World Journal of Neuroscience, 2012, 2, 234-247 WJNS
http://dx.doi.org/10.4236/wjns.2012.24036 Published Online November 2012 (http://www.SciRP.org/journal/wjns/)
The spatial and temporal relationship between oxidative
stress and neuronal degeneration in 3-nitropropionic acid
model
Thierry Delorme, Mohammad Najafi, Payman Nasr*
Department of Biological Sciences, Kent State University, Ashtabula, USA
Received 15 June 2012; revised 29 July 2012; accepted 14 August 2012
ABSTRACT
The current study investigates the role of oxidative
stress and calcium homeostasis in the development of
selective striatal lesions in metabolic impairment mo-
del caused by 3-nitropropionic acid (3NP). In this re-
port, we examined the distribution of oxidative stress
markers and the production of mitochondrial reac-
tive oxygen species in the presence of 3NP in male
Sprague-Dawley rats. Protein oxidation was assessed
using 3-nitrotyrosine immunoreactivity, while DNA
oxidative damage was evaluated by poly (ADP-ribose)
polymerase-1 activity. The Reactive Oxygen Species
(ROS) production was determined in isolated mito-
chondrial from striatum and cerebellum of two age
groups following 3NP and variable calcium concen-
tration. The results demonstrate that increased 3-
nitro-tyrosine level is the most robust in the striatum
and the least evident in the cerebellum following 4
days of 3NP treatment. No significant change in the
levels of poly ADP-ribosylated proteins was observed,
likely due to a rapid PARP-1 cleavage as detected by
the appearance of 50 kDa necrotic fragment. In mi-
tochondrial isolates, there was no immediate increase
in mitochondrial ROS following 3NP in either stria-
tum or cerebellum; however, calcium addition resulted
in a concentration dependent increase in reactive oxy-
gen species in striatal mitochondria of the older ani-
mals. These results suggest that in aging, mitochon-
dria become more susceptible to the generation of
ROS in conditions that cause a concurrent compro-
mised in mitochondrial calcium concentration. This
finding implicates mitochondria dysfunction as a key
cellular target in pathological states that are associ-
ated with metabolic impairment. The results also re-
inforce the notion that mitochondrial function in the
striatum and cerebellum respond differently to the
aging process, which may explain the variable re-
gional vulnerability in 3NP model.
Keywords: Energy Impairment; 3-Nitropropionic Acid;
Oxidative Stress; Neurodegeneration
1. INTRODUCTION
Numerous environmental toxins can compromise mito-
chondrial function and impair energy metabolism. One
such toxin is 3-nitropropionic acid (3NP) which is pro-
duced by a number of fungal and plant species [1-2].
3NP is an irreversible inhibitor of succinate dehydro-
genase (SDH), a component of mitochondrial complex II
and the Krebs cycle [3]. Severe 3NP poisoning results in
motor impairment accompanied with basal nuclei (stria-
tum) degeneration in humans, primates and rodents [4].
Since the discovery of 3NP as the causative agent in
sugarcane poisoning and striatal degeneration, many in-
vestigators have utilized this compound to reproduce the
cognitive and motor deficits seen in neurodegenerative
diseases such as Huntington’s disease in animal models
[5-8].
There are many reports of 3NP toxicity in humans due
to sugar cane infestation by Arthinium species. Over a
long period of storage, Arthinium species are capable of
producing 3NP. In 1970’s and 80’s, sugarcane, being a
common snack in China, resulted in more than 800 re-
ported cases of 3NP toxicity and 88 deaths [9]. In mild
cases of 3NP toxicity, the patients developed lethargy,
gastritis, nausea and vomiting. In severe cases, the sym-
ptoms consisted of severe seizures, coma, brain edema and
neurodegeneration in caudate putamen, globusp allidus and
lenticular nuclei [10,11].
In mammals, systemic administration of 3NP results in
progressive locomotor impairment and selective striatal
lesions [12-14]. In rats, the dorsolateral striatum is
generally considered homologous to the putamen in hu-
mans, a region directly involved in the control of move-
ment, while the ventromedial aspect of the rat striatum is
similar to the caudate nucleus [15]. Following systemic
3NP administration in rats, dorsolateral striatum is the
*Corresponding author.

T. Delorme et al. / World Journal of Neuroscience 2 (2012) 234-247 235
main site of neurodegeneration, whereas ventromedial
striatum is relatively spared. The majority of the neurons
in dorsolateral striatum are medium-sized spiny neurons
which receive strong glutamatergic inputs from the cere-
bral cortex and thalamus, as well as a dopaminergic input
from the substantia nigra. In addition, the striatum re-
ceives minor inputs from raphe nuclei, the globus pal-
lidus and subthalamic nucleus (for review see [16-18]).
The 3NP model presents a number of technical advan-
tages over other mitochondrial toxins such as malonate.
The main advantage of 3NP is that it can be administered
systemically and readily crosses the blood-brain barrier
[12]. Moreover, despite the inhibition of SDH throughout
the body, 3NP is highly selective in producing striatal
lesions similar to those seen in Huntington’s disease in
which the medium-sized spiny neurons in striatum are
the most vulnerable [19]. 3NP also causes more severe
neurotoxicity in the older experimental animals com-
pared to the younger ones [20,21]. In addition, 3NP is re-
latively inexpensive and simple to utilize. Taken together,
3NP offers a practical model to study neurodegenerative
disorders associated with metabolic impairment.
3NP administration results in uniform inhibition of
SDH activity throughout the brain [22,23], and the re-
asons for the selective vulnerability of striatal neurons
are not well understood. Growing evidence has impli-
cated secondary excitotoxicity, oxidative stress and cal-
cium homeostasis as key factors in the preferential stria-
tal degeneration in the 3NP model [24-28].
Striatum receives a strong glutamatergic input from
the cerebral cortex, which renders the striatum vulner-
able to excitotoxicity. In vitro, in the presence of 3NP,
N-Methyl-D-Aspartate (NMDA) receptor inhibition shifts
the cell death from predominantly necrotic to apoptotic
characteristics, but does not prevent the cell death [29].
In vivo, decortication protects the striatum against dege-
neration following systemic 3NP administration [19].
NMDA receptor activation is also closely linked to
oxidative stress via Nitric Oxide Synthase (NOS) activity
[30-32]. Nitric oxide is a simple diatomic gas with im-
portant physiological functions and is produced by NOS
in vivo [33-38]. Activation of NOS results in increased
production of nitric oxide, which in turn, in the presence
of an oxidative environment, readily reacts with super-
oxide radicals (2) to form peroxynitrite, a toxic oxi-
dant [39]. Tyrosine residues of proteins are particularly
susceptible to nitration by peroxynitrite, resulting in the
formation of 3-nitrotyrosine [40]. The level of 3-nitroty-
rosine can be detected in vivo and have been utilized as a
marker for protein oxidation in biological tissues [39,41].
Moreover, increased levels of 3-nitrotyrosine have pre-
viously been reported in a number of pathological con-
ditions including amyotrophic lateral sclerosis and Alz-
heimer’s disease [42-45].
O
Peroxynitrite and its byproducts can also damage
DNA, leading to the activation of the repair enzyme poly
(ADP-ribose) polymerase-1 (PARP-1) [46]. PARP-1
cleaves NAD+ into nicotinamide and ADP-ribose, with
subsequent polymerization of ADP-ribose on a set of
specific acceptor proteins, in turn altering their structure
and initiating the process of DNA repair. PARP-1 is a
highly energy-dependent enzyme, and its excessive acti-
vation rapidly depletes cellular NAD+ and ATP supply,
exacerbating the metabolic crisis inflicted by energy im-
pairment.
Others have demonstrated that excessive calcium up-
take by mitochondria stimulates the mitochondrial free
radical generation [47,48]. In vitro analysis demonstrates
that the addition of calcium to mitochondria isolates in
the presence of rotenone (complex I inhibitor) and anti-
mycin A (complex III inhibitor) results in significant in-
crease in ROS [49-52]. In addition, Jacquard and colle-
agues have demonstrated that impaired mitochondrial
calcium homeostasis is directly implicated in 3NP-in-
duced toxicity [53]. The neurotoxic effect of 3NP is re-
ported to be due to an improper sequestration of Ca2+ by
mitochondria and consequent calpain proteases activa-
tion [53].
In summary, the current report examines the role of
oxidative stress and calcium homeostasis in the develop-
ment of selective striatal lesion in metabolic impairment
model caused by 3NP. Protein oxidation was assessed
using 3-nitrotyrosine immunoreactivity, while DNA oxi-
dative damage was evaluated by poly (ADP-ribose) poly-
merase-1 activity. The reactive oxygen species produc-
tion was also determined in isolated mitochondrial from
striatum and cerebellum of two age groups following
3NP and variable calcium concentrations.
2. METHODOLOGY
2.1. Materials
3NP was purchased from Sigma-Aldrich (St. Louis, MO).
Protein levels were measured with Pierce BCA kit (Pierce
Chemical Company, Rockford, IL). The chemilumines-
cent substrate for Western blots, Super signal West Pico,
was also from Pierce. The anti-nitrotyrosine mouse mo-
noclonal IgG (clone 1A6 Cat# 05-233) was purchased
from Upstate biotechnology (Lake Placid, NY). The
PARP-1 cleavage detection kit (Cat# SK-003) and mono-
clonal anti-poly (ADP-ribose) antibody against ADP-
ribosylated proteins (Cat# SA-250) were obtained from
BioMol Research Laboratories (Plymouth, PA). All other
reagents were from Sigma-Aldrich (St. Louis, MO).
2.2. Animals
All experimental protocols involving animals were in
accordance with the guidelines published in the NIH

T. Delorme et al. / World Journal of Neuroscience 2 (2012) 234-247 236
Guide for the Care and Use of Laboratory Animals and
the Society for Neuroscience Guidelines for the Use of
Animals in Neuroscience Research. To study the in-
volvement of oxidative stress in 3NP toxicity, Sprague-
Dawley rats were intraperitoneally injected with 3-NP
(20 mg/kg/day) for 1, 2, 3 or 4 days, while the control
animals were treated with saline for 4 days. At the end of
the treatments, the animals were anesthetized with pento-
barbital (60 mg/kg), decapitated, and the brains were
removed immediately. One hemisphere was incubated in
4% paraformaldehyde for 24 hours at 4˚C followed by 24
hours incubation in 30% sucrose in phosphate buffer so-
lution (pH 7.4) for lesion analysis, and the other hemi-
sphere was dissected and the striatum, hippocampus and
cerebellum were removed, frozen on dry ice and stored
at –80˚C degrees for Western blot analysis
2.3. Western Blotting
The tissues of the region of interest (striatum, hippo-
campus and cerebellum) were homogenized in Tris-
buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.5)
containing protease inhibitor (l mM leupeptin, 25 mM
EDTA, 1 μM pepstatin A, 200 μM AEBSF) then centri-
fuged at 14,000× g for 5 minutes and supernatant was
collected. For PARP-1 cleavage products and ADP-
ribosylated proteins, 0.1% SDS, 1% Nonidet P-40 and
20% glycerol were added to the homogenizing buffer.
Protein concentrations were determined using the BCA
protein Assay, and equal samples were loaded on a
gradient SDS-PAGE gel (10 - 40 μg/ lane). For 3-nitroty-
rosine analysis, 10 μl of nitrotyrosineimmuno blotting
control (Cat# 05-233, Upstate Biotechnology) was uti-
lized as a positive control, while as a negative control,
the primary antibody was excluded in a sister blot. For
PARP-cleavage immunoblots, we utilized whole cell ex-
tracts of human HL60 leukemia cells containing intact
PARP-1 (Cat# SW101, BioMol Research Laboratories)
and whole cell extracts of human HL60 leukemia cells
undergoing apoptosis by the chemotherapeutic agent eto-
poside (Cat# SW-102, BioMol Research laboratories) as
positive controls.
SDS-PAGE was performed according to the method of
Laemmli [54] using a mini-gel apparatus (Bio-Rad, Her-
cules CA). Following SDS-PAGE, polypeptides were
transferred electrophoretically onto 0.45 μm nitrocellu-
lose membranes. The membranes were blocked for 60 -
120 minutes in 5% fat-free milk in TTBS and incubated
with the primary antibody in TTBS overnight at room
temperature. The membranes were then incubated with
peroxidase-conjugated goat anti-mouse or horse anti-
rabbit IgG (Jackson Immunoresearch Laboratories, West
Grove, PA) for 60 minutes at room temperature. The
blots were developed in SuperSignal West Pico chemi-
luminescent substrate (Pierce Chemical, Rockford, IL)
for 1 minute and exposed to Kodak T-Max X-ray film.
2.4. Lesion Analysis
For the fixed tissue, each brain hemisphere was sectioned
at 25 μm intervals using a cryostat instrument. Every 4th
section was mounted on slides and stained with cresyl
violet. The sections were imaged using a digital camera,
and the total striatal volume and the lesion volume were
measured and the percent spared tissue was calculated
for each animal. The lesion area was identified by absent
or pale cresyl violet staining. The cell loss in the lesion
area was confirmed by microscopic examination.
2.5. Mitochondrial Preparation
Isolated mitochondria were prepared as previously des-
cribed with slight modifications [55]. Briefly, Sprague-
Dawley rats (n = 6 per group) were anesthetized by
sodium pentobarbital (60 mg/kg), the brains were re-
moved, and the striatum and cerebellum were carefully
dissected. All the following steps in mitochondrial iso-
lation were performed at 4˚C. The dissected tissue was
minced in ice-cold homogenization buffer (250 mM
sucrose, 20 mM HEPES, 0.1% BSA, 1 mM EDTA, pH
7.2) and was rinsed with 10 ml homogenization buffer to
remove residual blood. Next, the minced tissue was pro-
cessed (8 strokes) using a hand-held tissue homogenizer
(Thomas Scientific). The resulting homogenate was cen-
trifuged for 3 min at 1300× g, the supernatant was re-
moved, centrifuged at 13,000× g for 10 min and the
pellet was placed in nitrogen cell bomb and exposed to
1000 psi for 5 minutes to disrupt synaptosomal mem-
branes. The pellets were resuspended in EGTA free
isolation buffer and centrifuged at 10,000 × g for 10 min.
Mitochondrial protein concentration was determined
using a Pierce BCA kit.
2.6. ROS Production
The mitochondrial ROS production assay was performed
immediately after mitochondrial isolation. ROS produc-
tion was measured using the indicator 2’, 7’-dichloro-
fluorescin diacetate (DCF-DA, Molecular Probes, Eugene,
OR) as previously described [55,56]. Briefly, 100 - 150
μg of isolated mitochondrial protein was incubated in a
total volume of 200 μl respiration buffer (215 mM man-
nitol, 75 mM sucrose, 1% BSA, 2 mM MgCl2, 2.5 mM
KH2PO4, 20 mM HEPES) at 37˚C for 15 min in the pre-
sence of 10 μM DCF-DA, which was made fresh before
each use. The relative amount of mitochondrial free radi-
cal generation in the presence of 3NP with or without
calcium (50 μM - 150 μM) was monitored by measuring
changes in fluorescence resulting from DCF-DA oxi-
dation product, DCF, using a CytoFluor 4000 fluoro-
metric plate reader (excitation 490 nm, emission 526 nm).

T. Delorme et al. / World Journal of Neuroscience 2 (2012) 234-247 237
Addition of H2O2 was utilized as a positive control and
increased DCF fluorescence in a linear style.
2.7. Statistical Analysis
For Western blot quantitative analysis, comparisons among
groups were made by two-way ANOVA followed by
Fisher’s PLSD t-test. The percent spared striatal tissue in
different groups was compared using an unpaired student
t-test. The data for DCF assay (t1 = 0 min vs. t2 = 15 min)
in each brain region was analyzed utilizing a 2-tailed
paired t-test, while the comparison in the rate of ROS
generation from different regions was performed by
2-tailed unpaired t-test. In each case, the t-test was
followed by a Bonferroni/Dunn test for multiple com-
parisons. All values are expressed as the mean values ±
the standard error of the mean of ‘n’ observations, and a
probability level of P < 0.05 was considered significant.
3. RESULTS
3.1. 3NP-Induced Lesions in the Rat Striatum
Four days after 3NP (20 mg/kg/day, i.p.) administration,
all animals demonstrated behavioral abnormalities asso-
ciated with 3NP toxicity, characterized by recumbence,
dystonia and lethargy similar to those previouslyde-
scribed [13,57-59]. The saline treated specimens did not
display behavioral impairment throughout the duration of
experiments. Upon examination with cresyl violet stain-
ing, 24 and 48 hours of 3NP treatment did not result in
any apparent cell loss in any regions of the brain. How-
ever, three days following the first 3NP injection, two
out of six rats had developed mild to moderate cell loss
in the lateral striatum. Four days following 3NP treat-
ment, one rat had moderate pathology, while the remain-
ing rats displayed more severe neuronal loss, typically
extending from lateral to dorsal and medial striatum
(Figures 1 (a) and (b)).
3.2. 3-Nitrotyrosine Levels
In 3NP-treated rats, hematoxylin and eosin staining
showed marked cellular alterations in the striatum cha-
racterized by nuclear shrinkage and the formation of
perinuclear spaces (Figure 2(a)). Treatment of rats with
3NP for 48 hours did not result in the elevation of 3-
nitrotyrosine levels. However, three days following 3NP
treatment, there was a mild elevation in 3-nitrotyrosine
immunoreactivity in the striatum of some animals;
nonetheless, upon quantitative Western blot analysis, the
averaged results did not statistically differ from that of
the saline control (n = 6, P = 0.08). Following 4 days of
3NP treatment, the levels of 3-nitrotyrosine in the stria-
tum were significantly greater than that of controls (n = 6,
P < 0.05). At this time point, there was a slight increase
(a)
% Spared Tissue
020 40 60 80100120
Saline
Day 1
Day 2
Day 3
Day 4*
(b)
Figure 1. Intraperitoneal administration of 3NP results in selec-
tive striatal lesion. (a) Selective striatal neurodegeneration first
appears in the lateral striatum and spreads throughout the dorsal
and medial striatum; (b) Quantitative analysis of the percent
spared tissue indicates a significant decrease in striatal spared
tissue 4 days following 3NP treatment (n = 6 per day, P < 0.05).
in 3-nitrotyrosine immunoreactivity in the hippocampus,
(n = 6, P = 0.09) (Figure 2(c)). Cerebellum did not ex-
hibit elevated 3-nitrotyrosine levels up to four days fol-
lowing 3NP treatment (Figures 2(b) and (c)).
3.3. Poly ADP-Ribosylation Levels
To investigate whether PARP-1 activation was asso-
ciated with 3NP-induced oxidative stress, we examined
the levels of poly-ADP ribosylated proteins in various
brain regions. There were occasional cells immuno-
stained for poly-ADP ribosylated proteins in striatum and
hippocampus following 4 days of 3NP treatment (data
not shown). However, Western blot analysis did not
indicate a significant increase in the total levels of poly
ADP-ribosylated proteins (Fi gure 3 ).
3.4. Poly (ADP-Ribose) Polymerase-1 Cleavage
To further investigate the nature of PARP-1 involvement
in the 3NP model, we utilized antibodies to detect
PARP-1 cleavage products since it has previously been
demonstrated that PARP-1 is cleaved during both apop-
totic and necrotic cell death [46,60-62]. Twenty-four
hours after 3NP administration, a weak 50 kDa band was

T. Delorme et al. / World Journal of Neuroscience 2 (2012) 234-247 238
(a) (b)
3-Nitrotyrosine Immu no reactivity
0
20
40
60
80
100
120
140
160
180
D1 D2 D3 D4
% Control
Str.
Hip
Cb *
(c)
Figure 2. 3-Nitrotyrosine levels following 3NP treatment. (a) H & E staining demonstrates histopathological char-
acteristics of 3NP administration in the striatum marked by cellular alterations in the striatum and nuclear shrink-
age and perinuclear spaces formation, while 3-nitrotyrosine immunoreactivity following 4 days of 3NP treatment
indicates a robust increase in levels of 3-nitrotyrosine in striatum. No other regions examined exhibited a signifi-
cant increase in 3-nitrotyrosine immunoreactivity as compared to the saline-treated controls; (b) Representative
Western blot results for 3-nitrotyrosine in various brain regions; (c) Quantitative analysis of 3-nitrotyrosine levels
in the corresponding region is illustrated as a percent of saline-treated controls (n = 6 per day, P < 0.05); (C = con-
trol, D = Day, 3NT = 3-nitrotyrosine, H & E = hematoxylin and eosin stain, Scale bar = 10 µm, str = striatum, hip
= hippocampus, cb = cerebellum).
present in the striatum. The 50 kDa fragment increased in
intensity on the second and third day but was reduced by
the end of the fourth day of treatment. Interestingly, in
hippocampus, the 50 kDa band did not appear until the
third day and was present by the end of fourth day fol-
lowing 3NP administration. PARP-1 cleavage products
were not observed in the cerebellum at any time points
following 3NP (Figure 4).
isolated mitochondria influences production of free ra-
dical species, we measured the generation of free radicals
in isolated mitochondria from striatum and cerebellum of
two- and ten-month old rats. In the older animals, 3NP
did not increase DCF fluorescence in either striatum or
cerebellum. In contrast, there was a reduction of DCF
signal in the striatum and cerebellum of younger animals
(Figure 5). The addition of two concentrations of Ca2+
(50 μM and 150 μM) to the isolated mitochondria pre-
paration from the striatum of older animals resulted in a
significant concentration-dependent increase in fluore-
3.5. Mitochondrial ROS Production
To investigate whether 3NP and calcium concentration in

T. Delorme et al. / World Journal of Neuroscience 2 (2012) 234-247 239
Figure 3. Analysis of poly ADP ribosylated proteins
following 3NP treatment. Representative Western blots
from various regions of brain demonstrate no major
change in poly-ADP ribosylated proteins in any of the
brain regions (n = 6 per day).
Figure 4. PARP-1 cleavage products following systemic
administration of 3NP. In the striatum, a 50 kDa PARP-1
cleavage fragment first appears one day following 3NP
treatment, intensifies on the second and third day and di-
minishes by the end of the fourth day, whereas in the hippo-
campus, a 50 kDa PARP-1 fragment first appears on the
third day post-3NP treatment and is sustained throughout the
fourth day. In the cerebellum, there is no apparent increase
in 50 kDa fragment up to 4 days following 3NP treatment.
0
20
40
60
80
100
120
140
160
180
200
Str . Y oungCb. Y oungStr . OldCb. Old
% Control DCF Fluorescence
(arb itrary units)
3NP 4mM
50 µM Ca
150 µM Ca
**
*
#
#
Figure 5. ROS production as detected by increased DCF fluo-
rescence signal. 3NP administration (4 mM) in mitochondrial
preparation for 15 minutes did not result in a significant change
in reactive oxygen species detected by DCF assay, while there
was a significant decrease in DCF fluorescence signal in the
striatum and cerebellum of the younger animals (#P < 0.01, n = 6).
The addition of calcium (50 µM and 150 µM) resulted in a
significant calcium concentration-dependent increase in ROS
production in the striatal mitochondrial preparation of the older
animals (*P < 0.05, **P < 0.01, n = 6). However, similar con-
centrations of calcium did not alter DCF fluorescence signals in
either the striatum or cerebellum of younger animals (Ca =
Calcium, Cb = Cerebellum, Str = Striatum).
scence signal. Although there was an increase in ROS
generation in cerebellar mitochondria in the aged animals,
the increase was subtler when compared to the striatum
(Figure 5). The striking finding was the lack of increase
in DCF fluorescence in the presence of Ca2+ in the
striatum of younger animals since mitochondria is the
principal source of calcium-dependent free radical pro-
duction [47,48,63].
4. DISCUSSION
The acquired knowledge from natural cases of 3NP toxi-
city propelled a number of scientific studies to cha-
racterize this compound. The effort led to the discovery
that chronic or acute administration of 3NP results in
selective striatal degeneration among experimental ani-
mals [19,20,25]. Moreover, behavioral studies in pri-
mates indicated that 3NP produces cognitive and motor
deficits reminiscent of those seen in Huntington’s disease
[7,64,65]. Several hypotheses have been proposed as the
possible mechanisms for 3NP toxicity. In 1987, Hamil-
ton and Gould observed the similarities between the
neuronal damage in 3NP and that of kainic acid in the rat
model. They proposed an excitotoxic mechanism [25].
Excitotoxicity has been associated with a number of pa-
thological conditions, such as ischemia and hypogly-
cemia, as well as several chronic neurodegenerative dis-
eases [66-69]. Furthermore, it has been demonstrated that
excitotoxicity due to overexposure to excitatory amino

T. Delorme et al. / World Journal of Neuroscience 2 (2012) 234-247 240
acids can produce lesions in the CNS similar to those
observed in Huntington’s disease [70-72]. L-glutamate is
the primary excitatory neurotransmitter in the central
nervous system [73]. High doses of intrastriatal admi-
nistration of glutamate result in rapid development of
neuropathological features that are characterized by swell-
ing of neuronal cell bodies and organelles, as well as nu-
clear pyknosis [66,74]. In addition, cell culture studies
have signified the role of glutamate receptors in 3NP
neurotoxicity following the reduction in intracellular en-
ergy levels and inhibition of ATP-dependent ion pumps
[24,29,75]. These findings conform with the “secondary
excitotoxicity” hypothesis stating that energy impairment
indirectly results in the opening of glutamate receptors,
influx of massive amount of calcium and overwhelming
the mitochondrial calcium buffering capacity, ultimately
leading to the activation of cell death pathways [24,29,
70,75-78]. Moreover, Liot and colleagues have estab-
lished temporal relationship of 3NP administration and
ROS production [79]. They documented an instant drop
in ATP, and several hours after the initial injection, a
significant increase in ROS production. A possible ex-
planation for this phenomenon was that the ATP drop
and ROS production, lead to depolarization of the plasma
membrane, possibly relieving the Mg2+ block of voltage-
gated NMDA receptors. This event leads to the opening
of the NMDA receptors, allowing massive Ca2+ influx,
ultimately resulting in substantial ROS production.
Other reports have implicated oxidative stress as a
possible pathway in 3NP neurotoxicity since following
systemic 3NP administration, there is an increase in pro-
tein oxidation markers in the striatum [80,81]. The ex-
posure of living organisms to free radicals and oxidants
is part of an intricate nature of aerobic respiration. Every
cell in the human body has mechanisms in place to
counteract or even utilize the production of free radicals
to conduct a variety of cellular functions. Thus, a correct
definition of oxidative stress as a pathological event is
considered necessary. An excellent definition for oxi-
dative stress was provided by Sies and Cadenas as “the
critical disturbances in prooxidant-antioxidant balance in
favor of the former” [82]. Consequently, the occurrence
of oxidative challenge in cells does not necessarily con-
stitute oxidative stress. However, when the noxious ex-
ternal or internal sources enhance the production of oxi-
dants, and the cellular defensive mechanisms against the
oxidants are either overwhelmed or compromised, we
approach a pathological oxidative environment which leads
to oxidative stress and subsequently oxidative damage
[83].
3-Nitrotyrosine formation has previously been re-
ported as a reliable marker for protein oxidation in the
3NP model [80,84-86] and other neurodegenerative dis-
eases [87-89]. 3-Nitrotyrosine is the product of reaction
between nitrogen species with tyrosine residues of pro-
teins [90]. The nitration of tyrosine residues alters the
conformation of proteins, resulting in a change of func-
tion such as the ability to be phosphorylated [91]. Since
the nitration of tyrosine residues is an irreversible pro-
cess, abnormal increases in 3-nitrotyrosine levels acutely
interfere with routine cellular functions [92]. One likely
source of elevated 3-nitrotyrosine is peroxynitrite (ONOO-),
a strong oxidant capable of oxidizing proteins and DNA.
In an oxidative environment, peroxynitrite can be readily
formed by the interaction between nitric oxide and
superoxide radicals [93]. Interestingly, the inhibition of
mitochondrial aerobic respiration, as seen in the 3NP
model, results in the loss of mitochondrial electro-che-
mical gradient and subsequently excessive generation of
superoxide radicals [94]. The increase in superoxide
radicals accompanied with the excess production of nitric
oxide may account for peroxynitrite and subsequent 3-
nitrotyrosine elevation. Our results demonstrate that the
levels of protein oxidation marker, 3-nitrotyrosine, sig-
nificantly increase in the striatum, the primary site of
neurodegeneration in 3NP model. However, in absence
of increased 3-nitrotyrosine prior to the formation of
striatal lesion, it is difficult to conclude that the increased
levels of 3-nitrotyrosine, and in turn, protein oxidation
are causative agents in 3NP-induced striatal degeneration.
The current results suggest that oxidative stress, as de-
tected by elevated levels of 3-nitrotyrosine, coincides but
does not necessarily precedes the selective striatal de-
generation. In addition, the delayed elevation of 3-nitro-
tyrosine in hippocampus is also consistent with the re-
ports demonstrating a delayed hippocampal lesion fol-
lowing 3NP treatment [95].
Although immunohistochemical analysis indicated
few cells stained strongly for poly ADP-ribosylated pro-
teins, quantitative analysis of poly ADP-ribosylated pro-
teins in the regions of interest (striatum, hippocampus
and cerebellum) revealed no significant quantitative in-
crease in poly ADP-ribosylated proteins. Proteolytic cle-
avage of PARP-1 has been widely utilized as specific
biochemical marker for DNA oxidation. Kaufmann and
colleagues first reported that PARP-1 is a substrate for
caspases 3 and 7 and suggested that the proteolytic clea-
vage of PARP-1 can be utilized as a biochemical hall-
mark of apoptosis [96]. PARP-1 cleavage by caspases
produces two major 89 and 24 kDa fragments as a result
of cleavage in N-terminal DNA binding domain [96,97].
Interestingly, Shah and colleagues demonstrated that
PARP-1 is also cleaved during the necrotic death, produ-
cing a major 50 kDa fragment [62]. Subsequent studies
by Gobeil and colleagues demonstrated that PARP-1 is
also a substrate for cathepsins B and G, two major lyso-
somal proteases [60,62]. Since in the necrotic cell death
the content of lysosomes are released into the cytosol

T. Delorme et al. / World Journal of Neuroscience 2 (2012) 234-247 241
[98], it is likely that the lack of a robust increase in poly
ADP-ribosylation in the striatum is due to the cleavage
of PARP-1 by lysosomal proteases and subsequent in-
ability of PARP-1 to polymerize the ADP-ribose poly-
mers on target proteins. Furthermore, PARP-1 is a highly
energy-dependent enzyme, which suggests that in meta-
bolic impairment paradigms, such as the 3NP model in
which both the Krebs cycle and the electron transport
chain are compromised, PARP-1 function may be in-
hibited systemically due to inadequate availability of en-
ergy resources. In our experimental paradigm, one day
post 3NP administration, Western blot analysis of PARP-1
cleavage products demonstrated a 50 kDa fragment in
the striatum, while the similar PARP-1 cleavage frag-
ment did not appear in the hippocampus until the third
day following 3NP. No major PARP-1 cleavage products
were detected in the cerebellum. These results are in-
teresting in the light of previous reports indicating that
the striatum is the first site of neurodegeneration fol-
lowed by hippocampal degeneration after longer treat-
ment with 3NP [19,22,99,100].
Although Calcium has no direct effect on oxidative
phosphorylation (for review see [52]), imbalance in cal-
cium homeostasis have been implicated in 3NP-induced
toxicity [53]. Calcium, a divalent cation, is the fifth most
common element in the mammalian body [101]. Calcium
can be used by cells as a second messenger to control a
plethora of cellular functions including proliferation,
contraction, secretion, neuronal excitation and cell death
among many others. In its insoluble form, calcium exists
as crystalline hydroxyapatite in bones and teeth, whereas
in its soluble form calcium is the prime inorganic second
messenger for regulation of the cellular functions. The
cytosolic level of Ca2+ is kept low (10 - 100 nM) but
normal physiological stimulations result in the level in-
creasing up to 500 - 1000 nM [102]. In neurons, calcium
determines the activity of adenylate cyclase and pho-
sphodiesterase through reversible interaction with calmo-
dulin [103]. Calcium also serves to regulate membrane
permeability, allowing neurotransmitter release as well
as diminishing neuromuscular excitability. Within cells,
only a small fraction of total cellular calcium (appro-
ximately 0.1%) is found free in the cytosol, the majority
of calcium is primarily bound to proteins and nucleic
acids or is sequestered in endoplasmic reticulum and
mitochondria [102]. Mitochondria have a high capacity
for transient calcium storage. In an excellent example of
evolutionary adaptation, unlike endoplasmic reticulum, the
mitochondrial Ca2+ levels under normal physiological
condition are similar to that of the cytosol [104,105]. The
main route of calcium entry into mitochondria is via the
Ca2+ uniporter. The Ca2+ influx into mitochondria via Ca2+
uniporter is driven by the mitochondrial membrane
potential which creates a negatively charged mito-chon-
drial matrix, attracting positively charged Ca2+ ions.
Interestingly, Ca2+ efflux from mitochondria is regulated
via Na+/Ca2+ pumps by which Na+ entry into mito-
chondria is coupled with Ca2+ efflux from mitochondria.
The Ca2+ influx into mitochondria is gradient-dependent;
the higher cytosolic calcium levels, the larger the rate of
calcium entry into the mitochondria. In contrast, the rate
of efflux of calcium via Na+/Ca2+ pumps is constant. In
other words, if the cytosolic Ca2+ levels increase, more
Ca2+ enters mitochondria, while the rate of efflux re-
mains the same, and as a result, cytosolic calcium levels
decrease, avoiding cytotoxic effects associated with large
cytosolic calcium concentration. When cytosolic Ca2+
levels decrease, the influx of Ca2+ via the uniporter also
decreases, while the rate of efflux remains constant, al-
lowing the cytosolic Ca2+ levels to increase and reach the
physiological concentration [106]. Considering the intri-
cate mechanisms in place to regulate Ca2+ concentration
in cells, it is expected that any major disturbances in this
system result in tissue injury and cell death. Numerous
reports have demonstrated that the disruption in cellular
Ca2+ homeostasis, either due to excitotoxicity or oxi-
dative stress, ultimately results in cellular demise [107-
111]. However, the isolated mitochondrial analysis as
reported in the current study suggest that aging results in
striking differences in mitochondrial free radical pro-
duction in response to calcium. This conclusion was
reached by demonstrating that calcium elevation in iso-
lated mitochondria from the older animals caused a sig-
nificant increase ROS generation as compared to that of
the younger animals. Although 3NP alone does not di-
rectly stimulate free radical production, the metabolic
impairment induced by 3NP interferes with the intra-
cellular Ca2+ homeostasis. This finding is of significant
importance since it suggests that in aging, mitochondria
become more susceptible to the generation of reactive
oxygen species, and in conditions that cause a concurrent
imbalance in mitochondrial calcium homeostasis, the ef-
fects may be magnified.
The current study implicates mitochondria dysfunc-
tion as a key cellular target in pathological states that are
associated with metabolic impairment. Moreover, the re-
sults also reinforce the notion that mitochondrial func-
tion in the striatum and cerebellum respond differently to
the aging process, which may explain the variable re-
gional vulnerability in 3NP model. Taken together, the
variable response of the isolated mitochondria from stri-
atum and cerebellum of aged animals to 3NP toxicity and
calcium overload suggest more acute changes in the older
specimens. This finding suggests an intrinsic difference
in the function of mitochondria in respect to age and
anatomical location. Consistent with this hypothesis, pre-
vious reports have indicated a decline in mitochondrial
function with age [112,113]. Others have shown oxidized

T. Delorme et al. / World Journal of Neuroscience 2 (2012) 234-247 242
hydroethidine, a molecule used to measure intracellular
superoxide anion presence, increases with age [114]. More-
over, age-related decline in levels of glutathione, a mole-
cule involved in the function of antioxidant enzymes, has
been documented [115]. The striking difference in mito-
chondria ROS generation in response to calcium addition
suggests that mitochondrial calcium homeostasis in the
older rats may play a significant role in ROS production.
This finding suggests the neurodegenerative processes in
3NP model may involve alternative pathways in younger
and older animals and offers further explanation for
likely mechanisms involved in the age-dependent vari-
ability seen in 3NP model.
Previous studies have shown that 3NP causes more
damage to the striatum than to other regions of the brain
[116,117]. However, 3NP causes similar levels of SDH
inhibition in different regions of the brain [22-23].
Striatal sensitivity cannot be only attributed to SDH inhi-
bition; therefore, other factors must be considered. One
possible mechanism that may account for the preferential
striatal vulnerability is suggested to be a selective break-
down of the striatal blood brain barrier, thereby allowing
a higher concentration of 3NP to reach the striatum [118-
119]. Other reports have implicated Ca2+ in the pre-
ferential striatal neurodegeneration. The typical mito-
chondrial response to Ca2+ administration is sustained mem-
brane depolarization, immediate increase in respiration,
and mitochondrial swelling [120]. Striatal mitochondria
seem to be more sensitive to intracellular Ca2+ levels
than their cortical counterparts [121]. A possible expla-
nation for the sensitivity of striatal mitochondria to intra-
cellular Ca2+ could be explained by a component of mito-
chondria membrane transition pore, cyclophilin D (CyP-D),
which its concentration is almost two fold greater in
striatum than in cortical mitochondria [121]. The link
between CyP-D and Ca2+ was demonstrated in a study of
liver mitochondria from mice, in which the Ca2+ sen-
sitivity of mice with CyP-D was compared to that of
mutant mice without CyP-D. The study also showed that
the mutant mice needed twice the amount of Ca2+, com-
pared to the wild-type, to open mitochondrial membrane
transition pore [122]. Another characteristic of the striatum
is that it is heavily innervated with glutamate inputs from
the cerebral cortex. However, glutamate alone cannot ac-
count for striatal sensitivity, because one would expect
other brain regions with heavy glutamatergic inputs to be
highly susceptible to 3NP (i.e. Purkinje cells in the cere-
bellum). Even though lesions have been observed in the
hippocampus, studies show extrastriatal regions do not
seem as sensitive to 3NP [21,123-126]. The striatum also
receive dopamine inputs from the substantianigra. Rey-
nolds and colleagues have demonstrated that removal of
dopamine input from one striatum prevented 3NP lesions
in the denervated striatum but not in the contralateral
striatum [126]. Furthermore, dopamine and 3NP have
been shown to increase the generation of hydroxyl radi-
cal; however, when administered together, they work
synergistically [127]. In addition, the endogenous dopa-
mine, in the presence of 3NP, decreases striatal mito-
chondria oxygen consumption and increases superoxide
levels in synaptosomes [128].
In summary, from studies to date, there is considerable
evidence suggesting that energy impairment is a common
biochemical mechanism underlying the etiology of a num-
ber of neurodegenerative disorders [129-130]. In the
current study, we utilized 3NP to model energy impair-
ment in rodents. The elevated level of 3-nitrotyrosine in
the striatum coincides with the development of striatal
lesion. However, it is unclear whether oxidative damage
is an effector or a consequence of the neurodegeneration.
The lack of robust increased levels of ADP-ribosylated
proteins is likely due to the cleavage and subsequent
deactivation of PARP-1 and does not exclude the role of
oxidative stress in 3NP toxicity. Although 3NP does not
directly increase the production of free radicals, the addi-
tion of calcium results in a significant increase in the
production of free radical species in the mitochondria of
older animals. The results also suggest that in aging,
mitochondria become more susceptible to the generation
of reactive oxygen species, which also may become grea-
ter in conditions that cause a concurrent imbalance in
mitochondrial calcium homeostasis.
5. ACKNOWLEDGEMENTS
“Authors thank and acknowledge Professor James W. Geddes for his
assistance, expertise and invaluable advices”.
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