Diversity of glycosphingolipid GM2 and cholesterol accumulation in NPC1 patient-specific iPSC-derived neurons

Michaela Trilck, Franziska Peter, Chaonan Zheng, Marcus Frank, Kostantin Dobrenis, Hermann Mascher, Arndt Rolfs, Moritz J. Frech

Research output: Contribution to journalArticle

6 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)52-61
Number of pages10
JournalBrain Research
Volume1657
DOIs
StatePublished - Feb 15 2017

Fingerprint

Type C Niemann-Pick Disease
Glycosphingolipids
Cholesterol
Neurons
Mutation
Hexosaminidase A
Staining and Labeling
Molecular Docking Simulation
G(M2) Ganglioside
Filipin
G(M3) Ganglioside
Gangliosides
Neuroglia
Neurodegenerative Diseases
High Pressure Liquid Chromatography
Phenotype
Cell Line

Keywords

  • Cholesterol
  • Glycosphingolipids
  • GM2
  • iPSC-derived neurons
  • iPSCs
  • Molecular docking
  • Niemann-Pick disease Type C1

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Developmental Biology
  • Clinical Neurology

Cite this

Diversity of glycosphingolipid GM2 and cholesterol accumulation in NPC1 patient-specific iPSC-derived neurons. / Trilck, Michaela; Peter, Franziska; Zheng, Chaonan; Frank, Marcus; Dobrenis, Kostantin; Mascher, Hermann; Rolfs, Arndt; Frech, Moritz J.

In: Brain Research, Vol. 1657, 15.02.2017, p. 52-61.

Research output: Contribution to journalArticle

Trilck, Michaela ; Peter, Franziska ; Zheng, Chaonan ; Frank, Marcus ; Dobrenis, Kostantin ; Mascher, Hermann ; Rolfs, Arndt ; Frech, Moritz J. / Diversity of glycosphingolipid GM2 and cholesterol accumulation in NPC1 patient-specific iPSC-derived neurons. In: Brain Research. 2017 ; Vol. 1657. pp. 52-61.
@article{2a461bbd179540d1b103d36a152f5ca6,
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.",
keywords = "Cholesterol, Glycosphingolipids, GM2, iPSC-derived neurons, iPSCs, Molecular docking, Niemann-Pick disease Type C1",
author = "Michaela Trilck and Franziska Peter and Chaonan Zheng and Marcus Frank and Kostantin Dobrenis and Hermann Mascher and Arndt Rolfs and Frech, {Moritz J.}",
year = "2017",
month = "2",
day = "15",
doi = "10.1016/j.brainres.2016.11.031",
language = "English (US)",
volume = "1657",
pages = "52--61",
journal = "Brain Research",
issn = "0006-8993",
publisher = "Elsevier",

}

TY - JOUR

T1 - Diversity of glycosphingolipid GM2 and cholesterol accumulation in NPC1 patient-specific iPSC-derived neurons

AU - Trilck, Michaela

AU - Peter, Franziska

AU - Zheng, Chaonan

AU - Frank, Marcus

AU - Dobrenis, Kostantin

AU - Mascher, Hermann

AU - Rolfs, Arndt

AU - Frech, Moritz J.

PY - 2017/2/15

Y1 - 2017/2/15

N2 - 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.

AB - 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.

KW - Cholesterol

KW - Glycosphingolipids

KW - GM2

KW - iPSC-derived neurons

KW - iPSCs

KW - Molecular docking

KW - Niemann-Pick disease Type C1

UR - http://www.scopus.com/inward/record.url?scp=85006272156&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85006272156&partnerID=8YFLogxK

U2 - 10.1016/j.brainres.2016.11.031

DO - 10.1016/j.brainres.2016.11.031

M3 - Article

C2 - 27923633

AN - SCOPUS:85006272156

VL - 1657

SP - 52

EP - 61

JO - Brain Research

JF - Brain Research

SN - 0006-8993

ER -