Promotion of the Unfolding Protein Response in Orexin/Dynorphin Neurons in Sudden Infant Death Syndrome (SIDS): Elevated pPERK and ATF4 Expression

Nicholas J. Hunt, Karen A. Waters, Rita Machaalani

We previously demonstrated that sudden infant death syndrome (SIDS) infants have decreased orexin immunoreactivity within the hypothalamus and pons compared to non-SIDS infants. In this study, we examined multiple mechanisms that may promote loss of orexin expression including programmed cell death, impaired maturation/structural stability, neuroinflammation and impaired unfolding protein response (UPR). Immunofluorescent and immunohistochemical staining for a number of markers was performed in the tuberal hypothalamus and pons of infants (1-10 months) who died from SIDS (n = 27) compared to age- and sex-matched non-SIDS infants (n = 19). The markers included orexin A (OxA), dynorphin (Dyn), cleaved caspase 3 (CC3), cleaved caspase 9 (CC9), glial fibrillary acid protein (GFAP), tubulin beta chain 3 (TUBB3), myelin basic protein (MBP), interleukin 1β (IL-1β), terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL), c-fos and the UPR activation markers: phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (pPERK), and activating transcription factor 4 (ATF4). It was hypothesised that pPERK and ATF4 would be upregulated in Ox neurons in SIDS compared to non-SIDS. Within the hypothalamus, OxA and Dyn co-localised with a 20 % decrease in expression in SIDS infants (P = 0.001). pPERK and ATF4 expression in OxA neurons were increased by 35 % (P = 0.001) and 15 % (P = 0.001) respectively, with linear relationships between the decreased OxA/Dyn expression and the percentages of co-localised pPERK/OxA and ATF4/OxA evident (P = 0.01, P = 0.01). No differences in co-localisation with CC9, CC3, TUNEL or c-fos, nor expression of MBP, TUBB3, IL-1β and GFAP, were observed in the hypothalamus. In the pons, there were 40 % and 20 % increases in pPERK expression in the locus coeruleus (P = 0.001) and dorsal raphe (P = 0.022) respectively; ATF4 expression was not changed. The findings that decreased orexin levels in SIDS infants may be associated with an accumulation of pPERK suggest decreased orexin translation. As pPERK may inhibit multiple neuronal groups in the pons in SIDS infants, it could also indicate that a common pathway promotes loss of protein expression and impaired functionality of multiple brainstem neuronal groups.