Infantile neuronal ceroid lipofuscinosis (INCL, or infantile Batten disease) is a hereditary progressive neurodegenerative disorder caused by mutations in a lysosomal enzyme (palmitoyl-protein thioesterase, PPT1) that removes fatty acids from modified cysteine residues in proteins. Little is understood about how deficiency in this enzyme causes neurodegeneration. In the period supported by this award, we have characterized a number of PPT1 mutations and produced knockout mice deficient in PPT1 and a homologous enzyme, PPT2. We found that residual PPT1 activity is the major determinant of age of onset of disease, that PPT1 knockout mice are an excellent model for INCL, and that PPT2 knockout mice have an NCL with unique extraneuronal features. In the current proposal we will delve deeper into the molecular basis of INCL. We will identify the physiological substrates of PPT1 and PPT2 through biochemical analysis of tissues derived from knockout mice. We will investigate the mechanism of action of cysteamine, the first rationally designed drug introduced for the treatment of INCL, through biochemical and metabolic labeling studies. We will also test the hypothesis that perturbations in membrane recycling in the presynaptic endosomal/lysosomal compartment lead to synaptic dysfunction and neuronal cell death in INCL. These studies will use electrophysiological recordings of spontaneous and evoked neurotransmitter release, fluorescent dye imaging, and ultrastructural analysis of presynaptic terminals in cortical and hippocampal cultures derived from PPT1 knockout and control mice. These studies will provide valuable insights into NCL and basic molecular and cellular mechanisms underlying neurodegenerative disorders. ? ?
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