Amyloid peptide, the main component of senile plaques, is a major biological characteristic of Alzheimer’s disease (AD). The aim of the present study conducted on human neuronal SK-N-BE cells was to evaluate whether oligomerized A β 1-40-induced cell damages was associated with lipid modifications. Under treatment with A β 1-40 (10 - 100 μM; 24 - 48 h), cell viability was recorded with the MTT test and by measuring LDH activity. Mitochondrial transmembrane potential and ATP production were assessed using flow cytometry and a luciferase-based ATP bioluminescence assay, respectively. Annexin V-CF647 staining assay for cell apoptosis detection was performed using flow cytometry. Potentially intracellular cytotoxic lipids (oxysterols: 7 α-hydroxycholesterol (7 α-OHC), 7 β-hydroxycholesterol (7 β-OHC), and 7-ketocholesterol (7KC), 24(S)-hydroxycholesterol; arachidonic acid (C20:4 n-6); VLCFAs (C22:0, C24:0, C24:6 and C26:0)) were measured using gas chromatography coupled with mass spectrometry. The cellular level of docosahexaenoic acid (C22:6 n-3), often altered in AD, was also quantified. In the presence of A β 1-40, the percentage of MTT-positive cells decreased and was associated with an increase in LDH activity. In addition, treatment with oligomerized A β 1-40 induced a decrease of mitochondrial transmembrane potential as well as an apoptotic cell death. Sterol analysis revealed a higher cholesterol level and a significant increase of cytotoxic oxysterols per cell (7KC + 7 β-OHC), and of the [(7 β-OHC + 7KC)/cholesterol] ratio, considered as a lipid peroxidation index, in A β 1-40-treated cells. An enhancement of C20:4 n-6, C22:6 n-3 and saturated VLCFAs was also observed. Therefore, A β 1-40-induced side effects are associated with intracellular accumulation of lipids, especially cholesterol, oxysterols (7 β-OHC, 7KC), C20:4 n-6, and saturated VLCFAs, which could in turn contribute to neurotoxicity.
Alzheimer’s disease (AD) is the most predominant dementia in the elderly. Aggregated amyloid deposits are the main components of senile plaques, which are characteristics of the AD brain [
At the moment, AD has been associated with several risk factors, and among them lipid alterations have been suspected [
Another finding relating lipid metabolism disorders to AD pathogenesis was the accumulation of saturated very long chain fatty acids (saturated VLCFAs: docosanoic acid (C22:0), tetracosanoic acid (C24:0) and hexacosanoic acid (C26:0)) in cortical regions of brains of AD patients with stages V-VI compared with those modestly affected (stages I-II) based on the neuropathological Braak classification [
With important roles attributed to Aβ in the development of AD due to its multiple neurotoxic activities [
As previously described, human neuronal cells (SK-N-BE) were seeded at 200,000 cells per well in 24-well microplates containing 1 mL of culture medium constituted by Dulbecco’s Modified Eagle Medium with L-glu- tamine (DMEM) (Lonza) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Pan Biotech) and 1% antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin) (Pan Biotech) [
The MTT assay was carried as previously described [
Cytotoxicity induced by Aβ1-40 was assessed by lactate dehydrogenase (LDH) leakage into the culture medium. The LDH activity was determined using a commercially available kit (Cayman Chemical Company). The assay is based on the conversion of lactate to pyruvate in the presence of LDH with parallel reduction of NAD. NADH formed from the above reaction is used by diaphorase to catalyze the reduction of tetrazolium salt to formazan which is proportional to the quantity of LDH released in the medium. A microplate reader was used at a wavelength of 490 nm and LDH activity was determined from the calibration curve. This LDH activity was adjusted to the number of cells per well, and was expressed as µU/mg of protein.
Intracellular ATP levels were measured using a luciferase-based ATP Bioluminescence Assay Kit CLS II (Roche Molecular Biochemicals). For ATP measurement, 100 µL of cell lysate was mixed with 50 µL of luciferase. Emitted bioluminescence was measured using a microplate reader. The protein of each treatment group was determined by the BCA Protein Assay Kit.
Variations of the transmembrane mitochondrial potential (ΔΨm) were measured with 3, 3’-dihexyloxacarbo- cyanine iodide (DiOC6 (3)) (Invitrogen), which allows the percentage of cells with low ΔΨm to be determined. With DiOC6 (3), mitochondrial depolarization is indicated by a decrease in green fluorescence collected through a 520/10-nm band pass filter. DiOC6 (3) was used at a 40 nM. Flow cytometric analyses were performed on a Galaxy flow cytometer (Partec). Ten thousand cells were acquired for each sample. Data were analyzed with Flomax software (Partec) or FlowJo software (Tree Star Inc.).
Apoptotic cell death was measured via Annexin V-CF647 (Millipore) staining followed by flow cytometry. Annexin V is a calcium-dependent phospholipid binding protein with high affinity for phosphatidylserine (PS), a membrane component normally localized to the internal face of the cell membrane and which is exposed on the cell surface upon induction of apoptosis. Annexin V, which is conjugated to CF647 (Abs/Em maxima: 650/665 nm), was excited by a red laser on a FACSalibur 4C flow cytometer (BD Biosciences) and the emission of fluorescence was collected with a 670 nm long pass filter. Five μL Annexin V-CF647 were added to the cells in the dark at 37˚C, in a humidified atmosphere containing 5% CO2. Around 15 min later, the stained cells were analyzed by flow cytometry. Ten thousand cells were acquired for each sample. Data were analyzed with Flomax software (Partec) or FlowJo software (Tree Star Inc.).
Cholesterol oxide derivatives (also called oxysterols), including 7α-OHC (mainly formed via CYP7A1 [
C22:0, C24:0, C26:0, C20:4, C22:6, and C24:6 were quantified using a HP7890A gas chromatograph equipped with an HP7683 injector and a HP5975C mass selective detector (Agilent Technologies). Chromatography was performed using an HP-5MS-fused silica capillary column (length: 30 m; inner diameter: 0.25 mm; film thickness: 0.25 mm; Agilent Technologies). The GC-MS conditions were as follows: carrier gas, helium at a flow rate of 1.1 mL/min; injector temperature, 250˚C, split mode; oven temperature, 140˚C increased at 5˚C/min to 300˚C and held for 10 min. The mass spectrometer was operated under negative chemical ionization mode with methane as the reactant gas. The ion source temperature and the quadrupole temperature were 150˚C and 106˚C, respectively. A SIM program was used for mass spectrometry with [M-181] (−) ions as quantification.
The ability of Aβ1-40 to induce neurotoxicity was estimated using i) the MTT test, which reflects mitochondrial activity and/or cell growth, and ii) by LDH activity. SK-NB-E cells were cultured without or with Aβ1-40 (10 - 100 µM, 24 - 48 h). A significant decrease in the percentage of MTT-positive cells was observed after 24 and 48 h of treatment with the two concentrations of Aβ1-40 used (
Data obtained with the MTT test support that mitochondrial activity and/or cell growth is affected under treatment
with Aβ1-40. To determine the impact of Aβ1-40 at the mitochondrial level, ATP production and mitochondrial transmembrane potential (ΔΨm) were measured. A significant increase of the percentage of DiOC6 (3) negative cells (with low ΔΨm) was observed with 100 µM at 24 h, and with 10 and 100 µM at 48 h of treatment with oligomerized Aβ1-40 (
The ATP level was measured on SK-N-BE cells treated with oligomerized Aβ1-40 (10 µM, 48 h). A significant increase in intracellular ATP supporting mitochondrial dysfunctions was revealed in treated cells compared to the control (
Annexin V-CF647 staining assay for cell apoptosis detection was performed using flow cytometry. A significant increase of the percentage of Annexin V positive cells was observed in cells treated with oligomerized Aβ1-40 (100 µM, 24 h) and with 10 and 100 µM at 48 h (P < 0.05) (
The effects of Aβ1-40 (10 µM, 48 h) on the intracellular levels of cholesterol, oxysterols (7α-OHC, 7β-OHC, 7KC, 24S-OHC) (
Cholesterol analysis revealed a significant increase (Mann-Whitney test; P < 0.05) in Aβ1-40-treated cells. In addition, oxysterol analysis in SK-N-BE-treated cells revealed a significant increase (Mann-Whitney test; P < 0.05) in 7β-OHC and in the sum (7β-OHC + 7KC), reflecting cholesterol autoxidation. Furthermore, the [(7β- OHC + 7KC)/cholesterol] ratio, considered as a lipid peroxidation index, was significantly enhanced (Mann- Whitney test; P < 0.05) in Aβ1-40-treated cells. However, no significant difference in the sum of cytotoxic oxysterols (7α-OHC + 7β-OHC + 7KC + 24S-OHC) was observed between untreated cells (control) and Aβ1-40- treated cells, whereas it was highest under treatment with Aβ1-40.
Considerable modifications in the intracellular levels of fatty acids were also revealed (
Amyloid peptide (Aβ), the main component of senile plaques, was shown to be neurotoxic in several studies but no data are available to evaluate the relation between this molecule and lipid metabolism disorders associated with AD pathogenesis [
On SK-N-BE cells, the neurotoxicity of oligomerized Aβ1-40 evaluated with the MTT test and LDH activity showed both a significant increase in the percentage of MTT-positive cells and an increase in LDH activity, which supports the ability of Aβ1-40 to induce cell death [
The significant increase in intracellular ATP observed in SK-N-BE cells treated with Aβ1-40 supports the hypothesis that stressed cells may require more energy to counteract various side effects resulting from stress conditions and to preserve their vital functions [
The increased intracellular level of cholesterol detected in Aβ1-40-treated cells supports the notion that the cellular stress triggered by Aβ1-40 can promote cholesterol synthesis and/or accumulation [
Although not significant, the enhancement of the intracellular level of 24S-OHC (a potent liver X receptor (LXR) agonist produced by enzymatic oxidation of cholesterol via CY46A1) [
On the other hand, analysis of intracellular fatty acids conducted on SK-N-BE cells treated with Aβ1-40 revealed a significant increase of AA (C20:4 n-3), C22:0 and DHA (C22:6 n-3) and the sum of VLCFAs ((C22:0 + C24:0 + C26:0). These results underline that Aβ1-40 could disrupt the metabolism of fatty acids and especially affect the peroxisomal β-oxidation of VLCFAs given that the β-oxidation or the synthesis of some of these lipids (C22:6 n-3, C24:0, and C26:0) occurs, at least in part, in the peroxisome [
The important accumulation of arachidonic acid (AA), the precursor of leukotrienes and prostaglandins, on SK-N-BE cells treated with Aβ1-40 also contributes new insights into the biological activities of this molecule. This finding is in agreement with data reporting that eicosanoids might participate in Aβ1-40 toxicity in neurons and that noncytokinic inflammation contributes to the development of AD [
In addition to its ability to trigger cell death, our data establish that Aβ1-40 favors a substantial cellular accumulation of lipids: cholesterol, oxysterols (especially those resulting from cholesterol autoxidation), and fatty acids. Since a marked accumulation of VLCFAs and DHA was observed, modifications of lipid metabolism, including peroxisomal dysfunctions, are suspected. It is suggested that the accumulation of cholesterol, oxysterols, and fatty acids could in turn contribute to the cytotoxic effects of Aβ1-40. Consequently, the identification of molecules capable of counteracting the different side effects of these lipids may be advantageous in preventing the neurotoxicity induced by Aβ1-40.