ABSTRACT
Increasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative diseases, including the rare inherited lysosomal storage disorders (LSDs) and the most common neurodegenerative diseases, such as Alzheimer's and Parkinson's disease (AD and PD). Although the triggers of the lysosomal impairment may involve the accumulated macromolecules or dysfunction of the lysosomal enzymes, the role of the lysosomal glycocalyx in the lysosomal (dys)function has not been studied. The goal of this work was to analyze whether there are changes in the lysosomal glycocalyx in a cellular model of a LSD Niemann-Pick type C disease (NPC). Using the ferrofluid nanoparticles we isolated lysosomal organelles from NPC1-null and CHOwt cells. The magnetically isolated lysosomal fractions were enriched with the lysosomal marker protein LAMP1 and showed the key features of NPC disease: 3-fold higher cholesterol content and 4-5 fold enlarged size of the particles compared with the lysosomal fractions of wt cells. These lysosomal fractions were further processed to isolate lysosomal membrane proteins using Triton X-114 and their N-glycome was analyzed by HILIC-UPLC. N-glycans presented in each chromatographic peak were elucidated using MALDI-TOF/TOF-MS. We detected changes in the N-glycosylation pattern of the lysosomal glycocalyx of NPC1-null versus wt cells which involved high-mannose and sialylated N-glycans. To the best of our knowledge this study is the first to report N-glycome profiling of the lysosomal glycocalyx in NPC disease cellular model and the first to report the specific changes in the lysosomal glycocalyx in NPC1-null cells. We speculate that changes in the lysosomal glycocalyx may contribute to lysosomal (dys)function. Further glycome profiling of the lysosomal glycocalyx in other LSDs as well as the most common neurodegenerative diseases, such as AD and PD, is necessary to better understand the role of the lysosomal glycocalyx and to reveal its potential contribution in lysosomal dysfunction leading to neurodegeneration.
Subject(s)
Glycocalyx/metabolism , Lysosomes/metabolism , Niemann-Pick Disease, Type C/metabolism , Polysaccharides/metabolism , Animals , CHO Cells , Cricetulus , Glycosylation , Niemann-Pick C1 Protein/geneticsABSTRACT
Proper function of lysosomes is particularly important in neurons, as they cannot dilute accumulated toxic molecules and aggregates by cell division. Thus, impairment of lysosomal function plays an important role in neuronal degeneration and in the pathogenesis of numerous neurodegenerative diseases. In this work we analyzed how inhibition and/or loss of the major lysosomal proteases, the cysteine cathepsins B and L (CtsB/L), affects lysosomal function, cholesterol metabolism and degradation of the key Alzheimer's disease (AD) proteins. Here, we show that cysteine CtsB/L, and not the aspartyl cathepsin D (CtsD), represent a major lysosomal protease(s) that control lysosomal function, intracellular cholesterol trafficking and AD-like amyloidogenic features. Intriguingly, accumulation of free cholesterol in late endosomes/lysosomes upon CtsB/L inhibition resembled a phenotype characteristic for the rare neurodegenerative disorder Niemann-Pick type C (NPC). CtsB/L inhibition and not the inhibition of CtsD led to lysosomal impairment assessed by decreased degradation of EGF receptor, enhanced LysoTracker staining and accumulation of several lysosomal proteins LC3II, NPC1 and NPC2. By measuring the levels of NPC1 and ABCA1, the two major cholesterol efflux proteins, we showed that CtsB/L inhibition or genetic depletion caused accumulation of the NPC1 in lysosomes and downregulation of ABCA1 protein levels and its expression. Furthermore, we revealed that CtsB/L are involved in degradation of the key Alzheimer's proteins: amyloid-ß peptides (Aß) and C-terminal fragments of the amyloid precursor protein (APP) and in degradation of ß-secretase (BACE1). Our results imply CtsB/L as major regulators of lysosomal function and demonstrate that CtsB/L may play an important role in intracellular cholesterol trafficking and in degradation of the key AD proteins. Our findings implicate that enhancing the activity or levels of CtsB/L could provide a promising and a common strategy for maintaining lysosomal function and for preventing and/or treating neurodegenerative diseases.
Subject(s)
Alzheimer Disease/metabolism , Amyloidogenic Proteins/metabolism , Cathepsin B/metabolism , Cathepsin L/metabolism , Cholesterol/metabolism , Lysosomes/metabolism , Niemann-Pick Disease, Type C/metabolism , Animals , CHO Cells , Cathepsin B/antagonists & inhibitors , Cathepsin L/antagonists & inhibitors , Cell Line, Tumor , Cricetulus , Homeostasis , ProteolysisABSTRACT
Alzheimer's disease (AD) and a rare inherited disorder of cholesterol transport, Niemann-Pick type C (NPC) share several similarities including aberrant APP processing and increased Aß production. Previously, we have shown that the AD-like phenotype in NPC model cells involves cholesterol-dependent enhanced APP cleavage by ß-secretase and accumulation of both APP and BACE1 within endocytic compartments. Since retrograde transport of BACE1 from endocytic compartments to the trans-Golgi network (TGN) is regulated by the Golgi-localized γ-ear containing ADP ribosylation factor-binding protein 1 (GGA1), we analyzed in this work a potential role of GGA1 in the AD-like phenotype of NPC1-null cells. Overexpression of GGA1 caused a shift in APP processing towards the non-amyloidogenic pathway by increasing the localization of APP at the cell surface. However, the observed effect appear to be independent on the subcellular localization and phosphorylation state of BACE1. These findings show that the AD-like phenotype of NPC model cells can be partly reverted by promoting a non-amyloidogenic processing of APP through the upregulation of GGA1 supporting its preventive role against AD.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Carrier Proteins/metabolism , Membrane Glycoproteins/metabolism , Niemann-Pick Disease, Type C/metabolism , Niemann-Pick Disease, Type C/pathology , Peptide Fragments/metabolism , Animals , CHO Cells , Carrier Proteins/genetics , Cricetulus , Humans , Intracellular Signaling Peptides and Proteins , Membrane Glycoproteins/genetics , Niemann-Pick C1 Protein , Subcellular Fractions/metabolism , Up-RegulationABSTRACT
Niemann-Pick type C disease (NPC) is an inherited disorder mainly caused by loss-of-function mutations in the NPC1 gene, that lead to intracellular cholesterol accumulation and disturbed cholesterol homeostasis. Similarly to Alzheimer's disease (AD), NPC is associated with progressive neurodegeneration and altered metabolism of amyloid precursor protein (APP). Liver X receptors (LXRs), the key transcriptional regulators of cholesterol homeostasis, were reported to play neuroprotective roles in NPC mice. We investigated the impacts of LXRs on APP metabolism in mutant CHO cells lacking the NPC1 gene (-NPC1 cells). Pharmacological activation of LXRs in -NPC1 cells tended to reduce the ratio of total secreted APP (sAPP) to full length APP (flAPP) levels and sAPPß levels as well as to increase the ratio of APP Cterminal fragments to flAPP levels, resulting in decreased levels of amyloid ß (Aß) peptides. -NPC1 cells treated with LXR agonist TO901317 (TO90) displayed a modest increase in cholesterol efflux to apolipoprotein A-I (apoA-I) but not to HDL3, or in the absence of extracellular cholesterol acceptors. The observed similar reduction of Aß levels upon TO90 treatment in the presence or in the absence of extracellular apoA-I indicated a cholesterol-efflux independent effect of TO90 on Aß levels. Furthermore, TO90 had no effect on the cholesterol synthesis rate in -NPC1 cells, while it reduced the rate of cholesterol esterification. The obtained results indicate that LXR activation may decrease Aß levels in NPC1- deficient conditions. The underlying mechanism of this action does not appear to be related to effects on cholesterol efflux or synthesis rates.
Subject(s)
Amyloid beta-Peptides/metabolism , Hydrocarbons, Fluorinated/pharmacology , Niemann-Pick Disease, Type C/metabolism , Orphan Nuclear Receptors/agonists , Peptide Fragments/metabolism , Sulfonamides/pharmacology , Animals , CHO Cells , Cholesterol/metabolism , Cricetulus , Liver X ReceptorsABSTRACT
Brain is one of the richest organs in lipid content. Phospholipids (glycerophospholipids and sphingolipids) are important building blocks of cell membranes, which provide an optimal environment for protein interactions, trafficking and function. Because of that, alterations in their cellular levels could lead to different pathogenic processes in the brain, such as in Alzheimer's disease (AD), the most common type of dementia among older populations. There is increasing evidence that phospholipid changes occur during pathogenic processes in AD. It is known that lipids are tightly connected with metabolism of the Amyloid Precursor Protein (APP), which produces Amyloid-beta peptide (Aß), the main component of senile plaques, which represent the main pathological hallmark of AD. However, the mechanism(s) of the lipid-effect on Aß metabolism and AD pathogenesis is still not completely understood. This review summarizes the current knowledge on phospholipid changes occurring during normal aging and discusses phospholipid changes in the human brain associated with different stages of AD, as well changes in the cerebrospinal fluid and blood/plasma, which are interesting potential biomarkers for AD diagnosis and disease monitoring. At the end, we have discussed future perspectives of phospholipid changes as potential biomarkers and as targets for development of novel treatment strategies against AD.
Subject(s)
Alzheimer Disease/metabolism , Biomarkers/metabolism , Brain/metabolism , Phospholipids/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/diagnosis , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Brain/pathology , Humans , Plaque, Amyloid/diagnosis , Plaque, Amyloid/metabolismABSTRACT
Low levels of amyloid-beta42 (Abeta42) and high total-tau (t-tau) or phosphorylated-tau (p181-tau) levels in cerebrospinal fluid (CSF) were shown to be characteristic for Alzheimer's disease (AD) patients and for mildly cognitively impaired (MCI) or non-demented individuals who will progress to AD. The goal of this study was to evaluate the benefit of CSF biomarker testing in a setting with no specialized dementia centers, in order to improve the accuracy of AD diagnosis and to identify individuals with incipient AD. Using ELISA assay we analyzed CSF Abeta42, t-tau and p181-tau levels among clinically diagnosed non-demented individuals, AD patients and individuals with uncertain dementia (n=36). CSF cut-off values of low Abeta42 (less than or equal to 530 pg/mL) and high t-tau (less than or equal to 350 pg/mL) or p181-tau (less than or equal to 52 pg/mL) were used to identify individuals with AD/MCI-CSF profile, regardless of clinical diagnosis. APOE genotyping was performed using PCR-RFLP method. In accord with previous studies we detected significantly decreased levels of CSF Abeta42 and increased tau and p181-tau levels in clinically diagnosed AD group vs. non-demented controls. CSF profiling identified individuals with a typical AD/MCI-CSF pattern in clinically referred non-demented group (9 percent) and among patients with uncertain dementia (41.7 percent). APOE epsilon4-allele was associated with the CSF biomarker changes typical for AD. This study shows that in a non-specialized setting CSF biomarker testing may be used as a screening tool for improving the accuracy of AD diagnosis and for predicting individuals with incipient Alzheimer's disease who need to receive further clinical follow-up.
Subject(s)
Alzheimer Disease/cerebrospinal fluid , Alzheimer Disease/diagnosis , Biomarkers/cerebrospinal fluid , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Amyloid beta-Peptides/cerebrospinal fluid , Apolipoproteins E/genetics , Cognitive Dysfunction/cerebrospinal fluid , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , Psychiatric Status Rating Scales , Statistics, Nonparametric , tau Proteins/cerebrospinal fluidABSTRACT
Niemann-Pick type C (NPC) is a progressive neurodegenerative lysosomal disease with altered cellular lipid trafficking. The metabolism of amyloid-ß (Aß) - previously mainly studied in Alzheimer's disease - has been suggested to be altered in NPC. Here we aimed to perform a detailed characterization of metabolic products from the amyloid precursor protein (APP) in NPC models and patients. We used multiple analytical technologies, including immunoassays and immunoprecipitation followed by mass spectrometry (IP-MS) to characterize Aß peptides and soluble APP fragments (sAPP-α/ß) in cell media from pharmacologically (U18666A) and genetically (NPC1 ( -/- ) ) induced NPC cell models, and cerebrospinal fluid (CSF) from NPC cats and human patients. The pattern of Aß peptides and sAPP-α/ß fragments in cell media was differently affected by NPC-phenotype induced by U18666A treatment and by NPC1 ( -/- ) genotype. U18666A treatment increased the secreted media levels of sAPP-α, AßX-40 and AßX-42 and reduced the levels of sAPP-ß, Aß1-40 and Aß1-42, while IP-MS showed increased relative levels of Aß5-38 and Aß5-40 in response to treatment. NPC1 ( -/- ) cells had reduced media levels of sAPP-α and Aß1-16, and increased levels of sAPP-ß. NPC cats had altered CSF distribution of Aß peptides compared with normal cats. Cats treated with the potential disease-modifying compound 2-hydroxypropyl-ß-cyclodextrin had increased relative levels of short Aß peptides including Aß1-16 compared with untreated cats. NPC patients receiving ß-cyclodextrin had reduced levels over time of CSF Aß1-42, AßX-38, AßX-40, AßX-42 and sAPP-ß, as well as reduced levels of the axonal damage markers tau and phosphorylated tau. We conclude that NPC models have altered Aß metabolism, but with differences across experimental systems, suggesting that NPC1-loss of function, such as in NPC1 ( -/- ) cells, or NPC1-dysfunction, seen in NPC patients and cats as well as in U18666A-treated cells, may cause subtle but different effects on APP degradation pathways. The preliminary findings from NPC cats suggest that treatment with cyclodextrin may have an impact on APP processing pathways. CSF Aß, sAPP and tau biomarkers were dynamically altered over time in human NPC patients.
Subject(s)
Amyloid beta-Peptides/metabolism , Niemann-Pick Disease, Type C/metabolism , 2-Hydroxypropyl-beta-cyclodextrin , Amyloid beta-Peptides/analysis , Amyloid beta-Peptides/cerebrospinal fluid , Amyloid beta-Protein Precursor/metabolism , Animals , Carrier Proteins/genetics , Cats , Cell Line, Tumor , Child , Child, Preschool , Chromatography, High Pressure Liquid , Female , Humans , Immunoprecipitation , Infant , Intracellular Signaling Peptides and Proteins , Male , Membrane Glycoproteins/genetics , Mutation, Missense/genetics , Niemann-Pick C1 Protein , Niemann-Pick Disease, Type C/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass Spectrometry , beta-Cyclodextrins/therapeutic use , tau Proteins/metabolismABSTRACT
Cholesterol accumulation in Niemann-Pick type C disease (NPC) causes increased levels of the amyloid-precursor-protein C-terminal fragments (APP-CTFs) and intracellular amyloid-ß peptide (Aß), the two central molecules in Alzheimer's disease (AD) pathogenesis. We previously reported that cholesterol accumulation in NPC-cells leads to cholesterol-dependent increased APP processing by ß-secretase (BACE1) and decreased APP expression at the cell surface (Malnar et al. Biochim Biophys Acta. 1802 (2010) 682-691.). We hypothesized that increased formation of APP-CTFs and Aß in NPC disease is due to cholesterol-mediated altered endocytic trafficking of APP and/or BACE1. Here, we show that APP endocytosis is prerequisite for enhanced Aß levels in NPC-cells. Moreover, we observed that NPC cells show cholesterol dependent sequestration and colocalization of APP and BACE1 within enlarged early/recycling endosomes which can lead to increased ß-secretase processing of APP. We demonstrated that increased endocytic localization of APP in NPC-cells is likely due to both its increased internalization and its decreased recycling to the cell surface. Our findings suggest that increased cholesterol levels, such as in NPC disease and sporadic AD, may be the upstream effector that drives amyloidogenic APP processing characteristic for Alzheimer's disease by altering endocytic trafficking of APP and BACE1.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/metabolism , Cholesterol/deficiency , Cholesterol/metabolism , Neurons/metabolism , Niemann-Pick Disease, Type C/metabolism , Androstenes/pharmacology , Animals , CHO Cells , Cricetinae , Endocytosis , Endosomes/metabolism , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/metabolism , Immunohistochemistry , Microscopy, Confocal , Neurons/drug effects , Niemann-Pick Disease, Type C/pathology , Rats , TransfectionABSTRACT
Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between ß-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer's disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1-/-), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1-/- cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1-/- cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1-/- cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1-/-. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/metabolism , Aspartic Acid Endopeptidases/metabolism , Cell Membrane/metabolism , Cholesterol/metabolism , Niemann-Pick Disease, Type C/metabolism , Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Aspartic Acid Endopeptidases/genetics , CHO Cells , Carrier Proteins , Cell Membrane/genetics , Cholesterol/genetics , Cricetinae , Cricetulus , Fluorescence Resonance Energy Transfer/methods , Gene Deletion , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Membrane Glycoproteins , Niemann-Pick C1 Protein , Niemann-Pick Disease, Type C/genetics , Niemann-Pick Disease, Type C/pathologyABSTRACT
The link between cholesterol and Alzheimer's disease has recently been revealed in Niemann-Pick type C disease. We found that NPC1(-/-) cells show decreased expression of APP at the cell surface and increased processing of APP through the beta-secretase pathway resulting in increased C99, sAPPbeta and intracellular Abeta40 levels. This effect is dependent on increased cholesterol levels, since cholesterol depletion reversed cell surface APP expression and lowered Abeta/C99 levels in NPC1(-)(/)(-) cells to the levels observed in wt cells. Finding that overexpression of C99, a direct gamma-secretase substrate, does not lead to increased intracellular Abeta levels in NPC1(-)(/)(-) cells vs. CHOwt suggests that the effect on intracellular Abeta upon cholesterol accumulation in NPC1(-)(/)(-) cells is not due to increased APP cleavage by gamma-secretase. Our results indicate that cholesterol may modulate APP processing indirectly by modulating APP expression at the cell surface and thus its cleavage by beta-secretase.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/metabolism , Carrier Proteins/genetics , Cholesterol/pharmacology , Membrane Glycoproteins/genetics , Niemann-Pick Disease, Type C/pathology , Animals , Antigens, Surface/metabolism , CHO Cells , Carrier Proteins/physiology , Cell Membrane/drug effects , Cell Membrane/metabolism , Cells, Cultured , Cholesterol/metabolism , Cricetinae , Cricetulus , Intracellular Signaling Peptides and Proteins , Membrane Glycoproteins/physiology , Niemann-Pick C1 Protein , Niemann-Pick Disease, Type C/genetics , Niemann-Pick Disease, Type C/metabolism , Protein Processing, Post-Translational/drug effects , Signal Transduction/drug effects , Signal Transduction/physiology , TransfectionABSTRACT
It has been suggested that cholesterol may modulate amyloid-beta (Abeta) formation, a causative factor of Alzheimer's disease (AD), by regulating distribution of the three key proteins in the pathogenesis of AD (beta-amyloid precursor protein (APP), beta-secretase (BACE1) and/or presenilin 1 (PS1)) within lipid rafts. In this work we tested whether cholesterol accumulation upon NPC1 dysfunction, which causes Niemann Pick type C disease (NPC), causes increased partitioning of APP into lipid rafts leading to increased CTF/Abeta formation in these cholesterol-rich membrane microdomains. To test this we used CHO NPC1(-/-) cells (NPC cells) and parental CHOwt cells. By sucrose density gradient centrifugation we observed a shift in fl-APP/CTF compartmentalization into lipid raft fractions upon cholesterol accumulation in NPC vs. wt cells. Furthermore, gamma-secretase inhibitor treatment significantly increased fl-APP/CTF distribution in raft fractions in NPC vs. wt cells, suggesting that upon cholesterol accumulation in NPC1-null cells increased formation of APP-CTF and its increased processing towards Abeta occurs in lipid rafts. Our results support that cholesterol overload, such as in NPC disease, leads to increased partitioning of APP/CTF into lipid rafts resulting in increased amyloidogenic processing of APP in these cholesterol-rich membranes. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer's disease and supports the role of lipid rafts in these processes.