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. 2019 Dec 30;10:52.
doi: 10.1186/s13229-019-0303-3. eCollection 2019.

Increased Ca 2+ Signaling in NRXN1α +/- Neurons Derived From ASD Induced Pluripotent Stem Cells

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Free PMC article

Increased Ca 2+ Signaling in NRXN1α +/- Neurons Derived From ASD Induced Pluripotent Stem Cells

Sahar Avazzadeh et al. Mol Autism. .
Free PMC article

Abstract

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a high co-morbidity of epilepsy and associated with hundreds of rare risk factors. NRXN1 deletion is among the commonest rare genetic factors shared by ASD, schizophrenia, intellectual disability, epilepsy, and developmental delay. However, how NRXN1 deletions lead to different clinical symptoms is unknown. Patient-derived cells are essential to investigate the functional consequences of NRXN1 lesions to human neurons in different diseases.

Methods: Skin biopsies were donated by five healthy donors and three ASD patients carrying NRXN1α +/- deletions. Seven control and six NRXN1α +/- iPSC lines were derived and differentiated into day 100 cortical excitatory neurons using dual SMAD inhibition. Calcium (Ca2+) imaging was performed using Fluo4-AM, and the properties of Ca2+ transients were compared between two groups of neurons. Transcriptome analysis was carried out to undercover molecular pathways associated with NRXN1α +/- neurons.

Results: NRXN1α +/- neurons were found to display altered calcium dynamics, with significantly increased frequency, duration, and amplitude of Ca2+ transients. Whole genome RNA sequencing also revealed altered ion transport and transporter activity, with upregulated voltage-gated calcium channels as one of the most significant pathways in NRXN1α +/- neurons identified by STRING and GSEA analyses.

Conclusions: This is the first report to show that human NRXN1α +/- neurons derived from ASD patients' iPSCs present novel phenotypes of upregulated VGCCs and increased Ca2+ transients, which may facilitate the development of drug screening assays for the treatment of ASD.

Keywords: Autism; Calcium signaling; Induced pluripotent stem cells; NRXN1α; Neurons; Transcriptome.

Conflict of interest statement

Competing interestsThe authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Derivation and validation of iPSCs. a Fibroblast outgrowth from the skin biopsy after 12 days of culturing. b IPSC colonies were visible and ready for collection after 24 days of reprogramming and became stable after few passaging (c). iPSCs were characterized and were stained positive for alkaline phosphatase (d) and pluripotent markers OCT4, SOX2, and NANOG and surface markers SSEA4 and TRA-1-60 and TRA-1-81 (bg). Spontaneous EB differentiation has shown the expression of markers for mesoderm (ASM, h), ectoderm (TUJ1, i), and endoderm (AFP, j). iPSCs also showed positive expression of proliferating marker Ki67 (k) and (m) phase marker PH3 (l). They were also quantified at mRNA level for the expression of OCT4 (n) and SOX2 (o). All representative images all from control line 4CCX1
Fig. 2
Fig. 2
Differentiation and validation of the cortical excitation neurons. ac ICC staining of day-100 neurons with MAP2 (a), SYN1/TUJ1 (b) for neuronal maturity, and with TBR1/CTIP2 for the identity of lower cortical pyramidal neurons (c). d Proportion of layer VI (TBR1+) and layer V/VI (CTIP2+) neurons in the cultures. e High mRNA expression (normalized to GAPDH [dotted line]) of excitatory post-synaptic markers (DLG4, SHANK1, SHANK2, SHANK3, SLC17A7, GRIN1, GRIN2A, GRIA1, GRIA4, GRIK1, GRIK3), neuronal markers (TUBB3, MAP2), upper cortical layers (BRN2, SATB2), and lower layer markers (TBR1, CTIP2) in day-100 cultures of control (black) and NRXN1α+/− (red) neurons. Results shown from two independent cortical neuronal differentiation (f). Representative waterfall traces of spontaneous Ca2+ transients in day 50 (blue) and 100 (pink) cultures from 200 s of recording. Neurons exhibited a significant increase in proportion (%) of spontaneous active cells (g), the frequency (mHz, h), and the amplitude (ΔF/F, i) of Ca2+ transients from day 50 to day 100. Statistical significance (**p < 0.01, ***p < 0.001, ****p < 0.0001) was evaluated using the Mann-Whitney U test). All representative images all from control line 4CCX1
Fig. 3
Fig. 3
Spontaneous calcium transients are voltage-gated calcium, action potential, and excitatory dependent. a Representative raster plots (activity of 100 cells in 300 s of recording, each dot represents Ca2+ transient activity, Line 4CX1), waterfall traces (3D representative of calcium transients in 300 s over defined threshold level, Line 4CX1), and their significant change showing the spontaneous Ca2+ transient activity of the cells and their response after the application of DL-AP5 (50 μM, a) and CNQX (50 μM, b), Nifedipine (50 μM, c), agatoxin (500 nM, d), and TTX (1 μM, e) (n = 2–3, 1CC1, 3VCX1, 4CX1). The red dotted line shows the point of drug application. All data summary are mean ± SEM
Fig. 4
Fig. 4
Spontaneous Ca2+ transient properties were significantly altered in the day 100 NRXN1α +/− neurons. a, b The representative images of control (a) and patient (b) cells loaded with Fluo-4 AM, displaying dense network of neurons in day 100 neuronal cultures. c, d Representative raster plots showed the spontaneous Ca2+ transient activity of the cells from control (c) and patient (d) cultures. e, f Representative waterfall traces of spontaneous Ca2+ transients in 250 cells over 300 s of recording. g The percentage of spontaneous Ca2+ transients remained unchanged between the two groups. h The frequency of spontaneous Ca2+ transients was significantly increased in the NRXN1α+/− deletion patient cells. i The duration of calcium transients was significantly longer in the NRXN1α+/− deletion patient cells. j The amplitude of spontaneous Ca2+ transient was significantly increased in the NRXN1α+/ deletion patient cells. Control n = 74 recordings/26 coverslips from 6 control iPSC lines (patient n = 47 recordings/21 coverslips) from 6 NRXN1α +/− iPSC lines (Additional file 1: Table S10). All data summary are mean ± SEM. Statistical significance (**p < 0.01, ***p < 0.001, ****p < 0.0001) was evaluated using the Mann-Whitney U test. Bar = 100 μm in a, b. Representative images are from control line 4CCX1 and patient ND1C1
Fig. 5
Fig. 5
The pathways of calcium and ion transport activity were significantly enriched in NRXN1α+/− iPSC-derived neurons. a Volcano plot of FDR on the y-axis and Log2 fold changes on the x-axis of DEGs in NRXN1α+/− neurons. Among the 530 DEGs, 254 were downregulated (in green) and 276 upregulated (in red) with FDR < 0.05. b Bivariate clustering of samples (columns) of the 530 DEGs. The color ranges from purple (low expression) to yellow (high expression) based on TPM values (http://rpubs.com/saharava/BivariateClustering). The arrows represent the up/downregulated genes in NRXN1α+/− neurons. c The heatmap represented all genes which were linked to the 21 pathways. The color showed the most overlapped genes in dark red to non-overlapped genes in white among the pathways. d Upregulated (right) and downregulated (left) genes were shown with Log2 fold expression. The VGCCs of CACNA1A, CACNA2D1, and CACNG2/3 were shared by most pathways
Fig. 6
Fig. 6
Strong association of calcium channel activity in NRXN1α+/− iPSC-derived neurons. a String KEGG analyses showing a significant network of VGCC association. Proteins in red are associated with calcium ion binding, in blue nodes with calcium ion transmembrane transporter activity, in green node with calcium channel activity, in yellow with voltage-gated calcium channel activity, and in purple with calcium signaling pathway. b STRING analyses of upregulated (not downregulated) genes in the NRXN1α+/− neurons showed association with “calcium ion transport” (red, 12 genes, FDR + 3.20E−03), “calcium ion transmembrane transport” (blue, 9 genes, FDR = 2.06E−02), “voltage-gated calcium channel activity” (green, 6 genes, FDR5.50E−04), calcium signaling pathway (pink, 8 genes, FDR = 4.89E−02), and “presynaptic depolarization and calcium channel opening” (light blue, 3 genes, FDR = 2.37E−02)

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