Tuberous sclerosis complex (TSC) occurs in approximately one of every 6,000 live births. Germline mutations are found in the TSC1 or TSC2 genes in 80% of TSC patients. Cortical lesions including tubers occur in most TSC patients and contribute to a range of neurological disorders. Up to 90% of TSC patients suffer from epileptic seizures, and many are refractory to pharmacological therapy, requiring surgical resection of epileptogenic tubers. It is unknown why some tubers are epileptogenic, while others are not. We propose to utilize an unparalleled repository of brain tissue that was resected to treat intractable epilepsy (collected through previous and current NIH grants) to test a novel hypothesis on the molecular basis of the epileptogenic potential of tubers. The central hypothesis of this research is that aberrant microRNA activity contributes to the epileptogenic potential of cortical tubers i TSC. MicroRNAs are short, non----coding nucleotides that provide post----transcriptional regulation of many genes, but their role in TSC is currently unknown. Our preliminary work reveals significant perturbations of microRNA expression in epileptogenic tubers, including dysregulated microRNAs predicted to target known epilepsy risk genes. We will compare microRNA expression profiles between epileptogenic tubers (seizure onset) and non----epileptogenic tubers using microarrays. Computational analysis and integration of epilepsy risk gene databases will identify target genes known to confer risk of epilepsy, and we will validate key microRNA target interactions using a human neural cell line model to ensure functional relevance to the human disease. Our secondary hypothesis is there are two subpopulations of epileptogenic tubers with different expression of microRNAs targeting the kynurenine pathway (KP) of tryptophan metabolism. Our previous studies showed that uptake of alpha[C---- 11]methyl----tryptophan (AMT) imaged with positron emission tomography (PET) for the identification of epileptogenic tubers reflects elevated KP activity Nearly all AMT positive ('hot') tubers are epileptogenic, but ~ 30% of epileptogenic tubers are AMT negative ('cold'). We will compare microRNA expression between hot and cold epileptogenic tubers to identify differences that account for KP activity. Expression unique to each AMT category will be identified, and target genes will be predicted. W expect to identify target genes and pathways that explain why some cold tubers are epileptogenic in the absence of elevated KP activity. The knowledge gained from these experiments is anticipated to provide the basis for improved diagnostic methods to identify epileptogenic tubers, including cases where AMT PET is negative. The proposed research is innovative because the role of microRNAs in TSC is currently unknown, and our approach in combining molecular neuroimaging with microRNA expression is also unique. The outcomes of this research are expected to expand our understanding of the molecular contributors to epilepsy in TSC, identify novel therapeutic targets, and improve diagnostic imaging.