CRISPR/Cas9-mediated <i>PHOX2B</i> functional knock-out in IMR32 neuroblastoma cells impairs neuronal excitability through dysregulation of ion channels genes.

Overview

1. Department of Medical Biotechnology and Translational Medicine (BIOMETRA), Università degli Studi di Milano, Milan, Italy
2. Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
3. Women and Children’s Health Research Institute, University of Alberta, Edmonton AB, Canada
4. National Research Council (CNR) - Institute of Neuroscience, Vedano al Lambro, MB, Italy
5. NeuroMi - Milan Center for Neuroscience, University of Milano Bicocca, Milan, Italy
6. National Research Council (CNR) - Institute of Biomedical Technologies, Segrate, MI, Italy
7. Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada

Abstract

Introduction: PHOX2B is a master transcriptional regulator of autonomic nervous system development whose mutations cause neurocristopathies including neuroblastoma and congenital central hypoventilation syndrome (CCHS). However, the identification and functional relevance of PHOX2B target genes remain incompletely defined, partly due to the lack of robust cellular models.

Methods: Through CRISPR/Cas9-engineering we established a PHOX2B functional knockout IMR32 neuroblastoma model, and combined transcriptomic, chromatin-binding, electrophysiological, and live-cell imaging analysis to investigate PHOX2B-dependent pathways. We then assessed the developmental relevance of the obtained results in iPSC−derived sympathetic neurons from CCHS patients carrying different PHOX2B mutations.

Results: PHOX2B functional knockout (PHOX2B-KO) cells exhibited morphological and molecular features indicative of enhanced neuronal maturation. RNA-seq and ChIP-seq analyses highlighted an unexpected direct role for PHOX2B in regulating the expression of ion channels, including KCNQ5, SCN3A and RYR2, primarily through transcriptional repression. Functionally, PHOX2B KO cells displayed significant alterations in intrinsic electrical properties, including depolarized resting membrane potential, reduced action potential amplitude, attenuated Ca2+ responses and profoundly altered K+ efflux dynamics. Functional rescue experiments confirmed that PHOX2B re-expression restores both transcriptional and electrophysiological phenotypes, thereby establishing a causal relationship between PHOX2B expression and the maintenance of intrinsic excitability homeostasis. Extending these findings to development, sympathetic neurons differentiated from CCHS patient-derived iPSCs displayed dysregulated maturation, transcriptional network rewiring, and altered expression of the same ion channel genes, supporting their relevance to disease.

Discussion: Together, our data establish the PHOX2B functional knockout neuroblastoma model as a valuable platform for functional genomics, enabling systematic analysis of PHOX2B targets and highlighting its critical role in coordinating transcriptional programs that shape neuronal excitability and maturation.

Reproduced under the paper's license (CC BY), from the paper cited above.

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Data

Datasets cited

• zenodo:20096956, at Zenodo; found in “Data availability statement”

Data availability statement

The RNA-sequencing data set generated and analyzed in this study has been deposited in the NCBI database under accession number GEO ID: GSE330047. The ChIP-sequencing datasets are available in the ZENODO repository under DOI: 10.5281/zenodo.20096956.

Reproduced under the paper's license (CC BY), from the paper cited above.

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Recorded: type, language, journal, volume, pages, dates, 14 authors, 8 keywords, 1 funder, 118 references.

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This paper

Cardani, S., Bertocchi, M., Donà, E., Chiesa, F., Cambria, C., Cuadros Gamboa, A. L., Piscitelli, E., Rancic, V., Gosgnach, S., Pagliardini, S., Antonucci, F., Fornasari, D., Di Lascio, S., & Benfante, R. (2026). CRISPR/Cas9-mediated <i>PHOX2B</i> functional knock-out in IMR32 neuroblastoma cells impairs neuronal excitability through dysregulation of ion channels genes. Frontiers in physiology, 17, 1844142. https://doi.org/10.3389/fphys.2026.1844142

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