Group Title: BMC Neuroscience
Title: Acute NMDA toxicity in cultured rat cerebellar granule neurons is accompanied by autophagy induction and late onset autophagic cell death phenotype
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Title: Acute NMDA toxicity in cultured rat cerebellar granule neurons is accompanied by autophagy induction and late onset autophagic cell death phenotype
Physical Description: Book
Language: English
Creator: Sadasivan, Shankar
Zhang, Zhiqun
Larner, Stephen
Liu, Ming
Zheng, Wenrong
Kobeissy, Firas
Hayes, Ronald
Wang, Kevin
Publisher: BMC Neuroscience
Publication Date: 2010
 Notes
Abstract: BACKGROUND:Autophagy, an intracellular response to stress, is characterized by double membrane cytosolic vesicles called autophagosomes. Prolonged autophagy is known to result in autophagic (Type II) cell death. This study examined the potential role of an autophagic response in cultured cerebellar granule neurons challenged with excitotoxin N-methyl-D-aspartate (NMDA).RESULTS:NMDA exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in both cell bodies and neurites as early as 3 hours post-treatment. Elevated levels of Beclin-1 and the autophagosome-targeting LC3-II were also observed following NMDA exposure. Prolonged exposure of the cultures to NMDA (8-24 h) generated MDC-, LC3-positive autophagosomal bodies, concomitant with the neurodegenerative phase of NMDA challenge. Lysosomal inhibition studies also suggest that NMDA-treatment diverted the autophagosome-associated LC3-II from the normal lysosomal degradation pathway. Autophagy inhibitor 3-methyladenine significantly reduced NMDA-induced LC3-II/LC3-I ratio increase, accumulation of autophagosomes, and suppressed NMDA-mediated neuronal death. ATG7 siRNA studies also showed neuroprotective effects following NMDA treatment.CONCLUSIONS:Collectively, this study shows that autophagy machinery is robustly induced in cultured neurons subjected to prolonged exposure to excitotoxin, while autophagosome clearance by lysosomal pathway might be impaired. Our data further show that prolonged autophagy contributes to cell death in NMDA-mediated excitotoxicity.
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General Note: M3: 10.1186/1471-2202-11-21
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Sadasivan et al. BMC Neuroscience 2010, 11:21
http://www.biomedcentral.com/1471-2202/11/21


BMeuroscience
Neuroscience


Acute NMDA toxicity in cultured rat cerebellar

granule neurons is accompanied by autophagy

induction and late onset autophagic cell death

phenotype

Shankar Sadasivanl*, Zhiqun Zhang2, Stephen F Larner 2, Ming C Liu2, Wenrong Zheng2, Firas H Kobeissyl,
Ronald L Hayes2, Kevin KW Wang1'2*


Abstract
Background: Autophagy, an intracellular response to stress, is characterized by double membrane cytosolic
vesicles called autophagosomes. Prolonged autophagy is known to result in autophagic (Type II) cell death. This
study examined the potential role of an autophagic response in cultured cerebellar granule neurons challenged
with excitotoxin N-methyl -D-aspartate (NMDA).
Results: NMDA exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC)
fluorescent dye-labeled autophagosome formation in both cell bodies and neurites as early as 3 hours post-
treatment. Elevated levels of Beclin-1 and the autophagosome-targeting LC3-II were also observed following NMDA
exposure. Prolonged exposure of the cultures to NMDA (8-24 h) generated MDC-, LC3-positive autophagosomal
bodies, concomitant with the neurodegenerative phase of NMDA challenge. Lysosomal inhibition studies also
suggest that NMDA-treatment diverted the autophagosome-associated LC3-II from the normal lysosomal
degradation pathway. Autophagy inhibitor 3-methyladenine significantly reduced NMDA-induced LC3-II/LC3-I ratio
increase, accumulation of autophagosomes, and suppressed NMDA-mediated neuronal death. ATG7 siRNA studies
also showed neuroprotective effects following NMDA treatment.
Conclusions: Collectively, this study shows that autophagy machinery is robustly induced in cultured neurons
subjected to prolonged exposure to excitotoxin, while autophagosome clearance by lysosomal pathway might be
impaired. Our data further show that prolonged autophagy contributes to cell death in NMDA-mediated
excitotoxicity.


Background
Autophagy is an intracellular pathway that is activated
in response to cell stress. It is a phenomenon where the
cytoplasmic organelles in the cell are engulfed by double
membrane vesicles called the autophagosomes and
delivered to the lysosomes where the organelles are bro-
ken down by lysosomal proteases and the amino acids
recycled back into the cell machinery to aid cell survival
[1,2]. Some of the key autophagy protein (Atg) identified
to be involved in this process are Atg4, Atg6, Atg8,

* Correspondence' shankarsadasivan@stjudeorg; kwang@banyanbiocomn
'Center for Neuroproteomics and Biomarkers Research, Department of
Psychiatry, the McKnight Brain Institute of the University of Florida,
Gainesville, FL 32610, USA


Atgl2 and Atg5 [3]. Autophagy has been reported to be
vital in the development of the central nervous system
[4,5]. It has also been documented to be constitutively
active in the healthy neurons and aid survival [6].
Researchers have used a number of tools to study and
interpret autophagy induction [7]. For example, an ele-
vated level of Bcl-2-binding protein Beclin-1 (Atg6) has
been documented to be indicative of autophagy induc-
tion. Another protein marker for autophagy induction
extensively studied, is the lipidated form of microtubule
associated protein light chain-3 (MAP-LC3) found on
the outer and to a lesser extent the inner membrane of
the double membrane of the autophagosome.


0 2010 Sadasivan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Biollet ed Central Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.






Sadasivan et al. BMC Neuroscience 2010, 11:21
http://www.biomedcentral.com/1471-2202/11/21


Programmed cell death among neurons in the central
nervous system is a regulated process. Neurons undergo
either apoptotic (type I) or autophagic (type II) cell
death or oncotic/necrotic (type III) depending on the
nature of insult [8,9]. Acute excitotoxic insults resulting
from the use of glutamate in primary culture has been
shown to induce both oncotic and apoptotic cell death
in neurons [10,11]. Increased excitation of the glutamate
receptors by its ligand has been shown to cause an
imbalance in the ionic gradient in neurons, resulting in
an increase in the calcium and sodium levels intracellu-
larly leading to oncosis. At the same time, this excessive
activation in neurons has also been demonstrated to
activate the endonucleases, causing internucleosomal
DNA fragmentation, thus resulting in apoptosis. Though
extensive studies have been conducted on apoptotic cell
death mechanisms, the biochemical mechanisms and the
exact definition of "autophagic cell death" is poorly
understood [12-16].
Autophagic vacuoles have been shown to accumulate
in affected neurons of several neurodegenerative diseases
such as Alzheimer's disease and Parkinson's disease.
Wang et al., (2006) recently demonstrated that the
induction of autophagy was associated with axonal
degeneration in Purkinje cells in Lurcher mice. More
recent experimental evidence has also shown the upre-
gulation of autophagy protein Beclin-1 (Atg-6) and/or to
LC3-II/LC3-I ratio increase in different rodent models
of traumatic brain injury (TBI) [17-20].
Excitotoxicity via overactivation of ionotropic gluta-
mate receptor subtype N-methyl-D-aspartate (NMDA)-
receptor, is one of the documented hallmark events that
occur following acute brain injury [21,22]. Hence we
sought to examine if autophagy is a general response
during excitotoxic NMDA challenge by using rat cere-
bellar granule neuronal (CGN) cultures in vitro. In addi-
tion, we hypothesize that autophagy and possible
autophagic cell death might also participate in NMDA
excitotoxicity.

Results
Acute NMDA exposure induces autophagy in cerebellar
granule neurons in culture
Rat cerebellar granule neurons (CGN) were treated with
or without NMDA (200 pM) in serum free medium
(SFM) to achieve excitotoxic and control conditions,
respectively. To assess the possible induction of autop-
hagy following acute NMDA exposure, the neurons
were stained with an antibody against microtubule asso-
ciated light chain-3 (LC3) protein, a known autophagy
protein marker, also called Atg-8 [23]. Neurons sub-
jected to NMDA exposure for 8 h exhibited increased
number of both regular sized and unusually large LC3
immunopositive autophagosomes inside the neuronal


cell body. Co-immunostaining with anti-NeuN antibody,
a protein marker of mature neurons was employed to
demonstrate that the increase in the LC3 positive autop-
hagosomes was indeed found in neurons following
NMDA treatment (Fig 1). The addition of autophago-
some inhibitor 3-methyladenine (3-MA) to NMDA-trea-
ted CGN suppressed the increase of LC-3 positive
staining. As a positive control, neurons subjected to
amino acid starvation (24 hours) showed robust forma-
tion of punctuate LC-3 positive autophagosomes, when
compared to untreated CGN. Acute exposure to NMDA
results in an increase in neurons with anti-LC3-positive
autophagosomes compared to control conditions, sug-
gesting an inherent autophagy response to NMDA
stress. This increase was quantified by counting the
number of neurons demonstrating anti-LC3 positive
regular sized autophagosomes as well as "unusually
large" autophagosomal bodies (defined as at least 5-time
the size of normal sized autophagosomes) (Fig 2).


MAP-LC3


NeuN


Merged


Ctrl 24h




Starv 24h


NMDA8h




NMDA+

U ..3-MA8h

Figure 1 NMDA excitotoxicity results in the induction of LC3-
positive autophagosomes in rat cerebellar granule neurons.
Representative laser confocal fluorescent micrographs of cerebellar
granule neurons in culture exposed to NMDA (200 pM) with or
without co-treatment with 3-MA. NeuN (red) was used to stain
mature neurons Arrowheads (yellow) represent the increased LC3
(green) staining of autophagosomes in the cell bodies of the
neurons co-localized with neuronal marker NeuN (red). Red
arrowheads represent the increased intensity of LC3 staining in the
neuronal cell bodies at 8 hours. Neurons treated with serum free
medium are labeled as controls (Ctrl) while neurons exposed to
amino acid starvation conditions depict positive controls (Starv) for
the induction of autophagy. All images were taken at 400x
,, 1 Scale bar represents 20 pm.


Page 2 of 11







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ST
T


a Cells with punctuate
LC3 staining
m Cells with aggregated
LC3 staining



,


Control Starvation NMDA8 h NMDA+ 3-MA
Figure 2 Quantification of punctate stains in LC3-positive
neurons. Punctate stains were quantified in different microscopic
fields (n 4) and compared to neurons exhibiting densely stained
LC3 immunopositive autophagosomes. The numbers were plotted
and statistical analysis was performed using Student's t-test. *p <
0.05 denotes i differences between different treatment
groups to serum free medium treated control neurons (Ctrl). p <
0.05 denotes statistical a in treatment groups when
compared to NMDA-treated neurons alone.



Similarly, the signal from the fluorescent dye mono-
dansylcadaverine (MDC) used to label acidic vesicles
such as autophagosomes [24] also showed a strong
increase in staining (yellow arrows) in both the cell
bodies and the neurites in cell cultures 6 h after NMDA
treatment (Fig 3).

Autophagy protein markers beclin-1 and LC3-11 are up-
regulated following early phase of NMDA exposure
Having established the induction of autophagy in neu-
rons exposed to NMDA, we sought to study protein
levels of the autophagy protein marker beclin-1 (Atg6).
We performed immunoblots on cell lysates obtained
from cultures following treatment with or without
NMDA at different time periods. The beclin-1 levels
appear to be increased in the NMDA-treated neurons
when compared to controls at time periods ranging from
3 h to 24 h post treatment (Fig 4A). Densitometric quan-
tification of Beclin-1 and normalization with GAPDH
level in the same samples demonstrated significant
increases in beclin-1 protein level at all time points after
NMDA exposure when compared to controls (Fig 4B).
In parallel, we also sought to examine if there was
increased levels of the autophagosomes associated lipi-
dated LC3-II form. Immunoblotting analysis of cell
lysates from NMDA-exposed culture was performed
using anti-LC3 antibody that detects both LC3-I and
LC-3-II. In control CGN cells, we detected the presence
of both LC3-I and LC3-II, in a LC3-II/LC3-I ratio of
about 0.60-0.65, in favor of the larger form (Fig 5). The
presence of endogenous levels of LC3-II here most likely
represents the basal level of autophagy that exists in all
resting cells. Upon NMDA treatment, importantly, a


Control


6h








12 h


NMDA


24 h




Figure 3 NMDA exposure induces the formation of MDC-
positive autophagosomes in cerebellar granule neurons.
Representative fluorescence micrographs of granule neurons
incubated with monodansylcadaverine (MDC) show an increase in
the labeling of the autophagosomes in both the neurites and the
cell bodies. Arrows and arrow heads (yellow) indicate the normal
punctate autophagosome staining at 6 and 12 hours in the cell
bodies and neurites, respectively. Red arrows indicate the dense
autophagosomes staining in the cell bodies following prolonged
exposure to NMDA (12 to 24 hours). All images were taken at 400x
.. .1 Scale bar represents 10 pm.



very rapid and robust increase of LC3-II was observed
(Fig 5A and 5B). We had previously established that
amino acid starvation could robustly induce autophagy
[25]. Thus, amino acid starvation of CGN was also used
here as positive controls. In fact, we observed an
increase of LC3-II levels at 6 h and 24 h after starvation
(Fig 5A and 5B). We also calculated LC3-II/LC3-I ratio
after various time points of NMDA treatment (6 h, 12 h
and 24 h) and they were 1.35-1.44, in favor of the lipi-
dated form (Fig 5C). This ratio in fact compared favor-
ably to those after starvation-induced autophagy in
CGN (LC3-II/LC3-I ratio of 1.14 after 6 h starvation
and 1.01 after 24 h starvation).

Autophagy inhibitor 3-Methyladenine (3-MA) effectively
suppresses NMDA-induced autophagy
Having observed NMDA-induced autophagy in CGN,
we examined the autophagy inhibitor 3-methyladenine


Page 3 of 11







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A CtrI. NMDA


Beclin-1 -o o
(60OkDa)


GAPDH


40
520
40


Z 0
5. 10 -




z z z
z
Figure 4 NMDA exposure of neurons results in an increase in
the beclin-1 levels in vitro. A) Lysates of neuronal cultures were
obtained at time periods of 3, 6, 12 and 24 hours, treated with or
without NMDA. These lysates were analyzed by immunoblots and
probed with the anti-beclin-1 antibody (n 3). B) Quantification of
the autophagy protein beclin-1 bands in the immunoblots was
plotted. The band intensities were normalized against the loading
control. Increases in the band intensities of the beclin-1 levels were
observed after NMDA-treated neuronal cultures as compared to
controls. The expressed values are means + S.E.M. (n 3; *p < 0.05).
GAPDH was used as a loading control.


(3-MA, 10 mM) for its ability to suppress the process of
autophagy under excitotoxic conditions. We investigated
whether the addition of 3-MA would inhibit the
increase of LC-3-II in NMDA-treated cells. Immunoblot
data indeed demonstrated a reduction of the LC3-II
levels (Fig 5). Consistent with the immunoblotting
results, a reduction in autophagy induction was further
confirmed using MDC staining. The MDC-positive
autophagosomes were relatively sparse and weak in
fluorescence intensity in CGN cultures co-treated with
NMDA+3-MA when compared to cultures treated with
NMDA (12 h) (Fig 6). We also noted that the intense
MDC staining normally observed at 16 hours post-
NMDA exposure was absent in the presence of 3-MA.

Interaction between NMDA challenge and LC3-11 flux and
turnover
It is conceivable that the NMDA induced LC3-II accumu-
lation as a result of either increased LC3-I to LC3-II con-
version or the result of autophagosome (thus LC3-II)
turnover inhibition. We addressed this issue with two
pharmacological tools: E64d, a cell permeable lyososomal
cysteine protease (cathepsin B, L) inhibitor, and lactacys-
tin, a proteasome inhibitor that blocks cytosolic protein
turnover via the ubiquitin-proteasome pathway. Our data
show that lysosomal inhibition (with E64d) potently ele-
vated LC3-II levels to 67.1 + 1.6 densitometric units from
41.3 + 1.6 units in controls possibly due to the inhibition


NMDA
Ctrl. +3MA
(A)


(N tD T ( D -


Starv.


LC34I
LC341l


60 1.60
1.40
00 *
m0 1 20 -
I40 I| 1.00 -


0.60
JP 4 0.65 1.44 1.35 1.35 e 00.6 0.72 0.60 14 1.01



Figure 5 NMDA challenge induced LC3-II accumulation which is 3-MA sensitive. A) Lysates were obtained from control neuronal cultures (at
24 h) and at 6, 12 and 24 hours from neuronal cultures treated with NMDA or NMDA+3-MA (left panels). For classic autophagy positive controls, 6
h and 24 h amino acid starvation in neurons were also included (right panels). These lysates subjected to immunoblotting were probed with anti-
LC3 and p-actin antibody. Representative immunoblots are shown (n = 4). p-actin was used as a loading control. B) LC3 immunoblot band
intensities were quantified densitometrically and the values plotted. The expressed values are means + S.E.M. (n = 4; *p < 0.05 compared to
controls; #p < 0.05 compared to NMDA-treated). C) LC3-11/LC3-1 ratio was also calculated plotted. The expressed values are means + S.E.M. (n = 4);
*NMDA groups or starvation groups I higher than control (*p < 0.05, Student paired T-test); #3-MA treatment groups I lower
than respective NMDA groups (#p < 0.05, Student paired t-test). The individual ratio value is also listed at the bottom of each bar.


Page 4 of 11


- km -am AMM aiiiiiiiiiiiiii,







Sadasivan et al. BMC Neuroscience 2010, 11:21
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Control


NMDA NMDA+3MA


MDC

12h


Figure 6 NMDA-induced autophagosome formation is
inhibited by 3-MA. Representative fluorescence micrographs show
an increase in Monodansylcadaverine (MDC; 0.05 mM) labeling of
autophagosomes in neurons increase following acute exposure to
NMDA (200 pM) compared to control conditions and co-treatment
of NMDA+3-MA. Yellow arrows and yellow arrow heads indicate the
presence of regularly sized autophagosomes in the cell bodies and
neurites respectively in 12 and 16 hours after NMDA exposure. The
red arrows in the image at 16 hours are indicative of the
accumulation of the unusually large MDC-positive autophagosomes
in the cell bodies of neurons following prolonged NMDA exposure.
Images are taken at 400x .. .1 Scale bar represents 20 pm.


of autophagosome turnover. NMDA treatment, to a lesser
extent, significantly increased LC3-II levels (to 56.0 1.2
units). Interestingly, under NMDA treatment conditions,
the cells appear refractory to further elevation with co-
treatment with E64d and lactacystin (Fig 7), suggesting
that the normal autophagosome clearance pathway may
no longer be in place. Thus, taken together, our data sug-
gest that normal LC3-II turnover by lysosome as well as
proteasome-pathways are inhibited by NMDA treatment.



so80

70 -

a 6so *



..i I 40 I
J '24030



20
Control Lacta E64d NMDA NILacta NIE64d
Figure 7 NMDA challenge interaction with LC3-11 flux and
turnover. A) Lysates were obtained from control neuronal cultures
(at 24 h), cultures treated with proteasome inhibitor lactasystin (20
pM) or with liposomal proteolysis inhibitor E64d (20 pM), or and 24
hours from neuronal cultures treated with NMDA (300 pM) or
NMDA+lactacystin or E64d preincubation for 2 h. LC3-11 immunoblot
band intensities were quantified densitometrically and the values
plotted. The expressed values are means + S.E.M. [n 3; *p < 0.05**
p < 0.01 (Student paired T-test) compared to controls; (p < 0.05]
shows difference between NMDA+ lactacystin or E64d vs. lactacystin
or E64d alone, respectively.


Cell death in NMDA-treated neurons was alleviated by 3-
MA
Neuronal cultures were divided into three treatment
groups: NMDA (200 pM), NMDA+3-MA and control.
Representative phase contrast images of the neurons at
16 hours following NMDA-treatment demonstrated
injured and dying neurons with shrunken cell bodies
and non-existing neurites when compared to healthy
control CGNs. In fact, NMDA-treated neurons showed
apoptotic cell morphology not seen in controls (Fig 8A).
In contrast, neurons co-treated with NMDA+3-MA
showed a significant sparing of neurites. While cell body
shrinkage was observed, membrane integrity, however,
was largely preserved. 3-MA co-treatment appears to
have neuroprotective effects for cells that have been
NMDA challenged. To further explore this issue,
NMDA-induced cell death was assayed by measuring
the lactate dehydrogenase (LDH) enzyme release into
the culture medium. The LDH release increases


(A)


Control


NMDA


NMDA+3MA


(B) 0.5 -
(B) 0.5 -0- Control

0.4 ->--NMDA I'
w *-m 4NMDA+3MA .
I 0.3
0 o.2
= ..

0
0 5 10 15 20 25
Time (hours)
Figure 8 Autophagy inhibitor 3-MA protects neurons against
NMDA excitotoxcity. A) Phase contrast images indicates the
changes in the morphology of neurons following 16 h treatment
with NMDA (200 pM) (middle panel), NMDA (200 pM) + 3-MA (10
mM) (right panel) and Control (left panel). With control CGN, arrows
(yellow) indicate healthy cell body morphology while arrow heads
(yellow) indicate healthy neurites. With NMDA treatment, red arrows
and red arrow heads indicate degenerative neurons and apoptotic
bodies, respectively. With NMDA+3 MA treatment, yellow arrow and
yellow arrow heads represent relatively preserved cell bodies and
neurites. Images are taken at 400x .. .1 Scale bar
represents 20 pm. B) LDH release recorded and plotted after
incubating cerebellar neurons in culture in NMDA (open diamond),
NMDA+3-MA (black square) and control (open circle). The expressed
values are means + S.E.M. (n = 6). *p < 0.05, ANOVA, NMDA group
is I higher than control (Ctrl); and 'p < 0.05, ANOVA,
NMDA+3 MA group is I lower than NMDA challenge
group).


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progressively from 6 h to 24 h after NMDA-treatment
while untreated control cultures have no significant
increase of LDH release over the same time period (up
to 24 h). Increases in LDH release following NMDA
exposure was significantly alleviated when the neuronal
cultures were co-treated with 3-MA. The levels of LDH
release between NMDA and NMDA+3-MA co-treated
cultures were significantly different at all time points
between 6 hours and 24 hours (#p < 0.05) (Fig 8B). This
confirms the neuroprotective effects of 3-MA against
NMDA-mediated exictotoxicity.

NMDA-induced caspase-3 activation is suppressed by 3-
MA
In our previous study [25], we had demonstrated the
activation of caspase-3 under conditions of prolonged
amino acid starvation-induced autophagy in PC-12 cells.
Here, we tested our hypothesis that the neuroprotective
effects of 3-MA may have been achieved through cas-
pase-3 suppression. To assess caspase-3 activation, we


examined the proteolysis of an endogenous caspase-3
substrate (all-spectrin) and caspase-3 enzymatic activity
assay [11,26]. The all-spectrin breakdown profile using
total anti-all-spectrin antibody showed an increase of
the caspase-3 generated spectrin breakdown product of
120 kDa (SBDP120) at 24 h following treatment of cere-
bellar neurons with NMDA in culture. Increases in the
calpain-generated SBDP150 and SBDP145 [17] were also
observed at 24 hours with NMDA-treated cultures, sug-
gesting the involvement of calpains (Fig 9A) as well.
Staurosporine (STS) treated cultures were used as posi-
tive controls for caspase-3 activation and SBDP120 gen-
eration. To further confirm that the 120 kDa band was
caspase-3 generated, immunoblots were analyzed using
anti-SBDP120 specific antibody developed in-house [27].
The blots confirmed the appearance of the SBDP120 at
24 hours in the NMDA-treated cultures but not in the
controls. 3-MA co-treatment suppressed the increased
SBDP120 levels to near normal (Fig 9A) levels. Densito-
metric analysis of the immunoblots showed a significant


Page 6 of 11


(A) NMDA
Ctrl +3MA


AMg M mII, all-spectlin
cc N4 CM)



Anti-all-Spectrin SBDP150
SBDP145
4 SBDP120


Anti-SBDP120 -., SBDP120

(B) 2, (C)





24h h 24h




Figure 9 NMDA-induced caspase-3 activation is suppressed by 3-MA. A) Representative immunoblot of all-spectrin breakdown profile
shows the presence of the caspase-3 specific ull-spectrin breakdown product (SBDP) of 120 kDa in NMDA-treated cultures after 24 hours
compared to the controls and NMDA+3-MA, or staursorpine (0.5 iM) (n 3). Representative immunoblot probed with anti-SBDP120 shows a
similar profile of the breakdown product in NMDA-treated cultures after 24 hours following treatmentbt not in controls or NMDA+3-MA co-
treatment (n 3). B) Densitometric analyses of the immunoblots probed with anti-SBDP 20 show a 1 increase in the all-spectrin
breakdown product of 120 kDa (SBDP120) after 24 hours following NMDA or STS treatment, when compared to control (*p < 0.05, paired
Student T-test). NMDA+3-MA co-treatment i 1 suppressed SBDP1 20, when compared to NMDA treatment alone (p < 0.05, paired
Student T-test). The expressed values are means + S.E.M. (n 3 C) Caspase-3 enzymatic assay was determined using the caspase substrate Ac-
Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) incubated with protease-inhibitor free lysates obtained from cultures treated with or
without NMDA and NMDA+3-MA co-treatment at 24 hours. The expressed values are means + S.E.M. (n 3). Caspase-3 activity in NMDA or STS
treatment are I higher than corresponding controls (*p < 0.05, paired Student T-test). NMDA+3-MA co-treatment i
suppressed caspase-3 activity, when compared to corresponding NMDA treatments alone (4p < 0.05, paired Student T-test).






Sadasivan et al. BMC Neuroscience 2010, 11:21
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reduction in the caspase-3 mediated SBDP120 levels in
NMDA+3-MA co-treated cultures as compared to
NMDA-treated cells (Fig 9B). Interestingly, calpain-
mediated SBDP150 and SBDP145 were not attenuated
by 3-MA. To assay the caspase-3 protease activity N-
acetyl-Asp-Glu-Val-Asp-AMC (7-amino-4-methylcou-
marin) (Ac-DEVD-AMC) was incubated with protease
inhibitor-free cell lysates under various conditions. Cas-
pase-3 activity was significantly increased in NMDA-
treated cultures at 12 and 24 hours when compared to
control cultures. On the other hand, this caspase-3
activity was significantly reduced by 3-MA co-treatment
when compared to NMDA-treatment alone (12 and 24
h) (Fig 9C).

ATG7 disruption results in neuroprotection following
NMDA exposure
Since 3-MA might have non-autophagy related effects,
ATG7 siRNA was generated and transfected into the
neurons in culture to further investigate the effects of
autophagy inhibition on NMDA neurotoxicity. First, we
observed that atg-7 siRNA partially but significantly
reduced Atg-7 protein levels as well as LC3-II levels
(downstream effect) at 72 h after siRNA treatment (Fig
10A). Scrambled ATG7 siRNA (negative control-) had
no effect-. We then further analyzed the effects of
ATG7 siRNA and scrambled ATG7 siRNA on NMDA
exposure induced cell death, as measured by LDH
release assay. Silencing the Atg7 protein expression in
neurons resulted in a significant (but partial) reduction
of LDH release in the medium compared to neurons
transfected with NMDA with scrambled ATG7 siRNA
or NMDA alone. ATG7 siRNA and scrambled siRNA
alone did not significantly increase LDH above control
cells (data not shown). Since NMDA toxicity has been
demonstrated to induce apoptotic cell death, we incu-
bated neurons with pan caspase inhibitor IDN6556 to
study if we achieved neuroprotection. LDH release assay
suggest comparable neuroprotection with ATG7 siRNA
as well with co-treatment with pan caspase-inhibitor
IDN6556 (20 pM) against NMDA mediated neurotoxi-
city (Fig 10B).

Discussion and Conclusions
Autophagy induction occurs in the central nervous sys-
tem under conditions of stress/starvation or protein
aggregating neurodegenerative diseases [5,28-30]. This
study has shown that acute excitotoxicity by NMDA
exposure can act as a stressor to induce autophagy in
cerebellar neurons. Glutamate excitotoxicity has pre-
viously been documented as one of the pathways of cell
death following experimental traumatic brain injury
[10,31,32]. Erlich and colleagues [33] demonstrated an
increase in beclin-1 expression in mice following


Control


ATG7 si Scramb si

mm Atg7


('- LOA-
. ('- LC3-11
S- (-LC3-II


o60
S50
40
S30
20
| 10


0.9
0.8
0.7
0.6
0.5
> E 0.4
- 0.3
I 0.2
5 0.1 -
0. o.o0


Control ATG7 siRNA Scramb siRNA







IiI'


Control NMDA N+casp3 N+ATG-7
inhibitor siRNA


N+
Scramb
siRNA


Figure 10 Knockdown of ATG7 results in neuroprotection
following NMDA-exposure A) ATG7 siRNA was transfected into
neurons 72 hours before NMDA treatment. Representative western
blot demonstrates the knockdown of the ATG7 in neurons (ATG7
siRNA) compared to controls and scrambled siRNA (Scramb siRNA).
Representative LC3 immunoblot also show reduction of LC3-11 band.
Quantification of the band intensities (n 3) representing the Atg7
protein levels in the granule neurons demonstrates a I
reduction in Atg 7 protein expression. Scrambled siRNA was used as
a negative control to compare te efficiency of Atg7 protein
suppression in neurons. *p < 0.05 ANOVA, Atg7 protein levels are
I lower in the siRNA paradigm compared to controls and
scrambled siRNA B) Lactate dehydrogenase (LDH) release from the
neurons into the medium was measured and quantified at 6 hours
post treatment (n 3). *p < 0.05, ANOVA, NMDA+caspase-3
inhibitor (IDN- and NMDA+ATG7 siRNA treatment groups are
I lower compared to NMDA treated neurons. Data is
represented as mean + S.E.M.


traumatic brain injury suggesting that autophagy is
upregulated around the regions of injury to support the
cells under duress and help dispose of damaged compo-
nents. Recently, there has also been suggestive evidence
for the involvement of autophagy in chronic neurode-
generative diseases such as Parkinson's disease and Hun-
tington disease [34-36].
In our experiments we observed an increase in the
autophagy protein LC3 immunostaining and the mono-
dansylcadaverine (MDC) positive autophagosomes fol-
lowing NMDA treatment as compared to control
samples (Figs 1, 2 and 3). The NMDA treatment also
increased the levels of LC3-I when compared to the


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controls at earlier time periods (3 and 6 h). This transi-
ent enhancement of the LC3-I protein levels in compari-
son to control indicates an enhanced capability of the
cells to launch an autophagic response (data not
shown). There was also an increase in LC3-II levels or
LC3-II/LC3-I ratio (from about 0.60 to 1.30) (Fig 5) fol-
lowing NMDA treatment, as measured by quantitative
immunoblots. This suggests that there may be a pool of
LC3-II being generated following NMDA exposure that
is translocated to the outer membrane of the autopha-
gosomes [37,38].
Evidence from other studies demonstrated the induc-
tion of autophagy and subsequent neuronal death in
spinal cord motor neurons and organotypic hippocam-
pal cultures, following glutamate receptor-mediated
injury [39,40]. According to one study, a buildup of
autophagosomes could be observed in the axonal term-
inals of neurons in Lurcher mice [41]. We extended
their findings by demonstrating that NMDA in cultured
neurons resulted in robust autophagosome formation
throughout the cell bodies and neurites. The presence
of unusually large stained autophagosomal bodies 24
hours following NMDA exposure, suggests a breakdown
in the turnover machinery of the autophagosomes. Also,
the presence of autophagosome accumulation in the
neurons at a time when neuronal death was observed,
points towards the fact that enhanced autophagy may be
pushing the cells towards autophagic cell death.
The NMDA-induced L3-II accumulation could be a
result of either LC3-I to LC-3-II conversion or it could
signify defects in LC3-II turnover. We found that both
lysosomal protease inhibition and proteasome pathway
inhibition significantly elevated LC3-II protein levels
compared to controls, suggesting that there are at least
two pathways of LC3-II turnover. NMDA treatment
conditions seem to render the cells refractory to further
elevation of LC3-II protein levels with co-treatment
with E64d and surprisingly with lactacystin. The signifi-
cance of proteasome involvement in LC3-II clearance
will need further investigation. Our data suggest the
proteasome system is a novel, alternative pathway for
clearance of LC3-II [either membrane-associated pool or
non-membrane-associated associated (cytosolic) pool]
under conditions of stress.
Since prolonged autophagy has been shown to result
in autophagic cell death (type II), we hypothesized that
this form of cell death may be a crucial component to
NMDA excitotoxicity. To test this hypothesis we
employed the autophagy inhibitor 3-MA and examined
whether autophagy inhibition could alleviate NMDA-
mediated neuronal death. The results showed effective
inhibition of the NMDA-induced increase of the lipi-
dated LC3-II protein, the latter being important in the
stabilization of the autophagosomal membrane. Also, a


strong decrease in the LC3 immunostaining and the
MDC-positive autophagosome staining with 3-MA co-
treatment following NMDA exposure were observed as
well. 3-MA co-treatment in neurons [45-47] also
resulted in significant protection against NMDA-
induced cell death (Fig 8). To avoid complete reliance
on interpretation of 3-MA treatment effects, we used
the siRNA approach to investigate the role of autophagy
in NMDA mediated neurotoxicity [47]. Employing
ATG7 siRNA significantly decreased LDH release (a
marker for compromised cell membrane integrity) from
neurons into the culture medium, suggesting its neuro-
protective effects (Fig 10).
Apoptotic and autophagy pathways are intricately
intertwined in the cell [42-44]. In our experiments we
observed that NMDA-induced caspase-3 enzyme activa-
tion and breakdown of spectrin were 3-MA sensitive.
Also, intervention with the pan caspase inhibitor IDN-
6556 (20 pM) resulted in partial but significant protec-
tion against NMDA-mediated excitotoxicity, suggesting
the involvement of other cell death pathways. These
observations suggest that autophagy and caspase-3 acti-
vation may be interlinked.
In summary, based on several lines of evidence, this
study shows that autophagosomes accumulated in neu-
rons when subjected to direct excitotoxic NMDA expo-
sure in a simple culture paradigm. Secondly, based on
(i) the presence of MDC- and LC3-densely stained
autophagosomes following NMDA treatment (but not
amino acid starvation paradigm) and (ii) the refractory
response of LC3-II to E64d treatment in the presence of
NMDA, it appears that NMDA exposure impairs or
diverts the LC3-II/autophagosomes from normal clear-
ance. Autophagy inhibition via 3-MA and ATG7 siRNA
provides neuroprotection against NMDA toxicity.
Taken together, we propose that NMDA neurotoxic
stress triggers the neurons into an autophagic response.
Impairment in autophagosomes clearance causes the
neurons to be 'stuck" in the early autophagosome accu-
mulation mode, which appears to be detrimental to cell
survival. In other words, autophagy under NMDA toxi-
city paradigm in fact contributes to the neuronal cell
injury/cell death process. Obviously, more research into
this direction will be needed to confirm this hypothesis.
It is also equally important to examine how widespread
this autophagic response phenomenon is in other neuro-
toxic conditions either in culture or in vivo.

Methods
Chemicals and Antibodies
N-methyl D aspartate (NMDA), 3-methyladenine (MA),
monodansylcadaverine (MDC), lysosomal protease inhi-
bitor E64D and proteasome blocker lactacystin were
purchased from Sigma Laboratories (St. Louis, MO).


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Prolong Antifade was purchased from Molecular Probes
(Eugene, OR). Fetal bovine serum and Dulbecco's modi-
fied eagle's medium (DMEM) was from Gibco labora-
tories (Grand Island, NY). Antibodies mouse
monoclonal anti-all-spectrin and rabbit polyclonal anti-
caspase-3 specific spectrin breakdown product of 120
kDa (SBDP120) [26,27] were made in-house as was pan
caspase inhibitor IDN-6556 [49]. Anti-p-actin antibody
was purchased from Sigma laboratories (St.Louis, MO),
anti-NeuN antibody was obtained from Chemicon
Laboratories (Temecula, CA) and anti-LC3 antibody
from Novus Biologicals (Littleton, CO).

Animals
Female pregnant Sprague Dawley rats (Harlan Labora-
tories) were received and housed in individual cages.
The animals were maintained on a 12 h light/dark cycle
with food and water ad libitum. All the experimental
procedures in the animals were performed in accor-
dance with the National Institute of Health Guide for
the Care and Use of Laboratory Animals and the proto-
cols were approved by the UF IACUC.

Cell cultures and treatments
Cerebellar cultures were obtained from dissociated cere-
bella of 6-8 day old Sprague Dawley rat pups (Harlan
Laboratories) and plated in Dulbecco's modified eagle's
medium (DMEM) supplemented with 25 mM glucose, 25
mM KC1, 10% fetal bovine serum on culture dishes
(Nunc plates, Fisher). 13p-arabinofuranosylcytosine (10
pM) was added to the culture medium 22 hours after
plating to prevent the proliferation on non-neuronal cells
for 48 hours. On the 8th day following harvesting, the
neurons were exposed to different treatment conditions
and subsequent experimental end points. The neurons
were treated with or without NMDA (200 pM) for differ-
ent time periods and the cells were eventually lysed with
triton based lysis buffer for protein immunoblots. The
other treatment condition involved a co-treatment of
NMDA with 3-methyladenine (3-MA, 10 mM). For fluor-
escent microscopy, the neurons were cultured on glass
coverslips coated with poly-l-lysine and treated in a simi-
lar manner as the cultures on plates. Positive control to
induce autophagy involved subjecting CGNs to amino
acid starvation by incubating them in Hank's buffer with
25 mM glucose (with calcium, magnesium) and vitamins.

Immunofluorescence
Cerebellar cells plated on coverslips were fixed using
freshly prepared 4% paraformaldehyde solution for 10
min at 4oC, washed in pure methanol and then permea-
bilized with lx Tris buffered saline -Tween-20 (TBST,
Sigma Laboratories, St. Louis, MO). Following TBST
washing, the cells were incubated in 5% normal goat


serum (NGS) at 37C for 30 minutes before incubating
with the primary antibody microtubule associated light
chain-3 (LC-3; Atg8; 1:1000) in 5% NGS overnight at 4
C. On the following day, the coverslips were washed 3
times with lx TBST and incubated with the Alexa Fluor
(Molecular Probes, Carlsbad, CA) red or green-conju-
gated secondary antibodies (1:3000) for 1 hour at 37C.
The coverslips were then washed with lx TBST and
then mounted with the mounting medium Vectashield
(Burlingame, CA) and observed under the microscope.

Autophagosome labeling with MDC
The neurons on the coverslips were incubated with a
fluorescent dye monodansylcadaverine (MDC; 0.05 mM)
in phosphate buffered saline (PBS) after 6, 12, 16 and 24
hour NMDA challenges at 37C for 10 minutes, to
observe for autophagosomes [50]. The cells were washed
2 times with PBS, mounted using Antifade solution
(Prolong Antifade) and immediately observed by Zeiss
fluorescence microscope. Following prolonged NMDA,
we observed unusually large autophagosomal bodies,
which are defined as having a size at least 5-time the
size of normal sized autophagosomes. For their quantifi-
cation of both average sized autophagosomes and unu-
sually large autophagosomal bodies, for each
experimental condition/well, 4 randomly selected fields
per well (400x magnification) are used and more than
20 cells in each field are analyzed with counted.

Western Blot Analysis
The cerebellar neuronal lysates were collected at differ-
ent time points after treatment with the appropriate
media using lysis buffer containing 1% (v/v) Triton X-
100, 5 mM EGTA, 5 mM EDTA, 150 mM NaCl and 20
mM Tris HCl (pH 7.4). The protein content was deter-
mined using DC Protein Assay (Bio-Rad, Hercules, CA)
and the protein concentration was standardized to 1 pg/
pL. Twenty micrograms of protein were subjected to
SDS-PAGE gel electrophoresis on 4-20% or 6% Tris-gly-
cine gels (Invitrogen, Carlsbad, CA) and then transferred
onto PVDF membrane on a semi-dry electro transfer-
ring unit (Bio-Rad). Following the transfer, the mem-
branes were blocked in 5% nonfat dry milk in lx Tris
buffered saline with Tween-20 (TBST) and probed over-
night with primary antibody at 4C. The following day,
the membranes were washed with TBST and probed
with either secondary peroxidase conjugated anti-rabbit
or the biotinylated anti-mouse antibody. Immunoreac-
tivity was detected by either using streptavidin alkaline
phosphatase conjugate tertiary antibody or enhanced
chemiluminescence (ECL) reaction (Amersham, Piscat-
away, NJ). Densitometric quantification of the bands
was performed using ImageJ software (version 1.29x;
NIH, Bethesda, MD).


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Lactate Dehydrogenase (LDH) Release Assay for Cell Death
Lactate dehydrogenase release assay was performed to
assess cell death by measuring the release of lactate
dehydrogenase into the medium from damaged cells
due to necrosis and secondary necrosis following apop-
tosis [51] or autophagic cell death. Culture medium, 25
pL, was collected after 0, 3, 6, 12, 16, 20 hours and 1
day in 96-well flat bottom plates. An equivalent volume
(25 pL) of detection reagent (CytoTox One Reagent,
Promega, Madison, WI), was added to each well con-
taining the culture medium and incubated for 30 min-
utes in the dark at room temperature. Absorbance was
measured (wavelength: 490 nm) using a colorimetric
microplate reader (SpectraMax Gemini, Molecular
Devices). Six replicates for each time point per experi-
ment were assayed and three such experiments were
performed. The arbitrary density unit values were
plotted against time.

Caspase-3 enzymatic activity assay
To assay for caspase-3 activity, control, NMDA-treated
and NMDA/3-MA co-treated granule neurons from
three different wells (12 and 24 h) were scraped in a
buffer containing 20 mM Tris-HC1 (pH 7.4 at 4oC), 150
mM NaC1, 1 mM dithiothreitol, 5 mM EDTA, 5 mM
EGTA, and 1% (vol/vol) Triton X-100 for 1 h. The
cleared lysates were mixed with 50% (vol/vol) glycerol.
Cell lysates were assayed with 100 pM acetyl-Asp-Glu-
Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-MCA;
Bachem Bioscience), 100 mM HEPES, 10% glycerol, 1
mM EDTA, 10 mM dithiothreitol. Fluorescence (excita-
tion, 380 + 15 nm; emission, 460 + 15 nm) was mea-
sured at 60 minutes using a fluorescent microplate
reader (SpectraMax Gemini EM, Molecular Devices) as
described previously [52].

ATG7 short interfering RNA (siRNA) preparation and
transfection
Small interfering RNA (siRNA) targeting rat ATG7 (5'-
GCAUCAUCUUUGAAGUGAA-3'; Sigma), or pooled
non-targeting control siRNA (5'-AUGAACGU-
GAAUUGCUCAA-3', 5'-UAAGGCUAUGAAGAGUAC-
3', 5'-AUGUAUUGGCCUGUAUUAG-3', and 5'-UAGC-
GACUAAACACAUCAA-3'; Dharmacon) [53] were pur-
chased commercially and used. Primary neurons were
transfected on day 4 of culture preparation, with 20 nmol
of ATG7 or control siRNA using Lipofectamine 2000
(Invitrogen), 72 hours before subjecting to NMDA chal-
lenge. The efficacy of ATG7 knockdown was assessed by
Western blot using an antibody against Atg7 (Sigma).

Statistical Analysis
One-way ANOVA with Tukey post hoc test was used to
draw comparisons between groups in the LDH assay.


Data was plotted as means S.E.M. (standard error of
the mean). A value of p < 0.05 was considered to be sig-
nificant. Student's t-test was performed to draw statisti-
cal comparisons between two treatment groups and a p
< 0.05 was considered to be statistically significant.


Abbreviations
3 MA' 3-methyladenine, MDC monodansylcadaverine, MAP-LC3' microtubule
associated protein-light chain 3

Acknowledgements
The authors wish to thank Drs William Dunn, Jr and Lucia Notterpek
(University of Florida) for their insights and opinions in this work This work
was supported by the Department of Defense grant DAMMED-03-1-0066
and NIH grant R01 NS049175-01-A1 KKW and RLH own stocks and receive
royalties from and are executive officers of Banyan Biomarkers Inc and as
such may benefit financially as a result of the outcome of this research or
work reported in this publication

Author details
'Center for Neuroproteomics and Biomarkers Research, Department of
Psychiatry, the McKnight Brain Institute of the University of Florida,
Gainesville, FL 32610, USA 2Center of Innovative Research, Banyan
Biomarkers, Inc, Alachua, FL 32615, USA

Authors' contributions
SS and KKW planned, designed and conducted experiments SS, KKW and
RLH wrote the manuscript RLH, SFL, ZZ, FHK, MCL and WZ helped perform
some of the experiments, interpretation of data and revise aspects of the
manuscript All authors have read and approved the final version of the
manuscript

Received: 2 December 2008
Accepted: 18 February 2010 Published: 18 February 2010

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doi:10.1186/1471-2202-11-21
Cite this article as: Sadasivan et al Acute NMDA toxicity in cultured rat
cerebellar granule neurons is accompanied by autophagy induction
and late onset autophagic cell death phenotype. BMC Neuroscience 2010
11 21


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