278 - Integrative Single Cell Multiomic Analysis of Mouse Neonatal Prefrontal Cortex Following Hyperoxia Exposure

Publication Number: 4274.278

Xingrao Ke, Children's Mercy Hospital, Kansas City, MO, United States; Wei Yu, Children's Mercy Kansas City, Kansas city, MO, United States; Carl Schwendinger-Schreck, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Melissa AH. Gener, UMKC School of Medicine, Kansas City, MO, United States; Sheng XIA, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Sherry M. Mabry, Children's Mercy, Kansas City, MO, United States; Heather Menden, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Venkatesh Sampath, Children's Mercy Kansas City, kansas city, KS, United States; Robert Lane, Physician Career Solution Institute, Parkville, MO, United States; Kaela M. Varberg, Dr., Shawnee, KS, United States

Background: Premature infants treated with life-saving oxygen therapy often develop bronchopulmonary dysplasia (BPD). Infants with BPD are at a high risk for abnormal neurodevelopment and cognitive impairment. Cognitive functions rely, in part, on prefrontal cortex (PFC) development. The PFC is critical for higher-order cognitive functions and may be vulnerable to neonatal hyperoxia (HOX). Prior studies show HOX disrupts gene expression related to cognitive decline, but the effects of HOX on PFC cellular composition, genes and signaling pathways remain unclear.

Objective: We hypothesized that neonatal HOX disrupts cellular composition, gene expression, and chromatin accessibility linked to cognitive regulation in the mouse PFC.

Design/Methods: C57Bl/6 mouse dams and pups were exposed to HOX (85% oxygen) or room air from postnatal day (P)1 to P14. At P14, PFC tissues were dissected and snap-frozen for single-cell multiomic analysis (RNA-seq and ATAC-seq). Oligodendrocyte progenitor cells (OPCs) were isolated from control pups at P5, cultured, and exposed to HOX for 24, 48, and 72 hours. Differentiation assays were performed on OPCs exposed to HOX or room air. Multiomic findings were validated by RNAscope and immunofluorescence using antibodies against OPC (SOX10, OLIG2) and mature oligodendrocyte (MBP, PLP1) markers on mouse and human PFC sections exposed to neonatal HOX.

Results: Neonatal HOX exposure reduced the numbers of myelin-form and mature oligodendrocytes in males. Neonatal HOX suppressed the expression and chromatin accessibility of the key myelination genes Ttr, Plp1, and Mbp in females and males. Conversely, neonatal HOX induced Olig2 expression in females and males, and Kdm3a induction was specific to females. Reductions in MBP and PLP1 and induction of OLIG2 were validated at the protein level. In males, neonatal HOX enhanced Netrin-1 signaling, whereas in females, it negatively impacted TP53-regulated metabolic genes. Finally, neonatal HOX inhibited OPC proliferation and differentiation in vitro.

Conclusion(s): Neonatal HOX exposure disrupts oligodendrocyte development in the PFC. Oligodendrocyte disruption is likely through dysregulation of transcription factors such as Olig2. The effects on myelination appear to be sex-specific and potentially mediated by Kdm3a upregulation in females. These disruptions may contribute to the cognitive deficits observed in HOX-exposed models. Future studies will include in vitro gain- and loss-of-function assays to further elucidate the underlying mechanisms of oligodendrocyte development.