DESCRIPTION (provided by applicant): Protein palmitoyl thioesterase 1 (PPT1) deficiency, or infantile neuronal ceroid lipofuscinosis (INCL), is a devastating human neurodegenerative disease characterized by lysosomal accumulation of autofluorescent granular osmophilic deposits. INCL is the most common class of several NCL diseases, which occur in 1:12,500 births in the United States. Individuals with mutations in the conserved serine hydrolase catalytic domain of PPT1 are subject to early blindness and progressive retardation, culminating in death by age 8-11 years. PPT1 catalyses the release of fatty-acyl chains from S-acylated substrates in vitro, but has little preference for S-acylated cysteine over acyl-CoA. Although dynamic palmitoylation is clearly important for many cytosolic and membrane proteins, PPT1 is believed to be exclusively lysosomal, implying it does not play a direct role in these well-characterized signaling pathways. Metabolic pulse-chase cysteine labeling has revealed the presence of accumulated lysosomal organic-soluble cysteine thioester metabolites in human immortalized PPT1-/- B-lymphoblast cell lines derived from INCL patients, yet a decade later, no detailed in vivo biochemical functions for this enzyme have been shown. In order to gain a better understanding of INCL pathogenesis, I propose to profile and chemically identify PPT1 substrates by comparative metabolomics. Using methods first developed in my sponsoring lab, metabolites (small molecules and peptides) will be harvested from cells and tissues by organic extraction, and then analyzed in broad scanning mode by LC-MS. PPT1+/+ and PPT1-/- metabolomes from patient-derived immortalized cell lines or PPT1 knockout (-/-) mice will be metabolically labeled with L-[13C3,15N]-cysteine, separating primary cysteine-thioester substrates from other secondary metabolites. Brain samples from PPT1-/- mice collected at progressive stages of neurodegeneration will be profiled and grouped into common pathways linked to PPT1-/- pathogenesis. The goal of this proposal is first identify PPT1 substrates, then to map the cascading secondary metabolite changes to identify important nodes critical for disease progression. Individuals with mutations in protein palmitoyl thioesterase 1 (PPT1) are subject to early blindness and progressive retardation, culminating in death by age 8-11 years. Significant effort has been focused on understanding the key events leading to PPT1-mediated neurodegeneration, yet no detailed in vivo biochemical functions for the enzyme are known. Using advanced analytical biochemistry, the primary substrates and secondary metabolite changes associated with disease pathogenesis will be identified.