Opioid-responsive microRNAs in neural stem cell fates: implications for neonatal opioid withdrawal syndrome infants
Restricted (Penn State Only)
- Author:
- Sullivan, Rhea
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 19, 2024
- Committee Members:
- Lisa Shantz, Program Head/Chair
Elizabeth Proctor, Outside Unit Member
Steven Hicks, Co-Chair & Dissertation Advisor
Jeffrey Sundstrom, Major Field Member
Robert Levenson, Co-Chair of Committee
Zissis Chroneos, Outside Field Member - Keywords:
- opioid
neonatal
neurodevelopment
microRNA
saliva
withdrawal
NOWS
NAS
iPSC
neural
stem cells
differentiation - Abstract:
- “Generation O” refers to the millions of children with prenatal opioid exposure (POE) during the ongoing opioid epidemic. Despite a growing body of clinical literature describing neurodevelopmental sequelae in these children—their biologic basis remains unknown. This is due, in part, to a lack of understanding of how human neurodevelopment is altered by chronic opioid exposure and withdrawal on a molecular level. In this dissertation, a novel human induced pluripotent stem cell-derived model of neonatal opioid exposure and withdrawal was developed to examine the impact of chronic morphine exposure and withdrawal on neural progenitor cell fates. Chronic morphine exposure and withdrawal significantly altered proportions of astrocytes, progenitors, and mature neurons. Notably, proportions of neuronal nuclear antigen+ (NEUN+) neurons decreased significantly with chronic morphine exposure, compared to vehicle controls. Cell fate perturbations were not associated with changes in proliferation or cytotoxicity. To identify a molecular mechanism underscoring these observations, the role of opioid-responsive microRNAs were studied in this model. Levels of miR-149, but not miR-23b, were significantly decreased in human neural progenitors after chronic morphine exposure, and decreased further in those that underwent withdrawal compared to vehicle exposed controls. Binding to putative miR-149 transcript target delta like non-canonical Notch ligand 1 (DLK1), a known regulator of neural progenitor differentiation, was confirmed via Argonaute 2-enriched RNA immunoprecipitations. Transfection of miR-149 mimic into neural progenitors prior to differentiation rescued resulting cell fate alterations in morphine-exposed Nestin+ (NES+) neural progenitors and NEUN+ neurons, in a DLK1-independent manner. The relevance of miR-149 to neonates experiencing withdrawal after chronic in utero opioid exposure was confirmed by decreased salivary levels of miR-149 compared to those of healthy infant controls between 24-96 hours after birth (n = 44, 28 unexposed and 28 infants with POE). Stratifying infants with POE by need for withdrawal treatment (i.e., morphine pharmacotherapy) revealed a further decrease in saliva levels of miR-149 in infants that required treatment. In a hierarchical linear regression model utilizing only infant demographic factors, miR-149 levels in neonatal saliva improved performance for the maximum morphine dose for symptom control. When infants with POE were stratified by the presence/absence of a neurodevelopmental delay diagnosis between 6-24 months (n = 21), levels of salivary miR-23b and let-7a at hospital discharge were significantly decreased in those with delays compared to those without delays. A logistic regression utilizing infant gestational age displayed a significant association with neurodevelopmental delay between 6 – 24 months. Addition of salivary let-7a levels (measured at discharge) via hierarchical regression significantly improved model performance. Separately adding levels of miR-23b also significantly improved model performance. These results indicate the potential clinical utility of neonatal miRNA levels for infant pharmacotherapy optimization and prediction of later neurodevelopmental delays. Further validation in a larger cohort is necessary. This dissertation identifies a role for an opioid-responsive miRNA in human neural progenitor differentiation and explores the utility of miRNAs as biomarkers for clinical outcomes related to POE.