Increased Activity and Altered Subcellular Distribution of Lysosomal Enzymes Determine Neuronal Vulnerability in Niemann-Pick Type C1-Deficient Mice

Abstract

Niemann-Pick disease type C (NPC), caused by mutations in the Npc1 or Npc2 genes, is a progressive neurodegenerative disorder characterized by intracellular accumulation/redistribution of cholesterol in a number of tissues including the brain. This is accompanied by a severe loss of neurons in selected brain regions. In this study, we evaluated the role of lysosomal enzymes, cathepsins B and D, in determining neuronal vulnerability in NPC1-deficient (Npc1 ؊/؊ ) mouse brains. Our results showed that Npc1 ؊/؊ mice exhibit an age-dependent degeneration of neurons in the cerebellum but not in the hippocampus. The cellular level/expression and activity of cathepsins B and D are increased more predominantly in the cerebellum than in the hippocampus of Npc1 ؊/؊ mice. In addition, the cytosolic levels of cathepsins, cytochrome c, and Bax2 are higher in the cerebellum than in the hippocampus of Npc1 ؊/؊ mice, suggesting a role for these enzymes in the degeneration of neurons. This suggestion is supported by our observation that degeneration of cultured cortical neurons treated with U18666A, which induces an NPC1-like phenotype at the cellular level, can be attenuated by inhibition of cathepsin B or D enzyme activity. These results suggest that the increased level/activity and altered subcellular distribution of cathepsins may be associated with the underlying cause of neuronal vulnerability in Npc1 ؊/؊ brains. Therefore , their inhibitors may have therapeutic potential in attenuating NPC

A novel intermediate mucolipidosis II/IIIαβ caused by GNPTAB mutation in the cytosolic N-terminal domain

“…Roles for cathepsin D have been suggested in neural development and degeneration, and maintenance of near-normal intracellular levels of this protease may confer a protective effect within the brain of these patients. [18][19][20] Delineation of this c.10A4C/p.K4Q GNPT-dependent intermediate ML has more than merely clinical and prognostic significance. The partial 'uncoupling' of physical and cognitive disability represents the apparently rare exception to the rule that nearly complete absence of GNPT activity yields the ML II phenotype and more residual activity results in ML IIIab.…”

Role of Cathepsin D in U18666A-induced Neuronal Cell Death

“…Mouse Hippocampal Neuronal Cultures-Primary hippocampal cultures were prepared from 16-or 17-day-old embryos of timed pregnant BALB/c mice as described previously (30,31). In brief, the pregnant mice were anesthetized with halothane and decapitated.…”

“…Samples were then denatured in modified Laemmli sample buffer (40 mM Tris-HCl, pH 6.8, 1% SDS, 4% 2-mercaptoethanol, 10% glycerol, and 0.002% bromphenol blue) and boiled for 2-5 min, and equal amounts of proteins (20 g) were separated by 4 -20% polyacrylamide gel electrophoresis (30). The proteins were subsequently transferred to nitrocellulose membranes, blocked with 5% nonfat milk, and incubated overnight at 4°C with anti-cathepsin D (1:200), anticleaved caspase-9 (1:200), anti-cleaved caspase-3 (1:1000), anti-AIF (1:300), anti-LC3 (1:1000), anti-Beclin-1 (1:200), anti-p62 (1:1000) or anti-Atg5 (1:1000) antibodies.…”

“…Membranes were then washed with TBST, incubated with appropriate secondary antibodies (1:5000), and visualized using an ECL detection kit. Blots were subsequently reprobed with anti-N cadherin (1:200), anti-GAPDH (1:1000), or anti-histone (1:1000) antisera as described earlier (30).…”

“…The protein amount was equalized after a protein assay with the BCA assay kit, and then cathepsin D activity was measured using the fluorogenic immunocapture activity assay kit as described earlier (30).…”