NeuroD family genes regulate the survival of neurons in the developing hippocampus

Abstract

NeuroD 1/2/6 transcription factors belong to the bHLH-containing protein family. They activate transcription and regulate various aspects of neuronal differentiation [1]. In the developing cortex and hippocampus, these three factors express overlapping patterns, indicating a partial redundancy of functions during development [2]. They aid in the creation of the brain’s main commissures, particularly the largest one, the corpus callosum. Agenesis, whether total or partial, is a frequently occurring comorbidity in congenital malformations amongst human beings. It is usually associated with the underdevelopment or absence of the hippocampus [3].

To examine the contribution of these factors in hippocampal formation development, we created allelic genetically modified mouse strains with inactivating mutations in all three genes. The results indicated that the inactivation of NeuroD1 leads to the dentate gyrus’ absence. In contrast, the absence of all three NeuroD genes results in the dentate gyrus’ and other hippocampal formation regions’ absence (CA1, CA2, CA3) at subsequent developmental stages.

To investigate the cellular mechanisms involved in the disruption of hippocampal formation development, it was hypothesized that NeuroD transcription factors may control either neuronal proliferation and differentiation or cell death. To test which of these hypotheses is correct, a series of BrdU injections were performed at the E15 stage of development. We examined several parameters related to proliferation, including the proportion of cells at various differentiation stages, cell cycle exit, cell cycle length, and the number of cells in the S phase of the cell cycle. The results showed no discernible differences in proliferation and cell cycle parameters between triple homozygous embryos and control littermates.

An analysis of programmed cell death, known as apoptosis, was conducted to test the alternative hypothesis. We used two methods to analyze cell death: firstly, we analyzed the activity of caspase-3 which is one of the proteins that activate apoptosis and, secondly, we analyzed the number of double-strand breaks using the TUNEL test. The findings revealed a significant increase in the number of cells positive for both caspase-3 and double-stranded DNA breaks in the developing hippocampus of triple mutants. The amount of apoptotic cells in the developing hippocampal formation relies on the gene dosage of NeuroD 1/2/6 alleles, as evident through their increment as the NeuroD gene dosage decreases. The double KO portrays an intermediate level of cell death, while the triple exhibits the highest level.

To determine the stage in which massive cell death occurs during neuronal differentiation, we modified the BrdU-chase assay. BrdU was injected during the E12 stage, and embryos were surveyed at various intervals (12, 18, and 24 hours) for analysis. This experiment permits estimation of the time period following exit from the mitotic cycle, during which the cell initiates the mechanism of apoptosis.

Keywords

NeuroD transcription factors / knockout / hippocampal formation / BrdU / cell death

References

[1] Bertrand N, Castro DS, Guillemot F. Proneural genes and the specification of neural cell types. Nature Reviews Neuroscience. 2002;3(7):517–530. doi: 10.1038/nrn874

[2] Bertrand N., Castro D.S., Guillemot F. Proneural genes and the specification of neural cell types // Nature Reviews Neuroscience. 2002. Vol. 3, N 7. P. 517–530. doi: 10.1038/nrn874

[3] Tutukova S, Tarabykin V, Hernandez-Miranda LR. The Role of Neurod Genes in Brain Development, Function, and Disease. Frontiers in Molecular Neuroscience. 2021;14:662774. doi: 10.3389/fnmol.2021.662774

[4] Tutukova S., Tarabykin V., Hernandez-Miranda L.R. The Role of Neurod Genes in Brain Development, Function, and Disease // Frontiers in Molecular Neuroscience. 2021. Vol. 14. P. 662774. doi: 10.3389/fnmol.2021.662774

[5] Pânzaru MC, Popa S, Lupu A, et al. Genetic heterogeneity in corpus callosum agenesis. Frontiers in Genetics. 2022;13:958570. doi: 10.3389/fgene.2022.958570

[6] Pânzaru M.-C., Popa S., Lupu A., et al. Genetic heterogeneity in corpus callosum agenesis // Frontiers in Genetics. 2022. Vol. 13. P. 958570. doi: 10.3389/fgene.2022.958570

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