Accumulation of sphingolipid activator proteins (SAPs) A and D in granular osmiophilic deposits in miniature Schnauzer dogs with ceroid-lipofuscinosis.

The neuronal ceroid-lipofuscinoses (NCL, Batten disease) are fatal inherited neurodegenerative diseases of children characterized by retinal and brain atrophy and the accumulation of electron-dense storage bodies in cells. Mutations in different genes underlie different major forms. The infantile disease (CLN-1, McKusick 256730) is distinguished by the storage of the sphingolipid activator proteins (SAPs) A and D in distinctive granular osmiophilic deposits (GRODs). This contrasts with the other major forms, where subunit c of mitochondrial ATP synthase is stored in various multilamellar profiles. Ceroid-lipofuscinoses also occur in dogs, including a form in miniature Schnauzers with distinctive granular osmiophilic deposit-like storage bodies. Antisera to SAPs A and D reacted to these storage bodies in situ. The presence of SAP D was confirmed by Western blotting and of SAP A by protein sequencing. Neither subunit c of mitochondrial ATP synthase nor of vacuolar ATPase is stored. This suggests that there are two families of ceroid-lipofuscinoses, the subunit c-storing forms, and those in which SAPs A and D, and perhaps other proteins, accumulate. Further work is required to determine whether other forms with granular osmiophilic deposits belong to the latter class and the genetic relationships between them and the human infantile disease.

Different patterns of hydrophobic protein storage in different forms of neuronal ceroid lipofuscinosis (NCL, Batten disease).

Since the discovery of mitochondrial ATP synthase subunit c storage in different forms of neuronal ceroid lipofuscinosis (NCL, Batten disease), it has been found that other hydrophobic proteins also accumulate in different forms. Costorage of subunit c of vacuolar ATPase is observed in "mnd/mnd" mice and in English Setters, Border Collies and Tibetan Terriers. A small amount is stored in the ovine disease and none in the human late-infantile disease. It is a storage body matrix component. An additional 8 kDa component immunoreactive to vacuolar ATPase subunit c antibodies is found in brain-derived storage bodies. The sphingolipid activator proteins, SAPs A and D, are stored in the human infantile disease and a form in Miniature Schnauzer dogs, but neither of the c subunits are. These results suggest two classes of NCL, the subunit c-storing diseases, related by a series of lesions in a subunit c-turnover pathway, and the SAP-storing diseases.

Immunocytochemical studies in the ceroid-lipofuscinoses (Batten disease) using antibodies to subunit c of mitochondrial ATP synthase.

Immunocytochemistry, using antibodies against subunit c of mitochondrial ATP synthase, has been carried out in the ovine, canine, late infantile, and adult forms of ceroid-lipofuscinosis. Intensity of staining varied depending on the particular disease, species, fixation regime, and the antibody used. Differential staining of storage cytosomes in neurons of affected sheep and those in the late infantile patient suggested exposure of different epitopes. This was supported by the variable staining using two different antibodies in ovine, late infantile, and adult onset (Kufs) diseases. Immunostaining of muscle in the late infantile, and muscle and ear cartilage in affected sheep can assist diagnosis but positive results may depend on the age of the patient, at least in the latter species. In these tissues there was immunostaining of structures not identified by histochemical or fluorescence microscopy in addition to storage cytosomes that could be identified by these means. Poor or no immunostaining occurred with canine tissues. At the ultrastructural level, storage cytosomes but not other organelles stained with the immunogold method.

Batten disease and the ATP synthase subunit c turnover pathway: raising antibodies to subunit c.

Analysis of storage bodies in the ceroid-lipofuscinoses (Batten disease) has demonstrated a high protein content suggestive of a proteinosis. Direct N-terminal sequencing has shown that subunit c of mitochondrial ATP synthase is specifically stored in the disease in sheep and cattle, and in the human late infantile and juvenile diseases, as well as in 3 breeds of dogs. No differences have been found between the stored subunit c and that in normal mitochondria. No other mitochondrial components are stored. Different proteins, sphingolipid activator proteins (SAPs or saposins) A and D, are stored in the infantile disease. Linkage studies have shown that different forms of ceroid-lipofuscinosis are coded for on different genes on different chromosomes. The genes for subunit c, its production, its insertion into mitochondria, and mitochondrial function are normal. This suggests that underlying the various forms of the disease is a family of lesions in the normal pathway of subunit c turnover, after its normal insertion into the ATP synthase complex. Antibodies to subunit c offer one way of mapping that pathway and detecting the sites of lesions. Specific antibodies have been raised against stored subunit c, using a liposomal adjuvant system which proved superior to classical adjuvants. These antibodies are also useful diagnostically, both in Western blotting and in immunocytochemistry.

Hematopoietic cell transplantation in fetal lambs with ceroid-lipofuscinosis.

Hematopoietic cells from the liver of normal 45-48-day-old fetal lambs (Hb type AA) were transplanted intraperitoneally into 58-60-day-old recipient fetuses (Hb type BB). The recipient fetuses resulted from mating homozygous ceroid-lipofuscinosis affected males with heterozygous, phenotypically normal, females. The sex of the donor fetus was also recorded. At age 2 1/2 months the recipient lambs with ceroid-lipofuscinosis were diagnosed by histopathology of brain biopsies. Monitoring of blood and bone marrow cells showed that an average of 9% of blood cells in ceroid-lipofuscinosis affected recipients were of donor origin. No differences were evident in the clinical course of disease, brain weight, or histopathology of organs between transplanted and non-transplanted lambs with ceroid-lipofuscinosis. Under the conditions of this experiment, transplantation of fetal hematopoietic cells was not beneficial.