Neuroprotective effects of toll-like receptor 4 antagonism in spinal cord cultures and in a mouse model of motor neuron degeneration.

Abstract

Sustained inflammatory reactions are common pathological events associated with neuron loss in neurodegenerative diseases. Reported evidence suggests that Toll-like receptor 4 (TLR4) is a key player of neuroinflammation in several neurodegenerative diseases. However, the mechanisms by which TLR4 mediates neurotoxic signals remain poorly understood. We investigated the role of TLR4 in in vitro and in vivo settings of motor neuron degeneration. Using primary cultures from mouse spinal cords, we characterized both the proinflammatory and neurotoxic effects of TLR4 activation with lipopolysaccharide (activation of microglial cells, release of proinflammatory cytokines and motor neuron death) and the protective effects of a cyanobacteria-derived TLR4 antagonist (VB3323). With the use of TLR4-deficient cells, a critical role of the microglial component with functionally active TLR4 emerged in this setting. The in vivo experiments were carried out in a mouse model of spontaneous motor neuron degeneration, the wobbler mouse, where we preliminarily confirmed a protective effect of TLR4 antagonism. Compared with vehicle- and riluzole-treated mice, those chronically treated with VB3323 showed a decrease in microglial activation and morphological alterations of spinal cord neurons and a better performance in the paw abnormality and grip-strength tests. Taken together, our data add new understanding of the role of TLR4 in mediating neurotoxicity in the spinal cord and suggest that TLR4 antagonists could be considered in future studies as candidate protective agents for motor neurons in degenerative diseases.

Full text

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INTRODUCTION
In pathological conditions, persistent
immune-stimulating signals may induce
aberrant microglial activation with ele-
vated cytokine release, eventually lead-
ing to neuronal injury. Toll-like receptors
(TLRs) recognize highly conserved struc-
tural motifs from either pathogens or
damaged and stressed tissues and are in-
volved in microglial response to physio-
logical and pathological signals. TLR4, in
particular, reportedly mediates neuropro-
tective (1,2) and neurotoxic (3–5) effects,
suggesting that TLR4 activation needs to
be tightly controlled, to regulate the
switch between its different roles in im-
mune surveillance or neuroinflammatory
propagation.
A well-known natural ligand of TLR4
is endotoxin/lipopolysaccharide (LPS),
one of the major cell wall components of
Gram-negative bacteria (6). LPS has po-
tent immunostimulatory effects (7),
mainly exerted through TLR4/MD2/
CD14-bearing cells (8,9). The membrane
anchor of LPS is a glucosamine-based
phospholipid called lipid A, which repre-
sents the endotoxic principle of LPS and
is responsible for its pathophysiological
effects (10) via TLR4 (11,12). There is con-
vincing evidence that LPS/TLR4 signal-
ing is involved in various human and ex-
perimental central nervous system (CNS)
diseases. In fact, increased expression of
microglial TLR4 was found in animal
models of Alzheimer’s disease (13) and
Parkinson’s disease (14). Altered levels of
the receptor have been reported in brain
M O L M E D 1 8 : 9 7 1 - 9 8 1 , 2 0 1 2 | D E PA O L A E T A L . | 9 7 1
Neuroprotective Effects of Toll-Like Receptor 4 Antagonism in
Spinal Cord Cultures and in a Mouse Model of Motor Neuron
Degeneration
Massimiliano De Paola,1Alessandro Mariani,1Paolo Bigini,2Marco Peviani,3,4Giovanni Ferrara,3
Monica Molteni,5Sabrina Gemma,5Pietro Veglianese,3Valeria Castellaneta,2Valentina Boldrin,3
Carlo Rossetti,6Chiara Chiabrando,1Gianluigi Forloni,3Tiziana Mennini,2and Roberto Fanelli1
1Department of Environmental Health Sciences, 2Department of Biochemistry and Molecular Pharmacology, 3Department of
Neuroscience, Mario Negri Institute for Pharmacological Research, Milan, Italy;4current affiliation: Department of Biology and
Biotechnology, University of Pavia, Pavia, Italy; 5Bluegreen Biotech, Mario Negri Institute for Pharmacological Research, Milan, Italy;
and 6Department of Biotechnology and Molecular Sciences, University of Insubria, Varese, Italy
Sustained inflammatory reactions are common pathological events associated with neuron loss in neurodegenerative dis-
eases. Reported evidence suggests that Toll-like receptor 4 (TLR4) is a key player of neuroinflammation in several neurodegener-
ative diseases. However, the mechanisms by which TLR4 mediates neurotoxic signals remain poorly understood. We investigated
the role of TLR4 in in vitro and in vivo settings of motor neuron degeneration. Using primary cultures from mouse spinal cords, we
characterized both the proinflammatory and neurotoxic effects of TLR4 activation with lipopolysaccharide (activation of mi-
croglial cells, release of proinflammatory cytokines and motor neuron death) and the protective effects of a cyanobacteria-
derived TLR4 antagonist (VB3323). With the use of TLR4-deficient cells, a critical role of the microglial component with functionally
active TLR4 emerged in this setting. The in vivo experiments were carried out in a mouse model of spontaneous motor neuron de-
generation, the wobbler mouse, where we preliminarily confirmed a protective effect of TLR4 antagonism. Compared with vehi-
cle- and riluzole-treated mice, those chronically treated with VB3323 showed a decrease in microglial activation and morpho-
logical alterations of spinal cord neurons and a better performance in the paw abnormality and grip-strength tests. Taken
together, our data add new understanding of the role of TLR4 in mediating neurotoxicity in the spinal cord and suggest that TLR4
antagonists could be considered in future studies as candidate protective agents for motor neurons in degenerative diseases.
Online address: http://www.molmed.org
doi: 10.2119/molmed.2012.00020
Address correspondence to Massimiliano De Paola, Department of Environmental
Health Sciences, Mario Negri Institute for Pharmacological Research, Via La Masa 19,
20156, Milan, Italy. Phone: +39-02-39014-521; Fax: +39-02-39014-735. E-mail:
massimiliano.depaola@marionegri.it.
Submitted January 23, 2012; Accepted for publication April 30, 2012; Epub
(www.molmed.org) ahead of print May 1, 2012.

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samples from Alzheimer’s disease (4)
and multiple sclerosis (15). Increased
CD14 (a TLR4 coreceptor) levels were
found in amyotrophic lateral sclerosis
(ALS) patients (16). Increased expression
of TLR4 was found in reactive glia in
human ALS spinal cord (17), suggesting
a possible role for the TLR/receptor for
advanced glycation end products
(RAGE) signaling pathways. The activa-
tion of these pathways in the spinal cord
may contribute to the progression of in-
flammation, resulting in motor neuron
injury. However, the specific role of
TLR4 in the spinal cord cell population,
and the mechanism(s) by which TLR4 ac-
tivation triggers inflammation and neu-
rotoxicity in this region, remain poorly
understood.
By using primary spinal cord cultures
from mouse embryos as an in vitro model
for the study of motor neuron injuries,
we investigated the effects induced by
TLR4 activation in neurons and glial
cells. Cultures from TLR4-deficient mice
were also used to understand the cell-
specific role of this receptor in mediating
motor neuron death. The effects of a
novel cyanobacteria-derived TLR4 antag-
onist (VB3323) were analyzed in this in
vitro setting and were compared with
those of a well-known commercially
available TLR4 antagonist, the LPS from
Rhodobacter sphaeroides (RsLPS).
VB3323 was also tested in vivo in a
mouse model of spontaneous motor neu-
ron degeneration. The wobbler mouse,
carrying a mutation in the vacuolar-
vesicular protein sorting S4 (Vps54) gene
(18) coding for a protein involved in the
retrograde transport of late endosomes
from the periphery to the Golgi apparatus
(19), shows early-onset selective motor
neuron death in the cervical spinal cord
(reviewed in [20]). Glial activation (21–23)
and upregulation of tumor necrosis factor
(TNF)-α (24) have been reported in the
cervical spinal cord of presymptomatic or
early symptomatic mice. Thus, the wob-
bler mouse represents a useful model for
testing candidate treatments aimed at
modifying the neuroinflammatory mecha-
nisms underlying the motor neuron loss.
MATERIALS AND METHODS
Primary Cell Cultures
Procedures involving animals and their
care were conducted in conformity with
the institutional guidelines that comply
with national (25,26) and international
laws and policies (27,28). Primary cul-
tures of motor neurons or neuron/glia
cocultures were obtained from the spinal
cord of 13-d-old C57 BL/6J (wild-type
[WT]) or C57BL/10ScNJ (bearing a TLR4-
null mutation, TLR4LPS-del; Charles River
Laboratories International, Calco, Italy)
mouse embryos, as previously described
(29). Briefly, ventral horns were dissected
from spinal cords, exposed to DNAse and
trypsin (Sigma-Aldrich, Milan, Italy) and
centrifuged through a bovine serum albu-
min (BSA) cushion. Cells obtained at this
step were a mixed neuron/glia population
and were centri fuged (800g for 15 min)
through a 6% iodixanol (OptiPrep™;
Sigma-Aldrich) cushion for motor neuron
enrichment. A sharp band (motor
neuron–enriched fraction) on the top of
the iodixanol cushion and a pellet (glial
fraction) were obtained. The glial feeder
layer was prepared by plating the glial
fraction at a density of 25,000 cells/cm2
into 12-well plates or into flasks, both
previously precoated with poly-L-lysine
(Sigma-Aldrich). Purified microglia were
obtained by adapting the protocols of
Hamby et al. (30) and Gingras et al. (31)
with minor modifications. Flasks contain-
ing confluent mixed glial cultures were
shaken overnight at 275 rpm in incuba-
tors. The supernatants (containing mi-
croglial cells) were collected and seeded
at a density of 20,000 cells/cm2into new
flasks for Western blot analysis, plates or
microslides (8-well μSlides; Ibidi GmbH,
Martinsried, Germany) for time-lapse
analysis. The purification yield of mi-
croglial cells with this protocol was about
98% (not shown). Astrocyte- enriched cul-
tures were obtained by treating the glial
cultures, from which microglia had been
previously harvested, with 60 mmol/L
L-leucine methyl ester (Sigma-Aldrich) for
90 min. To establish neuron/glia cocul-
tures, the motor neuron–enriched fraction
(obtained from the iodixanol-based sepa-
ration) was seeded at a density of 10,000
cells/cm2onto mature glial layers com-
posed of either mixed glial cells or puri-
fied astrocytes. In these cocultures, about
84 ± 5% (n = 6) of the neuronal cells (re-
vealed by neurofilament staining; Supple-
mentary Figure S1A) were SMI32-positive
cells with the typical motor neuron mor-
phology (Supplementary Figure S1B;
SMI32-positive cells merged with neuro-
filament staining in Supplementary Fig-
ure S1C; cell nuclei in blue). About 96 ±
2% (n = 3) of SMI32-positive neurons
were also stained by another antibody
specific for motor neurons, Hb9 (Supple-
mentary Figures S1D–F).
For combined mixed cocultures from
wild-type and TLR4LPS-delmouse embryos,
microglial cells were added (10% of the
astrocyte number) to motor neuron/
astrocyte cocultures on the third day in
vitro.
Motor Neuron Viability
The viability of motor neurons was as-
sayed as follows: only the SMI32-positive
cells, with typical morphology (triangular
shape, single well defined axon), large
bodies (>20 μm) and intact axons and den-
drites were counted, at a magnification of
200×, following the length of the coverslip
in four nonoverlapping pathways (29).
This number was normalized to the mean
of SMI32-positive cells counted in the ap-
propriate control wells. In a typical experi-
ment with cocultures, the number of
counted SMI32-positive cells in control
(untreated) wells was 70 ± 11 (n = 12).
Culture Treatments
Cocultures were exposed to the differ-
ent TLR4 ligands (diluted to the appropri-
ate concentrations in culture medium) on
the sixth day in vitro. Cultures maintained
with normal medium served as the control
condition. Motor neuron death was deter-
mined after 24-h incubation with 1 μg/mL
LPS (from Escherichia coli 0111:B4) or lipid
A (from E. coli F583; Sigma-Aldrich). The
protective effects of 20 μg/mL VB3323
(Bluegreen Biotech, Milan, Italy) or
20 μg/mL RsLPS (Sigma-Aldrich) were
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evaluated after simultaneous treatment
with the TLR4 agonists (LPS or lipid A).
For the other investigations (microglial ac-
tivation, cytokine release, TLR4 expres-
sion), we tested several exposure times to
the TLR4 ligands (from 0.5 to 24 h). Motor
neuron viability was assessed by counting
SMI32-positive cells in each treatment con-
dition, as previously reported (29). Treat-
ment effects were compared by one-way
analysis of variance (ANOVA) and the
Tukey or Dunnett test, or two-way
ANOVA and the Bonferroni posttest, using
GraphPad v5.01 (GraphPad Software).
VB3323
VB3323 is a highly (95%) purified
form of cyanobacterial LPS-like mole-
cule (CyP), extracted from the freshwa-
ter cyanobacterium Oscillatoria plank-
tothrix sp. by TRI reagent (Sigma-
Aldrich), as described previously
(32,33). The product was treated with
DNase (20 μg/mL) and RNase (10 μg/
mL) in 50 mmol/L Tris, 10 mmol/L
MgCl2, pH 7.5, for 2 h at room tempera-
ture before addition of proteinase K
(100 μg/mL) and incubation overnight
at 37°C. The sample was reextracted
with TRI reagent and then purified by
ion exchange chromatography.
Immunocytochemical and
Immunofluorescent Assays
Cells were fixed with 4% paraformal -
dehyde or methanol (for glial fibrillary
acidic protein [GFAP] staining). When in-
dicated, cells were permeabilized by 0.2%
Triton X-100 (Sigma- Aldrich). Staining
was carried out by overnight incubation
with the primary antibody, followed by
incubation with an appropriate fluores-
cent secondary antibody for immunofluo-
rescence (Dy-light; Rockland Immuno-
chemicals, Gilbertsville, PA, USA). Cell
nuclei were labeled with Hoechst 33258
by incubation with a 250 ng/mL solution.
Double or triple staining was done by
overnight incubation of the cultures sepa-
rately with each primary antibody. In
each experiment, some wells were
processed without the primary antibody
to verify the specificity of the staining.
Primary antibodies were as follows:
SMI32 (anti-nonphosphorylated neurofil-
ament H antibody, mouse, 1:6,000;
Covance, Princeton, NJ, USA), anti-
neurofilament 200 (1:500; Sigma-
Aldrich), Hb9 (1:200; Abcam, Cambridge,
MA, USA), GFAP (rabbit, 1:500; Santa
Cruz Biotechnology, Santa Cruz, CA,
USA), TLR4 (goat, 1:100; Santa Cruz
Biotechnology, Santa Cruz, CA, USA)
and CD11b (rat, 1:1,000; eBioscience, San
Diego, CA, USA). Appropriate fluores-
cent secondary antibodies conjugated to
different fluorochromes were used at
1:1,000 dilution. Biotinylated anti-mouse
secondary antibody (1:200; Vector Labo-
ratories, Burlingame, CA, USA) was used
to analyze motor neuron viability.
Microscopy and Live Imaging
Analysis
For the immunocytochemistry experi-
ments, pictures of stained cells were ob-
tained with a laser scanning microscope
(Olympus Fluoview BX61 microscope
with a FV500 confocal system; Olympus,
Milan, Italy), and images were analyzed
with ImageJ v1.43 (National Institutes of
Health). An optical microscope (Olym-
pus BX51) was used for the neurotoxic-
ity analysis. For time-lapse analysis, im-
ages were collected and analyzed with a
CellR imaging station (Olympus) cou-
pled to an inverted microscope (Olym-
pus IX 81) equipped with an incubator
to maintain constant temperature (37°C)
and CO2(5%) in at least three wells for
each condition. Images were acquired at
30-min intervals for 18 h. For this analy-
sis, purified microglial cultures were
seeded at a density of 15,000 cells/cm2
into eight-well microslides and main-
tained in culture for 1 wk. They were
then infected with lentiviral vectors
(2.0 × 109titer unit (T.U.)/ well) express-
ing green fluorescent protein (GFP) re-
porter, to allow cell tracking in live-cell
imaging experiments.
Viral Vector Production
High-titer viral vectors were produced
by transfecting 293T cells with pRSV-
Rev and pCMVdR8.74 plasmids (Trono
Laboratories, Lausanne, Switzerland) en-
coding gag/pol/rev proteins, pVSV-G
(BD Biosciences, Franklin Lakes, NJ,
USA), for expression of VSV-G protein
and pWPXLd transfer vector (Addgene
Plasmid 12257) encoding GFP reporter
gene under the EF1a ubiquitous pro-
moter. Briefly, 293T cells were plated at
70% confluence in Dulbecco’s modified
R E S E A R C H A R T I C L E
M O L M E D 1 8 : 9 7 1 - 9 8 1 , 2 0 1 2 | D E PA O L A E T A L . | 9 7 3
Figure 1. TLR4 expression on motor neurons from WT or TLR4LPS-delmice. Motor neuron/glia
cocultures from WT (A, B) or TLR4LPS-del(D, E) mouse embryos underwent immunocyto-
chemistry for SMI32 (A, D, green) and TLR4 (B, E, red). Images were acquired by a confocal
microscope. WT motor neurons showed a widespread TLR4 distribution colocalizing with
SMI32-positive dendrites and axons (C, merge). TLR4LPS-delmotor neurons gave only a very
weak, aspecific red fluorescence (E). Scale bar, 20 μm.

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Eagle’s medium, 10% fetal calf serum,
1% glutamine, 1% penicillin and 1%
streptomycin and maintained at 37°C in
a humidified 5% CO2incubator. Four
plasmids were cotransfected with an Ef-
fectene kit (Qiagen, Valencia, CA, USA)
according to the manufacturer’s instruc-
tions, and the medium was replaced
after 16 h. Supernatants containing the
viral particles were harvested 24 and
48 h later, filtered through a 0.45-μm fil-
ter and concentrated by ultracentrifuga-
tion at 50,000g at 4°C for 2 h. Viral pel-
lets were resuspended in sterile phos-
phate-buffered saline and stored at
–80°C until used. The titer of the viral
stocks was in the range of 1.0–2.0 × 109
T.U./mL, assessed by an endpoint dilu-
tion on HEK293 cells (34).
Quantitative Enzyme-Linked
Immunosorbent Assays
TNF-α, interleukin (IL)-6 and IL-1β
concentrations in cell culture super-
natants were quantified by solid-phase
sandwich enzyme-linked immunosor-
bent assay (ELISA) (eBioscience). Sam-
ples from each experiment were tested in
triplicate, according to the manufac-
turer’s instructions. The sensitivity of the
kits was 8 pg/mL for TNF-α and IL-1β
and 4 pg/mL for IL-6.
Immunoblotting
Microglial cultures were treated with
ice-cold lysis buffer, and the protein con-
9 7 4 | D E PA O L A E T A L . | M O L M E D 1 8 : 9 7 1 - 9 8 1 , 2 0 1 2
T L R 4 A N T A G O N I S M P R O T E C T S M O T O R N E U R O N S
Figure 2. TLR4 distribution and TLR4-mediated morphological alterations in purified microglia cultures. Purified microglial cultures were
treated with 1 μg/mL LPS alone or with 20 μg/mL VB3323, for 6 or 18 h. Cells were then double-stained by CD11b and TLR4. Nuclei were re-
vealed by Hoechst 33258 dye (blue). TLR4 was expressed in basal conditions (B) and appeared mostly unaltered during LPS treatment with
(H, N) or without (E, K) TLR4 antagonist. After 18 h, LPS induced morphological alterations in CD11b-positive cells (J) that were prevented by
cotreatment with VB3323 (M). (P) Morphometric parameters of CD11b-positive microglia were measured with Olympus DPSoft software. The
LPS-induced increase (***p < 0.001; one-way ANOVA and Tukey test) in the mean microglial cell area and perimeter was counteracted by
VB3323 cotreatment. At least 120 cells for each condition were analyzed from three independent experiments. (Q) Purified microglial cul-
tures were harvested after treatment, and cell lysates were processed by Western blot. In the autoradiograms, the anti-CD68 antibody rec-
ognizes two protein bands at different molecular weights (87–115 kDa), which were used for quantification by optical densitometry. Expres-
sion data were normalized relative to actin and are expressed as the percentage of the mean CD68 levels in untreated cells (control).
Data from three independent experiments were analyzed by one-way ANOVA and Tukey test. LPS doubled the CD68 level (***p < 0.001 ver-
sus control), and cotreatment with VB3323 prevented this. There was no stimulation by LPS in TLR4LPS-delcultures. Scale bar, 20 μm.

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centration from supernatants was deter-
mined by the Bradford method (Bio-Rad
Laboratories, Hercules, CA, USA). Pro-
teins were separated by 10% sodium do-
decyl sulfate– polyacrylamide gel elec-
trophoresis and then transferred to a
nitrocellulose membrane. Blots were
blocked and probed with the primary an-
tibody anti-CD68 (rat, 1:500 dilution;
AbD Serotec, Kidlington, UK). The mem-
branes were then washed and incubated
with horseradish peroxidase– conjugated
anti-rat secondary antibodies (1:5,000 di-
lution; Sigma-Aldrich). Blots were devel-
oped, and the densitometric analysis of
autoradiographic bands was done using
image analysis software (ImageJ v1.43;
National Institutes of Health [NIH], Be-
thesda, MD, USA). The integrated optical
density values (normalized for the total
protein content of each sample) were
used as individual data for the statistical
analysis.
In Vivo Treatment
Wobbler mice were originally ob-
tained from NIH genetics and then bred
at Charles River (Calco, Italy). Because
heterozygous mice do not show any
phenotypic difference compared with
homozygous healthy littermates, het-
erozygous founders were designed by
genotyping (23).
Thirty wobbler mice (fourth week of
life) and the same number of age-
matched healthy littermates were ran-
domly recruited in the following experi-
mental groups: (a) vehicle-treated mice;
(b) riluzole-treated mice; and (c) VB3323-
treated mice. The group of riluzole-
treated mice followed the treatment
schedule already reported by our group
(35). Riluzole was provided by Sanofi-
Aventis (Sanofi-Aventis Centre de
Récherche de Paris, Vitry Sur Seine,
Cedex, France). VB3323 was intraperi-
toneally injected three times a week at a
dose of 5 mg/kg/d and at a final concen-
tration of 0.5 mg/mL. All experimental
groups included the same numbers of
male and female mice, since no sex-
related differences were detected during
the symptom progression.
Behavioral Evaluations and Tissue
Preparation
Mice were weighed twice a week. Be-
havioral trials for wobbler mice were
done twice a week by the same operator
blinded to the treatment. Semiquantitative
score evaluation (paw abnormality) and
quantitative measurements (grip strength)
were done, as for previous protocols (35).
Immunohistochemistry and Histology
To ensure optimal quality for cryostat
sections, animals were transcardial per-
fused as previously described (36). For
Nissl staining, cryostatic sections of cer-
vical spinal cord (C2–C6) were serially
cut (30 μm thick) and processed by the
method reported by our group (37). The
mean number of neurons was calculated
for each animal and used for statistical
analysis. Free-floating sections (30 μm
thick) were incubated with GFAP (mouse
monoclonal antibody, Immunological
Science, Rome, Italy; dilution 1:1,000) or
CD11b (mouse monoclonal antibody OX
42; AbD Serotec; dilution 1:300), and
double-staining experiments were car-
ried out by using an antibody against
TNF-α (rat monoclonal antibody; HyCult
Biotech, Uden, the Netherlands; dilution
1:200). To exclude antibody cross-reac-
tions, each mouse monoclonal antibody
was tested with non-species–specific sec-
ondary antibody, and no signal was re-
vealed in this condition.
All supplementary materials are available
online at www.molmed.org.
RESULTS
Distribution of TLR4 in Spinal Cord Cells
In a first set of experiments, TLR4 ex-
pression was analyzed in the resident
R E S E A R C H A R T I C L E
M O L M E D 1 8 : 9 7 1 - 9 8 1 , 2 0 1 2 | D E PA O L A E T A L . | 9 7 5
Figure 3. Live imaging analysis of microglial activation induced by LPS. Purified microglial
cultures were incubated with a GFP-lentiviral construct for 72 h. The success of the infec-
tion was first confirmed by optical microscopy, and the cultures under the different experi-
mental conditions were then analyzed by a time-lapse recording. Pictures at different
time points from the recording of a LPS-treated (1 μg/mL) culture are reported, showing
cell debris phagocytosis by an activated microglial cell (white arrows). Scale bar, 40 μm.

Page 6

cell types of the spinal cord. Motor neu-
rons, in coculture with glia, were ex-
posed to 1 μg/mL LPS, with or without
20 μg/mL VB3323, for different times, to
check whether these agents altered the
expression of TLR4. Immunocytochem-
istry for SMI32 and TLR4 was then done.
As shown in Figure 1, immunopositivity
for TLR4 (red) was widespread in control
conditions (Figure 1B). SMI32-positive
motor neurons (Figure 1A, green) were
stained with the specific anti-TLR4 anti-
body (Figure 1C, merge). Treatment with
the TLR4 ligands did not change the flu-
orescent signal (not shown). In cocul-
tures from TLR4LPS-delembryos, used to
verify the specificity of immunostaining
with the TLR4 antibody, no signal was
obtained (Figure 1D–F).
We then analyzed the expression of
TLR4 on different glial cells, using puri-
fied glial cultures. In purified microglia
cultures, all cells were double-stained by
CD11b-specific (microglial marker, Fig-
ure 2A) and TLR4-specific (Figure 2B)
antibodies. The intensity and distribution
of TLR4 staining was not affected
by treatments with LPS, alone (Fig -
ures 2D–F, J–L) or in combination with
VB3323 (Figures 2G–I, M–O). TLR4 was
not detected in purified astrocyte cul-
tures, in either control conditions or after
LPS treatment (not shown).
Inhibition of LPS-Induced Microglial
Activation by the TLR4 Antagonist
VB3323
We initially characterized the effects
of LPS on the morphology of microglia
and astrocytes in purified cultures.
Cotreatment with VB3323 was also in-
vestigated, to verify whether TLR4 acti-
vation was involved in LPS-induced al-
terations.
Morphological and Biochemical
Alterations in Glial Cultures
Morphological changes in microglia
were investigated by immunocytochem-
istry or time-lapse analysis. In a first set
of experiments, purified microglial cells
were processed by immunocytochemistry
for CD11b, and perimeters and areas of
CD11b-positive cells were measured by an
imaging software tool. The exposure of
cultures to 1 μg/mL LPS for 18 h induced
the activation of microglia, which acquired
a reactive phenotype (Figures 2J–L). At
this time, increases in the mean area and
perimeter (Figures 2J, P) were evident in
treated cells, compared with both basal
(Figure 2A) and 6-h exposure (Figure 2D)
conditions. VB3323 counteracted these ef-
fects (Figures 2M, P).
To investigate whether these morpho-
logical changes were accompanied by
quantitative alterations in specific mi-
croglial biomarkers associated with
phagocytosis, immunoblot analysis for
CD68 was done on cell lysates from puri-
fied microglial cultures. Western blot ex-
periments confirmed that VB3323 pre-
vented the activation of microglia
induced by LPS. Optical densitometry
analysis of the CD68-specific bands
showed a significant increase of CD68 in
the LPS-treated cultures compared with
control (p < 0.001) or VB3323 treatment
(p < 0.01; Figure 2Q). As expected, LPS
did not affect the basal CD68 levels in
TLR4LPS-delmicroglia (Figure 2Q).
For easy cell tracking in time-lapse ex-
periments, microglial cells were infected
with GFP-expressing lentiviral vectors.
This step led to highly efficient transduc-
tion of microglia, as demonstrated by the
widespread GFP expression in the cul-
tures. Cultures were then treated with
1 μg/mL LPS, alone or in combination
with 20 μg/mL VB3323, and time-lapse
recording was done. In representative
video frames (Figure 3) after 10–15 h of
LPS exposure, striking morphological
changes were clearly visible in microglia,
toward an activated phenotype (increased
cell area). Phagocytic events were also
observed in LPS-treated cultures (for ex-
ample, cell debris processed by phagocy-
tosis indicated by the arrows in Figure 3),
but not in the control condition or when
VB3323 was present (not shown).
9 7 6 | D E PA O L A E T A L . | M O L M E D 1 8 : 9 7 1 - 9 8 1 , 2 0 1 2
T L R 4 A N T A G O N I S M P R O T E C T S M O T O R N E U R O N S
Figure 4. TLR4 ligands regulated cytokine release. Motor neuron/glia cocultures were
treated with 1 μg/mL LPS, alone or with 20 μg/mL VB3323 or RsLPS. Culture media were
collected at different times, and cytokine concentrations were measured by ELISA. (A)
Time course of TNF-α, IL-6 and IL-1β release induced by LPS. Concentrations of cytokines in
the culture media peaked between 18 and 24 h after exposure to LPS. (B) VB3323 or
RsLPS significantly reduced the release of TNF-α, IL-6 or IL-1β induced by LPS after 24 h.
***p < 0.001, **p < 0.01, *p < 0.05; one-way ANOVA and Tukey test.

Page 7

Reduction of LPS-Dependent Cytokine
Release by VB3323
The proinflammatory effect of LPS,
and the counteracting action of VB3323,
were further investigated in cocultures of
motor neurons and glia by analyzing the
release of cytokines in the culture
medium. Cultures were exposed to
1 μg/ mL LPS. Conditioned medium was
collected at different times after treat-
ment (3, 6, 18 and 24 h), and the concen-
trations of TNF-α, IL-1β and IL-6 were
measured by ELISA. The time course of
cytokine release showed that the highest
concentrations were reached 18–24 h
after treatment (Figure 4A). Cocultures
were treated with LPS and one TLR4
antagonist (20 μg/mL VB3323 or RsLPS),
and cytokine release was measured after
18 h of exposure. Both TLR4 antagonists
significantly reduced the cytokine release
triggered by LPS (Figure 4B). VB3323 re-
duced the release of the three cytokines
as follows: TNF-α by 47.5 ± 16.1% (p <
0.001 versus LPS), IL-1β by 59.7 ± 20.4%
(p < 0.001) and IL-6 by 29.3 ± 16.3% (p <
0.05). RsLPS had similar effects for
TNF-α and IL-6, but the reduction of
IL-1β release was significantly greater
(87.2 ± 14.1%, p < 0.05; see Figure 4B).
When the cytokine release was mea-
sured in culture medium from cocultures
of motor neurons and astrocytes (with-
out microglia), slightly but not statisti-
cally different levels were revealed.
Prevention of Motor Neuron
Degeneration by VB3323
We investigated whether, in addition
to the proinflammatory effects, TLR4 ac-
tivation also affected motor neuron via-
bility. Motor neuron/glia cocultures were
exposed to 1 μg/mL LPS, with or with-
out 20 μg/mL VB3323 or 20 μg/mL
RsLPS, for 24 h. Neurotoxicity was eval-
uated by comparing motor neuron via-
bility in treated and untreated cultures.
LPS reduced cell viability by 30.8 ± 11.9%
(p < 0.001 versus control; Figure 5A and
representative pictures of LPS-treated
motor neurons in Figures 5E and H).
This toxic effect was significantly coun-
teracted (Figure 5, Finteraction< 0.001) by
VB3323 (representative pictures in Fig-
ures 5F and I) or RsLPS, which both al-
most completely restored motor neuron
viability (91.3 ± 9.9% with p < 0.001 ver-
sus LPS, or 93.4 ± 7.9% with p < 0.001
versus LPS, respectively). We also tested
the effect of the toxic residue of LPS,
lipid A, in the same experimental setting.
Like the full-length endotoxin, 1 μg/mL
lipid A significantly reduced motor
neuron survival by 30% (Figure 5B),
without any dose-dependent effect at
higher concentrations (not shown).
VB3323 (20 μg/ mL) also counteracted
lipid A–induced motor neuron death
(see Figure 5B).
To evaluate whether LPS-induced
activation of glial cells was enough to
induce neurotoxicity, purified motor
neurons were cultured with a LPS-
conditioned glial layer pretreated with
1 μg/mL LPS for 24 h before cocultures
were established. Motor neuron survival
was significantly reduced by 35.1 ±
R E S E A R C H A R T I C L E
M O L M E D 1 8 : 9 7 1 - 9 8 1 , 2 0 1 2 | D E PA O L A E T A L . | 9 7 7
Figure 5. TLR4 modulation affected motor neuron survival. Viability of motor neurons di-
rectly (A and B) or indirectly (C) exposed to TLR4 agonists (1 μg/mL LPS or lipid A) with or
without an antagonist (20 μg/mL VB3323 or RsLPS), under different coculture conditions, is
shown. (A) Motor neuron/glia cocultures were exposed to LPS ± TLR4 antagonists for 24 h.
***p < 0.001; two-way ANOVA and Bonferroni posttest. Finteraction < 0.001. (B) Motor neuron/
glia cocultures were exposed to lipid A ± VB3323 for 24 h. **p < 0.01: one-way ANOVA and
Tukey test. C: Mixed glial layers were exposed to LPS ± VB3323 for 24 h. After removing the
TLR4 ligands, purified motor neurons were added to this conditioned glial layer. These co-
cultures were allowed to grow for 1 wk, and motor neuron viability was assessed. ***p <
0.001, *p < 0.05; two-way ANOVA and Bonferroni posttest. Data are mean number ± stan-
dard deviation (SD) of viable motor neurons normalized against control. At least nine wells
for each condition were analyzed from three independent experiments. (D–I) Representa-
tive pictures of SMI32-positive motor neurons maintained in control conditions (D, G) or
treated with LPS alone (E, H) or in combination with VB3323 (F, I). Scale bar (D–F), 40 μm;
scale bar (G–I), 20 μm.

Page 8

17.4% (p < 0.001 versus control), and
cotreatment with 20 μg/mL VB3323
completely counteracted this effect (Fig-
ure 5C). To investigate which of the
main glial populations mediated this
process, cocultures of motor neurons on
purified astrocytes were established.
When microglia were absent, LPS did
not affect motor neuron viability (not
shown).
Absence of TLR4 on Microglia Prevents
LPS Neurotoxicity
To verify the cell-specific role of TLR4
in LPS neurotoxic effects, we took motor
neurons or glial cells from TLR4LPS-del
mice. This step enabled us to establish co-
cultures of motor neurons, astrocytes and
microglia with different combinations of
cells lacking TLR4, which could be as-
sembled with a controlled cell composi-
tion. When TLR4LPS-delmotor neuron/ glia
cocultures were treated with LPS
(1 μg/mL), motor neuron viability was
not affected (Figure 6A). When cocultures
of TLR4LPS-delmotor neurons over WT
glia were used, LPS reduced viability by
20% (Figure 6B). This result suggests that
functionally active TLR4 on glial cells are
required for LPS to induce neurotoxicity.
We further distinguished the cell-specific
role of TLR4 in the different glial cell
types by establishing cocultures of puri-
fied microglia from TLR4LPS-delor WT
mice, plus WT motor neurons and astro-
cytes. When microglia were from
TLR4LPS-delmice, LPS (1 μg/mL) did not
affect motor neuron viability (Figure 6C).
Thus, TLR4-expressing microglia are re-
quired by LPS to exert neurotoxicity on
motor neurons.
Muscular Decay in Wobbler Mice Is
Reduced by VB3323
There were no significant differences
in the growth rate and in the body
weight progression among the three dif-
ferent experimental groups (not shown).
Four-week-old vehicle-treated wobbler
mice showed a rapid symptoms progres-
sion, as scored by the paw abnormality
degree and the forelegs grip-strength
measurements. Riluzole significantly im-
proved both behavioral trials until the
end of the clinical observation (10th wk
of life). VB3323-treated mice showed im-
provement in both behavioral scores
with earlier and stronger effects com-
pared with the riluzole-treated group
(Figures 7A, B).
Glial Activation and TNF-α α Expression
in the Cervical Spinal Cord of
Wobbler Mice Are Reduced by
VB3323
Compared to control mice (Figure
7C), the morphology of Nissl-stained
large neurons from vehicle-treated wob-
bler mice reveals a marked morphologi-
cal alteration (swollen cell bodies in
Fig ure 7D). Such alterations seem to be
reduced after treatment with riluzole
(Figure 7E) or VB3323 (Figure 7F). In
addition, the relevant process of chro-
matolysis observed in motor neurons of
untreated wobbler mice, which is char-
acterized by an almost overall loss of
cresyl-violet positive staining (see Fig-
ure 7D and representative neuronal cell
bodies in high-magnified pictures in
the panel below), is notably reduced
in riluzole-treated wobbler mice (Fig-
ure 7E). Interestingly, quite intense
staining for cresyl-violet is also ob-
served in VB3323-treated wobbler mice
(Figure 7F). In the cervical spinal cord
of 10-wk-old healthy mice, the im-
munoreactivity for the astrocyte marker
GFAP (red) was very low and no im-
munoreactivity for TNF-α (green sig-
nal) was revealed (Figure 7G). As previ-
ously reported (35,38), an increase in
GFAP and TNF-α staining was ob-
served in vehicle-treated wobbler mice
(Figure 7H). Treatment with riluzole re-
duced both GFAP and TNF-α im-
munoreactivity (Figure 7I). VB3323 in-
duced a greater reduction of GFAP
immunoreactivity, and TNF-α expres-
sion was reduced by a similar extent as
after riluzole treatment (Figure 7J).
Control mice did not show either
CD11b (blue) or TNF-α (green) positive
staining (Figure 7K). Both markers were
markedly increased in vehicle-treated
wobbler mice (Figure 7L) with a relevant
colocalization (light-blue staining). Both
riluzole (Figure 7M) and VB3323 (Fig-
ure 7N) clearly reduced the immunore-
activity for CD11b and TNF-α in wob-
bler mice. In VB3323-treated wobbler
mice, a reduced colocalization of the two
markers was also shown (light-blue in
Figure 7N).
9 7 8 | D E PA O L A E T A L . | M O L M E D 1 8 : 9 7 1 - 9 8 1 , 2 0 1 2
T L R 4 A N T A G O N I S M P R O T E C T S M O T O R N E U R O N S
Figure 6. LPS reduced motor neuron viability through functionally active TLR4 of microglia.
Motor neuron/glia cocultures were established by mixing the different neural populations
from the spinal cords of WT or TLR4LPS-delmouse embryos. Cocultures were then exposed to
1 μg/mL LPS, alone or together with 20 μg/mL VB3323, for 24 h. (A) LPS did not affect
motor neuron viability when both motor neuron and glial cells came from TLR4LPS-delmice.
(B) LPS significantly reduced viability of TLR4LPS-delmotor neurons in coculture with WT glia.
VB3323 prevented the neurotoxic effects of LPS. (C) LPS did not affect the viability of WT
motor neurons in cocultures with WT astrocytes and TLR4LPS-delmicroglia. Data are mean
number ± SD of viable motor neurons normalized against control. Nine wells for each con-
dition were analyzed from three independent experiments. *p < 0.05, one-way ANOVA
and Tukey test.

Page 9

DISCUSSION
In this work, we investigated the role
of TLR4 in in vitro and in vivo settings of
motor neuron degeneration. Primary
cell cultures from the mouse spinal cord
were used to investigate (a) the effects
of TLR4 activation in specific cell types,
(b) the different contribution of glial
populations in motor neuron toxicity
and (c) the protective effects of a spe-
cific TLR4 antagonist. LPS was used to
activate the TLR4-dependent pathways.
We showed, by multiple approaches
(immunocytochemistry, immunoblotting
and live-cell imaging analyses), that in
vitro LPS had a potent proinflammatory
effect on microglia, which acquired a re-
active and phagocytic phenotype, ac-
companied by functional activation, as
demonstrated by the massive increase in
TNF-α, IL-1β and IL-6 release in the cul-
ture medium. No effects were seen in
astrocyte cultures, in accordance with
the lack of receptor expression we
found for this population (15,39). Such
microglia-specific proinflammatory
changes may represent the leading
cause of neurotoxicity on motor neu-
rons. In fact, only in the presence of mi-
croglial cells was LPS able to reduce the
viability of motor neurons in cocultures.
Proinflammatory cytokines including
TNF-α (40), IL-1β (41) and IL-6 (42) are
known to contribute to the process of
motor neuron degeneration. Thus, a pri-
mary role for these cytokines in LPS-
induced neurotoxicity could be hypoth-
esized also in our model. By combining
cell types from spinal cords of control or
TLR4-deficient mice, we also specifically
demonstrated the primary role of mi-
croglial TLR4 in mediating LPS neuro-
toxicity. When TLR4 was lacking in mi-
croglia, in fact, LPS did not affect motor
neuron viability.
Although widely used to induce neu-
roinflammation in experimental models,
stimulation of neural TLR4 by LPS in
mammalian CNS diseases probably oc-
curs only with bacterial infections. LPS is
a high-molecular-weight hydrophilic
molecule that cannot cross the blood-
brain barrier (BBB) (43). We therefore
R E S E A R C H A R T I C L E
M O L M E D 1 8 : 9 7 1 - 9 8 1 , 2 0 1 2 | D E PA O L A E T A L . | 9 7 9
Figure 7. VB3323 treatment significantly improved the behavior performance in wob-
bler mice and reduced reactive astroglyosis. (A, B) Behavioral tests in wobbler mice
treated with vehicle (solid line), riluzole (dotted line) or VB3323 (broken line). Riluzole
was given in drinking water at the dose of 40 mg/kg/d; VB3323 was administered in-
traperitoneally three times a week at the dose of 5 mg/kg/d. The treatments were car-
ried out from the 4th to the 10th week of age. Each point represents the mean ± stan-
dard error of the mean of 10 mice. Statistical analysis for VB3323-treated wobbler mice:
**p < 0.01, ***p < 0.001, different from vehicle-treated wobbler mice; °p < 0.05, °°°p <
0.001, different from riluzole-treated wobbler mice. Riluzole-treated wobbler mice: ^p <
0.05, ^^p < 0.01, ^^^p < 0.001 different from vehicle-treated wobbler mice (Bonferroni
posttest). (C–F) Representative images showing the Nissl-positive stained large neurons
from the ventral horn of cervical spinal cord of 10-wk-old healthy mice (C), and age-
matched vehicle-treated wobbler mice (D), riluzole-treated wobbler mice (E) and
VB3323-treated wobbler mice (F); high magnified (1,000×) neuronal cell bodies are re-
ported in the lower panel for each treatment. (G–J) Colocalization experiments show-
ing the profile of immunoreactivity for GFAP (red), TNF-α (green) and their merge (yel-
low) from the ventral horn of cervical spinal cord of 10-wk-old healthy mice (G) and
age-matched vehicle-treated wobbler mice (H), in riluzole-treated wobbler mice (I)
and in VB3323-treated wobbler mice (J). (K–N) Colocalization experiments showing the
profile of immunoreactivity for CD11b (blue), TNF-α (green) and their merge (light
green) from the ventral horn of cervical spinal cord of 10-wk-old healthy mice (K), and
age-matched vehicle-treated wobbler mice (L), in riluzole-treated wobbler mice (M)
and in VB3323-treated wobbler mice (N). Scale bar (C–F, upper panels), 45 μm; scale
bar (C–F, lower panels), 10 μm; scale bar (G–N), 60 μm.

Page 10

also tested the effects of the hydrophobic
component of LPS, lipid A, which is re-
sponsible for most of the bioactivity of
endotoxin and might, because of its
lipophilic properties, cross the BBB. In-
terestingly, lipid A induced motor neu-
ron death to an extent similar to the full-
length endotoxin. Lipid A acts through
TLR4 (11,12), and, in accordance, cotreat-
ment with TLR4 antagonists counter-
acted its toxicity on motor neurons.
An LPS-like molecule extracted from
the cyanobacterium Oscillatoria Plank-
tothrix FP1 (CyP) dose-dependently in-
hibited TLR4-mediated inflammatory ef-
fects in vitro (32,33) and in vivo in a
model of lethal endotoxin shock (32).
TLR4 inhibition by CyP had an anticon-
vulsant effect on kainate-induced
seizures in mice (44). We investigated the
effect of a highly purified (95%) deriva-
tive of CyP, VB3323. It showed potent
antiinflammatory and neuroprotective
effects in spinal cord cultures, signifi-
cantly counteracting the effects of TLR4
stimulation by LPS (that is, microglial
morphological changes and release of
proinflammatory cytokines and motor
neuron toxicity). The effects of VB3323
were comparable to those of a well-
known, commercially available TLR4 an-
tagonist, the LPS from RsLPS, which is
commonly used to inhibit the TLR4-
dependent inflammatory mechanisms
(45–47).
Considering the antiinflammatory and
neuroprotective effects shown by VB3323
in vitro, it was of interest to test the com-
pound also in an in vivo setting of motor
neuron degeneration.
Inflammation is associated with motor
neuron death in ALS and has been well
characterized in animal models of motor
neuron degeneration. Marked activation
of microglia and astrocytes, and cytokine
overexpression (that is, TNF-α), were
commonly detected in the SOD1 trans-
genic mouse (48–50) and in the wobbler
mouse. The wobbler mouse shows early-
onset selective motor neuron death in the
cervical spinal cord (37) accompanied by
glial activation (21–23) and upregulation
of TNF-α (24) and the activation of both
jun N-terminal kinase (JNK) and p38 mi-
togen-activated stress kinase (p38MAPK)
in glial cells and neurons at the early
phases of symptom progression (38). The
beneficial effect shown by the chronic
treatment with a TNF-α binding protein
(rhTBP-1; 38) further strengthens the pri-
mary role of inflammation in motor neu-
ron degeneration in this model. Thus, we
considered the wobbler mouse as a valid
and useful model to preliminarily test
the effects of chronic treatment by
VB3323 on symptoms progression, motor
neuron protection and glial activation.
Because, similarly to ALS patients
(51–53), the wobbler mouse shows symp-
tom amelioration after riluzole adminis-
tration (35), the effect of VB3323 was
compared not only to vehicle-treated
mice, but also to riluzole, used as a refer-
ence drug. Chronic treatments with the
TLR4 antagonist induced a remarkable
reduction of gliosis and TNF-α produc-
tion in the cervical region of the wobbler
spinal cord. Notably, VB3323 also in-
duced significant improvements in
motor functional tests if compared with
either vehicle or riluzole. Whether or not
VB3323 has a direct effect in the CNS by
passing the BBB remains unknown, but,
as suggested also for other treatments
(riluzole, rhTBP-1), peripheral adminis-
tration of antiinflammatory compounds
might represents an effective pharmaco-
logical approach aimed at modifying the
pathological outcome of neurodegenera-
tive diseases.
CONCLUSION
These combined in vitro/in vivo inves-
tigations add new understanding on the
role of TLR4 in mediating neurotoxicity
in the spinal cord and suggest that TLR4
antagonists could be considered in future
studies as candidate protective agents for
motor neurons in degenerative diseases.
ACKNOWLEDGMENTS
This work was partially supported by
the Lombardy Region Found for the Pro-
motion of Institutional Agreements
(NEPENTE [Network lombardo di eccel-
lenza per lo sviluppo di farmaci di orig-
ine naturale diretti alla modulazione del
microambiente tissutale per la preven-
zione e terapia dei tumori e delle malattie
neurodegenerative] project ID 14501). A
fellowship to A Mariani was funded by
the Cariplo Foundation (grant 2009-2426)
(Milan, Italy). The authors acknowledge
Alessandro Soave for artwork and Judy
Baggott for the English revision.
DISCLOSURE
M Molteni was employed by Bluegreen
Biotech.
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