Fatty acid-based lipidomics and membrane remodeling induced by apoE3 and apoE4 in human neuroblastoma cells

    Fatty acid-based lipidomics and membrane remodeling induced by apoE3 and apoE4 in human neuroblastoma cells Paraskevi Prasinou, Ioannis Dafnis, Giorgia Giacometti, Carla Ferreri, Angeliki Chroni, Chryssostomos Chatgilialoglu PII: DOI: Reference:

S0005-2736(17)30225-0 doi:10.1016/j.bbamem.2017.07.001 BBAMEM 82533

To appear in:

BBA - Biomembranes

Received date: Revised date: Accepted date:

28 March 2017 19 June 2017 4 July 2017

Please cite this article as: Paraskevi Prasinou, Ioannis Dafnis, Giorgia Giacometti, Carla Ferreri, Angeliki Chroni, Chryssostomos Chatgilialoglu, Fatty acid-based lipidomics and membrane remodeling induced by apoE3 and apoE4 in human neuroblastoma cells, BBA - Biomembranes (2017), doi:10.1016/j.bbamem.2017.07.001

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Fatty acid-based lipidomics and membrane remodeling induced by apoE3 and apoE4 in human neuroblastoma cells

T

Paraskevi Prasinoua, Ioannis Dafnisb, Giorgia Giacomettia, Carla Ferreric, Angeliki Chronib and

SC R

a

IP

Chryssostomos Chatgilialoglua,c,*

Institute of Nanoscience and Nanotechnology, N.C.S.R. "Demokritos", 15310 Agia Paraskevi,

Athens, Greece b

Institute of Biosciences and Applications, N.C.S.R. “Demokritos”, 15310 Agia Paraskevi, Athens,

c

NU

Greece

Institute for the Organic Synthesis and Photoreactivity, Consiglio Nazionale delle Ricerche, 40129

MA

Bologna, Italy

* Corresponding author at: Consiglio Nazionale delle Ricerche, 40129 Bologna, Italy.

TE

D

E-mail address: [email protected] (C. Chatgilialoglu)

Abstract

CE P

Apolipoprotein E (apoE) is a major lipid carrier of the lipoprotein transport system that plays critical roles in various pathologies. Human apoE has three common isoforms, the apoE4 being associated with Alzheimer’s disease. This is the first study in the literature investigating the effects

AC

of apoE (apoE3 and apoE4 isoforms) on membrane fatty acid profile in neuroblastoma SK-N-SH cells. Fatty acid analyses were carried out by gas chromatography of the corresponding methyl esters (FAME). We observed the occurrence of membrane fatty acid remodeling in the presence of each of the two-apoE isoforms. ApoE3 increased the membrane level of stearic acid and dihomogamma-linolenic acid (DGLA), whereas apoE4 had opposite effects. Both apoE3 and apoE4, increased saturated and monounsaturated fatty acids (SFA and MUFA), omega-6/omega-3 ratio and decreased total polyunsaturated fatty acid (PUFA) amount, but with various intensities. Moreover, both apoE isoforms decreased membrane homeostasis indexes such as PUFA balance, unsaturation index and peroxidation index. Our results highlight membrane property changes connected to the apoE isoforms suggesting membrane lipidomics to be inserted in further model studies of apolipoproteins in health and disease.

1

ACCEPTED MANUSCRIPT

NU

SC R

IP

T

Graphical abstract

Highlights

opposite effects in neuroblastoma cells

MA

• ApoE3 increased stearic acid and dihomo-gamma-linolenic acid (DGLA), whereas apoE4 had

• Both apoE3 and apoE4 isoforms increased SFA, MUFA and omega-6/omega-3 ratio and

D

decreased total PUFA, but with various intensities

CE P

peroxidation indexes

TE

• Both apoE3 and apoE4 isoforms decreased PUFA balance as well as unsaturation and

Keywords

AC

membrane remodeling; fatty acids; lipidomics; apolipoprotein E isoforms Abbreviations

AD: Alzheimer’s disease, Aβ: amyloid beta peptide, apoE: apolipoprotein E, ARA: arachidonic acid, DHA: docosahexaenoic acid, DGLA: dihomo-gamma-linolenic acid, EPA: eicosapentanoic acid, FAME: fatty acid methyl esters, GC: gas chromatography, LA: linoleic acid, MUFA: monounsaturated fatty acids, PI: peroxidation index, PUFA: polyunsaturated fatty acids, SFA: saturated fatty acids, TFA: trans fatty acids, UI: unsaturation index

2

ACCEPTED MANUSCRIPT 1. Introduction Apolipoprotein E (apoE) is a major lipid carrier of the lipoprotein transport system that plays critical roles in atherosclerosis, dyslipidemia and various neuropathological processes. In particular,

T

liver primarily synthesizes plasma apoE, while central nervous system (CNS) apoE can be

IP

synthesized locally in the brain by astrocytes and to a lesser extent by microglia and neurons [1,2]. In the brain, apoE is crucial for cholesterol transport with a role in neuroplasticity-related

SC R

phenomena, since it intervenes in lipid changes and cholesterol dependent function [3]. ApoE is produced by injured and stressed neurons, being of critical importance for membrane repair and remodeling [4]. ApoE has been associated with Alzheimer’s disease (AD). In AD the accumulation

NU

of abnormally folded amyloid beta peptide (Aβ) and the hyper-phosphorylation of tau protein, forming amyloid plaques and intracellular neurofibrillary tangles (NFTs), respectively, induce the

MA

loss of neurons and synapses in the cerebral cortex and certain subcortical regions, brain atrophy and inflammation [5,6]. Several model studies pointed out the importance of the lipidation status of apoE since it can influence the protein conformation [7], the interaction of apoE with lipid

D

transporters that are involved in the cellular lipid homeostasis [8] and the affinity of apoE for Aβ

TE

peptides [9]. The apoE lipidation status can also alter its ability to promote the degradation and clearance of Aβ from brain [10].

CE P

Human apoE has three common isoforms that differ from each other by only one or two amino acids (apoE2 Cys-112/Cys-158, apoE3 Cys-112/Arg-158, and apoE4 Arg-112/Arg-158) and its polymorphism affect the risk for late-onset forms of Alzheimer's disease. The ε4 allele increases individual’s risk for AD as compared to the ε3 allele (the most frequently encountered worldwide),

AC

whereas the ε2 allele is considered to be protective [11-15]. Although apoE4 carriers account for almost the half of AD patients, its exact mode of action is unknown. Several hypotheses have been advanced to elucidate the association of apoE4 with AD and to probe the mechanism of this linkage. It has been proposed that apoE binds Aβ and induces changes in both Aβ conformation and levels. In addition, apoE can modulate Aβ accumulation and deposition in the brain in an isoformand lipidation status-dependent manner [16-18]. It has been demonstrated that apoE4 is much more susceptible to proteolysis than apoE3 and apoE2 and carboxyl-terminal truncated forms of apoE4 have been found in brains of AD patients and apoE4 transgenic mice [19-21]. Specific apoE4 fragments have been linked with early events in the pathogenesis of AD including neuroinflammation, tau pathology, and accumulation of Aβ42 in neurons accompanied with the induction of oxidative stress [20-23]. Other studies have associated the differential effects of apoE isoforms on AD pathogenesis with differences in their ability to affect neuronal repair and brain lipid transport or metabolism [17]. Furthermore, it has been shown that apoE can exert protective 3

ACCEPTED MANUSCRIPT effects against free radicals, but the response is isoform dependent. It appeared that, among the different isoforms, apoE4 is the less efficient in acting as a free radical scavenger and is more sensitive to attacks by free radicals [24,25]. In addition, massive disruption of the total membrane

T

lipid bilayers caused by Aβ1-40 was reported in the presence of apoE4 but not of apoE3,

IP

individuating apoE4 as promotive element for Aβ fibrillation and membrane oxidative damage [26]. In the current state of knowledge, a few data are available on the molecular effects of apoE

SC R

regarding lipid remodeling using in vitro experiments. Apolipoprotein E isoforms were found to enhance triglyceride-rich lipoprotein particle uptake, triglyceride utilization and cholesteryl ester hydrolysis in macrophages [27]. ApoE4-165 fragment was found to strongly reduce sphingomyelin

NU

(SM) levels in SK-N-SH cells, thus inducing changes in the micro-fluidity [28]. Incorporation of SM rather than PC in lipid emulsion models of lipoproteins reduced the binding of apoE to the

MA

emulsions and the apoE-mediated uptake of the emulsions by human hepatoma HepG2 cells. [29]. Very recently, the importance of cholesterol and phospholipid (PL) levels for the protein functioning in neuronal cells and their involvement in neurological diseases was assessed [30,31].

D

So far, information is lacking regarding the type or unsaturation degree of the fatty acids involved;

TE

however the relevance of PUFA omega-3, such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, for neuronal health is known [32,33].

CE P

Considering our interest in the diagnostic significance of fatty acid-based membrane lipidomics for human health [34], we planned a preliminary study to investigate whether fatty acid remodeling of cell membranes occurs in the presence of apoE proteins. Indeed, differences in the lipid organization induced by apoE isoforms could be synergic with other molecular changes of

AC

lipid supply and metabolic pathways, as listed above. Herein we report the fatty acid remodeling of human neuroblastoma cell line (SK-N-SH) exposed to the apoE3 and apoE4 isoforms. We show that the treatment with either apoE3 or apoE4 isoforms has induced significant changes in the organization of the membrane fatty acids, which predominantly influence membrane properties and peroxidation susceptibility.

2. Materials and methods Human neuroblastoma SK-N-SH cells were purchased from American Type Culture Collection – ATCC (Manassas, VA, USA). Fetal bovine serum (FBS) was purchased from Biosera (France). Minimum Essential Medium (Eagle), L-glutamine, nonessential amino acids, sodium pyruvate and sodium bicarbonate were from Biochrom AG (Berlin, Germany). Antibiotics (penicillin and streptomycin) and Phosphate-buffered saline (PBS) were purchased by Sigma Aldrich (St. Louis, MO, USA); 0.05% trypsin solution and potassium hydroxide were purchased by Merck Millipore 4

ACCEPTED MANUSCRIPT (Bedford, MA, USA). Chloroform, methanol and n-hexane, were purchased from Merck (HPLC grade) and used without further purification. Silica gel thin-layer chromatography was performed on Merck silica gel 60 plates (0.25 mm thickness) and the spots were detected by spraying the plate

T

with cerium ammonium sulfate reagent. Standard FAME references were commercially available

IP

from Supelco (USA) and all compounds were used without further purification.

SC R

2.1 Production and purification of apoE

The expression and purification of apoE4 and apoE3 was carried out according to protocols already described before [35,36]. BL21-Gold(DE3) cells were transformed with the expression vectors

NU

pET32-E43C [37] or pET32-E33C [38] carrying the gene sequence of apoE4 or apoE3, respectively, and cultured in LB medium containing 100 μg/ml ampicillinat 37 oC. Protein

MA

expression was induced with IPTG (final concentration 0.5mM) for 2 h. Cells were lysed in 20 mM Tris-HCl buffer, pH 8.0 containing 0.5 M NaCl, complete mini EDTA-free protease inhibitors cocktail and 0.1 mg/ml lysozyme (40 ml per 1 L of original culture) by using a French Press (SLM-

D

AMINCO, USA) and the lysate was centrifuged to remove cellular debris. For apoE3, lysis buffer

TE

was supplemented with 1 mM β-mercaptoethanol. The Trx-fused apoE isoforms in the supernatant were purified by Ni-NTA chromatography as follows: the supernatant was adjusted to contain 20

CE P

mM Tris-HCl, pH 8.0, 0.5 NaCl, and 5 mM imidazole and incubated with 4 ml (per 1L of original culture) Ni-NTA resin, under gentle stirring, at 4 °C overnight. The following day, the Ni-NTA suspension was loaded onto an empty chromatography column and washed with 10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5 or 10 mM imidazole. The Trx-fused apoE4 was eluted with 20 mM Tris-

AC

HCl, pH 8.0, 0.5 M NaCl containing 50–500 mM imidazole. The Trx-fused apoE3 was eluted with 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl containing 100–300 mM imidazole. The collected fractions were dialyzed extensively against 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, at 4 °C. Following dialysis, His-tagged 3C protease (prepared as described previously [37]) was added at a ratio of 1/70 (3C-protease/Trx-apoE, w/w) and cleavage of Trx from apoE isoforms was allowed to proceed for 18 h at 4 °C in 20 mM Tris-HCl buffer, pH 8.0 containing 0.5 M NaCl and 1 mM DTT. The apoE isoforms were separated by the cleaved tag by a second Ni-NTA resin affinity chromatography step. To facilitate the dissociation of non-covalent complexes among any uncut apoE, cut apoE and cut Trx the cleavage reaction protein solution was adjusted by adding 6 M urea (final concentration) and the solution was incubated with 1 ml of Ni-NTA resin (per 4 mg of protein) for 1 h at 4 °C. The suspension was loaded onto an empty chromatography column and the cut isoforms were found in the column flow-through. Each apoE solution was extensively dialyzed against 5 mM NH4HCO3, lyophilized, and stored at − 80 °C. Before analyses, lyophilized stocks of 5

ACCEPTED MANUSCRIPT proteins were dissolved in 6 M guanidine hydrochloride (GndHCl) in 50 mM sodium phosphate buffer pH 7.4 containing 1 mM DTT and refolded by extensive dialysis against 50 mM sodium phosphate buffer pH 7.4, 1 mM DTT. The refolded proteins were at least 95–98% pure, as

IP

T

estimated by SDS-PAGE.

2.2 Cell cultures

SC R

Human neuroblastoma SK-N-SH cells were cultured in Minimum Essential Medium Eagle medium supplemented with 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, 10% (v/v) FBS and antibiotics. Care was taken to use the same FBS

NU

batch in all experiments. Cells were plated on 100-mm Petri dishes at a density of 106 cells/dish and cultured for 24h at 37°C in the presence of 5% CO2. The cells were then washed and incubated with

MA

fresh serum-free medium in the presence or absence of 1 µM of lipid-free apoE3 or apoE4 for 24h. At the end of the treatment, cells were thoroughly washed using PBS and pelleted by centrifugation at 2500rpm x 10min x 4°C using a Heraeus, Labofuge 400R table centrifuge (Thermo Scientific,

TE

D

Rockford, IL, USA).

2.3 Lipid Extraction and GC Analysis

CE P

Cell (106) membrane PL were isolated using the well-established Folch method [38]. Briefly, the pellet was re-suspended in pure water and lipids were extracted with 2:1 chloroform:methanol. Thin layer chromatography using chloroform / methanol / water 65: 25: 4 was performed to determine the purity of the PL fraction [39]. The PL residue was transesterified with a 0.5 M KOH solution in

AC

methanol for 10 minutes at room temperature. The corresponding fatty acid methyl esters (FAME) were extracted with n-hexane and analyzed by Agilent 7890B GC system equipped with a flame ionization detector and a DB-23 (50%-Cyanopropyl)-methylpolysiloxane capillary column (60 m, 0.25 mm i.d., 0.25 µm film thickness).The initial temperature was 165°C, held for 3 min, followed by an increase of 1°C/min up to 195°C, held for 40 min, followed by a second increase of 10°C/min up to 240°C, held for 10 min. The carrier gas was hydrogen, held at a constant pressure of 16.482 psi. All the FAME were identified by comparison with the retention times of standard references either commercially available or obtained by synthesis, as already described elsewhere [40].

2.4 Statistical analysis Results are given as means ± SD. Six samples were included in each group. Statistical significance (P values) of the results was calculated by unpaired two-tailed Student's t test using GraphPad

6

ACCEPTED MANUSCRIPT PrismTM software version 6.01 for Windows (GraphPad Software Inc, La Jolla,CA, USA). The threshold for significance was set up at 0.05.

T

Table 1 Fatty acids obtained from SK-N-SH cell membrane phospholipids after incubation with 1

apoE3

n=6b

n=6b (p value)c

16:0

25.70 ± 0.45

26.60 ± 0.82 (0.0396)

9c-16:1

6.55 ± 0.72

6.91 ± 0.69 (0.4014)

18:0

9.72 ± 0.33

10.70 ± 0.58 (0.0049)

9.22 ± 0.14 (0.0043)

0.0002

9t-18:1

0.03 ± 0.01

0.04 ± 0.04 (0.4072)

0.06 ± 0.04 (0.1065)

0.5435

9c-18:1

36.76 ± 0.71

36.54 ± 0.59 (0.5668)

35.40 ± 0.36 (0.0018)

0.0024

11c-18:1

7.03 ± 0.20

7.47 ± 0.14 (0.0014)

7.23 ± 0.04 (0.0432)

0.0023

LA

1.51 ± 0.04

1.47 ± 0.03 (0.0653)

1.53 ± 0.04 (0.4581)

0.0134

DGLA

0.42 ± 0.00

0.47 ± 0.02 (<0.0001)

0.39 ± 0.02 (0.0200)

<0.0001

ARA

5.59 ± 0.12

4.95 ± 0.04 (<0.0001)

5.63 ± 0.12 (0.5377)

<0.0001

Ʃ mono-trans ARA

0.16 ± 0.06

0.12 ± 0.01 (0.1819)

0.14 ± 0.02 (0.5071)

0.0420

EPA

0.87 ± 0.06

0.69 ± 0.04 (0.0001)

0.83 ± 0.04 (0.1741)

<0.0001

DHA

5.66 ± 0.26

4.05 ± 0.13 (<0.0001)

4.46 ± 0.14 (<0.0001)

0.0004

a

apoE4

SC R

Control

apoE3 vs. apoE4

n=6b (p value)d

p valuee

26.91 ± 0.18 (0.0001)

0.4020

8.20 ± 0.38 (0.0006)

0.0025

NU

MA

D

TE

CE P

AC

FAME

IP

µM of apoE3 or apoE4 for 24h.a Controls are cells cultured for 24h in the absence of these proteins.

The values are obtained from the GC analysis of FAME, derived from membrane phospholipids as reported

in the experimental part. The values are reported as relative percentage (% rel) of the total fatty acid peak areas detected in the GC analysis, corresponding to >98% of the total peaks present in the chromatogram.

b

The values are given as mean ± SD and n is the number of samples per group. c Significance between control and ApoE3. d Significance between control and ApoE4. e Significance between ApoE3 and ApoE4.

7

ACCEPTED MANUSCRIPT Table 2 Total fatty acids methyl esters (FAME) per family and membrane homeostasis index obtained from the data of Table 1. apoE3

apoE4

apoE3 vs. apoE4

n=6 a

n=6 a (p value)b

n=6 a (p value)c

p valued

Total SFAe

35.42 ± 0.36

37.30 ± 0.75 (0.0003)

Total MUFAf

50.34 ± 0.16

50.92 ± 0.59 (0.0449)

PUFA ω-3g

6.53 ± 0.24

PUFA ω-6h

T

Control

0.0043

50.83 ± 0.08 (<0.0001)

0.7254

4.73 ± 0.16 (<0.0001)

5.29 ± 0.17 (<0.0001)

0.0002

7.51 ± 0.14

6.89 ± 0.06 (<0.0001)

7.55 ± 0.15 (0.6701)

<0.0001

Total PUFA

14.05 ± 0.24

11.62 ± 0.21 (<0.0001)

12.84 ± 0.31 (<0.0001)

<0.0001

Total TFAi

0.19 ± 0.08

0.17 ± 0.04 (0.5007)

0.20 ± 0.04 (0.7824)

0.1631

SFA/MUFA ratio

0.70 ± 0.01

0.73 ± 0.02 (0.0379)

0.71 ± 0.00 (0.2959)

0.0652

SFA/PUFA ratio

2.52 ± 0.07

3.21 ± 0.12 (<0.0001)

2.81 ± 0.08 (<0.0001)

<0.0001

EPA/ARA ratio

0.16 ± 0.01

0.15 ± 0.00 (0.1284)

0.0478

ω6/ω3 ratio

1.15 ± 0.05

1.46 ± 0.04 (<0.0001)

1.43 ± 0.03 (<0.0001)

0.1736

PUFA balancej

46.51 ± 1.10

40.72 ± 0.65 (<0.0001)

41.19 ± 0.43 (<0.0001)

0.1718

Unsaturation Index (UI)k

115.29 ± 1.52

102.76 ± 1.66 (<0.0001)

108.50 ± 1.41 (<0.0001)

<0.0001

Peroxidation Index (PI)l

76.49 ± 1.93

59.95 ± 1.38 (<0.0001)

66.77 ± 1.75 (<0.0001)

<0.0001

SC R

NU

MA

D

CE P

TE

0.14 ± 0.01 (0.0191)

AC

a

The values are obtained from the values of Table 1 and given as mean ± SD and n is the number of samples

per group. b

Significance between control and ApoE3.

c

Significance between control and ApoE4.

d

Significance between ApoE3 and ApoE4.

e

SFA= %16:0 + %18:0,

f

MUFA = %9c-16:1 + %9c-18:1 + %11c-18:1.

g

PUFA ω-3 = %EPA + %DHA.

h

PUFA ω-6 = %LA + %DGLA + %ARA.

i

TFA = %9t-18:1 + %Ʃ mono-trans ARA

j

PUFA balance = [(%EPA+ %DHA) / total PUFA] x100.

k l

IP

36.12 ± 0.21 (0.0022)

UI = (%MUFA x 1) + (%LA x 2) + (%DGLA x 3) + (%ARA x 4) + (%EPA x 5) + (%DHA x 6). PI = (%MUFA x 0.025) + (%LA x 1) + (%DGLA x 2) + (%ARA x 4) + (%EPA x 6) + (%DHA x 8).

8

ACCEPTED MANUSCRIPT 3. Results and discussion SK-N-SH cells were incubated in the presence or absence of lipid-free full-length apoE3 or apoE4 for 24 hours. For the whole duration of the study we took care that the FBS used is from the same

T

batch in order to avoid the external influence of the lipid supply. The choice of lipid-free apoE was

IP

made since it is known that the apoE secreted by brain cells (astrocytes, microglia, neurons) scavenges lipids from cells and can be lipidated in situ [1,2]. Therefore, the exogenously added

SC R

apoE isoforms are expected to behave similarly to apoE secreted from brain cells. After the treatment of cells, membrane fatty acid composition was determined following known methodologies for the membrane pellet formation, phospholipid isolation and transesterification to

NU

obtain fatty acid methyl esters, analyzed by gas chromatography (GC). The methods used in this work for GC analysis have been described elsewhere [40,41]. Six samples for each group were

MA

analyzed. Controls were cells incubated in the same conditions but in the absence of apoE proteins. Table 1 and Table 2 show the membrane fatty acid composition, the main membrane fatty acid families and the index of the SK-N-SH cells after 24h incubation in the presence or absence of

D

apoE3 or apoE4. Fatty acid quantities are reported as percentages of the main peak areas obtained

TE

from the GC analyses, calibrated and recognized with appropriate references, as already described [40,41].

CE P

In Figure 1 and Figure 2 the main fatty acid and families values are graphically represented as percentage differences from controls, together with their statistical significance against controls and between the two apoE isoforms. The main results can be summarized as follows:

AC

(i) Saturated fatty acids (SFA) were significantly increased after incubation of cells with apoE3 or apoE4 (p<0.001, p<0.01, respectively), but the increase was higher in the presence of apoE3 than apoE4 (p<0.01); (ii) Monounsaturated fatty acids (MUFA) were significantly increased after incubation with both apoE3 and apoE4 (p<0.05, p<0.0001, respectively), the major contribution for the latter being given by palmitoleic acid (p<0.001). (iii) Polyunsaturated fatty acids (PUFA) omega-6 were significantly diminished in the cell membranes after incubation with apoE3 (p<0.0001) but remained unperturbed in the presence of apoE4. (iv) PUFA omega-3 were significantly diminished in the membrane of cells treated with either apoE forms if compared with untreated samples (p<0.0001), but the decrease was of greater magnitude in the presence of apoE3 (p<0.001). The diminution of omega-3 affects also the PUFA balance indicator [42] respect to control (p<0.0001). 9

ACCEPTED MANUSCRIPT (v) Trans fatty acids (TFA), evaluated by a specific molecular library [43] tended to decrease after incubation of the cells with apoE3. On the contrary, the TFA content in cell

AC

CE P

TE

D

MA

NU

SC R

IP

T

membranes appeared to increase in the presence of apoE4.

Figure 1. Percentage differences compared to controls for each type of fatty acid in the SK-N-SH cell membranes following incubation with 1 µM apoE3 (black) or apoE4 (grey) after 24h. The values are given as mean ± SD (n=6). Each member of the fatty acid family is given in a row, the last column being the sum of the corresponding fatty acid family. Values significantly different from the control: (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p<0.0001; For specific values see Table 1. Black asterisks indicate the significance between control and apoE isoforms; Blue asterisks indicate the significance between the two apoE isoforms.

10

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Figure 2. Membrane homeostasis indexes in the SK-N-SH cells recorded after 24h treatment with 1

D

µM apoE3 (black) or apoE4 (grey). Upper row: total PUFA, -6/-3 ratio and PUFA balance;

TE

Middle row: SFA/MUFA, SFA/PUFA and EPA/ARA ratios; Lower row: unsaturation and peroxidation indexes. The values are given as mean ± SD (n=6). Values significantly different from

CE P

the control: (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p<0.0001; For specific values see Table 2. Black asterisks indicate the significance between control and apoE isoforms; Blue asterisks

AC

indicate the significance between the two apoE isoforms.

Deepening the evaluation of each fatty acid type present in the membrane phospholipids, interesting differences were detected between the two apoE isoform treatments. The saturated fatty acid stearic acid and the omega-6 DGLA (8c,11c,14c-20:3) were both decreased after incubation of cells with apoE4 (p<0.01 and p<0.05, respectively) and increased after incubation with apoE3 (p<0.01 and p<0.0001, respectively). The omega-6 ARA (5c,8c,11c,14c-20:4) was significantly diminished after incubation of cells with the apoE3 isoform (p<0.0001) and negligibly increased after incubation with apoE4 . These fatty acids were the only ones found with opposite trend for the two protein isoforms. Concerning the omega-3 fatty acids, EPA (5c,8c,11c,14c,17c-20:5) and DHA (4c,7c,10c,13c,16c,19c-22:6) levels were found to be lower after incubation of the cells with either of two apoE isoforms, as compared to untreated cells, but the decrease was of a greater extent after incubation with the apoE3 isoform as compared to apoE4 (p<0.0001 and p<0.001, respectively).

11

ACCEPTED MANUSCRIPT Expanding the vision to the whole membrane, these single fatty acid variations affect the corresponding indicators of the membrane status (Table 2 and Figure 2). Among them, the SFA/PUFA ratio in membranes was significantly increased after incubation with both isoforms

T

(p<0.0001), with apoE3 giving the higher entity of the increase compared to apoE4 (p<0.0001). The

IP

omega6/omega3 ratio (Figure 2) was increased after incubation of cells with either apoE isoforms too (p<0.0001), whereas both peroxidation and unsaturation index (PI and UI [44]) were lower than

SC R

controls (p<0.0001), with apoE3 inducing a greater decrease than apoE4 (p<0.0001). In general, the effects of the apoE isoforms were in the direction of decreasing membrane fluidity, due to SFA increase and PUFA loss. Other indicators of this significant decreasing trend, expressed as

NU

peroxidation index (PI) and unsaturation index (UI) [44] are shown in Table 2, being in both cases the difference from controls higher in the case of apoE3 than apoE4 (P<0.0001).

MA

Information about the fatty acid composition of membrane phospholipids and its changes in the presence of apolipoproteins are very important, and unknown so far, to understand the membrane properties and the cell predisposition to metabolic response originated from membrane constituents.

D

A first outcome is connected with known effects of fatty acids of different structures for the

TE

biophysical properties of cell membranes [45,46]. The increase in SFA and the decrease in PUFA, observed in the presence of both apoE isoforms, can influence membrane fluidity and permeability

CE P

as measured by the PI and UI indexes, consequently bringing to a more rigid packing of the bilayer. It is interesting to note that apoE3 resulted in a more consistent reduction of the PI and UI indexes, thus suggesting a different influence of the two-apoE isoforms on the properties of plasma membrane microenvironment. Indeed, biophysical and biological modifications induced by

AC

individual fatty acids deserve more attention in the apolipoprotein studies, as suggested by former observations on the palmitic acid-induced increase of de novo synthesis of ceramide in astroglia, known for being involved in Aβ production and in tau-protein hyperphosphorylation [47]. Notably, in the early stages of AD, sphingolipid balance is shifted towards ceramides accumulation and ceramides levels have been found to be increased in the brain, the CSF and the plasma of AD patients, as compared to the age-matched neurologically normal control subjects [48-50]. Moreover, the observed changes can have important consequences on the cellular signaling. In fact, the membrane phospholipids fatty acid types that change following apoE supplementation have direct connections with the signaling cascades mediated by eicosanoids, since they are omega-6 and omega-3 fatty acids that are precursor of signaling lipids after detachment from membranes by the action of phospholipase A2 [51]. The transformations of omega-6 fatty acids have different effects compared to omega-3 fatty acids, the former regulating cellular functions with inflammatory, atherogenic and prothrombotic effects, whereas the latter inhibiting the NF-kB activity directly, 12

ACCEPTED MANUSCRIPT binding peroxisome proliferator activated receptors (PPARs), thus controlling several genes involved in lipid metabolism and cytokine production [51]. More specifically, the transformation of the omega-6 DGLA has different outcomes in comparison to the omega-6 ARA, the former being

T

precursor of series-1 prostaglandins and other oxidation metabolites with anti-inflammatory and

IP

anti-proliferative effects [52]. It should also be mentioned that DGLA is the metabolic precursor of ARA and is converted to arachidonic by the activity of delta-5 desaturase. Therefore its biological

SC R

roles are determined by the partition into these two different pathways that must be evaluated in each specific condition. In this study, incubation of SK-N-SH cells with apoE3 increased the levels of DGLA and decreased the levels of ARA as compared to control cells. In contrast, incubation of

NU

cells with apoE4 had moderated diminution effect on DGLA levels and no significant effect on ARA levels. The effect of apoE3, but not of apoE4, on ARA could be attributed either to: i) either a

MA

metabolic negative influence on DGLA transformation (desaturase and elongase enzymes [34]), which correspondently increases in the glycerophospholipids of SK-N-SH cells, or ii) a different affinity of the two apoE isoforms for the two omega-6 containing lipids, influencing their

D

distribution to cells and availability for membrane phospholipid biosynthesis. On the other hand,

TE

multiple mechanisms can be synergically operative. It was described that apoE4, but not apoE3, triggers a significant elevation of cytokines (IL6 and IL8, both implicated in AD) in SHSY5Y

CE P

human neuroblastoma cells [53], through its connection with the formation of apoE-RelA transcriptional complex (RelA: v-rel avian reticuloendotheliosis viral oncogene homolog A/nuclear factor NF-kB p65 subunit - subunit of the NF-kB complex). As matter of facts, in our cell model apoE3 facilitates the increase of DGLA content in membrane phospholipids thus increasing the

AC

probability for protective molecular pathways. The role of DGLA and its transformations in AD have not been considered so far, and our results suggest a more comprehensive understanding of neurological functions including an evaluation of the balance among membrane fatty acid content and production of lipid mediators in neuronal cells [54]. Omega-6 fatty acid involvement in AD is already well documented. Altered neuroinflammatory markers and upregulation of the ARA cascades have already been widely described, supporting the association of arachidonic metabolism and cytokine production with neuronal damage in AD brains [55]. Moreover, the low ability of apoE4 to act as a radical scavenger is supported by our results of the differences of trans ARA isomers detected in neuronal membranes (see Table 1). Indeed, trans fatty acids are generated endogenously, resulting from the radical stress of sulfur-centered radicals that convert the cis double bonds of fatty acids [43]. In our results it appears that the apoE3 isoform is able to keep the amount of trans-ARA in a lower level than apoE4. This event could affect membrane lipid integrity and contribute to alter lipid organization and responses. 13

ACCEPTED MANUSCRIPT ApoE3 resulted in a higher reduction in the PI than apoE4 imparting to the cells greater stability against lipid oxidation. In a previous study we found that an apoE4 fragment, the apoE4-165, promotes amyloid-peptide beta 42 (Aβ42) accumulation in SK-N-SH cells and increases

T

intracellular reactive oxygen species formation [28]. ApoE4 fragments are produced in the onset

IP

and co-exist with the full length protein in the brain during the progression of AD [19,20]. The limited capacity of apoE4 to decrease the PI possibly facilitates the apoE4 fragments to induce

SC R

oxidative damage in neurons. Both may be in relation with the increased lipid peroxidation that has been found in post-mortem apoE4 brains [56,57]. Since the functions of lipoproteins in the brain and the recruitment of lipids by ApoE isoforms have been thoroughly investigated [58], our work

NU

expand the scenario to the observation of the membrane counterpart, which is certainly influenced by the different availability of fatty acids resulting also by the lipoprotein organization.

MA

The results provided by these in vitro experiments are the first reported and promising findings that connect molecular observations on membrane lipids to systemic effects observed in the brain cells and tissues and already noted in patients with neurodegenerative diseases. The

D

perspectives brought about these results can be useful for further targeted studies on the effects of

TE

omega-3 or other types of lipid supplementation or dietary regimes, taking into account that apoE4 carriers are reported with higher -6/-3 ratios [59]. Certainly, the combination of the clinical

CE P

observation and the follow-up of molecular events, provided by the membrane fatty acid-based lipidomics, would help to better understand and rationalize differences in the apoE3 and apoE4

5. Conclusions

AC

allele carriers that can prevent or induce the AD onset.

Our work is a model study of the membrane fatty acid remodeling or reorganization in SK-N-SH neuroblastoma cells in the presence of apoE3 and apoE4. Although it is established that the lipid binding proteins apoE3 and apoE4 display opposite AD onset and prognosis effects, with apoE4 being the major risk factor for AD, their effect on brain cells membrane fatty acid composition is largely unexplored. The fatty acid values and families estimated in this neuroblastoma cell line evidenced deep changes in membranes with known biophysical and biological implications. Herein, the membrane remodeling induced by apolipoproteins is for the first time reported, with the aim at encouraging further in vitro studies to better understand synergies between fatty acid-based membrane lipidomics and apolipoprotein lipid distribution, including the effects of different dietary conditions and lipid supply. It emerges that membrane participation is a crucial elements to be ascertained since molecular rearrangements of its fatty acids could contribute to the AD onset and progression. 14

ACCEPTED MANUSCRIPT

Acknowledgements This work was supported by the EU COST Action CM1201 "Biomimetic Radical Chemistry" and

T

the Marie Skłodowska-Curie European Training Network (ETN) ClickGene: Click Chemistry for

IP

Future Gene Therapies to Benefit Citizens, Researchers and Industry [H2020-MSCA-ETN-2014642023]. The infrastructure used for protein production for the current study is associated with the

AC

CE P

TE

D

MA

NU

SC R

Greek National Research Infrastructure in Structural Biology Instruct-EL.

15

ACCEPTED MANUSCRIPT References [1]

D.M. Holtzman, K.R. Bales, S. Wu, P. Bhat, M. Parsadanian, A.M. Fagan, L.K. Chang, Y. Sun, S.M. Paul, Expression of human apolipoprotein E reduces amyloid-β deposition in a

G.S. Getz, C.A. Reardon, Apoprotein E as a lipid transport and signaling protein in the blood,

[3]

SC R

liver and artery wall, J. Lipid Res. 50 (2009) S156–S161.

IP

[2]

T

mouse model of Alzheimer's disease, J. Clin. Invest. 103 (1999) R15–R21.

V. Hirsch-Reinshagen, S. Zhou, B.L. Burgess, L. Bernier, S.A. McIsaac, J.Y. Chan, G.H. Tansley, J.S. Cohn, M.R. Hayden, C.L. Wellington, Deficiency of ABCA1 impairs apolipoprotein E metabolism in brain, J. Biol. Chem. 279 (2004) 41197–41207. Q. Xu, A. Bernardo, D. Walker, T. Kanegawa, R.W. Mahley, Y. Huang. Profile and

NU

[4]

regulation of apolipoprotein E (apoE) expression in the CNS in mice with targeting of green

MA

fluorescent protein gene to the ApoE locus, J. Neurosci. 26 (2006) 4985–4994. [5] J. Kim, J.M. Basak, D.M. Holtzman, The role of apolipoprotein E in Alzheimer's disease, Neuron 63 (2009) 287–303.

C. Ballard, S. Gauthier, A. Corbett, C. Brayne, D. Aarsland, E. Jones, Alzheimer's disease,

D

[6]

[7]

TE

Lancet 377 (2011) 1019–1031.

C.A. Fisher, R.O. Ryan, Lipid binding-induced conformational changes in the N-terminal

[8]

CE P

domain of human apolipoprotein E, J. Lipid Res. 40 (1999) 93–99. L. Krimbou, M. Denis, B. Haidar, M. Carrier, M. Marcil, J. Genest, Molecular interactions between apoE and ABCA1 impact on apoE lipidation, J. Lipid Res. 45 (2004) 839–848. T. Tokuda, M. Calero, E. Matsubara, R. Vidal, A. Kumar, B. Permanne, B. Zlokovic, J.D.

AC

[9]

Smith, M.J. Ladu, A. Rostagno, B. Frangione, J. Ghiso, Lipidation of apolipoprotein E influences its isoform-specific interaction with Alzheimer's amyloid β peptides, Biochem. J. 348 (2000) 359–365. [10] Q. Jiang, C.D. Lee, S. Mandrekar, B. Wilkinson, P. Cramer, N. Zelcer, K. Mann, B. Lamb, T.M. Willson, J.L. Collins, J.C. Richardson, J.D. Smith, T.A. Comery, D. Riddell, D.M. Holtzman, P. Tontonoz, G.E. Landreth, ApoE promotes the proteolytic degradation of Aβ, Neuron 58 (2008) 681–693. [11] E.H. Corder, A.M. Saunders, W.J. Strittmatter, D.E. Schmechel, P.C. Gaskell, G. Small, A.D. Roses, J.L. Haines, M.A. Pericak-Vance, Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families, Science 261 (1993) 921–923. [12] W.J. Strittmatter, A.M. Saunders, D. Schmechel, M. Pericak-Vance, J. Enghild, G.S. Salvesen, A.D. Roses, Apolipoprotein E: high-avidity binding to beta-amyloid and increased

16

ACCEPTED MANUSCRIPT frequency of type 4 allele in late-onset familial Alzheimer disease, Proc. Natl. Acad. Sci. USA 90 (1993) 1977–1981. [13] M.C. Chartier-Hariln, M. Parfitt, S. Legrain, J. Pérez-Tur, T. Brousseau, A. Evans, C. Berr, O.

T

Vidal, P. Roques, V. Gourlet, et al., Apolipoprotein E, ɛ4 allele as a major risk factor for

chromosomal region, Hum. Mol. Genet. 3 (1994) 569–574.

IP

sporadic early and late-onset forms of Alzheimer's disease: analysis of the 19q13. 2

SC R

[14] R. Mayeux, R. Ottman, G. Maestre, C. Ngai, M.X. Tang, H. Ginsberg, M. Chun, B. Tycko, M. Shelanski, Synergistic effects of traumatic head injury and apolipoprotein-epsilon4 in patients with Alzheimer's disease, Neurology 45 (1995) 555–557.

NU

[15] D. Harold. et al. , Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer’s disease, Nat. Genet. 41 (2009) 1088–1093.

MA

[16] R. Deane, Z. Wu, A. Sagare, J. Davis, Y.S. Du, K. Hamm, F. Xu, M. Parisi, B. LaRue, H.W. Hu, P. Spijkers, H. Guo, X. Song, P.J. Lenting, W.E. Van Nostrand, B.V.Zlokovic, LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms,

D

Neuron 43(2004) 333–344.

TE

[17] G. Bu, Apolipoprotein E and its receptors in Alzheimer's disease: pathways, pathogenesis and therapy, Nat. Rev. Neurosci. 10 (2009) 333–344.

CE P

[18] L.M. Tai, S. Mehra, V. Shete, S. Estus, G W. Rebeck, G. Bu, M.J. La Du, Soluble apoE/A_ complex: mechanism and therapeutic target for APOE4-induced AD risk, Mol. Neurodegener. 9 (2014) 2.

[19] Y. Huang, X.Q. Liu, T. Wyss-Coray, W.J. Brecht, D.A. Sanan, R.W. Mahley, Apolipoprotein

AC

E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons, Proc. Natl. Acad. Sci. USA 98 (2001), 8838–8843. [20] F.M. Harris, W.J. Brecht, Q. Xu, I. Tesseur, L. Kekonius, T. Wyss-Coray, J.D. Fish, E. Masliah, P.C. Hopkins, K. Scearce-Levie, K.H. Weisgraber, L. Mucke, R.W. Mahley, Y. Huang, Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice, Proc Natl Acad Sci USA 100 (2003), 10966–10971. [21] W.J. Brecht, F.M. Harris, S. Chang, I. Tesseur, G.Q. Yu, Q. Xu, J. Dee Fish, T. WyssCoray,M. Buttini,L. Mucke , R.W. Mahley, Y. Huang, Neuron-specific apolipoprotein e4 proteolysis is associated with increased tau phosphorylation in brains of transgenic mice, J. Neurosci. 24 (2004) 2527–2534.

17

ACCEPTED MANUSCRIPT [22] I. Dafnis, A. Tzinia, E.C. Tsilibary, V.I. Zannis and A. Chroni, An apolipoprotein E4 fragment affects MMP9, TIMP1 and cytokine levels in brain cell lines, Neuroscience 210 (2012) 21–32.

T

[23] I. Dafnis, L. Argyri, M. Sagnou, A. Tzinia, E.C. Tsilibary, E. Stratikos, A.Chroni, The ability

IP

of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid peptide beta 42 is both isoform and size specific, Sci Rep. 6 (2016) Article no. 30654.

SC R

[24] Y. Christen, Oxidative stress and Alzheimer disease, Amer. J. Clin. Nutr. 71 (2000) 621s– 629s.

[25] M. Miyata, J.D. Smith, Apolipoprotein E allele-specific antioxidant activity and effects on

NU

cytotoxicity by oxidative insults and beta-amyloid peptides, Nat. Genet. 14 (1996) 55–61. [26] J. Legleiter, J.D. Fryer, D.M. Hltzmann, T. Kowalewski, The modulating effect of mechanical

MA

changes in lipid bilayers caused by Apo-E-containing lipoproteins on Aβ induced membrane disruption, ACS Chem. Neurosci. 2 (2011) 588–599. [27] B. Schwiegelshohn, J. F. Presley, M. Gorecki, T. Vogel, Y. A.Carpentier, F. R. Maxfield, R.

D

J. Deckelbaum. Effects of Aprotein E on intracellular metabolism of model troglyceride-roch

TE

particles are distinct from effects on cell particle uptake. J. Biol. Chem. 270 (1995) 1761– 1769.

CE P

[28] I. Dafnis, E. Stratikos, A. Tzinia, E.C. Tsilibary, V.I. Zannis, A. Chroni, An apolipoprotein E4 fragment can promote intracellular accumulation of amyloid peptide beta 42, J. Neurochem. 115 (2010) 873–884. [29] S. Morita, K. Okuhira, N. Tsuchimoto, A. Vertut-Doi, H. Saito, M. Nakano, T. Handa,

AC

Biochim. Biophys. Acta 1631 (2003), 169-176. [30] R.H. Mahley, Central Nervous System Lipoproteins: ApoE and Regulation of Cholesterol Metabolism, Arterioscler. Thromb. Vascular Biol. 36 (2016) 1305–1315. [31] X. Gao, W. A. CampbellIV, M. Chaibva, P. Jain, A. E. leslie, S. L. Frey, J. Legleiter. Cholesterol modifies Huntingtin binding to, disruption of, and aggregation on lipid membranes, Biochemistry, 55 (2016) 92-102. [32] P.L. Wood, Lipidomics of Alzheimer’s disease: current status, Alzheimers Res. Ther. 4 (2012), article 5. [33] C. R. Hooijmans, P. Pasker-de Jong, R. de Vries, M. Ritskes-Hoitinga, The effects of longterm omega-3 fatty acid supplementation on cognition and Alzheimer's pathology in animal models of Alzheimer's disease: a systematic review and meta-analysis. J. Alzheimer's Disease, 28 (2012) 191–209.

18

ACCEPTED MANUSCRIPT [34] C. Ferreri, C. Chatgilialoglu, Membrane Lipidomics for Personalized Health, Wiley and Sons, New York, 2015. [35] L. Argyri, V. Skamnaki, E. Stratikos, A. Chroni, A simple approach for human recombinant

T

apolipoprotein E4 expression and purification, Protein Expr. Purif. 79 (2011) 251–257.

IP

[36] D. Georgiadou, K. Stamatakis, E.K. Efthimiadou, G. Kordas, D. Gantz, A. Chroni, E. Stratikos, Thermodynamic and structural destabilization of apoE3 by hereditary mutations

SC R

associated with the development of lipoprotein glomerulopathy, J. Lipid Res. 54 (2013) 164– 176.

[37] A. Chroni, S. Pyrpassopoulos, A. Thanassoulas, G. Nounesis, V.I. Zannis, E. Stratikos,

NU

Biophysical analysis of progressive C-terminal truncations of human apolipoprotein E4: insights into secondary structure and unfolding properties, Biochemistry 47 (2008) 9071–

MA

9080.

[38] J. Folch, M. Lees, G.H. Sloane Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 (1957) 497–509.

D

[39] B. Fuchs, R. Süss, K. Teuber, M. Eibisch, J. Schiller, Lipid analysis by thin-layer

TE

chromatography-a review of the current state, J. Chromatogr. A 1218 (2011) 2754–2774. [40] G. Cohen, O. Shamni, Y. Avrahami, O. Cohen, E.C. Broner, N. Filippov-Levy, C.

CE P

Chatgilialoglu, C. Ferreri, N. Kaiser, S. Sasson, Beta cell response to nutrient overload involves phospholipid remodelling and lipid peroxidation, Diabetologia 58 (2015) 1333– 1343.

[41] A. Bolognesi, A. Chatgilialoglu, L. Polito, C. Ferreri, Membrane lipidome reorganization

AC

correlates with the fate of neuroblastoma cells supplemented with fatty acids, PLoS One 8 (2013), e55537.

[42] S.K. Abbott, P.A. Else, T.A. Atkins, A.J. Hulbert, Fatty acid composition of membrane bilayers: Importance of diet polyunsaturated fat balance. Biochim. Biophys. Acta 1818 (2012) 1309–1317. [43] C. Chatgilialoglu, C. Ferreri, M. Melchiorre, A. Sansone, A. Torreggiani, Lipid Geometrical Isomerism: From Chemistry to Biology and Diagnostics, Chem. Rev. 114 (2014) 255–284. [44] A.A. Puca, V. Novelli, C. Viviani, P. Andrew, F. Somalvico, N.A. Cirillo, C. Chatgilialoglu, C. Ferreri, Lipid profile of erythrocyte membranes as possible biomarker of longevity, Rejuven. Res. 11 (2008) 63–72. [45] G. Maulucci, O. Cohen, B. Daniel, A. Sansone, P.I. Petropoulou, S. Filou, A. Spyridonis, G. Pani, M. De Spirito, C. Chatgilialoglu, C. Ferreri, K.E. Kypreos, S. Sasson, Fatty acid-related

19

ACCEPTED MANUSCRIPT modulations of membrane fluidity in cells: detection and implications, Free Radic. Res. 50 (2016) S40–S50. [46] C.M.J. Holthuis, A.K. Menon. Lipid landscapes and pipelines in membrane homeostasis.

T

Nature 510 (2014) 48–57

IP

[47] S. Patil, J., Melrose, C. Chan, Involvement of astroglial ceramide in palmitic acid‐ induced Alzheimer‐ like changes in primary neurons, Eur. J. Neurosci. 26 (2007) 2131–2141.

SC R

[48] X. Han, M.H.D. Dw, J.R. Mckeel, J. Kelley, J.C. Morris, Substantial sulfatide deficiency and ceramide elevation in very early Alzheimer's disease: potential role in disease pathogenesis, J. Neurochem. 82 (2002) 809–818.

NU

[49] R.G. Cutler, J. Kelly, K. Storie, W.A. Pedersen, A. Tammara, K. Hatanpaa, J.C. Troncoso M.P. Mattson, Involvement of oxidative stress-induced abnormalities in ceramide and

MA

cholesterol metabolism in brain aging and Alzheimer's disease, Proc. Natl Acad. Sci. USA 101 (2004) 2070–2075.

[50] X. He, Y. Huang, B. Li, C.X. Gong, E.H. Schuchman, Deregulation of sphingolipid

D

metabolism in Alzheimer's disease, Neurobiol. Aging 31 (2010) 398–408.

(2008) 147-155.

TE

[51] G. Schmitz, J. Ecker, The opposing effects of n-3 and n-6 fatty acids. Progr. Lipid Res. 47

CE P

[52] X. Wang, H. Lin, Y. Gu. Multiple roles of dihomo-gamma-linolenic acid against proliferation diseases. Lipids Health Dis. 11 (2012), 25; doi: 10.1186/1476-511X-11-25. [53] V. Theendakara, C. A. Peters-Libeu, P. Spilman, K. S. Poksay, D. E. Bredesen, R. V. rao. Direct transcriptional effects of apolipoprotein E. J. Neurosci. 36 (2016) 685-700.

AC

[54] R.P. Bazinet, S. Layé, Polyunsaturated fatty acids and their metabolites in brain function and disease, Nat. Rev. Neurosci. 15 (2014) 771–785. [55] J.S. Rao, S.I. Rapoport, H.W. Kim, Altered neuroinflammatory, arachidonic acid cascade and synaptic markers in postmortem Alzheimer’s disease brain, Transl. Psychiatry 1 (2011), e31. [56] C. Ramassamy, D. Averill, U. Beffert, S.Bastianetto, L. Theroux, S. Lussier-Cacan, J.S. Cohn, Y. Christen, J. Davignon, R. Quirion, J. Poirier, Oxidative damage and protection by antioxidants in the frontal cortex of Alzheimer's disease is related to the apolipoprotein E genotype, Free Radic. Biol. Med. 27 (1999) 544–553. [57] C. Ramassamy , D. Averill , U. Beffert, L. Theroux, S. Lussier-Cacan , J.S. Cohn , Y. Christen, A. Schoofs, J. Davignon,

J. Poirier, Oxidative insults are associated with

apolipoprotein E genotype in Alzheimer's disease brain, Neurobiol. Dis. 7 (2000) 23–37. [58] H. Wang, R.H. Heckel, What are lipoproteins doing in the brain? Trends Endocrinol. Metab. 25 (2014) 8–14. 20

ACCEPTED MANUSCRIPT [59] R. Chouinard-Watkins, M. Plourde, Fatty acid metabolism in carriers of apolipoprotein E epsilon 4 allele: is it contributing to higher risk of cognitive decline and coronary heart

AC

CE P

TE

D

MA

NU

SC R

IP

T

disease?, Nutrients 6 (2014) 4452–4471.

21

ACCEPTED MANUSCRIPT Highlights • ApoE3 increased stearic acid and dihomo-gamma-linolenic acid (DGLA), whereas apoE4 had opposite effects in neuroblastoma cells

T

• Both apoE3 and apoE4 isoforms increased SFA, MUFA and omega-6/omega-3 ratio and

IP

decreased total PUFA, but with various intensities

SC R

• Both apoE3 and apoE4 isoforms decreased PUFA balance as well as unsaturation and

AC

CE P

TE

D

MA

NU

peroxidation indexes

22