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XI European Meeting on Glial Cell Function in Health and Disease

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July 3, 2013 - Hall A 3:15pm - 5:15pm
Symposium 4: Microglial Phagocytosis and Ros in Development and Neurodegeneration

Organizers:
Guy Brown (Cambridge)
Jau-Shyong Hong (USA)

3:15pm
S04-01

Inflammed microglia kill neurons by phagocytosing them

*Guy Brown 1
1 University of Cambridge, , Cambridge, United Kingdom
Abstract text :

It is generally assumed that microglia only phagocytose dead or dying neurons, however, we find that under inflammatory conditions microglia can also phagocytose live neurons and thereby kill them, a form of cell death we call ‘primary phagocytosis’ or ‘phagoptosis’.  We find that microglial phagocytosis of neurons is normally mediated by the exposure of the ‘eat-me’ signal phosphatidyserine (PS) on neurons, which is bound by the adapter protein MFG-E8, which induces phagocytosis via the vitronectin receptor on microglia. Reversible PS exposure on viable neurons can be induced by low levels of glutamate, oxidants, of activated microglia, but in the presence of activated microglia this neuronal PS exposure induces their phagocytosis.  Calreticulin exposure on live neurons can also induce their phagocytosis by microglia LRP.


We found inflammatory activation of neuronal-glial co-cultures with LPS, LTA, TNF-α or amyloid β results in progressive loss of neurons (without any apparent cell death), which is accompanied by microglial phagocytosis of neurons, and is prevented by blocking phagocytosis.  Neuronal loss induced by nanomolar amyloid β or LPS is absent in cultures from MFG-E8 knockout mice, but is reconstituted by adding wild-type MFG-E8.  LPS-induced neuronal loss in vivo is reduced by co-injection of phagocytosis inhibitors or in MFG-E8 knockout mice.  Microglial phagocytosis of otherwise viable neurons may contribute to inflammatory neuronal and synaptic loss in a variety of brain pathologies.


Fricker M, Oliva-Martin MJ, Brown GC (2012) Primary phagocytosis of viable neurons by microglia activated with LPS or Abeta is dependent on calreticulin/LRP phagocytic signalling. J Neuroinflammation. 9:196.


Fricker M, Neher JJ, Zhao JW, Théry C, Tolkovsky AM, Brown GC (2012) MFG-E8 Mediates Primary Phagocytosis of Viable Neurons during Neuroinflammation. J Neurosci. 32:2657-66.


Brown GC, Neher JJ (2012) Eaten alive! Cell death by primary phagocytosis: 'phagoptosis'. Trends Biochem Sci. 37:325-32.


Neher JJ, Neniskyte U, Brown GC (2012) Primary phagocytosis of neurons by inflamed microglia: potential roles in neurodegeneration. Front Pharmacol. 3:27.


Neniskyte U, Neher JJ, Brown GC. (2011) Neuronal death induced by nanomolar amyloid beta is mediated by primary phagocytosis of neurons by microglia. J Biol Chem. 286:39904-13.


Neher JJ, Neniskyte U, Zhao ZW, Bal-Price A, Tolkovsky AM & Brown GC (2011) “Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death”. J. Immunol. 186, 4973-83.



3:45pm
S04-02

Pattern recognition-related inflammatory oxidative insult from microglia mediates chronic neurodegeneration

*Huiming Gao 1 , Hui Zhou 2 , Jau-Shyony  Hong 2
1 Nanjing University, , Nanjing, China
2 NIEHS/NIH, Laboratory of Toxicology and Pharmacology, Research Triangle Park, United States
Abstract text :

Background: A diverse set of pattern recognition receptors (PRRs) on innate immune cells recognize pathogen-associated molecular patterns (PAMPs) and mediate the first line of host defence to combat numerous pathogens. Several microglial PRRs (e.g. RAGE) have been reported to participate in brain immune response and affect neuronal survival in models of neurodegenerative diseases.


Question: How microglial Mac-1 (macrophage antigen complex 1; a surface integrin receptor) functions as a PRR to recognize endogenous alarmin(s) and foreign PAMPs mediating microglial activation and neurodegeneration remains elusive.


Methods: Immunoprecipitation and binding experiments were used to identify alarmin HMGB1 (high-mobility group box 1) as a new ligand for microglial Mac1. Midbrain neuron-glia cultures stimulated with bacterial endotoxin lipopolysaccharide (LPS), neurotoxin 1-methyl-4-phenylpyridinium (MPP ), or pesticide rotenone were used as in vitro models of Parkinson's disease (PD) to examine effects of Mac1 activation on neurodegeneration.


Results: Microglial Mac1 recognized and bound HMGB1 (a DNA-binding protein with extracellular cytokine-like effects after passively released from necrotic cells or actively secreted from inflammatory cells). In the binding assay, more HMGB1 binding to wildtype microglial membranes than Mac1-null membranes suggests physical interaction of HMGB1 and Mac1. Co-immunoprecipitation of HMGB1 and Mac1 in HMGB1-treated wildtype microglia but not Mac1-null microglia confirmed HMGB1-Mac1 interaction. HMGB1 activated microglia and triggered translocation of cytosolic p47phox to membrane and release of superoxide. Such activation of NADPH oxidase (NOX2) was Mac1-dependent because HMGB1 failed to stimulate Mac1-null microglia to release superoxide. Genetic ablation of Mac1 or gp91phox (the catalytic subunit of NOX2) attenuated HMGB1-elicited chronic dopamine neurodegeneration in midbrain neuron-glia cultures. Furthermore, HMGB1 accumulated in culture medium after neuron-glia cultures were treated with 3 “PD-producing” toxins (LPS, rotenone, and MPP ). Neutralization of HMGB1 attenuated dopamine neurodegeneration triggered by these toxins. Microglial Mac-1 also recognized extracellular dsRNA and induced NOX2 activation contributing to virus-related neurodegeneration.  


Conclusions: Pattern recognition by Mac1 is an important mechanistic basis for inflammatory oxidative neurodegeneration. Targeting Mac1-NOX2 signalling cascade holds a therapeutic potential for PD.



4:15pm
S04-03

Phagocytosis executes delayed neuronal death after focal brain ischemia

*Jonas Neher 1
1 University of Tübingen, , Tübingen, Germany
Abstract text :

Delayed neuronal loss and brain atrophy after cerebral ischemia contribute to stroke and dementia pathology, but the mechanisms are poorly understood. Phagocytic removal of neurons is generally assumed to be beneficial and to occur only after neuronal death. However, here we report that inhibition of phagocytosis can prevent the delayed loss and death of functional neurons after brain ischemia. In vitro, glutamate-stressed neurons reversibly exposed the eat-me signal phosphatidylserine (PS) leading to their phagocytosis by microglia and this neuronal loss was prevented in the absence of microglia or if PS-recognition was blocked. Further, genetic deficiency in two phagocytic proteins that mediate PS-recognition, Mer receptor tyrosine kinase (MerTK) and Milk fat globule EGF-like factor 8 (MFG-E8), also prevented loss of glutamate-stressed neurons in vitro and these proteins were transiently expressed by microglia/macrophages after cerebral ischemia in vivo. Strikingly, deficiency in either protein strongly reduced brain atrophy through inhibiting phagocytosis of neurons and completely prevented long-term functional motor deficit after cerebral ischemia in vivo. Thus, phagocytosis of viable neurons contributes to brain pathology, and surprisingly blocking this process is strongly beneficial. Therefore, inhibition of specific phagocytic pathways may present a novel therapeutic target for preventing delayed neuronal loss after cerebral ischemia.



4:45pm
S04-04

Microglia in the developing brain: pruning synapses and sculpting neural circuits

*Rosa C. Paolicelli 1 , Yang Zahn 2 , Giulia  Bolasco 2 , Francesca Pagani 3 , Laura Maggi 3 , Davide Ragozzino 3 , Cornelius T. Gross 2
1 University of Zurich, , Zurich, Switzerland
2 EMBL, Mouse Biology Unit, Monterotondo, Italy
3 University La Sapienza, Department of Human Physiology and Pharmacology, Roma, Italy
Abstract text :

Microglia are phagocytic cells of myeloid origin that infiltrate the brain during development and play an important surveillance and scavenging function. In the past years many studies have explored the function of these immune cells of the brain in pathological contexts, elucidating how they promptly act to resolve injuries, by moving toward the site of damage, engulfing cellular debris and releasing a broad spectrum of cytokines. However, much less is known about the physiological role of microglia in the uninjured, developing brain, and about their contribution to synaptic remodelling. Emerging data are now revealing how microglia, by actively engulfing synaptic material during the early postnatal period, play a major role in synaptic pruning and are necessary for a proper refinement of neural circuits. Mice lacking the chemokine receptor Cx3cr1 show a transient reduction in microglia in the hippocampus during the early postnatal weeks and exhibit an excess of weak excitatory synapses, as a consequence of deficit in synaptic pruning. Interestingly, transient defects in synaptic connectivity are associated with long-lasting behavioural impairments in social interaction, and increased repetitive behaviour, hallmarks of autism spectrum disorders. These findings provide evidence for a critical role of microglia in refining  brain connectivity, and support the hypothesis that deficits in microglia-mediated synaptic pruning may contribute to some structural and behavioural features of autism.