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Effect of Choline-Containing Phospholipids on Transglutaminase Activity inPrimary Astroglial Cell CulturesV. Bramanti a; D. Bronzi b; D. Tomassoni c; G. Li Volti d; G. Cannavò e; G. Raciti d; M. Napoli f; A. Vanella d; A.Campisi d; R. Ientile e; R. Avola ag

a Department of Chemical Sciences, Section of Biochemistry and Molecular Biology, University of Catania,Catania, Italy b Department of Physiological Sciences, University of Catania, Catania, Italy c Department ofPharmacological Sciences and Public Health, Section of Human Anatomy, University of Camerino, Camerino,Italy d Departments of Biochemistry, Medical Chemistry and Molecular Biology, University of Catania, Catania,Italy e Departments of Biochemical, Physiological, and Nutritional Sciences, and of Hygiene and PreventiveMedicine, University of Messina, Messina, Italy f Botanic Department, University of Catania, Catania, Italy g

Italian National Institute of Biostructures and Biosystems (INBB), Rome, Italy

Online Publication Date: 01 November 2008

To cite this Article Bramanti, V., Bronzi, D., Tomassoni, D., Li Volti, G., Cannavò, G., Raciti, G., Napoli, M., Vanella, A., Campisi, A.,Ientile, R. and Avola, R.(2008)'Effect of Choline-Containing Phospholipids on Transglutaminase Activity in Primary Astroglial CellCultures',Clinical and Experimental Hypertension,30:8,798 — 807

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Clinical and Experimental Hypertension, 30:798–807, 2008Copyright © Informa Healthcare USA, Inc.ISSN: 1064-1963 print / 1525-6006 onlineDOI: 10.1080/10641960802563576

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LCEH1064-19631525-6006Clinical and Experimental Hypertension, Vol. 30, No. 8, October 2008: pp. 1–16Clinical and Experimental Hypertension

Effect of Choline-Containing Phospholipids on Transglutaminase Activity in Primary

Astroglial Cell Cultures

Effects of Choline-Containing Phospholipids on AstrocytesV. Bramanti et al. V. BRAMANTI,1 D. BRONZI,2 D. TOMASSONI,3 G. LI VOLTI,4 G. CANNAVÒ,5 G. RACITI,4 M. NAPOLI,6 A. VANELLA,4 A. CAMPISI,4 R. IENTILE,5 AND R. AVOLA1,7

1Department of Chemical Sciences, Section of Biochemistry and MolecularBiology, , University of Catania, Catania, Italy2Department of Physiological Sciences, University of Catania, Catania, Italy3Department of Pharmacological Sciences and Public Health, Section of HumanAnatomy, University of Camerino, Camerino, Italy 4Departments of Biochemistry, Medical Chemistry and Molecular Biology,University of Catania, Catania, Italy5Departments of Biochemical, Physiological, and Nutritional Sciences, and of Hygiene and Preventive Medicine, University of Messina, Messina, Italy6Botanic Department, University of Catania, Catania, Italy7Italian National Institute of Biostructures and Biosystems (INBB), Rome, Italy

The aim of the present investigation was to study the effects of choline and choline-containing phospholipids CDP-choline (CDPC) and L-alpha-glyceryl-phosphorylcholine(AGPC) on transglutaminase (TG) activity and expression in primary astrocyte cul-tures. TG is an important Ca2+-dependent protein that represents a normal constituentof nervous systems during fetal stages of development, playing a role in cell signaltransduction, differentiation, and apoptosis. Confocal laser scanning microscopy(CLSM) analysis showed an increase of TG activity in astrocyte cultures treated withcholine, CDPC, or AGPC at 0.1 mM or 1 mM concentrations. Comparatively, AGPCinduced the most conspicuous effects enhancing monodansyl-cadaverine fluorescenceboth in cytosol and in nuclei, supporting the evidence of the important role played byAGPC throughout differentiation processes tightly correlated to nucleus-cytosol cross- talkduring astroglial cells proliferation and development. Western blot analysis showedthat in 24h 1 mM AGPC and choline-treated astrocytes increased TG-2, whereas noeffect was observed in 24h 1 mM CDP-choline treated astrocytes. Our data suggest acrucial role of choline precursors during different stages of astroglial cell prolifera-tion and differentiation in cultures.

Submitted October 10, 2007; accepted November 15, 2007.Address correspondence to Prof. R. Avola, Department of Chemical Sciences, Section of

Biochemistry and Molecular Biology, University of Catania, Viale Andrea Doria, 6-95125 Catania,Italy; E-mail: ravola@unict.it

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Effects of Choline-Containing Phospholipids on Astrocytes 799

Keywords choline-containing phospholipids, astroglial cell cultures, tissuetransglutaminase, proliferation, differentiation

Introduction

Phospholipids are important components of mammalian cells, including neurons and glialcells, and exert different biological functions. They form lipid bilayers that providestructural integrity necessary for protein function, act as energy reservoirs, and serve asprecursors for various second messengers. A better knowledge of various aspects of phos-pholipid metabolism will contribute to understand the role of lipids in maintainingcell physiology and how alterations in their metabolism can contribute to various nervoussystem disorders. Choline is a quaternary amine obtained primarily from the diet andsynthesized in brain and liver as well. Choline is also a precursor in the biosynthesis of theneurotransmitter acetylcholine (ACh) and of phosphatidylcholine (PC), a major mem-brane constituent (1). Choline plays a crucial role as a key structural and functionalcomponent of cell membranes (2). Cellular membranes contain most of the choline in thebody, principally in the form of the phosphatide PC, of PC products or also as less abundantcholine-containing phospholipids (1).

Two choline-containing phospholipids, cytidine-5′-diphosphocholine (CDP-choline;citicoline, CDPC) and choline alphopscerate (L-alpha-glyceryl-phosphorylcholine,AGPC), are used in some countries as registered drugs for treating sequelae of neurologicaldisorders related to cerebrovascular disease (cerebral ischemia, stroke) or selected symp-toms of Parkinson’s and Alzheimer’s disease. CDPC, which is composed of choline andcytidine linked by a diphosphate bridge, is an essential intermediate in the synthesis ofendogenous PC through the Kennedy cycle (1). Choline alphoscerate is a semi-syntheticderivative of PC representing, among ACh precursors, a relatively new cholinergic drug.Pre-clinical investigations have shown that it increases the release of ACh in rat brain (3),facilitates learning and memory in experimental animals (4), and improves brain transduc-tion mechanisms (5) and counter age-dependent structural changes occurring in the ratfrontal cortex and hippocampus (6). The compound also contributes to anabolic processesresponsible for membrane phospholipid and glycerolipid synthesis, positively influencingmembrane fluidity (7).

Astroglial cells during proliferation and differentiation in primary cultures represent avaluable tool to study biochemical mechanisms involved in their development and matura-tion. Neuronal nicotinic cholinergic receptors are expressed by hippocampal astrocytes, andtheir activation produces rapid and transients Ca2+ currents (8). Tissue transglutaminase(TG-2) is an important Ca2+ -dependent protein, which represents a normal constituent ofcentral and peripheral nervous systems during fetal stages of development. TG-2 is amember of transglutaminases family. This enzyme catalyzes the formation of isopeptidebridges by calcium Ca2+-dependent cross-linking of the carboxamide moiety of a peptide-bound glutamine either to the ε-amino group of a peptide-bound lysine or to polyamines,with the liberation of ammonia (9,10). TG-2 is also the only member of TGs playing a rolein cell signal transduction, differentiation, and apoptosis (11–14).

The present study was designed to evaluate a possible protective, proliferative,or differentiative effect evoked by treatment for 24h with choline and with the cho-line-containing phsospholipids CDPC and AGPCat different concentrations in our invitro models of glial cells. Analysis was performed in primary astrocytes cultures at14, 21, and 35 or 39 days in vitro (DIV) exposed for 24h to choline HCl, CDPC, orAGPC.

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800 V. Bramanti et al.

Materials and Methods

Materials

Cell culture medium and sera were from Invitrogen (Milan, Italy). Monodansyl-cadaverine(DC), Bovine Serum Albumin (BSA), acetylcholine, and other chemicals of analyticalgrade were from Sigma (Milan, Italy). Choline chloride, CDPC, and AGPC were gentlygiven by MDM (Monza, Italy). TG-2 antibody CUB 7402 was obtained from LabVision(Fremont, California, USA). Horseradish peroxidase-conjugated antibody and anenhanced chemiluminescence (ECL)-developing system for immunoblots were purchasedfrom Amersham Pharmacia Biotech (Milan, Italy).

Cell Cultures

Primary astroglial cell cultures were performed from newborn albino rat brains (1–2-day-oldWistar strain) according to the methods previously reported (14,16–18). Briefly, cerebraltissues, after dissection and careful removal of the meninges, were mechanically dissoci-ated through sterile meshes of 82 mm-pore size (Nitex). Isolated cells were suspended inDulbecco’s Modified Eagle’s Medium (DMEM); supplemented with 20% (V/V) heat-inactivated photomultifetal bovine serum (FBS), 2 mM glutamine, streptomycin (50 mg/ml),and penicillin (50 U/ml); and plated at a density of 3 × 106 cells/100 mm dish and 0.5 × 105

cells/chamber in Lab-Tek multichambered slides. Cells were maintained at 37°C in a 5%CO2/95% air humidified atmosphere for five weeks, and the medium was removed everythree days. All efforts were made to minimize both the suffering and number of animalsused. All experiments conformed guidelines of the Ethical Committee of University ofCatania, Italy.

Drug Treatment

In order to synchronize cell cycle, at 12 or 19 or 33 DIV, the culture medium containingthe serum was removed and replaced with DMEM containing BSA (1 mg/1 ml). After24 hours, cell cultures were divided in five groups; four groups were exposed for differenttimes (6, 12, and 24 hours) at different concentration (0.1, 1, and 10 μM) of choline chlo-rure, CDPC or AGPC. One group of cells, used as a control, was maintained in mediumcontaining BSA for the same time of drug treated-cultures. Four replicate experimentswere performed for each sample.

Monodansyl-Cadaverine Labeling of Living Cells

TG-mediated monodansyl-cadaverine uptake into living cells after drug treatments at theconcentrations of 0.1, 1, and 10 μM was assayed according to Ientile et al. (19). Cultureswere then viewed by confocal laser scanning microscopy (CLSM).

Analysis by CLSM

Control treated cultures were examined by CLSM, as previously described (14,21).Briefly, optical fields were visualized using 403 1.0 NA oil immersion objective usinga Leica DM IRB inverted fluorescence microscope (Leica Microsystems, Heidelberg,Mannheim, Germany). Cultures were then were examined on a Leica TCS SP2 confocal

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Effects of Choline-Containing Phospholipids on Astrocytes 801

laser scanning microscope by a green fluorophore excitation at 488 nm, using He/Neonand Ar/Kr as laser sources. Fluorescence emitted from specimens was sent through aLeica acoustic optical turnable filter (AOTF) mechanism and pinhole to two differentphotomultiplier tubes (PMTs). Image outputs electronically generated by the same parametersettings (frame scanning, pinhole aperture, gain voltage, pixels, and spatial resolution)were measured and compared for intensity (treated vs. control), after non-specific fluores-cence subtraction. Finally, sequential optical sections, obtained using CLSM Z-axisstepping capability, were combined to form an extended depth of focus image, and stan-dard image processing was performed to enhance brightness and contrast, using LeicaConfocal Software (version 2.0 build 0585).

Western Blotting

After treatment, cells were harvested in cold PBS, collected by centrifugation, re-suspended in homogenizing buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1mM EDTA,0.1 mM phenylmethylsulfonyl fluoride, containing a 10 mg/ml concentration of aprotinin,leupeptin, and pepstatin), and sonicated on ice. Protein concentration of the homogenateswas diluted to 1 μg/ml using a reducing stop buffer [0.25 M Tris–HCl, pH 6.8, 5 mMEGTA, 25 mM dithiothreitol (DTT), 2% SDS, and 10% glycerol with bromophenol blueas the tracking dye]. Proteins (30 μg) were separated on 8.5% SDS–polyacrylamide gels,and transferred to nitrocellulose membranes. Blots were blocked overnight at 4°C with 5%non-fat dry milk dissolved in 20 mM Tris–HCl, pH 7.4, 150 mM NaCl 0.5% Tween 20.The TG-2 antigen was detected by incubation for 1 h with a mouse CUB 7402 antibody(1:1000 in PBS), followed by incubation for 1h with horseradish peroxidase-conjugatedanti-mouse IgG (1:1500 in PBS). The product of immune reaction was revealed bychemiluminescence with an ECL kit after autoradiography film exposure. Blots werescanned and quantified with an AlphaImager 1200 System (Alpha Innotech, San Leandro,California, USA).

Statistics

All values are presented as means ± SEM of data obtained from five different dishes. One-wayANOVA followed by Fisher’s PLSD test was used to compare values of densitometricanalysis of immunoblots. Statistical significance was expressed at p values of <0.05

Results

Primary astroglial cell cultures were characterized by using glial fibrillary acidic protein(GFAP) and glutamine synthetase activity, as previously reported (14,16). Approximately90% of cells used in our experiments were GFAP-positive, indicating that cultures wereenriched in astrocytes (data not shown). Astrocytes were treated at different times (data notshown) and concentrations with choline and choline-containing phospholipids (see Figures1 and 2). The times of culture exposure to drugs were chosen as those in which TGsreached maximum levels at 24 hours (data not shown).

TGs activity induced by choline and choline-containing precursors at the concentrationsof 0.1, 1, and 10 μM, was investigated by CLSM analysis by evaluating the distribution offluorescent DC incorporated in proteins of living cells. An intense DC-fluorescent patternwas observed in choline-treated cells at 0.1 μM (see Figure 1b) compared to controluntreated cultures at 14 DIV (see Figure 1a). The intensity of fluorescence induced by 1

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Figure 1. Confocal laser scanning microscope analysis of TG-2 activity assessed by DC incorporationin living astrocytes cultures at 14 DIV (a) left untreated, (b–d) treated for 24h with choline atthe concentrations of 0.1 μM, 1 μM, and 10 μM, respectively, or (e–g) treated with CDPC at theconcentrations of 0.1 μM, 1 μM, and 10 μM, respectively (×800).

Control

a

Choline 0.1 µM

b

Choline 1 µM

c

Choline 10 µM

d

CDPC 1 µM

f

CDPC 10 µM

g

CDPC 0.1 µM

e

Figure 2. Confocal laser scanning microscope analysis of TG-2 activity assessed by DC incorpora-tion in living astrocytes cultures at 14 DIV (a) left untreated, or (b–d) treated for 24h with AGPC atthe concentrations of 0.1 μM, 1 μM, and 10 μM, respectively (×800).

AGPC0.1 µM

b

AGPC1 µM

c

AGPC10 µM

d

Control

a

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Effects of Choline-Containing Phospholipids on Astrocytes 803

μM choline (see Figure 1c) was similar to that obtained in control cultures and slightlydecreased at 10 μM (see Figure 1d). Fluorescence distribution was prevalently perinuclearboth in untreated and in 0.1 μM choline-treated cells, whereas it appeared in the nuclearcompartment using a 1 μM choline concentration (see Figure 1c).

A dose-dependent increase of TG-2 activity was observed in CDPC-treated astrocytes(see Figures 1e–1g) in comparison to untreated cells at 14 DIV (see Figure 1a). Atmaximum, CDPC concentration level of fluorescence remained high, but cells appearedwrinkled (see Figure 1g). The increase of fluorescence was found primarily along astro-cyte cytosolic prolungaments, when 14 DIV cell cultures were exposed to CDPC 1 μM(see Figure 1f) compared to untreated astrocytes (see Figure 1a).

Treatment for 24h with AGPC at 0.1 and 1 μM concentration induced showed anincrease of TGs activity (see Figures 2b and 2c) compared to control cells (see Figure 2a).In 1 μM α-GPC-treated astrocytes at 14 DIV, the fluorescence was distributed both in thecytosol and in the nuclear compartments. Fluorescence decreased when astroglial cell cul-tures were treated with A-GPC at concentration 10 μM (see Figure 2d).

Figures 3–5 summarize the results of choline and choline-containing phospholipidstreatment on TG-2 expression evaluated by Western blot analysis in astroglial cell culturesat 14, 21, and 35 DIV. Treatment with 1 μM choline or AGPC at 14 DIV for 24h increasedTG-2 expression compared to untreated control cultures, whereas treatment with the sameconcentration and for the same time with CDPC was without effect (see Figure 3). Treat-ment for 24h with 1 μM of the three compounds tested slightly reduced TG-2 expressionin astroglial cell cultures at 21 DIV (see Figure 4).

Figure 5 shows comparatively the results of the effects of treatment with 1 μM AGPCon TG-2 expression in 14 DIV (see Figure 5a) and in 35 DIV (see Figure 5b) astrocyte

Figure 3. Western blots of TG-2 expression in astrocytes cultures at 14 DIV treated for 24 h withcholine, CDPC, or AGPC at the concentration of 1 μM (upper panel) and Western blot densitometry(lower panel). Densitometry values are expressed in arbitrary units (AU) of means ± SEM) of valuesobtained from five different dishes. *p < 0.05 vs. control.

Control

AGPCCDPC

Choline

80 kDa

Actin

00,20,40,60,81

1,21,41,61,8

Control AGPC CDPC Choline

A.U.

*

*

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804 V. Bramanti et al.

cultures. As shown, AGPC addition to 35 DIV cell cultures remarkably decreased TG-2expression in comparison with effects elicited by the same treatment in 14 DIV astrocytecultures. In particular, a 20% fetal calf serum (FCS) addition to cell cultures at 14 (seeFigure 5a) and 35 DIV (see Figure 5b) was accompanied by a significant rise in immu-noreactivity compared with BSA (1 mg/ml) addition, the results of which represent theuntreated control cultures.

Discussion

The present study has evaluated the effects evoked by choline and by two choline-containingphospholipids (CDPC and AGPC) in primary astrocyte cultures at different stages ofgrowth, development and maturation (14, 21, and 35 DIV). The main purpose of thisinvestigation was to analyze the modulation of the activity and expression of TG-2 as amarker of astroglial cells differentiation.

Furthermore, it well known that CDPC plays important roles in the generation ofphospholipids involved in the membrane formation and repair and as also beneficial phys-iological action on cellular functions (20). In addition, choline alphoscerate is acholinergic precursor, which has shown to be effective in countering cognitive symptomsin forms of dementia disorders of degenerative, vascular, or combined origin (21). Theobservation that treatment with choline alphoscerate attenuates the extent of glial reaction inthe hippocampus of SHR suggests also that the compound may afford neuroprotection inthis animal model of vascular brain damage (21). The effects of association of cholinergicprecursors choline or choline alphoscerate with the cholinesterase inhibitor rivastigmineon acetylcholine levels and [(3)H]hemicholinium-3 binding were recently assessed in ratfrontal cortex, hippocampus, and striatum (22).

Figure 4. Western blots of TG-2 expression in astrocytes cultures at 21 DIV treated for 24 h withcholine, CDPC, or AGPC at the concentration of 1 μM (upper panel) and Western blot densitometry(lower panel). Densitometry values are expressed in arbitrary units (AU) of means ± SEM) of valuesobtained from five different dishes. *p < 0.05 vs. control.

Control

AGPCCDPC

Choline

80 kDa

Actin

00,20,40,60,81

1,21,41,61,8

Control AGPC CDPC Choline

A.U.

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Effects of Choline-Containing Phospholipids on Astrocytes 805

On the other hand, our findings demonstrate by CLSM analysis an intense DC-fluorescence in 0.1 μM choline-treated cells prevalently at perinuclear level, whereas at1 μM fluorescence was also found in the nuclear compartment. This suggests an importantinvolvement of TGs activity not only during cell differentiation, but also during astroglialcell proliferation in cultures maintained in the presence of cholinergic modulators. Anincreased fluorescence localized prevalently along cytosolic prolungaments in 1 μMCDPC-treated astrocyte cultures compared to untreated ones was observed, indicating theoccurrence of an intense TG-2 activity during in different biochemical pathways andphysiological processes consequent to neuroprotection effects evoked by CDPC .

Treatment for 24h with 1 μM AGPC at 1 μM induced a raised TG-2 activity tocompare to control astrocytes at 14 DIV. This treatment induced an intense fluorescenceaccumulation both in cytosol and nuclear compartments of cultured astrocytes at 14 DIV.This finding supports the evidence of the important role played by AGPC during differen-tiation processes tightly correlated to nucleus-cytosol cross-talk in proliferating astroglialcells in a particular stage of development in vitro.

Figure 5. Western blots of TG-2 expression in astrocytes cultures (left-hand panels) at (a) 14 DIVand (b) 35 DIV treated for 24h with AGPC and Western blot densitometry (right-hand panels).Astrocyte cultures maintained in the presence of bovine serum albumine (BSA) were considered asuntreated controls or were maintained in the presence of 20% fetal calf serum (FCS). Densitometryvalues are expressed in arbitrary units (AU) of means ± SEM) of values obtained from five differentdishes. *p < 0.05 vs. control.

A.U.

00,40,81,21,6

BSA14DIV

FCS14DIV

2,02,42,8

BSA 14 DIV

FCS 14 DIV

AGPC 14 DIV

80 kDa

Actin

a

80 kDa

AGPC 35 DIV

BSA 35 DIV

FCS 35 DIV

Actin

bAGPC 35 DIV

00,511,522,533,544,5

A.U.

BSA35 DIV

FCS35 DIV

*

AGPC 14 DIV

*

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806 V. Bramanti et al.

Data on the influence of the three treatments on TG-2 expression evaluated by Western-blot analysis in astrocytes cultures at 14, 21, and 35 DIV showed that the treatment for 24hwith 1μM choline and AGPC enhanced TG-2 expression in astrocyte cultures at 14 DIVcompared to untreated ones, whereas 1 μM CDPC treatment for 24h was without effect. Theunaffected TG-2 expression by CDPC indicates a reduced protein biosynthesis, even thougha marked DC fluorescence distribution evidences the enzymatic TGs activity independentlyon protein expression. In astroglial cell culture at 21 DIV 1 μM choline, CDPC and AGPCslightly decreased TG-2 expression as well as AGPC addition in 35 DIV cultures. Thesefindings suggest an up- and down-regulation of TG-2 expression during different stages ofmaturation and differentiation in our cell culture model. Moreover, these data indicate unaf-fected involvement of cholinergic transmission mechanisms in astroglial cells, in theabsence of neuron-glia cross-talking important in neurotransmission modulation.

In conclusion, our findings suggest that choline, CDPC, and AGPC may play a role atthe molecular level during biochemical and physiological events involved in differentstages of astroglial cell maturation in starved serum-free cultures. In addition, it is wellknown (24) that beneficial activity of exogenous CDP-choline, choline, and α-GPChas also been postulated or reported for dyskinesia, Parkinson's disease, cardiovasculardisease, aging, Alzheimer's disease, learning and memory, and cholinergic stimulation.

In particular, our in vitro model may be considered a tool to investigate also the effects ofcholine and choline-containing phospholipids as possible therapeutic agents in pathological sit-uations such as ischemia and/or hypoxia, where these molecules may have beneficial effects.

Acknowledgments

This work is dedicated to the memory of Dr. Mariano Trognoni, who expired while theseexperiments were in progress.

Declaration of Interest

This research was supported by a grant from MDM S.p.A. (Monza, Italy) to ProfessorRoberto Avola.

The authors report no conflicts of interest. The authors alone are responsible for thecontent and writing of the paper.

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