Altered proteolysis in fibroblasts of Alzheimer patients with predictive implications for subjects at risk of disease.

There is great interest in developing reliable biomarkers to support antemortem diagnosis of late-onset Alzheimer's disease (AD). Early prediction and diagnosis of AD might be improved by the detection of a proteolytic dysfunction in extracts from cultured AD fibroblasts, producing altered isoelectrophoretic forms of the enzyme transketolase (TK-alkaline bands). The TK profile and apolipoprotein E (APOE) genotype were examined in fibroblasts from 36 clinically diagnosed probable late-onset sporadic AD patients and 38 of their asymptomatic relatives, 29 elderly healthy individuals, 12 neurological non-AD patients, and 5 early-onset AD patients. TK alterations occurred in (i) several probable AD patients regardless of age-of-onset and severity of disease; (ii) all early-onset AD patients and APOE ε 4/4 carriers; and (iii) nearly half of asymptomatic AD relatives. Normal subjects and non-AD patients were all negative. Notably, culture conditions promoting TK alterations were also effective in increasing active BACE1 levels. Overall, the TK assay might represent a low-cost laboratory tool useful for supporting AD differential diagnosis and identifying asymptomatic subjects who are at greater risk of AD and who should enter a follow-up study. Moreover, the cultured fibroblasts were confirmed as a useful in vitro model for further studies on the pathogenetic process of AD.

Age-related macular degeneration and cognitive impairment show similarities in changes of neutral lipids in peripheral blood mononuclear cells.

Starting from previous studies showing that patients with cognitive deficit present neutral lipids (NLs) accumulation in cytoplasm of their peripheral blood mononuclear cells (PBMCs) and considering that there is epidemiological evidence linking age-related macular degeneration (AMD) to cognitive deficit, the first purpose of this study was to test whether neutral lipids also accumulated in PBMCs from AMD subjects. Moreover, the impact of statin use on AMD was explored and whether such use in AMD subjects was associated with NLs accumulation in PBMCs. The study was conducted on 222 subjects: 136 AMD (36 of which - 26.5% - using statins], 48 cognitive deficit (20 of which - 41.7% - using statins) and 38 healthy controls (4 of which -10.1% - using statins), AMD lesions were assessed from color fundus photographs. Mini-mental state examination (MMSE), demographics, lifestyle factors and medical history were collected at interview. MMSE score was categorized as normal (24-30), and impaired (<24), NLs content was evaluated by oil red 0 (ORO) staining method. ORO determination showed that neutral lipids were generally absent or very low (score between 0 and 1) in healthy controls while most of PBMCs from cognitive deficit and AMD had ORO staining levels scoring 2-4. Post hoc analysis (Bonferroni) in a one-way ANOVA revealed that ORO score was significantly higher in cognitive deficit and AMD subjects compared to healthy controls and in cognitive deficit compared to AMD. Bonferroni-test also showed that AMD subjects had significantly lower total cholesterol (TC) levels compared to healthy controls while high density lipoprotein-cholesterol (HDL-C) did not reach statistical significance. The results also revealed a significant higher number of statin-users in AMD compared to healthy controls. Likewise when cognitive deficit vs healthy controls was analyzed, the number of statin users were found to be significant higher in cognitive deficit than in healthy controls. There were no significant differences in statin use between AMD and cognitive deficit. Compared to healthy controls, statin use in cognitive deficit and AMD groups was significantly associated with ORO scores of 2-4. This data supports the hypothesis that AMD and cognitive deficit share similar complex pathophysiology and risk factors including NLs accumulation in their PBMCs, although this does not necessarily imply that one disease causes the other. In addition, they provide further evidence that statin use may increase the risk of AMD.

Cognitive decline and depressive symptoms in late-life are associated with statin use: evidence from a population-based study of Sardinian old people living in their own home.

OBJECTIVES: This study was designed to provide further insights into the effects of dyslipidemia (Dys-y) and use of statins (St-y) on cognitive functions and mood in older people. METHODS: Three hundred and twenty-nine subjects aged > or = 65 years were screened for cognitive dysfunction using mini mental state examination (MMSE). The geriatric depression scale (GDS) was used to detect depression. Interview questionnaires surveyed activities of daily living (ADL) and instrumental ADL (IADL), as well as other functional disabilities. The presence of neutral lipids (NLs) in cytoplasm of peripheral blood mononuclear cells (PBMCs) was determined with the Oil red O (ORO) staining. RESULTS: There was no significant difference in MMSE and GDS scores between normal (Dys-n) and Dys-y. However, when Dys-y subjects were divided into St-y and non-statin users (St-n), significant differences emerged in the scores of MMSE and GDS: St-y had lower MMSE and higher GDS than St-n. Multiple correspondence analysis and logistic regression provided further evidence that elderly St-y were much more likely to suffer of cognitive impairment and depression than St-n. Another interesting finding was that the intensity of NL-PBMCs measured by ORO staining was greater in subjects with altered MMSE compared with cognitively normal subjects. In addition St-y had higher ORO score than St-n. DISCUSSION: This is an observational study and cannot, therefore, prove a causal relationship between St-y in the elderly and a higher cognitive decline, nevertheless it provides substantial indications that caution should be exercised in the provision of statins in elderly subjects to avoid accelerated memory loss.

Changes in cholesterol metabolism-related gene expression in peripheral blood mononuclear cells from Alzheimer patients.

BACKGROUND: Cholesterol homeostasis dysfunction has been reported to have role in the pathogenesis of Alzheimer disease (AD). Therefore, changes in cholesterol metabolism in blood components may help to develop new potential AD biomarkers. In this study changes in cholesterol metabolism-related gene expression genes were evaluated in peripheral blood mononuclear cells (PBMCs) from AD subjects, their first degree relatives (FDR) and two groups of age matched controls (C1 > 80 years, C2 < 60 years). The expression of three genes related to APP processing was also determined. RESULTS: Results showed significantly different behavior (P = 0.000) in the expression of all analyzed genes among the 4 groups. An inverse correlation emerged between the age of controls and the propensity of their PBMCs to express selected genes. Moreover, when gene expression was evaluated in PBMCs from AD patients and compared with that of PBMCs from healthy subjects of the same age, LDL-R and APP mRNAs were most abundant in AD as compared C1 whereas SREBP-2 and particularly nCEH were present at much lower mRNA levels in AD-PBMCs. This study describes for the first time a differential expression profile of cholesterol and APP related genes in PBMCs from AD patients and their FDR. CONCLUSIONS: We suggest that the expressions of cholesterol homeostasis and APP processing related genes in PBMC could be proposed as possible biomarkers to evaluate AD risk. In addition, gene expression in PBMC could be also used for diagnosis and development of therapeutic strategies as well as for personalized prediction in clinical outcome of AD.

Cholesterol esterification during differentiation of mouse erythroleukemia (Friend) cells.

Cholesterol is an essential constituent of all mammalian cell membranes and its availability is therefore a prerequisite for cellular growth and other functions. Several lines of evidence are now indicating an association between alterations of cholesterol homeostasis and cell cycle progression. However, the role of cholesterol in cell differentiation is still largely unknown. To begin to address this issue, in this study we examined changes in cholesterol metabolism and in the mRNA levels of proteins involved in cholesterol import and esterification (multi-drug resistance, MDR-3) and acylCoA: cholesterol acyltransferase (ACAT) and cholesterol export (caveolin-1) in Friend virus-induced erythroleukemia cells (MELC), in the absence or in the presence of the chemical inducer of differentiation, hexamethylene bisacetamide (HMBA). FBS-stimulated growth of MELC was accompanied by an immediate elevation of cholesterol synthesis and cholesterol esterification, and by an increase in the levels of MDR-3 and ACAT mRNAs. A decrease in caveolin-1 expression was also observed. However, when MELC were treated with HMBA, the inhibition of DNA synthesis caused by HMBA treatment, was associated with a decrease in cholesterol esterification and in ACAT and MDR-3 mRNA levels and an increase in caveolin-1 mRNA. Detection of cytoplasmic neutral lipids by staining MELC with oil red O, a dye able to evidence CE but not FC, revealed that HMBA-treatment also reduced growth-stimulated accumulation of cholesterol ester to approximately the same extent as the ACAT inhibitor, SaH. Overall, these results indicate for the first time a role of cholesterol esterification and of some related genes in differentiation of erythroid cells.

Evidence for a proatherogenic biochemical phenotype in beta thalassemia minor and intermedia.

The purpose of this study was to focus on pathophysiological mechanisms linking ß-thalassemia intermedia (ß-TI) and minor (ß-TMI) with cardiovascular risk. Iron status, prooxidant-antioxidant balance and lipid profiles in serum, and lipid content in peripheral blood mononuclear cells (PBMCs) were evaluated in 20 ß-TMI subjects, 22 ß-TI patients and in 30 nonthalassemic blood donors. The mRNA levels of some genes involved in the regulation of iron and cholesterol metabolism were also determined. In ß-TI and in ß-TMI, serum iron, prooxidant-antioxidant ratio, transferrin saturation and erythropoietin levels were higher, while transferrin and hepcidin were lower compared to controls. Hepcidin and interleukin-1α mRNA levels were found to be reduced in ß-TI- and ß-TMI-PBMCs, while those of tumor necrosis factor alpha were increased. A reduction in high-density lipoprotein cholesterol in serum and an accumulation of neutral lipids coupled with increased mRNA levels of acetyl-coenzyme A:cholesterol acyltransferase and decreased neutral cholesterol ester hydrolase in PBMCs were also observed in ß-TI and ß-TMI compared to controls. Taken together, these findings provide experimental support for the idea that not only ß-TI patients but also ß-TMI have a proatherogenic biochemical phenotype which may contribute to increase their cardiovascular disease risk.

Glucose-6-phosphate-dehydrogenase deficiency as a risk factor for pterygium.

PURPOSE: Glucose-6-phosphate dehydrogenase (G6PD) is an important site of metabolic control in the pentose phosphate pathway (PPP), providing reducing power (NADPH) and pentose phosphates. The purpose of this study was to investigate the possible involvement of G6PD deficiency (G6PD-) in the pathogenesis of pterygium. METHODS: Erythrocyte G6PD activity was evaluated in 123 pterygium patients and in 112 age-matched control patients. Enzyme activity, mRNA, rate of growth, green autofluorescence, response to oxidative stress, and cholesterol metabolism were determined in pterygium fibroblasts (PFs) and in normal conjunctival fibroblasts (NCFs) isolated from G6PD normal (NCFs+ and PFs+) and G6PD- (NCFs- and PFs-) patients. RESULTS: Higher prevalence of G6PD- was found in patients affected by primary pterygium than in control subjects, both men and women, suggesting that this enzymatic defect may be a predisposing factor for pterygium. G6PD activity was significantly lower in NCFs- than in NCFs+, but not in PFs- than in PFs+. In PFs-, G6PD mRNA levels were significantly higher than in PFs+. Growth-stimulated NCFs- grew at half the rate of NCFs+, although PFs- and PFs+ grew at the same rate. Increased green autofluorescence and susceptibility to oxidative stress were observed in PFs (+/-) and in NCFs-, but not in NCFs+. Moreover, ex vivo PFs (+/-) accumulated more lipids than corresponding NCFs. CONCLUSIONS: The results of this study, although restricted to a limited group of subjects (i.e., those of Sardinian ancestry), suggest that G6PD- not only does not protect against pterygium, but may even be considered a risk factor for the development of this disorder.

Accumulation of neutral lipids in peripheral blood mononuclear cells as a distinctive trait of Alzheimer patients and asymptomatic subjects at risk of disease.

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.

Altered cholesterol ester cycle in skin fibroblasts from patients with Alzheimer's disease.

Intracellular cholesterol metabolism was reported to modulate amyloid-beta (Abeta) generation in Alzheimer's disease (AD). Results presented herein demonstrated that, like brain cells, cultured skin fibroblasts from AD patients contained more cholesterol esters than fibroblasts from healthy subjects. Particularly, Oil Red-O, Nile Red, and filipin staining highlighted higher levels of neutral lipids which responded to inhibitors of acyl-coenzyme A:cholesterol acyl-transferase (ACAT-1), associated with an increase in free cholesterol. ACAT-1 mRNA levels increased significantly in AD fibroblasts, whereas those of sterol regulatory element binding protein-2, neutral cholesterol ester hydrolase, and ATP-binding cassette transporter member 1 were markedly down-regulated. Instead, mRNA levels of low-density lipoprotein receptor, hydroxy-methyl-glutaryl-coenzyme A reductase, caveolin-1, and amyloid-beta protein precursor (AbetaPP) were virtually unchanged. Notably, mRNA levels of both beta-site AbetaPP-cleaving enzyme 1 (BACE1) and neprilysin were significantly down-regulated. An increase in Abeta(40) and Abeta(42) immunostaining and a decrease in BACE1 active form were also found in AD versus control fibroblasts. Altogether, these findings support the hypothesis that the derangement of cholesterol homeostasis is a systemic alteration involving central but also peripheral cells of AD patients, and point to cholesterol ester levels in AD fibroblasts as an additional metabolic hallmark useful in the laboratory and clinical practice.

Antiprion activity of cholesterol esterification modulators: a comparative study using ex vivo sheep fibroblasts and lymphocytes and mouse neuroblastoma cell lines.

Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC(50)] range, 1.4 to 40 microM) were comparable to those of antiprion reference compounds (EC(50) range, 0.6 to 10 microM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.

Modulation of cholesterol homeostasis by antiproliferative drugs in human pterygium fibroblasts.

PURPOSE: The authors have previously shown that the growth of cultured fibroblasts obtained from primary pterygia was associated with an increase in cholesterol esterification, suggesting that alterations of cholesterol homeostasis may be involved in the development and progression of this disorder. This investigation was conducted to determine whether antiproliferative agents such as pioglitazone (PIO) and everolimus (EVE) may inhibit proteins involved in the cholesterol ester cycle and the proliferation of pterygium fibroblasts (PF). METHODS: Quiescent normal conjunctival fibroblasts and PFs were treated with or without inhibitors of cell proliferation (PIO and EVE) or with inhibitors of cholesterol esterification-progesterone (Pg) and Sandoz compound (SaH)-and then were stimulated to growth by 10% fetal calf serum (FCS). Cell proliferation was assessed by counting cells. Trypan blue uptake was used to determine cell viability. mRNA and protein levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. RESULTS: PIO and EVE significantly abolished the increase in cholesterol esters, acyl-coenzyme A cholesterol acyltransferase (ACAT1), and multidrug resistance protein (MDR1) mRNA observed in growing cells. Each inhibitor upregulated ATP-binding cassette-A1 (ABCA1), neutral cholesterol ester hydrolase (NCEH) mRNA, and caveolin-1 expression in a manner similar to that of specific inhibitors of cholesterol esterification such as Pg and SaH. CONCLUSIONS: Intracellular modifications of cholesterol homeostasis may be relevant to pterygium development. Moreover, antiproliferative agents such as PIO and EVE may represent a potential topical medication in the prevention and inhibition of pterygium growth at an early stage, probably by modulation of cholesterol ester metabolism.

Fibroblasts isolated from human pterygia exhibit altered lipid metabolism characteristics.

To determine whether the fibrovascular proliferation observed in pterygium, may be, at least in part, mediated by an increased activity of cholesterol metabolism. The correlation between lipid metabolism and rate of growth was studied in human normal conjunctival (NCF) and primary pterygium fibroblasts (PFs) in primary culture. The expression of two proliferation markers (Ki-67 and p53) was evaluated by immunohistochemical staining techniques. Proliferation was evaluated by [(3)H]thymidine incorporation and by immunohistochemical assays. Lipid metabolism was evaluated by (14)C-oleate incorporated into cholesterol esters as well as by oil red O staining. Moreover, the cultures of pterygium fibroblasts were supplemented with two antiproliferative drugs in order to confirm the effective alterations in cholesterol metabolism related to proliferation. Immunohistochemistry of frozen sections from primary pterygium demonstrated an increased staining in Ki-67 and p53 compared with staining observed in normal conjunctiva. A dramatically increased activity of intracellular cholesterol metabolism was demonstrated in pterygium fibroblasts obtained from four different patients. This finding was confirmed by the reduction of cholesterol metabolism in pterygium fibroblasts treated with antiproliferative drugs. Collectively, these data support the hypothesis that alterations of cholesterol metabolism are involved in the development of pterygia. This finding may represent a target of new therapeutic approaches for treatment and prevention of pterygium.