Thomas Möller

Microglia Biology in Health and Disease

Microglia cells are resident central nervous system (CNS) leukocytes that regulate innate immunity and participate in adaptive immune responses in CNS tissue. However, microglia cells also appear to play an important role during normal function of the mature nervous system. In response to injury, ischemia, and inflammatory stimuli, microglia cells assume an activated phenotype associated with proliferation, migration to the site of injury, phagocytosis of cellular debris, and elaboration (Power and Proudfoot 2001) of both neurotoxic and neurotrophic factors. Recent reports strongly suggest that regulating microglia function may be a fruitful future therapeutic target for the prevention of neurological dysfunction in a variety of CNS injuries and chronic diseases. Thus, developing a thorough understanding of extracellular signals that activate microglia as well as a complete catalogue of microglia responses to activating stimuli in both the healthy and diseased state are crucial scientific endeavors. This review presents the current understanding of the biology of microglia during normal CNS function as well as in response to CNS injury or neurodegenerative disease. In addition, microglia modulate both the activation and down-regulation of the adaptive immune response in the CNS. Evidence that microglia cells play a primary role in regulating CNS immune responses will also be discussed.

A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington's disease

Huntingtons disease (HD) is an inherited neurodegenerative disorder characterized by both neurological and systemic abnormalities. We examined the peripheral immune system and found widespread evidence of innate immune activation detectable in plasma throughout the course of HD. Interleukin 6 levels were increased in HD gene carriers with a mean of 16 years before the predicted onset of clinical symptoms. To our knowledge, this is the earliest plasma abnormality identified in HD. Monocytes from HD subjects expressed mutant huntingtin and were pathologically hyperactive in response to stimulation, suggesting that the mutant protein triggers a cell-autonomous immune activation. A similar pattern was seen in macrophages and microglia from HD mouse models, and the cerebrospinal fluid and striatum of HD patients exhibited abnormal immune activation, suggesting that immune dysfunction plays a role in brain pathology. Collectively, our data suggest parallel central nervous system and peripheral pathogenic pathways of immune activation in HD.

Astrocyte-Derived ATP Induces Vesicle Shedding and IL-1β Release from Microglia

ATP has been indicated as a primary factor in microglial response to brain injury and inflammation. By acting on different purinergic receptors 2, ATP is known to induce chemotaxis and stimulate the release of several cytokines from these cells. The activation of purinergic receptors 2 in microglia can be triggered either by ATP deriving from dying cells, at sites of brain injury or by ATP released from astrocytes, in the absence of cell damage. By the use of a biochemical approach integrated with video microscopy experiments, we investigated the functional consequences triggered in microglia by ATP released from mechanically stimulated astrocytes, in mixed glial cocultures. Astrocyte-derived ATP induced in nearby microglia the formation and the shedding of membrane vesicles. Vesicle formation was inhibited by the ATP-degrading enzyme apyrase or by P2X7R antagonists. Isolation of shed vesicles, followed by IL-1β evaluation by a specific ELISA revealed the presence of the cytokine inside the vesicular organelles and its subsequent efflux into the extracellular medium. IL-1β efflux from shed vesicles was enhanced by ATP stimulation and inhibited by pretreatment with the P2X7 antagonist oxidized ATP, thus indicating a crucial involvement of the pore-forming P2X7R in the release of the cytokine. Our data identify astrocyte-derived ATP as the endogenous factor responsible for microvesicle shedding in microglia and reveal the mechanisms by which astrocyte-derived ATP triggers IL-1β release from these cells.

The serine hydrolase ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors

The endocannabinoid 2-arachidonoylglycerol (2-AG) regulates neurotransmission and neuroinflammation by activating CB1 cannabinoid receptors on neurons and CB2 cannabinoid receptors on microglia. Enzymes that hydrolyze 2-AG, such as monoacylglycerol lipase, regulate the accumulation and efficacy of 2-AG at cannabinoid receptors. We found that the recently described serine hydrolase α-β-hydrolase domain 6 (ABHD6) also controls the accumulation and efficacy of 2-AG at cannabinoid receptors. In cells from the BV-2 microglia cell line, ABHD6 knockdown reduced hydrolysis of 2-AG and increased the efficacy with which 2-AG can stimulate CB2-mediated cell migration. ABHD6 was expressed by neurons in primary culture and its inhibition led to activity-dependent accumulation of 2-AG. In adult mouse cortex, ABHD6 was located postsynaptically and its selective inhibition allowed the induction of CB1-dependent long-term depression by otherwise subthreshold stimulation. Our results indicate that ABHD6 is a rate-limiting step of 2-AG signaling and is therefore a bona fide member of the endocannabinoid signaling system.

Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14

We have identified desmoglein-2 (DSG-2) as the primary high-affinity receptor used by adenoviruses Ad3, Ad7, Ad11 and Ad14. These serotypes represent key human pathogens causing respiratory and urinary tract infections. In epithelial cells, adenovirus binding of DSG-2 triggers events reminiscent of epithelial-to-mesenchymal transition, leading to transient opening of intercellular junctions. This opening improves access to receptors, for example, CD46 and Her2/neu, that are trapped in intercellular junctions. In addition to complete virions, dodecahedral particles (PtDds), formed by excess amounts of viral capsid proteins, penton base and fiber during viral replication, can trigger DSG-2–mediated opening of intercellular junctions as shown by studies with recombinant Ad3 PtDds. Our findings shed light on adenovirus biology and pathogenesis and may have implications for cancer therapy.

Complement 5a controls motility of murine microglial cells in vitro via activation of an inhibitory G-protein and the rearrangement of the actin cytoskeleton

Microglial cells respond to most pathological events by rapid transformation from a quiescent to an activated phenotype characterized by increased cytotoxicity and motile activity. To investigate the regulation of microglial motility by different inflammatory mediators, we studied cultured murine microglia by time-lapse video microscopy and a computer-based motility assay. Microglial cells exhibited a high resting motility. The acute application of complement 5a (C5a) immediately induced intense ruffling of microglial membranes followed by lamellipodia extension within few seconds, while formyl-Met-Leu-Phe-OH, bacterial endotoxin (lipopolysaccharide) or inflammatory cytokines did not increase motility. This process was accompanied by a rapid rearrangement of the actin cytoskeleton as demonstrated by labelling with fluorescein isothiocyanate-phalloidin and could be inhibited by cytochalasin B. A GTP-binding protein was involved in the signal cascade, since pertussis toxin inhibited motility and actin assembly in response to C5a. Chemotactic migration in a gradient of C5a was also completely blocked by pertussis toxin and cytochalasin B. The C5a-induced motility reaction was accompanied by an increase in intracellular calcium ([Ca2+]i) as measured by a Fluo-3 based imaging system. Ca2+ transients were, however, not a prerequisite for triggering the increase in motility; motility could be repeatedly evoked by C5a in nominally Ca(2+)-free solution, while Ca2+ signals occurred only upon the first stimulation. Moreover, conditions mimicking intracellular Ca2+ transients, like incubation with thapsigargin or Ca2+ ionophore A23187, were not able to induce any motility reaction, suggesting that Ca2+ transients are not necessary for, but are associated with, microglial motility. Motile activity was shown to be restricted to a defined concentration range of [Ca2+]i as revealed by lowering [Ca2+]i with BAPTA-AM or increasing [Ca2+]i with A23187. Since complement factors are released at pathological sites, this signal cascade could serve to increase motility and to direct microglial cells to the lesioned or damaged area by means of a G-protein-dependent pathway and via the rearrangement of the actin cytoskeleton.

Assessing disease onset and progression in the SOD1 mouse model of ALS.

&NA; SOD1 transgenic mice are the most widely used animal model of amyotrophic lateral sclerosis (ALS). In addition to providing valuable insights into the pathogenesis of ALS, these animals are used intensively in many laboratories as an in vivomodel for investigating novel therapeutic interventions towards this devastating motorneuron disease. Such pre‐clinical studies require objective and reliable quantification of the clinical phenotype of individual mice, most importantly of the neuromuscular abnormalities. Here we compare four parameters of the clinical phenotype: motor signs, body weight, rotarod performance and paw grip endurance for their usefulness in monitoring the SOD1 mouse model. We found that paw grip endurance is a sensitive and inexpensive alternative to the widely used rotarod test.

Thrombin‐Induced Activation of Cultured Rodent Microglia

Abstract: 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 Ca2+ increase in microglial cells, which persisted in Ca2+‐free media. It was blocked by thapsigargin, indicating that thrombin caused a Ca2+ release from internal stores. Preincubation with pertussis toxin did not alter the thrombin‐induced [Ca2+]i signal, whereas it was blocked by hirudin, a blocker of thrombins proteolytic activity. Incubation with thrombin led to the production of nitric oxide and the release of the cytokines tumor necrosis factor‐α, interleukin‐6, interleukin‐12, the chemokine KC, and the soluble tumor necrosis factor‐α 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.

Astrocytes in culture produce anandamide and other acylethanolamides

Anandamide (arachidonylethanolamide) is an endocannabinoid that belongs to the acylethanolamide lipid family. It is produced by neurons in a calcium-dependent manner and acts through cannabinoid CB1 receptors. Other members of the acylethanolamide lipid family are also produced by neurons and act through G-protein-coupled receptors: homo-γ-linolenylethanolamide (HEA) and docosatetraenylethanolamide (DEA) act through CB1 receptors, palmitylethanolamide (PEA) acts through CB2-like receptors, and oleylethanolamide (OEA) acts through receptors that have not yet been cloned. Although it is clear that anandamide and other acylethanolamides play a major role in neuronal signaling, whether astrocytes also produce these lipids is unknown. We developed a chemical ionization gas chromatography/mass spectrometry method that allows femtomole detection and quantification of anandamide and other acylethanolamides. Using this method, we unambiguously detected and quantified anandamide, HEA, DEA, PEA, and OEA in mouse astrocytes in culture. Stimulation of mouse astrocytes with ionomycin, a calcium ionophore, enhanced the production of anandamide, HEA, and DEA, whereas PEA and OEA levels were unchanged. Endothelin-1, a peptide known to act on astrocytes, enhanced the production of anandamide, without affecting the levels of other acylethanolamides. These results show that astrocytes produce anandamide, HEA, and DEA in a calcium-dependent manner and that anandamide biosynthesis can be selectively stimulated under physiologically relevant conditions. The relative levels of acylethanolamides in astrocytes from rat and human were different from the relative levels of acylethanolamides in mouse astrocytes, indicating that the production of these lipids differs between species. Because astrocytes are known to express CB1 receptors and inactivate endocannabinoids, our finding strongly suggests the existence of a functional endocannabinoid signaling system in these cells.

Increased cytotoxic potential of microglia from ALS-transgenic mice.

Amyotrophic lateral sclerosis is a fatal, adult‐onset motor neuron disease. A subset of cases is caused by mutations of superoxide dismutase 1 (SOD1) gene. The mechanisms how the mutations in this ubiquitous enzyme mediate the highly selective motor neuron degeneration, however, remain poorly understood. Recent results from transgenic animal models suggest a “non‐cell autonomous” mechanism; i.e., cells other than neurons play an active role in motor neuron death. To investigate a possible effect of mtSOD1 on microglial cells, we compared primary cultured microglia from mtSOD1‐transgenic mice and nontransgenic litter controls at neonatal (3 days) and adult (60 days) age. We found that mtSOD1 expression increases the production of TNF‐α and attenuates IL‐6‐release by LPS‐activated adult microglia. Neonatal microglia, however, showed no differences. Our findings suggest an increased cytotoxic potential of adult mtSOD1 microglia, which only becomes apparent after microglial activation.