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CRISPR/Cas9-mediated Knockout of the Neuropsychiatric Risk Gene KCTD13 Causes Developmental Deficits in Human Cortical Neurons Derived from Induced Pluripotent Stem Cells

Molecular Neurobiology volume 57, pages 616–634 (2020)Cite this article

Abstract

The human KCTD13 gene is located within the 16p11.2 locus and copy number variants of this locus are associated with a high risk for neuropsychiatric diseases including autism spectrum disorder and schizophrenia. Studies in zebrafish point to a role of KCTD13 in proliferation of neural precursor cells which may contribute to macrocephaly in 16p11.2 deletion carriers. KCTD13 is highly expressed in the fetal human brain and in mouse cortical neurons, but its contribution to the development and function of mammalian neurons is not completely understood. In the present study, we deleted the KCTD13 gene in human-induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 nickase. Following neural differentiation of KCTD13 deficient and isogenic control iPSC lines, we detected a moderate but significant inhibition of DNA synthesis and proliferation in KCTD13 deficient human neural precursor cells. KCTD13 deficient cortical neurons derived from iPSCs showed decreased neurite formation and reduced spontaneous network activity. RNA-sequencing and pathway analysis pointed to a role for ERBB signaling in these phenotypic changes. Consistently, activating and inhibiting ERBB kinases rescued and aggravated, respectively, impaired neurite formation. In contrast to findings in non-neuronal human HeLa cells, we did not detect an accumulation of the putative KCTD13/Cullin-3 substrate RhoA, and treatment with inhibitors of RhoA signaling did not rescue decreased neurite formation in human KCTD13 knockout neurons. Taken together, our data provide insight into the role of KCTD13 in neurodevelopmental disorders, and point to ERBB signaling as a potential target for neuropsychiatric disorders associated with KCTD13 deficiency.

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Acknowledgments

We would like to thank Dr. Michael Brendel (Boehringer Ingelheim Pharma GmbH & Co. KG) for help with statistical analysis of neurite formation assays, and Dr. Jinwei Zhang (University of Dundee) for validation of anti-KCTD13 antibodies for immunoprecipitation.

Funding

The research leading to these results has received support (iPSC line from a healthy donor) from the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115439. Resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution. This publication reflects only the author's views and neither the IMI JU nor EFPIA nor the Europeam Commission are liable for any use that may be made of the information therein.

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Affiliations

  1. CNS Diseases Research, Boehringer Ingelheim Pharma GmbH & Co KG, 88397, Biberach an der Riss, Germany

    Valeria Kizner, Maximilian Naujock, Sandra Fischer, Stefan Jäger, Selina Reich, Ines Schlotthauer, Cornelia Dorner-Ciossek & Frank Gillardon

  2. Target Discovery Research, Boehringer Ingelheim Pharma GmbH & Co KG, 88397, Biberach an der Riss, Germany

    Kai Zuckschwerdt, Tobias Hildebrandt & Nathan Lawless

  3. Cardio-metabolic Diseases Research, Boehringer Ingelheim Pharma GmbH & Co KG, 88397, Biberach an der Riss, Germany

    Tobias Geiger

  4. MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee, DD1 5EH, UK

    Thomas Macartney

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  1. Valeria Kizner
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Correspondence to Frank Gillardon.

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Boehringer Ingelheim Pharma GmbH & Co. KG supported this work only by providing financial support in the form of authors’ salaries (all affiliated with Boehringer Ingelheim Pharma GmbH & Co. KG) and research materials. Data collection and analysis, study design, decision to publish, or preparation of the manuscript for this work were performed independently. The authors declare that they have no conflict of interest.

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ESM 2
figure 8

Supplementary Fig. S1, related to Fig. 2 Supportive data for unaltered stemness of KCTD13 KO iPSCs. a–c Bar graphs showing similar mRNA expression levels of stemness marker genes (NANOG, SOX2, OCT4) assessed by qRT-PCR. n = 3 replicates each. (PNG 134 kb)

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Supplementary Fig. S2, related to Fig. 3 Additional data supporting decreased proliferation of KCTD13 KO NPCs. a Cell viability (number of non-condensed Hoechst+ nuclei) of BrdU+ cells was unchanged in all NPC lines. Total n = 30 wells per NPC line. b Representative confocal images of BrdU (green) and Hoechst 33342 (blue) nuclear stainings in NPC cultures. Scale bar, 50 μm. c Percentage of EdU+ NPCs is decreased in KCTD13 KO cultures. Total n = 28–29 wells per NPC line; One-way ANOVA followed by Dunnett’s multiple comparison test; ****p < 0.0001. d Gated cell population used for cell cycle analysis is similar between genotypes. e Percentage of NPCs in G0/G1 phase and f in G2/M phase are comparable between lines. Total n = 28–31 measurements per NPC line from 5 independent experiments. g Enriched canonical pathways in KCTD13 KO versus WT NPCs as detected by Ingenuity Pathway Analysis based on RNA sequencing data. (PNG 1.20 mb)

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Supplementary Fig. S3, related to Fig. 5a Treatment with the RhoA inhibitor Rhosin (50 μM) does not rescue decreased percentage of MAP2+ neurons with roots in KCTD13 KO cultures. Phenotypic changes: One-way ANOVA followed by Dunnett’s multiple comparison test; **p = 0.0013, ***p = 0.0008. Treatment effect: Unpaired t test: **p = 0.0082 (KCTD13+/+ vs. 50 μM Rhosin), **p = 0.0027 (KCTD13−/− vs. 50 μM Rhosin). b Rhosin does not rescue decreased number of roots per MAP2+ neuron with roots. Phenotypic changes: One-way ANOVA followed by Dunnett’s multiple comparison test; ***p = 0.0007 (KCTD13+/+ vs. KCTD13+/- ), ***p = 0.0003 (KCTD13+/+ vs. KCTD13−/−). Treatment effect: Unpaired t test: *p = 0.0244, **p = 0.0019 (KCTD13−/− vs. 50 μM Rhosin), **p = 0.0012 (KCTD13+/+ vs. 50 μM Rhosin). Total n = 14–18 wells per genotype. (PNG 294 kb)

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Supplementary Fig. S4, related to Fig. 6 Representative Western blot images showing similar protein levels of total ERBB2 (a) and total ERBB4 (b) in KCTD13 KO versus WT iPSC-derived neurons. c Treatment with the potent specific ERBB2 inhibitor Tucatinib (1 μM) has no significant effect on neurite elongation either in WT or KCTD13 KO neurons. Total n = 16–20 wells per neuron line from 1 experiment. Phenotypic changes: Unpaired t test; ***p = 0.0008. d Treatment with NRG1b peptide also rescues decreased percentage of MAP2+ neurons with roots. Total n = 63–90 wells per neuron line from 3 to 4 independent experiments. Phenotypic changes: One-way ANOVA followed by Dunnett’s multiple comparison test; *p = 0.0446, ****p < 0.0001. Treatment effect: Unpaired t-test: *p = 0.0289, ***p = 0.0003. e Treatment with the ERBB1 ligand EGF (3, 10, 30 nM) impairs neurite elongation in WT and KCTD13 KO neurons. Total n = 16–24 wells per neuron line from 1 experiment. Phenotypic changes: Unpaired t test; ****p < 0.0001. Treatment effect: one-way ANOVA followed by Dunnett’s multiple comparison test, **p = 0.0062, ****p = 0.0001. (PNG 514 kb)

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Supplementary Fig. S5, related to Fig. 6e Confirmation of the deficit in neurite formation and its rescue by NRG1b treatment in a second homozygous KCTD13 KO clone (KCTD13−/−_2) compared to an isogenic subclone of the parental wildtype clone (KCTD13+/+_2). Mean neurite length is significantly decreased in homozygous KCTD13 KO neurons derived from iPSCs and is significantly increased by single administration of NRG1b peptide (50 nM). Unpaired t test; **p = 0.002 (KCTD13+/+_2, Vehicle vs. NRG1b), **p = 0.0014 (KCTD13+/+_2 vs. KCTD13−/−_2), ****p < 0.0001. Total n = 21–24 wells per experimental group. (PNG 485 kb)

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Supplementary Fig. S6, related to Fig. 7d Analysis of mean spike rate using multi-electrode-arrays revealed a significant decrease in iPSC-derived KCTD13 deficient neuron cultures. Reduced neuronal network activity was not rescued by repeated administration of NRG1b peptide at 50 nM during each medium exchange. Total n = 8 wells per neuron line from 1 experiment; Phenotypic changes: One-way ANOVA followed by Dunnett’s multiple comparison test; ***p = 0.0009 (KCTD13+/+ vs. KCTD13+/- ), ***p = 0.0003 (KCTD13+/+ vs. KCTD13−/−). (PNG 433 kb)

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Kizner, V., Naujock, M., Fischer, S. et al. CRISPR/Cas9-mediated Knockout of the Neuropsychiatric Risk Gene KCTD13 Causes Developmental Deficits in Human Cortical Neurons Derived from Induced Pluripotent Stem Cells. Mol Neurobiol 57, 616–634 (2020). https://doi.org/10.1007/s12035-019-01727-1

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Keywords

  • KCTD13
  • 16p11.2
  • CRISPR/Cas9
  • Induced pluripotent stem cells
  • Neurodevelopment
  • Neuropsychiatric disorders