillermo Bueno-Gil1,2, Isabel Espadas1,2, Eva Rodríguez-Traver1,2, Jaime Kulisevsky2,3, Antonia Gutierrez2,4, José C Dávila2,4, Rosa A González-Polo2,5, José M Fuentes2,5, Pablo Mir2,6, Carlos Vicario1,2, Rosario Moratalla1,2. 1. Instituto Cajal, CSIC, Madrid, Spain. 2. CIBERNED, Madrid, Spain. 3. Movement Disorders Unit, Neurology Dpt, Hospital Sant Pau (IIB-Sant Pau), Univ. Autònoma de Barcelona, Barcelona, Spain. 4. Dpto. de Biología Celular, Genética y Fisiología, Facultad de Ciencias, IBIMA, Universidad de Málaga, Málaga, Spain. 5. Dpto. de Bioquímica, Biología Molecular y Genética F. Enfermería y T.O., Univ. de Extremadura, Cáceres, Spain. 6. Neurology Dpt, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain.
Abstract
BACKGROUND: Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme β-glucocerebrosidase-1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S-GBA1 mutation on cellular homeostasis and vulnerability in a patient-specific cellular model of PD. METHODS: We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy-lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry. RESULTS: The N370S mutation produced a significant reduction in β-glucocerebrosidase-1 protein and enzyme activity and β-glucocerebrosidase-1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death. CONCLUSIONS: Our results support a connection between the loss of β-glucocerebrosidase-1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1-PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1-PD shares common features with lipid-storage diseases.
BACKGROUND: Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme β-glucocerebrosidase-1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S-GBA1 mutation on cellular homeostasis and vulnerability in a patient-specific cellular model of PD. METHODS: We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy-lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry. RESULTS: The N370S mutation produced a significant reduction in β-glucocerebrosidase-1 protein and enzyme activity and β-glucocerebrosidase-1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death. CONCLUSIONS: Our results support a connection between the loss of β-glucocerebrosidase-1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1-PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1-PD shares common features with lipid-storage diseases.
Authors: Anwesha Sanyal; Mark P DeAndrade; Hailey S Novis; Steven Lin; Jianjun Chang; Nathalie Lengacher; Julianna J Tomlinson; Malú G Tansey; Matthew J LaVoie Journal: Mov Disord Date: 2020-02-08 Impact factor: 10.338
Authors: Daniel Macías-García; María Teresa Periñán; Laura Muñoz-Delgado; María Valle Jimenez-Jaraba; Miguel Ángel Labrador-Espinosa; Silvia Jesús; Dolores Buiza-Rueda; Carlota Méndez-Del Barrio; Astrid Adarmes-Gómez; Pilar Gómez-Garre; Pablo Mir Journal: NPJ Parkinsons Dis Date: 2021-07-16