acid-phosphatase and Neuronal-Ceroid-Lipofuscinoses

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a neurodegenerative disorder caused by defective function of the lysosomal membrane glycoprotein CLN3. The activity of the lysosomal acid phosphatase (LAP/ACP2) was found to be significantly increased in the cerebellum and brain stem of Cln3-targeted mice during the early stages of postnatal life. Histochemical localization studies revealed an increased LAP/ACP2 staining intensity in neurons of the cerebral cortex of 48-week-old Cln3-targeted mice as compared with controls. Additionally, the expression of another lysosomal membrane protein LAMP-2 was increased in all brain areas. Knockdown of CLN3 expression in HeLa cells by RNA interference also resulted in increased LAP/ACP2 and LAMP-2 expression. Finally in fibroblasts of two juvenile neuronal ceroid lipofuscinosis patients elevated levels of LAP/ACP2 were found. Both activation of gene transcription and increased protein half-life appear to contribute to increased LAP/ACP2 protein expression in CLN3-deficient cells. The data suggest that lysosomal dysfunction and accumulation of storage material require increased biogenesis of LAP/ACP2 and LAMP-2 positive membranes which makes LAP/ACP2 suitable as biomarker of Batten disease.

Topics: Acid Phosphatase; Animals; Biomarkers; Brain; Cerebral Cortex; Female; Fibroblasts; HeLa Cells; Humans; Intracellular Membranes; Lysosomal-Associated Membrane Protein 2; Lysosomes; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Chaperones; Neuronal Ceroid-Lipofuscinoses; Transcriptional Activation; Up-Regulation

Acid phosphatases in brain and cultured lymphoblasts from patients affected with neuronal ceroid-lipofuscinoses (NCL) were studied by starch gel electrophoresis. After electrophoresis the gel was incubated with 4-methyl umbelliferyl phosphate at pH 4.5 and the fluorescent reaction product was visualized under ultraviolet light. Control brain showed a single band with mobility of about 1 cm while NCL patients showed two additional fast moving bands. In the late-infantile, and in the adult form (Kufs disease), the middle band was prominent while the fast moving band was predominant in juvenile NCL. In long-term lymphoblasts, controls showed a single band of acid phosphatase activity while both juvenile and late-infantile NCL showed two additional fast moving bands. Obligate heterozygotes showed reduced levels of the fast moving bands. Fluorometric assay of acid phosphatase using 4-methylumbelliferyl phosphate as substrate showed a 2-fold increase in activity in the patients. The increased acid phosphatase activity is completely inhibited by tartrate. Lymphocyte hexosamnidase activities were unchanged in NCL patients lymphoblasts. Studies on brains of NCL patients and on cultured lymphoblasts from families with late-infantile and juvenile form of NCL showed that abnormal acid phosphatase is characteristic of NCL.

Topics: Acid Phosphatase; Adolescent; Adult; beta-N-Acetylhexosaminidases; Brain; Cell Line; Cells, Cultured; Child, Preschool; Family; Gaucher Disease; Humans; Infant; Lymphocytes; Mucopolysaccharidosis III; Neuronal Ceroid-Lipofuscinoses; Substrate Specificity

Fibroblasts cultured from patients with various forms of neuronal ceroid-lipofuscinosis (NCL; Batten disease) showed variably decreasing cathepsin B activity with increasing passage number and months in culture in the presence of fetal calf serum. Cathepsin H activity and that of a wide range of lysosomal hydrolases was unaffected by these conditions. Cathepsin B activity was assayed either colorimetrically (N alpha-benzoyl-DL-Arg-beta-naphthylamide; BANA), fluorimetrically (Z-Arg-Arg-methylcoumarin), or autoradiographically, following NaDodSO4-12.5% polyacrylamide gel electrophoresis ([125]Tyr-Ala-Lys-Arg-CH2Cl) and was found to be lysosomal in localization. Fractionation of disrupted fibroblasts on a Percoll gradient showed evidence of abnormally buoyant lysosomes in some NCL patients, and these tended to be low in cathepsin B but rich in other lysosomal hydrolases. Our data do not support a primary defect in cathepsin B as the basic defect in NCL. However, a possible explanation for various studies implicating a protease defect in NCL is that cathepsin B was highly sensitive to inactivation by peroxides and aldehydes. Thus hydrogen peroxide (0.3 mM) or 4-hydroxynonenal (1 nM) inactivated cathepsin B without inhibiting cathepsin H or lysosomal hydrolases such as alpha-L-fucosidase. Since peroxides and 4-hydroxynonenal have been shown to accumulate in NCL tissue (despite apparently normal peroxidase activity), we tested the possibility of a defect in the removal of peroxidized lipids from phospholipids as the primary defect in NCL. The nociceptive peptide bradykinin (BK) normally initiates a cascade involving receptor-mediated phospholipase C activation and release of arachidonate and prostanoids from cultured skin fibroblasts. Release of [3H]arachidonate by BK was deficient in NCL fibroblasts, suggesting that the primary defect in NCL could involve the deficiency of a specific phospholipase A2 activity.

Topics: Acid Phosphatase; Arachidonic Acid; Arachidonic Acids; Cathepsin B; Cells, Cultured; Child; Child, Preschool; Cysteine Endopeptidases; Fibroblasts; Humans; Hydrogen Peroxide; Hydrolases; Kinetics; Lysosomes; Neuronal Ceroid-Lipofuscinoses; Skin