The Unfolded Protein Response (UPR) in the brain and placenta after nicotinic and hypoxic exposures, and within Sudden Infant Death Syndrome (SIDS)

Abstract

INTRODUCTION The Unfolded Protein Response (UPR) is a function of the gene expression process whereby misfolded proteins are removed. The UPR has multiple functions, including regulation of autophagy, transcription and translation, and certain apoptotic pathways. There are 3 main arms of the UPR, regulated by Protein kinase R-like endoplasmic reticulum kinase (PERK); Inositol-requiring enzyme 1 (IRE1); and Activating Transcription Factor 6 (ATF6). The UPR is known to be activated in the presence of SIDS risk factors hypoxia and cigarette smoke (CS) exposure. Previous work from this laboratory identified abnormalities in the pPERK pathway in several brain regions of SIDS infants. This thesis aimed to expand on this finding, testing the hypothesis that the other 2 pathways are also dysregulated widely in SIDS infant brains. The study therefore included additional brain regions of SIDS infants. Impact on the UPR of exposure to risk factors CS and hypoxia were investigated by additional studies in a piglet model and human placenta. METHODS Three methods were used: 1- immunohistochemistry (IHC), 2- western blotting, and 3- microarray analysis. The two tissue sources were brain and placenta, and the three datasets used were: 1- A dataset of SIDS infants (n=28) and non-SIDS infants (n=12). 2- A piglet model wherein piglets were either exposed to intermittent hypercapnic-hypoxia (IHH) for either 1,2,3 or 4 days total prior to collection (n=7,6,4,7 respectively), or exposed to nicotine (n=7). Controls for IHH were exposed to air for either 1 or 4 days (n=5 and 4, respectively), and controls for nicotine were exposed to saline only (n=6). Naïve controls were not exposed to the IHH, the nicotine, or the experimental stress (n=5). 3- Human placental tissue from women exposed to CS (n=8), women who experienced pre-eclampsia (PE, n=8), and women who had normal pregnancies (n=8). The UPR markers examined included pPERK, pIRE1α and ATF6α, alongside the neuropeptide Orexin-B. The microarray analysis included a wide range of cell death/apoptosis and stress markers. RESULTS SIDS infants showed more UPR staining than non-SIDS infants; those who were found prone or exposed to CS showed higher ATF6 in several nuclei, while those who bed-shared showed higher pPERK. IHH piglets showed an increase in pPERK and a decrease in ATF6 across several brain regions after 1 day of exposure, an increase in ATF6 after 2&3 days, and a decrease in pPERK after 3&4 days. Piglets exposed to nicotine showed a decrease in both ATF6 and pPERK. PE placentas had higher pPERK and pIRE1, and the PE and CS placentas all had reduced expression of apoptotic inhibitory marker IκBα. CONCLUSION The three arms of the UPR are dysregulated somewhat in the brain of SIDS infants. In our exposure models, SIDS risk factors maternal CS and hypoxia (prone sleeping and/or bedsharing) were also linked to dysregulation of the UPR.