Inflammation driven molecular alterations in disorders of the perinatal brain

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Casey, Sophie
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University College Cork
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Introduction: Inflammatory insults during the perinatal period are known to disrupt normal neurodevelopmental processes. Neonatal hypoxic ischemic encephalopathy (HIE) may occur with or without infection and is characterised by a significant immunoinflammatory response. Infection sensitises the neonatal brain to further hypoxic-ischemic injury. HIE with and without infection affects approximately 3 per thousand of all live births in the developed world. Despite the advent of therapeutic hypothermia (TH), almost half of affected neonates die or are left with lifelong disabilities or disorders. Autism Spectrum Disorder (ASD) is one such disorder and affects approximately 1.5% of the population in the developed world. Epidemiological and animal studies suggest that maternal immune dysregulation may contribute to the development of ASD. Rapid identification of neonates and infants at risk of HIE and ASD is vital, but many miss the critical therapeutic windows due to subjective and suboptimal diagnostic techniques. Circulating inflammation-associated markers such as microRNA (miRNA) and cytokines may hold the key to rapid diagnosis, providing an insight into maternal, foetal and neonatal injury cascades. Blood represents a non-invasive, and rapidly accessible medium in which to examine aberrant expression of these inflammation-driven molecular markers. In addition, miRNAs and cytokines are stably expressed in the blood, so may represent robust biomarker candidates. Furthermore, while many studies of miRNA expression have been performed in clinical cases of HIE, their functional role is still largely unknown. The primary hypothesis of this thesis is that examination of temporal inflammatory profiles may improve our understanding of the pathophysiology of early-life neuronal injury and subsequent development of long-term disabilities, and may provide novel biomarkers, therapeutic targets, or candidates capable of improving long-term outcomes. The current thesis aims to identify early molecular biomarkers of HIE and ASD in clinical and preclinical models and investigate the functional role of altered miRNAs in vitro. To investigate this, we aimed to examine temporal profiles of miRNA and cytokine alterations in a large animal model of HIE, with and without inflammatory sensitisation. Following this, we aimed to identify rapidly detectable miRNA and cytokine biomarkers of HIE, with and without inflammatory sensitisation. Lastly, we aimed to investigate the role of altered miRNA biomarkers through identification and examination of their functional targets, and to examine the mid-gestational cytokine profiles of mothers who give birth to offspring with ASD in a large clinical cohort. Methods: The current thesis utilised a large cohort of HIE neonates recruited to the Validation of Biomarkers in Hypoxic Ischemic Encephalopathy (BiHIVE) 1 & 2 studies (NCT02019147) and a clinically relevant large animal model of HIE with and without inflammation-sensitisation – the neonatal porcine model. To achieve a moderate-severe level of hypoxia-ischemia (HI) in piglets, inspired oxygen was reduced to 4% for approximately 30 minutes and titrated to achieve the desired level of neuronal injury. To achieve inflammation-sensitisation, piglets received an infusion of lipopolysaccharide (LPS) prior to HI. Blood was drawn from piglets at regular intervals for 48-72h following injury. Whole blood was used for analysis of miRNA content, while serum was preferable for analysis of cytokines. The multi-national Screening for Pregnancy Endpoints (SCOPE) cohort was utilised for the examination of circulating midgestational cytokine markers of ASD following diagnosis at neurodevelopmental follow-up. Cohorts from Cork, Ireland and Auckland, New Zealand were examined. Techniques employed throughout the current thesis are state-of-the-art, and were performed by both the PhD candidate and collaborating co-authors. Neurodevelopmental outcomes for neonatal study subjects were measured using Apgar/Sarnat staging, clinical signs (Chapter 3), early intervention services teams and child psychiatrists (Chapter 6). Outcomes for animal subjects were measured using clinical signs and Haemotoxylin & Eosin (H&E)/Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining of porcine brain tissues (Chapters 3 and 4). Further techniques featured in Chapter 3 include ribonucleic acid (RNA) Sequencing (RNA-Seq), Haemotoxylin & Eosin (H&E) staining of porcine brain tissues, magnetic resonance spectroscopy (MRS), RNA isolation from whole blood, complementary deoxyribonucleic acid (cDNA) synthesis, quantitative real-time polymerase chain reaction (qRT-PCR), in silico analysis, cell culture of SH-SY5Y cells and primary cultures of embryonic day (E) 14 rat midbrain, in vitro transfection of miRNA inhibitors, immunocytochemistry and fluorescent microscopy. Techniques used throughout the remainder of the thesis were performed solely by the PhD candidate. Techniques employed in Chapters 4 and 6 include electrochemiluminescent Mesoscale Discovery sandwich enzyme-linked immunosorbent (ELISA) immunoassays for analysis of serum cytokines. Finally, Chapter 5 used cell cultures of SH-SY5Y cells, in vitro transfection of miRNA inhibitors, green fluorescent protein (GFP) based reporter assays, immunocytochemistry, and fluorescent microscopy. All statistical analyses were conducted using IBM SPSS Statistics 24/26 and GraphPad Prism 7/8. Graphical representations of data were generated using GraphPad Prism 8. Results: miRNA Dysfunction in the Porcine Model of HIE Eleven candidate miRNAs were determined from a combination of results generated from microarray and RNA-Seq from a clinical cohort of hypoxic-ischemic neonates. These were then examined in the porcine model over 72h. Six miRNAs - miR-128, miR-148a, miR-151a, miR-181a, miR-181b and miR-374a, were upregulated in whole blood 1h after HI. Three - miR-374a, miR-181a and miR-181b, were specifically upregulated in moderate-severely injured piglets. miRNA levels at 1h correlated with histopathological and MRS-measured Lactate/Creatine (Lac/Cr) neuropathological outcomes. Inhibition of miR-181a in vitro resulted in increased neurite growth and increased expression of one member of the target bone morphogenetic protein (BMP) signalling pathway - BMPR2. Cytokine Dysfunction in the Porcine Model of Inflammation-Sensitised HIE Eight candidate cytokines were examined in serum samples from the inflammation-sensitised porcine model over 48h. Interleukin (IL) 6, tumor necrosis factor (TNF) α and Tau displayed a sustained inflammatory response following LPS exposure with and without hypoxia. Neuron-specific enolase (NSE) increased slowly following HI. LPS + Hypoxia-ischemia (LPS-HI) piglets displayed late increases in both NSE and C-reactive protein (CRP). TNFα and IL-6 allowed discrimination between animals who were exposed to inflammation-sensitised HI and those who were exposed to HI alone 6h following a moderate-severe HIE-like insult. miRNA Function In Vitro We previously identified the BMP signalling pathway as a predicted downstream target of miR-374a and miR-181a. The effect of miR-374a and miR-181a manipulation on the BMP-small mothers against decapentaplegic (SMAD) signalling pathway was investigated in vitro. miR-374a inhibition increased expression of BMP2 and BMPR2 but did not alter transcription of SMAD. Likewise, miR-181a inhibition was previously found to increase BMPR2 expression and did not alter SMAD transcription in vitro. Midgestational Cytokine Dysfunction in ASD Eight candidate cytokines were examined in serum samples retrieved at 15 and 20 weeks’ gestation in mothers of children affected by ASD. IL-17A concentrations were downregulated at 20 weeks’ gestation in mothers of children who progressed to develop ASD. IL-16, eotaxin, monocyte chemoattractant protein (MCP) 1, interferon (IFN) γ, IL-1β, IL-6 and IL-8 were unchanged in both groups at both 15 and 20 weeks. Conclusions: The current thesis has identified dysregulated expression of inflammation-driven molecular markers of the perinatal brain disorders HIE and ASD. Circulating levels of miRNAs (miR-128 miR-148a, miR-151a, miR-181a, miR-181b and miR-374a) and cytokines (IL-6, TNFα) were rapidly raised in response to HIE with and without inflammation-sensitisation in the porcine model. IL-17A was also dysregulated at 20 weeks’ gestation in mothers of ASD-affected children. These molecular markers may aid in rapid diagnosis, prognosis, and therapeutic decision-making for these time-sensitive disorders. Moreover, they may allow for discrimination of complex inflammation-sensitised HIE from classic HIE. Furthermore, evidence is beginning to suggest that HIE-associated miRNAs may play a functional role in the regulation of essential BMP-SMAD signalling. The current thesis puts forward novel information regarding the temporal profiles of these circulating molecular markers and begins to explore the functional roles of HIE-associated miRNAs. We hope this work will aid in the development of early blood-based biomarkers of disorders with an inflammatory milieu like HIE and ASD, and pave the way for more functional analysis of these markers.
Hypoxic ischemic encephalopathy , Autism spectrum disorder , Cytokine , Inflammation , MicroRNA
Casey, S. 2021. Inflammation driven molecular alterations in disorders of the perinatal brain. PhD Thesis, University College Cork.