ABSTRACT
Niemann-Pick type C (NPC) disease is an autosomal recessive disorder caused by mutations of NPC1 and NPC2 genes. Progressive neurodegeneration that accompanies NPC is fatal, but the underlying mechanisms are still poorly understood. In the present study, we characterized the association of autophagic-lysosomal dysfunction with cholesterol accumulation in Npc1(-/-) mice during postnatal development. Brain levels of lysosomal cathepsin D were significantly higher in mutant than in wild-type mice. Increases in cathepsin D occurred first in neurons and later in astrocytes and microglia and were both spatially and temporally associated with intracellular cholesterol accumulation and neurodegeneration. Furthermore, levels of ubiquitinated proteins were higher in endosomal/lysosomal fractions of brains from Npc1(-/-) mice than from wild-type mice. Immunoblotting results showed that levels of LC3-II were significantly higher in brains of mutant than wild-type mice. Combined LC3 immunofluorescence and filipin staining showed that LC3 accumulated within filipin-labeled cholesterol clusters inside Purkinje cells. Electron microscopic examination revealed the existence of autophagic vacuole-like structures and multivesicles in brains from Npc1(-/-) mice. These results provide strong evidence that cholesterol accumulation-induced changes in autophagy-lysosome function are closely associated with neurodegeneration in NPC.
Subject(s)
Autophagy , Brain/metabolism , Cholesterol/metabolism , Lysosomes/metabolism , Niemann-Pick Diseases/metabolism , Proteins/genetics , Animals , Brain/cytology , Brain/growth & development , Brain/pathology , Cathepsin B/metabolism , Cathepsin D/metabolism , Child , Filipin/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Lysosomes/ultrastructure , Mice , Mice, Inbred BALB C , Mice, Knockout , Microtubule-Associated Proteins/metabolism , Niemann-Pick C1 Protein , Niemann-Pick Diseases/physiopathology , Proteins/metabolism , Purkinje Cells/metabolism , Purkinje Cells/ultrastructureABSTRACT
Niemann-Pick C disease (NPC) is an irreversible neurodegenerative disorder without current treatment. It is thought to result from deficient intracellular cholesterol and/or ganglioside trafficking. We have investigated the effects of allopregnanolone treatments on survival, weight loss, motor function, magnetic resonance imaging (MRI), and neuropathology in the mouse model of NPC (Npc1(-/-) mice). We confirmed previous results showing that a single injection of 250 microg of allopregnanolone on postnatal day 7 significantly extended the life span of Npc1(-/-) mice. This caused a marked difference in the weight curves of the treated mice but no statistical difference in the Rota-Rod performance. T2-weighted MRI and diffusion tensor imaging (DTI) of treated mice showed values of signal intensity and fractional anisotropy closer to those of wild-type mice than those of untreated Npc1(-/-) mice. Neuropathology showed that day-7 treatment markedly suppressed astrocyte reaction and significantly reduced microglial activation. Furthermore, the steroid treatment also increased myelination in brains of Npc1(-/-) mice. Similar effects of allopregnanolone treatment were observed in Npc1(-/-), mdr1a(-/-) double-mutant mice, which have a deficient blood-brain barrier, resulting in increased steroid uptake. The effects on survival and weight loss of a single injection on day 7 followed by injections every 2 weeks were also evaluated in Npc1(-/-) mice, and the beneficial effects were found to be greater than with the single injection at day 7. We conclude that allopregnanolone treatment significantly ameliorates several symptoms of NPC in Npc1(-/-) mice, presumably by effects on myelination or neuronal connectivity.
Subject(s)
Anesthetics/administration & dosage , Demyelinating Diseases/drug therapy , Niemann-Pick Diseases/drug therapy , Pregnanolone/administration & dosage , 2',3'-Cyclic-Nucleotide Phosphodiesterases/metabolism , Age Factors , Animals , Anisotropy , Antigens, Differentiation/metabolism , Body Weight/drug effects , Brain/drug effects , Brain/pathology , Cell Count/methods , Cell Survival/drug effects , Demyelinating Diseases/etiology , Disease Models, Animal , Drug Administration Schedule , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry/methods , Magnetic Resonance Imaging/methods , Membrane Transport Proteins/deficiency , Mice , Mice, Inbred BALB C , Mice, Knockout , Motor Activity/drug effects , Niemann-Pick Diseases/complications , Niemann-Pick Diseases/genetics , Niemann-Pick Diseases/pathology , Time FactorsABSTRACT
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.
Subject(s)
Brain/pathology , Nerve Degeneration/pathology , Niemann-Pick Diseases/genetics , Niemann-Pick Diseases/pathology , Phosphatidylinositol 3-Kinases/metabolism , Proteins/genetics , Animals , Biomarkers/metabolism , Blotting, Western , Brain/metabolism , Brain Chemistry , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Crosses, Genetic , Cytoplasm/chemistry , Cytoplasm/metabolism , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Heterozygote , Homozygote , Immunohistochemistry , Intracellular Signaling Peptides and Proteins , Mice , Mice, Inbred BALB C , Mice, Knockout , Models, Biological , NF-kappa B/analysis , NF-kappa B/chemistry , NF-kappa B/metabolism , Nerve Degeneration/genetics , Nerve Degeneration/metabolism , Niemann-Pick C1 Protein , Phosphorylation , Protein Subunits/metabolismABSTRACT
Inflammatory responses involving microglia, the resident macrophages of the brain, are thought to contribute importantly to the progression of Alzheimer's disease (AD) and possibly other neurodegenerative disorders. The present study tested whether the mevalonate-isoprenoid biosynthesis pathway, which affects inflammation in many types of tissues, tonically regulates microglial activation. This question takes on added significance given the potential use of statins, drugs that block the rate-limiting step (3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase)) in mevalonate and cholesterol synthesis, in AD treatment. Both mevastatin and simvastatin caused a concentration- and time-dependent activation of microglia in cultured rat hippocampal slices. This response consisted of a transformation of the cells from a typical resting configuration to an amoeboid, macrophage-like morphology, increased expression of a macrophage antigen, and up-regulation of the cytokine tumor necrosis factor-alpha. Evidence for proliferation was also obtained. Statin-induced microglial changes were blocked by mevalonate but not by cholesterol, indicating that they were probably due to suppression of isoprenoid synthesis. In accord with this, the statin effects were absent in slices co-incubated with geranylgeranyl pyrophosphate, a mevalonate product that provides for the prenylation of Rho GTPases. Finally, PD98089, a compound that blocks activation of extracellularly regulated kinases1/2, suppressed statin-induced up-regulation of tumor necrosis factor-alpha but had little effect on microglial transformation. These results suggest that 1) the mevalonate-isoprenoid pathway is involved in regulating microglial morphology and in controlling expression of certain cytokines and 2) statins have the potential for enhancing a component of AD with uncertain relationships to other features of the disease.
