-
inal
neada
CytokineNeurodegenerationInammatoryPesticide
ash murecytokine, granulocyte macrophage colony stimulating
factor (GM-CSF), would
aled th
Neurobiology of Disease 43 (2011) 99112
Contents lists available at ScienceDirect
Neurobiology
.e ldisease (PD) (Carvey et al., 2006; Cicchetti et al., 2009;
Liu et al., 2003).Parallel work in rodents has demonstrated that
administration of thepesticides, paraquat and rotenone, provoked a
loss of substantia nigrapars compacta (SNc) dopamine (DA) neurons,
coupledwith histologicaland behavioral symptoms reminiscent of PD
(Cannon et al., 2009; DiMonte, 2003; Litteljohnet al., 2009; Sherer
et al., 2007; Somayajulu-Nituet al., 2009). The neurodegenerative
process induced by paraquat, aswell as other well-established DA
toxins, such as MPTP, involves theactivation of microglial cells
and liberation of oxidative radicals, as well
coupled with mitochondrial dysfunction, and alterations of
apoptoticregulators within the SNc (Fitzmaurice et al., 2003;
Hartmann et al.,2002; Jenner, 1998; Knott et al., 2000; Nagatsu and
Sawada, 2007;Tatton et al., 2003).
Given that the neurodegenerative process in PD occurs over
aprolonged period of time, there may be a critical window
whereintherapeutic intervention may halt or even reverse disease
progres-sion. In particular, the trophic cytokines, glial derived
neurotrophicfactor (GDNF) and brain derived neurotrophic factor
(BDNF), haveas pro-inammatory cytokines, such as interfenecrosis
factor- (TNF-) (McCoy et al., 20Indeed, the deleterious impact of
paraquat w
Corresponding author at: 1125 Colonel By Drive, OttFax: +1 613
520 4052.
E-mail address: [email protected] (S. Hayley).Available
online on ScienceDirect (www.scienced
0969-9961/$ see front matter 2011 Elsevier Inc.
Aldoi:10.1016/j.nbd.2011.02.011at chronic exposure to., heavy
metals and evenogression of Parkinson's
neuroinammatory response (Mangano andHayley, 2009; Purisai et
al.,2007). These studies are in line with post-mortem analyses of
PD brain,which revealed signs of neuroinammation and oxidative
damage,pesticides and other environmental toxins (e.gimmune
infections) are involved in the prParaquatGM-CSFParkinson's
diseaseToxinTrophic factor
Introduction
Epidemiological studies have revemodest but signicant
neurodegenerative effect that was markedly augmented with LPS
priming. Centralinfusion of GM-CSF into the substantia nigra pars
compacta (SNc) prevented the loss of SNc dopamineneurons to a
degree comparable to that of glial derived neurotrophic factor.
Importantly, systemicadministration of GM-CSF also had
neuroprotective consequences, suggesting that the trophic cytokine
cancross the blood brain barrier to promote neuronal survival.
Indeed, GM-CSF acted to inhibit the LPS andparaquat induced
microglial response, while augmenting astrocyte immunoreactivity
within the SNc.Moreover, GM-CSF blunted the paraquat induced
reduction of brain derived neurotrophic factor within
thehippocampus, as well as in culturedmesencephalic neurons.
Although paraquat reducedmesencephalic levelsof the anti-apoptotic
protein, Bcl-2, GM-CSF had no effect in this regard. Hence, GM-CSF
appears to affectinammatory and/or neuroplastic factors within the
SNc that may be linked to neurodegeneration, as well asin other
brain regions (hippocampus), which could be important for co-morbid
non-motor symptoms in PD.These data suggest that peripheral GM-CSF
administration might hold promise as a treatment of PD.
2011 Elsevier Inc. All rights reserved.
pre-treated with the bacterial endotoxin, lipopolysaccharide
(LPS), andthis effect was associated with a heightened
microglial-dependentron- (IFN-) and tumor08; Mount et al., 2007).as
augmented in rodents
been considered2000; Porritt et aGDNF have beenmodels of PD
(E1996; Levivier eSimilarly, BDNF wcoupled with it(Taliaz et al.,
20
awa, Ontario, Canada K1S 5B6.
irect.com).
l rights reserved.haride (LPS). As previously observed, paraquat
provoked aKeywords:inhibit the neurodegenerative effects of the
pesticide, paraquat, administered either alone or followingpriming
with the bacterial endotoxin, lipopolysaccAccepted 27 February
2011Available online 4 March 2011
the hematopoietic trophicGranulocyte macrophage-colony
stimulatdopaminergic cell loss in an environment
E.N. Mangano, S. Peters, D. Litteljohn, R. So, C. BethuInstitute
of Neuroscience, Carleton University, 1125 Colonel By Drive,
Ottawa, Ontario, Can
a b s t r a c ta r t i c l e i n f o
Article history:Received 20 October 2010Revised 26 January
2011
Parkinson's disease (PD) hinammatory agents, whicassociated with
toxin expos
j ourna l homepage: wwwg factor protects against substantia
nigratoxin model of Parkinson's disease
, J. Bobyn, M. Clarke, S. Hayley K1S 5B6
been linked to exposure to a variety of chemical (e.g.,
pesticides) anday act cumulatively over time. Finding novel means
of limiting pathologywould have tremendous clinical importance. To
this end, we assessed whether
of Disease
sev ie r.com/ locate /ynbd ipossible therapeutic agents for PD
(Howells et al.,l., 2005; Yasuhara et al., 2007). In fact, both
BDNF andshown to be neuroprotective in virtually all
animalslamboli, 2005; Huang et al., 2010; Hung and Lee,t al., 1995;
Shults et al., 1995; Zhang et al., 2007).as shown to impart
antidepressant effects that were
s positive actions upon hippocampal neurogenesis10); an
important nding given the high degree of
-
100 E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112depressive co-morbidity evident in PD and the fact the we
previouslyfound paraquat to induce depressive-like behaviors and
enhancehippocampal monoamine activity (Litteljohn et al., 2009).
Unfortu-nately, clinical trials using GDNF have failed to
consistently yieldbenecial clinical outcomes, stemming perhaps from
surgical com-plications and side-effects [GDNF is unable to cross
the blood brainbarrier (BBB)] (Lang et al., 2006), and the limited
diffusion of GDNF inhuman brain (i.e., leading to reduced
bioavailability in nigrostriatalbrain regions) (Nutt et al., 2003;
Salvatore et al., 2006).
Granulocyte-macrophage colony stimulating factor (GM-CSF) is
ahematopoietic cytokine produced mainly by peripheral immune
cells(T-lymphocytes, macrophages, and NK cells) and also by
centralastrocytes (Davignon et al., 1988; Guillemin et al., 1996;
Hercus et al.,2009). Its basic function is to promote the
differentiation andmaturation of peripheral innate immune cells,
including neutrophilsand dendritic cells. The role of GM-CSF in the
brain is not fullyappreciated; however, unlike GDNF, GM-CSF is able
to cross theBBB and accumulate at physiologically relevant levels
within thebrain parenchyma (Franzen et al., 2004; McLay et al.,
1997;Thomson and Lotze, 2003). Additionally, systemic exposure to
GM-CSF can promote functional recovery following traumatic spinal
cordinjury (Bouhy et al., 2006; Huang et al., 2009) and ischemia
(Nakagawaet al., 2006; Schabitz et al., 2008).
It has been suggested that GM-CSF may be neuroprotective
bypromoting microglia to adopt a dendritic cell-like state (Fischer
andReichmann, 2001; Santambrogio et al., 2001; Schermer and
Humpel,2002), facilitating the release of BDNF (Hayashi et al.,
2009) andincreasing the expression of anti-apoptotic proteins such
as Bcl-2(Huang et al., 2007; Kim et al., 2009). To this end,
elucidating the pro-survivalmechanisms of GM-CSF against
DA-targeting toxinsmay offernew therapeutic targets for PD. Thus,
we sought to assess whethercentral or peripheral administration of
GM-CSF would prevent theneurodegenerative consequences induced by
exposure to paraquatalone or with LPS pre-treatment and whether
GM-CSF's pro-survivaleffects are linked to changes in the glial
microenvironment. In light ofthe potentially important role for
BDNF in both PD and its co-morbidpathology (e.g. depression and
cognitive disturbances), it was also ofinterest to assess whether
the toxin treatments affected this trophicfactor and whether GM-CSF
could inuence such changes.
Materials and methods
C57BL/6 male mice were purchased from Charles River
Laboratories(LaPrairie, Quebec, Canada) at 1012 weeks of age and
subsequentlysingle-housed in standard polypropylene cages (272114
cm), andmaintained on a 12-h light/dark cycle. Mice were housed in
atemperature (21 C) and humidity controlled room, and providedwith
an ad libitum diet of Ralston Purina mouse chow and water.Animals
were acclimatized for a period of 1 week prior to thecommencement
of experimental procedures. All experimental testparadigms were
approved by the Carleton University Committee forAnimal Care and
adhered to the guidelines outlined by the CanadianCouncil for the
Use and Care of Animals in Research.
Experiment 1: assessment of the benecial effects of
supra-nigraladministration of GDNF and GM-CSF following
LPS-paraquat inducedpathology
Given that GM-CSF can diminish apoptotic cell death and
stimulatethe release of BDNF, we sought to determine if a direct
infusion of GM-CSF into the SNcwould protect DA neurons from the
neurodegenerativeeffects of LPS and paraquat to a degree comparable
to GDNF. To this end,mice underwent stereotaxic surgery (described
in an ensuing section)and cannulae were implanted just above the
SNc. Following 1 week ofconvalescence, animals were infused with a
single dose of either saline
or LPS (0.1 g/2.0 l) and given a 2 day rest period. Thereafter,
micereceived intraperitoneal (i.p.) injections of saline or
paraquat (SigmaAldrich; 10 mg/kg), 3 times aweek for 3
consecutiveweeks (aparadigmpreviously found to reliably impact the
survival of SNc DA neurons;Mangano and Hayley, 2009). At the
beginning of each week (i.e.,immediately prior to the 1st, 4th and
7th paraquat or saline injection),mice (n=1620/group) received
supra-SNc infusion (using the samecannulae that previously
delivered LPS or saline) of saline, GDNF (R&DSystems; 1 g/2 l)
or GM-CSF (R&D Systems; 10 ng/2 l) into the SNc.Finally, in
order to assess SNcDAneuronal survival, 5 days following thenal
paraquat or saline injection, a subset of mice (n=810/group)were
perfused with 4% paraformaldehyde (PFA), brains post-xed for24 h
and then cryoprotected for 3 days in a solution comprising
20%sucrose, 0.1 M PBS and 0.02% sodium azide (pH 7.4).
Immunohistochemical procedures: SNc DA neuronal survival
andmicroglial reactivity
Frozen brains were cryostat-sectioned and free-oating
coronalsections (20 m) of the SNc were mounted onto gelatin-coated
slides.Anti-tyrosine hydroxylase (TH) antibody along with 1% cresyl
violet(Sigma-Aldrich) as a counter-stain was used to detect SNc
DAneurons. The primary antibody (TH) was diluted in a
solutioncontaining 1% BSA, 0.5% Triton X-100, 0.05% Tween 20, and
0.05%sodium azide in 0.01 M PBS, pH 7.3; and sections were
incubatedovernight at room temperature (1:3000, ImmunoStar, Hudson,
WI,USA). The primary antibodies were visualized using a biotin
rabbitanti-mouse (1:500, Cedarlane) secondary and horseradish
peroxi-dase-conjugated streptavidin tertiary (1:500, Cedarlane).
Sectionswere then further incubated with diaminobenzidine (DAB;
Sigma-Aldrich) for 10 min, counterstained with cresyl violet
(Sigma-Aldrich)and dehydrated with serial alcohol washes before
cover-slipping withclearene (Surgipath).
Survival of SNc DA-producing cells was determined by way
ofserial section analysis of the number of TH+ cells within the
SNc, atbregma levels ranging from 3.08 to 3.40. Using a double
blindprocedure, 810 animals were analyzed per group and the number
ofTH+ cells was counted across multiple bregma levels.
Midbrainsections from each bregma level were compared across
treatmentgroups to assess the extent of DA neuronal loss induced by
paraquatalone or primed with LPS, and to determine if GM-CSF or
GDNFprevented such effects. In order to ensure that a genuine loss
of DAneurons occurred rather than simply a phenotypic suppression,
thetotal number of surviving SNc neurons (TH+ and TH) was
countedusing cresyl violet staining.
Striatal sections were stained using TH+ antibody (1:1000)
andphotomicrographs were obtained for each animal using the
sameexposure time. Image J software was used to determine
thebackground threshold for each striatal section and the total
numberof white (background) and black (TH+) pixels. All images
wereconverted into an 8-bit format, where the grayscale varied from
0 to255. The area of interest was selected and the upper and
lowerthreshold values were used across all images to separate the
featuresof interest from the background. The upper and lower
thresholdswere determined using an automatic thresholding option, a
modiedversion of the IsoData method. This algorithm divides the
image intothe object of interest and background by taking an
initial threshold(histogram-derived), followed by the averages of
the pixels at orbelow the threshold and pixels above are computed.
The averages ofthose two values are computed, the threshold is
incremented and theprocess is repeated until the threshold is
larger than the compositeaverage. The data were then presented as a
histogram with thenumber of black (object of interest) and white
(background) pixelspresent.
Morphological changes of microglia are generally taken to
indicatesome degree of change in their activation states (Mangano
and
Hayley, 2009). Indeed, differing degrees of compacted soma,
-
101E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112thickening of processes, and enhanced proliferation are
normallytaken to reect a state of activation, wherein microglia are
capable ofsubstantial release of inammatory and oxidative factors
(Nimmer-jahn et al., 2005). Hence, the morphological state of
microglia wasassessed histologically using CD11b (complement
receptor markerpresent on microglia) antiserum (1:1000; AbD
Serotec, Raleigh, NC,USA; overnight incubation at 4 C). The primary
antibody was dilutedin 1% BSA, 0.03% Triton X-100, 0.05% Tween 20
and 0.05% sodiumazide in 0.01 M PBS (pH 7.3). Thereafter, sections
were washed with0.01 M PBS and incubated with biotin rabbit
anti-rat secondary(1:500, Cedarlane) and horseradish
peroxidase-conjugated strepta-vidin tertiary (1:500, Cedarlane)
antibodies. Sections were once againvisualized by incubation with
DAB (Sigma-Aldrich) for 10 min anddehydrated with serial alcohol
washes before cover-slipping withclearene (Surgipath).
A semi-quantitative rating scale previously published by our
lab(Mangano and Hayley, 2009) was used to verify the degree
ofmicroglial reactivity. The morphological change in microglia
wasanalyzed using a 03 rating scale. Specially, a rating of 0 was
given ifthe SNc microglia appeared quiescent (highly ramied with
manylong thin primary and secondary branches). A rating of 1
reected anintermediate reactivity state in which less than 10 cells
within the SNccould be considered moderately activated (thickened,
short processeswith a compact soma). A rating of 2 was given if the
majority of themicroglial cells appeared to be intermediately
activated withoccasional highly activated cells (amoeboid and
macrophage-likeappearance, circular in shape lacking processes) and
a rating of 3 wasgiven only when the majority of cells displayed
the most highlyactivated amoeboid shape.
Western blot analyses of hippocampal BDNF levels
It has become clear that, besides the frank nigrostriatal
neuronaldamage, PD is associated with pathology involving brain
regionsimportant for neuroplasticity (Bruck et al., 2004; Jokinen
et al., 2009).Hence, it was of interest to assess whether paraquat
would affecthippocampal BDNF levels. Moreover, it is of clinical
importance toevaluate whether GM-CSF administration can reverse
such effects. Tothis end, a subset of the mice from Exp. 1
(n=810/group) wereeuthanized by rapid decapitation (again 5 days
after the nalinjection) followed by micropunch dissection of the
hippocampus.Tissue samples were stored at 80 C until Western blot
analyses(Hayley et al., 1999; Hayley et al., 2004).
All chemicals were obtained from Sigma-Aldrich (St. Louis,
MO)and all antibodies were obtained from Santa Cruz
Biotechnology(Santa Cruz, CA) unless otherwise indicated. Samples
were dilutedwith lysis and protease inhibitor buffer up to the
desired proteinconcentration, yielding whole cell lysate (50 g) in
20 l and 20 lloading buffer (5% glycerol, 5% -mercaptoethanol, 3%
SDS and 0.05%bromophenol blue). To denature the proteins, the 40 l
sample washeated in boiling water for 5 min. Sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the
separating buffer[370 mM Tris-base (pH 8.8), 3.5 mM SDS], and the
stacking buffer[124 mM Tris-base (pH 6.8), 3.5 mM SDS], were placed
in runningbuffer (25 mM Tris-base, 190 mM glycine, 3.5 mM SDS); and
samples,alongwith the Precision Plus Protein Standards Dual Color
(Bio-Rad,Hercules, CA, Cat #161-0374), were loaded into the
Acrylamide gel(12.5%) for molecular weight determination at 120
V.
After electrophoresis, proteins were transferred overnight, at 4
Cand 180 mA, in transfer buffer (25 mM Tris-base, 192 mM
Glycine,20% methanol) onto PVDF (Bio-Rad, Cat #162-177).
Thereafter,membranes were blocked for 1 h with gentle shaking in a
solutionof non-fat dry milk (5% w/v) dissolved in TBS-T buffer (10
mM Tris-base (pH 8.0), 150 mM sodium chloride, 0.5% Tween-20).
Themembranes were then incubated with a rabbit anti-BDNF
primary
antibody (1:500) diluted in blocking solution at room
temperature for1 h. Any unbound antibody was removed using three
washes of 15 mlTBS-T at room temperature. Membranes were incubated
on a shakerfor 1 h at room temperature with HRP (horseradish
peroxidase)conjugated anti-rabbit (1:2000) secondary antibody and
washedagain with TBS-T. Finally, BDNF was visualized with a
chemilumines-cent substrate (Perkin Elmer, Waltham, MA, cat
#NEL102001EA; for1 min) and briey exposed on a Kodak lm.
Experiment 2: assessment of the benecial effects of
systemicadministration of GM-CSF following LPS-paraquat induced
pathology
Mice underwent stereotaxic surgery identical to Experiment 1
andreceived a single supra-nigral infusion of either saline or LPS
(same doseused in Experiment 1), followed 2 days later with i.p.
saline or paraquattreatment (10 mg/kg; 3 times per week for 3
weeks). Paralleling theintra-SNc infusions delivered in the
previous experiment, mice receivedi.p. injections of GM-CSF
(R&D Systems; 2 g) once per week for theduration of the 3 week
paraquat regimen. All mice were sacriced5 days after the nal
paraquat or saline injection and brains wereperfused for
immunohistochemical analyses.
Immunohistochemical analyses of SNc neuronal loss
Stereological analysis of SNc sections (60 m) was used
todetermine DA degeneration for Experiment 2. Briey, the SNc
wasoutlined under 2.5 magnication and TH+ neurons counted using a60
oil immersion objective. The SNc was sampled in a systematicrandom
fashion according to the optical fractionator method outlinedby
MicroBrightField Inc. Cells were quantied in 3-dimensionalcounting
frames using a counting grid size of 9090 m and acounting frame
size of 6060 m with a 15 m dissector height and3 m upper and lower
guard zones. Only the portion of the SNcipsilateral to the infusion
site was quantied. All analyses wereconducted with the counter
blind to the treatment conditions. StriatalDA terminal staining was
conducted in a manner identical to that ofExperiment 1.
DA neurons were sampled throughout the rostralcaudal axis ofthe
SNc in every 2nd section, labeled with TH and counterstained with1%
cresyl violet. Free oating sections were blocked in 0.3%
hydrogenperoxide solution for 30 min, washed in 10 mM PBS and
thenincubated overnight at 4 C in mouse anti-TH primary
antibody(1:500 in 10 mM PBSwith 2% BSA, 1% sodium azide and 0.5%
Triton-X,Immunostar Inc.). This was followed by incubation in
secondary biotinSP-conjugated Afnipure goat anti-mouse IgG (1:200
in 10 mM PBSwith 2% BSA, 1% sodium azide and 0.5% Triton-X, Jackson
Immunor-esearch Laboratories) for 2 h at room temperature and,
lastly,incubation in tertiary peroxidase-conjugated streptavidin
(1:200 in10 mM PBS with 2% BSA and 0.3% Triton-X, Jackson
ImmunoresearchLaboratories).
Following antibody incubations, sections were rinsed 3 times
for5 min in 10 mM PBS. To visualize the antibody complex, sections
wereincubated for 5 min in 1 ml (per well) of DAB (0.2 mg/ml) in 50
mMTris HCl solution. Sections were incubated for an additional 15
minfollowing addition of 50 l of 6% hydrogen peroxide (in
distilledwater) per well. Sections were mounted on glass slides,
air-driedovernight, counterstained with 1% cresyl violet, and
dehydrated withserial alcohol washes. Slides were then
cover-slipped and stored atroom temperature. TH+ cells were
quantied as outlined above andrepresentative photomicrographs
depicting TH+ neurons in the SNcwere taken using an Olympus BX52
microscope under magnicationof the 10 objective for qualitative
analysis. The same free-oatingsections used to determine TH loss
were also used to conrm thatneurons were actually lost and TH
expression not simply reduced.Hence, the total number of SNc
neurons was determined using the
cresyl violet counterstain.
-
102 E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112Assessment of glial changes
Free-oating sections were incubated overnight at 4 C with
eithermouse anti-GFAP (1:1000; Chemicon) to visualize astrocytes or
ratanti-mouse CD11b (1:1000, Serotec) to visualize microglia.
Allantibodies were diluted in a solution containing 10 mM PBS with
1%sodium azide and 0.3% Triton-X. Sections were then washed with10
mM PBS 3 times prior to incubating with their respectivesecondary
biotinylated antibodies, namely goat anti-mouse and goatanti-rat
(1:200, Jackson Immunoresearch Laboratories), for 2 h atroom
temperature. Following 3 washes with 10 mM of PBS, thesections were
then incubated in tertiary peroxidase-conjugatedstreptavidin
(1:200, Jackson Immunoresearch Laboratories) for 2 hat room
temperature. The antibody complexes were visualized byincubating
with DAB for 10 min. Sections were mounted on glassslides,
air-dried overnight, cover-slipped and stored at room temper-ature.
Both astrocytic and microglial reactivity were
qualitativelyassessed using a double blind procedure.
Representative photomicro-graphs were taken on an Olympus BX52
microscope using a 40objective.
General stereotaxic surgical procedures for Experiments 1 and
2
All mice underwent stereotaxic surgery wherein indwelling
cannu-lae (26-gauge stainless steel) were implanted just above the
SNc(bregma: anterior-posterior 3.16 mm, lateral1.2 mm, ventral4.0
mm). Central administration of LPS or saline was achieved using
aHarvard Apparatus Pico Plus syringe pump. A total of 2 l of uid
wasdelivered to the SNc over a period of 5 min from polyethylene
tubingconnected to a Hamilton microliter syringe. Infusions and
injectionswere administered between 0830 h and 1400 h to minimize
thepotentially confounding effects of circadian variation. It is
importantto note that animals were unrestrained during the infusion
procedure,allowing the mice to move freely about their home cage in
order tominimize stress. Also,mice had a 1 week convalescence
period betweencannulae implantation and administration of any
treatment. This is animportant point to address given that
inammatory processes aremarkedly affected by wounds resulting from
surgical procedures andthe stress associated with restraint.
Experiment 3: in vitro assessment of the effects of paraquat and
GM-CSF
Primary mesencephalic neuronglia cultures were prepared
frombrains of embryonic day 18 (E18) C57Bl/6 mice. Whole brains
wereextracted aseptically and the mesencephalon was isolated.
Afterremoving the blood vessels andmeninges, pooledmidbrain
tissuewasdissociated using a papain dissociation kit (Worthington
BiochemicalCorp.). Briey, 500 l of papain solution was added to
pooled tissueand mechanically homogenized and placed in an
incubator for 1 h at37 C. Cultures were maintained at 37 C in a
humidied atmosphereof 5% CO2, 95% air. Following dissociation, the
cells were triturated 5times and centrifuged at 300g for 5 min. The
cells were then re-suspended in a papain/digestion inhibitor
solution and counted usinga hemacytometer (BrightLine; 394485).
Cells were diluted insupplemented Neurobasal A media containing 2%
B27, 1% N2,416 M L-glutamine, 41.6 U/ml penicillin and 41.6 g/ml
streptomy-cin. Thereafter, cells were seeded in either 6-well
plates (25.0104/ml) or 4-well slides (5000 cells/ml) pre-coated for
1 h with poly-D-lysine (20 g/ml; Sigma) and bronectin (20 g/l;
Sigma).
Two days following seeding, the media was replaced with
freshsupplemented Neurobasal A media alone or containing GM-CSF(250
ng/ml), paraquat (30 M) or paraquat (30 M)+GM-CSF(250 ng/ml), and
incubated for 6 h. Importantly, the optimal paraquatconcentration
and exposure time were empirically determined byperforming dose
(0.3300 M) and time (124 h) course analyses. A
+dose and exposure time that reliably induced 3040% loss of
THneuronswas chosen as this degree of cell death paralleled that
observedunder in vivo conditions in the aforementioned experiments.
Immedi-ately following the drug exposure, all treated cells were
replaced withfresh supplementedmedia and those that had been
incubatedwithGM-CSF received fresh GM-CSF for a further 24 h.
Thereafter, mesencephaliccultures were scraped (for western blot
analysis) or xed with 4% PFAfor 24 h for quantication of cell
death. Hence, mesencephalic neuronswere exposed to paraquat for 6 h
and GM-CSF for 30 h in total.
Immunocytochemistry
Cells were xed with 4% PFA for 1 h at room temperature in
sterilePBS. Non-specic staining was blocked using 10% normal
serumdiluent (containing 0.5% Triton X-100, 0.03% sodium azide in
PBS) for1 h. The total neurons were visualized using NeuN (1:800;
Chemicon)and DA neurons using TH (1:800; Chemicon) antibodies. The
cultureswere incubatedwith these primary antibodies (diluted in the
blockingsolution) overnight at 4 C. The following day the cultures
werewashed 3 times with PBS for 10 min and incubated for 1-h
withsecondary antibodies (1:200; Alexa 488 and Alexa 555). All
imageswere analyzed using an up-right Nikon microscope.
Western blot
Western blot procedures were identical to those already
describedfor Experiment 1, with two exceptions: 1) mesencephalic
culturesinstead of hippocampal tissue were processed, and 2) in
addition towhole cell lysate fractions for BDNF, sub-cellar
fractions wereobtained for Bax and Bcl-2 proteins. Given the
evidence that suggestsGM-CSF can affect apoptotic mitochondrial
proteins, it was of interestto determine any changes in pro- and
anti-apoptotic factors withinthe cytosolic and mitochondrial
fractions. Specically, the pro-apoptotic factor, Bax, is
translocated from the cytosol to themitochondrial membrane
following activation, whereas the anti-apoptotic Bcl-2 is
permanently associated with the mitochondrialmembrane. To this end,
subcellular fractionationwas performed usinga kit purchased from
Calbiochem (QIA88), which separated culturesinto cytosol and
mitochondrial sub-fractions in order to determineBcl-2 and Bax
protein concentrations. Briey, neuronal cell pelletswere suspended
in 200 l of 1 cytosol extraction buffer containingprotease
inhibitor cocktail and DTT. The cells were homogenizedusing a
tissue grinder, centrifuged (700g for 10 min and 10,000g for30 min)
to obtain the cytosolic fraction. The pelletswere re-suspendedin
100 l of mitochondrial extraction buffer containing
proteaseinhibitors and sonicated in order to obtain the
mitochondrial fraction.Thereafter, sampleswere dilutedwith lysis
andprotease inhibitor buffer(as described earlier) up to the
desired protein concentration: cytosolicfractions (50 g) and
mitochondrial fraction (50 g) in 20 l and 20 lloading buffer (5%
glycerol, 5% -mercaptoethanol, 3% SDS and 0.05%bromophenol blue).
Finally, all sampleswere loadedonto an acrylamidegel, transferred
to PDVF and visualized using chemiluminescenceprocedures that were
once again identical to those of the earlier BDNFexperiment. Table
1 provides a list of the antibody dilutions and gelspecics. All
antibodies were obtained from Santa Cruz Biotechnology(Santa Cruz,
CA) except for anti-rabbit-HRP, whichwas purchased fromSigma
Aldrich.
Statistical data analysis
All data were analyzed by ANOVA followed by Fisher's
plannedcomparisons where appropriate. Data were evaluated using a
Stat-View (version 6.0) statistical software package available from
the SASInstitute, Inc. For the in vitro study, protein expression
was quantiedby densitometry (AlphaEase FC v.3.1.2, Alpha Innotech,
Co., SanLeandro, CA) and, unless otherwise indicated, all results
are expressed
as meansS.E.M of at least three independent experiments.
-
Results
Experiment 1: Supra-nigral injection of GM-CSF or GDNF
attenuatedLPS-paraquat induced degeneration of SNc dopamine neurons
andstriatal terminals
The ANOVAs revealed that the number of surviving TH+ neuronsat
several levels of the SNc varied as a function of the LPS,
paraquat,and growth factor (GM-CSF or GDNF) treatments {F7,
52=6.727,F7, 33=3.072, F7, 46=3.278 and F7, 35=4.548 psb0.001;
bregmalevels 3.08, 3.16, 3.28 and 3.40, respectively}. As depicted
in
Fig. 1, paraquat treatment alone caused approximately 30% loss
ofTH+ neurons across SNc levels. Consistent with our previous
report(Mangano and Hayley, 2009), priming the SNc with LPS 2-days
priorto commencing the pesticide regimen resulted in a greater loss
of TH-expressing neurons (~4050% yet, this was not signicantly
differentfrom the 30% loss induced by paraquat alone). Moreover,
centraladministration of either GM-CSF or GDNF completely prevented
theneuronal loss induced by paraquat alone or following LPS
priming.Indeed, the follow up statistical comparisons conrmed that
paraquatand LPS+paraquat administration signicantly reduced the
survivalof TH+ neurons across all four bregma levels and infusion
of eitherGM-CSF or GDNF prevented these reductions (pb0.01).
To determine whether the loss of TH+ neurons reected a
genuineneurodegenerative effect or simply a phenotypic suppression,
all SNcsections were counterstained with cresyl violet and the
total numberof surviving neurons was quantied across bregma levels
for each ofthe treatment groups. Not surprisingly, the ANOVA
revealed asignicant difference in the total number of cresyl violet
stainedneurons as a function of the treatments {F7, 52=5.009, F7,
33=3.102 ,F7, 46=3.036 and F7, 35=4.380 psb0.001; bregma
levels3.08, 3.16,
Table 1List of antibody dilutions and gel specics used for
Western blotting.
Primary antibody Catalogue # % SDS-PAGE gel Dilutions
anti-Bcl-2 HRP sc-492 HRP 15 1:250anti- Bax HRP sc-493 HRP 15
1:100anti-BDNF sc-546 12.5 1:500anti-Actin HRP sc-47778 HRP
1:5000anti-rabbit-HRP A6154 1:2000
103E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112Fig. 1. Central administration of GM-CSF and GDNF protected
SNc neurons. Dopaminergic neSNc. The top representative
photomicrographs illustrate the degree of dopaminergic
neuronexposure alone (10 mg/kg; 3 per week for 3 weeks) or
following LPS priming (0.01 g/2 ltreatment (* pb0.05). However, a
single supra-nigral injection of GDNF (1 g/2 l) or GM-CSFinduced by
paraquat alone and with the addition of LPS. Data are expressed as
meanSEMuron loss was assessed using TH (1:1000) antibody staining
across several levels of theloss induced by LPS and paraquat, as
well as the effects of GDNF and GM-CSF. Paraquat) of the SNc 2 days
earlier induced a signicant loss of TH+ neurons, relative to
saline(10 ng/2 l) once per week during the paraquat regimen
prevented the SNc neuron loss
; n=810.
-
3.28 and 3.40, respectively}. Specically, in a manner identical
tothat observed for TH+ neurons, paraquat reduced the total number
ofSNc neurons and once again, this effect was somewhat
augmented(albeit not signicantly) by LPS priming and was absent in
mice thatreceived the GM-CSF or GDNF treatments (pb0.01; see
Supplemen-tary data Figure). However, this effect appeared to be
restricted to theTH+ neurons, as the number of TH- cresyl violet
stained neurons werenot signicantly affected by any of the
treatments.
It is important to underscore that cell countswere conducted
usingaccepted manual procedures across multiple bregma levels of
the SNc(as in Hayley et al., 2004). However in the second
experiment of thismanuscript (to be presented shortly) we used more
recentlyestablished stereological assessments to quantify cell
loss. Thisdifference stems from the fact that we only had access to
a properstereology set-up at the time when the second study was
conducted.Nonetheless, we believe the current manual cell counts to
be fullyvalid and accurate. In fact, we found the two methods to
yield verysimilar results with regards to the basic
neurodegenerative effects ofparaquat. Yet, we fully acknowledge the
limitations of the manualsystem used in the experiment.
As was the case for the SNc, the LPS, paraquat and growth
factortreatments altered TH+ immunoreactivity within the
striatum{F7, 33=32.195 pb0.001}. In this regard, mice exposed to
paraquatalone or primed with LPS displayed reduced striatal TH+
staining(pb0.05); and, once again, infusion of either GM-CSF of
GDNFprevented this effect, see Fig. 2.
Supra-nigral injection of GM-CSF inuenced LPS and paraquat
induced
Specically, semi-quantitative ratings conrmed that paraquat
inducedamodest elevation of CD11b+ staining in comparisonwithmice
treatedonly with saline, and prior LPS infusion further enhanced
this effect{F7, 32=4.937 pb0.001}. The majority of "activated"
microglia receiveda score of 1, as they displayed an intermediate
level of reactivitycharacterized by shortened, thick dendritic
processes. A few cells,particularly in response to LPS and paraquat
treatment, appeared to beclumped together with more compact soma
characteristic of a reactivestate, and thus received a score of 2
(Fig. 3). Surprisingly, a single supra-nigral injection of GDNF at
the beginning of each week of the paraquatregimen did not
appreciably inuence the impact of paraquat or LPSupon CD11b+
immunoreactivity. However, the weekly GM-CSF infu-sionsdid cause a
change in the state ofmicroglial activation,wherein thetrophic
cytokine greatly attenuated the morphological changes ofmicroglia
that were provoked by paraquat and LPS, causing themicroglia to be
more ramied and received lower ratings (see Fig. 3).
In contrast to the augmentedmicroglial response (at least in
terms ofmorphology), astrocytic expression within the SNc (as
indicated byGFAP+ immunoreactivity) was modestly reduced in mice
exposed toLPS+paraquat, see Fig. 4. Although a somewhat surprising
nding, wedid previously nd a similar attenuated GFAP response in
LPS primedmice later exposed to paraquat (Mangano and Hayley,
2009). Impor-tantly, a single infusion of either of the growth
factors, GDNF or GM-CSF,once per week throughout the paraquat
regimen appeared to beassociatedwith a reverse of the LPS+paraquat
inducedGFAP reduction.
Supra-nigral infusion of GM-CSF modied the paraquat
inducedreduction of mature hippocampal BDNF
paricrne oGM
104 E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112Fig. 2. Central administration of GM-CSF and GDNF attenuated
the impact of LPS andanalyzing pixel intensity of sections stained
with TH (1:1000). The representative photomber staining. Indeed,
mice exposed to paraquat (10 mg/kg; 3 per week for 3 weeks) aloloss
of striatal terminals (* pb0.05). A single supra-nigral injection
of GDNF (1 g/2 l) orglial changes in the SNc
LPS and paraquat induced changes in microglial morphology
thatparalleled the loss of TH+ neurons within the SNc. As
previouslyobserved (Mangano and Hayley, 2009), paraquat
administered alone orin LPS primed mice enhanced microglial
reactivity (as indicated bymorphological changes and increased
CD11b density) within the SNc.of TH+ber immunoreactivity. Data are
expressed as meanSEM; n=810.aquat upon striatal dopaminergic
terminals. Striatal DA ber loss was determined byographs illustrate
that paraquat with or without LPS priming induced a reduction of
TH+
r following priming 2-days earlier with LPS (0.01 g/2 l)
displayed approximately 35%-CSF (10 ng/2 l) once per week during
the paraquat regimen totally prevented this lossAs already alluded
to, disturbances of neurotrophin-mediatedplasticitymightbe
importantnot only for the cognitive decits observedin PD, but also
the other frequent co-morbid psychiatric symptoms, suchas anxiety
and depression (Litteljohn et al., 2009). To this end, it
isimportant that the paraquat and GM-CSF treatments provoked
altera-tions of hippocampal BDNF {F2, 10=8.597 pb0.01}, see Fig.
5.
-
105E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112Specically, the mature 1314 kDa form of BDNF was reduced
withinthe hippocampus following paraquat administration, relative
to vehicletreatment (pb0.05); however, GM-CSF infusion attenuated
this effect,such that the growth factor did not differ from
controls. Owing to tissuelimitations, the Western blot analyses
were restricted to only thevehicle, paraquat and GM-CSF+paraquat
treatment groups.
Experiment 2: systemic administration of GM-CSF had
neuroprotectiveconsequences
As in Experiments 1, the ANOVA revealed a signicant
differencebetween the treatment groups in terms of the number of
survivingTH+ neurons within the SNc {F3, 12=79.701 pb0.0001}. The
follow-up comparisons indicated that chronic paraquat exposure
signi-cantly reduced the number of TH+ SNc neurons (~30%
reduction)relative to vehicle-treated mice (pb0.01), and this
effect was furthermodestly but non-signicantly increased when
animals were pre-treated with LPS before exposure to the pesticide
(N40% reduction).Paralleling the results of supra-nigral infusion
in Experiment 1, i.p.GM-CSF injection appeared to attenuate the
impact of LPS andparaquat, such that the number of surviving SNc
TH+ neurons didnot signicantly differ from those that received
vehicle alone, seeFig. 6. Once again, the impact of paraquat
appeared to be restrictedto DA neurons; indeed, although the total
number of cresyl violetstained neurons was reduced, no such
reduction was observed forspecic TH- cresyl violet neurons (data
not shown).
Fig. 3. Central GDNF and GM-CSF modulated the impact of LPS and
paraquat upon SNcimmunostaining within the SNc of mice that
received supra-nigral saline or LPS infusion togGM-CSF (10 ng/2 l).
The above photomicrographs taken using a 40 objective
demonstratethe SNc. Interestingly, although GM-CSF infusion blunted
the impact of LPS and paraquat (marker. This was conrmed using a
semi-quantitative rating scale (graph below); paraquat aappear to
alter this effect, GM-CSF treated mice were comparable from saline
treated contrAgain paralleling the earlier ndings, LPS, paraquat
and GM-CSFtreatments affected TH+ immunoreactivity within the
striatum{F3, 14=31.592 pb0.0001}. As depicted in Fig. 6, mice
exposed toparaquat alone or primed with LPS displayed reduced
striatal TH+
immunostaining (pb0.05).As was the case for central infusion of
GM-CSF, systemic adminis-
tration of the trophic cytokine appeared to blunt the
CD11b+changesinduced by LPS and paraquat (Fig. 7). Indeed,
semi-quantitative ratingrevealed that CD11b+cell morphological
signs of activation varied asa function of the treatments {F3,
11=14.65 pb0.01}. As can be seen inFig. 7 and conrmed by the
planned comparisons, paraquat exposurealone or in the context of
LPS pre-treatment increased ratings ofmicroglia reactivity
(relative to saline treatment; pb0.01), whereas GM-CSF treated
animals were indistinguishable from saline controls. Onceagain, as
was observedwith central GM-CSF infusion, systemic exposureto the
cytokine augmented GFAP immunoreactivity, whereas LPS andparaquat
appeared tomoderately suppress the astrocyticmarker (Fig. 8).
Experiment 3: GM-CSF neuroprotection was associated with
normalizedBDNF but not Bcl-2 or Bax levels
To evaluate the mechanism utilized by GM-CSF to protect SNc
DAneurons from paraquat toxicity, primary mesencephalic
neurongliamixed cultures were assessed following paraquat exposure.
A doseresponse curve and time dependent evaluation of paraquat
toxicityrevealed the dose that provoked a degree of cell death
similar to that
microglia. The representative photomicrographs depict microglia
(CD11b+; 1:1000)ether with systemic paraquat treatment, in presence
or absence of GDNF (1 g/2 l) orthat paraquat and LPS+parquat
treatments elevated CD11b+ immunostaining withinin terms of
CD11b+staining intensity), GDNF had no such inuence on the
microglialnd LPS+paraquat hadmarked effects onmicroglia morphology.
Although GDNF did notols.
-
106 E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112observed in vivo, see Fig. 9. Specically, the dose (0.350 M)
and time(312 h) course analyses revealed that exposure to 30 M
ofparaquat for 6 h provided optimal conditions, reliably inducing
3040% loss of TH+ neurons. Indeed, further experiments
conductedusing these parameters revealed signicant differences in
the numberof surviving TH+ neurons in mesencephalic cultures as a
function ofpesticide exposure {F3, 16=13.048 pb0.0001}. As shown in
Fig. 9,paraquat provoked a signicant loss of cultured mesencephalic
TH+
neurons (pb0.01); however, again paralleling the in vivo ndings,
thiseffect was completely prevented by co-treatment with GM-CSF(250
ng/ml).
Given that GM-CSF has been reported to impart
protectiveconsequences through its effects upon trophic and
apoptotic path-ways (Huang et al., 2007), the trophic factor, BDNF,
and the pro- andanti-apoptotic factors, Bax and Bcl-2,
respectively, were assessedusing Western blot. To this end, the
ANOVAs revealed signicantdifferences between the treatment groups
for BDNF and Bcl-2 (but notBax) protein levels within the
mesencephalic cultures {F2, 4=6.412,F2, 9=3.881, respectively,
pb0.05}. As depicted in Fig. 9 andconrmed by the follow up
comparisons, paraquat signicantlyreduced levels of BDNF and Bcl-2
within the cultured neurons
Fig. 4. Central GDNF and GM-CSF modulated the impact of LPS and
paraquat upon SNcimmunostaining within the SNc of mice that
received supra-nigral saline or LPS infusion togGM-CSF (10 ng/2 l).
The above photomicrographs (A) show that GFAP+
immunoreactivitparaquat, relative to saline treatment. This can be
better observed with the higher magnicaCSF infusions appeared to
reverse the effects of LPS and paraquat and in fact, GM-CSF
treat(pb0.05). Co-treatment with GM-CSF restored BDNF protein
expres-sion within the mesencephalic cultures; however, the growth
factordid not appear to affect Bcl-2 protein expression.
Discussion
Inammatory and oxidative processes, together with reducedtrophic
support, are widely considered to be essential players in
thepathological processes of PD (Bossers et al., 2009; Chauhan et
al.,2001; Guerini et al., 2009; Howells et al., 2000; Masaki et
al., 2003).Indeed, accumulating evidence suggests that an
augmentedmicroglialresponse, possibly driven by pro-inammatory
cytokines such as IFN- and TNF- contributes to the progression of
PD (Hirsch and Hunot,2009; Whitton, 2007). In this regard, the
release of oxidative species,along with enhanced inammatory enzyme
activity (e.g., COX-2) andthe induction of various MAP kinase
pathways likely representproximal mediators of DA neuronal
pathology (Gao et al., 2003;Hunot et al., 2004). In contrast to the
more pro-inammatory role ofmicroglia, astrocytes have more
frequently been associated with aprotective, buffering capacity and
the release of trophic factors (Chenet al., 2006). At the same
time, several reports detected reductions of
astrocytes. The representative photomicrographs depict astrocyte
(GFAP+; 1:1000)ether with systemic paraquat treatment, in presence
or absence of GDNF (1 g/2 l) ory was moderately diminished by LPS
priming followed 2 days later with exposure totion (40) images in
the below photomicrographs (B). Once again, the GDNF and GM-ment
appeared to increase GFAP staining slightly above that of saline
treated controls.
-
107E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112anti-oxidants, particularly glutathione, as well as the
trophic factor,BDNF, occur in the PD brain (Fitzmaurice et al.,
2003; Murer et al.,2001). Whatever the case, increasing attention
has been devoted toutilizing novel means of interfering with the
pro-death process inanimal models of PD. To this end, we report
that both central andperipheral administration of the trophic
cytokine, GM-CSF, protectedSNc DA neurons from LPS and paraquat
toxicity, andmodulated BDNF,microglia and astrocyte responses to
these toxins.
Neurotrophic factors are essential for the maintenance and
survivalof neuronal populations. Consequently, both BDNF andGDNF
have beenextensively studied as potential therapeutic agents for PD
patients(Peterson and Nutt, 2008). BDNF is normally present in
relatively high
Fig. 5. Central administration of GM-CSF inuenced the effects of
paraquat onhippocampal BDNF expression. The Western blot revealed
that paraquat promoted amarked reduction of hippocampal BDNF
protein. Four representative bands are shownfrom the three
treatment groups. Clearly, paraquat caused a reduction of BDNF in
eachof the animals exposed, whereas the addition of GM-CSF (10 ng/2
l) infusion generallyattenuated this effect (at least in 3 of the 4
animals). The bar graph on the bottomconrmed that these effects
were statistically signicant (*pb0.05, relative to salinetreated).
All integrated density scores were normalized against b-actin to
control forany variations in loading. Data are expressed as
meanSEM; n=4.concentrations within SNc DA neurons and is
responsible for supplyingnutritive support, as well as promoting
plastic and pro-survivalprocesses that have the potential to
inhibit or possibly remediateneuronal pathology (Baquet et al.,
2005;Murer et al., 2001). The trophicfactor has well known
pro-survival and differentiation effects onmesencephalic DA
neurons, and was reported to protect against MPTPand 6-OHDA insults
(Hung and Lee, 1996; Shults et al., 1995). Inagreement with the
nding that BDNF mRNA levels are reduced withinthe SNc of
post-mortem PD brain (Mogi et al., 1999; Salehi andMashayekhi,
2009)we presently report that paraquat diminished BDNFlevels in
mesencephalic cultures. Moreover, in vitro application of GM-CSF
blunted this effect andprevented the loss of
culturedmesencephalicTH+ neurons induced by paraquat. These ndings
are consistent withreports showing that GM-CSF can augment BDNF to
combat spinal cordinjury (Bouhy et al., 2006; Hayashi et al.,
2009); and, taken together,suggest that BDNF could be contributing
to some of the neuroprotectiveactions of GM-CSF in response to
diverse injury-inducing stimuli.
In addition to BDNF, GDNF acts on dopaminergic neurons toenhance
their morphological differentiation and survival (Lin et al.,1993),
which has led to much excitement regarding the potential ofGDNF as
a therapeutic agent for PD. Indeed, numerous rodent andprimate
studies reported that GDNF attenuated the neurodegenera-tive
effects of MPTP and further boosted DA functioning of the
existingneurons (Gash et al., 1996; Kearns and Gash, 1995; Tomac et
al.,1995). Importantly, the GDNF-induced restoration of DA
functioningwas apparent even months after trophic support was
terminated(Grondin et al., 2002; Kirik et al., 2004; Maswood et
al., 2002). Despitethe overwhelming evidence supporting the benets
of GDNF, it haslargely failed in human clinical trials due to
complications arising fromthe fact that GDNF is unable to cross the
BBB. Indeed, intracerebro-ventricular injection of GDNF was
reported to cause problems relatedto (limited) diffusion into the
brain parenchyma and the manifesta-tion of debilitating
side-effects (Kordower et al., 1999; Nutt et al.,2003).
To circumvent the fact that trophic factors generally do not
cross theBBB, new techniques are currently being developed to
facilitate centralpenetration (Juillerat-Jeanneret and Schmitt,
2007; Pardridge, 2002);these include encapsulation or breaking GDNF
into small bioactivefragments (Peleshok and Saragovi, 2006).
However, nding novelneurotrophic factors that easily cross the BBB
(and thus avoid suchcomplications entirely) may provide a new class
of therapeutic agentsfor PD. In this regard, GM-CSF is one of three
cytokines belonging to afamily of colony stimulating factors that
have trophic effects and arecapable of crossing the BBB and
accumulating in the brain parenchymaat reasonable levels (Franzen
et al., 2004; McLay et al., 1997; Thomsonand Lotze, 2003). The
exact mechanism by which GM-CSF permeatesthe BBB is presently
unclear; however, based on experiments whichtagged GM-CSF prior to
systemic administration to rodents, it is clearthat this cytokine
can, indeed, gain entry into the brain (McLay et al.,1997).
GM-CSF has well documented clinical efcacy in the
periphery,including its use as an immune restorative agent in
certain cancertreatments (owing to its ability to stimulate
leukocyte production);however, minimal attention has been devoted
to elucidating thetrophic cytokine's potential as a neurotrophic
agent. Albeit, a fewreports have indicated that GM-CSF had
neurorestorative functions inthe spinal cord and even assisted in
cerebral ischemic recovery (Ha etal., 2005; Hayashi et al., 2009;
Nakagawa et al., 2006; Schabitz et al.,2008). There is also some
evidence to suggest that GM-CSF canpromote the release of BDNF from
microglia (Bouhy et al., 2006;Hayashi et al., 2009). In addition,
GM-CSF has been shown to down-regulate the IFN- mediated induction
of MHC-II expression onmicroglia (Hayashi et al., 1993). This is
important since IFN- wasrecently shown to play a primary role in
the degeneration of DAneurons following paraquat or MPTP exposure
(Mount et al., 2007).These ndings, in combination with the fact
that GM-CSF mightpromote a phenotypic shift in microglial
functioning towards a moresupportive and less inammatory role
(Bouhy et al., 2006; Re et al.,2002), prompted us to hypothesize
that GM-CSFmight protect againstDA acting toxins by modulating the
inammatory balance betweenmicroglial and astrocytic cells.
Consistent with reports showing that GM-CSF might affectneuronal
survival by inducing microglia to adopt a dendritic cell-like
morphology (Reddy et al., 2009), exposure to the growth factor
inthe current study appreciably diminished the impact of LPS
andparaquat upon microglia (as indicated by staining
intensity).However, the well established neurotrophin, GDNF, did
not appre-ciably affect SNc microglia staining (at least according
to CD11b+ cellmorphology and proliferation), despite the fact that
the growth factorprevented DA neuron loss to a degree comparable to
that of GM-CSF.These ndings raise the possibility that the two
trophic factors mightbe exerting some differential effects within
the SNc.
In contrast to the heightened microglia response induced by
LPSand paraquat, these insults seemed to reduce the SNc
astrocyticresponse (as indicated by GFAP+ immunostaining).
Interestingly,both GM-CSF and GDNF reversed the astrocytic
reduction induced bythe toxins. This might be particularly
important given that anastrocyte reduction would likely result in
diminished availability oftrophic support (e.g., BDNF and GDNF
release) and reduced glutamatebuffering capabilities (Malipiero et
al., 1990). Although GM-CSFreceptors are certainly expressed on
astrocytes (Guillemin et al.,1996; Malipiero et al., 1990), they
have also been found on neurons
throughout the brain, including DA neurons of the SNc (Kim et
al.,
-
108 E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
991122009). Hence, while GM-CSF might conceivably be
promotingneuronal survival through direct modulatory effects upon
microgliaand astrocytes, it is also possible that these glial
changes are simplysecondary to the direct impact of the trophic
cytokine upon DAneurons (or other yet to be identied processes
involved in theneurodegenerative response).
The capacity of GM-CSF to promote anti-apoptotic
signaling(Schabitz et al., 2008) could be important in its ability
to attenuatetoxin induced DA neuronal loss. Indeed, a recent study
found that GM-CSF promoted Bcl-2 expression following MPTP exposure
(Kim et al.,2009). However, in the present investigation, the
growth factor didnot inuence Bcl-2 expression in primary
mesencephalic cultures.This discrepancy may stem from the fact that
the previous studyassessed Bcl-2 within PC12 cells (Kim et al.,
2009), whereas wepresently assessed primarymesencephalic levels of
the anti-apoptoticfactor. It also might be the case that the nature
of toxin used (MPTP vs.paraquat) is relevant in this regard.
Fig. 6. Systemic GM-CSF treatment protected nigrostriatal
neurons (both SNc soma and stdopamine (DA) neurons was determined
using stereological cell counts of TH+ (1:1000)degree of TH+ cell
loss induced by LPS and paraquat, as well as the protective effects
of GM2 days prior to commencing the paraquat regimen (10 mg/kg; 3
per week for 3 weeks)mod25% (paraquat alone) to around 40%
(*pb0.05). Moreover, as shown in the top right photombres
(*pb0.05), however, LPS priming did not further augment this effect
whatsoever. The bapparent protective effects of systemic (i.p.)
administration of GM-CSF (2 g). Finally, lowmof the location of
TH+terminal loss within the region. Data are expressed as
meanSEM;In light of the fact that depression and other co-morbid
symptomstypically occur together with the motor disturbances in PD
(Farabaughet al., 2009), it was particularly interesting that
paraquat reducedhippocampal BDNF expression. Indeed, substantial
evidence hasindicated that reduced BDNF levels can contribute to
depressive-likebehavioral effects, aswell as decits in hippocampal
neuroplasticity andcognitive functioning (Anisman et al., 2008;
Heldt et al., 2007; Schmidtand Duman, 2007). Similarly, other
recent studies showed that BDNFadministration in mice promoted a
range of anti-depressant and anti-anxiety-like effect (e.g., as
assessed in forced swim, anhedoniaparadigms), as well as promoting
hippocampal neurogenesis (Gourleyet al., 2008; Schmidt and Duman,
2010).
While the precise mechanisms and neural substrates underlyingthe
co-morbid non-motor symptoms in PD have yet to be fullyestablished,
it is possible that inammatory and/or neuroplasticprocesses in
stressor-sensitive cognitive and emotional brain regions(e.g.,
hippocampus, prefrontal cortex, locus coeruleus) are important.
riatal terminals) from LPS and paraquat induced dopaminergic
cell death. The loss ofneurons through the SNc. The top left
representative photomicrographs illustrate the-CSF. Once again,
priming the SNc with a single supra-nigral injection LPS (0.01 g/2
l)estly (albeit not signicantly) enhanced the degree of neuronal
loss from approximatelyicrographs, mice exposed to the paraquat
regimen displayed a signicant loss of striatalottom bar graphs show
the quantication of SNc soma and striatal terminal loss and
theagnication (2.5) images of the striatum are shown on the bottom
to give a better idean=5.
-
Indeed, histological pathology, including accumulation of
-synu-clein, occurredwithin the hippocampus of PD patients
(Bertrand et al.,2003; Galvin et al., 1999). Moreover, paraquat
altered hippocampal
monoamine activity, and promoted depressive- and-
anxiety-likeresponses in mice (Litteljohn et al., 2009). The
present investigationprovides evidence that paraquat, and possibly
other environmental
Fig. 7. Systemic GM-CSF administration modulated the effects of
LPS and paraquat upon SNc microglia. A. Paraquat and LPS+paraquat
robustly induced microglia (CD11b+)immunostaining within the SNc.
Higher magnication (40 Objective; B) better illustrates the
morphological differences between the different groups. Furthermore
differences inmicroglial morphology were conrmed using a
semi-quantitative rating scale (C; see methods for details on the
microglial rating procedures).
109E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112Fig. 8. Systemic GM-CSF administration modulated the effects
of LPS and paraquat upon SNceffects of LPS and paraquat on
microglial cells, LPS primed mice that received paraquat
displaParalleling the aforementioned effects apparent with central
GM-CSF administration, systempopulation. Panel B reveals higher
magnication (40) images to give a better idea of morastrocytes. A.
The representative photomicrographs (20) reveal that in contrast to
theyed a reduction of astrocyte (GFAP+) staining, whereas paraquat
alone had little effect.ic injection of the trophic cytokine
blunted the impact of LPS and paraquat upon the glialphology of the
GFAP+cells.
-
110 E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112toxins, could potentially affect emotional and cognitive
processing bycausing trophic factor reductions in hippocampal
pathways acting inparallel with the nigrostriatal system.
Importantly, GM-CSF appearedto reverse the paraquat-induced
hippocampal BDNF reduction,suggesting that its protective effects
were not specic for nigrostriatalfunctioning but also translated
across brain regions.
Although the current ndings do not fully elucidate a mechanismof
action, it is clear that the neuroprotective effects of both
systemicand central GM-CSF administration are accompanied by a
modulationof the neuroinammatory glial responses provoked by LPS
andparaquat. This is important in light of the accumulating
evidencesuggesting an important role for glial-driven inammation in
thedeath of DA neurons. However, disturbances of
mitochondrialfunctioning, generation of oxidative radicals and
promotion ofapoptotic factors are probably the most proximal events
in theneurodegenerative process. In this regard, our data did not
support arole for the classical Bax and Bcl-2 apoptotic pathways in
the presentLPS-paraquat model or the protective effects of GM-CSF.
Yet, it isparticularly notable that paraquat reduced BDNF within
both thehippocampus, as well as cultured mesencephalic neurons, and
thatGM-CSF moderately reversed both of these effects. In
summary,paraquat (particularly in the context of inammatory priming
withLPS) could conceivably contribute to motor and non-motor
(e.g.,depression, cognitive disturbance) PD symptoms by
enhancinginammatory processes and altering neuroplasticity, and
GM-CSFmight have important mitigating effects in this regard.
Fig. 9. GM-CSF restored hippocampal BDNF expression inmidbrain
cultures exposed to paraq(30 M) exposure for 6 h induced a
signicant reduction in BDNF and Bcl-2 levels in primaryml)
partially prevented the reduction of BDNF but had no effect on
Bcl-2 levels within the(varying from3 to 12 h)were conducted in
order to determinewhich dose and time of paraquhours reliably
caused a 40% reduction in midbrain DA neurons. (C) Paralleling the
aforemenmidbrain cultures (pb0.05) and once again, GM-CSF reversed
this effect (bottom right bar gSupplementarymaterials related to
this article can be found onlineat doi:
10.1016/j.nbd.2011.02.011.
Acknowledgments
This work was supported by funds from the Canadian Institutes
ofHealth Research (CIHR) and Parkinson's Society Canada. S.H. is
aCanadian Research Chair in Behavioural Neuroscience.
References
Anisman, H., et al., 2008. Neurotransmitter, peptide and
cytokine processes in relationto depressive disorder: comorbidity
between depression and neurodegenerativedisorders. Prog. Neurobiol.
85, 174.
Baquet, Z.C., et al., 2005. Brain-derived neurotrophic factor is
required for theestablishment of the proper number of dopaminergic
neurons in the substantianigra pars compacta. J. Neurosci. 25,
62516259.
Bertrand, E., et al., 2003. Degenerative axonal changes in the
hippocampus andamygdala in Parkinson's disease. Folia Neuropathol.
41, 197207.
Bossers, K., et al., 2009. Analysis of gene expression in
Parkinson's disease: possibleinvolvement of neurotrophic support
and axon guidance in dopaminergic celldeath. Brain Pathol. 19,
91107.
Bouhy, D., et al., 2006. Delayed GM-CSF treatment stimulates
axonal regeneration andfunctional recovery in paraplegic rats via
an increased BDNF expression byendogenous macrophages. FASEB J. 20,
12391241.
Bruck, A., et al., 2004. Hippocampal and prefrontal atrophy in
patients with early non-demented Parkinson's disease is related to
cognitive impairment. J. Neurol.Neurosurg. Psychiatry 75,
14671469.
Cannon, J.R., et al., 2009. A highly reproducible rotenone model
of Parkinson's disease.Neurobiol. Dis. 34, 279290.
uat. (A) As depicted by the representative blots and conrmed by
densitometry, paraquatmidbrain tissue (*pb0.05). When applied
concomitant with paraquat, GM-CSF (250 ng/midbrain. (B) A dose
response (varying from 3 to 50 M of paraquat) and time courseat
exposurewould yield approximately 40% ofmidbrain neurons. 30 Mof
Paraquat for 6tioned in vivo data, paraquat exposure reduced the
number of TH+ neurons within theraph). Data are expressed as
meanSEM; n=23 (BDNF) and n=45 (Bcl-2).
-
111E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112Carvey, P.M., et al., 2006. Progressive dopamine neuron loss
in Parkinon's disease: themultiple hit hypothesis. Cell Transplant.
15, 239250.
Chauhan, N.B., et al., 2001. Depletion of glial cell
line-derived neurotrophic factor insubstantia nigra neurons of
Parkinson's disease brain. J. Chem. Neuroanat. 21,277288.
Chen, P.S., et al., 2006. Valproate protects dopaminergic
neurons in midbrain neuron/glia cultures by stimulating the release
of neurotrophic factors from astrocytes.Mol. Psychiatry 11,
11161125.
Cicchetti, F., et al., 2009. Environmental toxins and
Parkinson's disease: what have welearned from pesticide-induced
animal models? Trends Pharmacol. Sci. 30,475483.
Davignon, J.L., et al., 1988. Selective production of
interleukin 3 (IL3) and granulocyte-macrophage colony-stimulating
factor (GM-CSF) in vitro by murine L3T4+ T cells:lack of
spontaneous IL3 and GM-CSF production by Ly-2-/L3T4- lpr subset.
Eur. J.Immunol. 18, 13671372.
Di Monte, D.A., 2003. The environment and Parkinson's disease:
is the nigrostriatalsystem preferentially targeted by neurotoxins?
Lancet Neurol. 2, 531538.
Eslamboli, A., 2005. Assessment of GDNF in primate models of
Parkinson's disease:comparison with human studies. Rev. Neurosci.
16, 303310.
Farabaugh, A.H., et al., 2009. Pattern of depressive symptoms in
Parkinson's disease.Psychosomatics 50, 448454.
Fischer, H.G., Reichmann, G., 2001. Brain dendritic cells and
macrophages/microglia incentral nervous system inammation. J.
Immunol. 166, 27172726.
Fitzmaurice, P.S., et al., 2003. Nigral glutathione deciency is
not specic for idiopathicParkinson's disease. Mov. Disord. 18,
969976.
Franzen, R., et al., 2004. Nervous system injury: focus on the
inammatory cytokine'granulocyte-macrophage colony stimulating
factor'. Neurosci. Lett. 361, 7678.
Galvin, J.E., et al., 1999. Axon pathology in Parkinson's
disease and Lewy body dementiahippocampus contains alpha-, beta-,
and gamma-synuclein. Proc. Natl. Acad. Sci. U.S. A. 96,
1345013455.
Gao, H.M., et al., 2003. Critical role for microglial NADPH
oxidase in rotenone-induceddegeneration of dopaminergic neurons. J.
Neurosci. 23, 61816187.
Gash, D.M., et al., 1996. Functional recovery in parkinsonian
monkeys treated withGDNF. Nature 380, 252255.
Gourley, S.L., et al., 2008. Acute hippocampal brain-derived
neurotrophic factor restoresmotivational and forced swim
performance after corticosterone. Biol. Psychiatry64, 884890.
Grondin, R., et al., 2002. Chronic, controlled GDNF infusion
promotes structural andfunctional recovery in advanced parkinsonian
monkeys. Brain 125, 21912201.
Guerini, F.R., et al., 2009. BDNF Val66Met polymorphism is
associated with cognitiveimpairment in Italian patients with
Parkinson's disease. Eur. J. Neurol. 16,12401245.
Guillemin, G., et al., 1996. Granulocyte macrophage colony
stimulating factorstimulates in vitro proliferation of astrocytes
derived from simian mature brains.Glia 16, 7180.
Ha, Y., et al., 2005. Synthes Award for Resident Research on
Spinal Cord and SpinalColumn Injury: granulocyte macrophage colony
stimulating factor (GM-CSF)prevents apoptosis and improves
functional outcome in experimental spinal cordcontusion injury.
Clin. Neurosurg. 52, 341347.
Hartmann, A., et al., 2002. FADD: A link between TNF family
receptors and caspases inParkinson's disease. Neurology 58,
308310.
Hayashi, M., et al., 1993. Granulocyte-macrophage colony
stimulating factor inhibitsclass II major histocompatibility
complex expression and antigen presentation bymicroglia. J.
Neuroimmunol. 48, 2332.
Hayashi, K., et al., 2009. Activation of dendritic-like cells
and neural stem/progenitorcells in injured spinal cord by GM-CSF.
Neurosci. Res. 64, 96103.
Hayley, S., et al., 1999. Sensitization to the effects of tumor
necrosis factor-alpha:neuroendocrine, central monoamine, and
behavioral variations. J. Neurosci. 19,56545665.
Hayley, S., et al., 2004. Regulation of dopaminergic loss by Fas
in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of
Parkinson's disease. J. Neurosci. 24,20452053.
Heldt, S.A., et al., 2007. Hippocampus-specic deletion of BDNF
in adult mice impairsspatial memory and extinction of aversive
memories. Mol. Psychiatry 12,656670.
Hercus, T.R., et al., 2009. The granulocyte-macrophage
colony-stimulating factorreceptor: linking its structure to cell
signaling and its role in disease. Blood 114,12891298.
Hirsch, E.C., Hunot, S., 2009. Neuroinammation in Parkinson's
disease: a target forneuroprotection? Lancet Neurol. 8, 382397.
Howells, D.W., et al., 2000. Reduced BDNF mRNA expression in the
Parkinson's diseasesubstantia nigra. Exp. Neurol. 166, 127135.
Huang, X., et al., 2007. GM-CSF inhibits apoptosis of neural
cells via regulating theexpression of apoptosis-related proteins.
Neurosci. Res. 58, 5057.
Huang, X., et al., 2009. GM-CSF inhibits glial scar formation
and shows long-termprotective effect after spinal cord injury. J.
Neurol. Sci. 277, 8797.
Huang, R., et al., 2010. Gene therapy using lactoferrin-modied
nanoparticles in arotenone-induced chronic Parkinson model. J.
Neurol. Sci. 290, 123130.
Hung, H.C., Lee, E.H., 1996. The mesolimbic dopaminergic pathway
is more resistantthan the nigrostriatal dopaminergic pathway to
MPTP and MPP+toxicity: role ofBDNF gene expression. Brain Res. Mol.
Brain Res. 41, 1426.
Hunot, S., et al., 2004. JNK-mediated induction of
cyclooxygenase 2 is required forneurodegeneration in amouse model
of Parkinson's disease. Proc. Natl. Acad. Sci. U.S. A. 101,
665670.
Jenner, P., 1998. Oxidative mechanisms in nigral cell death in
Parkinson's disease. Mov.Disord. 13 (Suppl 1), 2434.Jokinen, P., et
al., 2009. Impaired cognitive performance in Parkinson's disease is
relatedto caudate dopaminergic hypofunction and hippocampal
atrophy. ParkinsonismRelat. Disord. 15, 8893.
Juillerat-Jeanneret, L., Schmitt, F., 2007. Chemical modication
of therapeutic drugs ordrug vector systems to achieve targeted
therapy: looking for the grail. Med. Res.Rev. 27, 574590.
Kearns, C.M., Gash, D.M., 1995. GDNF protects nigral dopamine
neurons against 6-hydroxydopamine in vivo. Brain Res. 672,
104111.
Kim, N.K., et al., 2009. Granulocyte-macrophage
colony-stimulating factor promotessurvival of dopaminergic neurons
in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine-induced murine
Parkinson's disease model. Eur. J. Neurosci. 29, 891900.
Kirik, D., et al., 2004. Localized striatal delivery of GDNF as
a treatment for Parkinsondisease. Nat. Neurosci. 7, 105110.
Knott, C., et al., 2000. Inammatory regulators in Parkinson's
disease: iNOS, lipocortin-1, and cyclooxygenases-1 and 2. Mol.
Cell. Neurosci. 16, 724739.
Kordower, J.H., et al., 1999. Clinicopathological ndings
following intraventricular glial-derived neurotrophic factor
treatment in a patient with Parkinson's disease. Ann.Neurol. 46,
419424.
Lang, A.E., et al., 2006. Randomized controlled trial of
intraputamenal glial cell line-derived neurotrophic factor infusion
in Parkinson disease. Ann. Neurol. 59,459466.
Levivier, M., et al., 1995. Intrastriatal implantation of
broblasts genetically engineeredto produce brain-derived
neurotrophic factor prevents degeneration of dopami-nergic neurons
in a rat model of Parkinson's disease. J. Neurosci. 15,
78107820.
Lin, L.F., et al., 1993. GDNF: a glial cell line-derived
neurotrophic factor for midbraindopaminergic neurons. Science 260,
11301132.
Litteljohn, D., et al., 2009. Interferon-gamma deciency modies
the motor and co-morbid behavioral pathology and neurochemical
changes provoked by thepesticide paraquat. Neuroscience 164,
18941906.
Liu, B., et al., 2003. Parkinson's disease and exposure to
infectious agents and pesticidesand the occurrence of brain
injuries: role of neuroinammation. Environ. HealthPerspect. 111,
10651073.
Malipiero, U.V., et al., 1990. Production of hemopoietic
colony-stimulating factors byastrocytes. J. Immunol. 144,
38163821.
Mangano, E.N., Hayley, S., 2009. Inammatory priming of the
substantia nigrainuences the impact of later paraquat exposure:
Neuroimmune sensitization ofneurodegeneration. Neurobiol. Aging 30,
13611378.
Masaki, T., et al., 2003. Association between a polymorphism of
brain-derivedneurotrophic factor gene and sporadic Parkinson's
disease. Ann. Neurol. 54,276277.
Maswood, N., et al., 2002. Effects of chronic intraputamenal
infusion of glial cell line-derived neurotrophic factor (GDNF) in
aged Rhesus monkeys. Neurobiol. Aging 23,881889.
McCoy, M.K., et al., 2008. Intranigral lentiviral delivery of
dominant-negative TNFattenuates neurodegeneration and behavioral
decits in hemiparkinsonian rats.Mol. Ther. 16, 15721579.
McLay, R.N., et al., 1997. Granulocyte-macrophage
colony-stimulating factor crosses thebloodbrain and bloodspinal
cord barriers. Brain 120 (Pt 11), 20832091.
Mogi, M., et al., 1999. Brain-derived growth factor and nerve
growth factorconcentrations are decreased in the substantia nigra
in Parkinson's disease.Neurosci. Lett. 270, 4548.
Mount, M.P., et al., 2007. Involvement of interferon-gamma in
microglial-mediated lossof dopaminergic neurons. J. Neurosci. 27,
33283337.
Murer, M.G., et al., 2001. Brain-derived neurotrophic factor in
the control human brain,and in Alzheimer's disease and Parkinson's
disease. Prog. Neurobiol. 63, 71124.
Nagatsu, T., Sawada, M., 2007. Biochemistry of postmortem brains
in Parkinson's disease:historical overview and future prospects. J.
Neural Transm. 113120 (Suppl.).
Nakagawa, T., et al., 2006. Intracarotid injection of
granulocyte-macrophage colony-stimulating factor induces
neuroprotection in a rat transient middle cerebral arteryocclusion
model. Brain Res. 1089, 179185.
Nimmerjahn, A., et al., 2005. Resting microglial cells are
highly dynamic surveillants ofbrain parenchyma in vivo. Science
308, 13141318.
Nutt, J.G., et al., 2003. Randomized, double-blind trial of
glial cell line-derivedneurotrophic factor (GDNF) in PD. Neurology
60, 6973.
Pardridge, W.M., 2002. Targeting neurotherapeutic agents through
the blood-brainbarrier. Arch. Neurol. 59, 3540.
Peleshok, J., Saragovi, H.U., 2006. Functional mimetics of
neurotrophins and theirreceptors. Biochem. Soc. Trans. 34,
612617.
Peterson, A.L., Nutt, J.G., 2008. Treatment of Parkinson's
disease with trophic factors.Neurotherapeutics 5, 270280.
Porritt, M.J., et al., 2005. Inhibiting BDNF expression by
antisense oligonucleotideinfusion causes loss of nigral
dopaminergic neurons. Exp. Neurol. 192, 226234.
Purisai, M.G., et al., 2007. Microglial activation as a priming
event leading to paraquat-induced dopaminergic cell degeneration.
Neurobiol. Dis. 25, 392400.
Re, F., et al., 2002. Granulocyte-macrophage colony-stimulating
factor induces anexpression program in neonatal microglia that
primes them for antigenpresentation. J. Immunol. 169, 22642273.
Reddy, P.H., et al., 2009. Granulocyte-macrophage
colony-stimulating factor antibodysuppresses microglial activity:
implications for anti-inammatory effects inAlzheimer's disease and
multiple sclerosis. J. Neurochem. 111, 15141528.
Salehi, Z., Mashayekhi, F., 2009. Brain-derived neurotrophic
factor concentrations in thecerebrospinal uid of patients with
Parkinson's disease. J. Clin. Neurosci. 16, 9093.
Salvatore, M.F., et al., 2006. Point source concentration of
GDNF may explain failure ofphase II clinical trial. Exp. Neurol.
202, 497505.
Santambrogio, L., et al., 2001. Developmental plasticity of CNS
microglia. Proc. Natl.Acad. Sci. U. S. A. 98, 62956300.
-
Schabitz, W.R., et al., 2008. A neuroprotective function for the
hematopoietic proteingranulocyte-macrophage colony stimulating
factor (GM-CSF). J. Cereb. Blood FlowMetab. 28, 2943.
Schermer, C., Humpel, C., 2002. Granulocyte macrophage-colony
stimulating factoractivatesmicroglia in rat cortex organotypic
brain slices. Neurosci. Lett. 328, 180184.
Schmidt, H.D., Duman, R.S., 2007. The role of neurotrophic
factors in adult hippocampalneurogenesis, antidepressant treatments
and animal models of depressive-likebehavior. Behav. Pharmacol. 18,
391418.
Schmidt, H.D., Duman, R.S., 2010. Peripheral BDNF Produces
Antidepressant-Like Effects inCellular and Behavioral Models.
Neuropsychopharmacology 35, 23782391.
Sherer, T.B., et al., 2007. Mechanism of toxicity of pesticides
acting at complex I:relevance to environmental etiologies of
Parkinson's disease. J. Neurochem. 100,14691479.
Shults, C.W., et al., 1995. BDNF attenuates the effects of
intrastriatal injection of 6-hydroxydopamine. NeuroReport 6,
11091112.
Somayajulu-Nitu, M., et al., 2009. Paraquat induces oxidative
stress, neuronal loss insubstantia nigra region and parkinsonism in
adult rats: neuroprotection and
amelioration of symptoms by water-soluble formulation of
coenzyme Q10. BMCNeurosci. 10, 88.
Taliaz, D., Stall, N., Dar, D.E., Zangen, A., 2010. Knockdown of
brain-derivedneurotrophic factor in specic brain sites precipitates
behaviors associated withdepression and reduces neurogenesis. Mol.
Psychiatry 15 (1), 8092.
Tatton, W.G., et al., 2003. Apoptosis in Parkinson's disease:
signals for neuronaldegradation. Ann. Neurol. 53 (Suppl. 3), S61S70
(discussion S70-2).
Thomson, A.W., Lotze, M.T. (Eds.), 2003. The Cytokine Handbook.
Academic Press,London.
Tomac, A., et al., 1995. Protection and repair of the
nigrostriatal dopaminergic systemby GDNF in vivo. Nature 373,
335339.
Whitton, P.S., 2007. Inammation as a causative factor in the
aetiology of Parkinson'sdisease. Br. J. Pharmacol. 150, 963976.
Yasuhara, T., et al., 2007. Glial cell line-derived neurotrophic
factor (GDNF) therapy forParkinson's disease. Acta Med. Okayama 61,
5156.
Zhang, H.T., et al., 2007. Immunohistochemical distribution of
NGF, BDNF, NT-3, andNT-4 in adult rhesus monkey brains. J.
Histochem. Cytochem. 55, 119.
112 E.N. Mangano et al. / Neurobiology of Disease 43 (2011)
99112
Granulocyte macrophage-colony stimulating factor protects
against substantia nigra dopaminergic cell loss in an
environment...IntroductionMaterials and methodsExperiment 1:
assessment of the beneficial effects of supra-nigral administration
of GDNF and GM-CSF following LPS-paraquat...Immunohistochemical
procedures: SNc DA neuronal survival and microglial
reactivityWestern blot analyses of hippocampal BDNF
levelsExperiment 2: assessment of the beneficial effects of
systemic administration of GM-CSF following LPS-paraquat induced
path...Immunohistochemical analyses of SNc neuronal lossAssessment
of glial changesGeneral stereotaxic surgical procedures for
Experiments 1 and 2Experiment 3: in vitro assessment of the effects
of paraquat and GM-CSFImmunocytochemistryWestern blotStatistical
data analysis
ResultsExperiment 1: Supra-nigral injection of GM-CSF or GDNF
attenuated LPS-paraquat induced degeneration of SNc dopamine
neurons...Supra-nigral injection of GM-CSF influenced LPS and
paraquat induced glial changes in the SNcSupra-nigral infusion of
GM-CSF modified the paraquat induced reduction of mature
hippocampal BDNFExperiment 2: systemic administration of GM-CSF had
neuroprotective consequencesExperiment 3: GM-CSF neuroprotection
was associated with normalized BDNF but not Bcl-2 or Bax levels
DiscussionAcknowledgmentsReferences