P2-137: High membrane cholesterol enlarges early endosomes and increases clathrin-dependant APP endocytosis and Abeta production

2009 ◽  
Vol 5 (4S_Part_10) ◽  
pp. P301-P302
Author(s):  
Jack-Christophe Cossec ◽  
Anne Simon ◽  
Randal X. Moldrich ◽  
Christophe Leterrier ◽  
Claire Perruchini ◽  
...  
Keyword(s):  
Membrane Cholesterol ◽  
Early Endosomes ◽  
Abeta Production

scholarly journals Inefficient HIV-1 trans Infection of CD4 + T Cells by Macrophages from HIV-1 Nonprogressors Is Associated with Altered Membrane Cholesterol and DC-SIGN

2018 ◽  
Vol 92 (13) ◽  
Author(s):  
Diana C. DeLucia ◽  
Charles R. Rinaldo ◽  
Giovanna Rappocciolo
Keyword(s):  
T Cells ◽  
Hiv Infection ◽  
Disease Progression ◽  
B Lymphocytes ◽  
Cholesterol Content ◽  
Therapeutic Strategies ◽  
Membrane Cholesterol ◽  
Data Support ◽  
Early Endosomes ◽  
Hiv 1

ABSTRACT Professional antigen-presenting cells (APC; myeloid dendritic cells [DC] and macrophages [MΦ]; B lymphocytes) mediate highly efficient HIV-1 infection of CD4 + T cells, termed trans infection, that could contribute to HIV-1 pathogenesis. We have previously shown that lower cholesterol content in DC and B lymphocytes is associated with a lack of HIV-1 trans infection in HIV-1-infected nonprogressors (NP). Here, we assessed whether HIV-1 trans infection mediated by another major APC, MΦ, is deficient in NP due to altered cholesterol metabolism. When comparing healthy HIV-1 seronegatives (SN), rapid progressors (PR), and NP, we found that monocyte-derived MΦ from NP did not mediate HIV-1 trans infection of autologous CD4 + T cells, in contrast to efficient trans infection mediated by SN and PR MΦ. MΦ trans infection efficiency was directly associated with the number of DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)-expressing MΦ. Significantly fewer NP MΦ expressed DC-SIGN. Unesterified (free) cholesterol in MΦ cell membranes and lipid rafting was significantly lower in NP than PR, as was virus internalization in early endosomes. Furthermore, simvastatin (SIMV) decreased the subpopulation of DC-SIGN + MΦ as well as cis and trans infection. Notably, SIMV decreased cell membrane cholesterol and led to lipid raft dissociation, effectively mimicking the incompetent APC trans infection environment characteristic of NP. Our data support that DC-SIGN and membrane cholesterol are central to MΦ trans infection, and a lack of these limits HIV-1 disease progression. Targeting the ability of MΦ to drive HIV-1 dissemination in trans could enhance HIV-1 therapeutic strategies. IMPORTANCE Despite the success of combination antiretroviral therapy, neither a vaccine nor a cure for HIV infection has been developed, demonstrating a need for novel prophylactic and therapeutic strategies. Here, we show that efficiency of MΦ-mediated HIV trans infection of CD4 + T cells is a unique characteristic associated with control of disease progression, and it is impaired in HIV-infected NP. In vitro treatment of MΦ from healthy donors with SIMV lowers their cholesterol content, which results in a strongly reduced trans infection ability, similar to the levels of MΦ from NP. Taken together, our data support the hypothesis that MΦ-mediated HIV-1 trans infection plays a role in HIV infection and disease progression and demonstrate that the use of SIMV to decrease this mechanism of virus transfer should be considered for future HIV therapeutic development.

scholarly journals Mutations in the juxtamembrane segment of the cholesterol-binding site of APP alter its processing and promotes production of shorter, less toxic Aβ peptides

2020 ◽  
Author(s):  
Linda Hanbouch ◽  
Béatrice Schaack ◽  
Amal Kasri ◽  
Gaëlle Fontaine ◽  
Eleni Gkanatsiou ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Site ◽  
Amyloid Β ◽  
Neuronal Cultures ◽  
Membrane Cholesterol ◽  
Aβ Peptides ◽  
App Processing ◽  
Juxtamembrane Region ◽  
Early Endosomes ◽  
Cholesterol Binding

AbstractBackgroundThe brains of patients with Alzheimer’s disease (AD) reveal increased cellular membrane levels of cholesterol. Correspondingly, we previously showed that elevating levels of membrane cholesterol in neuronal cultures recapitulates early AD phenotypes including excessive cleavage of amyloid β (Aβ) peptides from the amyloid precursor protein (APP). Here we aimed to evaluate how the presence of a cholesterol-binding site (CBS) in the transmembrane and juxtamembrane regions of APP regulates its processing.MethodsWe generated seven single and two double APP mutants at amino acid positions 22, 26, 28, 29, 33, 39 of the Aβ sequence changing the charge and/or hydrophobicity of the targeted amino acids. HEK293T cells were transfected with APP constructs and secreted Aβ peptides were measured using ELISA and mass spectrometry (MS). APP processing in normal and high cholesterol condition, and endocytosis were assessed in stably expressing APPwt and APPK28A HEK293T clones. Finally, we measured the binding of synthetic peptides derived from the Aβ sequence to cholesterol-rich exosomes purified from control HEK293T cells.ResultsMost mutations triggered a reduction in the production of Aβ40 and Aβ42 peptides, whereas only juxtamembrane mutants resulted in the generation of shorter Aβ peptides. We confirmed by mass spectrometry this specific change in the profile of secreted Aβ peptides for the most characteristic APPK28A mutant. A transient increase of plasma membrane cholesterol enhanced the production of Aβ40 by APPWT, an effect absent with APPK28A. The enzymatic activity of α-, β- and γ-secretases remained unchanged in cells expressing APPK28A. Similarly, APPK28A subcellular localization in early endosomes did not differ to APPWT. Finally, WT but not CBS mutant Aβ derived peptides bound to cholesterol-rich exosomes.ConclusionsTaken together, these data reveal a major role of the juxtamembrane region of APP in binding to cholesterol and accordingly in the regulation of APP processing. Binding of cholesterol to K28 could staple APP to the juxtamembrane region thereby permitting access to γ-secretase cleavage at positions 40-42. The APPK28 mutant would lie deeper in the membrane, facilitating the production of shorter Aβ peptides and unveiling this specific region as a novel target for reducing the production of toxic Aβ species.

Acidification of rat liver lysosomes: quantitation and comparison with endosomes

1993 ◽  
Vol 265 (4) ◽  
pp. C901-C917 ◽  
Author(s):  
R. W. Van Dyke
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Rat Liver ◽  
Proton Pumps ◽  
Intact Cells ◽  
Early Endosomes ◽  
Medium Permeability ◽  
Electrogenic Proton Pump ◽  
Secondary Lysosomes ◽  
Permeability Studies

Both lysosomes and endosomes are acidified by an electrogenic proton pump, although studies in intact cells indicate that the steady-state internal pH (pHi) of lysosomes is more acid than that of endosomes. We undertook the present study to examine in detail the acidification mechanism of purified rat liver secondary lysosomes and to compare it with that of a population of early endosomes. Both endosomes and lysosomes exhibited ATP-dependent acidification, but proton influx rates were 2.4- to 2.7-fold greater for endosomes than for lysosomes because of differences in both buffering capacity and acidification rates, suggesting that endosomes exhibited greater numbers or rates of proton pumps. Lysosomes, however, exhibited a more acidic steady-state pHi due in part to a slower proton leak rate. Changes in medium Cl- increased acidification rates of endosomes more than lysosomes, and the lysosome ATP-dependent interior-positive membrane potential was only partially eliminated by high-Cl- medium. Permeability studies suggested that lysosomes were less permeable to Na+, Li+, and Cl- and more permeable to K+ and PO4(2-) than endosomes. Na-K-adenosine-triphosphatase did not appear to regulate acidification of either vesicle type. Endosome and lysosome acidification displayed similar inhibition profiles to N-ethylmaleimide, dicyclohexyl-carbodiimide, and vanadate, although lysosomes were somewhat more sensitive [concentration producing 50% maximal inhibition (IC50) 1 nM] to bafilomycin A1 than endosomes (IC50 7.6 nM). Oligomycin (1.5-3 microM) stimulated lysosome acidification due to shunting of membrane potential. Overall, acidification of endosomes and lysosomes was qualitatively similar but quantitatively somewhat different, possibly related to differences in the density or rate of proton pumps as well as vesicle permeability to protons, anions, and other cations.

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