Variant Proteins in Ovine Ceroid-Lipofuscinosis

Abstract

The neuronal ceroid-lipofuscinoses are inherited neurodegenerative diseases. In humans three major forms have been described on the basis of age of onset and clinical course of the disease. These are the infantile, late-infantile and juvenile neuronal ceroidlipofuscinoses. Similar diseases have been described in several domestic animals. The main features of these diseases are brain atrophy and the intracellular accumulation of fluorescent "lipopigment". The stored material is mainly hydrophobic protein; primarily subunit c of mitochondrial F0F1-ATPase in the late-infantile and juvenile human forms and in all the known animal forms, and saposins in the human infantile form. Linkage analyses of the major human forms demonstrated that they arise from mutations in different genes. The molecular defects underlying these diseases are still unknown. This study has been carried out on the ovine model of neuronal ceroidlipofuscinosis, which closely resembles the late-infantile and juvenile human forms. Quantitative two dimensional polyacrylamide gel electrophoresis was used to search for disease related protein variation in homogenates of liver, pancreas and brain cortex and in liver and pancreas subcellular fractions. Although there was substantial accumulation of subunit c in affected tissues, no other consistent disease-related protein variation could be detected by two dimensional electrophoresis of liver or pancreas homogenates. Affected brain homogenates contained higher amounts of a protein tentatively identified as glial fibrillary acidic protein. In affected liver accumulation of light chain ferritin was found in the microsomal fraction and partial loss of senescence marker protein occurred in the soluble fraction. Light chain ferritin and senescence marker protein were directly identified by microsequencing. Two unknown proteins, at approximately pI 5.0,21 kDa, and pI 5.2, 19 kDa, respectively accumulated in the liver microsomal fraction of affected animals. The tentatively identified serum retinol binding protein was present in affected liver at twice the amount in controls, while an unknown mitochondrial protein, approximately pI 4.4,29 kDa, was found in half the normal amount in affected liver. Rapidly turning over proteins, obtained by in vitro translation of total and poly(A).+RNA of one control and one affected liver were analyzed by two dimensional electrophoresis. A number of differences between the translation products of control and affected RNA were found, some of which may be considered disease-related. Of possible interest was the finding of an acidic, low molecular weight protein, present exclusively in the translation products of total RNA from affected liver. The analysis by one dimensional polyacrylamide gel electrophoresis of the subcellular distribution of subunit c in affected and control liver showed accumulation of subunit c in the mitochondria-enriched fraction of affected tissue. Preliminary results indicated that the accumulation of subunit c seemed to occur in the mitochondrial matrix. Very little subunit c was found in the affected lysosome-enriched fraction while in the control lysosome-enriched fraction no subunit c was detected. These results suggest that subunit c accumulated in mitochondria, and secondarily in lysosomes as a consequence of autophagy. However the greater abundance of subunit c in the mitochondria-enriched fraction may have been an artifact due to contamination of this fraction with storage bodies. The subcellular distribution of acid phosphatase, which is a characteristic of storage bodies, seemed to support this view, as affected mitochondria-enriched fractions showed an acid phosphatase activity higher than the control mitochondria-enriched fractions. Two dimensional electrophoresis was used to separate and compare the proteins of storage body preparations from different forms of neuronal ceroid lipofuscinosis. One family of proteins, migrating at approximately pI 4,7-10 kDa, was found in all the storage body preparations. Sheep liver storage body proteins, separated by 2-D PAGE, were also compared with the proteins present in subcellular fractions from normal liver. Sheep liver storage bodies contained a subset of mitochondrial proteins with possible minor contributions by proteins from other subcellular fractions. The family of 7-10 kDa proteins was among the mitochondrial proteins selectively accumulated in storage bodies. A proposed mechanism of formation of storage bodies involves autophagy of mitochondria by lysosomes, with consequent accumulation of a subset of slowly degraded mitochondrial proteins, including subunit c. Biosynthetic labelling of control and affected skin fibroblasts, and analysis of the biosynthetically labelled proteins by one dimensional polyacrylamide gel electrophoresis, showed that subunit c was one of the most slowly synthesised and slowly degraded proteins in both control and affected fibroblasts.