(47) Sexually Differentiated Gene Expression Profiles of the Hippocampal Neurons Following Neonatal Hypoxia Ischemia

Background: The hippocampus, crucial for learning and memory, is negatively affected by neonatal hypoxia ischemia (HI). We previously identified sex-specific differences in the tyrosine kinase B receptor (TrkB) pathway within the hippocampus after HI in mice. However, the molecular responses of cell cluster type- and sex-specific transcriptomic responses to HI and TrkB agonist [7,8-dihydroxyflavone (DHF)] treatment remains unexplored.

Objectives: In this study, by using single nucleus RNA sequencing (snRNA-seq), we determined cell cluster type- and sex-specific transcriptomic responses to HI and DHF treatment.

Design/Methods: Sham surgery or HI was induced in postnatal day (P) 9 mice followed by vehicle control or DHF treatment daily for three days (n=3-4). Only moderately injured hippocampi were selected for snRNA-seq at P12. Python, JupytrLab and Wilcoxon differential expression analysis were used to identify sex-specific differences in response to HI and DHF for each cluster. The top 250 differentially expressed genes with adjusted P-value ≤ 0.05 were utilized in over-representation analysis and functionally annotated by Metascape.

Results: Among the 29 hippocampal cell clusters identified, females exhibited a greater number of gene expression changes than males in excitatory and inhibitory neurons (EN, IN) post- HI. Expression of the TrkB gene, Ntrk2, was significantly higher in HI female EN, Lamp5 and SST IN clusters compared with HI male counterparts. Moreover, Ntrk2 decreased in male ENs post-HI. In enrichment analysis, HI induced genes associated with the neuron projection development pathway regardless of sex and treatment in all neurons. However, after HI, female IN clusters showed sex-specific enrichment in Ntrk2-related synaptic plasticity pathways such as learning and synapse assembly. Following treatment, although males exhibited a greater number of gene expression changes than females across multiple cell types, the pathways associated with differentially expressed genes didn`t differ remarkably between HI and treatment groups in both sexes, only exception was that female ENs and SST INs displayed sex-specific enrichment in the cell survival pathway (PI3K/Akt).

Conclusion: SnRNA-seq helps define sex-specific pathways in neonatal hippocampal neurons. HI upregulates genes associated with Ntrk2 related synaptic plasticity and DHF enhances cell-survival pathways in SST INs only in females.