Analyzing the Gene Expression Network in Developing Hippocampus from the Perspective of NEIL3 Deficiency

Background

The hippocampus, a crucial region of the brain, is well-regarded for its key roles in memory consolidation, information processing, and emotional regulation. In the field of neuroscience research, the gene regulatory mechanisms of the hippocampus are considered critical for its normal development, synaptic plasticity, and functional adaptability. However, although analyses of gene expression differences have identified key genes involved in hippocampal circuit organization and function, broader gene expression patterns and higher-order functions are still not fully understood.

NEIL3 is a DNA glycosylase widely present in the developing central nervous system, including the hippocampal neurogenesis area and the dorsal ventricular zone. Previous studies have shown that NEIL3 plays a significant role in the survival of neural precursor cells, post-stroke neurogenesis, and hippocampal adult neurogenesis. Based on this background, the authors of this paper decided to further explore the role of NEIL3 in hippocampal development.

Source of the Research

This research was conducted by Anna M. Bugaj, Nicolas Kunath, and Vidar Langseth Saasen from the Norwegian University of Science and Technology (NTNU) and was published in the journal “Progress in Neurobiology” (2024, Vol. 235, Article 102599). The paper was published online on March 24, 2024.

Research Process

Experimental Subjects and Sample Processing

The experiment used wild-type (C57BL/6N) and NEIL3-/- mice, with a total of 42 transcriptome samples covering two major developmental stages: p8 (juvenile) and 3 months (adult). Samples were taken from the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus.

Tissue Collection

Mice were euthanized by isoflurane anesthesia followed by an overdose of pentobarbital sodium, followed by intracardial perfusion with saline and fixation of brain tissue with 4% paraformaldehyde. The fixed brain tissues were then sectioned and subjected to immunohistochemical staining.

RNA Sequencing Process

RNA samples were extracted using an RNA extraction kit and transcriptome sequencing was performed by BGI Genomics. Samples were sequenced on the BGISEQ-500 platform, generating approximately 4.92GB of read data. Bioinformatics analysis was completed in the research laboratory.

Data Preprocessing and Weighted Gene Co-expression Network Analysis (WGCNA)

A weighted gene co-expression network analysis (WGCNA) was performed on the 15,000 most variable genes. Genes were grouped using hierarchical clustering, modules were constructed, and the correlation between modules and developmental stages was evaluated using a module-feature relationship map.

Main Research Results

Delayed Hodgkin Maturation Phenomenon

The study found that NEIL3 deficiency significantly delayed the maturation of neurons in all subregions of the hippocampus (CA1, CA3, and DG), but did not have a significant impact on the total number of mature neurons in adult mice. Immunohistochemical analysis showed that the proportion of mature neurons in NEIL3-deficient mice was significantly lower than in wild-type mice at P26.

Differential Gene Expression Analysis

RNA sequencing and principal component analysis revealed that age, brain region, and genotype were the main determinants of variation. NEIL3 deficiency significantly affected the overall gene expression of the developing hippocampus, especially in the juvenile state, suggesting that NEIL3 plays an important role in hippocampal development.

Different Gene Co-expression Networks

WGCNA analysis revealed significant differences in the connectivity and module specificity of the gene co-expression networks between wild-type and NEIL3-/- mice. Eleven different modules were identified in wild-type mice through hierarchical clustering analysis, but the distribution of these modules was completely altered in NEIL3-/- mice, demonstrating the importance of NEIL3 in maintaining the structure and dynamics of gene networks.

Module Analysis

Specific modules were identified in the transcriptome of the wild-type hippocampus, containing highly interactive gene networks at different developmental stages. Some key hub genes in these modules were upregulated in the juvenile dentate gyrus and adult CA1/CA3, while hub genes in other modules were specifically upregulated in adult hippocampal regions.

Impact of NEIL3 on Module Connectivity and Specificity

NEIL3 deficiency resulted in a general decrease in module connectivity, especially in specific modules such as module 1-tan, module 5-black, and module 6-magenta. Analysis of these modules indicated that NEIL3 drives the formation of gene co-expression networks specific to juveniles by influencing key hub genes within these modules.

Conclusion

This paper further explores the molecular mechanisms of NEIL3 in hippocampal development by combining WGCNA with DGE analysis. The results show that NEIL3 deficiency significantly affects the connectivity and specificity of the gene co-expression network in the hippocampus, revealing a potential link between gene connectivity and functional connectivity, providing a systematic-level analysis for understanding hippocampal function. This study lays the foundation for future studies on hippocampal function and related neuropathological states.

Highlights of the Study

1. Clarified the key role of NEIL3 in hippocampal neuron maturation.
2. Identified specific gene modules related to hippocampal development through weighted gene co-expression network analysis.
3. Revealed the impact of NEIL3 deficiency on the structure and functionality of gene co-expression networks.
4. Provided a comprehensive system-level framework to assess the potential correlation between gene connectivity and hippocampal network functional connectivity.

This paper illustrates the unique role of NEIL3 in hippocampal development and, through meticulous molecular and cellular-level analysis, reveals the intricate regulatory mechanisms of gene expression during neural development. The study not only enhances our understanding of the complexity of hippocampal neural networks but also provides important references for future research on the hippocampus in neuro-pathological states.