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Cited by 226Alzheimer's disease research and treatmentsPluripotent Stem Cells ResearchDementia and Cognitive Impairment Research

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Neurons derived from sporadic Alzheimer’s disease iPSCs reveal elevated TAU hyperphosphorylation, increased amyloid levels, and GSK3B activation

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Ochalek, Anna, Mihalik, Balázs, Avci, Hasan X., Chandrasekaran, Abinaya, Téglási, Annamária, Bock, István, Giudice, Maria Lo, Táncos, Zsuzsanna, Molnár, Kinga, László, Lajos, Nielsen, Jørgen E., Holst, Bjørn, Freude, Kristine, Hyttel, Poul, Kobolák, Julianna, Dinnyés, András·2017·Alzheimer's Research & Therapy
DOI 10.1186/s13195-017-0317-zPMC PMC5709977
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Abstract

Alzheimer’s disease (AD) is the most common type of dementia, affecting one in eight adults over 65 years of age. The majority of AD cases are sporadic, with unknown etiology, and only 5% of all patients with AD present the familial monogenic form of the disease. In the present study, our aim was to establish an in vitro cell model based on patient-specific human neurons to study the pathomechanism of sporadic AD. We compared neurons derived from induced pluripotent stem cell (iPSC) lines of patients with early-onset familial Alzheimer’s disease (fAD), all caused by mutations in the PSEN1 gene; patients with late-onset sporadic Alzheimer’s disease (sAD); and three control individuals without dementia. The iPSC lines were differentiated toward mature cortical neurons, and AD pathological hallmarks were analyzed by RT-qPCR, enzyme-linked immunosorbent assay, and Western blotting methods. Neurons from patients with fAD and patients with sAD showed increased phosphorylation of TAU protein at all investigated phosphorylation sites. Relative to the control neurons, neurons derived from patients with fAD and patients with sAD exhibited higher levels of extracellular amyloid-β 1–40 (Aβ 1–40 ) and amyloid-β 1–42 (Aβ 1–42 ). However, significantly increased Aβ 1–42 /Aβ 1–40 ratios, which is one of the pathological markers of fAD, were observed only in samples of patients with fAD. Additionally, we detected increased levels of active glycogen synthase kinase 3 β, a physiological kinase of TAU, in neurons derived from AD iPSCs, as well as significant upregulation of amyloid precursor protein (APP) synthesis and APP carboxy-terminal fragment cleavage. Moreover, elevated sensitivity to oxidative stress, as induced by amyloid oligomers or peroxide, was detected in both fAD- and sAD-derived neurons. On the basis of the experiments we performed, we can conclude there is no evident difference except secreted Aβ 1–40 levels in phenotype between fAD and sAD samples. To our knowledge, this is the first study in which the hyperphosphorylation of TAU protein has been compared in fAD and sAD iPSC-derived neurons. Our findings demonstrate that iPSC technology is suitable to model both fAD and sAD and may provide a platform for developing new treatment strategies for these conditions.

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Figures

Fig. 1 Immunofluorescence characterization of the induced pluripotent stem cells (iPSCs) derived neural progenitor cells. a Representative images of neural progenitor cells (NPCs) from healthy control individuals (ctrl-1–ctrl-4), patients with early-onset familial Alzheimer’s disease (fAD-1–fAD-4), and patients with late-onset sporadic Alzheimer’s disease (sAD-1–sAD-6) stained with Nestin (green), paired box 6 (PAX6; red), and 4′,6-diamidino-2-phenylindole (DAPI; blue). All three groups exhibited similar progenitor marker expression. scale bar = 30 μm. b Flow cytometric analysis of cultured NPCs. Dot plots demonstrate similar expression pattern of PAX6 and Nestin in control, fAD, and sAD iPSC line-derived NPC cultures with similar passage numbers (p6)
Fig. 1 Immunofluorescence characterization of the induced pluripotent stem cells (iPSCs) derived neural progenitor cells. a Representative images of neural progenitor cells (NPCs) from healthy control individuals (ctrl-1–ctrl-4), patients with early-onset familial Alzheimer’s disease (fAD-1–fAD-4), and patients with late-onset sporadic Alzheimer’s disease (sAD-1–sAD-6) stained with Nestin (green), paired box 6 (PAX6; red), and 4′,6-diamidino-2-phenylindole (DAPI; blue). All three groups exhibited similar progenitor marker expression. scale bar = 30 μm. b Flow cytometric analysis of cultured NPCs. Dot plots demonstrate similar expression pattern of PAX6 and Nestin in control, fAD, and sAD iPSC line-derived NPC cultures with similar passage numbers (p6)CC BY · PubMed Central
Fig. 2 Neuronal differentiation from control and Alzheimer’s disease induced pluripotent stem cells (AD-iPSCs). a Representative immunofluorescence images show expression of neuronal markers at day 70 of terminal differentiation (TD70): tubulin β 3 class III (TUBB3; green, left panel), microtubule-associated protein 2 (MAP2; red, left panel), TAU (red, middle panel), and neurofilament, heavy polypeptide 200 kDa (NF200; red, right panel). Scale bar = 50 μm. b Differentiation of iPSCs into various neuronal subtypes at TD70 was confirmed by the presence of specific markers: vesicular acetylcholine transporter (VACHT) (cholinergic neurons), glutamic acid decarboxylases 2 and 1 (GAD2/1) (GABAergic neurons), TH (dopaminergic neurons), and vesicular glutamate transporter 1/2 (VGLUT1/2) (glutamatergic neurons). Scale bars = 40 μm and 10 μm as indicated. c Gene expression of neuronal markers from ctr-1, early-onset familial Alzheimer’s disease (fAD-1), and sporadic Alzheimer’s disease (sAD-1) lines at TD70 obtained by qPCR. The expression values were normalized to GAPDH and calculated as a relative amount of messenger RNA versus expression value of neural progenitor cells, which was set to 1. Data are reported as mean ± SEM of three independent measurements. AD-iPSC neurons demonstrate expression pattern similar to that of neuronal cells derived from control iPSCs. d Ultrastructure of a synapse at TD35 with synaptic vesicles (Sv) and synaptic cleft with synaptic junctions (tight junctions, black arrowheads). Docking synaptic vesicles are also observable (white arrowheads). Scale bar = 100 nm. DAPI 4′,6-Diamidino-2-phenylindole
Fig. 2 Neuronal differentiation from control and Alzheimer’s disease induced pluripotent stem cells (AD-iPSCs). a Representative immunofluorescence images show expression of neuronal markers at day 70 of terminal differentiation (TD70): tubulin β 3 class III (TUBB3; green, left panel), microtubule-associated protein 2 (MAP2; red, left panel), TAU (red, middle panel), and neurofilament, heavy polypeptide 200 kDa (NF200; red, right panel). Scale bar = 50 μm. b Differentiation of iPSCs into various neuronal subtypes at TD70 was confirmed by the presence of specific markers: vesicular acetylcholine transporter (VACHT) (cholinergic neurons), glutamic acid decarboxylases 2 and 1 (GAD2/1) (GABAergic neurons), TH (dopaminergic neurons), and vesicular glutamate transporter 1/2 (VGLUT1/2) (glutamatergic neurons). Scale bars = 40 μm and 10 μm as indicated. c Gene expression of neuronal markers from ctr-1, early-onset familial Alzheimer’s disease (fAD-1), and sporadic Alzheimer’s disease (sAD-1) lines at TD70 obtained by qPCR. The expression values were normalized to GAPDH and calculated as a relative amount of messenger RNA versus expression value of neural progenitor cells, which was set to 1. Data are reported as mean ± SEM of three independent measurements. AD-iPSC neurons demonstrate expression pattern similar to that of neuronal cells derived from control iPSCs. d Ultrastructure of a synapse at TD35 with synaptic vesicles (Sv) and synaptic cleft with synaptic junctions (tight junctions, black arrowheads). Docking synaptic vesicles are also observable (white arrowheads). Scale bar = 100 nm. DAPI 4′,6-Diamidino-2-phenylindoleCC BY · PubMed Central
Fig. 3 Characterization of amyloid-β (Aβ) secretion in control and Alzheimer’s disease induced pluripotent stem cell (AD iPSC)-derived neurons at terminal differentiation day 42 (TD42), TD56, and TD70. a The amount of secreted Aβ1–40 from control-, early-onset familial Alzheimer’s disease (fAD)- (fAD-1–fAD-4), and sporadic Alzheimer’s disease (sAD)- (sAD-1–sAD-6)-iPSC-derived neurons. b The amount of Aβ1–42 released from control- and AD-iPSC-derived neurons. c The ratio of Aβ1–42/Aβ1–40 from neurons derived from control and AD lines. Aβ1–40 and Aβ1–42 secreted from neural cells into the medium were measured at day 4 after the last medium change. The extracellular Aβ levels determined (in picomolar concentrations) were normalized to total protein content of cell lysates. Data represent mean ± SEM (n = 3). Dunnett’s test was performed to evaluate the significance of groups compared with control at the same time point of differentiation (*p < 0.05)
Fig. 3 Characterization of amyloid-β (Aβ) secretion in control and Alzheimer’s disease induced pluripotent stem cell (AD iPSC)-derived neurons at terminal differentiation day 42 (TD42), TD56, and TD70. a The amount of secreted Aβ1–40 from control-, early-onset familial Alzheimer’s disease (fAD)- (fAD-1–fAD-4), and sporadic Alzheimer’s disease (sAD)- (sAD-1–sAD-6)-iPSC-derived neurons. b The amount of Aβ1–42 released from control- and AD-iPSC-derived neurons. c The ratio of Aβ1–42/Aβ1–40 from neurons derived from control and AD lines. Aβ1–40 and Aβ1–42 secreted from neural cells into the medium were measured at day 4 after the last medium change. The extracellular Aβ levels determined (in picomolar concentrations) were normalized to total protein content of cell lysates. Data represent mean ± SEM (n = 3). Dunnett’s test was performed to evaluate the significance of groups compared with control at the same time point of differentiation (*p < 0.05)CC BY · PubMed Central
Fig. 4 Characterization of amyloid precursor protein (APP) and amyloid precursor protein carboxy-terminal fragment (APP-CTF) expression during neuronal differentiation. Representative immunoblots of (a) APP and APP-CTF in control neurons (ctrl-1), early-onset familial Alzheimer’s disease (fAD)-derived neurons (fAD-1–fAD-4), and (b) sporadic Alzheimer’s disease (sAD)-derived neurons (sAD-1–sAD-6) at terminal differentiation day 14 (TD14), TD28, TD42, TD56, and TD70. Quantification of (c) APP and (d) APP-CTF signals at TD70 was normalized to GAPDH. Data are presented as mean ± SEM (n = 3). Dunnett’s test was performed to evaluate the significance of groups compared with control (*p < 0.05)
Fig. 4 Characterization of amyloid precursor protein (APP) and amyloid precursor protein carboxy-terminal fragment (APP-CTF) expression during neuronal differentiation. Representative immunoblots of (a) APP and APP-CTF in control neurons (ctrl-1), early-onset familial Alzheimer’s disease (fAD)-derived neurons (fAD-1–fAD-4), and (b) sporadic Alzheimer’s disease (sAD)-derived neurons (sAD-1–sAD-6) at terminal differentiation day 14 (TD14), TD28, TD42, TD56, and TD70. Quantification of (c) APP and (d) APP-CTF signals at TD70 was normalized to GAPDH. Data are presented as mean ± SEM (n = 3). Dunnett’s test was performed to evaluate the significance of groups compared with control (*p < 0.05)CC BY · PubMed Central
Fig. 5 Western blot analysis of total TAU and phosphorylated TAU (pTAU) protein. a Schematic representation of human TAU isoform (441 amino acids) with the functional projection and microtubule-binding domains. Projection domains including a proline-rich region and N-terminal part interact with cytoskeletal elements and are involved in signal transduction. Microtubule-binding domains with a C-terminal part regulate the microtubule polymerization and bind to proteins such as presenilin 1 (PSEN1). Epitopes of pTAU antibodies analyzed in this study are indicated on the scheme. b–f Densitometric analysis of TAU phosphorylated at different epitopes: S262, S396, S202/T205, T181, and S400/T403/S404. All samples were analyzed at day 70 of terminal differentiation. The amount of pTAU relative to total TAU levels in the lysates was measured. GAPDH as the loading control was used to normalize the data. All values are the mean ± SEM (n = 3). Dunnett’s test was performed to evaluate the significance of groups compared with control (*p < 0.05)
Fig. 5 Western blot analysis of total TAU and phosphorylated TAU (pTAU) protein. a Schematic representation of human TAU isoform (441 amino acids) with the functional projection and microtubule-binding domains. Projection domains including a proline-rich region and N-terminal part interact with cytoskeletal elements and are involved in signal transduction. Microtubule-binding domains with a C-terminal part regulate the microtubule polymerization and bind to proteins such as presenilin 1 (PSEN1). Epitopes of pTAU antibodies analyzed in this study are indicated on the scheme. b–f Densitometric analysis of TAU phosphorylated at different epitopes: S262, S396, S202/T205, T181, and S400/T403/S404. All samples were analyzed at day 70 of terminal differentiation. The amount of pTAU relative to total TAU levels in the lysates was measured. GAPDH as the loading control was used to normalize the data. All values are the mean ± SEM (n = 3). Dunnett’s test was performed to evaluate the significance of groups compared with control (*p < 0.05)CC BY · PubMed Central
Fig. 6 Analysis of glycogen synthase kinase 3β (GSK3B) and the active form of GSK3B in neuronal culture. a Representative immunoblotting shows the phosphorylation of GSK3B at Ser9 (inactive form of the kinase [102]) and GSK3B in control neurons (ctrl-1), early-onset familial Alzheimer’s disease (fAD) neurons (fAD-1–fAD-4), and (b) sporadic Alzheimer’s disease (sAD) neurons (sAD-1–sAD-6) at all time points of neuronal terminal differentiation (TD14–TD70). c Quantification of the active GSK3B form at TD70 was presented as a percentage of nonphosphorylated GSK3B at Ser9. d Densitometric analysis of TAU phosphorylated at the T231 epitope at TD70. All values were normalized to GAPDH and are presented as mean ± SEM (n = 3). Dunnett’s test was performed to evaluate the significance of groups compared with control (*p < 0.05)
Fig. 6 Analysis of glycogen synthase kinase 3β (GSK3B) and the active form of GSK3B in neuronal culture. a Representative immunoblotting shows the phosphorylation of GSK3B at Ser9 (inactive form of the kinase [102]) and GSK3B in control neurons (ctrl-1), early-onset familial Alzheimer’s disease (fAD) neurons (fAD-1–fAD-4), and (b) sporadic Alzheimer’s disease (sAD) neurons (sAD-1–sAD-6) at all time points of neuronal terminal differentiation (TD14–TD70). c Quantification of the active GSK3B form at TD70 was presented as a percentage of nonphosphorylated GSK3B at Ser9. d Densitometric analysis of TAU phosphorylated at the T231 epitope at TD70. All values were normalized to GAPDH and are presented as mean ± SEM (n = 3). Dunnett’s test was performed to evaluate the significance of groups compared with control (*p < 0.05)CC BY · PubMed Central
Fig. 7 Effect of hydrogen peroxide (H2O2) and amyloid-β1–42 (Aβ1–42) oligomer treatment on neuronal viability. a Viability of induced pluripotent stem cell (iPSC)-derived neurons from control individuals (ctrl-1–ctrl-4), patients with early-onset familial Alzheimer’s disease (fAD) (fAD-1–fAD-4), and patients with sporadic Alzheimer’s disease (sAD) (sAD-1–sAD-6) at day 28 of terminal differentiation (TD28) and TD56 after 24 h of treatment with 30 μM H2O2 (left panel) and 60 μM H2O2 (right panel). b Neuronal survival of control and Alzheimer’s disease (AD) clones at TD28 and TD56 cultured 24 h in the presence of 5 μM Aβ1–42 oligomer solution. Neuronal survival was represented as a percentage of control. Viability of the cultures following H2O2 and Aβ treatment was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. Values are presented as the mean ± SEM (n = 3). Dunnett’s test was performed to evaluate the significance of groups compared with control at the same time point of differentiation (*p < 0.05)
Fig. 7 Effect of hydrogen peroxide (H2O2) and amyloid-β1–42 (Aβ1–42) oligomer treatment on neuronal viability. a Viability of induced pluripotent stem cell (iPSC)-derived neurons from control individuals (ctrl-1–ctrl-4), patients with early-onset familial Alzheimer’s disease (fAD) (fAD-1–fAD-4), and patients with sporadic Alzheimer’s disease (sAD) (sAD-1–sAD-6) at day 28 of terminal differentiation (TD28) and TD56 after 24 h of treatment with 30 μM H2O2 (left panel) and 60 μM H2O2 (right panel). b Neuronal survival of control and Alzheimer’s disease (AD) clones at TD28 and TD56 cultured 24 h in the presence of 5 μM Aβ1–42 oligomer solution. Neuronal survival was represented as a percentage of control. Viability of the cultures following H2O2 and Aβ treatment was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. Values are presented as the mean ± SEM (n = 3). Dunnett’s test was performed to evaluate the significance of groups compared with control at the same time point of differentiation (*p < 0.05)CC BY · PubMed Central

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  1. Ferreira ST, Lourenco MV, Oliveira MM, De Felice FG. Soluble amyloid-β oligomers as synaptotoxins leading to cognitive impairment in Alzheimer’s disease. Front Cell Neurosci. 2015;9:191.

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Metadata

Authors
Ochalek, A., Mihalik, B., Avci, H. X., Chandrasekaran, A., Téglási, A., Bock, I., Giudice, M. L., Táncos, Z., Molnár, K., László, L., Nielsen, J. E., Holst, B., Freude, K., Hyttel, P., Kobolák, J., & Dinnyés, A.
Affiliations
  • BioTalentum (Hungary) (HU)
  • University of Szeged (HU)
  • Eötvös Loránd University (HU)
  • University of Copenhagen (DK)
  • Bioneer (Denmark) (DK)
  • Magyar Agrár- és Élettudományi Egyetem (HU)
Corresponding author
Dinnyés, András
ORCID
Venue
Alzheimer's Research & Therapy
Publisher
Springer Science and Business Media LLC
Published
2017-12
Volume
9
Issue
1
Pages
90-90
ISSN
1758-9193 (ISSN-L)
DOI
10.1186/s13195-017-0317-z
PMC
PMC5709977
OpenAlex
W2769598455
Cited by
225
References
104
Citations by year
2026: 16 · 2025: 27 · 2024: 28 · 2023: 19 · 2022: 29 · 2021: 25 · 2020: 38 · 2019: 29 · 2018: 13
FWCI
16.19
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Alzheimer s Research & Therapy (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • ELTE Digital Institutional Repository (EDIT) (Eötvös Loránd University) (submittedVersion, other-oa)
Funders
  • EU FP7, STEMMAD (PIAPP-GA-2012-324451)
  • EU FP7, EpiHealth (HEALTH-2012-F2-278418)
  • EU FP7, EpiHealthNet (PITN-GA-2012-317146)
  • Richter Gedeon Research Grant (RG-IPI-2013_TP7/026)
  • Innovation Fund Denmark (BrainStem 4108-00008B, NeuroStem 4096-00001B)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
Keywords
sporadic Alzheimer's disease, familial Alzheimer's disease, iPSC-derived neurons, tau hyperphosphorylation, amyloid-beta, amyloid precursor protein, oxidative stress, amyloid oligomers
MeSH terms
Glycogen Synthase Kinase 3 beta, Alzheimer Disease, Animals, Cell Differentiation, Cell Line, Transformed, Gene Expression Regulation, Humans, Hydrogen Peroxide, Mutation, Nerve Tissue Proteins, Neurons, Phosphorylation, RNA, Messenger, Time Factors, Amyloid beta-Peptides, Amyloid beta-Protein Precursor, tau Proteins, Oxidative Stress, Presenilin-1, Induced Pluripotent Stem Cells

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