PROJECT SUMMARY Despite fewer deaths as a result of the Safe Sleep campaign, the Sudden Infant Death Syndrome (SIDS) remains the leading cause of post-neonatal infant mortality in the United States (~0.39/1000 live births). Our research strongly implicates biological abnormalities within the brainstem that underlie an infant's vulnerability to SIDS, including abnormalities in the serotonin (5-HT) neurotransmitter systems. MicroRNA (miRNA) biology has emerged rapidly with clear evidence that miRNAs play a critical role in regulation of protein expression, including in the regulation of proteins involved in the development and function of the central nervous system (CNS) and in the regulation of the 5-HTergic system within the CNS. We recently performed an unbiased sequencing screen of microRNAs in a dissected area of the medulla concentrated with 5-HT neurons to identify differentially expressed miRNAs in SIDS compared to controls. In this proposal we focus on two of the miRNAs identified with altered expression – miR-34a-5p (upregulated in SIDS) and miR-122-5p (downregulated in SIDS) and two mRNAs that these miRNAs target, hyperpolarization-activated cyclic nucleotide gated 3 (HCN3) and cytoplasmic polyadenylation element binding protein 1 (CPEB1), respectively. These specific miRNAs were selected based on the reported roles that HCN channels and CPEB1 play in 5-HT function and inflammation, respectively, the latter of interest given evidence of an effect of inflammation on 5-HT metabolism. Here we test the following hypotheses, directly in SIDS tissue and serum that: 1) cell-specific dysregulation of miR-34a-5p and miR-122- 5p in the medulla of SIDS, results in aberrant expression of their respective mRNA targets, HCN3 and CPEB1 (Specific Aims 1 and 2) and, 2) that aberrant levels of medullary miR-34a-5p and miR-122-5p are reflected within the serum of SIDS infants and serve as potential biomarkers of SIDS pathogenesis (Specific Aim 3). We will use our unique database of SIDS and non-SIDS controls, multiplexed in-situ hybridization techniques, machine learning quantitative software, and qPCR directly in human SIDS and control tissue and serum. Data generated from this R21 will be utilized to further develop novel hypotheses and animal models aimed at this important regulatory system.