The infantile neuronal ceroid lipofuscinosis (INCL), a rare (one in 100 000 births) but one of the most lethal inherited neurodegenerative storage disorders of childhood, is caused by inactivating mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. PPT1 cleaves thioester linkages in s-acylated (palmitoylated) proteins and facilitates their degradation and/or recycling. Thus, PPT1-deficiency leads to an abnormal intracellular accumulation of s-acylated proteins causing INCL pathogenesis. Although neuronal apoptosis is the suggested cause of neurodegeneration in this disease, the molecular mechanism(s) remains poorly understood. We recently reported that one of the major pathways of neuronal apoptosis in PPT1-knockout (PPT1-KO) mice that mimic INCL, is mediated by endoplasmic reticulum (ER) stress-induced caspase-12 activation. ER stress also increases the production of reactive oxygen species (ROS), disrupts Ca(2+) homeostasis and increases the potential for destabilizing mitochondrial membrane. Mitochondrial membrane destabilization activates caspase-9 present in this organelle, and can mediate apoptosis. We report here that the levels of superoxide dismutase (SOD), most likely induced by ROS, in human INCL as well as PPT1-KO mouse brain tissues are markedly elevated. Moreover, we demonstrate that activated caspase-3 and cleaved-PARP, indicative of apoptosis, are also increased in these tissues. Using cultured neurospheres from PPT1-KO and wild-type mouse fetuses, we further demonstrate that the levels of ROS, SOD-2, cleaved-caspase-9, activated caspase-3 and cleaved-PARP are elevated. We propose that: (i) ER stress due to PPT1-deficiency increases ROS and disrupts calcium homeostasis activating caspase-9 and (ii) caspase-9 activation mediates caspase-3 activation and apoptosis contributing to rapid neurodegeneration in INCL.