Characterizing Three Component Heteromeric Complexes Mediated by Otoferlin, a Multivalent Membrane Fusion Protein, Using a Novel Single-Molecule Assay (original) (raw)
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The beta-amyloid peptide (Aβ) involved in Alzheimer's disease (AD) has been described to associate/aggregate on the cell surface disrupting the membrane through pore formation and breakage. However, molecular determinants involved for this interaction (e.g., some physicochemical properties of the cell membrane) are largely unknown. Since cholesterol is an important molecule for membrane structure and fluidity, we examined the effect of varying cholesterol content with the association and membrane perforation by Aβ in cultured hippocampal neurons. To decrease or increase the levels of cholesterol in the membrane we used methyl-β-cyclodextrin (MβCD) and MβCD/cholesterol, respectively. We analyzed if membrane fluidity was affected using generalized polarization (GP) imaging and the fluorescent dye di-4-ANEPPDHQ. Additionally membrane association and perforation was assessed using immunocytochemistry and electrophysiological techniques, respectively. The results showed that choleste...
Seminars in Cell & Developmental Biology, 2009
Amyloid-β (Aβ) peptide accumulation in the brain is central to the pathogenesis of Alzheimer's disease (AD). Aβ is produced through proteolytic processing of a transmembrane protein, β-amyloid precursor protein (APP), by βand γ-secretases. Mounting evidence has demonstrated that alterations in APP cellular trafficking and localization directly impact its processing to Aβ. Members of the lowdensity lipoprotein receptor family, including LRP, LRP1B, SorLA/LR11, and apoER2, interact with APP and regulate its endocytic trafficking. Additionally, APP trafficking and processing are greatly affected by cellular cholesterol content. In this review, we summarize the current understanding of the roles of lipoprotein receptors and cholesterol in APP trafficking and processing and their implication for AD pathogenesis and therapy.
Cholesterol depletion reduces aggregation of amyloid-beta peptide in hippocampal neurons
Neurobiology of Disease, 2006
A key event in the pathogenesis of Alzheimer's disease is the conversion of soluble amyloid AB-peptide into toxic aggregates. Here, we studied the effect of cholesterol depletion on the formation of insoluble AB. We found that reduction of neuronal cholesterol by¨25% reduced the neuronal formation of insoluble AB without affecting the secretion of soluble AB. Moreover, we demonstrate that AB-oligomers from Alzheimer's disease brains associate with a detergent-resistant membrane fraction in a cholesterol-dependent manner. These results suggest a key role for cholesterol in aggregation of AB. D
BMC structural …, 2006
Background: Soluble Alzheimer's Aβ oligomers autoinsert into neuronal cell membranes, contributing to the pathology of Alzheimer's Disease (AD), and elevated serum cholesterol is a risk factor for AD, but the reason is unknown. We investigated potential connections between these two observations at the membrane level by testing the hypothesis that Aβ(1-42) relocates membrane cholesterol. Results: Oligomers of Aβ(1-42), but not the monomeric peptide, inserted into cholesterolcontaining phosphatidylcholine monolayers with an anomalously low molecular insertion area, suggesting concurrent lipid rearrangement. Membrane neutron diffraction, including isomorphous replacement of specific lipid hydrogens with highly-scattering deuterium, showed that Aβ(1-42) insertion was accompanied by outward displacement of membrane cholesterol, towards the polar surfaces of the bilayer. Changes in the generalised polarisation of laurdan confirmed that the structural changes were associated with a functional alteration in membrane lipid order. Conclusion: Cholesterol is known to regulate membrane lipid order, and this can affect a wide range of membrane mechanisms, including intercellular signalling. Previously unrecognised Aβdependent rearrangement of the membrane sterol could have an important role in AD. Background Alzheimer's Disease (AD) is associated with neurofibrillary tangles containing tau proteins, and with extracellular amyloid plaques containing fibrils formed by Aβ(1-40), Aβ(1-42) and Aβ(1-43), amyloid-β (Aβ) peptides generated by intracellular proteolytic cleavage of Amyloid Precursor Protein (APP), specifically the neuronal membrane APP homologue, APLP-1. Although Aβ peptides, espe
Neuronal membrane cholesterol loss enhances amyloid peptide generation
Journal of Cell Biology, 2004
R are found in the same membrane environment. Much higher levels of BACE 1-APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.
Cholesterol reduction impairs exocytosis of synaptic vesicles
Journal of Cell Science, 2010
Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Ca v 2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons.
Aging Cell, 2011
Beta amyloid (bA) plays a central role in the pathogenesis of the most common and devastating neurodegenerative disorder, Alzheimer's disease (AD). The mechanisms of bA neurotoxicity remain controversial, but include dysregulation of calcium homeostasis and oxidative stress. A large body of data suggest that cholesterol plays a significant role in AD. In mixed cultures containing hippocampal neurons and astrocytes, we have shown that neurotoxic bA peptides (1-42 and 25-35) cause sporadic cytosolic calcium ([Ca 2+ ] c) signals in astrocytes but not in neurons, initiating a cascade that ends in neuronal death. We now show, using the cholesterol-sensitive fluorescent probe, Filipin, that membrane cholesterol is significantly higher in astrocytes than in neurons and mediates the selective response of astrocytes to bA. Thus, lowering [cholesterol] using mevastatin, methyl-b-cyclodextrin or filipin prevented the bA-induced [Ca 2+ ] c signals, while increased membrane [cholesterol] increased bA-induced [Ca 2+ ] c signals in both neurons and astrocytes. Addition of bA to lipid bilayers caused the appearance of a conductance that was significantly higher in membranes containing cholesterol. Increasing membrane [cholesterol] significantly increased bA-induced neuronal and astrocytic death. We conclude that a high membrane [cholesterol] promotes bA incorporation into membranes and increased [Ca 2+ ] c leading to cell death.
Cholesterol-dependent balance between evoked and spontaneous synaptic vesicle recycling
The Journal of Physiology, 2007
Cholesterol is a prominent component of nerve terminals. To examine cholesterol's role in central neurotransmission, we treated hippocampal cultures with methyl-β-cyclodextrin, which reversibly binds cholesterol, or mevastatin, an inhibitor of cholesterol biosynthesis, to deplete cholesterol. We also used hippocampal cultures from Niemann-Pick type C1-deficient mice defective in intracellular cholesterol trafficking. These conditions revealed an augmentation in spontaneous neurotransmission detected electrically and an increase in spontaneous vesicle endocytosis judged by horseradish peroxidase uptake after cholesterol depletion by methyl-β-cyclodextrin. In contrast, responses evoked by action potentials and hypertonicity were severely impaired after the same treatments. The increase in spontaneous vesicle recycling and the decrease in evoked neurotransmission were reversible upon cholesterol addition. Cholesterol removal did not impact on the low level of evoked neurotransmission seen in the absence of synaptic vesicle SNARE protein synaptobrevin-2 whereas the increase in spontaneous fusion remained. These results suggest that synaptic cholesterol balances evoked and spontaneous neurotransmission by hindering spontaneous synaptic vesicle turnover and sustaining evoked exo-endocytosis.
Journal of Neurochemistry, 2009
It has been suggested that cellular cholesterol levels can modulate the metabolism of the amyloid precursor protein (APP) but the underlying mechanism remains controversial. In the current study, we investigate in detail the relationship between cholesterol reduction, APP processing and γsecretase function in cell culture studies. We found that mild membrane cholesterol reduction led to a decrease in Aβ 40 and Aβ 42 in different cell types. We did not detect changes in APP intracellular domain or Notch intracellular domain generation. Western blot analyses showed a cholesterol-dependent decrease in the APP C-terminal fragments and cell surface APP. Finally, we applied a fluorescence resonance energy transfer (FRET)-based technique to study APP-Presenilin 1 (PS1) interactions and lipid rafts in intact cells. Our data indicate that cholesterol depletion reduces association of APP into lipid rafts and disrupts APP-PS1 interaction. Taken together, our results suggest that mild membrane cholesterol reduction impacts the cleavage of APP upstream of γ-secretase and appears to be mediated by changes in APP trafficking and partitioning into lipid rafts.