The sudden infant death syndrome (SIDS) is the leading cause of postneonatal infant mortality in the United States today. Under the auspices of this grant which has been continuously funded for 24 years, we have reported a deficiency of the neurotransmitter serotonin (5-HT) and its biosynthetic enzyme, tryptophan hydroxylase (TPH2), in regions of the medulla oblongata that modulate cardiorespiratory function during sleep (the medullary 5-HT system) in four independent datasets. This deficiency is also associated with abnormalities in 5-HT receptors, transporter, and cell number. Based upon these findings, we propose that SIDS is a disorder of 5-HT deficiency in the medullary 5-HT system which causes an inability to restore homeostasis following life-threatening challenges, e.g., asphyxia, during a sleep period and leads to sudden death in the critical first year of life when homeostatic systems are not fully mature. In the present cycle, we performed state-of-the-art proteomics to identify candidate proteins which could provide novel insight into the cause(s) and pathogenesis of the medullary 5-HT deficiency in SIDS. We discovered several proteins that differed significantly in abundance between the SIDS cases and controls that have never before been considered in the context of SIDS brainstem pathology. These proteins include two families that we have chosen to pursue in the next cycle because they are directly relevant to 5-HT neurotransmission: 1) 14-3-3 proteins which are involved in signal transduction, including in regulation of TPH2; and 2) certain synaptic proteins, including 3-synuclein, actin, and spectrin. Our over-riding hypothesis is that an important subset of SIDS is due to alterations in key proteins related to 5-HT regulation and synaptic transmission in the medullary 5-HT system. We will analyze the proteins of interest in the same cases in order to determine how they inter-relate to each other and to the medullary 5-HT system in normative development and SIDS using immunocytochemical, western blotting, and other tissue techniques. We will also use hypothesis-driven proteomics to analyze comprehensively proteins upstream of 5-HT synaptic pathways and the 14-3-3 regulatory network in SIDS cases compared to controls to gain insight into the underlying basis of the observed protein alterations. The proposed study has the potential to determine the underlying basis of the medullary 5-HT deficiency in SIDS-knowledge which is essential to the future development of a means to identify and treat living infants at risk. PUBLIC HEALTH RELEVANCE: The sudden infant death syndrome (SIDS) is the leading cause of postneonatal infant mortality and the third leading cause of infant mortality overall in the United States today. Our research suggests that the majority of SIDS cases is associated with a deficiency of the neurotransmitter serotonin in brainstem regions that influence breathing and autonomic function during sleep. Our goal is to determine the fundamental basis of this deficiency by state-of-the-art techniques in human brainstem analysis. Such knowledge is essential to the future development of a means to identify and treat living infants at risk.