1. Neuroscience
Download icon

Mechanisms of hyperexcitability in Alzheimer’s disease hiPSC-derived neurons and cerebral organoids vs isogenic controls

  1. Swagata Ghatak
  2. Nima Dolatabadi
  3. Dorit Trudler
  4. XiaoTong Zhang
  5. Yin Wu
  6. Madhav Mohata
  7. Rajesh Ambasudhan
  8. Maria Talantova
  9. Stuart A Lipton  Is a corresponding author
  1. The Scripps Research Institute, United States
  2. Scintillon Institute, United States
  3. University of California, San Diego, United States
Research Article
Cite this article as: eLife 2019;8:e50333 doi: 10.7554/eLife.50333
Voice your concerns about research culture and research communication: Have your say in our 7th annual survey.
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
AD neurons show enhanced excitability compared to isogenic control neurons.

(A) Spontaneous action potentials (sAP) at resting membrane potential (RMP). WT/WT hiPSC-derived cerebrocortical neuron data in black, M146V/WT and APPswe/WT in red. (B) Quantification of sAP frequency. (C) Evoked APs in neurons hyperpolarized to −60 mV. Single traces (insets). (D–H) Neuronal membrane and AP properties. Quantification of resting membrane potential (RMP, (D), AP threshold (APthreshold, (E); AP height (F); width at AP half height (G); AP decay slope (H). (I). Quantification of cell capacitance (Cm), reflecting neuronal size. Data are mean ± SEM. Statistical significance analyzed by ANOVA with post hoc Dunnett’s test. Exact p values for comparison to WT are listed in the bar graphs in this and subsequent figures. Unless otherwise stated, total number of neurons quantified is listed above the bars in this and subsequent electrophysiology figures.

Figure 1—source data 1

Excel files containing data shown as summary bar graph in Figure 1B,D–I.

https://cdn.elifesciences.org/articles/50333/elife-50333-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
AD hiPSC-derived neuronal cultures express cortical neuronal markers and aberrant Aβ levels compared to isogenic controls.

(A) DNA sequencing to confirm the presence of mutations in hiPSC-derived neural progenitor cells with WT/WT, M146V/WT, and APPswe/WT genotypes. (B) hiPSC-derived neurons showing FOXG1 and Tuj1 expression. (C) hiPSC-derived neurons showing FOXG1 and CTIP2 expression. (D) Ratio of Aβ42/Aβ40 quantified by ELISA from cultures at 5 weeks. (E) Quantification of Aβ levels normalized to total protein from lysates at 5 weeks. Data are mean ± SEM. Statistical significance analyzed by ANOVA with post hoc Dunnett’s test for multiple comparisons. Number of independent experiments listed above the bars.

Figure 1—figure supplement 2
ΔE9/WT neurons show enhanced excitability compared to isogenic control neurons.

(A) Spontaneous action potentials (sAP) at resting membrane potential (RMP). WT/WT hiPSC-derived cerebrocortical neuron data in black, ΔE9/WT in red. (B) Quantification of sAP frequency. (C) Evoked APs in neurons hyperpolarized to −60 mV. Single traces (insets). (D–H) Neuronal membrane and AP properties. Quantification of resting membrane potential (RMP, (D), AP threshold (APthreshold, (E); AP height (F); width at AP half height (G); AP decay slope (H). (I) Quantification of cell capacitance (Cm), reflecting overall neuronal size. Data are mean ± SEM. Statistical significance analyzed by two-tailed unpaired Student’s t-test.

Figure 2 with 2 supplements
AD neurons show differences in morphology and sodium channel properties compared to WT neurons.

WT/WT hiPSC-derived cerebrocortical neuron data in black, M146V/WT and APPswe/WT in red at 5 weeks of culture. (A) Representative images of cells expressing the neuronal marker Tuj1. (B) Quantification of area covered by neurites expressing Tuj1 normalized to total number of neurons. (C) Quantification of somal area normalized to total number of neurons. Total number of random fields of neuronal cultures analyzed in three separate experiments is listed above the bars in B,C. (D) Representative tracings of neurites from WT/WT, APPswe/WT, and M146V/WT AD lines. Scale bar: 100 µm. (E) Quantification of total neurite length. (F) Quantification of total number of neurite branchpoints. Total number of neurons analyzed in three separate neuronal cultures is listed above the bars in E,F. (G) Representative sodium and potassium currents recorded from neurons clamped at −70 mV. (H) Current densities. (I) Sodium current (INa) rise time (τrise) and decay time (τdecay). Data are mean ± SEM. Statistical significance analyzed by ANOVA with post hoc Dunnett’s test.

Figure 2—source data 1

Excel files containing data shown as summary bar graph in Figure 2E,F,H,I.

https://cdn.elifesciences.org/articles/50333/elife-50333-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Changes in MAP2 and Tuj1 expression.

(A) Representative images of neurons expressing MAP2 (red) and Tuj1 (green). (B) Quantification of area covered by neurites expressing MAP2 normalized to total number of Tuj1 and DAPI-positive neurons in nine random fields from three different cultures. Data are mean ± SEM. Statistical significance analyzed by ANOVA with post hoc Dunnett’s test (p<0.0001).

Figure 2—figure supplement 2
Changes in Tuj1 expression and sodium channel properties in ΔE9/WT neurons.

(A) Representative images of neurons expressing Tuj1. (B) Quantification of area covered by neurites expressing Tuj1 normalized to total number of neurons. (C) Quantification of somal area normalized to total number of neurons. Total number of random fields of neuronal cultures analyzed in three separate experiments is listed above the bars in B,C. (D) Representative sodium and potassium currents recorded from neurons voltage-clamped at −70 mV. Magnified sodium current traces shown at right. WT/WT hiPSC-derived cerebrocortical neuron data in black, ΔE9/WT AD neurons in red. (E) Sodium (INa) and potassium (IK) current densities. (F) INa rise time (τrise) and decay time (τdecay). Data are mean ± SEM. Statistical significance analyzed by two-tailed unpaired Student’s t-test.

Figure 3 with 2 supplements
Developmental timeline of AD neurons compared to isogenic control neurons.

WT/WT hiPSC-derived cerebrocortical neuron data in black, M146V/WT and APPswe/WT AD neurons in red. (A–C) Representative evoked APs and sodium/potassium currents recorded from WT/WT (A), M146V/WT (B), and APPswe/WT (C) hiPSC-derived cerebrocortical neurons in culture for 2 weeks (Left), 4 weeks (Middle) and 6 weeks (Right). (D) Sodium (INa) and potassium (IK) current densities. (E) Quantification of cell capacitance (Cm). Note that potassium current density and cell size were significantly greater in M146V/WT compared to WT/WT at the 2 week timepoint, but at later timepoints there was no difference in potassium current density but the cell capacitance of AD neurons significantly decreased. Data are mean ± SEM. Statistical significance analyzed by ANOVA with post-hoc Dunnett’s test.

Figure 3—source data 1

Excel files containing data shown as summary bar graph in Figure 3D (2 weeks, 4 weeks and 6 weeks); Figure 3E (2 weeks, 4 weeks and 6 weeks).

https://cdn.elifesciences.org/articles/50333/elife-50333-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Developmental timeline of ΔE9/WT neurons compared to isogenic control neurons.

(A,B) Representative evoked APs and sodium/potassium currents recorded from WT/WT (A) and ΔE9/WT (B) hiPSC-derived cerebrocortical neurons in culture for 2 weeks (left), 4 weeks (middle) and 6 weeks (right). (C) Sodium (INa) and potassium (IK) current densities. (D) Quantification of cell capacitance (Cm). Data are mean ± SEM. Statistical significance analyzed by two-tailed unpaired Student’s t-test.

Figure 3—figure supplement 2
Synaptic development in hiPSC-derived AD neurons vs WT neurons.

(A) Representative images showing presynaptic (synapsin; green) and postsynaptic (homer; red) staining in WT/WT, M146V/WT, and APPswe/WT cultures at 2 weeks, 4 weeks and 6 weeks. (B) Quantification of number of synapses, represented by colocalization of synapsin and homer puncta per µm of neurite length. Data are mean ± SEM. Statistical significance analyzed by ANOVA with post hoc Dunnett’s test. Total number of neurites analyzed for each condition listed above each bar for 4–5 experiments.

Figure 4 with 2 supplements
AD neurons show disrupted excitatory synaptic transmission compared to isogenic control neurons.

(A) Representative spontaneous excitatory postsynaptic currents (sEPSC) recorded at −70 mV from WT/WT, M146V/WT and APPswe/WT hiPSC-derived cerebrocortical neurons in culture for 5 weeks. (B,C) Quantification of sEPSC parameters. Quantification of mean amplitude (B) and quantification of mean frequency (C). (D) Representative miniature excitatory postsynaptic currents (mEPSCs) recorded at −70 mV from WT/WT, M146V/WT and APPswe/WT hiPSC-derived cerebrocortical neurons at 5 weeks in culture. (E,F) Cumulative probability of mEPSC amplitude (inset: quantification of mean amplitude, E) and mEPSC inter-event interval (inset: quantification of mean frequency, -F). (G) Representative images of VGLUT1 immunostaining. (H) Quantification of VGLUT1 intensity normalized to Tuj1 intensity. Total number of random fields of neuronal cultures analyzed in three separate experiments is listed above the bars. (I) Representative western blot images showing VGLUT1 protein levels. (J) Ratio of VGLUT1/GAPDH normalized to the value of WT VGLUT1/GAPDH. Data are mean ± SEM. Statistical significance analyzed by ANOVA with post-hoc Dunnett’s test, or, for immunoblot analysis, by a Kruskal Wallis test followed by Dunn’s multiple comparisons (Deyts et al., 2016). Number of independent experiments listed above bars.

Figure 4—source data 1

Excel files containing data shown as summary bar graph in Figure 4B,C.

https://cdn.elifesciences.org/articles/50333/elife-50333-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
ΔE9/WT neurons show increased excitatory synaptic activity compared to isogenic control neurons.

WT/WT hiPSC-derived cerebrocortical neuron data in black, ΔE9/WT in red. (A) Representative images showing presynaptic (synapsin; green) and postsynaptic (homer; red) staining in WT/WT and ΔE9/WT cultures. (B) Quantification of number of synapses, represented by colocalization of synapsin (green) and homer (red) puncta per µm of neurite length. Total number of neurites analyzed in 4–5 experiments for each condition listed above bars. (C) Representative spontaneous excitatory postsynaptic current (sEPSC) recorded at −70 mV from WT/WT and ΔE9/WT hiPSC-derived cerebrocortical neurons in culture for 5 weeks. (D,E) Quantification of sEPSC parameters. Quantification of mean amplitude (D) and quantification of mean frequency (E). (F) Representative miniature excitatory postsynaptic currents (mEPSCs) recorded from WT/WT and ΔE9/WT hiPSC-derived cerebrocortical neurons at 5 weeks in culture. (G,H) Cumulative probability of mEPSC amplitude (inset: quantification of mean amplitude) and mEPSC inter-event interval (inset: quantification of mean frequency). (I) Representative images of VGLUT1 immunostaining. (J) Quantification of VGLUT1 intensity normalized to Tuj1 intensity. Total number of random fields of neuronal cultures analyzed in three separate experiments is listed above the bars. (K) Representative western blot images showing expression of VGLUT1. (L) Ratio of VGLUT1/GAPDH normalized to the value of WT VGLUT1/GAPDH. Data are mean ± SEM. Statistical significance analyzed by Student’s t test or, for immunoblot analysis, Mann Whitney U test. Number of independent experiments listed above bars in D, E, G, H, J and L.

Figure 4—figure supplement 2
Depletion of readily releasable pool in AD and WT neurons.

(A) Representative EPSC recorded at −70 mV upon application of 500 mM sucrose containing hypertonic solution in WT/WT, M146V/WT, and APPswe/WT hiPSC-derived AD cerebrocortical neurons in culture for 5 weeks. (B) Quantification of the readily releasable pool size (nC). Data are mean ± SEM. Statistical significance analyzed by ANOVA with post hoc Dunnett’s test.

Figure 5 with 2 supplements
AD neurons show diminished inhibitory synaptic transmission compared to isogenic control neurons.

WT/WT hiPSC-derived cerebrocortical neuron data in black, M146V/WT and APPswe/WT in red. (A) Representative spontaneous inhibitory postsynaptic currents (sIPSCs) recorded at 0 mV from WT/WT, M146V/WT, and APPswe/WT hiPSC-derived cerebrocortical neurons in culture for 5 weeks. (B,C) Quantification of sIPSC amplitude and frequency. (D) Representative miniature inhibitory postsynaptic currents (mIPSCs) recorded at 0 mV from WT/WT, APPswe/WT, and M146V/WT hiPSC-derived cerebrocortical neurons at 5 weeks in culture. (E,F) Cumulative probability of mIPSC amplitude (inset: quantification of mean amplitude) and mIPSC inter-event interval (inset: quantification of mean frequency). (G) Representative images of VGAT immunostaining. (H) Quantification of VGAT intensity normalized to Tuj1 intensity. Total number of random fields of neuronal cultures analyzed in three separate experiments is listed above the bars. (I) Representative western blot images showing expression of VGAT. (J) Ratio of VGAT/GAPDH normalized to the value of WT VGAT/GAPDH. Data are mean ± SEM. Statistical significance analyzed by ANOVA with post-hoc Dunnett’s test, or by Kruskal Wallis test followed by Dunn’s test for western blot quantification). Number of independent experiments listed above bars.

Figure 5—figure supplement 1
APPswe/WT and M146V/WT AD cultures contain decreased number of inhibitory neurons compared to WT/WT cultures.

(A) Representative images of GABA immunostaining in WT/WT, M146V/WT, and APPswe/WT neurons cultured for 5 weeks. (B) Quantification of percentage of GABA-positive inhibitory neurons. (C) Representative images of PV immunostaining in WT/WT, M146V/WT, and APPswe/WT neurons. (D) Quantification of percentage of PV-positive neurons. Data are mean ± SEM. Statistical significance analyzed by ANOVA with post hoc Dunnett’s test. Total number of random fields of neuronal cultures analyzed in 3–4 separate experiments is listed above the bars.

Figure 5—figure supplement 2
ΔE9/WT neurons show decreased inhibitory synaptic activity compared to isogenic control neurons.

(A) Representative spontaneous inhibitory postsynaptic currents (sIPSCs) recorded at 0 mV from WT/WT and ΔE9/WT hiPSC-derived cerebrocortical neurons in culture for 5 weeks. (B,C) Quantification of sIPSC parameters. Quantification of mean amplitude and quantification of mean frequency. (D) Representative miniature inhibitory postsynaptic currents (mIPSCs) recorded from WT/WT and ΔE9/WT hiPSC-derived cerebrocortical neurons at 5 weeks in culture. (E,F) Cumulative probability of mIPSC amplitude (inset: quantification of mean amplitude) and mIPSC inter-event interval (inset: quantification of mean frequency. (G) Representative images of VGAT immunostaining. (H) Quantification of VGAT intensity normalized to Tuj1 intensity. Total number of random fields of neuronal cultures analyzed in three separate experiments is listed above the bars. (I) Representative western blot images showing expression of VGAT. (J) Ratio of VGAT/GAPDH normalized to the value of WT VGAT/GAPDH. Data are mean ± SEM. Statistical significance analyzed by Student’s t test or, for immunoblot analysis, Mann Whitney U test. Number of independent experiments listed above bars.

γ-Secretase inhibitor or BACE1 inhibitor prevent the hyperexcitability of AD neurons.

(A) Spontaneous action potentials (sAP) at resting membrane potential (RMP) in the presence of γ-secretase inhibitor (GSi) for 2 days or BACE1 inhibitor (BACEi) for 4 days. WT/WT (left, in black) hiPSC-derived cerebrocortical neuron data, M146V/WT (center, in red), and APPswe/WT (right, in red). (B) Quantification of sAP frequency. (C) Representative sodium and potassium currents recorded from neurons clamped at −70 mV. (D) Current densities. (E) Representative spontaneous excitatory postsynaptic currents (sEPSCs) recorded at −70 mV from WT/WT and ΔE9/WT hiPSC-derived cerebrocortical neurons in culture for 5 weeks. (F,G) Quantification of sEPSC parameters. Quantification of mean amplitude (F) and quantification of mean frequency (G). Data are mean ± SEM. Statistical significance analyzed by ANOVA with post hoc Dunnett’s test for multiple comparisons and by Student’s t test for comparison between two groups.

Figure 6—source data 1

Excel files containing data shown as summary bar graph in Figure 6B (WT/WT, M146V/WT, APPswe/WT); Figure 6D (WT/WT, M146V/WT, APPswe/WT).

https://cdn.elifesciences.org/articles/50333/elife-50333-fig6-data1-v2.xlsx
Figure 7 with 1 supplement
AD cerebral organoids show increased synchronous burst activity compared to WT.

(A) Representative immunostaining images of Nestin, TBR2, and CTIP2 in 2-month-old WT/WT, M146V/WT, and APPswe/WT organoids showing cortical layer formation. (B) Representative Aβ immunostaining in WT/WT, M146V/WT, and APPswe/WT cerebral organoids; lower panel shows 3D projection of Aβ immunostaining. Note increased Aβ deposition in 2-month-old AD organoids. (C) Ratio of Aβ42/Aβ40 quantified by ELISA from cultures at 2 months. (D) Quantification of Aβ levels normalized to total protein from lysates at 2 months. (E) Golgi staining of neurons in 200 µm sections of organoids. (F) Quantification of total dendrite length. Total number of neurons analyzed in 3–4 separate experiments is listed above the bars. (G) Representative raster plots of MEA recordings in WT/WT, M146V/WT, and APPswe/WT cerebral organoids at 2 months. (H) Quantification of mean firing rate. Data are mean ± SEM. Statistical significance analyzed by ANOVA with post-hoc Dunnett’s test. Number of independent experiments listed above the bars.

Figure 7—source data 1

Excel files containing data shown as summary bar graph in Figure 7F,H.

https://cdn.elifesciences.org/articles/50333/elife-50333-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
AD cerebral organoids show increased VGLUT1 levels and decreased VGAT levels compared to WT.

(A) Representative VGLUT1 and VGAT staining in 2-month-old WT/WT, M146V/WT, and APPswe/WT organoids. (B) Quantification of ratio of VGLUT1 to VGAT intensity normalized to Tuj1 intensity. Data are mean ± SEM. Statistical significance analyzed by Kruskal-Wallis test with post-hoc Dunn’s test. Number of independent experiments listed above bars.

Tables

Key resources table
Reagents or
resource
DesignationSource or
reference
IdentifiersAdditional information
Cell cultureMatrigelCorningCat # 354230
Cell culturemTeSR 1STEMCELL
Technologies
Cat # 85850
Cell cultureDorsomorphinTocrisCat # 3093
Cell cultureA83-01StemgentCat # 04–0014
Cell culturePNU74654TocrisCat # 3534
Cell cultureDMEM/F12ThermoFisherCat # 10565018
Cell cultureN2ThermoFisherCat # 17502048
Cell cultureB27ThermoFisherCat # 17504044
Cell cultureFGFR and DCat # 4114-TC
Cell culturep-ornithineMillipore SigmaCat # 27378490
Cell culturelamininTrevigenCat # 340001001
Cell cultureKnockout Serum ReplacementThermoFisherCat # 10828028
Cell cultureβ-MercaptoethanolThermoFisherCat # 21985023
Cell cultureBDNFPeprotechCat # AF-450–02
Cell cultureGDNFPeprotechCat # AF-450–10
Cell cultureES-FBSThermoFisherCat # 16141079
Organoid cultureCerebral Organoid KitSTEMCELL TechnologiesCat # 08570
Critical commercial
assay
V-PLEX Plus Aβ
Peptide Panel
1 (6E10) Kit
Meso Scale
Discovery
Cat # K15200
ElectrophysiologyK-GluconateMillipore SigmaCat # P1847
ElectrophysiologyCs-GluconateHello BioCat # HB4822
ElectrophysiologyKClMillipore
Sigma
Cat # 60128
ElectrophysiologyCsClMillipore
Sigma
Cat # C4036
ElectrophysiologyMgCl2FlukaCat # 63020
ElectrophysiologyHEPESMillipore
Sigma
Cat # H4034
ElectrophysiologyEGTAMillipore
Sigma
Cat # 3889
ElectrophysiologyMg-ATPMillipore
Sigma
Cat # A9187
ElectrophysiologyHBSSThermoFisherCat # 14175079
ElectrophysiologyCaCl2Millipore
Sigma
Cat # C5080
ElectrophysiologyGlycineMillipore
Sigma
Cat # G2879
ElectrophysiologyTetrodotoxin
citrate
Hello BioCat # HB 1035
Electrophysiologyβ-Secretase
Inhibitor IV
EMD MilliporeCat # 565788
ElectrophysiologyCompound EEMD MilliporeCat # 565790
Immunocytochemistry (ICC)PFAAlfa AesarCat # 43368
ICCBSAMillipore
Sigma
Cat # A0336
ICCTriton X-100Millipore
Sigma
Cat # T8532
ICC antibodyDAPIInvitrogenCat # D1306;
RRID:AB_2629482
1:500
ICC antibodyChicken polyclonal Anti-Tuj1AbcamCat # ab41489;
RRID:AB_727049
1:200
ICC antibodyMouse monoclonal Anti-Synapsin 1Synaptic
Systems
Cat # 106 011;
RRID:AB_2619772
1:500
ICC antibodyRabbit polyclonal
Anti-Homer 1
Synaptic
Systems
Cat # 160 003;
RRID:AB_887730
1:200
ICC antibodyMouse monoclonal
Anti-VGAT
Synaptic
Systems
Cat # 131 011;
RRID:AB_887868
1:200
ICC antibodyRabbit polyclonal Anti-VGLUT1EMD MilliporeCat # ABN1647;
RRID:AB_2814811
1:500
ICC antibodyRabbit polyclonal
Anti-GABA
SigmaCat # A2052;
RRID:AB_477652
1:100
ICC antibodyMouse monoclonal Anti-ParvalbuminEMD MilliporeCat # MAB1572;
RRID:AB_2174013
1:100
ICC antibodyRabbit polyclonal
Anti-FOXG1
AbcamCat # ab18259;
RRID:AB_732415
1:250
ICC antibodyRat monoclonal Anti-CTIP2AbcamCat # ab18465;
RRID:AB_2064130
1:200
ICC antibodyRabbit polyclonal
Anti-TBR2
AbcamCat # ab23345;
RRID:AB_778267
1:300
ICC antibodyMouse monoclonal
Anti-Nestin
AbcamCat # ab22035;
RRID:AB_446723
1:200
ICC antibodyAnti-Amyloid β 1–16BiolegendCat # 803001;
RRID:AB_2564653
1:2000
Immunoblot antibodyMouse monoclonal
Anti-VGAT
Synaptic
Systems
Cat # 131011;
RRID:AB_887868
1:500
Immunoblot antibodyRabbit polyclonal
Anti-VGLUT1
Synaptic SystemsCat # 135303;
RRID:AB_887875
1:1000
Software and
Algorithms
Clampex v10.6 (pClamp)Molecular
Devices
RRID:SCR_011323
Software and AlgorithmsClampfit v10.6 (pClamp)Molecular
Devices
RRID:SCR_011323
Software and
Algorithms
Mini AnalysisSynapstosofthttp://www.synaptosoft.com/MiniAnalysis/;
RRID:SCR_002184
Software and
Algorithms
ImageJNIHhttps://imagej.nih.gov/ij/;
RRID:SCR_003070
Software and
Algorithms
Prism7GraphPadhttp://www.graphpad.com;
RRID:SCR_002798

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)