Young microglia restore amyloid plaque clearance of aged microglia

Alzheimer′s disease (AD) is characterized by deposition of amyloid plaques, neurofibrillary tangles, and neuroinflammation. In order to study microglial contribution to amyloid plaque phagocytosis, we developed a novel ex vivo model by co‐culturing organotypic brain slices from up to 20‐month‐old, amyloid‐bearing AD mouse model (APPPS1) and young, neonatal wild‐type (WT) mice. Surprisingly, co‐culturing resulted in proliferation, recruitment, and clustering of old microglial cells around amyloid plaques and clearance of the plaque halo. Depletion of either old or young microglial cells prevented amyloid plaque clearance, indicating a synergistic effect of both populations. Exposing old microglial cells to conditioned media of young microglia or addition of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was sufficient to induce microglial proliferation and reduce amyloid plaque size. Our data suggest that microglial dysfunction in AD may be reversible and their phagocytic ability can be modulated to limit amyloid accumulation. This novel ex vivo model provides a valuable system for identification, screening, and testing of compounds aimed to therapeutically reinforce microglial phagocytosis.     

Carnosic acid, a pro-electrophilic compound, inhibits LPS-induced activation of microglia

In the previous studies, we reported that carnosic acid (CA) protects cortical neurons by activating the Keap1/Nrf2 pathway, which activation is initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type CA. Here, we found that the pro-electrophilic CA inhibited the in vitro lipopolysaccharide (LPS)-induced activation of cells of the mouse microglial cell line MG6. LPS induced the expression of IL-1β and IL-6, typical inflammatory cytokines released from microglial cells. CA inhibited the NO production associated with a decrease in the level of inducible NO synthase. Neither CA nor LPS affected cell survival at the concentrations used here. These actions of CA seemed to be mediated by induction of phase 2 genes (gclc, gclm, nqo1 and xct). We propose that an inducer of phase 2 genes may be a critical regulator of microglial activation. Thus, CA is a unique pro-electrophilic compound that provides both a protective effect on neurons and an anti-inflammatory one on microglia through induction of phase 2 genes.     

Evidence of tricellulin expression by immune cells, particularly microglia

Tight junctions (TJs) are elaborate structures located on the apical region of epithelial cells that limit paracellular permeability. Tricellulin is a recently discovered TJ protein, which is concentrated at the structurally specialized tricellular TJs but also present at bicellular contacts between epithelial cells, namely in the stomach. Interestingly, several TJ proteins have been found in other than epithelial cells, as astrocytes, and tricellulin mRNA expression was reported in mature dendritic cells. These findings prompted us to look for tricellulin expression in both epithelial and immune cells in the stomach, as well as in microglia, the brain resident immunocompetent cells. Immunohistochemical analysis of human stomach tissue sections revealed peroxidase staining at three-corner contact sites, as well as at the contact between two adjacent epithelial cells, thus evidencing the expression of tricellulin not only at tricellullar but at bicellular junctions as well. Such analysis, further revealed tricellulin immunostaining in cells of the monocyte/macrophage lineage, scattered throughout the lamina propria. Cultured rat microglia exhibited a notorious tricellulin staining, consistent with an extensive expression of the protein along the cell, which was not absolutely coincident with the lysosomal marker CD68. Detection of mRNA expression by real-time PCR provided supportive evidence for the expression of the TJ protein in microglia. These data demonstrate for the first time that microglia express a TJ protein. Moreover, the expression of tricellulin both in microglia and in the stomach immune cells point to a possible role of this new TJ protein in the immune system.     

Physiological function of microglia

Broad interest in the rapidly advancing field of microglial involvement in forming neural circuits is evident from the fresh findings published in leading journals. This special issue of Neuron Glia Biology contains a special collection of research articles and reviews concerning the new appreciation of microglial function in the normal physiology of the brain that extends beyond their traditionally understood role in pathology.     

Lectin staining of sheep microglia

The B₄-isolectin from Griffonia simplicifolia is known to stain microglial cells in a variety of species. The present report describes a lectin staining method that has been modified to facilitate staining of resting microglia, as well as perivascular cells, in vibratome sections of normal sheep brain. This modified method employs tissue fixed in formaldehyde or paraformaldehyde and requires incubating sections with Triton X-100 prior to staining.     

老年大鼠脊髓小胶质细胞分选新方法及功能特征观察*

目的: 建立分离纯化老年大鼠小胶质细胞的改良方法,并初步观察老年大鼠脊髓小胶质细胞的生物学特性。方法: 以年轻SD大鼠(2月龄)为对照组,采用胰酶、胰酶替代物和机械网搓法等不同的制备方法,制备大鼠小胶质细胞的单细胞悬液,通过检测细胞纯度、存活率,观察细胞形态特征,分析细胞的炎性功能特征等,确定老年大鼠(20月龄)小胶质细胞的分离纯化方法,观察老年大鼠脊髓小胶质细胞功能特征。结果: 胰酶消化所得细胞的存活率低(年轻大鼠83%,老年大鼠60%);机械网搓法虽得到的存活率较高(95%),但是细胞获取率最低(年轻大鼠((0.207±0.020)×10⁶,老年大鼠(0.243±0.023)×10⁶);采用胰酶替代物解离、密度梯度离心方法分选出的老年大鼠脊髓小胶质细胞数量多、活性好、存活率高,细胞纯度可达85%以上,我们采用此方法分选纯化不同年龄大鼠脊髓小胶质细胞,与年轻大鼠相比,老年鼠脊髓组织量大,所需消化液多,但消化时间缩短;与年轻大鼠小胶质细胞相比,老年大鼠脊髓小胶质细胞其胞体较大较圆,突起少且粗短,形态上偏向于激活状态,老年大鼠小胶质细胞促炎因子IL-1β表达降低(P＜0.05),而抗炎因子IL-10(P＜0.01)表达升高。结论: 成功建立胰酶替代物解离结合密度梯度离心法从大鼠脊髓组织中分离纯化小胶质细胞,老年大鼠脊髓内小胶质细胞整体表现出抗炎表型。     

Glycine modulates membrane potential,cell volume,and phagocytosis in murine microglia

Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1–4). In BV-2 cells, glycine (5 mM) led to a rapid Na⁺-dependent depolarization of membrane potential (V _mem). Furthermore, glycine increased CV by about 9 %. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13 %. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor α-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na⁺ with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs.     

Thrombin-induced activation of cultured rodent microglia

Microglia are the resident immune cells of the CNS. Upon brain damage, these cells are rapidly activated and function as tissue macrophages. The first steps in this activation still remain unclear, but it is widely believed that substances released from damaged brain tissue trigger this process. In this article, we describe the effects of the blood coagulation factor thrombin on cultured rodent microglial cells. Thrombin induced a transient Ca(2+) increase in microglial cells, which persisted in Ca(2+)-free media. It was blocked by thapsigargin, indicating that thrombin caused a Ca(2+) release from internal stores. Preincubation with pertussis toxin did not alter the thrombin-induced [Ca(2+)](i) signal, whereas it was blocked by hirudin, a blocker of thrombin's proteolytic activity. Incubation with thrombin led to the production of nitric oxide and the release of the cytokines tumor necrosis factor-alpha, interleukin-6, interleukin-12, the chemokine KC, and the soluble tumor necrosis factor-alpha receptor II and had a significant proliferative effect. Our findings indicate that thrombin, a molecule that enters the brain at sites of injury, rapidly triggered microglial activation.     

Physiological roles of microglia during development

In all the species examined thus far, the behavior of microglia during development appears to be highly stereotyped. This reproducibility supports the notion that these cells have a physiological role in development. Microglia are macrophages that migrate from the yolk sac and colonize the central nervous system early during development. The first invading yolk-sac macrophages are highly proliferative and their role has not yet been addressed. At later developmental stages, microglia can be found throughout the brain and tend to preferentially reside at specific locations that are often associated with known developmental processes. Thus, it appears that microglia concentrate in areas of cell death, in proximity of developing blood vessels, in the marginal layer, which contains developing axon fascicles, and in close association with radial glial cells. This review describes the main features of brain colonization by microglia and discusses the possible physiological roles of these cells during development.     

小胶质细胞活化对癫痫发病的影响

目的探讨抑制小胶质细胞活化对癫痫发作的影响及对脑神经元的保护作用。方法将大鼠随机分为4组：生理盐水对照组（NS组）,海人酸致痫组（KA组）,美满霉素预处理＋海人酸组（MC＋KA组）,单纯美满霉素组（MC组）。采用免疫组化法观察各组大鼠造模后脑内Ox-42、c—fos和caspase-3免疫反应性。RT—PCR或Westernblot法检测造模后Apaf-1mRNA含量和caspase-3蛋白表达量。结果在KA致痫后2h大脑皮质及海马部位有明显的小胶质细胞活化、增殖,同时C—los蛋白的表达在致痫组也较对照组显著增强,Mc干预致痫组可明显减弱上述效应。Apaf-1mRNA的含量及caspase-3蛋白含量和免疫反应在造模后24h时间点四组之间无明显差异,在48h和72h时间点,KA组明显高于对照组（P〈0．05）,MC预处理则明显拮抗其高表达（P〈O．05）。结论KA致痫不仅通过提高神经元的兴奋性而且也通过激活小胶质细胞共同导致癫痫发作,二者相互协同作用,可进一步促进神经元凋亡,而MC能通过抑制小胶质细胞活化在癫痫中发挥神经保护作用。     

Role of microglia in CNS inflammation

There is increasing confusion about the meaning of the terms inflammation, neuroinflammation, and microglial inflammation. We aim in this review to achieve greater clarity regarding these terms, which are essential for our understanding of the role of microglia in CNS inflammatory conditions. The important concept of sterile inflammation is explained against the backdrop of classical inflammation, and its key differences from what researchers refer to when they use the terms neuroinflammation and microglial inflammation are illustrated. We propose to replace the term "neuroinflammation" with "microglial activation" or "CNS pseudo-inflammation", if microglial activation does not suffice. In addition, we recommend abandoning the terms "microglial inflammation" and "inflamed microglia" because of the lack of a clear concept behind them.     

异常活化的小胶质细胞的特征与功能

神经系统的各种损伤均可以引起小胶质细胞的激活.小胶质细胞是神经系统中发挥免疫功能的细胞,与T淋巴细胞、B淋巴细胞、自然杀伤细胞,以及其他的循环系统来源的白细胞共同参与神经损伤后的免疫反应.一般情况下,激活的小胶质细胞可以中止或者降低受累组织或细胞的生化代谢紊乱.但是持续激活的小胶质细胞释放的大量炎性因子能够对正常组织造成损害,加重神经损伤.关于小胶质细胞激活的病理生理学研究在最近几年取得较大进展,本文将主要介绍小胶质细胞在病理情况下激活的过程、特征,以及激活后发挥的功能及其调节机制.     

Expression of the 2',3'-cAMP-adenosine pathway in astrocytes and microglia

Many organs express the extracellular 3',5'-cAMP-adenosine pathway (conversion of extracellular 3',5'-cAMP to 5'-AMP and 5'-AMP to adenosine). Some organs release 2',3'-cAMP (isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'- and 3'-AMP and convert these AMPs to adenosine (extracellular 2',3'-cAMP-adenosine pathway). As astrocytes and microglia are important participants in the response to brain injury and adenosine is an endogenous neuroprotectant, we investigated whether these extracellular cAMP-adenosine pathways exist in these cell types. 2',3'-, 3',5'-cAMP, 5'-, 3'-, and 2'-AMP were incubated with mouse primary astrocytes or primary microglia for 1 h and purine metabolites were measured in the medium by mass spectrometry. There was little evidence of a 3',5'-cAMP-adenosine pathway in either astrocytes or microglia. In contrast, both cell types converted 2',3'-cAMP to 2'- and 3'-AMP (with 2'-AMP being the predominant product). Although both cell types converted 2'- and 3'-AMP to adenosine, microglia were five- and sevenfold, respectively, more efficient than astrocytes in this regard. Inhibitor studies indicated that the conversion of 2',3'-cAMP to 2'-AMP was mediated by a different ecto-enzyme than that involved in the metabolism of 2',3'-cAMP to 3'-AMP and that although CD73 mediates the conversion of 5'-AMP to adenosine, an alternative ecto-enzyme metabolizes 2'- or 3'-AMP to adenosine.     

Monitoring in vivo function of cortical microglia

Microglia, the innate immune cells of the brain, are becoming increasingly recognized as an important player both in the context of physiological brain function and brain pathology. To fulfill their executive functions microglia can modify their morphology, migrate or move their processes in a directed fashion, and modify the intracellular Ca²⁺ dynamics leading to modifications in gene expression, phagocytosis, release of cytokines and other inflammation markers, etc. Here we describe the recently developed tools enabling in vivo monitoring of morphology and Ca²⁺ signaling of microglia and show how these techniques may be used for examining microglial function in healthy and diseased brain.     

Mutant SOD1(G93A) microglia are more neurotoxic relative to wild-type microglia

Recent studies suggest that microglia over-expressing mutant human superoxide dismutase (mSOD1(G93A)) may contribute to motoneuron death in a transgenic mouse model of familial amyotrophic lateral sclerosis. To further assess the relative neurotoxicity of wild-type microglia, mSOD1(G93A) microglia, and microglia over-expressing wild-type human SOD1, we used primary cultures of microglia and motoneurons in the presence and absence of lipopolysaccharide stimulation. Following activation with lipopolysaccharide, mSOD1(G93A) microglia released more nitric oxide, more superoxide, and less insulin-like growth factor-1 than wild-type microglia. In microglia/motoneuron co-cultures, mSOD1(G93A) microglia induced more motoneuron death and decreased neurite numbers and length compared with wild-type microglia. Mutant SOD1(G93A) microglia also induced more motoneuron injury than microglia over-expressing wild-type human SOD1 in microglia/motoneuron co-cultures. Motoneuron survival was inversely correlated with nitrate + nitrite concentrations in mSOD1(G93A) co-cultures, suggesting the important role of nitric oxide in microglia-induced motoneuron injury. Thus, relative to wild-type microglia, mSOD1(G93A) microglia were more neurotoxic and induced more motoneuron injury than similarly treated wild-type microglia.     

Degradation of oxidized extracellular proteins by microglia

In living organisms a permanent oxidation of protein oxidation occurs. The degradation of intracellular oxidized proteins is intensively studied, but knowledge about the fate of oxidatively modified extracellular proteins is still limited. We studied the fate of exogenously added oxidized proteins in microglial cells. Both primary microglial cells and RAW cells are able to remove added oxidized laminin and myelin basic protein from the extracellular environment. Moderately oxidized proteins are degraded most efficiently, whereas strongly oxidized proteins are taken up by the microglial cells without an efficient degradation. Activation of microglial cells enhances the selective recognition and degradation of moderately oxidized protein substrates by proteases. Inhibitor studies also revealed an involvement of the lysosomal and the proteasomal system in the degradation of extracellular proteins. These studies let us conclude that microglial cells are able to remove oxidized proteins from the extracellular environment in the brain.