The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1β secretion. Both caspase-1 and IL-1β contribute to disease progression in the mouse SOD1^G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1^G93A mice microglia do not express NLRP3, and SOD1^G93A protein generated IL-1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1^G93A mice. We show that both aggregated and soluble SOD1^G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1β cleavage, ASC speck formation, and the secretion of IL-1β in a dose- and time-dependent manner. Importantly, SOD1^G93A was unable to induce IL-1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1β secretion. Microglial NLRP3 upregulation was also observed in the TDP-43^Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1^G93A-mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.     

Appearance of phagocytic microglia adjacent to motoneurons in spinal cord tissue from a presymptomatic transgenic rat model of amyotrophic lateral sclerosis

Microglial activation occurs early during the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent evidence indicates that the expression of mutant Cu²⁺/Zn²⁺ superoxide dismutase 1 (SOD1) in microglia contributes to the late disease progression of ALS. However, the mechanism by which microglia influence the neurodegenerative process and disease progression in ALS remains unclear. In this study, we revealed that activated microglia aggregated in the lumbar spinal cord of presymptomatic mutant SOD1^H46R transgenic rats, an animal model of familial ALS. The aggregated microglia expressed a marker of proliferating cell, Ki67, and phagocytic marker proteins ED1 and major histocompatibility complex (MHC) class II. The motoneurons near the microglial aggregates showed weak choline acetyltransferase (ChAT) immunoreactivity and contained reduced granular endoplasmic reticulum and altered nucleus electron microscopically. Furthermore, immunopositive signals for tumor necrosis factor‐α (TNFα) and monocyte chemoattractant protein‐1 (MCP‐1) were localized in the aggregated microglia. These results suggest that the activated and aggregated microglia represent phagocytic features in response to early changes in motoneurons and possibly play an important role in ALS disease progression during the presymptomatic stage. © 2010 Wiley‐Liss, Inc.     

Immune reactivity in a mouse model of familial ALS correlates with disease progression

OBJECTIVE: The cause of motor neuron death in ALS is incompletely understood. This study aims to define the potential involvement of nonneuronal immune-inflammatory factors in the destruction of motor neurons in mutant superoxide dismutase-1 (SOD1) transgenic mice as a model of ALS. BACKGROUND: The presence of activated microglia, IgG and its receptor for Fc portion (FcgammaRI), and T lymphocytes in the spinal cord of both patients with ALS and experimental animal models of motor neuron disease strongly suggests that immune-inflammatory factors may be actively involved in the disease process. METHODS: The expression of immune-inflammatory factors was followed in both human mutant (G93A) SOD1 transgenic mice and human wild-type SOD1 transgenic mice, at different ages (40, 80, and 120 days). Fixed, frozen, free-floating sections of the lumbar spinal cord were stained with antibodies against CD11b, IgG, FcgammaRI, intercellular adhesion molecule-1 (ICAM-1), CD3, and glial fibrillary acidic protein. RESULTS: The earliest change observed was the upregulation of ICAM-1 in the ventral lumbar spinal cord of 40-day-old mutant SOD1 mice. IgG and FcgammaRI reactivities were detected on motor neurons as early as 40 days and on microglial cells at later stages. Microglial activation was first evident in the ventral horn at 80 days, whereas reactive astrocytes and T cells became most prominent in 120-day-old mutant SOD1 mice. CONCLUSION: The upregulation of proinflammatory factors during early presymptomatic stages as well as the expansion of immune activation as disease progresses in mutant SOD1 transgenic mice suggest that immune-inflammatory mechanisms could contribute to disease progression.     

Treatment with minocycline after disease onset alters astrocyte reactivity and increases microgliosis in SOD1 mutant mice

Several reports have demonstrated that attenuation of microglial activation by minocycline, an antimicrobial drug with anti-inflammatory properties, delays disease progression in a mouse model of ALS. However, the negative results obtained in recent clinical trials raised some questions regarding the role of inflammatory response and glial cells as a therapeutic target in ALS. To investigate this controversy we took advantage of a mouse model for live imaging of neuroinflammatory responses in ALS (GFAP-luc/ SOD1^G93A reporter mouse) and analyzed in real time the effects of minocycline treatment initiated at different stages of the disease. To our surprise, unlike neuroprotection that is conferred when minocycline is administered pre-symptomatically, treatment with minocycline initiated after the disease onset significantly altered glial responses and exaggerated neuroinflammation. Further analysis revealed that the late minocycline treatment was associated with significant induction of the end-stage GFAP-biophotonic signals, expression levels of connexin 43, a major protein of astrocytic gap junction and markers of microglial activation, such as Iba1 and CD68. The results of our study suggest that when administered at later stages of disease, once microglial cells are chronically reactive, minocycline may not have anti-inflammatory properties, and contrary to expectations, may alter astrocyte reactivity and increase microgliosis. Finally, our results further suggest the existence of close interactions/communication between activated microglia and astrocytes in late stage ALS.     

Extracellular mutant SOD1 induces microglial‐mediated motoneuron injury

Through undefined mechanisms, dominant mutations in (Cu/Zn) superoxide dismutase‐1 (mSOD1) cause the non‐cell‐autonomous death of motoneurons in inherited amyotrophic lateral sclerosis (ALS). Microgliosis at sites of motoneuron injury is a neuropathological hallmark of ALS. Extracellular mutant SOD1 (mSOD1) causes motoneuron injury and triggers microgliosis in spinal cord cultures, but it is unclear whether the injury results from extracellular mSOD1 directly interacting with motoneurons or is mediated through mSOD1‐activated microglia. To dissociate these potential mSOD1‐mediated neurotoxic mechanisms, the effects of extracellular human mSOD1^G93A or mSOD1^G85R were assayed using primary cultures of motoneurons and microglia. The data demonstrate that exogenous mSOD1^G93A did not cause detectable direct killing of motoneurons. In contrast, mSOD1^G93A or mSOD1^G85R did induce the morphological and functional activation of microglia, increasing their release of pro‐inflammatory cytokines and free radicals. Furthermore, only when microglia was co‐cultured with motoneurons did extracellular mSOD1^G93A injure motoneurons. The microglial activation mediated by mSOD1^G93A was attenuated using toll‐like receptors (TLR) 2, TLR4 and CD14 blocking antibodies, or when microglia lacked CD14 expression. These data suggest that extracellular mSOD1^G93A is not directly toxic to motoneurons but requires microglial activation for toxicity, utilizing CD14 and TLR pathways. This link between mSOD1 and innate immunity may offer novel therapeutic targets in ALS. © 2009 Wiley‐Liss, Inc.     

Relationship of microglial and astrocytic activation to disease onset and progression in a transgenic model of familial ALS

Transgenic mice that highly over-express a mutated human CuZn superoxide dismutase (SOD1) gene [gly⁹³→ala; TgN(SOD1-G93A)G1H line] found in some patients with familial ALS (FALS) have been shown to develop motor neuron disease that is characterized by motor neuron loss in the lumbar and cervical spinal regions and a progressive loss of motor activity. The mutant Cu,Zn SOD exhibits essentially normal SOD activity but also generates toxic oxygen radicals as a result of an enhancement of a normally minor peroxidase reaction. Consequently, lipid and protein oxidative damage to the spinal motor neurons occurs and is associated with disease onset and progression. In the present study, we investigated the time course of microglial (major histocompatibility-II antigen immunoreactivity) and astrocytic (glial fibrillary acidic protein immunoreactivity) activation in relation to the course of motor neuron disease in the TgN(SOD1-G93A)G1H FALS mice. Four ages were investigated: 30 days (pre-motor neuron pathology and clinical disease); 60 days (after initiation of pathology, but pre-disease); 100 days (approximately 50% loss of motor neurons and function); and 120 days (near complete hindlimb paralysis). Compared to non-transgenic littermates, the TgN(SOD1-G93A)G1H mice showed significantly increased numbers of activated astrocytes (P < 0.01) at 100 days of age in both the cervical and lumbar spinal cord regions. However, at 120 days of age, the activation lost statistical significance. In contrast, microglial activation was significantly increased several-fold at both 100 and 120 days. We hypothesize that astrocytic activation may exert a trophic influence on the motor neurons that is insufficiently maintained late in the course of the disease. On the other hand, the sustained, intense microglial activation may conceivably contribute to the oxidative stress and damage involved in the disease process. If true, then agents which inhibit microglia may help to limit disease progression. GLIA 23:249–256, 1998. © 1998 Wiley-Liss, Inc.     

Aberrant neuregulin 1 signaling in amyotrophic lateral sclerosis

Neuregulin 1 (NRG1) is a neuron-derived trophic molecule that supports axoglial and neuromuscular development through several alternatively spliced isoforms; its possible role in the pathogenesis and progression of amyotrophic lateral sclerosis (ALS) is not known. We analyzed the relationship of NRG1 isoform expression to glial cell activation and motor neuron loss in spinal cords of ALS patients and during disease progression in the superoxide dismutase 1 (SOD1) ALS mouse model. Microgliosis, astrocytosis, and motor neuron loss were observed in the ventral horns in ALS patients and were increased in SOD1 mice along with disease progression. Type III (membrane-bound) NRG1 expression was reduced in parallel with motor neuron loss, but Type I (secreted) NRG1 expression was increased and was associated with glial activation. Increased NRG1 receptor activation was observed on activated microglia in both ALS patients and in SOD1 mice. This activation was observed at the time of disease onset and before upregulation of NRG1 gene expression in the mice. The downregulation of membrane-bound Type III NRG1 forms may reflect motor neuron loss, but increased signaling by secreted-type NRG1 isoforms could contribute to disease pathogenesis through glial cell activation. NRG1 might, therefore, represent a novel therapeutic target against disease progression in ALS.     

Regulatory T lymphocytes from ALS mice suppress microglia and effector T lymphocytes through different cytokine-mediated mechanisms

Activated microglia and infiltrating lymphocytes are neuropathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motoneuron disease. Although both cell types play pivotal roles in the ALS pathogenic process, the interactions between microglia and lymphocytes, specifically regulatory CD4(+)CD25(High) T lymphocytes (Tregs) and cytotoxic CD4(+)CD25(-) T lymphocytes (Teffs), have not been addressed. When co-cultured with mSOD1 adult microglia, mSOD1 Tregs suppressed the cytotoxic microglial factors NOX2 and iNOS through an IL-4-mediated mechanism, whereas Teffs were only minimally effective; IL-4 inhibitory antibodies blocked the suppressive function of mSOD1 Tregs, and conditioned media from mSOD1 Tregs or the addition of IL-4 reduced microglial NOX2 expression. During the stable disease phase, the total number of Tregs, specifically the numbers of CD4(+)CD25(High)IL-4(+), CD4(+)CD25(High)IL-10(+) and CD4(+)CD25(High)TGF-β(+) Tregs, were increased in ALS mice compared with WT mice; Tregs isolated during this phase reduced Teff proliferation. In contrast, during the rapidly progressing phase, the number of mSOD1 Tregs decreased while the proliferation of mSOD1 Teffs increased. The combination of IL-4, IL-10, and TGF-β was required to inhibit the proliferation of mSOD1 Teffs by mSOD1 Tregs that were isolated during the slow phase, while inhibition of mSOD1 Teffs by mSOD1 Tregs during the rapid phase, as well as WT Teffs, was not dependent on these factors. Thus, mSOD1 Tregs at the slow phase suppressed microglial toxicity and SOD1 Teff proliferation through different mechanisms; microglial activation was suppressed through IL-4 whereas mSOD1 Teffs were suppressed by IL-4, IL-10 and TGF-β. These data suggest that mSOD1 Tregs contribute to the slowly progressing phase in ALS mice and may offer a novel therapeutic option for ALS patients.     

一种连续获取大量高纯度小胶质细胞的培养方法

目的 建立一种简易、高产量高纯度的连续获得小胶质细胞的培养方法.方法 原代培养新生1～3 d Wistar大鼠大脑皮层中混合胶质细胞,第8～9天采用振摇法和玻璃吸管吹打法分离获得Yield 1小胶质细胞,按1∶2传代培养剩余贴壁的混合胶质细胞,分别继续培养10～12 d、12～14 d后应用同样方法获得Yield 2、Yield 3小胶质细胞;采用流式细胞仪分析所获小胶质细胞CD11b/c、CD45、CD80、CD86、GFAP的表达,免疫荧光染色和CCK-8法分别检测小胶质细胞CD11b/c的表达和增殖情况,应用扫描电镜观察其超微结构,应用吞噬红色乳胶微球实验评价其吞噬功能.结果 本实验所用培养方法连续稳定地获得了大量高纯度的小胶质细胞;流式细胞仪检测结果显示Yield 1与Yield 3小胶质细胞CD11b/c、CD45、CD80、CD86表达阳性;免疫荧光染色显示Yield 1、Yield 2、Yield 3小胶质细胞CD11b/c表达均阳性;CCK-8法结果显示3代小胶质细胞增殖活力相比较差异无统计学意义(P＞0.05);扫描电镜显示3代小胶质细胞形态均呈胞体饱满、胞突细长等细胞形貌特点.相同条件下Yield 1、Yield 3小胶质细胞之间吞噬功能比较差异无统计学意义(P＞0.05).结论 以本实验方法可以连续获得大量高纯度的小胶质细胞,且这些不同代次小胶质细胞的抗原表型、增殖活力及吞噬功能无明显差异,可为相关的实验研究提供细胞来源.

Abstract：

Objective To establish an easy culture method of successively getting high purity and yield of microglia. Methods Cortices of neonatal Wistar rats (1-3 days old) were employed in this experiment. The first-generation microglial cells were isolated from the mixed glial culture by mechanical means (gently shaking and blowing with pipette). After the mixed glial cells being passaged at a density third generations ofmicroglial cells were harvested. CD1 lb/c, CD45, CD80, CD86 and GFAP were employed as the identification markers in detecting the phenotypes and purity of different generation of microglial cells by scanning electron microscope and flow cytometry. Immunofluorescence staining and CCk8 vitality measurement were used to judge the expression of CD11b/c and detect the proliferation of microglia cells. Microglial phagocytotic function was evaluated by phagocytosis of fluorescent microspheres. Results High yield and purity of microglial cells were stably obtained in this experiment. CD11b/c, CD45, CD80 and CD86 positive expressions were noted in the first and third generations of microglial cells by flow cytometry; CD1 1b/c positive expression was noted in the first,second and third generations of microglial cells by immunofluorescence staining. No obvious differences in the 3 different generations of microglia cells were found on proliferation ability by CCk8 vitality measurement, and on morphology and phenotypes by scanning electron microscope; no obvious differences in the first and third generations of microglia cells were found on phagocytic ability (P＞0.05).Conclusion High yield and purity of microglial cells can successively obtain through the above method;no significant differences are noted among different generations of microglia cells on purity, morphology,phenotypes, proliferation activity and phagocytic ability.     

A simple magnetic separation method for high-yield isolation of pure primary microglia

Microglial cells play a dynamic role in the brain beyond their established function of immune surveillance. Activated microglia play key roles in neural development, neuroinflammation, neural repair and neurotoxicity. They are particularly important in several neurodegenerative diseases in which sustained microglial activation contributes to the progression of neurodegenerative processes. Consequently, understanding microglial function in CNS health and disease has become an area of active research in recent years. However, a significant obstacle to progress in this field has been the inherent difficulties in obtaining large amounts of primary microglial cells to routinely perform mechanistic studies and characterize signaling pathways regulating the dynamics of microglial activation. Herein, we describe a novel column-free magnetic separation protocol for high-yield isolation of primary microglia from mouse postnatal mixed glial cultures. The procedure is based on optimized culture conditions that enable high microglial cell densities in confluent mixed glial cultures followed by highly efficient recovery of pure microglia by magnetic separation. The novel column-free magnetic separation system utilizes tetrameric antibody complexes (TAC) with dual specificity for CD11b-PE labeled microglia and dextran magnetic nanoparticles. An FcR blocker (anti-CD16/32) is added to enhance the purity of the microglial separation by preventing non-specific labeling of other cell types. This procedure yields on average >3×10? microglial cells per mouse pup, with a remarkable purity of 97% and recovery of around 87% of microglia from the mixed glial population. Importantly, the microglia obtained by this method are fully functional and respond like cells obtained by conventional isolation techniques.     

Neuroinflammation in amyotrophic lateral sclerosis: role of glial activation in motor neuron disease

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS) are characterised by the appearance of reactive microglial and astroglial cells, a process referred to as neuroinflammation. In transgenic mouse models of mutant SOD1-associated familial ALS, reactive microglial cells and astrocytes actively contribute to the death of motor neurons. The biological processes that drive this glial reaction are complex and have both beneficial and deleterious effects on motor neurons. Therapeutic interventions targeting these cells are being explored. An improved understanding of the biological processes that cause neuroinflammation will help to define its medical importance and to identify the therapeutic potential of interfering with this reaction.     

Persistent activation of p38 mitogen-activated protein kinase in a mouse model of familial amyotrophic lateral sclerosis correlates with disease progression

The p38 mitogen-activated protein kinase (p38MAPK) is activated via phosphorylation in neurones and glial cells by a variety of stimuli including oxidative stress, excitotoxicity, and inflammatory cytokines. Activated p38MAPK can in turn induce phosphorylation of cytoskeletal proteins and activation of cytokines and nitric oxide, thus contributing to neurodegeneration. We investigated the expression and distribution of p38MAPK in the spinal cord of transgenic mice expressing a superoxide dismutase 1 mutation (SOD1G93A), a model of familial amyotrophic lateral sclerosis (ALS). Accumulation of p38MAPK was found by immunoblotting in the spinal cord of G93A mice during the progression of disease, but no changes were detected in its mRNA levels. Immunostaining for phosphorylated p38MAPK in lumbar spinal cord sections of SOD1G93A mice at the presymptomatic and early stages of disease showed an increased labeling in motor neurones that colocalized with phosphorylated neurofilaments in vacuolized perikarya and neurites, as detected by confocal microscopy. As the disease progressed, activated p38MAPK also accumulated in hypertrophic astrocytes and reactive microglia, as demonstrated by colocalization with GFAP and CD11b immunostaining, respectively. These data suggest that activation of p38MAPK in motor neurons and then in reactive glial cells may contribute, respectively, to the development and progression of motor neuron pathology in SOD1G93A mice.     

Following nerve injury neuregulin-1 drives microglial proliferation and neuropathic pain via the MEK/ERK pathway

Following peripheral nerve injury microglia accumulate within the spinal cord and adopt a proinflammatory phenotype a process which contributes to the development of neuropathic pain. We have recently shown that neuregulin-1, a growth factor released following nerve injury, activates erbB 2, 3, and 4 receptors on microglia and stimulates proliferation, survival and chemotaxis of these cells. Here we studied the intracellular signaling pathways downstream of neuregulin-1-erbB activation in microglial cells. We found that neuregulin-1 in vitro induced phosphorylation of ERK1/2 and Akt without activating p38MAPK. Using specific kinase inhibitors we found that the mitogenic effect of neuregulin-1 on microglia was dependant on MEK/ERK1/2 pathway, the chemotactic effect was dependant on PI3K/Akt signaling and survival was dependant on both pathways. Intrathecal treatment with neuregulin-1 was associated with microgliosis and development of mechanical and cold pain related hypersensitivity which was dependant on ERK1/2 phosphorylation in microglia. Spinal nerve ligation results in a robust microgliosis and sustained ERK1/2 phosphorylation within these cells. This pathway is downstream of neuregulin-1/erbB signaling since its blockade resulted in a significant reduction in microglial ERK1/2 phosphorylation. Inhibition of the MEK/ERK1/2 pathway resulted in decreased spinal microgliosis and in reduced mechanical and cold hypersensitivity after peripheral nerve damage. We conclude that neuregulin-1 released after nerve injury activates microglial erbB receptors which consequently stimulates the MEK/ERK1/2 pathway that drives microglial proliferation and contributes to the development of neuropathic pain.     

小胶质细胞在SOD1-G93A转基因小鼠腰髓中的变化

目的观察小胶质细胞在SOD1-G93A转基因小鼠不同时期腰髓中的变化,探讨小胶质细胞活化与肌萎缩侧索硬化(ALS)疾病进展的关系。方法以国际公认的SOD1-G93A转基因小鼠,应用免疫组化、激光共聚焦显微镜及Westernblot方法 ,分别观察SOD1-G93A转基因小鼠症状前期、症状期、终末期及其同窝对照腰髓小胶质细胞形态数量及特异性标记物表达的变化情况。结果 SOD1-G93A转基因小鼠腰髓在症状前期(60天)已出现小胶质细胞数量增多及特异性标记物CD11b表达升高,随病程进展,症状期小胶质细胞增多、活化显著,终末期达高峰。结论随SOD1-G93A转基因小鼠病程进展小胶质细胞增生明显,小胶质细胞的活化可能参与ALS运动神经元损伤。     

Animal models of amyotrophic lateral sclerosis]

Dominant mutation in the gene of superoxide dismutase 1 (SOD1) leads amyotrophic lateral sclerosis (ALS), an adult-onset progressive fatal motor neuron disease. Recent research progress in ALS has been made by the use of transgenic mouse model of familial ALS, which expresses mutant form of SOD1 and recapitulates the phenotype and pathology of motor neuron disease. There is accumulating evidence indicating non-cell-autonomous motor neuron death in ALS mouse model. In this symposium, I review the recent advance of ALS research focusing on the development of animal models, the role of glial cells in ALS, and therapeutic intervention of rodent models and discuss their prospect.     

A Nitroalkene Benzoic Acid Derivative Targets Reactive Microglia and Prolongs Survival in an Inherited Model of ALS via NF-κB Inhibition

Motor neuron degeneration and neuroinflammation are the most striking pathological features of amyotrophic lateral sclerosis (ALS). ALS currently has no cure and approved drugs have only a modest clinically therapeutic effect in patients. Drugs targeting different deleterious inflammatory pathways in ALS appear as promising therapeutic alternatives. Here, we have assessed the potential therapeutic effect of an electrophilic nitroalkene benzoic acid derivative, (E)-4-(2-nitrovinyl) benzoic acid (BANA), to slow down paralysis progression when administered after overt disease onset in SOD1^G93A rats. BANA exerted a significant inhibition of NF-κB activation in NF-κB reporter transgenic mice and microglial cell cultures. Systemic daily oral administration of BANA to SOD1^G93A rats after paralysis onset significantly decreased microgliosis and astrocytosis, and significantly reduced the number of NF-κB-p65-positive microglial nuclei surrounding spinal motor neurons. Numerous microglia bearing nuclear NF-κB-p65 were observed in the surrounding of motor neurons in autopsy spinal cords from ALS patients but not in controls, suggesting ALS-associated microglia could be targeted by BANA. In addition, BANA-treated SOD1^G93A rats after paralysis onset showed significantly ameliorated spinal motor neuron pathology as well as conserved neuromuscular junction innervation in the skeletal muscle, as compared to controls. Notably, BANA prolonged post-paralysis survival by ~30%, compared to vehicle-treated littermates. These data provide a rationale to therapeutically slow paralysis progression in ALS using small electrophilic compounds such as BANA, through a mechanism involving microglial NF-κB inhibition.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00953-z.Key Words: ALS, NF-κB-p65, microglia, BANA.     

Temporal gene expression patterns in G93A/SOD1 mouse.

Amyotrophic lateral sclerosis (ALS) is a generally fatal degenerative disorder of motor neurons that has no known cure. Many pathological processes have been implicated. However, the early, initiating events in the disease are poorly understood. We performed multivariate analyses of gene expression of 21 selected genes from categories including glutamate neurotoxicity, oxidative stress, neuroinflammation, aberrant metal ion regulation, apoptosis, and abnormal microglial function on G93A SOD1 mice. These animals develop symptoms of motor neuron dysfunction at about 12 weeks of age, and die at age 18 to 20 weeks. We analyzed animals at both presymptomatic and symptomatic stages. Differential regulation of several genes involved in neuroinflammation, including TNF-alpha, IL- RA, CD86, CD200R and Groalpha, was observed in presymptomatic mice, aged 6-9 weeks, while expression of genes representative of other processes was not altered until the animals reached symptomatic stages. Analysis of expression of inflammatory genes and microglia related genes together also revealed a highly significant change in mutant mice relative to wildtype at 6-9 weeks. These changes were due to the presence of the mutant gene, and not simply to overexpression of a SOD1 gene. These findings are discussed in relation to possible roles of microglia function in the development of ALS.     

Microglial reactivity to beta-amyloid is modulated by astrocytes and proinflammatory factors

The brains of Alzheimer's disease (AD) patients present activated glial cells, amyloid plaques and dystrophic neurites. The core of amyloid plaques is composed of aggregated amyloid peptide (Abeta), a peptide known to activate glial cells and to have neurotoxic effects. We evaluated the capability of glial cells to mediate Abeta(1-42) cytotoxicity in hippocampal cultures. Conditioned media obtained from microglial cultures exposed to Abeta induced apoptosis of hippocampal cells. This pro-apoptotic effect was not observed in hippocampal cultures exposed to conditioned media obtained from mixed glial (astrocytes and microglia) cultures that had been exposed to Abeta. Microglia exposed to Abeta responded with reactive morphological changes, induction of iNOS, elevated nitric oxide production and decreased reductive metabolism. All these responses were attenuated by the presence of astrocytes. This astrocyte modulation was however, not observed when glial cells were exposed to proinflammatory factors (LPS+Interferon-gamma) alone or in combination with Abeta. Our results suggest that astrocytes and proinflammatory molecules are determining factors in the response of microglia to Abeta.