Open Journal of Apoptosis, 2012, 1, 9-18 Published Online July 2012 (
Astrocytes Prevent Ethanol Induced Apoptosis of Nrf2
Depleted Neurons by Maintaining GSH Homeostasis
Madhusudhanan Narasimhan1,2*, Marylatha Rathinam1, Dhyanesh Patel1,
George Henderson1,2, Lenin Mahimainathan1,2*
1Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, USA
2South Plains Alcohol and Addiction Research Center, Texas Tech University Health Sciences Center, Lubbock, USA
Email: {*lenin.mahimainathan, *madhu.narasimhan}
Received May 14, 2012; revised June 27, 2012; accepted July 12, 2012
Glutathione (GSH), a major cellular antioxidant protects cells against oxidative stress injury. Nuclear factor erythroid
2-related factor 2 (NFE2L2/Nrf2) is a redox sensitive master regulator of battery of antioxidant enzymes including
those involved in GSH antioxidant machinery. Earlier we reported that ethanol (ETOH) elicits apoptotic death of pri-
mary cortical neurons (PCNs) which in partly due to depletion of intracellular GSH levels. Further a recent report from
our laboratory illustrated that ETOH exacerbated the dysregulation of GSH and caspase mediated cell death of cortical
neurons that are compromised in Nrf2 machinery (Narasimhan et al., 2011). In various experimental models of neu-
rodegeneration, neuronal antioxidant defenses mainly GSH has been shown to be supported by astrocytes. We therefore
sought to determine whether astrocytes can render protection to neurons against ETOH toxicity, particularly when the
function of Nrf2 is compromised in neurons. The experimental model consisted of co-culturing primary cortical astro-
cytes (PCA) with Nrf2 downregulated PCNs that were exposed with 4 mg/mL ETOH for 24 h. Monochlorobimane
(MCB) staining followed by FACS analysis showed that astrocytes blocked ETOH induced GSH decrement in
Nrf2-silenced neurons as opposed to exaggerated GSH depletion in Nrf2 downregulated PCNs alone. Similarly, the
heightened activation of caspase 3/7 observed in Nrf2-compromised neurons was attenuated when co-cultured with as-
trocytes as measured by luminescence based caspase Glo assay. Furthermore, annexin-V-FITC staining followed by FACS
analysis revealed that Nrf2 depleted neurons showed resistance to ETOH induced neuronal apoptosis when co-cultured
with astrocytes. Thus, the current study identifies ETOH induced dysregulation of GSH and associated apoptotic events
observed in Nrf2-depleted neurons can be blocked by astrocytes. Further our results suggest that this neuroprotective
effect of astrocyte despite dysfunctional Nrf2 system in neurons could be compensated by astrocytic GSH supply.
Keywords: Astrocyte-Neuron Co-Culture; Nrf2; Ethanol; Oxidative Stress; GSH
1. Introduction
Glutathione (GSH) is the most abundant intracellular
non-protein thiol and anti-oxidant in the body with a
concentration of approximately 2 - 3 mM in brain [1]. It
is vital for guarding normal healthy metabolism as well
as defense against a range of disease and toxicity mecha-
nisms by appropriately controlling cellular redox levels,
most notably in the central nervous system (CNS) [1,2].
Various studies have demonstrated that maintenance of
intracellular GSH pool is important for limiting oxida-
tive-stress induced neuronal injury [3-5]. Several mecha-
nisms underlying GSH synthesis in neurons have been
proposed ranging from availability of a rate-limiting sub-
strate, cysteine to γ-glutamyl cycle that inturn includes a
number of enzymes and transporter molecules that all
play a vital role in GSH homeostasis [3,6].
In the last two decades of cellular antioxidant system
discoveries, a novel concept of “indirect antioxidants”
has been gaining popularity. The factors comprising “In-
direct antioxidants” chiefly acts through augmenting cel-
lular antioxidant capacity by binding to antioxidant re-
sponse elements (ARE) in the promoter of a gene and
enhancing its expression. In this class of molecules,
transcription factor NF-E2-related factor 2 (NFE2L2/
Nrf2) is demonstrated to be a master regulator activating
several antioxidative cytoprotective gene clusters [7].
Activation of Nrf2/ARE pathways has been shown to be
neuroprotective when induced by electrophilic drugs [8],
growth factors such as bFGF [9], and ethanol [4]. The
importance of Nrf2 was further validated using Nrf2
knockout studies in which Nrf2 null animals were found
to be more sensitive to various neurotoxins and deve-
loped oxidative stress dependent pathological symptoms
[10,11]. Notably, enzymes involved in GSH homeostasis
such as γ-glutamyl transpeptidase (GGT), glutathione
*Corresponding author.
opyright © 2012 SciRes. OJApo
synthase, glutathione reductase, γ-glutamyl cysteine li-
gase, and multi-drug resistance associated proteins are
targets of Nrf2 [7].
Typically, neuronal function in brain is mediated in
part by the complex interactions among different cell
types including astrocytes. Astrocytes are glial cells that
tile up 25% to 50% of brain volume outnumbering neu-
rons by over 5:1 [12-14]. These specialized cells lie in
close proximity to neurons serving multiple functions
ranging from energetic, antioxidant, signaling, plasticity
to several others which are required for normal function-
ing of the nervous system [15-17]. Cortical astrocytes
display higher basal and stimulated level of ARE-medi-
ated gene expression than neurons [18,19]. Further corti-
cal astrocytes have been demonstrated to possess strong-
er and highly regulated Nrf2 dependent GSH homeostasis
machinery [20,21]. Initial step in maintenance of neuron
GSH homeostasis is efflux of GSH by the astrocyte [22]
and the outcome of which is neuroprotection against
oxidative damage [23].
Astrocytes can provide direct neurotrophic support to
neurons [24]. Reports from numerous laboratories have
illustrated that primary neurons co-cultured with astro-
cytes showed reduced neurotoxicity as compared to neu-
ronal cultures against various neurotoxicants includeing
ethanol [5,23,25]. However, some studies suggest that
neurons become more susceptible to neurotoxicants in
the presence of astrocytes [26,27]. Since, astrocytes me-
diate both positive and negative responses in neuronal
cells, neuroscience views normal brain functioning as an
outcome of how information processing is accomplished
based on neuron-astrocyte interactions. This inturn, is
dependent on the diverse physiological and biological
responses that is evoked by exogenous-endogenous va-
riables in the individual cellular compartments.
Recent report from our laboratory has shown that
ETOH depletes intracellular GSH levels in isolated cor-
tical neuronal cultures without astrocytes [4] (Narasim-
han et al., 2011). Further the same study demonstrated
that ETOH induced GSH depletion and associated cell
death was exaggerated in Nrf2 downregulated primary
cortical neurons. In another independent study from our
laboratory, we have shown that co-culturing cortical as-
trocytes with fetal cortical neurons blocked ethanol medi-
ated oxidative stress and normalized GSH homeostasis
and subsequent cell death [23]. Thus, in the current study,
we have extended our previous observations and attempt-
ed to address whether astrocytes can protect neurons
even when the latter is compromised in Nrf2 system.
2. Materials and Methods
2.1 Materials
Minimum Essential Media (MEM), Dulbecco Minimum
Essential Medium (DMEM), Hank’s Balanced Salt Solu-
tion (HBSS), Fetal Bovine Serum (FBS) were obtained
from Invitrogen (Carlsbad, CA). Horse Serum (HS), tryp-
sin, DNase, antibiotics, poly-D-lysine, uridine, monochlo-
robimane, were purchased from Sigma (St. Louis, MO).
Annexin-V FITC apoptosis detection kit was obtained
from BD Biosciences (San Jose, CA). Fisher-Costar cell
culture inserts for co-culture was from Fisher Scientific
(Pittsburgh, PA). Smart Pool siRNA against Nrf2 and
non-targeting siRNA pool was purchased from Dhar-
macon Inc., (Lafayette, CO). Caspase-Glo 3/7 assay kit
was obtained from Promega Corporation (Madison, WI).
siPORT amine was from Ambion Inc. (Austin, TX).
2.2. Primary Cortical Neuron (PCN) Cultures
PCNs were prepared from E16-E17 timed pregnant
Sprague Dawley rats as described earlier [28]. Briefly,
embryos from amniotic sac were carefully taken out and
cerebral cortex from fetus was mechanically dissociated
in HBSS. The cells were suspended in MEM containing
10% FBS and 10% HS and were grown in a poly-D-ly-
sine coated plates. Cells were maintained in a humidified
atmosphere of 95% air and 5% CO2. After 1 day in vitro
(DIV), the cells were given “inhibitory” feeding with
uridine (10 mg/mL) containing MEM supplemented with
10% HS to suppress the growth of astrocytes and enrich
the cultures for neurons. This is a well-established and
documented primary neuronal culture system, which is
essentially free of glia. Dual immunostaining with MAP2
(for neurons) and GFAP (for astrocytes) were performed
and the isolation procedure reproducibly adopted yielded
~95% enriched neuronal culture [4,29]. Handling of ani-
mals was carried out according to the National Institutes
of Health guidelines for the use and care of laboratory
animals. The procedures involving isolation of cortical
neurons and astrocytes from the embryos were approved
by Institutional Animal Care and Use Committee.
2.3. Small Interfering RNA (siRNA)
4 DIV PCNs were transfected with 100 nM of smart-
pool siRNA against Nrf2 or non-targeting siRNA pool.
Briefly, 5 µL of siPORT amine and 100 nM of either
smartpool siRNA against Nrf2 or non-targeting siRNA
pool were diluted in Opti-MEM separately. After com-
plex formation for 20 mins according to manufacturer’s
instructions (Ambion), the transfection mixture was gen-
tly added to PCNs and returned to incubator for 24 h. For
the experiments involving ETOH, 24 h post transfection
of siRNA the cells were exposed to ETOH for additional
24 h and processed for various downstream applications
such as FACS analysis for detection of MCB, Annexin V
FITC/PI, and the caspase glo assay.
Copyright © 2012 SciRes. OJApo
2.4. Ethanol Treatment of PCNs
On 5 DIV, PCNs were treated with ETOH (4 mg/mL) for
24 h in an incubator saturated with ETOH to maintain
media ETOH (monitored using Analox AM1 alcohol
analyzer) [23]. The in vitro experiments involving ETOH
in the current study uses a clinically relevant dose, which
is at or below to that used in other studies to elicit a range
of neurotoxic responses including brain apoptotic res-
ponses in various neuron culture, mouse and rat models
2.5. Primary Cortical Astrocytes (PCA) Cultures
PCAs were prepared from the cerebral hemispheres of
2-day old rat neonates as described by [31]. Briefly, brain
cortices from the new-born pups were aseptically re-
moved, trypsin digested and DNase treated to dissociate
the cells. The cells were then filtered through a 0.25 µM
sieve to remove neurons and the filtered cells containing
astrocytes were resuspended in DMEM supplemented
with 10% FBS, antibiotics and seeded in 75 cm2 tissue
culture flasks at a density of 6 × 106 cells. The cultures
were fed every 3 days and at confluency, the astrocytes
were split on day 6 and plated onto 100 mm petri dish.
Approximately 95% of the cells were identified as astro-
cytes based on the positivity for glial fibrillary acidic
protein (GFAP) staining showing the purity of astrocytic
composition of the culture (data not shown).
2.6. Co-Culture of PCA and Nrf2 siRNA
Transfected PCNs
The transwell insert used in our study is made of polyes-
ter membrane of 10 m in thickness with a pore size and
diameter of 0.4 m and 24 mm respectively. These in-
serts with straight pore structure is suitable and widely
employed in transport studies, chemotaxis, co-culture,
and microbial pathogenesis studies. For co-culture setting,
the cortical astrocytes prepared as above were gently
tyrpsinized and replated onto cell-culture inserts at a
density of 2.5 × 105 cells/well. The insert plated with
astrocytes were further maintained for 7 days to allow it
to form a confluent layer across the surface of the insert.
Independently, PCNs prepared from E16-E17 as above
on 4 DIV were transfected either with smart pool siRNA
against Nrf2 or scrambled siRNA pool. On the 5DIV,
inserts containing confluent astrocyte cultures were placed
in wells containing either Nrf2 siRNA or scramble siRNA
transfected neurons and exposed to ETOH (Figure 1(a)).
Following 24 h incubation the cells were processed for
various downstream applications such as GSH measure-
ment, caspase 3/7 glo activity assay and apoptosis.
2.7. GSH Measurement by Flow Cytometry
Free GSH measurement in live cells was determined by
flow cytometry using monochlorobimane, a non-fluore-
scent reagent which reacts with GSH to form a highly
fluorescent derivative [4,32]. At the end of treatment,
PCNs were incubated with 10 µM of MCB for 30 min in
cell culture incubator, cells scraped, washed, and resus-
pended in cold PBS. Acquisition and analysis were per-
formed on a FACS flow cytometer with excitation and
emission settings of 360 nm and 460 nm, respectively.
2.8. Caspase-Glo 3/7 Assay
Caspases-3/7 activities were estimated using Caspase-glo
3/7. Briefly, at the end of treatment, PCNs were washed
with PBS and 300 µL of caspase-glo 3/7 reagent was
added to each well and the cells were scraped and col-
lected in a microfuge tube in dark. The cell lysate was
incubated in dark for 30 min and the resultant lumines-
cence was read in a Glomax luminometer (Promega).
RLU was recorded and results were expressed as fold
change in caspase 3/7 activity from control.
2.9. Annexin-V Staining and FACS Analysis
Apoptosis was measured using Annexin V binding fol-
lowed by flow cytometry analysis.
Briefly, both detached and attached cells were har-
vested and centrifuged at low speed (1000 g) for 5 min at
the end of the experiment. The cell pellet was washed
with cold PBS and resuspended in binding buffer con-
taining annexin V-FITC and propidium iodide (PI). The
cells were gently vortexed and incubated in the dark for
15 min. Untreated cells that were either unstained or
stained with PI or stained with annexin V-FITC were
included along with the experimental samples to correct
the background fluorescent signal arising due to the dyes.
Data were collected on a flow cytometer and analyzed
using Cell Quest (BD) software.
2.10. Statistical Analysis
Data are presented as means ± s.e.m. Statistical differ-
ences were determined using one-way ANOVA followed
by Student-Newman-Keuls post-hoc analysis and a value
of P < 0.05 was considered as statistically significant.
3. Results and Discussion
3.1. Co-Culturing Nrf2 Compromised Neurons
with Astrocytes Showed Resistance to
ETOH Induced GSH Dysregulation
Generally neurons and astrocytes function as interde-
pendent networks and astrocytes play a critical role in
optimizing the function of neurons and protect against
diverse insults [15,17]. Report from our laboratory dem-
onstrates that astrocytes enable protection of neurons
against ETOH by a mechanism that is likely to be based
Copyright © 2012 SciRes. OJApo
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Schematic representation of astrocyte-neuron co-culture set up (a). PCNs transfected either with
scramble or Nrf2 siRNA in astrocyte co-culture were exposed to ETOH (4 mg/mL) for 24 h. At
the end of the experiment, neuronal cells were subjected to flow cytometric determination of
MCB staining to measure cellular GSH. (a) A representative FACS diagram is shown in panel (b)
and panel (c) depict percentage of cells positive for MCB staining. Data are mean ± s.e.m (n = 3)
and one-way ANOVA was performed to establish statistical significance. *: P < 0.05 and ns re-
present not significant.
Figure 1. Astrocyte co-culture prevented ETOH induced GSH depletion in Nrf2 silenced primary cortical neurons.
on GSH efflux [23]. Of note, unpublished data from our
laboratory suggests that co-culture of neurons with as-
trocytes deficient in Nrf2, a redox-sensitive regulatory
factor controlling GSH homeostasis were unable to offer
protection to neurons with intact Nrf2 against ETOH
toxicity. Also, perturbation of Nrf2 levels in pure neu-
ronal cultures remarkably sensitizes neurons to ETOH-
induced GSH loss, ROS activation and cell death [4].
Thus we questioned as to whether ETOH exposure to
Nrf2 compromised neurons has any bearing in the GSH
antioxidation based health of neurons when co-cultured
with astrocytes possessing intact Nrf2. To address this,
we utilized Nrf2 targeted primary cortical neurons that
are co-cultured with primary cortical astrocytes (PCAs)
and assessed the effect of ETOH (4 mg/mL) on intracel-
lular GSH levels in Nrf2 silenced neurons by staining
with MCB followed by FACS analysis. Under native
state, MCB is non-fluorescent while it only fluoresces
upon conjugation to GSH in the cells. Exposure of
ETOH to scramble transfected neuron cultures displayed
no change in GSH levels when co-cultured with astro-
cytes (Scr vs Scr + ETOH; Figures 1(b) and (c)). No-
tably the same concentration of ETOH significantly de-
pleted intracellular GSH levels in PCNs without astro-
cyte co-culture setting [4]. Importantly, ETOH exposure
to Nrf2 silenced neurons showed a significant increase in
GSH levels when compared to Nrf2 downregulated neu-
rons (siNrf2 + ETOH vs. siNrf2; Figure 1(c)) as opposed
to exaggerated GSH depletion in Nrf2 downregulated
primary cortical neurons without astrocyte co-culture [4].
Astroglial cells have been reported to afford extracellular
antioxidant protection via GSH efflux as they possess
predominant fraction of GSH in brain [33,34]. It has been
shown that selective activation of astrocytic Nrf2 pro-
tected mice from MPTP-induced Parkinsonism by acti-
vating anti- oxidant response pathways [35].
In our previous report, flow cytometric measurement
of GSH resulted in a striking heterogeneity of GSH pro-
file viz. high, medium, low GSH containing neurons
based on differential MCB staining [4]. However in the
current study which involved an astrocyte/neuron co-
culture setting, we did not observe GSH heterogeneity in
neurons. This could be attributed to copious supply of
GSH from astrocytes to neurons as GSH efflux from as-
trocyte is considered to be an initial step in maintenance
of neuron GSH homeostasis [23]. It is noteworthy that
GSH concentration in neurons was reported to double in
24 h when co-cultured with astroctyes [36]. Hence it is
very much likely that the medium and low GSH popula-
tion of neurons was supplied with sufficient GSH from
astrocytes such that homogeneity in intracellular GSH
levels was achieved resulting in uniform population of
GSH containing neurons. Thus these results demonstrate
that ETOH induced GSH dysregulation in Nrf2-comp-
romised neurons could be controlled when co-cultured
with astrocytes via GSH efflux from the latter [5,23].
Notably, an ATP-dependent, multidrug-resistant protein
1 (MRP1) in astrocytes have been reported to mediate ex-
port of GSH, GSSG and glutathione conjugates [37]. How-
ever, the precise mechanism of how GSH effluxes from
astrocytes in the current setting remains to be elucidated.
3.2. Co-Culture of Astrocytes Restrained ETOH
Induced Activation of Caspase3/7 Activity in
Nrf2 Silenced PCNs
Progressive cell death is a pathological hallmark of neu-
rodegenerative diseases which in general, is initiated by a
cascade of highly organized biochemical, morphological
and signaling events involving activation of caspases, a
family of cysteine-containing, aspartate specific prote-
ases [38]. Growing body of evidences employ the mea-
sure of caspase activity as one of the primary methods to
quantify neuronal apoptosis [39]. Among this family of
proteases, caspase-3 and caspase-7 are the effector cas-
pases that executes cell death [40] and thus measurement
of effector caspases 3/7 activity is widely gaining popu-
lar as index of proapoptotic response [4,41,42]. We have
demonstrated that ETOH exposure (4 mg/mL) for 24 h
resulted in a significant increase in caspase 3/7 activity,
the effect which was further exacerbated when Nrf2 was
downregulated in PCNs [4]. Thus, in the current study
we sought to address whether ETOH induced caspase 3/7
activity in Nrf2 downregulated PCNs could be prevented
by co-culturing with astrocytes. Treating scramble
siRNA-transfected neurons with ETOH when co-cultured
with astrocytes did not show any significant activation in
caspase 3/7 activity (col. 2 vs. col. 1; Figure 2). Interest-
ingly, ETOH treatment of neurons that are deficient in
Nrf2 when co-cultured with astrocytes also failed to
show any activation in caspase 3/7 activity (col 4 vs. col.
3; Figure 2 ). A classical study at the single cell level has
demonstrated that execution phase of apoptosis ensues
mainly due to depletion of GSH [43]. Induction of oxida-
tive stress has been shown to activate caspase-3 and 7 re-
sulting in enhancing sensitivity to apoptosis in neurons
[44,45]. Thus, the observed prevention of ETOH-induced
caspase 3/7 activity in Nrf2 depleted neurons could be
attributed to abundant supply of GSH from astrocytes that
are likely to limit ROS-mediated caspase 3/7 activation.
At this juncture, it is important to note that a total down-
regulation of target gene using knockdown strategy in
any primary culture setting is very challenging. In our
experimental setting, we could achieve differential
knockdown efficiency of Nrf2 ranging between 50% -
PCNs transfected either with scramble or Nrf2
siRNA in astrocyte co-culture were exposed to
ETOH (4 mg/mL) for 24 h. At the end of the ex-
periment, caspase 3/7 activity in neurons was as-
sessed by Caspase-Glo assay. Data expressed as
fold change in caspase3/7 activity are mean + s.e.m
(n = 6). Statistical significance was determined by
one-way ANOVA; ns represent not significant.
Figure 2. Nrf2 depleted neurons displayed resistance to
ETO H-induced caspase 3/7 activation w hen co-culture d wit h
Copyright © 2012 SciRes. OJApo
Copyright © 2012 SciRes. OJApo
80% [4]. Thus, we cannot discount any involvement of
residual Nrf2 based antioxidant ac- tivity in neurons that
could have also partially contri- buted along with astro-
cytic supply of GSH to curtail ETOH induced ROS me-
diated activation of caspase. Nevertheless, this result
suggests that normal astrocytes can avert ETOH induced
activation of caspase 3/7 asso- ciated apoptotic signaling
events in Nrf2-silenced neurons.
Nrf2-silenced neurons due to astrocyte co-culture was
indeed reflecting in controlled apoptosis, we assessed
FACS analysis followed by Annexin-V-FITC staining of
Nrf2-silenced neurons treated with and without ETOH in
astrocyte co-culture set up. In this assay, annexin-V posi-
tive cells represent a true state of balance of pro- and
anti-apoptotic factors in which greater the positivity,
more the shift in balance towards proapoptotic factors
typifying early signs of programmed cell death. Neither
scramble siRNA nor Nrf2 silenced neurons treated with
ETOH when co-cultured with astrocytes displayed any
appreciable increase in annexin V-FITC positive (siNrf2
+ ETOH vs. siNrf2; Figures 3(a) and (b)). Earlier report
from our laboratory has shown that adenovirus medi-
ated overexpression of Nrf2 in isolated pure neurons
prevented ETOH induced apoptosis [4]. However, this
study suggests that even when there is a partial loss of
Nrf2 in neurons, astroctyes with intact Nrf2 can curtail
3.3. Astrocytes Co-Culture Protected Nrf2
Silenced PCNs from ETOH Induced
It is well accepted that astrocytes provide metabolic and
trophic support essential for the survival and function of
neurons [16,24]. Nrf2 based ARE activation in neighbor-
ing astrocytes have been shown to protect neurons
against oxidative insult [46]. To determine whether the
caspase 3/7 inhibitory effect observed in ETOH exposed
PCNs transfected either with scramble or Nrf2 siRNA in astrocyte co-culture
were exposed to ETOH (4 mg/mL) for 24 h. Neuronal apoptosis was esti-
mated by Annexin-V-FITC staining followed by flow cytometric analysis.
(a) Representative FACS diagram is shown in panel (a) and panel (b) depict
percentage of cells positive for Annexin-V staining. Data are mean ± s.e.m
(n = 5) and statistical analysis was determined by one-way ANOVA; ns rep-
resent not significant.
Figure 3. Astrocytes protected Nrf2 silenced neurons from ETOH induced apoptotic death.
neuronal apoptosis. In line with our findings, it has been
demonstrated that in a mouse model of PD, astrocytic
restricted expression of Nrf2 rendered protection against
MPTP mediated neurotoxicity [35]. Furthermore, activa-
tion of Nrf2 mediated ATF3 in astrocytes is suggested to
afford protection against neurons caused by oxidative
stress by modulating redox status and glutathione levels
[47]. In the nervous system, ATF-3 is a stress-inducible
gene and a transcriptional repressor and considered as a
marker of cellular stress [48-50]. ATF3 prevents SCG
neuronal cell death and induces neurite elongation via
upregulation of Hsp27 after NGF withdrawal [51].
Thus astrocytes could boost antioxidant status in Nrf2
depleted neurons and affords neuronal cytoprotection
against ETOH by two probable mechanisms: 1) by func-
tioning as a stress/shock-absorber thus protecting itself
against ETOH by activating Nrf2 dependent ATF3 as a
counter- response and 2) at the same time generating
GSH in an Nrf2 dependent process and enhancing the
availability of GSH to neurons.
In conclusion, the current study identified a neuro-
protective role for cortical astrocytes against ETOH even
under a condition when neurons are partially depleted of
Nrf2 based antioxidant machinery (Figure 4). Another
important finding of our study was that this neuropro-
tective effect of astrocyte despite partially compromised
neuronal Nrf2 could be achieved by GSH supply, a cen-
tral component mediating redox signaling and cell death
progression. Studies are underway to identify the appro-
priate molecular identity of how GSH is transported from
astrocytes to neurons.
4. Acknowledgements
This work was supported by RO1 AA010114 (to G.I.H).
We thank the flow cytometry and optical imaging core
facility of UTHSCSA which is supported by NIH-NCI
(a) Normal neurons that have low basal Nrf2 levels when exposed to ETOH display a disturbed GSH based redox homeostasis
resulting in apoptosis culminating in onset of neurodegeneration [4]; (b) Under conditions when Nrf2 is repressed in neurons,
ETOH induced dysregulation in GSH-related apoptotic death is exaggerated resulting in worsening of the neurodegenerative
process [4]. Up until second trimester which is when astrocytes colonize the developing CNS, neurons will be highly sensitive
to recreational neurotoxicants including, ETOH, a state reflected in Fetal Alcohol Spectrum Disorder (FASD) ((a) and (b)); (c)
When Nrf2 compromised neurons are co-cultured with astrocytes, the redox based apoptotic responses to ETOH in neurons is
blocked by elevation of neuron GSH levels that is supplied from astrocytes.
Figure 4. Schematic view of astrocytic protection of Nrf2 compromised neuron s against ETOH toxicity.
Copyright © 2012 SciRes. OJApo
P30 CA54174 (Cancer Therapy & Research Center),
NIH-NIA P30 AG013319 (Nathan Shock Center) and
(NIH-NIA P01AG19316).
[1] R. Dringen, “Metabolism and Functions of Glutathione in
Brain,” Progress in neurobiology, Vol. 62, No. 6, 2000,
pp. 649-671. doi:10.1016/S0301-0082(99)00060-X
[2] H. L. Martin and P. Teismann, “Glutathione—A Review
on Its Role and Significance in Parkinson’s Disease,”
FASEB Journal: Official Publication of the Federation of
American Societies for Experimental Biology, Vol. 23,
No. 10, 2009, pp. 3263-3272.
[3] K. Aoyama, M. Watabe and T. Nakaki, “Regulation of
Neuronal Glutathione Synthesis,” Journal of Pharmaco-
logical Sciences, Vol. 108, No. 3, 2008, pp. 227-238.
[4] M. Narasimhan, L. Mahimainathan, M. L. Rathinam, A.
K. Riar and G. I. Henderson, “Overexpression of Nrf2
Protects Cerebral Cortical Neurons from Ethanol-Induced
Apoptotic Death,” Molecular Pharmacology, Vol. 80, No.
6, 2011, pp. 988-999. doi:10.1124/mol.111.073262
[5] L. T. Watts, M. L. Rathinam, S. Schenker and G. I. Hen-
derson, “Astrocytes Protect Neurons from Ethanol-Induced
Oxidative Stress and Apoptotic Death,” Journal of Neu-
roscience Research, Vol. 80, No. 5, 2005, pp. 655-666.
[6] R. Dringen, B. Pfeiffer and B. Hamprecht, “Synthesis of
the Antioxidant Glutathione in Neurons: Supply by As-
trocytes of CysGly as Precursor for Neuronal Gluta-
thione,” The Journal of Neuroscience: The Official Jour-
nal of the Society for Neuroscience, Vol. 19, No. 2, 1999,
pp. 562-569.
[7] Y. J. Surh, J. K. Kundu and H. K. Na, “Nrf2 as a Master
Redox Switch in Turning on the Cellular Signaling In-
volved in the Induction of Cytoprotective Genes by Some
Chemopreventive Phytochemicals,” Planta Medica, Vol.
74, No. 13, 2008, pp. 1526-1539.
[8] T. Satoh, S. I. Okamoto, J. Cui, Y. Watanabe, K. Furuta,
M. Suzuki, K. Tohyama and S. A. Lipton, “Activation of
the Keap1/Nrf2 Pathway for Neuroprotection by Elec-
trophilic [Correction of Electrophillic] Phase II Inducers,”
Proceedings of the National Academy of Sciences of the
United States of America, Vol. 103, No. 3, 2006, pp. 768-
773. doi:10.1073/pnas.0505723102
[9] M. R. Vargas, M. Pehar, P. Cassina, L. Martinez-Palma, J.
A. Thompson, J. S. Beckman and L. Barbeito, “Fibroblast
Growth Factor-1 Induces Heme Oxygenase-1 via Nuclear
Factor Erythroid 2-Related Factor 2 (Nrf2) in Spinal Cord
Astrocytes: Consequences for Motor Neuron Survival,”
The Journal of Biological Chemistry, Vol. 280, No. 27,
2005, pp. 25571-25579. doi:10.1074/jbc.M501920200
[10] M. J. Calkins, R. J. Jakel, D. A. Johnson, K. Chan, Y. W.
Kan and J. A. Johnson, “Protection from Mitochondrial
Complex II Inhibition in Vitro and in Vivo by Nrf2- Me-
diated Transcription,” Proceedings of the National Aca-
demy of Sciences of the United States of America, Vol.
102, No. 1, 2005, pp. 244-249.
[11] A. Y. Shih, S. Imbeault, V. Barakauskas, H. Erb, L. Jiang,
P. Li and T. H. Murphy, “Induction of the Nrf2-Driven
Antioxidant Response Confers Neuroprotection during
Mitochondrial Stress in Vivo,” The Journal of Biological
Chemistry, Vol. 280, No. 24, 2005, pp. 22925-22936.
[12] A. Bignami, “Glial Cells in Central Nervous System,” In:
P. J. Magistretti, Ed., Discussions in Neuroscience, El-
sevier, Amsterdam, 1991, pp. 1-45.
[13] H. K. Kimelberg and M. D. Norenberg, “Astrocytes,”
Scientific American, Vol. 260, No. 4, 1989, pp. 66-76.
[14] J. O’kusky and M. Colonnier, “A Laminar Analysis of the
Number of Neurons, Glia, and Synapses in the Adult
Cortex (Area 17) of Adult Macaque Monkeys,” The
Journal of Comparative Neurology, Vol. 210, No. 3, 1982,
pp. 278-290. doi:10.1002/cne.902100307
[15] M. Pekny and M. Nilsson, “Astrocyte Activation and
Reactive Gliosis,” Glia, Vol. 50, No. 4, 2005, pp. 427-434.
[16] C. E. Schmidt and J. B. Leach, “Neural Tissue Engineer-
ing: Strategies for Repair and Regeneration,” Annual Re-
view of Biomedical Engineering, Vol. 5, 2003, pp. 293-
[17] M. V. Sofroniew and H. V. Vinters, “Astrocytes: Biology
and Pathology,” Acta Neuropathologica, Vol. 119, No. 1,
2010, pp. 7-35. doi:10.1007/s00401-009-0619-8
[18] D. A. Johnson, G. K. Andrews, W. Xu and J. A. Johnson,
“Activation of the Antioxidant Response Element in Pri-
mary Cortical Neuronal Cultures Derived from Trans-
genic Reporter Mice,” Journal of Neurochemistry, Vol.
81, No. 6, 2002, pp. 1233-1241.
[19] T. H. Murphy, J. Yu, R. Ng, D. A. Johnson, H. Shen, C.
R. Honey and J. A. Johnson, “Preferential Expression of
Antioxidant Response Element Mediated Gene Expres-
sion in Astrocytes,” Journal of Neurochemistry, Vol. 76,
No. 6, 2001, pp. 1670-1678.
[20] J. B. Schulz, J. Lindenau, J. Seyfried and J. Dichgans,
“Glutathione, Oxidative Stress and Neurodegeneration,”
European Journal of Biochemistry/FEBS, Vol. 267, No.
16, 2000, pp. 4904-4911.
[21] A. Y. Shih, D. A. Johnson, G. Wong, A. D. Kraft, L.
Jiang, H. Erb, J. A. Johnson and T. H. Murphy, “Coordi-
nate Regulation of Glutathione Biosynthesis and Release
by Nrf2-Expressing Glia Potently Protects Neurons from
Oxidative Stress,” The Journal of Neuroscience: The Of-
ficial Journal of the Society for Neuroscience, Vol. 23,
No. 8, 2003, pp. 3394-3406.
[22] X. F. Wang and M. S. Cynader, “Astrocytes Provide
Cysteine to Neurons by Releasing Glutathione,” Journal
of Neurochemistry, Vol. 74, No. 4, 2000, pp. 1434-1442.
[23] M. L. Rathinam, L. T. Watts, A. A. Stark, L. Mahi-
mainathan, J. Stewart, S. Schenker and G. I. Henderson,
Copyright © 2012 SciRes. OJApo
“Astrocyte Control of Fetal Cortical Neuron Glutathione
Homeostasis: Up-Regulation by Ethanol,” Journal of Neu-
rochemistry, Vol. 96, No. 5, 2006, pp. 1289-1300.
[24] E. D. Martin, A. Araque and W. Buno, “Synaptic Regula-
tion of the Slow Ca2+-Activated K+ Current in Hippo-
campal CA1 Pyramidal Neurons: Implication in Epilep-
togenesis,” Journal of Neurophysiology, Vol. 86, No. 6,
2001, pp. 2878-2886.
[25] K. V. Rao, K. S. Panickar, A. R. Jayakumar and M. D.
Norenberg, “Astrocytes Protect Neurons from Ammonia
Toxicity,” Neurochemical Research, Vol. 30, No. 10,
2005, pp. 1311-1318. doi:10.1007/s11064-005-8803-2
[26] D. R. Brown, “Neurons Depend on Astrocytes in a Co-
culture System for Protection from Glutamate Toxicity,”
Molecular and Cellular Neurosciences, Vol. 13, No. 5,
1999, pp. 379-389. doi:10.1006/mcne.1999.0751
[27] L. F. Romao, O. Sousa Vde, V. M. Neto and F. C. Gomes,
“Glutamate Activates GFAP Gene Promoter from Cul-
tured Astrocytes through TGF-Beta1 Pathways,” Journal
of Neurochemistry, Vol. 106, No. 2, 2008, pp. 746-756.
[28] V. Ramachandran, L. T. Watts, S. K. Maffi, J. Chen, S.
Schenker and G. Henderson, “Ethanol-Induced Oxidative
Stress Precedes Mitochondrially Mediated Apoptotic Death
of Cultured Fetal Cortical Neurons,” Journal of Neuro-
science Research, Vol. 74, No. 4, 2003, pp. 577-588.
[29] M. Narasimhan, M. Rathinam, A. Riar, D. Patel, S.
Mummidi, H. S. Yang, N. H. Colburn, G. I. Henderson
and L. Mahimainathan, “Programmed Cell Death 4 (PD-
CD4): A Novel Player in Ethanol-Mediated Suppression
of Protein Translation in Primary Cortical Neurons and
Developing Cerebral Cortex,” Alcoholism, Clinical and
Experimental Research, 2012.
[30] J. W. Olney, T. Tenkova, K. Dikranian, Y. Q. Qin, J.
Labruyere and C. Ikonomidou, “Ethanol-Induced Apop-
totic Neurodegeneration in the Developing C57BL/6 Mouse
Brain,” Developmental Brain Research, Vol. 133, No. 2,
2002, pp. 115-126. doi:10.1016/S0165-3806(02)00279-1
[31] K. D. Mccarthy and J. De Vellis, “Preparation of Separate
Astroglial and Oligodendroglial Cell Cultures from Rat
Cerebral Tissue,” The Journal of Cell Biology, Vol. 85,
No. 3, 1980, pp. 890-902. doi:10.1083/jcb.85.3.890
[32] S. K. Maffi, M. L. Rathinam, P. P. Cherian, W. Pate, R.
Hamby-Mason, S. Schenker and G. I. Henderson, “Glu-
tathione Content as a Potential Mediator of the Vulner-
ability of Cultured Fetal Cortical Neurons to Ethanol-In-
duced Apoptosis,” Journal of Neuroscience Research,
Vol. 86, No. 5, 2008, pp. 1064-1076.
[33] R. Dringen, L. Kussmaul, J. M. Gutterer, J. Hirrlinger and
B. Hamprecht, “The Glutathione System of Peroxide De-
toxification Is Less Efficient in Neurons than in Astro-
glial Cells,” Journal of Neurochemistry, Vol. 72, No. 6,
1999, pp. 2523-2530.
[34] M. L. Schroeter, K. Mertsch, H. Giese, S. Muller, A.
Sporbert, B. Hickel and I. E. Blasig, “Astrocytes Enhance
Radical Defence in Capillary Endothelial Cells Consti-
tuting the Blood-Brain Barrier,” FEBS Letters, Vol. 449,
No. 2-3, 1999, pp. 241-244.
[35] P. C. Chen, M. R. Vargas, A. K. Pani, R. J. Smeyne, D. A.
Johnson, Y. W. Kan and J. A. Johnson, “Nrf2-Mediated
Neuroprotection in the MPTP Mouse Model of Parkin-
son’s Disease: Critical Role for the Astrocyte,” Pro-
ceedings of the National Academy of Sciences of the
United States of America, Vol. 106, No. 8, 2009, pp.
2933-2938. doi:10.1073/pnas.0813361106
[36] S. J. Heales and J. P. Bolanos, “Impairment of Brain Mi-
tochondrial Function by Reactive Nitrogen Species: The
Role of Glutathione in Dictating Susceptibility,” Neuro-
chemistry International, Vol. 40, No. 6, 2002, pp. 469-474.
[37] J. Hirrlinger, J. B. Schulz and R. Dringen, “Glutathione
Release from Cultured Brain Cells: Multidrug Resistance
Protein 1 Mediates the Release of GSH from Rat Astro-
glial Cells,” Journal of Neuroscience Research, Vol. 69,
No. 3, 2002, pp. 318-326. doi:10.1002/jnr.10308
[38] K. A. Jellinger and C. H. Stadelmann, “The Enigma of
Cell Death in Neurodegenerative Disorders,” Journal of
Neural Transmission, No. 60, 2000, pp. 21-36.
[39] S. Kothakota, T. Azuma, C. Reinhard, A. Klippel, J. Tang,
K. Chu, T. J. Mcgarry, M. W. Kirschner, K. Koths, D. J.
Kwiatkowski and L. T. Williams, “Caspase-3-Generated
Fragment of Gelsolin: Effector of Morphological Change
in Apoptosis,” Science, Vol. 278, No. 5336, 1997, pp.
294-298. doi:10.1126/science.278.5336.294
[40] J. G. Walsh, S. P. Cullen, C. Sheridan, A. U. Luthi, C.
Gerner and S. J. Martin, “Executioner Caspase-3 and
Caspase-7 Are Functionally Distinct Proteases,” Proceed-
ings of the National Academy of Sciences of the United
States of America, Vol. 105, No. 35, 2008, pp. 12815-
12819. doi:10.1073/pnas.0707715105
[41] M. C. Kowalczyk, Z. Walaszek, P. Kowalczyk, T. Kinjo,
M. Hanausek and T. J. Slaga, “Differential Effects of
Several Phytochemicals and Their Derivatives on Murine
Keratinocytes in Vitro and in Vivo: Implications for Skin
Cancer Prevention,” Carcinogenesis, Vol. 30, No. 6, 2009,
pp. 1008-1015. doi:10.1093/carcin/bgp069
[42] K. Kumagai, S. Imai, F. Toyoda, N. Okumura, E. Isoya,
H. Matsuura and Y. Matsusue, “17β-Oestradiol Inhibits
Doxorubicin-Induced Apoptosis via Block of the Vol-
ume-Sensitive Cl Current in Rabbit Articular Chondro-
cytes,” British Journal of Pharmacology, Vol. 166, No. 2,
2012, pp. 702-720.
[43] R. Franco, M. I. Panayiotidis and J. A. Cidlowski, “Glu-
tathione Depletion Is Necessary for Apoptosis in Lym-
phoid Cells Independent of Reactive Oxygen Species
Formation,” The Journal of Biological Chemistry, Vol.
282, No. 42, 2007, pp. 30452-30465.
[44] H. G. Lee, Y. J. Lee and J. H. Yang, “Perfluorooctane
Sulfonate Induces Apoptosis of Cerebellar Granule Cells
via a ROS-Dependent Protein Kinase C Signaling Path-
way,” Neurotoxicology, Vol. 33, No. 3, 2012, pp. 314-
Copyright © 2012 SciRes. OJApo
Copyright © 2012 SciRes. OJApo
320. doi:10.1016/j.neuro.2012.01.017
[45] K. Sathishkumar, X. Xi, R. Martin and R. M. Uppu,
“Cholesterol Secoaldehyde, an Ozonation Product of
Cholesterol, Induces Amyloid Aggregation and Apoptosis
in Murine GT1-7 Hypothalamic Neurons,” Journal of
Alzheimers Disease: JAD, Vol. 11, No. 3, 2007, pp. 261-
[46] A. D. Kraft, D. A. Johnson and J. A. Johnson, “Nuclear
Factor E2-Related Factor 2-Dependent Antioxidant Re-
sponse Element Activation by Tert-Butylhydroquinone
and Sulforaphane Occurring Preferentially in Astrocytes
Conditions Neurons Against Oxidative Insult,” The Jour-
nal of Neuroscience: The Official Journal of the Society
for Neuroscience, Vol. 24, No. 5, 2004, pp. 1101-1112.
[47] K. H. Kim, J. Y. Jeong, Y. J. Surh and K. W. Kim, “Ex-
pression of Stress-Response ATF3 Is Mediated by Nrf2 in
Astrocytes,” Nucleic Acids Research, Vol. 38, No. 1, 2010,
pp. 48-59. doi:10.1093/nar/gkp865
[48] B. P. Chen, G. Liang, J. Whelan and T. Hai, “ATF3 and
ATF3 Delta Zip. Transcriptional Repression versus Acti-
vation by Alternatively Spliced Isoforms,” The Journal of
Biological Chemistry, Vol. 269, No. 22, 1994, pp. 15819-
[49] T. Hai, C. D. Wolfgang, D. K. Marsee, A. E. Allen and U.
Sivaprasad, “ATF3 and Stress Responses,” Gene Expres-
sion, Vol. 7, No. 4-6, 1999, pp. 321-335.
[50] G. Liang, C. D. Wolfgang, B. P. Chen, T. H. Chen and T.
Hai, “ATF3 Gene. Genomic Organization, Promoter, and
Regulation,” The Journal of Biological Chemistry, Vol.
271, No. 3, 1996, pp. 1695-1701.
[51] S. Nakagomi, Y. Suzuki, K. Namikawa, S. Kiryu-Seo and
H. Kiyama, “Expression of the Activating Transcription
Factor 3 Prevents c-Jun N-Terminal Kinase-Induced
Neuronal Death by Promoting Heat Shock Protein 27 Ex-
pression and Akt Activation,” The Journal of Neurosci-
ence: The Official Journal of the Society for Neuroscience,
Vol. 23, No. 12, 2003, pp. 5187-5196.
ETOH, ethanol; PCN, primary cortical neurons; PCA,
primary cortical astrocytes; Nrf2/NFE2L2, nuclear factor
erythroid 2-related factor 2; GSH, glutathione; MCB,
monochlorobimane; siNrf2, small interfering RNA against
Nrf2; Scr, Scrambled siRNA.