Innate immunity is a specific and organized immunological program engaged by peripheral organs and the CNS to maintain homeostasis after stress and injury. In neurodegenerative disorders, its putative deregulation, featured by inflammation and activation of glial cells resulting from inherited mutations or viral/bacterial infections, likely contributes to neuronal death. However, it remains unclear to what extent environmental factors and innate immunity cooperate to modulate the interactions between the neuronal and non-neuronal elements in the perturbed CNS. In the present study, we addressed the effects of acute and chronic administration of lipopolysaccharide (LPS), a Gram-negative bacterial wall component, in a genetic model of neurodegeneration. Transgenic mice expressing a mutant form of the superoxide dismutase 1 (SOD1G37R) linked to familial amyotrophic lateral sclerosis were challenged intraperitoneally with a single nontoxic or repeated injections of LPS (1 mg/kg). At different ages, SOD1G37R mice responded normally to acute endotoxemia. Remarkably, only a chronic challenge with LPS in presymptomatic 6-month-old SOD1G37R mice exacerbated disease progression by 3 weeks and motor axon degeneration. Closely associated with the severity of disease is the stronger and restricted upregulation of the receptor of innate immunity Toll-like receptor 2 and proinflammatory cytokines in degenerating regions of the ventral spinal cord and efferent fiber tracts of the brain from the LPS-treated SOD1G37R mice. This robust immune response was not accompanied by the establishment of acquired immunity. Our results provide solid evidence that environmental factors and innate immunity can cooperate to influence the course of disease of an inherited neuropathology.
- innate immunity
- amyotrophic lateral sclerosis
- superoxide dismutase 1
- proinflammatory cytokines
- transgenic mice
The innate immune response is a rapid and coordinated cascade of reactions by cells of the host to pathogens and insults (Akira et al., 2001; Nguyen et al., 2002). In the CNS, the accuracy of this system can protect neurons by favoring remyelination and trophic support afforded by glial cells. Conversely, its deregulation might be harmful for neuronal integrity and might trigger neurodegeneration (Nguyen et al., 2002; Wyss-Coray and Mucke, 2002). Actually, the dual nature of the innate immune response relies on the fine-tuned regulation of microglial cells, the resident macrophages of the CNS (Nguyen et al., 2002; Wyss-Coray and Mucke, 2002).
Interestingly, numerous proinflammatory genes are induced in the CNS of presymptomatic mice expressing a mutant form of superoxide dismutase 1 (SOD1G37R) linked to amyotrophic lateral sclerosis (ALS), the most common form of human motor neuron disease (Nguyen et al., 2001b, 2002). ALS is an adult-onset neurological disorder characterized by the selective degeneration of motor neurons, culminating in paralysis and death within 3-5 years (Cleveland and Rothstein, 2001; Julien, 2001). Missense mutations in the gene coding for the Cu-Zn SOD1, located on chromosome 21, account for ∼20% cases of familial ALS (Rosen et al., 1993; Cudkowicz et al., 1997). The SOD1 protein is a cytosolic metalloenzyme catalyzing the conversion of superoxide anions to hydrogen peroxide (Fridovich, 1986). Transgenic mice expressing mutant SOD1 develop motor neuron disease resembling ALS, through a gain of unidentified deleterious properties (Wong et al., 1995; Bruijn et al., 1997). Several mechanisms have been proposed to account for such toxicity including excitotoxicity (Bruijn et al., 1997), disruption of the calcium homeostasis (Morrison et al., 1996; Roy et al., 1998), cytoskeletal abnormalities (Wong et al., 1995), Fas ligand (FasL)-mediated death (Raoul et al., 2002), and deregulation of Cdk5 (cyclin-dependent kinase 5) (Nguyen et al., 2001a).
Despite these findings, the toxicity of SOD1 mutants linked to human ALS remains poorly understood. SOD1 is a ubiquitously expressed protein, and therefore, it is possible that cells other than motor neurons play a role in ALS (Clement et al., 2003). Indeed, a restricted expression of mutant SOD1G37R to neurons in transgenic mice was not sufficient to provoke motor neuron disease (Pramatarova et al., 2001; Lino et al., 2002). Neither did the selective expression of mutant SOD1 in astrocytes provoke pathology despite astrocytosis (Gong et al., 2000). Compatible with this view of non-cell autonomy are studies reporting dysfunction of astrocytes, inflammatory processes, and activation of microglia and other immune cells in ALS patients and mice (Bruijn et al., 1997; Nguyen et al., 2001b, 2002).
The use of antiinflammatory compounds has recently conferred protection in models of neurodegeneration, including ALS (Drachman and Rothstein, 2000; Kriz et al., 2002; Schenk, 2002). However, it remains unclear whether these compounds mediate their beneficial effects directly on neurons or through the modulation of microglial activity and innate immunity. In addition, the modulation of neurodegenerative processes by environmental factors remains unexplored. In the present study, we triggered the innate immunity of SOD1G37R mice with systemic administration of lipopolysaccharide (LPS), a potent activator of microglia. Our results demonstrate that chronic activation of innate immunity by systemic LPS is noxious to motor neurons bearing SOD1G37R linked to ALS. We advance a model linking environmental factors and innate immunity that, potentially, might be extrapolated to sporadic cases of neurodegeneration.
Materials and Methods
Generation of SOD1G37R mice and protocol for LPS injection. The inbred C57BL/6 SOD1G37R mice (line 29) used in this study have a life span of 10-12 months (Nguyen et al., 2000). C57BL/6 SOD1G37R mice (line 42) exhibit a life span of 5-6 months (Wong et al., 1995). The mouse genotypes were determined by Southern blotting of tail DNA. The SOD1G37R mice were housed at room temperature (21°C) and in a light-controlled environment with ad libitum access to food and water. To avoid any potential interference with the effects of LPS, mice were kept in a pathogen-free facility. To trigger an acute innate immune response in the CNS, presymptomatic 3-, 6-, and 9-month-old SOD1G37R mice received a single intraperitoneal injection of LPS (1 mg/kg of body weight; from Escherichia coli; serotype 055:B5; Sigma, Saint Louis, MO) diluted in 100 μl of vehicle (Veh) solution (sterile pyrogen-free saline) or vehicle solution. At different times after the systemic injections (from 30 min to 24 hr), animals were deeply anesthetized via an intraperitoneal injection of a mixture of ketamine hydrochloride (91 mg/ml) and xylazine (9 mg/ml) and then rapidly perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 m borax buffer, pH 9.5, at 4°C. Brains and spinal cords were rapidly removed from the animals, postfixed for 2-8 d, and then placed in a solution containing 10% sucrose diluted in 4% paraformaldehyde-borax buffer overnight at 4°C. The frozen tissues were mounted on a microtome (Reichert-Jung; Cambridge Instruments, Deerfield, IL) and cut into 20 μm coronal or longitudinal (spinal cord) sections from the olfactory bulb to the end of the spinal cord. The slices were collected in a cold cryoprotectant solution (0.05 m sodium phosphate buffer, pH 7.3, 30% ethylene glycol, 20% glycerol) and stored at -20°C.
Another group of presymptomatic 6-month-old SOD1G37R mice received intraperitoneal LPS or vehicle injections once every 2 weeks for a duration of 3 months. Around month 9 (42-43 weeks of age), the chronically LPS-treated SOD1G37R mice exhibited the first signs of paralysis, and the injections were stopped. The end stage of disease was observed 3 weeks later (45-46 weeks; 10 months of age), which corresponds to the life span of these mice. At this stage, in situ hybridization, staining, and axonal counts were performed. At the same time, Veh-SOD1 or SOD1 mice that did not show signs of paralysis were killed to have a matched control group for the equivalent analysis. Some of the Veh-SOD1 or SOD1 were left until they exhibited the paralytic phenotype (3-5 weeks after the LPS-SOD1), which permitted the calculation of their life span. Thus, the analysis of the Veh-SOD1 and SOD1 mice was performed strictly before the onset and not at the end stage of disease.
In situ hybridization and histological preparations. The riboprobes used in this study are listed in Table 1. In situ hybridization using 35S-labeled cRNA probes and quantification of the signals were accomplished as described previously (Laflamme et al., 1999; Nadeau and Rivest, 2000). Double-labeling procedures were similar to those described in other studies published by our group (Laflamme et al., 1999; Nadeau and Rivest, 2000, 2002). Neuronal death was detected by the Fluoro-Jade B (FJB) method (see below). Briefly, every sixth section of the entire rostrocaudal extent of each brain and spinal cord was mounted onto poly-l-lysine-coated slides, dried under vacuum for 2 hr, dehydrated through graded concentrations of alcohol (50, 70, and 100%; 1 min), rehydrated through graded concentrations of alcohol (100, 70, and 50%; 1 min each) and 1 min in distillated water. They were then dipped and shacked into potassium permanganate (0.06%) for 10 min, rinsed 1 min in distilled water, and dipped and shacked in a solution containing Fluoro-Jade B [Fluoro-Jade B (0.0004%; Histochem, Jefferson, AR) plus acetic acid (0.1%; catalog #A-6404; Sigma) plus 4′,6′-diamidino-2-phenylindole (DAPI) (0.0002%; catalog #D-1306; Molecular Probes, Eugene, OR)] for 20 min. The slides were thereafter rinsed three times in distilled water (1 min each), dried, dipped in xylene three times (2 min each), and coverslipped with distrene plasticizer xylene. FJB is novel fluorescent dye that has high affinity for dying neurons and does not discriminate between apoptosis and necrosis (Schmued et al., 1997). This explains our choice to use this dye, because it is still unclear whether unusual apoptosis or necrosis is the dominant form of cell death in ALS (Migheli et al., 1999; Pasinelli et al., 2000; Vukosavic et al., 2000).
Morphological and morphometric analyses. Mice were killed by overdose of chloral hydrate, perfused with 0.9% NaCl and then with fixative (3% v/v glutaraldehyde in PBS buffer, pH 7.4). Tissue samples were immersed in fixative overnight, rinsed in phosphate buffer, and then postfixed in 1% phosphate-buffered osmium tetroxide. After three washes with phosphate buffer, each sample was dehydrated in a graded series of ethanol and embedded in Epon. The thin sections of spinal cord and L5 ventral root were stained with toluidine blue and examined under a light microscope. The counting of axons in the L5 ventral root was performed with the Image-1 software from Universal Imaging Corporation (West Chester, PA).
Western blots. Mice were killed by intraperitoneal injection of chloral hydrate. Total protein extracts of spinal cord were obtained by homogenization in SDS-urea β-mercaptoethanol, 0.5% SDS, 8 m urea in phosphate buffer, pH 7.4, with a mixture of protease inhibitors (PMSF, leupeptin, pepstatin, and aprotinin). The protein concentration was estimated by the Bradford procedure (Bio-Rad, Hercules, CA). Proteins were fractionated on 12-15% SDS-PAGE and blotted on a nitrocellulose or polyvinylidene difluoride membrane for Western blot analysis. Membranes were incubated with antibodies against SOD1 (Biodesign; Santa Cruz Biotechnology, Santa Cruz, CA), α-tubulin (B512; Sigma), and actin (MAB 1501; Chemicon, Temecula, CA). The Western blots were revealed by Renaissance, a Western blot chemiluminescence kit from NEN (Boston, MA).
Statistical analysis. The statistical analyses for the different dependent variables were performed by a two-way ANOVA followed by a Bonferroni-Dunn test procedure as post hoc comparison. Please see the figures for more specific details.
SOD1G37R mice exhibited a normal innate immune response after an acute systemic LPS injection
A single systemic LPS injection caused a robust increase in the expression of Toll-like receptor (TLR)2 mRNA across the brain and spinal cord of both normal [wild-type (WT)] and SOD1G37R mice (Fig. 1). The expression wave of this transcript was similar in the CNS of both mouse strains at all of the times evaluated (i.e., 30 min, 3, 6, and 24 hr). These small, scattered positive cells that were found across the brain, L5 segment, and cervical part of the spinal cord are microglial cells (see Fig. 3) (Laflamme et al., 2001). Indeed, an antibody directed against the ionized calcium-binding adapter molecule 1 (iba1) was used to stain microglia that were positive for TLR2 transcript in the CNS of LPS-treated mice (Laflamme et al., 2001). This was also the case for numerous other genes involved in the innate immune system, such as IκBα [inhibitory protein of nuclear factor-κB (NF-κB)] (index of NF-κB activity), tumor necrosis factor-α (TNF-α), and CD14 (data not shown). Microglia are therefore the main group of cells expressing most of the genes involved in the control of the innate immune response in the CNS after LPS administration.
Semiquantitative analyses revealed similar increases in expression levels of TLR2 mRNA in different regions of the CNS in both WT and SOD1G37R groups of mice challenged acutely with LPS (Fig. 1D,E). The area postrema (AP) and spinal L5 segment of LPS-treated SOD1G37R mice did not exhibit a different hybridization signal when compared with WT animals. These results indicate that SOD1G37R are not more sensitive to the endotoxin and, therefore, exhibit a normal cerebral innate immune response after acute endotoxemia.
Chronic systemic injections of LPS exacerbate disease progression and motor axon degeneration in SOD1G37R mice
Remarkably, a chronic systemic injection of an equal dose of LPS in presymptomatic 6-month-old SOD1G37R mice had a significant effect on their life span. Figure 2A shows the survival curve of SOD1G37R mice (line 29) treated chronically with Veh or LPS. The SOD1G37R mice treated with vehicle (n = 16) used as control exhibited an average life span of 49.4 ± 2.7 weeks and a median life probability of 49.0 weeks, which is not different from the life span of the nontreated SOD1G37R mice (Nguyen et al., 2000, 2001a). The median life probability is defined as the age at which the probability of survival is 50%. SOD1G37R mice treated chronically with LPS (n = 13) exhibited an average life span of 45.5 ± 2.2 weeks and a median life probability of 46.0 weeks. These results demonstrate that repeated intraperitoneal injections of a nontoxic dose of LPS in presymptomatic 6-month-old SOD1G37R mice exacerbated disease progression by 3 weeks.
To verify whether this acceleration in disease progression is caused by a more severe loss of motor axons, we examined at light microscopy and counted the number of axons in L5 ventral roots of SOD1G37R mice treated chronically with LPS or vehicle and killed at 10 months of age. At this age, the L5 ventral roots of LPS-treated SOD1G37R mice were smaller when compared with those dissected from Veh-SOD1G37R mice (Fig. 2B). Furthermore, LPS-treated SOD1G37R mice (n = 5) had 358 ± 48 axons, whereas Veh-treated SOD1G37R mice (n = 4) had 478 ± 40 (Table 2). Thus, at this age, there was a more severe loss of motor axons in SOD1G37R mice that received repeated injections with the endotoxin. These data indicate that exacerbation of disease progression is caused by accelerated degeneration of motor axons. It is noteworthy that the chronic injection of LPS in WT mice did not cause neurodegeneration (1033 ± 46; n = 4) as confirmed by axonal counts (Table 2).
Inflammatory response in chronically treated mice
We next assessed the transcriptional activation of the receptor of innate immunity TLR2 and the proapoptotic cytokine TNF-α, in the SOD1G37R mice challenged chronically with LPS. TLR2 is induced during disease progression of SOD1G37R mice and especially, in late stages, where massive neurodegeneration occurs (Gurney et al., 1994; Nguyen et al., 2001a,b). The physiological role for such induction remained unexplained. During endotoxemia, TLR expression has been suggested to be neuroprotective by favoring the elimination of pathogens (Akira et al., 2001). Similarly, a chronic expression of TLR2 might also trigger microglial apoptosis and thereby prevent the detrimental effects of subsequent inflammation (Aliprantis et al., 1999, 2000; Laflamme et al., 2001).
The innate immune response was much more pronounced in chronically LPS-treated SOD1G37R mice than in Veh-treated SOD1G37R mice (Figs. 3, 4, 5). The robust expression of TLR2 mRNA (Fig. 3D,G) is associated with strong hybridization signals for the genes encoding the proapoptotic cytokines TNF-α andinterleukin-12 (IL-12) in degenerating efferent fiber tracts of the brain (Fig. 3B,C) and in degenerating ventral spinal horns (Fig. 4A, rows 2-4). Degenerating neurons were labeled via FJB staining that clearly overlapped with the hybridization signal (Figs. 3E,F, 4, row 5). Indeed, positive FJB fibers were found in the reticular formation just above the olivary complex (Fig. 3E) and numerous other regions receiving projections from the spinal cord. The immune response and microglial activation were therefore highly associated with the degenerative groups of neurons, a phenomenon that was clearly exacerbated by chronic treatment with the bacterial cell wall component (Fig. 5).
In addition, a higher number of FJB-positive astrocytes were found in the spinal cord of chronically LPS-treated mutant SOD1 mice (Fig. 4B). Disturbance in functions of astrocytes is one of the first pathological changes observed in patients and rodent models with ALS (Rothstein et al., 1995; Bristol and Rothstein, 1996; Bruijn et al., 1997) that can lead to excitotoxicity-evoked motor neuron death (for review, see Cleveland and Rothstein, 2001; Julien, 2001).
Absence of adaptive immunity in the CNS of LPS-treated SOD1G37R mice
To determine whether upregulated innate immunity transfers to the adaptive form, we performed in situ hybridization on spinal cord and brain tissues using highly sensitive probes for interferon-γ (IFN-γ) and IL-12, two cytokines essential for the transfer from the innate to the acquired immunity. Figures 3C and 4A, row 3, show an upregulation of IL-12 in degenerating CNS regions of the chronically LPS-treated SOD1G37R mice when compared with chronically Veh-treated SOD1G37R mice. However, in situ hybridization failed to detect positive signal for IFN-γ transcript in the brain and spinal cord of SOD1G37R mice treated acutely or chronically with the endotoxin. Immunohistochemistry using antibodies directed against CD4+ or CD8+ also failed to provide anatomical evidence of infiltrating T cells in the CNS of both vehicle- and LPS-treated SOD1G37R mice (data not shown). The robust innate immune reaction is therefore not associated with an adaptive immune response.
Accelerated neurodegeneration in chronically LPS-treated SOD1G37R mice is not attributable to an upregulation in levels of superoxide dismutases 1
Emerging evidence indicates that LPS can induce SOD1, which subsequently might play an important role in mediating the immune response (Marikovsky et al., 2003). Central to the understanding of mechanisms causing accelerated neurodegeneration in chronically LPS-treated SOD1G37R mice is whether LPS upregulates expression of the transgene SOD1G37R. Indeed, transgenic mice having higher levels of SOD1G37R (line 42) exhibit a more aggressive pathology with previous onset (Wong et al., 1995). To address this question, protein levels for both endogenous mouse SOD1 (mSOD1) and human mutant SOD1G37R (hSOD1) were determined in SOD1G37R mice in response to acute or chronic injection of LPS. As shown in Figure 6, mSOD1 and hSOD1 levels in spinal cord and spleen were unchanged 24 hr after acute injection of LPS when compared with animals treated with vehicle solution (Veh). Similar effects were found 48 hr postinjection (data not shown). In addition, SOD1G37R mice chronically challenged with LPS did not display increased levels of both enzymes. These results clearly indicate that toxicity caused by chronic LPS treatment is not caused by enhanced SOD1 expression.
Activation of innate immunity featured by the expression of TLR2 and several other inflammatory genes takes place in transgenic mice expressing SOD1G37R linked to ALS (Nguyen et al., 2001b). The results presented here demonstrate that repeated LPS injections exacerbated the pathogenesis and neuronal death processes of ALS caused by SOD1 mutation. Although SOD1G37R mice responded normally to an acute systemic injection of LPS, chronic stimulation of innate immune response with the bacterial cell wall component led to sustained induction of TLR2 receptor strictly within parenchymal microglial cells. This upregulation of TLR2 was not accompanied by enhanced expression of both endogenous and transgene SOD1 (Fig. 6). Most importantly, the degree of TLR2 induction correlated perfectly with degenerating motor neurons and motor axons, as demonstrated by the overlap between FJB, the in situ hybridization signals for the different immune transcripts, and axonal counts. As a consequence, the chronically LPS-treated SOD1G37R mice exhibited accelerated disease progression, motor axon degeneration, and a life span shortened by 3 weeks. Interestingly, chronically LPS-treated SOD1G37R mice exhibited a more important loss of astrocytes than Veh-treated or nontreated SOD1G37R mice. Thus, the profound innate immune reaction by microglial cells in the degenerating area is clearly detrimental for the cerebral tissue. These results are compatible with a non-cell-autonomous mechanism of motor neuron death in ALS mice (Clement et al., 2003).
The endotoxin LPS is able to activate microglia, because these cells are of myeloid lineage and express CD14 and TLR4 receptors (Lacroix et al., 1998; Laflamme and Rivest, 2001; Lehnardt et al., 2002). These receptors and proinflammatory cytokines are involved in controlling the innate immune response and, potentially, in orchestrating the transfer to an adaptive immune response (Nguyen et al., 2002). In the periphery, cytokines acting directly on macrophages are immediately produced on TLR activation, whereas those that mediate the transition from innate to adaptive immune response appear after a few hours. Binding of pathogen-associated molecular patterns to their respective TLRs leads to the release of IL-12, a cytokine that is involved in the transition from innate to the adaptive immunity. Indeed, macrophage-derived IL-12 stimulates the differentiation of a subset of T lymphocytes (CD4+) into T helper 1 cells that produce IFN-γ (Moser and Murphy, 2000; Nguyen et al., 2002). One of the mechanism by which the deregulated innate immune response may induce the selective killing of motor neurons is by establishing and orchestrating an adaptive immune response (Nguyen et al., 2002; Wyss-Coray and Mucke, 2002). This latter assumption is supported by studies on human patients with neurodegenerative disorders including ALS, reporting the expression of molecules of adaptive immunity, such as major histocompatibility complex (MHC) class I, MHC class II, and human histocompatibility leukocyte antigen in brains, spinal cords, CSF, and sera (Kriz et al., 2002; Nguyen et al., 2002; Schenk, 2002; Wyss-Coray and Mucke, 2002).
Although a robust signal for the gene encoding IL-12 was found in the CNS of LPS treated-SOD1G37R mice, the cerebral tissue of these animals did not exhibit positive signal for IFN-γ transcript. Immunohistochemistry also failed to detect infiltrating CD4+ and CD8+ cells in the brain and spinal cord of SOD1G37R mice treated chronically with vehicle or LPS. These results do not support the concept that deregulation of innate immunity is associated with a specific transfer to an adaptive immune response in ALS mice. Alternatively, defects in the fine interplay between innate and acquired immunity or in the transfer from innate to adaptive response may be toxic for the CNS of the LPS-treated SOD1G37R and SOD1G37R mice. For instance, upregulation of the local adaptive immune response in SOD1G93A mice with Copaxone (glatiramer acetate) vaccination eliminates destructive self-compounds associated with motor neuron death resulting in protection of motor neurons and extension of the life span of the animals. In addition, numerous studies have reported a protective role of the fine-tuned adaptive immune response (Warrington et al., 2000; Bieber et al., 2001; Kriz et al., 2002; Mitsunaga et al., 2002; Nguyen et al., 2002; Schenk, 2002; Wyss-Coray and Mucke, 2002).
The proapoptotic cytokine TNF-α is likely to play a determinant role in this model. The endotoxin LPS is able to trigger transcriptional activation of the gene encoding TNF-α in microglial cells across the CNS, and TNF-α gene expression progressively increased in the spinal cord of SOD1G37R mice (Nadeau and Rivest, 2000; Nguyen et al., 2001b). High TNF-α levels are also found in the CSF of ALS patients (Poloni et al., 2000). This cytokine shares with TLR2 common downstream effector kinases of the NF-κB pathway, which is critical for activating most genes involved in the innate immune response (Nguyen et al., 2002). Recent data also provided evidence supporting the participation of FasL-Fas receptor (FasR) in the selective killing of embryonic motor neurons derived from mutant SOD1 mice (Raoul et al., 2002). The FasR belongs to the superfamily of TNF receptors (TNFRs) that include TNFRI (p55) and TNFRII (p75NGFr) by which the cytokine stimulates signaling events (Locksley et al., 2001). The generation of SOD1 mice lacking TLR2, TNF-α, TNFRI, TNFRII, FasL, and FasR will be essential to clarify the roles of these molecules in the control of the innate immune response in this model of neurodegeneration. In addition, determining the endogenous ligand of TLR2 would help in deciphering the mechanistic details of its actions within activated microglia.
For a long time, the CNS was considered to be a privileged organ from an immunological point of view because of its inability to mount an immune response and process antigens. An accumulating body of evidence indicates that the CNS shows a well organized innate immune reaction in response to systemic bacterial infection and cerebral injury. This response rejuvenates the idea that environmental/immunological challenges might be an etiological factor in sporadic cases of neurodegeneration. It also indicates that primary causes of such neurodegeneration could originate outside the CNS. Indeed, the mechanisms that underlie 90% of ALS cases, sporadic Parkinson's disease, and Alzheimer's disease remain elusive (Nguyen et al., 2002; Wyss-Coray and Mucke, 2002).
Actually, sporadic ALS may result from gene-environment interactions. Factors such as insecticide, heavy metals, and dietary glutamate-fat intake have been proposed as etiological causes for ALS. In addition to lifestyle, viral and bacterial infections may induce motor neuron disease. This assumption is supported by cases of motor dysfunction observed in individuals afflicted by infections. In the same line, viral and bacterial components associated with infiltration of peripheral immune cells in the CNS of sporadic ALS cases have been reported (Nguyen et al., 2002). Nevertheless, because of the low number of cases, epidemiological studies have failed to consistently incriminate any specific environmental factors in this disease. Beyond the toxicity of chronic administration of LPS in the mutant SOD1 mice, our study constitutes a simple example of genetic modulation in mammals by a peripheral bacterial challenge during neurodegeneration. In light of studies reporting viral and bacterial infections in a wide diversity of neurodegenerative disorder including ALS, we provide evidence here that environmental factors and innate immunity can cooperate to influence the course of disease of an inherited neuropathology. Chronic activation of microglial cells and deregulated innate immunity have profound and detrimental effects on neuronal survival. The study of components of innate immune response and environmental factors such as infections therefore may require more attention and revision, especially in sporadic cases of neurodegeneration evolving over months and years.
This work was supported by the Canadian Institutes of Health Research (CIHR) and the Robert Packard Center for ALS Research at Johns Hopkins. M.D.N. was a recipient of a K. M. Hunter-CIHR scholarship and holds a long-term fellowship from the Human Frontier Science Program Organization. J.-P.J. holds CIHR Senior Investigator awards and a chair in neurodegenerative diseases. S.R. is a CIHR scientist and holds a chair in neuroimmunology. The technical help of Pascale Hince, Nathalie Laflamme, and Amélie Lapointe is gratefully acknowledged. We are grateful to Drs. D. L. Price (John Hopkins University, Baltimore, MD) and D. W. Cleveland (University of California, San Diego, La Jolla, CA) for the kind gift of SOD1G37R mice (line 29), Dr. Y. Imai (National Institute of Neuroscience, Kodaira, Tokyo, Japan) for the gift of iba1 antisera, Dr. A. Israel (Institut Pasteur, Paris, France) for the mouse IκBα cDNA, Dr. D. Radzioch (McGill University, Montréal, Québec, Canada) for the plasmid containing the mouse TNF-α cDNA, Dr. I. Campbell (The Scripps Research Institute, La Jolla, CA) for the mouse IFN-γ cDNA, Dr. K. Pahan (University of Nebraska, Lincoln, NE) for the mouse IL-12p40 cDNA, and Dr. Li-Huei Tsai for hosting the last steps of this study at Harvard Medical School.
Correspondence should be addressed to Dr. Serge Rivest, Laboratory of Molecular Endocrinology, Laval University Medical Center Research Center and Department of Anatomy and Physiology, Laval University, 2705 Boulevard Laurier, Sainte-Foy, Québec, G1V 4G2 Canada. E-mail:.
M. D. Nguyen's present address: Harvard Medical School, Department of Pathology, Howard Hughes Medical Institute, Harvard University, Boston, MA 02115.
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