Measurements of neurite extension and nucleokinesis in an iPSC-derived model system following microtubule perturbation

Mol Biol Cell. 2025 Jan 1;36(1):mr1. doi: 10.1091/mbc.E24-02-0061. Epub 2024 Nov 27.

Abstract

In neurons, patterns of different microtubule types are essential for neurite extension and nucleokinesis. Cellular model systems such as rodent primary cultures and induced pluripotent stem cells (iPSC)-derived neurons have provided key insights into how these patterns are created and maintained through the action of microtubule-associated proteins, motor proteins, and regulatory enzymes. iPSC-derived models show tremendous promise but lack benchmarking and validation relative to rodent primary cultures. Here we have characterized a recent iPSC-derived model, in which doxycycline-induced expression of Neurogenin-2 drives consistent transdifferentiation into the neuronal state (EBiSC-NEUR1 neurons, referred to as NGN2 neurons below). We developed a suite of open-access, semiautomated methods to measure neurite extension and nucleokinesis of NGN2 neurons, which compare favorably to published data from other models. Then, we challenged NGN2 neurons with a panel of drugs that perturb microtubule physiology. NGN2 neurons extension and nucleokinesis were significantly perturbed by two microtubule-targeting drugs, namely a taxane (paclitaxel) and a vinca alkaloid (DZ-2384). In contrast, inhibition of microtubule severing (spastazoline) or of deacetylation (trichostatin A) had a limited effect on nucleokinesis only. Our results support the primary importance of microtubule dynamics in neuronal development and demonstrate the power of NGN2 neurons as a model system.

MeSH terms

  • Animals
  • Basic Helix-Loop-Helix Transcription Factors
  • Cell Differentiation / drug effects
  • Cell Transdifferentiation / drug effects
  • Cells, Cultured
  • Humans
  • Induced Pluripotent Stem Cells* / cytology
  • Induced Pluripotent Stem Cells* / drug effects
  • Induced Pluripotent Stem Cells* / metabolism
  • Microtubule-Associated Proteins / metabolism
  • Microtubules* / drug effects
  • Microtubules* / metabolism
  • Nerve Tissue Proteins / metabolism
  • Neurites* / drug effects
  • Neurites* / metabolism
  • Neurites* / physiology
  • Neuronal Outgrowth / drug effects
  • Neurons* / cytology
  • Neurons* / drug effects
  • Neurons* / metabolism
  • Paclitaxel / pharmacology

Substances

  • Nerve Tissue Proteins
  • Paclitaxel
  • NEUROG2 protein, human
  • Microtubule-Associated Proteins
  • Basic Helix-Loop-Helix Transcription Factors