title: Diversity of glycosphingolipid GM2 and cholesterol accumulation in NPC1 patient-specific iPSC-derived neurons.
abstract: Niemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. On the cellular level NPC1 mutations lead to an accumulation of cholesterol and gangliosides. As a thorough analysis of the severely affected neuronal cells is unfeasible in NPC1 patients, we recently described the cellular phenotype of neuronal cells derived from NPC1 patient iPSCs carrying the compound heterozygous mutation c.1836A>C/c.1628delC. Here we expanded the analysis to cell lines carrying the prevalent mutation c.3182T>C and the novel mutation c.1180T>C, as well as to the determination of GM2 and GM3 gangliosides in NPC1 patient-specific iPSC-derived neurons and glia cells. Immunocytochemical detection of GM2 revealed punctated staining pattern predominantly localized in neurons. Detection of cholesterol by filipin staining showed a comparable staining pattern, colocalized with GM2, indicating a deposit of GM2 and cholesterol in the same cellular compartments. Accumulations were not only restricted to cell bodies, but were also found in the neuronal extensions. A quantification of the GM2 amount by HPLC-MS/MS confirmed significantly higher amounts in neurons carrying a mutation. Additionally, these cells displayed a lowered activity of the catabolic enzyme Hex A, but not B4GALNT1. Molecular docking simulations indicated binding of cholesterol to Hex A, suggesting cholesterol influences the GM2 degradation pathway and, subsequently, leading to the accumulation of GM2. Taken together, this is the first study showing an accumulation of GM2 in neuronal derivatives of patient-specific iPSCs and thus proving further disease-specific hallmarks in this human in vitro model of NPC1.
authors.affiliation: Albrecht-Kossel-Institute for Neuroregeneration (AKos), University Medicine Rostock, Gehlsheimer Straße 20, 18147 Rostock, Germany; Institute of Neurogenetics, University of Luebeck, Maria-Goeppert-Str. 1, 23562 Luebeck, Germany. Electronic address: michaela.trilck@neuro.uni-luebeck.de.
authors.forename: Michaela
authors.initials: M
authors.lastname: Trilck
authors.affiliation: Albrecht-Kossel-Institute for Neuroregeneration (AKos), University Medicine Rostock, Gehlsheimer Straße 20, 18147 Rostock, Germany. Electronic address: franziska.peter2@uni-rostock.de.
authors.forename: Franziska
authors.initials: F
authors.lastname: Peter
authors.affiliation: Albrecht-Kossel-Institute for Neuroregeneration (AKos), University Medicine Rostock, Gehlsheimer Straße 20, 18147 Rostock, Germany; Leibniz Institute for Catalysis, University of Rostock, Rostock, Germany. Electronic address: cnzheng2009@163.com.
authors.forename: Chaonan
authors.initials: C
authors.lastname: Zheng
authors.affiliation: Medical Biology and Electron Microscopy Center, University Medicine Rostock, Strempelstraße 14, 18057 Rostock, Germany. Electronic address: marcus.frank@med.uni-rostock.de.
authors.forename: Marcus
authors.initials: M
authors.lastname: Frank
authors.affiliation: Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Rose F. Kennedy Center for Research on Intellectual and Developmental Disabilities, 1410 Pelham Parkway South, Bronx, NY 10461, USA. Electronic address: kostantin.dobrenis@einstein.yu.edu.
authors.forename: Kostantin
authors.initials: K
authors.lastname: Dobrenis
authors.affiliation: pharm-analyt Labor GmbH, Ferdinand-Pichler-Gasse 2, 2500 Baden, Austria. Electronic address: hermann.mascher@pharm-analyt.com.
authors.forename: Hermann
authors.initials: H
authors.lastname: Mascher
authors.affiliation: Albrecht-Kossel-Institute for Neuroregeneration (AKos), University Medicine Rostock, Gehlsheimer Straße 20, 18147 Rostock, Germany. Electronic address: arndt.rolfs@med.uni-rostock.de.
authors.forename: Arndt
authors.initials: A
authors.lastname: Rolfs
authors.affiliation: Albrecht-Kossel-Institute for Neuroregeneration (AKos), University Medicine Rostock, Gehlsheimer Straße 20, 18147 Rostock, Germany. Electronic address: moritz.frech@med.uni-rostock.de.
authors.forename: Moritz J
authors.initials: MJ
authors.lastname: Frech
mesh_terms: D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D005678:G(M2) Ganglioside / Q000378:metabolism*; D005679:G(M3) Ganglioside / Q000378:metabolism; D054818:Hexosaminidase A / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism* / Q000473:pathology; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D009154:Mutation; D058953:Neural Stem Cells / Q000378:metabolism / Q000473:pathology; D009457:Neuroglia / Q000378:metabolism / Q000473:pathology; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D000077296:Niemann-Pick C1 Protein; D052556:Niemann-Pick Disease, Type C / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
chemical_list: D002352:Carrier Proteins; D005679:G(M3) Ganglioside; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D005678:G(M2) Ganglioside; D002784:Cholesterol; D054818:Hexosaminidase A
keywords: Cholesterol; GM2; Glycosphingolipids; Molecular docking; Niemann-Pick disease Type C1; iPSC-derived neurons; iPSCs