Molecular Pathology of Subunit C Storage in Neuronal Ceroid Lipofuscinoses

Abstract

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative childhood diseases, characterised by seizures, blindness, dementia and premature death. The majority of NCLs accumulate FoF1, ATP synthase subunit c in autofluorescent,lysosome-derived storage bodies. Disease loci for eight variants have been mapped and enzyme defects identified in four of these. The relationship between the genetic defects, subunit c storage and neurodegeneration is unknown. The present study used a naturally-occurring sheep NCL (ovine ceroid lipofuscinosis; OCL) to study the molecular pathology of subunit c storage. The subcellular distribution of subunit c and storage bodies in liver was investigated using differential centrifugation and digitonin fractionation of mitochondrial-enriched fractions. The fractions were assessed by enzyme markers, western blots and one- and two-dimensional electrophoresis. Subunit c accumulated in the mitochondrial inner membrane vesicle fraction (IMV) in the absence of other storage body proteins. ATPase activity was not changed and no other FoF, subunits accumulated. The state of subunit c in mitochondrial fractions was studied using differential solubilisation and in vitro degradation assays. There was a substantial decrease in the solubility of subunit c by Triton X-100 in the OCL IMV fractions. The degradation of subunit c was assessed in vitro at pH 4, 5 and 7.4. Degradation was greatest under acidic conditions. Little degradation was seen at pH 7.4. There was a significant decrease in the rate of subunit c degradation at pH 4 when OCL fractions were used as a source of subunit c. Complete loss of subunit c was not seen even when OCL subunit c was incubated with subcellular fractions from normal liver. These results suggest that subunit c accumulates in the mitochondria in a state that is resistant to degradation by normal cellular processes prior to accumulation in lysosome-derived storage bodies. The accumulation of subunit c in OCL was also shown in primary neuron cultures from fetal OCL brain. Neurons from OCL and normal ovine fetal brain were cultured and analysed for the accumulation of autofluorescent material and subunit c over 2l days. The accumulation of autofluorescent bodies was demonstrated and quantified using confocal microscopy. To demonstrate that the autofluorescent material was accumulating in lysosomes, cells were probed with the acidotropic probe, Lyso Tracker RedTM. All autofluorescent storage bodies codistributed with lysosomes. A similar experiment using the mitochondrial probe, Mito Tracker RedTM showed no colocalisation between mitochondria and autofluorescent bodies. The accumulation of subunit c was shown both by immunohistochemistry and by Western blotting of culture extracts. Immunocytochemistry showed that the majority of the subunit c co-localised in cells with neuronal morphology. There were apparently no differences in the survival or growth of subunit c-loaded OCL neurons after 90 days in culture compared to normal control neurons, suggesting that subunit c is not directly toxic to neurons.