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Abnormal cathepsin B activity in Batten disease
1Joseph P. Kennedy, Jr. Mental Retardation Research Center, Department of Pediatrics, University of Chicago, Illinois 60637.
Insights
Neuronal ceroid-lipofuscinosis (NCL) fibroblasts show reduced cathepsin B activity, but this is not the primary defect. Impaired lipid metabolism and phospholipase A2 deficiency may underlie NCL.
Area of Science:
- Biochemistry
- Cell Biology
- Neuroscience
Background:
- Neuronal ceroid-lipofuscinosis (NCL), also known as Batten disease, is a group of inherited neurodegenerative disorders.
- Previous studies suggested a potential protease defect, specifically involving cathepsin B, in NCL pathogenesis.
- Lysosomal dysfunction is a hallmark of NCL, impacting cellular waste disposal and neuronal health.
Purpose of the Study:
- To investigate the role of cathepsin B activity in NCL fibroblasts.
- To explore potential mechanisms for altered protease activity in NCL.
- To identify the primary molecular defect underlying NCL.
Main Methods:
- Culturing fibroblasts from NCL patients and assessing cathepsin B and H activity over time.
- Utilizing colorimetric, fluorimetric, and autoradiographic methods for enzyme assays.
- Analyzing lysosomal localization and buoyancy using Percoll gradient fractionation.
- Testing the sensitivity of cathepsin B to peroxides and aldehydes (hydrogen peroxide, 4-hydroxynonenal).
- Investigating bradykinin-induced release of [3H]arachidonate in NCL fibroblasts.
Main Results:
- Fibroblasts from NCL patients exhibited decreased cathepsin B activity with increasing passage number, while cathepsin H and other lysosomal hydrolases remained unaffected.
- Abnormally buoyant lysosomes with reduced cathepsin B were observed in some NCL fibroblasts.
- Cathepsin B was found to be sensitive to inactivation by hydrogen peroxide and 4-hydroxynonenal, which accumulate in NCL tissues.
- NCL fibroblasts showed deficient release of [3H]arachidonate in response to bradykinin.
Conclusions:
- The data do not support a primary defect in cathepsin B as the cause of NCL.
- Cathepsin B inactivation by accumulating peroxides and aldehydes may explain previous findings linking protease defects to NCL.
- A deficiency in the removal of peroxidized lipids from phospholipids, potentially involving phospholipase A2, is proposed as the primary defect in NCL.
Abstract:
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.