Alterations in Stress Response Patterns During in vitro Neuronal Development

Vesselina Semkova1, Andreas Till1, Michaela Segschneider2, Catherine Bell3, Magnus Ingelman-Sundberg3, Simone Haupt2, Oliver Brüstle1

1 Institute of Reconstructive Neurobiology, University of Bonn, Germany
2 LIFE&BRAIN GmbH, Bonn, Germany
3 Karolinska Institute, Department of Physiology and Pharmacology, Stockholm, Sweden

Human neurodevelopment comes with alterations in metabolic and cell signaling pathways. Correlation of these changes to compound toxicity can be used to identify critical developmental periods and neurodegenerative mechanisms. We present detailed characterization of an in vitro model of human neurodevelopment using hiPSC-derived long-term self-renewing neuroepithelial stem (lt-NES) cells. We identified developmental windows characterized by decreasing stress susceptibility to the oxidative stress (OS) inducer tert-butyl-hydroperoxide (TBHP) with differentiation. Conversely, microarray analysis showed upregulation of target genes of the antioxidant response Keap1-Nrf2-ARE pathway after 6 weeks of differentiation, along with a decrease in the Keap1/Nrf2 protein ratios. Nrf2 pharmacological inhibition reduced cell survival at high TBHP doses, confirming its role in the increasing stress resistance. Autophagic and proteasomal pathways, too, showed differential regulation during lt-NES cell differentiation, suggesting an interplay between proteostasis and OS-response. The proteostatic stress response indicated that the 20S-Proteasome is a major contributor to the OS-response in mature neurons. Pre-treatment of neurons with MG132 followed by TBHP stress-induction indicated that the proteasome is essential for the ability of lt-NES neurons to withstand acute stress, while blocking autophagy by Bafilomycin did not affect the acute response. Gene expression analysis of the Nrf2-downstream targets NQO1 and TXNRD1 and Western blot analysis of the Keap1 and Nrf2 protein levels suggested that the proteasome is regulating Keap1-Nrf2 pathway activation under acute OS conditions by modulating the Keap1/Nrf2 protein ratio. Taken together, our data show how neurodevelopmental changes converge to control the OS-response with Nrf2 pathway being pivotally affected by proteasomal activity.