Project description:Microglia is emerging as a key player in the progression of neurodegenerative diseases. In pediatric neurodegenerative diseases like mucopolysaccharidosis type III (MPSIII), the progressive accumulation of abnormal glycosaminoglycans (GAGs) induces a severe neuroinflammation by triggering a microglial production of pro-inflammatory cytokines and chemokines via TLR4-dependent pathway.We have already shown the essential role of microglia in the development of neuroinflammation but the extent of its contribution in the neuropathology of MPSIII still remains unclear. Microglia interacts with other cells in the CNS via several mechanisms, including extracellular vesicle (EV) release. Although EVs have been shown to be implicated in the pathogenesis of adult neurodegenerative diseases, no reports have addressed the proteins and small RNAs profile of EVs released by microglia in the context of MPS and the effect of these EVs on neuronal functions. Here, we isolated EVs sub-populations from conditioned media of the murine BV-2 microglial cell line treated with GAG purified from urines of MPSIII patients and from age-matched unaffected children. By label-free quantitative proteomic using LC-MS/MS, we showed that MPS III-EVs are enriched in proteins involved in the inflammatory response and more specifically in neuroinflammation. On the other hand, proteins involved in axonal guidance, myelination or synaptogenesis were less abundant in MPSIII-EVs. RNA sequencing analysis revealed that MPSIII-EVs are highly enriched in 4 miRNAs - miR-155-5p, miR-146a-5p, miR-221-3p and miR-100-5p, also involved in neuroinflammation and neurodevelopment pathways. Moreover, we showed by immunofluorescence on primary cortical neurons isolated from wild type pups that MPSIII microglia-EVs reduce neurites total area, impair dendritic arborization, with a higher number of immature dendritic spines and also cause an increase in soma swelling, thus inducing a phenotype close to that observed in MPSIII brain mice. Theses findings uncover that MPSIII microglia-EVs can deliver a specific molecular message to surrounding naive cells and thus propagate neuroinflammation and deprive neurons of neurodevelopmental molecules.This work is the first report on the content of EVs released by MPSIII microglia and reveals a disease-associated signature, providing a framework for future studies on biomarkers to evaluate efficiency of emerging therapies.

Project description:Sanfilippo syndrome type B (MPS III B) is an autosomal recessive, neurodegenerative disease of children, characterized by profound mental retardation and dementia. The primary cause is mutation in the NAGLU gene, resulting in deficiency of N-acetylglucosaminidase and lysosomal accumulation of heparan sulfate. In the mouse model of MPS III B, neurons and microglia display the characteristic vacuolation of lysosomal storage of undegraded substrate, but neurons in the medial entorhinal cortex (MEC) display accumulation of several additional substances. We used whole genome microarray analysis to examine differential gene expression in MEC neurons isolated by laser capture microdissection from Naglu -/- and Naglu +/- mice. Neurons from the lateral entorhinal cortex (LEC) were used as tissue controls. The highest increase in gene expression (6- to 7-fold between mutant and control) in MEC and LEC neurons was that of Lyzs, which encodes lysozyme, but accumulation of lysozyme protein was seen in MEC neurons only. Because of a report that lysozyme induced the formation of hyperphosphorylated tau (P-tau) in cultured neurons, we searched for P-tau by immunohistochemistry. P-tau was found in MEC of Naglu -/- mice, in the same neurons as lysozyme. In older mutant mice, it was also seen in the dentate gyrus, an area important for memory. Electron microscopy of dentate gyrus neurons showed cytoplasmic inclusions of paired helical filaments - P-tau aggregates characteristic of tauopathies, a group of age-related dementias that includes Alzheimer disease. Our findings indicate that the Sanfilippo syndrome type B should also be considered a tauopathy. Two-condition experiment, Naglu-/- (affected) vs. Naglu+/- (unaffected control) of neurons from two adjacent brain regions: medial entorhinal cortex (MEC) and lateral entorhinal cortex (LEC). Biological replicates: 3 MEC Naglu-/-, 3 MEC Naglu+/-, 3 LEC Naglu-/- and 3 LEC Naglu+/-. Comparisons were made between 3 pairs of mutant and control female littermates for MEC and LEC neurons with dye switch duplicates, for a total of 12 microarrays.

Project description:Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impacts EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. We hypothesize that microglia-derived EVs have unique molecular profiles that are determined by microglial activation state. Using size-exclusion chromatography to purify EVs from BV2 microglia, combined with proteomic (label-free quantitative mass spectrometry or LFQ-MS) and transcriptomic (mRNA and non-coding RNA seq) methods, we obtained comprehensive molecular profiles of microglia-derived EVs. LFQ-MS identified several classic EV proteins (tetraspanins, ESCRT machinery, and heat shock proteins), in addition to over 200 proteins not previously reported in the literature. Unique mRNA and microRNA signatures of microglia-derived EVs were also identified. After treating BV2 microglia with lipopolysaccharide (LPS), interleukin-10, or transforming growth factor beta, to mimic pro-inflammatory, anti-inflammatory, or homeostatic states, respectively, LFQ-MS and RNA seq revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, LPS treatment had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Furthermore, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally-relevant inflammatory signals. These comprehensive microglia-EV molecular datasets represent important resources for the neuroscience and glial communities, and provide novel insights into the role of microglia-derived EVs in neuroinflammation.

Project description:Toxoplasma gondii (T. gondii), a prolific protozoan parasite, forms cysts within neurons of the central nervous system that maintain infection for the lifetime of the host. Astrocytes are fundamental to neuronal health by providing nutrients and structural support and help regulate neurotransmitters by continuous communication with neurons. It is not yet known how infection and the presence of intracellular cysts, disrupts the crucial relationship between these cells. Extracellular vesicles (EVs) function in intracellular communication and can contain proteins, lipids, DNA, miRNA, and other RNA subtypes. EVs are produced by all cells and play an important role in neuronal-astrocyte interactions, including the regulation of glutamate receptors on astrocytes. Previous work has demonstrated that Toxoplasma infection reduces astrocytic expression of the primary glutamate transporter, GLT-1. Here we tested if cyst infection of neurons alters the production and content of EVs. EVs were isolated from uninfected and infected primary murine cortical neurons and their size, concentration, and characterization were confirmed with nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), ELISA, western blot, liquid chromatography (LC)-mass spectrometry (MS)/MS, and microRNA sequencing. Analysis reveals that infection of neurons reduced neuronal production of EVs and altered their protein and miRNA content. In addition to changes in host protein content, EVs from infected neurons contained the Toxoplasma proteins GRA1, GRA2, GRA7, MAG1 and MAG2. Following incubation of neuronal EVs with primary astrocytes, GRA7 protein could be observed within intracellular EVs and the nuclei of GRA7+EV-containing cells. EVs from infected neurons altered gene expression of astrocytes resulting in an increase in pro-inflammatory transcriptional signatures, along with a downregulation of GLT-1 protein expression with similar transcriptional changes found in astrocytes in vivo. These results demonstrate the ability of a parasitic infection in the brain to alter EV production and the fundamental communication between neurons and astrocytes.

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Extracellular vesicles from microglial cells activated by abnormal heparan sulfate oligosaccharides from Sanfilippo patients impair neuronal dendritic arborization

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