Glucocorticoids Alter Human Cortical Neurogenesis by Regulating ZBTB16 Expression

Background Introduction

During pregnancy, glucocorticoids (GCs) are crucial steroid hormones for the development of the central nervous system (CNS). GCs regulate the development of fetal organs, particularly the lungs and brain. However, when GC levels exceed the physiological range or time window, such as due to the therapeutic use of synthetic glucocorticoids (sGCs) or maternal endocrine and stress-related diseases, they can affect fetal development. Additionally, maternal stress and depression reduce placental metabolism of cortisol, and synthetic glucocorticoids such as betamethasone and dexamethasone can easily cross the placenta, increasing fetal exposure levels. Therefore, sGCs are commonly administered between the 22nd and 33rd weeks of pregnancy for high-risk preterm births to promote fetal lung maturation and improve survival rates.

The clinical and societal importance of GC use during pregnancy, as well as the potential long-term effects of GCs on fetal development, has prompted research teams to explore the mechanisms by which glucocorticoids influence neurogenesis and their impact on subsequent cognition and brain structure.

Source of the Paper

The study was conducted by researchers Anthi C. Krontira, Cristiana Cruceanu, and Leander Dony from institutions such as the Max Planck Institute of Psychiatry, Karolinska Institutet, and Ludwig-Maximilians-University. The paper was published in the journal Neuron on May 1, 2024.

Research Process and Main Results

Research Process

The study used human cerebral organoids (HCOs) and mouse embryos to investigate the effects of GCs on neurogenesis in different cell types. The specific research procedures are as follows:

1. Experimental Design:

   • HCOs were treated with 100nm dexamethasone (Dex) for 7 days starting on Day 43 to simulate clinical treatment doses. The time window from Day 40 to 50 was selected because different types of progenitor cells are most active in neurogenesis during this period in HCOs.

2. Cell Type Analysis:

   • Changes in cell type and number were validated using immunofluorescence and flow cytometry analysis (FCA), with Pax6 and Eomes marking different types of progenitor cells (radial glia and basal progenitors).
   • Single-cell RNA sequencing (scRNA-seq) was used to analyze changes in RNA levels under different treatment conditions.

3. Gene Regulation Analysis:

   • Zbtb16 was overexpressed (OE) in mouse fetal brains using in utero electroporation (IUE), and cell type and quantity were analyzed through brain tissue slices.

4. Exploration of Molecular Mechanisms:

   • The expression pattern of Zbtb16 during brain development was analyzed by examining its mRNA and protein expression in HCOs and mouse embryos to elucidate its regulatory mechanisms.

5. Genome-Wide Association Study (GWAS) Data Analysis:

   • Mendelian randomization analysis combined with genetic data from the UK Biobank was used to explore the association between regulatory SNPs and brain structure and cognitive performance.

Main Research Results

1. GCs Increase Pax6+Eomes+ Basal Progenitor Cells and Upper Layer Neurons:

   • In Dex-treated HCOs, Pax6+Eomes+ basal progenitor cells significantly increased (by 11% to 25.7%). These cells are typically enriched in species with gyrified brains. The number of upper layer neurons (Satb2+ neurons) also significantly increased, whereas there were no significant changes in deep layer neurons (Tbr1+ neurons and Bcl11b+ neurons).

2. Importance of Zbtb16 Under the Influence of GCs:

   • The action of GCs is mediated by upregulating Zbtb16. The physiological expression pattern of Zbtb16 was observed not only in HCOs but also in mouse embryos. Overexpression of Zbtb16 can mimic the effects of Dex, increasing the number of Pax6+Eomes+ basal progenitor cells and upper layer neurons. Using CRISPR-Cas9 to knock out one allele of Zbtb16 (zbtb16+/−) showed that Dex-induced increases in Pax6+Eomes+ basal progenitor cells were significantly inhibited.

3. Mouse Model Validation:

   • Zbtb16 overexpression not only had an effect in HCOs but also significantly increased Pax6+Eomes+ basal progenitor cells and enhanced regenerative capabilities and neurogenesis in mouse embryos after electroporation.

4. Long-Term Effects and Gene Association Study:

   • Gene association and Mendelian randomization analyses revealed that Zbtb16-associated SNP (rs648044) is linked to educational attainment, cognitive performance, and changes in brain structure. The A allele of rs648044, associated with higher GC-induced Zbtb16 levels, correlates with higher cognitive performance, educational achievement, and lower neuroticism.

Conclusions, Significance, and Research Highlights

GCs increase the number of Pax6+Eomes+ basal progenitor cells, which have neurogenic potential characteristic of gyrified brain species, and upper layer neurons by upregulating Zbtb16 during neurogenesis. This mechanism was validated not only in HCOs but also in mouse models, showing cross-species consistency in this regulatory pathway. Additionally, results from gene association studies relating to long-term cognitive function and brain structure further support the beneficial effects of GCs on brain development through Zbtb16, providing molecular and cellular-level clues for understanding the benefits of early sGC use.

Important Findings in the Study

• The mechanism of GCs in fetal brain involves primarily the regulation of Zbtb16.
• Pax6+Eomes+ basal progenitor cells play a key role in the brain development of species with gyrified brains.
• The importance of Zbtb16 in brain development is highlighted not only during neurogenesis but also in terms of long-term cognitive performance and brain structure changes.

Additional Valuable Information

This study provides scientific evidence for the timing and potential long-term benefits of early sGC administration, which is of significant importance for optimizing clinical treatment guidelines. Furthermore, these findings offer new perspectives and experimental models for future related research. By elucidating the molecular mechanisms and long-term effects of GCs, this study provides important references for neurodevelopmental science and clinical medicine.