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These findings identify myeloid MVs as a marker and therapeutic target of brain inflammation.

Microvesicles (MVs) released into the brain microenvironment are emerging as a novel way of cell-to-cell communication. We have recently shown that microglia, the immune cells of the brain, shed MVs upon activation but their possible role in microglia-to-neuron communication has never been explored. To investigate whether MVs affect neurotransmission, we analysed spontaneous release of glutamate in neurons exposed to MVs and found a dose-dependent increase in miniature excitatory postsynaptic current (mEPSC) frequency without changes in mEPSC amplitude. Paired-pulse recording analysis of evoked neurotransmission showed that MVs mainly act at the presynaptic site, by increasing release probability. In line with the enhancement of excitatory transmission in vitro, injection of MVs into the rat visual cortex caused an acute increase in the amplitude of field potentials evoked by visual stimuli. Stimulation of synaptic activity occurred via enhanced sphingolipid metabolism. Indeed, MVs promoted ceramide and sphingosine production in neurons, while the increase of excitatory transmission induced by MVs was prevented by pharmacological or genetic inhibition of sphingosine synthesis. These data identify microglia-derived MVs as a new mechanism by which microglia influence synaptic activity and highlight the involvement of neuronal sphingosine in this microgliato-neuron signalling pathway.

Alzheimer's disease (AD) is characterized by extracellular amyloid-b (Ab) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Ab species, rather than insoluble fibrils, are the most toxic forms of Ab. Preventing soluble Ab formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of soluble Ab species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Ab forms trafficked to MVs after Ab internalization into microglia. MV neurotoxicity was neutralized by the Ab-interacting protein PrP and anti-Ab antibodies, which prevented binding to neurons of neurotoxic soluble Ab species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease. Cell Death and Differentiation (2014) 21, 582-593; doi:10.1038/cdd.2013.180; published online 13 December 2013Alzheimer's disease (AD) is the major cause of dementia in humans. Neuronal loss and cognitive decline occurring in AD patients are traditionally linked to the accumulation in the brain of extracellular plaques consisting of short amyloid-b (Ab) peptides of 39-42 amino acids, generated by amyloidogenic cleavage of the amyloid precursor protein. 1 Among Ab peptides, Ab 1-42 and pyroglutamate-modified Ab very rapidly aggregate and initiate the complex multistep process that leads to mature fibrils and plaque. 2,3 Although association of amyloid plaques with AD has long been assumed, Ab load does not correlate with neuronal loss 4,5 and high plaque burden does not necessarily lead to dementia in humans. 6,7 Accordingly, recent evidence clearly showed that the amyloid load reaches a plateau early after the onset of clinical symptoms in AD patients 8 and does not substantially increase in size during clinical progression. 9 These observations agree with the current view that small, soluble pre-fibrillar Ab species, rather than plaques formed by insoluble Ab fibrils, are the most toxic forms of Ab. 10 These cause synaptic dysfunction and spine loss, and correlate most closely with the severity of human AD. 5,8,11 Recent biochemical studies indicated that natural sphingolipids and gangliosides, whose metabolism has been shown to be altered in AD patients, 12 destabilize and rapidly resolubilize long Ab fibrils to neurotoxic species. 13 These studies also showed that phospholipids stabilize toxic oligomers...

Recent evidence indicates synaptic dysfunction as an early mechanism affected in neuroinflammatory diseases, such as multiple sclerosis, which are characterized by chronic microglia activation. However, the mode(s) of action of reactive microglia in causing synaptic defects are not fully understood. In this study, we show that inflammatory microglia produce extracellular vesicles (EVs) which are enriched in a set of miRNAs that regulate the expression of key synaptic proteins. Among them, miR-146a-5p, a microglia-specific miRNA not present in hippocampal neurons, controls the expression of presynaptic synaptotagmin1 (Syt1) and postsynaptic neuroligin1 (Nlg1), an adhesion protein which play a crucial role in dendritic spine formation and synaptic stability. Using a Renilla-based sensor, we provide formal proof that inflammatory EVs transfer their miR-146a-5p cargo to neuron. By western blot and immunofluorescence analysis we show that vesicular miR-146a-5p suppresses Syt1 and Nlg1 expression in receiving neurons. Microglia-to-neuron miR-146a-5p transfer and Syt1 and Nlg1 downregulation do not occur when EV–neuron contact is inhibited by cloaking vesicular phosphatidylserine residues and when neurons are exposed to EVs either depleted of miR-146a-5p, produced by pro-regenerative microglia, or storing inactive miR-146a-5p, produced by cells transfected with an anti-miR-146a-5p. Morphological analysis reveals that prolonged exposure to inflammatory EVs leads to significant decrease in dendritic spine density in hippocampal neurons in vivo and in primary culture, which is rescued in vitro by transfection of a miR-insensitive Nlg1 form. Dendritic spine loss is accompanied by a decrease in the density and strength of excitatory synapses, as indicated by reduced mEPSC frequency and amplitude. These findings link inflammatory microglia and enhanced EV production to loss of excitatory synapses, uncovering a previously unrecognized role for microglia-enriched miRNAs, released in association to EVs, in silencing of key synaptic genes.Electronic supplementary materialThe online version of this article (10.1007/s00401-017-1803-x) contains supplementary material, which is available to authorized users.