Accumulation of neutral lipids in peripheral blood mononuclear cells as a distinctive trait of Alzheimer patients and asymptomatic subjects at risk of disease - PubMed (original) (raw)
doi: 10.1186/1741-7015-7-66.
Antonella Mandas, Giacomo Diaz, Claudia Abete, Pier Luigi Cocco, Fabrizio Angius, Annalisa Brundu, Nico Muçaka, Maria Elena Pais, Antonio Saba, Luigi Barberini, Cristina Zaru, Manuela Palmas, Paolo F Putzu, Alessandra Mocali, Francesco Paoletti, Paolo La Colla, Sandra Dessì
Affiliations
- PMID: 19883495
- PMCID: PMC2777188
- DOI: 10.1186/1741-7015-7-66
Accumulation of neutral lipids in peripheral blood mononuclear cells as a distinctive trait of Alzheimer patients and asymptomatic subjects at risk of disease
Alessandra Pani et al. BMC Med. 2009.
Abstract
Background: Alzheimer's disease is the most common progressive neurodegenerative disease. In recent years, numerous progresses in the discovery of novel Alzheimer's disease molecular biomarkers in brain as well as in biological fluids have been made. Among them, those involving lipid metabolism are emerging as potential candidates. In particular, an accumulation of neutral lipids was recently found by us in skin fibroblasts from Alzheimer's disease patients. Therefore, with the aim to assess whether peripheral alterations in cholesterol homeostasis might be relevant in Alzheimer's disease development and progression, in the present study we analyzed lipid metabolism in plasma and peripheral blood mononuclear cells from Alzheimer's disease patients and from their first-degree relatives.
Methods: Blood samples were obtained from 93 patients with probable Alzheimer's disease and from 91 of their first-degree relatives. As controls we utilized 57, cognitively normal, over-65 year-old volunteers and 113 blood donors aged 21-66 years, respectively. Data are reported as mean +/- standard error. Statistical calculations were performed using the statistical analysis software Origin 8.0 version. Data analysis was done using the Student t-test and the Pearson test.
Results: Data reported here show high neutral lipid levels and increased ACAT-1 protein in about 85% of peripheral blood mononuclear cells freshly isolated (ex vivo) from patients with probable sporadic Alzheimer's disease compared to about 7% of cognitively normal age-matched controls. A significant reduction in high density lipoprotein-cholesterol levels in plasma from Alzheimer's disease blood samples was also observed. Additionally, correlation analyses reveal a negative correlation between high density lipoprotein-cholesterol and cognitive capacity, as determined by Mini Mental State Examination, as well as between high density lipoprotein-cholesterol and neutral lipid accumulation. We observed great variability in the neutral lipid-peripheral blood mononuclear cells data and in plasma lipid analysis of the subjects enrolled as Alzheimer's disease-first-degree relatives. However, about 30% of them tend to display a peripheral metabolic cholesterol pattern similar to that exhibited by Alzheimer's disease patients.
Conclusion: We suggest that neutral lipid-peripheral blood mononuclear cells and plasma high density lipoprotein-cholesterol determinations might be of interest to outline a distinctive metabolic profile applying to both Alzheimer's disease patients and asymptomatic subjects at higher risk of disease.
Figures
Figure 1
MMSE vs ORO intensity in PBMCs from control 1 and AD. Pearson correlation test revealed a significant inverse correlation between these variables in AD patients but not in control 1.
Figure 2
Neutral lipid accumulation in PHA-stimulated PBMCs from control 1 subjects and AD patients. Immediately after separation, cells were incubated with PHA for the time periods indicated. After harvesting, cells were washed, fixed by soaking in 10% formalin, stained with ORO for NL, and counter-stained with Mayer's hematoxylin for nuclei. Cells were then examined by light microscopy and two different fields per sample were imaged. Red ORO intensity was measured in these two fields using NIH Image J software. Panel A shows ORO staining images for one control 1 (84 years old; ORO score 0 at 0 time) and one AD (85 years old; ORO score 3 at 0 time), which are representative of about thirty subjects for each group. Panel B shows red intensity expressed as mean pixels ± SE/cm2, as described in Materials and Methods. *P < 0.01, compared with control 1 by Student's t test.
Figure 3
Plasma lipid profile in control 1subjects and AD patients. Data are expressed as a mean ± SE of the enrolled subjects. *TC P = 0.116; HDL P = 0.000; %HDL/TC P = 0.002; TC female P = 0.227; D HDL female P = 0.000; % HDLC/TC female P = 0.002; TC male P = 0.221; HDL male P = 0.003; %HDLC/TC male, P = 0.297 of AD vs the corresponding control 1 by Student's t test.
Figure 4
HDL-C vs ORO intensity and MMSE vs TC in PBMCs from control 1 and AD. A. HDL-C vs ORO intensity; B. %HDL-C vs ORO intensity; C. MMSE vs TC. The test revealed a significant inverse correlation between HDL-C and %HDL/TC vs ORO in AD patients but not in control 1. No correlation was observed between MMSE vs TC in both groups.
Figure 5
ACAT-1 protein expression in control 1 and AD PBMCs. Western blotting with DM10 was used to monitor ACAT-1 protein content. The anti-ACAT-1 DM10 antibody specifically recognized a single protein band from PBMC extracts with an apparent molecular weight of about 50 kDa. No other protein signal(s) was detectable. A. Representative ACAT-1 immunoblot. B. Results of densitometric analysis by Scion Image software of ACAT-1 protein immunoreactivity relative to β-actin in 20 AD patients and 20 control 1. Data values are represented as mean ± SE *P = 0.000 vs control 1.
Figure 6
Lipid synthesis and efflux in PBMCs with different ORO scores. Freshly isolated PBMCs (four for each ORO score group) were plated at a density of 10,000 cell/cm2 in six well plates and incubated with PHA for the time periods indicated. Total cellular lipid synthesis (A) and cholesterol ester synthesis (B) were evaluated by incubating cells for six hours in medium containing [14C]acetate at a final concentration of 2 μCi/ml. After incubation, cells were washed with PBS and lipids extracted with acetone. Lipid subclasses were separated out by thin layer chromatography (TLC) and [14C]acetate incorporation into the various lipid fractions was measured. Efflux (C) from the cells into the medium at each time point was expressed as the percentage of radioactivity in medium/total radioactivity (cells + medium). Data shown are the mean ± SE of four subjects from each score group, *P < 0.05.
Figure 7
Plasma lipid profile in control 2 and AD-FDR. Data are expressed as a mean ± SE of the enrolled subjects. *TC P = 0.784; HDL P = 0.154; %HDL/TC P = 0.04; TC female P = 0.185; D HDL female P = 0.304; % HDLC/TC female P = 0.05; TC male P = 0.490; HDL male P = 0.215; %HDLC/TC male, P = 0.316 of AD-FDR vs the corresponding control 2 by Student's t test.
Figure 8
Neutral lipid accumulation in PHA-stimulated PBMCs from control 2 and AD-FDR subjects. Immediately after separation cells were incubated with PHA for the indicated time periods. After harvesting, cells were washed, fixed by soaking in 10% formalin, stained with ORO for NL, and counter-stained with Mayer's hematoxylin for nuclei. Cells were then examined by light microscopy and two different fields per sample were imaged. Red ORO intensity was measured in these two fields by using NIH Image J software. Panel A shows images of ORO staining of one control 2 (47 years old) and two AD-FDR one (48 years old) with ORO score 0 and the other one (37 years old) with ORO score 3 at 0 time. These images are representative of at least 30 control 2, 15 AD-FDR with ORO score between 0 and 1, and 15 with ORO score between 2 and 3. Panel B shows red intensity expressed as mean pixels ± SE/cm2 of triplicate wells obtained by utilizing four created different regions of interest (ROIs). *P < 0.01, compared with control 2 by Student's t test.
Figure 9
ACAT-1 protein expression in PBMCs from control 2 and AD-FDR subjects. Western blotting with DM10 was used to monitor the ACAT-1 protein content. The anti-ACAT-1 DM10 antibody specifically recognized a single protein band from cell extracts of PBMCs, with an apparent molecular weight of 50 kDa. No other protein signal(s) was detectable. A. Representative immunoblot of ACAT-1 of two control 2; two AD-FDR with ORO score between 0 and 1 (FDR-1) and two AD-FDR with ORO score between 2-3 (FDR-2). B. Results of densitometric analysis by Scion Image software of ACAT-1 protein.
References
- Govaerts L, Schoenen J, Bouhy D. Pathogenesis of Alzheimer's disease: molecular and cellular mechanisms. Rev Med Liege. 2007;62:209–216. -PubMed
- Giannakopoulos P, Kövari E, Gold G, von Gunten A, Hof PR, Bouras C. Pathological substrates of cognitive decline in Alzheimer's disease. Front Neurol Neurosci. 2009;24:20–29. -PubMed
- Blacker D, Albert MS, Basset SS, Go RCP, Harrell LE, Folstein MF. Reliability and validity of NINCDS-ADRDA criteria for Alzheimer's disease. The National Institute of Mental Health Genetics Initiative. Arch Neurol. 1994;51:1198–1204. -PubMed
- DeCarli C, Kaye JA, Horwitz B, Rapoport SI. Critical analysis of the use of computer assisted transverse axial tomography to study human brain in aging and dementia of the Alzheimer type. Neurology. 1990;40:872–883. -PubMed
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