Journal of Neuroscience Research 92:8694 (2014)

Effect of Lipoic Acid and

a-Glyceryl-Phosphoryl-Choline
on Astroglial Cell Proliferation and
Differentiation in Primary Culture
S. Grasso,1 V. Bramanti,1,2 D. Tomassoni,3 D. Bronzi,1 G. Malfa,2,4 E. Traini,2,5
M. Napoli,6 M. Renis,4 F. Amenta,5 and R. Avola1,2*
1

Department of Bio-Medical Sciences, Section of Biochemistry, University of Catania, Catania, Italy

International PhD Course in Neurobiology, Department of Chemical Sciences, Section of Biochemistry,
University of Catania, Catania, Italy
3
School of Bioscience and Biotechnology, University of Camerino, Camerino, Italy
4
Department of Drug Sciences, University of Catania, Catania, Italy
5
School of Pharmacy, Section of Human Anatomy, University of Camerino, Camerino, Italy
6
Department of Biological, Geological and Environmental Sciences, University of Catania, Catania, Italy
2

Lipoic acid plays a crucial role as antioxidant and

metabolic component of enzymes involved in glucose
metabolism of different cell types. Choline alphoscerate
(a-glyceryl-phosphoryl-choline [aGPC]) is a semisynthetic
derivative of phosphatidylcholines representing, among
acetilcholine precursors, a cholinergic drug. In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 or 21 DIV astrocyte
cultures treated with 50 lM (1)lipoic acid or (1/2)lipoic
acid and/or 10 mM aGPC for 24 hr. In addition, we evaluated the possible genoprotective effect by analysis of
DNA status detected by alkaline comet assay. The addition of single drugs [(1)lipoic acid, (1/2)lipoic acid, or
aGPC] induced an upward modulation of the expression of biomarkers used in our study. On the contrary, the
cotreatment with either (1)lipoic acid 1 aGPC or (1/2)lipoic 1 aGPC surprisingly showed no significant modification or even a downregulation of the above-mentioned
biomarkers. This latter finding demonstrated no additional
effect after the cotreatment with both drugs with respect
to the single treatments alone. Further studies are necessary to clarify the specific mechanism evoked by the
processing of these neuroprotective agents in our in vitro
models. Finally, these preliminary findings may represent
a good tool with which to clarify the antioxidant and
metabolic roles played by lipoic acid in proliferating
and differentiating astroglial cell cultures, during an interactive cross-talk between glial and neuronal cells, after
brain lesions or damage correlated with oxidative stress
that may occur in some degenerative diseases. VC 2013
Wiley Periodicals, Inc.

Key words: a-lipoic acid; aGPC; cytoskeletal proteins;

cyclin D1; MAP-kinase; astroglial cell cultures

cord. They are the most abundant cells of the brain.

Astrocytes function to maintain the homeostatic environment of the CNS and also play an important role in
immune regulation, acting as a source of chemokines,
cytokines, and effector molecules. The idea that astrocytes
have active roles in the modulation of neuronal activity
and synaptic neurotransmission is now widely accepted
(Bramanti et al., 2012).
a-Lipoic acid (ALA), also known as thioctic acid, was
first isolated from bovine liver in 1950 (Reed, 2001).
Lipoic acid contains two thiol groups, which may be oxidized or reduced. As with the thiol antioxidant glutathione, ALA is part of a redox pair, being the oxidized
partner of the reduced form dihydrolipoic acid (DHLA).
Unlike glutathione, for which only the reduced form is
an antioxidant, both the oxidized and reduced forms of
lipoic acid are antioxidants.
ALA is reduced in vivo to its dithiol form, DHLA,
which also possesses biological activity. DHLA is a potent
reducing agent with the capacity to reduce the oxidized
forms of several important antioxidants, including vitamin
C and glutathione (Jones et al., 2002). Endogenously synthesized ALA is covalently bound to specific proteins,
which function as cofactors for mitochondrial dehydrogenase enzyme complexes.
S. Grasso and V. Bramanti contributed equally to this work.
Contract grant sponsor: MDM S.p.A. (to R. Avolas research group).
*Correspondence to: Prof. Roberto Avola, Full Professor of Biochemistry, Department of Bio-Medical Sciences, Section of Biochemistry, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. E-mail:
ravola@unict.it
Received 19 June 2013; Revised 23 July 2013; Accepted 25 July 2013

Astrocytes, also known collectively as astroglia, are

characteristic star-shaped glial cells in the brain and spinal
C 2013 Wiley Periodicals, Inc.
V

Published online 26 October 2013 in Wiley Online Library

(wileyonlinelibrary.com). DOI: 10.1002/jnr.23289

Effect of ALA and aGPC on Astrocyte Cultures

Because of an asymmetric carbon having four different attached groups, ALA exists as two enantiomers, the
R-enantiomer [(1)lipoic acid] and the S-enantiomer
[(2)lipoic acid]. Naturally occurring lipoic acid is the
R-form, but synthetic lipoic acid is a racemic mixture of
R-enantiomer and S-enantiomer [(2/1)lipoic acid].
Both forms seem to have different potencies; it was previously shown that the R-enantiomer is more potent than
the S-enantiomer in its ability to stimulate glucose uptake
in L6 myotubes, (Estrada et al., 1996) as well as to
increase insulin-stimulated glucose uptake in obese
Zucker rats (Khanna et al., 1999).
ALAs antioxidant properties consist of the following: 1) its capacity to scavenge reactive oxygen species
(ROS) directly; 2) its ability to regenerate endogenous
antioxidants, such as glutathione and vitamins E and C;
and 3) its metal-chelating activity, resulting in reduced
ROS production. Largely because of its antioxidant properties, ALA has recently been reported to afford protection against oxidative injury in various disease processes,
including neurodegenerative disorders (Packer et al.,
1997; Evans and Goldfine, 2000; Smith et al., 2004; Salinthone et al., 2008).
ALA has neuroprotective effects in neuronal cells.
One possible mechanism for the antioxidant effect of ALA
is its metal-chelating activity (Ou et al., 1995). In a further
study, Muller and Krieglstein (1995) have tested whether
pretreatment with ALA can protect cultured neurons
against injury caused by cyanide, glutamate, or iron ions.
Neuroprotective effects were significant only when the
pretreatment with ALA occurred for >24 hr. The authors
conclude that neuroprotection occurs only after prolonged
pretreatment with ALA and probably is due to the radicalscavenging properties of endogenously formed DHLA.
Changes in cholinergic function are implicated in
the pathogenesis of learning and memory alterations
occurring in adult-onset cholinergic dysfunction, including dementia disorders (Gottfries et al., 1994). Many of
these precursors were disregarded early because their efficacy was not clearly demonstrated. This is not true for
choline alphoscerate (aGPC), a cholinergic precursor
available in the pharmaceutical market of several countries, which has been studied both in preclinical paradigms
and in clinical trials.
aGPC is a semisynthetic derivative of lecithin. After
oral administration, it is converted to phosphorylcholine,
a metabolically active form of choline able to reach cholinergic nerve terminals, where it increases acetylcholine
synthesis, levels, and release (Amenta and Tayebati, 2008;
Bramanti et al., 2008a). Mechanisms of action aGPC are
mainly two. In fact, the compound interferes with brain
phospholipid metabolism and increases brain choline and
acetylcholine levels and release (Amenta et al., 2001;
Amenta and Tayebati, 2008).
A restorative role of aGPC in the central cholinergic system was also documented by studies performed in
old rodents. In these investigations, the compound was
able to counter age-related changes in brain acetylcholine
synthesizing (choline acetyltransferase) and degrading
Journal of Neuroscience Research

87

(acetylcholinesterase) enzymes (Amenta et al., 1994) and

some subtypes of muscarinic cholinergic receptors
(Amenta et al., 1994; Muccioli et al., 1996).
It is well known (Bramanti et al., 2010a) that glial
cells are also involved in providing neurotrophic signals to
neurons required for their survival, proliferation, and differentiation. Glial fibrillary acidic protein (GFAP) is expressed
in the central nervous system in astrocytes. It is involved in
many important CNS processes, including cell communication and functioning of the bloodbrain barrier.
Concerning cell cycle study, it is well known that
cyclin D is a member of the cyclin protein family that is
involved in regulating cell cycle progression. Another
crucial cell cycle marker is ornithine decarboxylase
(ODC). It is an enzyme involved in polyamine metabolism. Instead, a well-known signalling transduction pathway marker is MAP-kinase.
Because ALA plays a pivotal role as antioxidant and
metabolic component of some enzymatic complexes
involved in glucose metabolism of different cell types, the
present study evaluated the expression of some proliferation and differentiation markers in 15 or 21 DIV astrocyte
cultures treated with 50 lM (1)lipoic acid or (1/2)
lipoic acid and/or 10 mM aGPC for 24 hr. In particular,
the expression changes of some astroglial differentiation,
proliferation, and signalling transduction pathway biomarkers, in 15 or 21 DIV astrocyte cultures, were evaluated via molecular biology techniques. In addition, we
evaluated the possible genoprotective effect by analysis of
DNA status detected by the alkaline comet assay.
MATERIALS AND METHODS
Astroglial Cell Cultures
Primary cultures of astrocytes were prepared from newborn albino rat brains (12-day-old Wistar strain) as previously
described (Prezzavento et al., 2007; Bramanti et al., 2008b). In
particular, cerebral tissues, after dissection and careful removal of
the meninges, were mechanically dissociated through sterile
meshes of 82-mm pore size (Nitex). Isolated cells were suspended in Dulbeccos modified Eagles medium (DMEM), supplemented with 20% (v/v) heat-inactivated fetal bovine serum
(FBS), 2 mM glutamine, streptomycin (50 mg/ml), and penicillin
(50 U/ml) and plated at a density of 3 3 106 cells/100-mm dish
and 0.5 3 105 cells (Campisi et al., 2008). The low initial plating
density of dissociated cells was meant to favor the growth of
astrocytes with only very little oligodendroglial and microglial
contamination. Cells were maintained at 37 C in a 5% CO2/
95% O2 air humidified atmosphere for 2 weeks, and the medium
was removed every 3 days. Astroglial cells were characterized at
15 DIV, i.e., when confluent, by immunofluorescent staining
with the glial marker GFAP, as previously reported (Bramanti
et al., 2007). All efforts were made to minimize both the suffering and the number of animals used. All experiments conformed
to guidelines of the Ethical Committee of University of Catania.
Drug Treatment
ALA is a potent antioxidant that is in clinical trials for diabetic neuropathy. Many reagents, when dissolved in growth

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Grasso et al.

media, release hydrogen peroxide. This can confound the study

of their protective effects in cell cultures by producing a prooxidant effect. Catalase is an enzyme that breaks down hydrogen
peroxide, so the freshly dissolved ALA was treated with catalase
to remove hydrogen peroxide before applying to the cultures as
follows. Astrocyte cultures at 15 DIV were mantained under
following experimental conditions: lipoic acid was dissolved in
0.5 M dimethylsulfoxide (DMSO) and diluted in culture
medium at a concentration of 1 mM. Catalase (1,000 U/ml)
was added, and the solution was incubated at 37 C for 30 min.
This solution was then diluted 1:10 in the culture medium in
order to obtain 50 lM lipoic acid final concentration. As evaluated in doseresponse analysis (data not showed), the best concentration of (1/2)lipoic acid or (1)lipoic acid was 50 lM.
Treatments included 1) control untreated astrocyte cultures; 2)
50 lM (1/2)lipoic acid- or 50 lM (1)lipoic acid-treated astrocyte cultures; 3) 10 mM a-GPC-treated astrocyte cultures; and
4) 50 lM (1/2)lipoic acid 1 10 mM a-GPC- or 50 lM
(1)lipoic acid 1 10 mM a-GPC-treated astrocyte cultures.
Determination of Cell Viability
Cell viability was evaluated by the 3-[4,5-dimethylthiazol-2-yl)22,5-diphenyl]tetrazolium bromide (MTT) reduction
assay. This cell proliferation assay was used as a quantitative colorimetric method for measurements of cellular cytotoxicity.
Briefly, MTT was added to each well with a final concentration
of 1.0 mg/ml and incubated for 1 hr in a CO2 incubator. The
dark blue formazan crystals formed in intact cells were extracted
with 250 ll DMSO, and the absorbance was read at 595 nm
with a microtiter plate reader (Bio-Tek Instruments, Winooski,
VT). Results were expressed as the percentage MTT reduction
vs. control cells.
Immunocytochemical Analysis
The cells were fixed with 4% paraformaldehyde in 0.1 M
Tris saline buffer (TBS) for 20 min. Nonspecific antibody reactions were blocked with 5% normal goat serum for 1 hr at
room temperature. Next, fixed cells were incubated overnight
at 4 C with primary antibodies directed against GFAP (1:200;
Chemicon, Temecula, CA). After three washes, cells were
incubated with secondary antibody conjugate with fluorescein5-isothiocyanate (FITC) for 1 hr at room temperature (1:200;
Chemicon; Bronzi et al., 2010).
Western Blot Analysis for Cytoskeletal Proteins
Quantitative Western blots were performed for GFAP
and vimentin as reported by Bramanti et al. (2008c) and Tomassoni et al. (2010). Briefly, after treatment, cells were harvested
in cold PBS, collected by centrifugation, resuspended in a
homogenizing buffer (50 mM Tris-HCl, pH 6.8, 150 mM
NaCl, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride,
and 10 lg/ml of aprotinin, leupeptin, and pepstatin) and sonicated on ice. Protein concentration of the homogenates was
diluted to 1 mg/ml with 23 reducing stop buffer (0.25 M TrisHCl, pH 6.8, 5 mM EGTA, 25 mM dithiothreitol [DTT], 2%
sodium dodecyl sulfate [SDS], and 10% glycerol with bromophenol blue as the tracking dye). Proteins (30 lg) were sepa-

rated on 10% SDS-polyacrylamide gels and transferred to

nitrocellulose membranes. Blots were blocked overnight at 4 C
with 5% nonfat dry milk dissolved in 20 mM Tris-HCl, pH
7.4, 150 mM NaCl, 0.5% Tween 20. GFAP, vimentin, Cyclin
D1, O.D.C., MAP-Kinase and beta-actin expression was
detected by incubation, respectively, with specific monoclonal
antibodies for GFAP, vimentin, cyclin D1, O.D.C. and MAPkinase (GFAP: MAB360 clone: GA5 Chemicon; vimentin:
mAb clone V9 MAB3400 Chemicon; Cyclin D1 C55882ML, Sigma Aldrich; O.D.C. clone ODC-29, O1136-2ML
Sigma Aldrich; MAP-Kinase M5670-2ML, Sigma Aldrich;
beta-actin A1978-200UL, Sigma Aldrich; dilutions 1:1,000 for
GFAP, vimentin, cyclin D1, O.D.C. and MAP-kinases). A secondary antibody-enzyme conjugate, which recognizes the primary antibody is added to find locations where the primary
antibody bound. Protein expression was visualized with a
chemiluminescence ECL kit after autoradiography film exposure. Blots were scanned and quantified in Image J. The intensity of single bands was referred to the intensity of b-actin band
used as a reference protein.
Comet Assay
The comet assay, a gel electrophoretic technique particularly advantageous to assess the presence of DNA fragmentation
in every single cell, was performed on astroglial cells treated as
previously reported according to a modification of the protocol
reported by Singh and collaborators. In 10 ml cell suspension,
65 ml of low-melting-point agarose (LMA) is added to 0.7%.
The incorporated cells are arranged on a slide previously coated
with an agarose thin layer of normal melting point agarose
(NMA) at 1%. The sandwich structure is completed by overlaying a further layer of LMA to 0.7% (85 ml).

Lysis of cells. The slides are immersed in lysis solution at pH 10 (N-lauryl-sarcosine 1%, NaCl 2.5 M, 100 mM
Na2EDTA, Triton X-100 1%, DMSO 10%) for 1 hr at 4 C.
The strongly alkaline conditions of the lysis induce protein
denaturation, including histone, and hydrolysis of RNA. The
presence of the hydroxyl group in position 20 on the ribose
causes the formation of unstable intermediates that are cyclically
further hydrolyzed with formation of a mixture of nucleoside
monophosphates 20 and 30 . The absence of this group in the
DNA molecule protects the latter from hydrolysis, allowing
only denaturation. Moreover, the presence of DMSO, destabilizing the double helix, allowed us to obtain the DNA separated
into two filaments.
Electrophoresis. Finished lysis slides are positioned
in the electrophoretic tank containing, for the alkaline comet,
running buffer at pH > 13 (300 mM NaOH, 1 mM Na2EDTA)
and left for 20 min in the dark, in order to facilitate unwinding
of the DNA. After this step, the electrophoresis runs for 30 min
at constant voltage (0.7V/cm). At the end of the stroke, the
slides are washed twice with the neutralization solution to pH
7.5 (0.4 M Tris-HCl) and scored using a Leica fluorescence
microscope (Leica, Wetzlar, Germany) interfaced with a computer. Dedicated software (Leica-QWIN) allowed the analysis
and the quantification of DNA damage by measuring the level
of DNA damage as percentage of the fragmented DNA
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Effect of ALA and aGPC on Astrocyte Cultures

(TDNA), because this is considered to be the most comprehensive and meaningful comet parameter.

89

RESULTS
Intracellular localization of astroglial markers and relative
morphological modifications induced by drug treatment
were evaluated by immunocytochemical analysis (Fig. 1).
To assess the viability of astroglial cells in cultures treated
with ALA, dextrorotatory enantiomer/raceme was performed by MTT analysis with tetrazolium salts; we
observed that ALA had no harmful effects on cell metabolic activity (data not shown). Data concerning immunocytochemical analysis evaluated for GFAP show that all
of the treatments are positive for the above-mentioned
astroglial marker (Fig. 1). In addition, immunocytochemical analysis highlights that the protein is found in the
cytoplasm and in subcellular compartments, and a typical
polygonal flat shaped astrocyte was clearly observed
(Fig. 1).
Preliminary studies (data not shown) have indicated
that (1)lipoic acid was particularly active in the expression of biomarkers in astroglial cultures of astrocytes at 15

DIV during the proliferative phase in our in vitro model.

Instead, treatment with the (1/2)lipoic acid in astroglial
cultures at 21 DIV, the period of astrocytes differentiation, highlighted a significant increase of the expression of
the biomarkers GFAP, vimentin, cyclin D1, MAP-kinase,
and ODC. Therefore, we decided to present in this article
the more significant data that highlight the effect of dextrorotatory enantiomer and the racemic, respectively, in
cultured astrocytes at 15 and 21 DIV.
The results obtained by Western blot analysis show
that GFAP (Fig. 2) and vimentin (Fig. 3) expression
increased after treatment with (1)lipoic acid and aGPC
alone in 15 DIV astrocyte cultures compared with
untreated control ones. In 21 DIV astrocyte cultures, the
treatment with (1/2)lipoic acid showed a slight increase
of GFAP expression (Fig. 2) and no significant modification of vimentin expression (Fig. 3); instead, the treatment with aGPC alone or with (1/2)lipoic acid both
together significant decreased it.
In 15 DIV astrocyte cultures, the treatment with
(1)lipoic acid alone induced a highly significant
enhancement of cyclin D1 expression (Fig. 4), a wellknown proliferation marker, with respect to the values of
untreated control ones. Conversely, no significant modification of cyclin D1 expression in aGPC-treated astrocyte cultures was observed (Fig. 4); instead, the treatment
with (1)lipoic acid and aGPC together significant
decreased it. In 21 DIV astrocyte cultures, the treatment
with (1/2)lipoic acid alone or in combination with

Fig. 1. Immunocytochemical analysis for GFAP after (1/2)lipoic acid

(B), aGPC (C), (1/2)lipoic acid 1 aGPC (D), (1)lipoic acid (E),
and (1)lipoic acid 1 aGPC (F) in astrocyte cultures. A: Control.
Data show that all of the treatments are positive for the above-

mentioned astroglial marker. ALA, a-lipoic acid; GFAP, glial fibrillar

acidic protein; aGPC, choline alphoscerate or a-glyceryl-phosphorylcholine. [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]

Statistical Analysis
All values are presented as mean 6 SEM of data obtained
from five different dishes. Students t-test was used to compare
values of densitometric analysis of immunoblots. Statistical significance was acceptyed at p < 0.05

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Grasso et al.

Fig. 2. Western blotting analysis data for GFAP expression after thye following treatments. Astrocyte cultures at 21 DIV: control (1), (1/
2)lipoic acid treatment (2), aGPC treatment (3), (1/2) lipoic
acid 1 aGPC treatment (4); astrocyte cultures at 15 DIV: control (5),
(1)lipoic acid treatment (6), aGPC treatment (7), (1)lipoic
acid1aGPC (8). Each value expressed as A.U. (arbitrary units) is the
average 6 SEM of values from five different dishes. Statistical significance was accepted at *p<0.05 vs control. GFAP, glial fibrillar acidic
protein; aGPC, choline alphoscerate or a-glyceryl-phosphoryl-choline.

aGPC showed no significant modification of cyclin D1

expression (Fig. 4). A significant enhancement of cyclin
D1 expression was observed in aGPC-treated cultures
(Fig. 4).
In 15 DIV astrocyte cultures, the treatment with
(1)lipoic acid alone induced a highly significant increase
of ODC expression, a well-known marker associated
with increased cell growth (Fig. 5). The treatment with
aGPC alone or in combination with (1)lipoic acid
showed a marked decrement of ODC expression (Fig. 5).
In 21 DIV astrocyte cultures, surprisingly, the treatment
with (1/2)lipoic acid in combination with aGPC significantly increased ODC expression (Fig. 5); instead, no
significant modification was observed after addition of
(1/2)lipoic acid or aGPC alone (Fig. 5).
Moreover, the MAP-kinase expression evaluated by
Western blot analysis was enhanced after treatment with
(1)lipoic acid alone in 15 DIV astrocyte cultures; no significant modification after treatment with aGPC alone
was observed, and a decrement of marker expression after
treatment with (1)lipoic acid 1 aGPC together was
observed (Fig. 6). In 21 DIV astrocyte cultures, the treatment with (1/2)lipoic acid or aGPC alone induced an
enhancement of MAP-kinase expression, but no significant modification of the marker expression in (1/2)
lipoic acid in combination with aGPC was observed

Fig. 3. Western blotting analysis data for vimentin expression after the
following treatments. Astrocyte cultures at 21 DIV: control (1), (1/
2)lipoic acid treatment (2), aGPC treatment (3), (1/2)lipoic
acid 1 aGPC treatment (4); astrocyte cultures at 15 DIV: control (5),
(1)lipoic acid treatment (6), aGPC treatment (7), (1)lipoic
acid 1 aGPC (8). Each value expressed as A.U. (arbitrary units) is the
average 6 SEM of values from five different dishes. Statistical significance was accepted at * p<0.05 vs control. aGPC, choline alphoscerate or a-glyceryl-phosphoryl-choline.

(Fig. 6). The analysis of DNA status by alkaline comet

assay showed significant modifications induced by the
treatment with (1)lipoic acid.
DISCUSSION
The proliferation and differentiation of astroglial cells in
primary cultures represents a valuable tool to study biochemical mechanisms involved in their development and
maturation. As is well known, glial cells are involved in
providing neurotrophic signals to neurons required for
their survival, proliferation, and differentiation (Bramanti
et al., 2010a). In particular, astrocytes perform many functions, including biochemical support of endothelial cells
that form the bloodbrain barrier, provision of nutrients
to the nervous tissue, maintenance of extracellular ion
balance, and a role in the repair and scarring process of
the brain and spinal cord following traumatic injuries.
In addition to their physiological involvement,
astrocytes play an important role in pathological conditions of the nervous system. Several lines of evidence
indicate that glia influences the growth, migration, and
differentiation of neurons, but the effect of neuronal cells
on astrocytes is far from being well understood. Increasing
evidence has accumulated that neurons are modulators of
astrocyte gene expression and differentiation (Bramanti
et al., 2010b). In addition to the physiological functions
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Effect of ALA and aGPC on Astrocyte Cultures

91

Fig. 4. Western blotting analysis data for cyclin D1 expression after

the following treatments. Astrocyte cultures at 21 DIV: control (1),
(1/2)lipoic acid treatment (2), aGPC treatment (3), (1/2)lipoic
acid 1 aGPC treatment (4); astrocyte cultures at 15 DIV: control (5),
(1)lipoic acid treatment (6), aGPC treatment (7), (1)lipoic
acid 1 aGPC (8). Each value expressed as A.U. (arbitrary units) is the
average 6 SEM of values from five different dishes. Statistical significance was accepted at *p<0.05 vs control. aGPC, choline alphoscerate or a-glyceryl-phosphoryl-choline.

Fig. 5. Western blotting analysis data for O.D.C. expression after the
following treatments. Astrocyte cultures at 21 DIV: control (1), (1/
2)lipoic acid treatment (2), aGPC treatment (3), (1/2)lipoic
acid 1 aGPC treatment (4); astrocyte cultures at 15 DIV: control (5),
(1)lipoic acid treatment (6), aGPC treatment (7), (1)lipoic
acid 1 aGPC (8). Each value expressed as A.U. (arbitrary units) is the
average 6 SEM of values from five different dishes. Statistical significance was accepted at *p<0.05 vs control. ODC, ornithine decarboxylase; aGPC, choline alphoscerate or a-glyceryl-phosphoryl-choline.

of protein-bound ALA, there is increasing scientific and

medical interest in potential therapeutic uses of pharmacological doses of free ALA (Kramer et al., 2001).
It is well known that lipoic acid is a neuroprotective
antioxidant agent able to act by scavenging ROS and
stimulation of glutathione synthesis. In fact, it seems that
ALA stimulating glutathione synthesis plays a pivotal antioxidant role, particularly in astroglial cells, maintained
under stressed conditions induced by glutamate excitotoxicity. Moreover, ALA and DHLA exhibit many antioxidant properties, including the ability to recycle GSH and
ascorbate, scavenge ROS and reactive nitrogen species,
and chelate transition and heavy metals. Several cell culture studies as well as in vivo studies in animal and human
populations demonstrate that ALA may be effective in a
variety of pathological conditions, especially those that are
associated with oxidative stress. In addition, it is particularly interesting that ALA/DHLA redox coupling significantly modulates critical protein thiol groups, thereby
activating certain signal transduction pathways. It is also
known that a number of transcription factors (e.g.,
nuclear factor-jB [NFjB], activator protein 1 [AP-1],
stimulatory protein [SP-1], and NFe2-related factors
[Nrf1, Nrf2]) also act in a redox-sensitive manner.
In the literature are data concerning novel therapeutic approaches for different neurodegenerative diseases

associated with oxidative stress, and, in particular, the

raceme ALA is usually utilized in clinical trials, even if the
(1)lipoic acid is the preferred isomer in biological systems. ALA is unique among natural antioxidants in its
ability to fulfill all of these requirements, making it a
potentially highly effective therapeutic agent for a number
of conditions in which oxidative damage has been
implicated.
Largely because of its antioxidant properties, ALA
has recently been reported to afford protection against
oxidative injury in various disease processes, including
neurodegenerative disorders (Packer et al., 1997; Evans
et al., 2000). Although the ability of ALA to scavenge
ROS directly appears to be responsible, at least partially,
for its neuroprotective effects, it remains unknown
whether the neuroprotecitve effects of ALA might also
occur through other mechanisms, such as induction of
the endogenous antioxidants and phase 2 enzymes in neuronal cells, and, in addition, the increasing endogenous
defences might afford protection against oxidative/electrophilic neuronal cell injury.
Particularly interesting are the findings concerning
the different effects evoked by the treatment with (1/2)
lipoic acid or (1)lipoic acid, evidencing a better stimulation role by (1)lipoic acid on astroglial proliferation and
differentiation biomarkers with respect to the addition of

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Grasso et al.

Fig. 6. Western blotting analysis data for MAP-kinase expression after

the followqing treatments. Astrocyte cultures at 21 DIV: control (1),
(1/2)lipoic acid treatment (2), aGPC treatment (3), (1/2)lipoic
acid 1 aGPC treatment (4); astrocyte cultures at 15 DIV: control (5),
(1)lipoic acid treatment (6), aGPC treatment (7), (1)lipoic
acid 1 aGPC (8). Each value expressed as A.U. (arbitrary units) is the
average 6 SEM of values from five different dishes. Statistical significance was accepted at *p<0.05 vs control. aGPC, choline alphoscerate or a-glyceryl-phosphoryl-choline.

(1/2)lipoic acid. Previous findings (data not shown)

from doseresponse analysis showed that the best concentration of (1/2)lipoic acid or (1)lipoic acid was 50 lM.
For aGPC, it is well known that it is a cholinergic
precursor, which has been shown to be effective in countering cognitive symptoms in forms of dementia disorders

Fig. 7. Genotoxic evaluation by comet assay. The analysis of DNA status by alkaline comet assay showed significant modifications induced by
the treatment with (1)lipoic acid resembling possible genoprotective
effect. (1) Ctrl; (2) (1/2)lipoic acid; (3) aGPC; (4) (1)lipoic acid;
(5) aGPC 1 (1/2)lipoic acid; (6) aGPC 1 (1)lipoic acid. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]

of degenerative, vascular, or combined origin (Bramanti

et al., 2008c). The observation that treatment with aGPC
attenuates the extent of glial reaction in the hippocampus
of spontaneously hypertensive rats (SHR) suggests also
that the compound may afford neuroprotection in this
animal model of vascular brain damage and has an influence on astroglial dynamics (Bramanti et al., 2008c). Furthermore, the neurochemical profile of aGPC drug
induces an initial short stimulatory effect on acetylcholine
release paralleled by a significant and long-lasting increase
of acetylcholine synthesis, resulting in an increased storage
of acetylcholine and a rising secretion upon stimulation
(Bramanti et al., 2008c).
A neuroprotective effect of treatment with aGPC
on hippocampal microanatomy and glial reaction, which
represents an early sign of brain damage, was documented
in SHR used as an animal model of brain vascular injury
(Tomassoni et al., 2006). Among cholinergic precursors
tested, aGPC elicited the most relevant stimulation on
vesicular acetylcholine transporter and choline transporter
in the same model of brain vascular injury, suggesting that
it represents a strong enhancer of central cholinergic neurotransmission (Tayebati et al., 2013).
In the present study, to clarify the antioxidant role
of (1)lipoic acid in the astroglial compartment, we investigated the effect of both (1)lipoic acid and (1/2)lipoic
acid alone or in combination with aGPC on astroglial
cell proliferation and differentiation in primary cultures.
In addition, particular attention was devoted to the
involvement of both compounds on the expression of
some different biomarkers related to the astroglial cytoskeletal network, cell cycle, and signal transduction pathways in our in vitro model.
With regard to the results obtained in the astroglial
cell cultures treated with lipoic acid or aGPC alone or
both in combination, it is particularly important to underscore that the addition of single drugs [(1)lipoic acid, (1/
2)lipoic acid, or aGPC] induced an upweard modulation
of the expression of biomarkers used in our study. In particular, data concerning Western blot analysis in 15 DIV
astrocyte cultures show that GFAP (Fig. 2) and vimentin
(Fig. 3) expression increased after treatment with
(1)lipoic acid and aGPC alone compared with untreated
control ones. This demonstrates that young cultures (15
DIV) with their proliferative and differentiative activity
responded better to the neuroprotective effect of each
treatment, especially with (1)lipoic acid treatment, as
well documented by enhanced cytoskeletal protein
expression.
In fact, GFAP has beens proposed to play a crucial
role in astrocyteneuron interactions as well as in cellcell
communication. Furthermore, GFAP synthesis is considered an important element of the developmental program
of astrocyte differentiation and is part of the reactive
response to almost any CNS injury (Bramanti et al.,
2010a). GFAP has also been shown to be important in
repair after CNS injury, more specifically for its role in
the formation of glial scars in a multitude of locations
throughout the CNS, including the eye and brain. During
Journal of Neuroscience Research

Effect of ALA and aGPC on Astrocyte Cultures

the formation of GFAP networks in some reactive astrocytes, vimentin may act as a cytoskeleton-associated protein (Harrison et al., 1992).
In addition, the evidence that 15 DIV astroglial cultures treated with (1)lipoic acid alone showed an upregulation of cell cycle markers probably is due to the
neuroprotective effect of the drug inducing a marked proliferating stimulatory effect. In fact, in proliferating cells,
cyclin D-Cdk4/6 complex accumulation is of great
importance for cell cycle progression. Namely, cyclin DCdk4/6 complex partially phosphorylates Rb, which is
able to induce expression of some genes important for
S-phase progression. Cyclin D is regulated by the
downstream pathway of mitogen receptors via the Ras/
MAP kinase and the b-catenin-Tcf/LEF pathways and
PI3K.
On the contrary, the cotreatment with both
(1)lipoic acid1 aGPC, or (1/2)lipoic1 aGPC surprisingly showed no significant modification or even a downregulation of the above-mentioned biomarkers (GFAP,
vimentin, cyclin D1, ODC, and MAP-kinases). This latter finding demonstrated no additional effect after the
cotreatment with both drugs with respect to the single
treatments alone. This finding requires further investigation to clarify the specific mechanism evoked by the
processing of these neuroprotective agents in our in vitro
models.
A possible explanation of this unexpected results
may depend on the fact that lipoic acid acting as an
insulin mimetic agent and stimulating the available
receptors would elicit a mechanism of action that could
interfere in common signal transduction pathways, causing their desensitization and thereby leading to a downregulation of the expression of the biomarkers studied. In
genotoxic evaluation by comet assay, we observed that
the analysis of DNA status showed a significant modification induced by the treatment with (1)lipoic acid resembling a possible genoprotective effect.
In conclusion our results indicate a significant
increase of GFAP and vimentin expression in 15 DIV
astrocyte cultures after treatment with (1)lipoic acid or
aGPC alone. In addition, an increase of proliferative
marker expression in 15 DIV astrocyte cultures treated
with (1)lipoic acid alone was observed. The antioxidant
role played by ALA and aGPC may well be correlated
with the proliferative and differentiative state of astroglial
cell cultures in our in vitro model, as demonstrated by
upward and downward modulation of the expression of
the astroglial biomarkers investigated. This suggests that
the neuroprotective action against oxidative stress mimicked by ALA and aGPC may aid the proliferating and
differentiating activity of astroglial cells in primary cultures. Finally, these preliminary findings may represent a
good tool for clarifying the antioxidant and metabolic
role played by lipoic acid in proliferating and differentiating astroglial cell cultures, during an interactive cross-talk
between glial and neuronal cells, after brain lesions or
damage correlated with the oxidative stress that may
occur in some neurodegenerative diseases, such as AlzheiJournal of Neuroscience Research

93

mers and Parkinsons diseases, Huntingtons chorea,

stroke, and ictus.
ACKNOWLEDGMENTS
The authors greatly appreciated the skillful cooperation of
Dr. Roberto Gabriele from M.D.M. S.p.A. Monza
(Monza e Brianza, Italy). The authors declare that they
have no current conflicts of interest with regard to the
contents of this article.
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