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

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 pathology.Niemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral disorder caused by mutations in the Npc1 or Npc2 gene. NPC1 is a membrane protein that contains a sterol-sensing domain and resides primarily in late endosomes/lysosomes, whereas NPC2 is a soluble protein that resides primarily in lysosomes.^1,2,3,4 The loss of function of either protein results in intracellular accumulation of unesterified cholesterol and glycosphingolipids within the endosomal-lysosomal (EL) system in a number of tissues including the brain. In addition, there is evidence that homeostatic responses to exogenously supplied cholesterol and activation of cholesterol esterification are severely impaired in cells lacking functional NPC1. These defects in cholesterol accumulation/homeostasis trigger abnormal liver and spleen function as well as widespread neurological deficits including ataxia, dystonia, seizures, and dementia that eventually lead to premature death.^5,6,7,8,9 Interestingly, BALB/cNctr-Npc^N/N mice, which do not express NPC1 protein because of a spontaneous deletion/insertion mutation in the Npc1 gene, have been shown to recapitulate pathological features associated with NPC disease. These Npc1^−/− mice are asymptomatic at birth but gradually develop tremor and ataxia, dying prematurely at ∼3 months.^10,11,12,13 As in the human disease, Npc1^−/− mice show accumulation of unesterified cholesterol in the EL system and exhibit activation of microglia and astrocytes as well as degradation of the myelin sheath throughout the central nervous system. Progressive loss of neurons is particularly evident in the prefrontal cortex, thalamus, brainstem, and cerebellum but not in the hippocampal formation.^{13,14,15,16,17,18} However, at present, very little is known about the underlying mechanisms associated with the vulnerability of select populations of neurons in Npc1^−/− mice.A number of earlier studies have shown that the EL system, the major site of cholesterol accumulation in NPC pathology, consists of two dynamic interrelated cellular pathways: the endocytic pathway and the lysosomal system. Under normal conditions, the EL system serves as an important site for intracellular protein turnover and proteolytic processing of certain proteins mediated by lysosomal hydrolases termed cathepsins.^19,20,21 After their synthesis in the endoplasmic reticulum, cathepsins bind to the insulin-like growth factor-II (IGF-II)/mannose 6-phosphate (M6P) receptor on the trans face of the Golgi complex and are transported in vesicles to the EL system.^22,23,24 The importance of lysosomal enzymes in the proper functioning of the EL system is underscored by the fact that altered synthesis, sorting, or targeting of lysosomal enzymes is the molecular basis of more than 40 inherited disorders associated with extensive neurodegeneration, mental retardation and often progressive cognitive decline.^19,25,26,27There is evidence that increased endosome volumes and/or levels of cathepsins, such as cathepsins B and D, can mediate cell death by inducing lysosomal destabilization and enzyme leakage into cell cytosol, as is observed during oxidative stress²⁸ and experimental brain ischemia in primates.²⁹ Conversely, a number of recent studies have shown that lysosomal enzyme expression/levels can be up-regulated in the absence of cell death as a compensatory mechanism to repair damage/injury.^30,31,32,33 Thus, it seems that lysosomal enzymes are not only involved in the degeneration of neurons but also in the protection of neurons against toxicity in a variety of experimental as well as pathological paradigms. Although the EL system, the major site of cholesterol accumulation in NPC1-deficient cells, has been suggested to play a critical role in the development of NPC pathology,^6,7,8 very little is known about the significance of lysosomal cathepsins in determining neuronal vulnerability associated with the disease. To address this issue, we measured age-related changes in the levels, distribution, and activity of cathepsins B and D in the hippocampus and cerebellum of Npc1^−/− and age-matched control mice. In parallel, we evaluated the levels and distribution of the IGF-II/M6P receptor in Npc1^−/− and control mice to establish whether factors regulating cathepsin bioavailability can also influence the development of pathological changes. In addition, using cultured mouse cortical neurons we determined the significance of cathepsins B and D in the degeneration of neurons after accumulation of cholesterol. Our results reveal that alterations in the levels/activity as well as subcellular distribution of the lysosomal enzymes may be one of the underlying mechanisms associated with the selective neuronal vulnerability observed in NPC pathology.