Postnatal Changes in Cathepsin D in Rat Neural Tissue

Abstract

Cathepsin D, an aspartyl endopeptidase, was analyzed in cortex from forebrain and cerebellum, spinal cord, and optic and sciatic nerves, and in the liver of rats from 1 to 120 days of age. Cathepsin D was quantitated in tissue extracts by measurement of enzyme specific activity on a substrate of [methyl‐14C]‐methylated hemoglobin and by radioimmunoassay. Immunocytochemistry was used to ascertain the identity of the mixed cell types that contributed to the cathepsin D detected. As quantitated by radioimmunoassay, immunoreactive cathepsin D varied between 0.2 and 1 ng/μg of total protein. Maximum activity occurred at approximately the 15th postnatal day; the least amount of immunoreactive cathepsin D was found at 30 or 60 days of age. A subsequent increase of varying magnitude occurred at postnatal day 120. There was good correspondence between immunoreactive enzyme and enzyme specific activity, which ranged from 1 to 4 ng/μg of total protein, and the activities determined by the two methods provided similar, but not identical, developmental profiles. Cathepsin D was demonstrated by immunocytochemistry to be present in most neurons, in all choroid plexus epithelium, and in certain oligodendrocytes from the first postnatal day. Cathepsin D was present in oligodendrocytes in cord lateral funiculi and optic nerve by the first postnatal day, and by the sixth postnatal day many oligodendrocytes were abundantly stained. In contrast, oligodendrocytes in the corpus callosum and in the cerebellar white matter did not contain demonstrable cathepsin D until postnatal days 10 and 15, respectively. These results indicate a role for cathepsin D during the postnatal development of rat CNS and suggest that this proteinase may be involved in the steps of myelination.

Alterations of the posttranslational processing of a lysosomal enzyme in C6 glioma cells

“…The increase of CD seen here was similar to the increase of specific activity of two other lysosomal enzymes from C6 cells grown under comparable conditions (Wallace and McCormick, 1982). Previous studies (Whitaker et al, 1980;Snyder and Whitaker, 1983) from whole tissue have shown that CD measured by RIA parallels that of CD specific activity. It is known from in vitro translation studies of mouse and porcine spleen CD mRNA (Erickson and Blobel, 1979;Rosenfeld et al, 1982) that the initial product of translation is a glycoprotein with an M, of greater than 50 kD.…”

“…For radioimmunoassay (RIA), the cells were washed once with PBS, removed by scraping, pelleted by centrifugation, and washed twice with PBS. Cell pellets were prepared for RIA as described previously (Snyder and Whitaker, 1983).…”

“…The RIA for cathepsin D has been described in detail previously (Whitaker, 1980;Snyder and Whitaker, 1983). The amount of DNA was determined by the method of Burton (1956); protein, by the BCA protein assay method (Smith et al, 1985); and the activity of cysteine proteinase cathepsin B, according to Barrett and Kirschke (1977) with the substrate Benzoyl-(DL)-arg-arg napthalamide (Enzyme Systems Products, Livermore, CA).…”

Changes Induced in Astrocyte Cathepsin D by Cytokines and Leupeptin

“…After the culture period, CM was removed and 1 ml of 0.1 M Tris-acetate, pH 7.2, containing 0.1 % Triton X-100 was added and the sample sonicated (Whitaker et al, 1983a,b). Cathepsin D protein was then quantitated by a double antibody radioimmunoassay using absorbed rabbit (R 164) antirat cathepsin D (Snyder et al, 1983;Whitaker et al, 1983a,b). The immunoreactive cathepsin D and the cathepsin D enzyme activity was related to the total protein concentration determined by the Bradford method (BioRad).…”

“…Because there is no known intra-cellular inhibitor olf cathepsin D in mammals, the cellular level of the enzyme and its response to external stimuli should have a direct relationship to the activity of cathepsin D. The normal nervous system also reflects the wide but uneven distribution of cathepsin D among its cell types (Whitaker el. al., 1981;Snyder and Whitaker, 1983;Whitaker and Crowley, 1983;Snyder et al, 1985). The normal controls and pathological events influencing cathepsin D content among cells in neural tissue are unknown.…”