Stefan Lazic1*, Marija Lazic1, Luka Bojic1, Jelena Pejic1, Vanda Balint1, Simona Lapcevic1, Mirjana Novkovic1, Mila Ljujic1, Milena Stevanovic2, Danijela Drakulic1, Danijela Stanisavljevic Ninkovic1, Marija Mojsin1, Milena Milivojevic1, Isidora Petrovic1 and Marija Schwirtlich1
1Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia
2Serbian Academy of Sciences and Arts
stefan.lazic [at] imgge.bg.ac.rs
Abstract
Various pathological conditions, resulting from oxygen deprivation during development and adulthood, impact neurogenesis, which in turn impairs different cognitive functions and contributes to the onset of age-related neurodegenerative diseases. Therapeutic options remain limited, and many neuroprotective agents that appear promising in preclinical research have not succeeded in human clinical trials. The present study aimed to evaluate how the hypoxia-mimicking agent CoCl2 influences the neurogenic potential of human neural precursor cells (NPCs), reflecting early embryonic development stages and adult neurogenesis.
We employed next-generation sequencing and bioinformatic analysis to functionally characterize the transcriptome and assess treatment effects in NPCs. Six cDNA libraries were generated, with three from each experimental group of cells treated with either 300 µM CoCl2 or deionized water for 48 hours. Transcriptomic analysis identified 2,925 differentially expressed genes, of which 1,065 were upregulated and 1,860 downregulated in treated NPCs. Functional analysis showed enrichment of upregulated genes in processes such as hypoxia-related pathways, metabolic reprogramming, and transmembrane transport. Furthermore, downregulated genes were associated with cell cycle progression and axon guidance. Notably, genes related to immunity and apoptotic cell death were unaffected. Additionally, downregulated genes were identified in gene sets from neural stem and progenitor cells across different regions of the human brain, suggesting that hypoxia may affect specific characteristics and functions of these cells. Finally, by combining results from quantitative real-time PCR and double immunocytochemical analysis of cells differentiated for 10 days post-treatment, we showed that cobalt chloride affects the neuronal differentiation of NPCs in multiple ways: it decreases the number of differentiated neurons but also enhances their maturity.
Taken together, our findings provide a basis for understanding the molecular mechanisms in the human brain in response to hypoxic stress. The key hub genes, core regulatory programs, and pathways identified in our study are designated as potential targets for neuroprotective strategies following hypoxia-related brain injuries.
Keywords: Hypoxia, neurogenesis, neurodegeneration, transcriptome, NT2/D1.
Acknowledgement: This research was supported by Ministry of Science, Technological Development and Innovation of the Republic of Serbia (No: 451-03-33/2026-03/200042). The authors would like to thank Sasa Todorovic and Aleksandra Vitkovac for all the help and support with RNA sequencing and initial data processing.