Deregulation of the phosphatidylinositol-3 kinase signaling cascade is associated with neurodegeneration in Npc1-/- mouse brain

Niemann-Pick type C (NPC) disease is caused by mutations to genes that encode proteins critical to intracellular lipid homeostasis. The events underlying NPC progressive neurodegeneration are poorly understood but include neurofibrillary tangles of the type found in Alzheimer's disease. Here we investigated possible contributions of a phosphatidylinositol-3 kinase cascade [PI3K, Akt, glycogen synthase kinase-3beta (GSK-3beta)] that is linked to apoptosis and various degenerative conditions. Brain concentrations of phosphorylated Akt, which phosphorylates and inactivates GSK-3beta, were significantly elevated in Npc1-/- mice relative to Npc1+/+ mice. Accordingly, levels of inactive GSK-3beta were 50 to 100% higher in mutant brains than in controls. Increases in inactive GSK-3beta occurred early in postnatal development, well before neuronal loss, and were most prominent in structures with intracellular cholesterol accumulation, suggesting a contribution to subsequent degeneration. Perturbations of nuclear factor (NF)-kappaB, which is regulated by GSK-3beta, occurred in Npc1-/- mouse brains. Nuclear concentrations and DNA binding activity of NF-kappaB's transactivation subunit, p65, were significantly reduced in Npc1-/- mice compared to Npc1+/+ mice. Cytoplasmic levels of the p50 subunit and its precursor, p105, were higher in Npc1-/- mice. These results suggest that excessive activity in the PI3K-Akt pathway depresses GSK-3beta, thereby disrupting the formation and/or nuclear import of p50/p65 NF-kappaB dimers and contributing to neuronal degeneration.     

Microarray expression analysis and identification of serum biomarkers for Niemann-Pick disease, type C1

Niemann-Pick disease type C (NPC) is a lysosomal storage disorder characterized by liver disease and progressive neurodegeneration. Deficiency of either NPC1 or NPC2 leads to the accumulation of cholesterol and glycosphingolipids in late endosomes and early lysosomes. In order to identify pathological mechanisms underlying NPC and uncover potential biomarkers, we characterized liver gene expression changes in an Npc1 mouse model at six ages spanning the pathological progression of the disease. We identified altered gene expression at all ages, including changes in asymptomatic, 1-week-old mice. Biological pathways showing early altered gene expression included: lipid metabolism, cytochrome P450 enzymes involved in arachidonic acid and drug metabolism, inflammation and immune responses, mitogen-activated protein kinase and G-protein signaling, cell cycle regulation, cell adhesion and cytoskeleton remodeling. In contrast, apoptosis and oxidative stress appeared to be late pathological processes. To identify potential biomarkers that could facilitate monitoring of disease progression, we focused on a subset of 103 differentially expressed genes that encode secreted proteins. Further analysis identified two secreted proteins with increased serum levels in NPC1 patients: galectin-3 (LGALS3), a pro-inflammatory molecule, and cathepsin D (CTSD), a lysosomal aspartic protease. Elevated serum levels of both proteins correlated with neurological disease severity and appeared to be specific for NPC1. Expression of Lgals3 and Ctsd was normalized following treatment with 2-hydroxypropyl-β-cyclodextrin, a therapy that reduces pathological findings and significantly increases Npc1(-/-) survival. Both LGALS3 and CTSD have the potential to aid in diagnosis and serve as biomarkers to monitor efficacy in therapeutic trials.     

A novel mouse model of Niemann-Pick type C disease carrying a D1005G-Npc1 mutation comparable to commonly observed human mutations

We have identified a point mutation in Npc1 that creates a novel mouse model (Npc1(nmf164)) of Niemann-Pick type C1 (NPC) disease: a single nucleotide change (A to G at cDNA bp 3163) that results in an aspartate to glycine change at position 1005 (D1005G). This change is in the cysteine-rich luminal loop of the NPC1 protein and is highly similar to commonly occurring human mutations. Genetic and molecular biological analyses, including sequencing the Npc1(spm) allele and identifying a truncating mutation, confirm that the mutation in Npc1(nmf164) mice is distinct from those in other existing mouse models of NPC disease (Npc1(nih), Npc1(spm)). Analyses of lifespan, body and spleen weight, gait and other motor activities, as well as acoustic startle responses all reveal a more slowly developing phenotype in Npc1(nmf164) mutant mice than in mice with the null mutations (Npc1(nih), Npc1(spm)). Although Npc1 mRNA levels appear relatively normal, Npc1(nmf164) brain and liver display dramatic reductions in Npc1 protein, as well as abnormal cholesterol metabolism and altered glycolipid expression. Furthermore, histological analyses of liver, spleen, hippocampus, cortex and cerebellum reveal abnormal cholesterol accumulation, glial activation and Purkinje cell loss at a slower rate than in the Npc1(nih) mouse model. Magnetic resonance imaging studies also reveal significantly less demyelination/dysmyelination than in the null alleles. Thus, although prior mouse models may correspond to the severe infantile onset forms of NPC disease, Npc1(nmf164) mice offer many advantages as a model for the late-onset, more slowly progressing forms of NPC disease that comprise the large majority of human cases.     

Intracellular accumulation of amyloidogenic fragments of amyloid-beta precursor protein in neurons with Niemann-Pick type C defects is associated with endosomal abnormalities

Niemann-Pick type C disease (NPC) is characterized by neurodegeneration secondary to impaired cholesterol trafficking and excessive glycosphingolipid storage. Abnormal cholesterol and ganglioside metabolism may influence the generation and aggregation of amyloidogenic fragments (ie, C99 and Abeta) from amyloid-beta precursor protein (APP), crucial factors causing neurodegeneration in Alzheimer's disease. To reveal whether abnormal accumulation and aggregation of APP fragments also occurs in NPC, we studied their expression in cultured cortical neurons treated with U18666A, a compound widely used to induce NPC defects, and also in brain tissues from NPC patients. U18666A treatment resulted in increased intraneuronal levels of C99 and insoluble Abeta42, which were distributed among early and late endosomes, in compartments distinct from where endogenous cholesterol accumulates. Analyses of NPC brains revealed that C99 or other APP C-terminal fragments (APP-CTF), but not Abeta42, accumulated in Purkinje cells, mainly in early endosomes. In contrast, in hippocampal pyramidal neurons, the major accumulated species was Abeta42, in late endosomes. Similar to what has been shown in Alzheimer's disease, cathepsin D, a lysosomal hydrolase, was redistributed to early endosomes in NPC Purkinje cells, where it co-localized with C99/APP-CTF. Our results suggest that endosomal abnormalities related to abnormal lipid trafficking in NPC may contribute to abnormal APP processing and Abeta42/C99/APP-CTF deposition.     

Autophagy in Niemann-Pick C disease is dependent upon Beclin-1 and responsive to lipid trafficking defects

Niemann-Pick C (NPC) disease is an autosomal recessive lipid storage disorder characterized by a disruption of sphingolipid and cholesterol trafficking that produces cognitive impairment, ataxia and death, often in childhood. Most cases are caused by loss of function mutations in the Npc1 gene, which encodes a protein that localizes to late endosomes and functions in lipid sorting and vesicle trafficking. Here, we demonstrate that NPC1-deficient primary human fibroblasts, like npc1(-/-) mice fibroblasts, showed increased autophagy as evidenced by elevated LC3-II levels, numerous autophagic vacuoles and enhanced degradation of long-lived proteins. Autophagy because of NPC1 deficiency was associated with increased expression of Beclin-1 rather than activation of the Akt-mTOR-p70 S6K signaling pathway, and siRNA knockdown of Beclin-1 decreased long-lived protein degradation. Induction of cholesterol trafficking defects in wild-type fibroblasts by treatment with U18666A increased Beclin-1 and LC3-II expression, whereas treatment of NPC1-deficient fibroblasts with sphingolipid-lowering compound NB-DGJ failed to alter the expression of either Beclin-1 or LC3-II. Primary fibroblasts from patients with two other sphingolipid storage diseases, NPC2 deficiency and Sandhoff disease, characterized by sphingolipid trafficking defects also showed elevation in Beclin-1 and LC3-II levels. In contrast, Gaucher disease fibroblasts, which traffic sphingolipids normally, showed wild-type levels of Beclin-1 and LC3-II. Our data define a critical role for Beclin-1 in the activation of autophagy because of NPC1 deficiency, and reveal an unexpected role for lipid trafficking in the regulation of this pathway in patients with several sphingolipid storage diseases.     

Participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease)

In cathepsin D-deficient (CD-/-) and cathepsins B and L double-deficient (CB-/-CL-/-) mice, abnormal vacuolar structures accumulate in neurons of the brains. Many of these structures resemble autophagosomes in which part of the cytoplasm is retained but their precise nature and biogenesis remain unknown. We show here how autophagy contributes to the accumulation of these vacuolar structures in neurons deficient in cathepsin D or both cathepsins B and L by demonstrating an increased conversion of the molecular form of MAP1-LC3 for autophagosome formation from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). In both CD-/- and CB-/-CL-/- mouse brains, the membrane-bound LC3-II form predominated whereas MAP1-LC3 signals accumulated in granular structures located in neuronal perikarya and axons of these mutant brains and were localized to the membranes of autophagosomes, evidenced by immunofluorescence microscopy and freeze-fracture-replica immunoelectron microscopy. Moreover, as in CD-/- neurons, autofluorescence and subunit c of mitochondrial ATP synthase accumulated in CB-/-CL-/- neurons. This suggests that not only CD-/- but also CB-/-CL-/- mice could be useful animal models for neuronal ceroid-lipofuscinosis/Batten disease. These data strongly argue for a major involvement of autophagy in the pathogenesis of Batten disease/lysosomal storage disorders.     

Npc1 haploinsufficiency promotes weight gain and metabolic features associated with insulin resistance

A recent population-based genome-wide association study has revealed that the Niemann-Pick C1 (NPC1) gene is associated with early-onset and morbid adult obesity. Concurrently, our candidate gene-based mouse growth study performed using the BALB/cJ NPC1 mouse model (Npc1) with decreased Npc1 gene dosage independently supported these results by suggesting an Npc1 gene-diet interaction in relation to early-onset weight gain. To further investigate the Npc1 gene in relation to weight gain and metabolic features associated with insulin resistance, we interbred BALB/cJ Npc1(+/-) mice with wild-type C57BL/6J mice, the latter mouse strain commonly used to study aspects of diet-induced obesity and insulin resistance. This breeding produced a hybrid (BALB/cJ-C57BL/6J) Npc1(+/-) mouse model with increased susceptibility to weight gain and insulin resistance. The results from our study indicated that these Npc1(+/-) mice were susceptible to increased weight gain characterized by increased whole body and abdominal adiposity, adipocyte hypertrophy and hepatic steatosis in the absence of hyperphagia. Moreover, these Npc1(+/-) mice developed abnormal metabolic features characterized by impaired fasting glucose, glucose intolerance, hyperinsulinemia, hyperleptinemia and dyslipidemia marked by an increased concentration of cholesterol and triacylglycerol associated with low-density lipoprotein and high-density lipoprotein. The overall results are consistent with a unique Npc1 gene-diet interaction that promotes both weight gain and metabolic features associated with insulin resistance. Therefore, the NPC1 gene now represents a previously unrecognized gene involved in maintaining energy and metabolic homeostasis that will contribute to our understanding concerning the current global epidemic of obesity and type 2 diabetes mellitus.     

Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease

Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol in the endolysosomes. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that nonenzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1(-/-) mouse model and found several cholesterol oxidation products that were elevated in Npc1(-/-) mice, were detectable before the onset of symptoms, and were associated with disease progression. Nonenzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with the age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.     

C1型尼曼-匹克症小鼠脊髓胶质细胞的活性变化

目的通过观察C1型尼曼-匹克症小鼠不同脊髓节段星形胶质细胞和小胶质细胞的活性变化,探讨Npc1基因突变对脊髓发育的影响。方法 Npc1~(+/-)小鼠交配繁殖产生Npc~(1-/-)(n=3)和Npc1~(+/+)小鼠(n=3),PCR检测新生小鼠的基因型;选取35日龄的Npc1~(-/-)和Npc1~(+/+)小鼠,采用免疫荧光方法观察对比脊髓不同节段(颈、胸、腰、骶)星形胶质细胞和小胶质细胞的活性变化。采用免疫双染色检测胶质细胞中炎性因子的表达情况,采用免疫印迹方法检测白细胞介素-1β(IL-1β)、SMI31和磷酸化的tau蛋白表达情况。结果在35日龄Npc1~(-/-)小鼠脊髓的各个节段,其背角和腹角的星形胶质细胞和小胶质细胞的活性均明显增强(P0.05),并伴随细胞炎性因子IL-1β表达量的显著增加;同时,脊髓神经丝蛋白和骨架蛋白tau蛋白发生超磷酸化。结论 Npc1基因突变引起脊髓神经胶质细胞发生病理性变化,可能是脊髓神经元病理性损伤的重要原因。     

Temporal and cell-specific deletion establishes that neuronal Npc1 deficiency is sufficient to mediate neurodegeneration

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder caused by mutations in the NPC1 or NPC2 genes. Loss of function mutations in either gene disrupt intracellular lipid trafficking and lead to a clinically heterogeneous phenotype that invariably includes neurological dysfunction and early death. The mechanism by which impaired lipid transport leads to neurodegeneration is poorly understood. Here we used mice with a conditional null allele to establish the timing and cell type that underlie neurodegeneration due to Npc1 deficiency. We show that global deletion of Npc1 in adult mice leads to progressive weight loss, impaired motor function and early death in a time course similar to that resulting from germline deletion. These phenotypes are associated with the occurrence of characteristic neuropathology including patterned Purkinje cell loss, axonal spheroids and reactive gliosis, demonstrating that there is not a significant developmental component to NPC neurodegeneration. Furthermore, we show that these same changes occur when Npc1 is specifically deleted only in neurons, establishing that neuronal deficiency is sufficient to mediate central nervous system (CNS) disease. In contrast, astrocyte-specific deletion does not impact behavioral phenotypes, CNS histopathology or synaptic function. We conclude that defects arising in neurons, but not in astrocytes, are the determining factor in the development of NPC neuropathology.     

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.     

Endosomal/lysosomal processing of gangliosides affects neuronal cholesterol sequestration in Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is a severe neurovisceral lysosomal storage disorder caused by defects in NPC1 or NPC2 proteins. Although numerous studies support the primacy of cholesterol storage, neurons of double-mutant mice lacking both NPC1 and an enzyme required for synthesis of all complex gangliosides (β1,4GalNAc transferase) have been reported to exhibit dramatically reduced cholesterol sequestration. Here we show that NPC2-deficient mice lacking this enzyme also exhibit reduced cholesterol, but that genetically restricting synthesis to only a-series gangliosides fully restores neuronal cholesterol storage to typical disease levels. Examining the subcellular locations of sequestered compounds in neurons lacking NPC1 or NPC2 by confocal microscopy revealed that cholesterol and the two principal storage gangliosides (GM2 and GM3) were not consistently co-localized within the same intracellular vesicles. To determine whether the lack of GM2 and GM3 co-localization was due to differences in synthetic versus degradative pathway expression, we generated mice lacking both NPC1 and lysosomal β-galactosidase, and therefore unable to generate GM2 and GM3 in lysosomes. Double mutants lacked both gangliosides, indicating that each is the product of endosomal/lysosomal processing. Unexpectedly, GM1 accumulation in double mutants increased compared to single mutants consistent with a direct role for NPC1 in ganglioside salvage. These studies provide further evidence that NPC1 and NPC2 proteins participate in endosomal/lysosomal processing of both sphingolipids and cholesterol.     

Oxidative stress in Niemann–Pick disease,type C

Niemann–Pick disease, type C (NPC) is a neurodegenerative lysosomal storage disorder due to impaired intracellular cholesterol and lipid transport. Increased oxidative stress has been reported in human NPC1 mutant fibroblasts and in tissues from Npc1 mutant mice. However, oxidative stress in NPC patients has not been established. In this study, we demonstrated increased oxidative stress in NPC patients. Evaluation of serum from 37 NPC patients, compared to control values, showed significant decreases (p < .01) in both the fraction of reduced coenzyme Q10 (CoQ10) and trolox equivalent antioxidant capacity (TEAC). Both findings are consistent with increased oxidative stress in NPC. Supplementation with CoQ10 was not effective in correcting the decreased fraction of reduced CoQ10. Increased oxidative stress may be a contributing factor to the pathology of NPC, and demonstration of increased oxidative stress in NPC patients provides both a rationale and the biomarkers necessary to test the efficacy of antioxidant therapy in NPC.     

Autophagy regulates the processing of amino terminal huntingtin fragments

The N-terminus of mutant huntingtin (htt) has a polyglutamine expansion and forms neuronal aggregates in the brain of Huntington's disease (HD) patients. Htt expression in vitro activates autophagy, but it is unclear whether autophagic/lysosomal pathways process htt, especially N-terminal htt fragments. We explored the role of autophagy in htt processing in three cell lines, clonal striatal cells, PC12 cells and rodent embryonic cells lacking cathepsin D. Blocking autophagy raised levels of exogenously expressed htt1-287 or 1-969, reduced cell viability and increased the number of cells bearing mutant htt aggregates. Stimulating autophagy promoted htt degradation, including breakdown of caspase cleaved N-terminal htt fragments. Htt expression increased levels of the lysosomal enzyme cathepsin D by an autophagy-dependent pathway. Cells without cathepsin D accumulated more N-terminal htt fragments and cells with cathepsin D were more efficient in degrading wt htt than mutant htt in vitro. These results suggest that autophagy plays a critical role in the degradation of N-terminal htt. Altered processing of mutant htt by autophagy and cathepsin D may contribute to HD pathogenesis.     

Loss of CB1 receptors leads to decreased cathepsin D levels and accelerated lipofuscin accumulation in the hippocampus

Early onset of age-related changes in the brain of cannabinoid 1 receptor knockout (Cnr1^−/−) mice suggests that cannabinoid 1 (CB1) receptor activity significantly influences the progression of brain aging. In the present study we show that lack of CB1 receptors leads to a significant increase in lipofuscin accumulation and a reduced expression and activity of cathepsin D, lysosomal protease implicated in the degradation of damaged macromolecules, in the hippocampus of 12-month-old mice. The impaired clearance of damaged macromolecules due to the low cathepsin D levels and not enhanced oxidative stress may be responsible for the lipofuscin accumulation because macromolecule oxidation levels were comparable between the genotypes within the same age group. The altered levels of autophagy markers p62 and LC3-II suggest that autophagy is upregulated in CB1 knockout mice. Increased autophagic flux in the absence of CB1 receptors is probably a compensatory mechanism to partially counteract decreased lysosomal degradation capacity. Together, these results suggest that CB1 receptor activity affects lysosomal activity, degradation of damaged macromolecules and thus it may influence the course and onset of brain aging.     

Pulmonary function and pathology in hydroxypropyl-beta-cyclodextin-treated and untreated Npc1⁻/⁻ mice

Lung dysfunction is an important part of the pathology of the neurodegenerative disorder, Niemann-Pick C1 (NPC1). We have studied the pulmonary disease in the Npc1(NIH/NIH) mouse model. On histology, we find large numbers of alveolar foamy macrophages but no alveolar proteinosis. Lung weight as percent of body weight was markedly increased; using the flexiVent small animal ventilator (SCIREQ, Inc.), we find inspiratory capacity, elastance and hysterisivity to be increased while resistance was not changed. Cholesterol measurements show a doubling of lung cholesterol levels. Collagen is also increased. Treatment of Npc1(-/-) mice with hydroxypropyl-β-cyclodextrin (HPBCD), despite efficacious effects in brain and liver, results in little difference from age-matched controls (using a CNS-expressed transgene to extend the life expectancy of the Npc1(-/-) mice) for these variables.     

CLC-3 deficiency leads to phenotypes similar to human neuronal ceroid lipofuscinosis

BACKGROUND: CLC-3 is a member of the CLC chloride channel family and is widely expressed in mammalian tissues. To determine the physiological role of CLC-3, we generated CLC-3-deficient mice (Clcn3-/- ) by targeted gene disruption. RESULTS: Together with developmental retardation and higher mortality, the Clcn3-/- mice showed neurological manifestations such as blindness, motor coordination deficit, and spontaneous hyperlocomotion. In histological analysis, the Clcn3-/- mice showed a pattern of progressive degeneration of the retina, hippocampus and ileal mucosa, which resembled the phenotype observed in cathepsin D knockout mice. The defect of cathepsin D results in a lysosomal accumulation of ceroid lipofuscin containing the mitochondrial F1F0 ATPase subunit c. In immunohistochemistry and Western blot analysis, we found that the subunit c was heavily accumulated in the lysosome of Clcn3-/- mice. Furthermore, we detected an elevation in the endosomal pH of the Clcn3-/- mice. CONCLUSIONS: These results indicated that the neurodegeneration observed in the Clcn3-/- mice was caused by an abnormality in the machinery which degrades the cellular protein and was associated with the phenotype of neuronal ceroid lipofuscinosis (NCL). The elevated endosomal pH could be an important factor in the pathogenesis of NCL.     

Altered levels and distribution of IGF-II/M6P receptor and lysosomal enzymes in mutant APP and APP + PS1 transgenic mouse brains

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor participates in the trafficking of lysosomal enzymes from the trans-Golgi network or the cell surface to lysosomes. In Alzheimer's disease (AD) brains, marked up-regulation of the lysosomal system in vulnerable neuronal populations has been correlated with altered metabolic functions. To establish whether IGF-II/M6P receptors and lysosomal enzymes are altered in the brain of transgenic mice harboring different familial AD mutations, we measured the levels and distribution of the receptor and lysosomal enzymes cathepsins B and D in select brain regions of transgenic mice overexpressing either mutant presenilin 1 (PS1; PS1(M146L+L286V)), amyloid precursor protein (APP; APP(KM670/671NL+V717F)) or APP+PS1 (APP(KM670/671NL+V717F)+PS1(M146L+L286V)) transgenes. Our results revealed that levels and expression of the IGF-II/M6P receptor and lysosomal enzymes are increased in the hippocampus and frontal cortex of APP and APP+PS1, but not in PS1, transgenic mouse brains compared with wild-type controls. The changes were more prominent in APP+PS1 than in APP single transgenic mice. Additionally, all beta-amyloid-containing neuritic plaques in the hippocampal and cortical regions of APP and APP+PS1 transgenic mice were immunopositive for both lysosomal enzymes, whereas only a subset of the plaques displayed IGF-II/M6P receptor immunoreactivity. These results suggest that up-regulation of the IGF-II/M6P receptor and lysosomal enzymes in neurons located in vulnerable regions reflects an altered functioning of the endosomal-lysosomal system which may be associated with the increased intracellular and/or extracellular A beta deposits observed in APP and APP+PS1 transgenic mouse brains.     

Spatial and temporal distribution of intracellular free cholesterol in brains of a Niemann-Pick type C mouse model showing hyperphosphorylated tau protein. Implications for Alzheimer's disease

Niemann-Pick type C (NPC) disease is a fatal hereditary neurovisceral disorder with diagnostically relevant intracellular accumulation of cholesterol in non-brain tissue, for example the spleen and fibroblasts. In the brain, many ballooned neurons are seen. Using filipin microfluorodensitometry, significant accumulations of free cholesterol in specified neurons have been described in NPC patients. The present study demonstrates spatial and temporal accumulation of free cholesterol in the brains of homozygous NPC (-(npc)/-(npc)) mice, a widely acknowledged mouse model, and in primarily cultured neurons therefrom. Intraneuronal storage of free cholesterol was already prominent at a pre-clinical stage in various grey matter areas of the murine cerebral cortex. Hippocampal areas showed differential development of the pathological distribution of free cholesterol. The pyramidal cells in the CA3 sector of Ammon's horn were affected much earlier than in CA1. Some of the deeper cerebral nuclei were affected only slightly, even at the final stage. Neurons (E15-E17) cultured in a cholesterol-free medium also showed massive accumulation of intracellular free cholesterol. In addition, brains from the murine NPC model for Alzheimer's disease (AD)-like changes in the microtubule-associated protein tau were tested using the Gallyas silver technique and AT8-immunolabelling, since both human diseases are accompanied by intraneuronal tangles made up of tau protein aggregations. Although the analysis failed to show classical silver-stainable tangles of the AD type in the NPC mice, tau protein phosphorylated at epitopes considered to represent early stages of AD was found. This further strengthens the concept that an alteration in cholesterol metabolism may play an important role in AD. The NPC mouse model may thus serve as a tool to analyse the role of cholesterol in initial changes of tau that eventually lead to the formation of tangles in both NPC and AD.