þÿ

CHANGES IN CHOLESTEROL METABOLISM IN PERIPHERAL CELLS OF

ALZHEIMER DISEASE PATIENTS AND THEIR RELATIVES

Alessandra Pani

, Paolo La Colla

, Claudia Abete

, Claudia Mulas

, Marirosa Putzolu

, Claudia Norfo

, Sergio Laconi

, Anna Borgia

, Cristina Zaru

, Manuela Palmas

Paolo F. Putzu

, Alessandra Mocali

Francesco Paoletti

and Sandra Dessì

Department of Biomedical Sciences and Technologies, University of Cagliari,

Blood Transfusion Center, ASL7, Iglesias,

Alzheimer Center, ASL8, Cagliari and

Department of Experimental Pathology and Oncology, University of Florence, Italy

ABSTRACT

Background. Previous epidemiological and experimental studies indicated cholesterol

as a central player in Alzheimer disease (AD). Here, we utilized skin fibroblasts and

PBMC as possible ex vivo models for the study of dysfunctions of cholesterol

homeostasis which may be related to AD development.

Methods. We analyzed cholesterol homeostasis using colorimetric, thin layer

chromatography (TLC), and histologic technique in ex vivo cultures of skin fibroblasts

and PBMCs from patients with probable AD and their first-degree relatives.

Additionally, healthy age-matched individuals served as controls.

Findings. As compared to controls, skin fibroblasts and PBMCs from AD patients,

displayed an evident alteration of cholesterol metabolism; namely an anomalous

accumulation of cholesterol esters in their cytoplasm. No change in intracellular free

cholesterol was observed. Cellular overloading of cholesterol esters was dramatically

increased after specific growth stimulation of the different cell types. Cholesterol ester

accumulation was negatively correlated to plasma levels of high density lipoprotein

cholesterol (HDL-C) and positively correlated with severity of cognitive symptoms

measured by Mini-Mental State Examination (MMSE). Inhibitors of cholesterol

esterification, such as progesterone and SaH, as well as a potent inhibitor of cell

proliferation, RAD, were able to prevent accumulation of cholesterol esters.

Interpretation. Changes of cholesterol esters in the peripheral compartment may be

indicative of a systemic alteration of intracellular cholesterol homeostasis, which in turn

might create a cellular milieu favourable to the production of ß-amyloid in the brain.

Pathways that control cholesterol esterification might represent promising targets for

novel diagnostic and therapeutic AD approaches.

INTRODUCTION

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive

deterioration of memory and cognition. Neural damage is thought to result from

deposition of

-amyloid peptide (A

), a low molecular weight fragment derived from

the amyloid precursor protein (APP) after proteolytic cleavage by the

-secretases.

1,2

Nature Precedings : hdl:10101/npre.2007.1246.1 : Posted 23 Oct 2007

and 3

. Many studies over the last decade have provided a mass of information about

structural and biochemical co-factors favouring A

deposition in AD patients.

Particular attention has been devoted to changes in cholesterol metabolism.

5, 6,7 and 8

However, these studies carried out in animal models or in cell culture systems have

often yielded conflicting results. Several "in vitro" studies have reported a reduced

secretion of A

in cells that have been depleted of cholesterol

4,6,9

. In line with these

findings some, "in vivo" studies have shown that an environment low in cholesterol

favours the production of the non-amyloidogenic, soluble fragments of (

APPs).

9,10

On the basis of such findings it has been suggested that an high cholesterol content in

rafts, where

-secretases are located, could promote the co-clustering of these enzymes

and APP, thereby promoting the production of APP-derived amyloidogenic cleavage

fragments. However, contrasting with this picture, some "in vivo" evidence would

indicate that low

brain cholesterol levels can in fact promote AD development.

Moreover, some clinical evidence does not support the notion that high brain

cholesterol levels are associate with AD development. In fact, a reduction rather than an

increase in membrane cholesterol was observed in the hippocampus of AD patients by

Ledesma and collaborators

, while

Abad-Rodriguez and collaborators,

demonstrated

that a reduction of membrane

cholesterol in hippocampal neurons from AD patients

resulted in a higher ß-secretase APP co-localization in rafts associated with an elevated

production of A

. Since a comprehensive view of the complex relationship between

cholesterol metabolism and AD is still lacking, it is difficult, at present, to reconcile all

these conflicting reports. However, it is reasonable to suggest that, similarly to other

metabolic and biochemical alterations found in AD patients, alterations in cholesterol

metabolism, rather than being confined to the brain tissue, might be systemic in AD

patients. Indeed, a number of abnormalities in metabolic and biochemical processes

described in AD brains have also been found in cultured skin fibroblasts derived from

AD patients,

14,15

thus supporting the view that AD might be seen as a systemic disorder

and that fibroblasts and other peripheral cells might be useful to identify and to test

hypotheses on brain pathological mechanisms leading to AD. Starting from these

considerations, in the present work we investigated cholesterol metabolism in skin

fibroblasts and in peripheral blood mononuclear cells (PBMCs) from AD patients and

their first-degree relatives as possible ex vivo models for the identification of alterations

in cholesterol homeostatic regulatory pathways that may be relevant to AD

development and progression.

METHODS

Participants. In a first phase of study, we took advantage of a pre-existing bank of skin

fibroblasts collected as part of a project of "Ricerca Finalizzata Alzheimer", funded by

the Ministero della Salute through Regione Toscana and entitled "Identification and

validation of peripheral markers of altered proteolysis in AD patients. All individuals

included in this study were enrolled at the Azienda USL 4, Geriatric Unit of Prato

Hospital (Italy). The dermal biopsies were obtained, following informed written

Nature Precedings : hdl:10101/npre.2007.1246.1 : Posted 23 Oct 2007

consent, from the upper forearm of the subjects by a 2-mm punch after local anesthesia

with 2% xylocaine, according to the guidelines established and approved by the local

Ethical Committee of Azienda USL 4, Prato (Italy).

We analysed fibroblasts from six

non-AD controls (six females, mean age 67.0 ± 6.1 years) and seven AD patients (five

females, two males, mean age 73.8 ± 9.4 years). In a second phase, blood samples were

obtained from 62 patients with probable AD (range: 6491 years; mean ± S.E. = 78.6

0.8), predominantly women (49/62) and from 78 first-degree relatives (range: 26-87

years; mean ± S.E = 47.0

1.3) (55/78 women), recruited at the Geriatric Service of

Alzheimer Center, USL 8, Cagliari (Italy). Informed written consent was obtained from

all the subjects or, when necessary, from their legal guardians under local institutional

review board supervision and approval (# 351, 14 Feb 2003) of local Ethical Committee

of USL 8, Cagliari, Italy. Routine clinical and laboratory evaluation, including magnetic

resonance imaging, was performed to exclude other causes of cognitive impairment.

The Reisberg Global Deterioration Scale (GDS) was used to indicate the severity of the

cognitive impairment in AD patients. Abnormal GDS levels start from level 3 and

maximal deterioration grade corresponds to level 7. Patient evaluation included medical

history, physical and neurological examinations, laboratory blood tests to rule out

metabolic causes of dementia (thyroid hormones, Vitamin B12, and erythrocyte

sedimentation rate), and a neuroimaging (computed tomography and/or magnetic

resonance) of the brain. In addition, all patients received neuropsychological tests

including the Mini-Mental State Examination (MMSE) (mean ±S.E. = 18.9±0.5).

Patients with neoplastic or hematological disorders, recent infections or surgery, severe

hepatic or renal failure, myocardial infarction or cranial trauma in the previous 6

months, or who had received statins, antineoplastic, corticosteroid, or

immunosuppressive drug treatments were not included in the present study.

Additionally, 114 individuals: 30 volunteers aged between 65-87 years (22/30 women)

with no cognitive impairment, as determined by clinical interview and by a normal

MMSE score (29.4 ± 0.6) and 84 blood donors, recruited at the Centro Trasfusionale,

USL7, Iglesias, Italy, (35/84 women) aged between 19 and 69 years;

mean±S.E.=42.5±1.3, with no personal or family history of neurological or psychiatric

disorders, served as controls.

Cell cultures. Skin fibroblasts were isolated by dermal biopsies obtained from the

upper forearm by a 2-mm punch after local anesthesia with 2% xylocaine. Biopsies

were washed with sterile PBS supplemented with penicillin and streptomycin, minced

to pieces, transferred to culture dishes and overlaid with a coverslip. Cultures were

incubated at 37°C in 5% CO

humidified atmosphere with DMEM containing 4.5 g/l

glucose and 10% FCS for 18-21 days to obtain primary cultures of human diploid

fibroblasts. These were then subcultured

seeded onto 60-mm plastic Petri dishes

(28 cm

) in 10% FCSDMEM and grown to confluence All experiments were

completed using fibroblasts between passages two to three in culture. For "in vitro"

kinetic experiments, cells were plated at a density of 10.000 cell/cm

in 6 well plates

and then incubated for 48 h in DMEM with 0.2% FCS to synchronize cells at a

quiescent state. Quiescent fibroblasts were then diluted in complete growth medium

Nature Precedings : hdl:10101/npre.2007.1246.1 : Posted 23 Oct 2007

with 10% FCS, either in the presence or in the absence of 20 nM of 40-O-(2-

hydroxyethyl) rapamycin (RAD) (supplied by Novartis Pharma AG, Basel,

Switzerland). In addition, 10

M of Progesterone (PG) (Sigma-Aldrich,

St. Louis, MO)

and 4

M of the acyl amide ACAT inhibitor SaH (Novartis Pharma AG, Basel) was

added to some cultures as a broad-spectrum regulators

of cholesterol ester cycle.

Preliminary experiments were carried out to find the minimal effective dosages with the

least effect on cell viability. Cells were harvested at indicated time points after

treatments. Cell proliferation was assessed by direct cell counting with a

hemocytometer. The trypan blue exclusion test was used to assess cell viability.

Blood samples. Blood samples were centrifuged at 2200 rpm for 15 min to separate

plasma. Plasma was removed, transferred to centrifuge tubes and stored at 80C° until

analyses. The buffy coat was collected and the peripheral blood mononuclear cells

( PBMCs ) were isolated by density-gradient centrifugation (Lymphoprep; density,

1.077 g/L; Nycomed Pharma, Oslo, Norway) at 1200 rpm for 10 minutes at 20°C, and

washed twice with Hanks balanced salt solution (HBSS). Purified PBMCs were then

seeded onto 24-well plates at 1.0 × 10

/mL in RPMI 1640 medium with the standard

supplements: (10% fetal calf serum (FCS), 200 mM L-glutamine, and 100 U/mL

penicillin-streptomycin) and, where indicated, growth-stimulated with 10 µg/ml

phytohemagglutinin (PHA; Sigma) in the presence or in the absence of 20 nM of RAD.

Plasma lipid profile.Total (TC) and HDL cholesterol (HDL-C) content was determined

in plasma by routine colorimetric enzymatic procedures (Sclavo Diagnostics

International S.r.l. Sovicille, Italy).

Cholesterol esterification and lipid cell content. Cholesterol esterification was

evaluated by incubating cells for 6 hours in medium containing [1-

C]oleic acid

(Dupont, NEN 55 mCi/mmol), bound to bovine serum albumin (BSA) at a final

concentration of 2

Ci/ml. After incubation cells were washed with PBS and lipids

extracted with acetone. Lipid subclasses were separated by thin layer chromatography

(TLC ) and incorporation of [14C]oleate into cholesterol esters was measured. For lipid

cell content determinations, free (FC) and esterified cholesterol (CE) were separated by

TLC and their mass determined by enzymatic assay methods.

Neutral lipid staining.

To visualize the degree of cytoplasmic neutral lipid

accumulation,

cultured fibroblasts and freshly isolated PBMCs were washed three times

with PBS and fixed by soaking in 10% formalin. Cells were then treated with isopropyl

alcohol (60%), washed, stained with oil red O (ORO) (a lipid-soluble dye that stains

cholesterol esters but not free cholesterol) and counterstained with Mayer's

hematoxylin. Stained cells were examined by light microscopy and digital images were

recorded. For quantification of mean neutral lipid content in skin fibroblasts, red color

intensity was measured in single cells using the software Scion Image (Scion Corp.,

Fredrick, Maryland, USA). Values were expressed as the mean of red color intensity in

each cell, determined in at least 30 single cells in six random microscopic fields.

Neutral lipids in ORO stained PBMCs calculated as above were further graded into 5

Nature Precedings : hdl:10101/npre.2007.1246.1 : Posted 23 Oct 2007

classes: 0 (0), 1 (+), 2 (++), 3 (+++) and 4 (++++) based on mean color intensity

measurements.

Statistical analysis. Data are reported as mean ± standard error (SE). Statistical

calculations were performed using the statistical

analysis software of Origin 7.0 version

(Microcal, Inc, Northampton, MA, USA). A value of P<0.05 was considered to be

statistically

significant. Correlation analysis were performed by correlation test of

Microsoft Excel for Statistics.

Funding source. This study was supported by grants of Regione Autonoma della

Sardegna and of Monte dei Paschi di Siena.

RESULTS

A number of abnormalities in metabolic and biochemical processes described in AD

brains have been found in cultured skin fibroblasts derived from AD patients.

14,15

Prompted by our previous findings of a tight correlation between cell growth and

cholesterol ester accumulation

16,17,18 and 20

in various cell systems and by studies

demonstrating abnormally high levels of intracellular lipid droplets (mainly cholesterol

esters) in cell-based and animal models of AD

21, 22, 23, and 24

, we initially sought to explore

a possible relationship between growth capability and the extent of cholesterol

esterification in skin fibroblasts obtained from AD patients (AD fibroblasts). Direct cell

counting revealed a significantly higher mytogenic response of AD fibroblasts as

compared to control fibroblasts 72 hours after 10% FCS stimulation (5x10

vs. 3x10

cells/ml). As expected, the increase in cell number correlated with a parallel increase in

the rate of cholesterol esterification (data not shown). Noteworthy, total cholesterol was

significantly higher in AD fibroblasts as compared to controls (59,3

2.0

cholesterol/mg cell protein vs. 45.0

1.5

g cholesterol/mg cell protein ); this

increment was mainly accounted by an increased CE content in AD fibroblasts and

resulted in a striking decrease in the FC /CE ratio in these cells (2.5

0 3, vs 11.2

1.2). Analysis by ORO staining revealed that unstimulated control fibroblasts contain

very low basal levels of cytoplasmic neutral lipid droplets

(figure 1), confirming that,

under normal conditions, intracellular cholesterol esters are maintained at low levels,

possibly by a cyclic process

of de-esterification and re-esterification, as originally

suggested by Goldstein and Brown

. On the other hand, ORO staining of AD-

fibroblasts confirmed that this regulatory mechanism is altered in AD patients, as

evidenced by the greatly expanded cytoplasmic CE pool in these cells (figure 1 A and

B). Moreover, the finding that, three different inhibitors of cholesterol esterification

were able to reduce lipid droplet accumulation in AD fibroblasts (figure 1B) constitutes

a strong evidence that this expanded CE pool is, at least partly, due to an increased

esterification activity. Taken together, these results suggest that the mechanism(s)

involved in the regulation of cholesterol esterification may be systemically altered in

AD-patients.

Nature Precedings : hdl:10101/npre.2007.1246.1 : Posted 23 Oct 2007

Figure 1. Neutral lipids in fibroblasts from controls and AD patients. The upper panel (A)

shows analysis of ORO-stainings performed on six controls and seven AD different fibroblast

cell cultures. Bars indicate the mean ± SE of red color intensity/cell, determined in each culture

well in at least 30 single cells in six random microscopic fields.

p < 0.05 vs. control values.

Panel B shows lipid droplet accumulation in 48 hours FCS stimulated AD fibroblasts incubated

in the presence or in the absence of SaH. PG, and RAD. Shown are results from 1 control and 1

AD patient which are representative of six controls and seven AD different fibroblast cell lines.

If this hypothesis is correct, than at least in theory, one should expect that this systemic

alteration could also be reflected by changes of lipid levels in the plasma. To test such a

possibility we thus studied TC and HDL-C levels in plasma and neutral lipid content in

PBMCs from 62 patients with probable AD as well as from 30 age-matched healthy

controls. Levels of TC were moderately decreased in the plasma from AD patients as

compared to controls but this was not statistically significant. By contrast, plasma HDL-

C levels were significantly decreased, in AD patients

(figure 2A) as compared to controls. This paralleled with increased levels of CE in AD-

PBMCs (figure 2B) indicating that abnormally low levels of HDL-C coupled with

cytoplasmic accumulation of CE might be relevant in AD pathogenesis.

If examined individually, the majority of AD-PBMCs (53%) had ORO staining levels

(calculated as stated in Methods) that scored between 3 and 4, while the majority of

PBMCs from controls (58 %) scored between 0 and 1 (figure 3A). In addition,

correlation test revealed that in AD patients,

ORO staining levels

in PBMCs were

negatively correlated with HDL-C levels

in plasma

(r=-0.44, P< 0.05) (figure 3B)

Nature Precedings : hdl:10101/npre.2007.1246.1 : Posted 23 Oct 2007

Although the significance, if any, of observed correlation between low HDL-C levels

and AD risk remains to be established,

the data presented above lead to suggest

that the

low HDL-C levels in the plasma of AD patients may be a consequence of a decreased

removal rate of cholesterol from tissues, possibly as a result of the increased rate of

cholesterol esterification and intracellular lipid droplet accumulation.

Interestingly,

the

intensity of ORO staining in PBMCs of AD was significantly and directly correlated

with the severity of cognitive alteration, determined by the MMSE scores (r = -0.41;

p < 0.05) (figure 3 C). This last finding would suggest that, rather than being just an

epiphenomenon, this metabolic alteration might have some clinical relevance and/or

perhaps contribute to the pathogenesis of this disease.

Figure 2. Plasma cholesterol and neutral lipids in PBMCs from AD patients (AD-PBMCs).

Panel A shows TC and HDL-C in plasma of 62 AD patients and 30 age-matched controls.

*P<0.05 vs controls. Panel B shows neutral lipid droplet accumulation as determined by ORO

method in quiescent (0h) and 48 hours PHA stimulated AD-PBMCs incubated in the presence

or in the absence of RAD.

Nature Precedings : hdl:10101/npre.2007.1246.1 : Posted 23 Oct 2007

Figure 3. Correlation between neutral lipid in PBMCs and HDL-C levels in plasma. Panel A.

Numbers of control and AD blood samples, distributed according to the intensity of the ORO

bound lipids, and sub-divided into five classes: (0), 1(+), 2 (++), 3 (+++) and 4 (++++) based

on intensity

measurements. Panel B Correlations between HDL-C and ORO intensity in AD

and age-matched controls. Panel C Correlations between MMSE scores and ORO intensity in

AD and age-matched controls.

It is well known that individuals who have had a late onset-AD case in their family may

run an increased risk of developing AD themselves.

However, with the exception of

apolipoprotein E polymorphisms,

27 and 28

no accepted indicator is so far available

estimate risk in AD relatives.

It will be thus worth investigating whether altered

cholesterol metabolic profile might serve for the pre-clinical identification of a subset

of AD relatives who might be at major risk of developing AD later in their lifetime.

therefore

studied HDL-C levels in plasma and the content of ORO lipid droplets in

PBMCs in 78 first-degree relatives of AD patients and compared the data with those

obtained from 84 age-matched healthy controls. When compared to both the AD and

control groups, a greater variability was observed in the data obtained from first-degree

AD relatives (mean age: 47.0

1.3), and a partial overlap was present with the data

from a control group (mean age: 42. 5

1.3) (figure 4A). However, 41% of PBMCs

from AD relatives had ORO staining levels that scored between 2 and 3, as compared to

only 18% of PBMCs from controls (Figure 4B).

Figure 4. Correlation between neutral lipid in PBMCs and HDL-C levels in plasma of first

degree relatives of AD patients. Panel A. Total and HDL-C levels. Panel B. Numbers of control

and AD first degree relatives blood samples, distributed according to the intensity of the ORO

bound lipids, and sub-divided into five classes: (0), 1(+), 2 (++), 3 (+++) and 4 (++++) based

Nature Precedings : hdl:10101/npre.2007.1246.1 : Posted 23 Oct 2007

on intensity

measurements. Panel C. Correlations between HDL-C and ORO intensity in AD

first degree relatives and age-matched controls.

In addition, similarly to what observed in AD patients,

plasma

HDL-C levels were

significantly correlated with

ORO intensity

(r=-0.39, P< 0.05) in

AD relatives

(figure

4C), indicating that some AD relatives tend to display a cholesterol metabolic pattern

similar to that of typical AD patients. Interestingly, three unaffected relatives (two

brothers and 1 sister) of comparable age of three AD patients did not show relevant

accumulation of neutral lipid in their PBMCs (figure 5).

Figure 5. Comparison of ORO staining between three AD patients and three unaffected

relatives (two brothers and 1 sister) of comparable age.

DISCUSSION

Many experimental and clinical studies indicate that

cholesterol plays a role in AD

1,5, 23

The exact nature of this role, however, remains elusive, since available data are often

conflicting and in most studies no

information on intracellular cholesterol pools (free

vs. esterified) and/or lipoprotein fractions was available. High levels of total plasma

cholesterol have been variably associated with either increased

7,29.30 and 31

or decreased

risk of AD, while other studies have found

no association at all. Early work that served

as a rationale for the use of statins as novel "quick and easy" therapy for AD

, had to

be entirely re-examined when three independent prospective studies, altogether

accounting for about 30,000 person-years at risk for AD, found no protective effect of

statins against AD development. The rationale to the use of statins in AD was based on

the hypothesis that high cholesterol levels accelerate the production of ß-amyloid by

shifting APP metabolism from alpha to beta cleavage products. According to this

hypothesis, statins, blocking cholesterol synthesis, would cause a reduction in neuronal

membrane free-cholesterol content, thus inhibiting the activity of

-secretases. While

this theoretical arguments were initially supported by some in vitro observations, a

recent epidemiological study found that statins use was associated with a greater risk of

AD.

To explain this surprising result, the authors of this study have suggested that

Nature Precedings : hdl:10101/npre.2007.1246.1 : Posted 23 Oct 2007

statins might exacerbate AD progression by inhibiting cholesterol synthesis at the

neuronal level, thus altering cholesterol-dependent synaptic integrity

and

neurotransmission. In an attempt to understand the reasons for these apparently

conflicting reports and to have a larger view of the relationship between cholesterol and

AD, in the present study we investigated and compared the overall cholesterol

metabolism in patients with probable AD and age-matched healthy controls. We

evaluated lipid profiles in the plasma compartment as well as intracellular cholesterol

pools in skin fibroblasts and/or PBMCs. Unlike fibroblasts and PBMCs from controls,

which were virtually devoid of lipid droplets, cells from AD patients accumulated a

large number of lipid droplets (mainly cholesterol esters). This accumulation was

associated with lower levels of plasma HDL-C, while no change in plasma TC levels

was observed between AD patients and age-matched controls. Interestingly, the extent

of neutral lipid accumulation in cells was inversely correlated with HDL-C levels in

plasma. The combination of low plasma HDLC and cellular lipid accumulation is

intriguing in the light of the proposed role of HDL in unloading cholesterol from

tissues, including brain, and transporting it back to the liver In this respect, low HDL-C

levels would seem to indicate that lipid export from cells is defective in AD patients,

possibly leading to increased amounts of cholesterol ester storage in the cytoplasm. This

view is corroborated by studies showing that HDL-C and ApoA-I (the main

apolipoprotein of HDL) levels, are significantly lower in the plasma of AD patients in

comparison to the controls

34,35

and that deficiency in ATP-binding cassette ABCA1

the major regulator of cholesterol efflux, increases

-amyloid deposition. Low HDL-C

levels in plasma and a reduction of LDL receptor expression in PBMC were also found

in Down syndrome patients which, in spite of high risk for AD, show a low prevalence

of coronary artery disease.

In the present study we found that skin fibroblasts from AD patients, although showing

a normal content of free cholesterol, display an evident alteration of cholesterol

metabolism; namely an anomalous accumulation of cholesterol esters in their

cytoplasm. This intracellular overloading with cholesterol esters was dramatically

increased after mitogenic stimulation, while two different inhibitors of cholesterol

esterification, such as progesterone and SaH, as well as a potent inhibitor of cell

proliferation, RAD, were able to prevent it. We hypothesize that these observed changes

in cholesterol esters in the peripheral compartment may be indicative of a systemic

alteration of intracellular cholesterol homeostasis, which in turn might create a cellular

milieu favourable to the production of ß-amyloid in the brain.

In agreement with this hypothesis, Kovacs et al. found that cholesterol-ester levels are

directly correlated to A

production: a decrease in the content of lipid droplets led to a

decrease in the production of A

, whereas abnormally high levels of lipid droplets led

to an increase in amyloid beta production.

21,24

The Authors also showed that drugs

known to inhibit the activity of acyl-coenzyme A:cholesterol acyltransferase (ACAT),

the enzyme that catalyses the formation of cholesterol esters, are potent modulators of A

generation

21,24

. Moreover, a RNAi-induced, 50% reduction of ACAT expression

resulted in a significant suppression of A

production (

40%) confirming that

Nature Precedings : hdl:10101/npre.2007.1246.1 : Posted 23 Oct 2007

reduction of cellular ACAT activity represents a realistic and specific approach for the

modulation A

generation. Therefore,

inhibitors of this enzyme may turn out to be an

extremely important tool in the treatment of AD.

Another interesting finding of our study was that neutral lipid accumulation in PBMCs

correlated positively with the severity of dementia, expressed by the MMSE score.

These observations not only indicate that a progressive systemic alteration in

cholesterol metabolism accompanies the development of mental deterioration, thus

implying measurements of neutral lipids in peripheral cells, together with HDL-C in

plasma, may be useful in the monitoring of AD progression, but also suggests that

assessment of cholesterol metabolisms in peripheral cells among asymptomatic AD

relatives might serve to identify those that carry a higher risk of developing the disease

at older ages

To our knowledge, these findings are the first ex vivo demonstration that

alterations of cholesterol metabolism, which are associated with AD and parallel disease

severity, are also present in peripheral cells, and support biochemical, epidemiological,

and genetic literature arguing for a deep link between cholesterol and AD.

1,5, 23

Focusing

on the relationship between cholesterol and APP metabolism would be important for

etiological as well as for therapeutic purposes, as it might help to understand whether

early pharmacological interventions on cholesterol levels could provide a relatively safe

mean to reduce A

accumulation both in brain and in endothelial compartment. These

data confirm that new therapeutic approaches targeting lipid metabolism may be useful

to slow down AD progression.

In summary, further studies are necessary to elucidate the mechanisms responsible for

the alterations of cholesterol homeostasis in AD patients and in their first degree

relatives, and to unveil possible cause-effect relationships between APP cleavage and

these alterations. Similarly, additional work is needed to fully explore the potential

implication of this association for AD susceptibility. Nonetheless, the data reported in

the present paper suggest that accumulation of cholesterol esters in peripheral cells,

together with a decrease of HDL-C in the plasma might serve to identify a distinctive

profile associated with increased susceptibility to AD.

In addition, the lack of an AD

diagnostic has limited so far the development of specific drugs and the assessment of

the response of peripheral tissues to disease-modifying therapies. Fibroblasts and/or

PBMCs from AD patients could represent an excellent in vitro model to test the toxicity

as well as the efficacy of agents capable of restoring the control of cholesterol

homeostasis toward the profile of the normal phenotype.

Acknowledgements. The Authors thank volunteers, blood donors and AD patient

relatives that with enthusiasm have accepted to participate to this study. Appreciation is

extended to Anna Saba and Martina Ciuffo for technical assistance.

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