Delineating Brainstem CircRNAs role in Neonatal sepsis
Authors: Alves Michele , Ayyalaraju Anika , Otero Jose 
Published: May 2024

Background: Neonatal sepsis remains one of the leading causes of mortality in newborns. Despite advances in newborn care, the literature still deprived of mechanistic insights into neonatal sepsis. Our prior findings revealed that systemic inflammation induced by TLR4 (LPS) or TLR1/2 (PAM3CKS4) ligands destabilizes breathing in neonates and impairs inspiratory rhythmogenesis following hypoxia. Circular RNAs (circRNAs) are emerging as important mechanistic mediators in neuronal activity. CircRNA constitutes a class of closed non-coding RNAs with a closed-loop structure. Brain-expressed circRNAs are enriched during synaptogenesis and many, occurs independently of the linear transcripts. Given the abundance of circRNAs in the brain, understanding their potential functional role in health and disease represents an emerging need. Our study delineates changes that neonatal sepsis evokes on brainstem circRNAs and their epigenetic modifications. Methods:CD1 mice were i.p. treated at postnatal day 5 (PD5) with LPS, or PAM3CSK4 (PAM), or saline (control). Following euthanasia, brainstems were dissected out and kept in -80C until further processing. Total RNAs were digested with Rnase R to remove linear RNAs. Labeled circRNAs were hybridized onto the Arraystar Mouse circRNA Array V2 or Mouse circRNA Epitranscriptomic Arrays and scanned by the Agilent Scanner G2505C. All data analyses were performed in RStudio Version 2022. Results: Our data revealed, in total, 13890 circRNAs expressed in the brainstem of PD5 pups. We found hundreds of circRNAs differentially expressed (DE) as a result of systemic inflammation. Strikingly, LPS induced a very low level of circRNA modulation, whereas PAM3CSK4 enriched 184 circRNAs relative to controls. Gene ontology (GO) analysis demonstrated the enrichment of mitochondrial-metabolic processes, translation, t-RNA, and RNA aminoacylation due to LPS-inducing inflammation. PAM3CSK4 administration led enrichment of processes associated with neurogenesis, neuronal transmission, and synapse organization. Additionally, KEGG pathways identified synapse processes, including GABAergic, dopaminergic, and glutamatergic enriched by circRNAs upregulated in PAM3CSK4. Epigenetic brainstem circRNA modifications in neonatal sepsis were found due to either LPS or PAM3CSK4. A greater number of hypomethylated than hypermethylated m6A and m5c circRNA modifications were promoted by PAM3CSK4. LPS augmented hypermethylation of m6A and hypomethylation of m5c modifications. Conclusions: Brainstem expressed circRNAs were specifically regulated by the inflammatory stimuli. Neonatal sepsis increases susceptibility to circRNAs modulation in brainstem neuronal processes. Our findings indicate that circRNAs are positioned to regulate brainstem-related functions. NIH/NHLBI R01HL132355 and GF316909 Mangurian Foundation Neuro Research for JJO. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.