Neuroprotective Effects of Toll-Like Receptor 4 Antagonism in Spinal Cord Cultures and in a Mouse Model of Motor Neuron Degeneration
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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 vivosettings 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 cyanobacteriaderived 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.
In pathological conditions, persistent immune-stimulating signals may induce aberrant microglial activation with elevated cytokine release, eventually leading to neuronal injury. Toll-like receptors (TLRs) recognize highly conserved structural motifs from either pathogens or damaged and stressed tissues and are involved in microglial response to physiological and pathological signals. TLR4, in particular, reportedly mediates neuroprotective (1,2) and neurotoxic (3, 4, 5) effects, suggesting that TLR4 activation needs to be tightly controlled, to regulate the switch between its different roles in immune 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 potent 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 represents the endotoxic principle of LPS and is responsible for its pathophysiological effects (10) via TLR4 (11,12). There is convincing evidence that LPS/TLR4 signaling is involved in various human and experimental 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 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 activation of these pathways in the spinal cord may contribute to the progression of inflammation, resulting in motor neuron injury. However, the specific role of TLR4 in the spinal cord cell population, and the mechanism(s) by which TLR4 activation triggers inflammation and neurotoxicity 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 antagonist (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 neuron 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 ). Glial activation (21, 22, 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 wobbler mouse represents a useful model for testing candidate treatments aimed at modifying the neuroinflammatory mechanisms 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 cultures 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 albumin (BSA) cushion. Cells obtained at this step were a mixed neuron/glia population and were centrifuged (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 containing confluent mixed glial cultures were shaken overnight at 275 rpm in incubators. The supernatants (containing microglial cells) were collected and seeded at a density of 20,000 cells/cm2 into new flasks for Western blot analysis, plates or microslides (8-well µSlides; Ibidi GmbH, Martinsried, Germany) for time-lapse analysis. The purification yield of microglial cells with this protocol was about 98% (not shown). Astrocyte-enriched cultures 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 cocultures, the motor neuron-enriched fraction (obtained from the iodixanol-based separation) was seeded at a density of 10,000 cells/cm2 onto mature glial layers composed of either mixed glial cells or purified astrocytes. In these cocultures, about 84 ± 5% (n = 6) of the neuronal cells (revealed by neurofilament staining; Supplementary Figure S1A) were SMI32-positive cells with the typical motor neuron morphology (Supplementary Figure S1B; SMI32-positive cells merged with neurofilament staining in Supplementary Figure 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 (Supplementary Figures S1D-F).
For combined mixed cocultures from wild-type and TLR4LPS-del mouse 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 assayed as follows: only the SMI32-positive cells, with typical morphology (triangular shape, single well defined axon), large bodies (>20 µm) and intact axons and dendrites were counted, at a magnification of 200µ, 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 appropriate control wells. In a typical experiment with cocultures, the number of counted SMI32-positive cells in control (untreated) wells was 70 ± 11 (n = 12).
Cocultures were exposed to the different TLR4 ligands (diluted to the appropriate concentrations in culture medium) on the sixth day in vitro. Cultures maintained with normal medium served as the control condition. Motor neuron death was determined 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 evaluated after simultaneous treatment with the TLR4 agonists (LPS or lipid A). For the other investigations (microglial activation, cytokine release, TLR4 expression), 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 condition, as previously reported (29). Treatment 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 is a highly (95%) purified form of cyanobacterial LPS-like molecule (CyP), extracted from the freshwater cyanobacterium Oscillatoria planktothrix 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 temperature 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% paraformaldehyde or methanol (for glial fibrillary acidic protein [GFAP] staining). When indicated, 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 fluorescent secondary antibody for immunofluorescence (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 separately 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 neurofilament 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 fluorescent secondary antibodies conjugated to different fluorochromes were used at 1:1,000 dilution. Biotinylated anti-mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) was used to analyze motor neuron viability.
Microscopy and Live Imaging Analysis
For the immunocytochemistry experiments, pictures of stained cells were obtained 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 (Olympus BX51) was used for the neurotoxicity analysis. For time-lapse analysis, images were collected and analyzed with a CellR imaging station (Olympus) coupled to an inverted microscope (Olympus 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 analysis, purified microglial cultures were seeded at a density of 15,000 cells/cm2 into eight-well microslides and maintained in culture for 1 wk. They were then infected with lentiviral vectors (2.0 × 109 titer unit (T.U.)/well) expressing green fluorescent protein (GFP) reporter, 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) encoding 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 promoter. Briefly, 293T cells were plated at 70% confluence in Dulbecco’s modified Eagle’s medium, 10% fetal calf serum, 1% glutamine, 1% penicillin and 1% streptomycin and maintained at 37°C in a humidified 5% CO2 incubator. Four plasmids were cotransfected with an Effectene kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions, 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 filter and concentrated by ultracentrifugation at 50,000g at 4°C for 2 h. Viral pellets were resuspended in sterile phosphate-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 dilution on HEK293 cells (34).
Quantitative Enzyme-Linked Immunosorbent Assays
TNF-α, interleukin (IL)-6 and IL-1β concentrations in cell culture supernatants were quantified by solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) (eBioscience). Samples from each experiment were tested in triplicate, according to the manufacturer’s instructions. The sensitivity of the kits was 8 pg/mL for TNF-α and IL-1β and 4 pg/mL for IL-6.
Microglial cultures were treated with ice-cold lysis buffer, and the protein concentration from supernatants was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated by 10% sodium do-decyl sulfate-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. Blots were blocked and probed with the primary antibody anti-CD68 (rat, 1:500 dilution; AbD Serotec, Kidlington, UK). The membranes were then washed and incubated with horseradish peroxidase-conjugated anti-rat secondary antibodies (1:5,000 dilution; Sigma-Aldrich). Blots were developed, and the densitometric analysis of autoradiographic bands was done using image analysis software (ImageJ v1.43; National Institutes of Health [NIH], Bethesda, 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 obtained 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, heterozygous founders were designed by genotyping (23).
Thirty wobbler mice (fourth week of life) and the same number of age-matched healthy littermates were randomly recruited in the following experimental groups: (a) vehicle-treated mice; (b) riluzole-treated mice; and (c) VB3323-treated mice. The group of riluzoletreated 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 intraperitoneally injected three times a week at a dose of 5 mg/kg/d and at a final concentration 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. Behavioral 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 perfused as previously described (36). For Nissl staining, cryostatic sections of cervical 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 carried out by using an antibody against TNF-α (rat monoclonal antibody; HyCult Biotech, Uden, the Netherlands; dilution 1:200). To exclude antibody cross-reactions, each mouse monoclonal antibody was tested with non-species-specific secondary antibody, and no signal was revealed in this condition.
All supplementary materials are available online at www.molmed.org .
Distribution of TLR4 in Spinal Cord Cells
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 investigated, to verify whether TLR4 activation was involved in LPS-induced alterations.
Morphological and Biochemical Alterations in Glial Cultures
Morphological changes in microglia were investigated by immunocytochemistry 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 effects (Figures 2M, P).
To investigate whether these morphological changes were accompanied by quantitative alterations in specific microglial biomarkers associated with phagocytosis, immunoblot analysis for CD68 was done on cell lysates from purified microglial cultures. Western blot experiments confirmed that VB3323 prevented 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-del microglia (Figure 2Q).
Reduction of LPS-Dependent Cytokine Release by VB3323
When the cytokine release was measured in culture medium from cocultures of motor neurons and astrocytes (without microglia), slightly but not statistically different levels were revealed.
Prevention of Motor Neuron Degeneration by VB3323
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 ng/mL LPS for 24 h before cocultures were established. Motor neuron survival was significantly reduced by 35.1 ± 17.4% (p < 0.001 versus control), and cotreatment with 20 µg/mL VB3323 completely counteracted this effect (Figure 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
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 different experimental groups (not shown). Four-week-old vehicle-treated wobbler mice showed a rapid symptoms progression, as scored by the paw abnormality degree and the forelegs grip-strength measurements. Riluzole significantly improved both behavioral trials until the end of the clinical observation (10th wk of life). VB3323-treated mice showed improvement in both behavioral scores with earlier and stronger effects compared with the riluzole-treated group (Figures 7A, B).
Glial Activation and TNF-α Expression in the Cervical Spinal Cord of Wobbler Mice Are Reduced by VB3323
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 (Figure 7N) clearly reduced the immunoreactivity for CD11b and TNF-α in wobbler mice. In VB3323-treated wobbler mice, a reduced colocalization of the two markers was also shown (light-blue in Figure 7N).
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 specific 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 reactive and phagocytic phenotype, accompanied by functional activation, as demonstrated by the massive increase in TNF-α, IL-1β and IL-6 release in the culture 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 neurons. In fact, only in the presence of microglial 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 primary role for these cytokines in LPS-induced neurotoxicity could be hypothesized 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 microglial TLR4 in mediating LPS neurotoxicity. When TLR4 was lacking in microglia, in fact, LPS did not affect motor neuron viability.
Although widely used to induce neuroinflammation in experimental models, stimulation of neural TLR4 by LPS in mammalian CNS diseases probably occurs only with bacterial infections. LPS is a high-molecular-weight hydrophilic molecule that cannot cross the blood-brain barrier (BBB) (43). We therefore also tested the effects of the hydrophobic component of LPS, lipid A, which is responsible for most of the bioactivity of endotoxin and might, because of its lipophilic properties, cross the BBB. Interestingly, lipid A induced motor neuron death to an extent similar to the full-length endotoxin. Lipid A acts through TLR4 (11,12), and, in accordance, cotreatment with TLR4 antagonists counteracted its toxicity on motor neurons.
An LPS-like molecule extracted from the cyanobacterium Oscillatoria Planktothrix FP1 (CyP) dose-dependently inhibited TLR4-mediated inflammatory effects in vitro (32,33) and in vivo in a model of lethal endotoxin shock (32). TLR4 inhibition by CyP had an anticonvulsant effect on kainate-induced seizures in mice (44). We investigated the effect of a highly purified (95%) derivative of CyP, VB3323. It showed potent antiinflammatory and neuroprotective effects in spinal cord cultures, significantly 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 antagonist, the LPS from RsLPS, which is commonly used to inhibit the TLR4-dependent inflammatory mechanisms (45, 46, 47).
Considering the antiinflammatory and neuroprotective effects shown by VB3323 in vitro, it was of interest to test the compound 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 transgenic mouse (48, 49, 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, 22, 23) and upregulation of TNF-α (24) and the activation of both jun N-terminal kinase (JNK) and p38 mitogen-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 primary role of inflammation in motor neuron 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, 52, 53), the wobbler mouse shows symptom amelioration after riluzole administration (35), the effect of VB3323 was compared not only to vehicle-treated mice, but also to riluzole, used as a reference drug. Chronic treatments with the TLR4 antagonist induced a remarkable reduction of gliosis and TNF-α production in the cervical region of the wobbler spinal cord. Notably, VB3323 also induced 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 administration of antiinflammatory compounds might represents an effective pharmacological approach aimed at modifying the pathological outcome of neurodegenerative diseases.
These combined in vitro/in vivo investigations 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.
M Molteni was employed by Bluegreen Biotech.
This work was partially supported by the Lombardy Region Found for the Promotion of Institutional Agreements (NEPENTE [Network lombardo di eccellenza per lo sviluppo di farmaci di origine naturale diretti alla modulazione del microambiente tissutale per la prevenzione 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.
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