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.

Project description:Extracellular vesicles (EVs) are small membrane-derived vesicles that shuttle proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the CNS. Exosomes are small EVs (40-150 nm) that are derived from multi-vesicular bodies (MVBs) of the endo-lysosomal pathway, formed by inward budding of the limiting membrane into the MVB lumen and released into the extracellular space upon fusion of the MVB with the plasma membrane (PM). Previous research revealed certain effector molecules to be required for exosome release. For instance, RAB GTPases have been shown to control exosome release in a cell- and tissue-specific manner. In addition, exosome release appears to be evoked by membrane depolarization in conjunction with calcium influx and to depend on members of the SNARE family of proteins. However, the specific cellular and molecular factors that regulate neuronal exosome release and segregate it from the release of neurotransmitter vesicles are currently unknown. Here, we used a combination of molecular biology, patch-clamp electrophysiology and pH-sensitive dye imaging to examine the effect of the neuronal growth and differentiation factors basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) on neuronal EV release. We found that bFGF-treatment of cultured hippocampal neurons increased the abundance of EVs in the culture medium as measured by Western blot and nanoparticle tracking analysis (NTA), without affecting the number or size of neuronal MVBs. The effect of bFGF depended on calcium and on receptor tyrosine kinase (RTK) activity as the calcium chelating agent BAPTA and the tyrosine kinase inhibitor genistein both abolished the bFGF-induced increase in EV release. In accord with these results, untreated neurons had a low rate of spontaneous and stimulus-evoked MVB-PM fusion events as measured by pH-sensitive dye imaging in conjunction with patch-clamp electrophysiology, but treatment with bFGF significantly increased the number of neurons exhibiting MVB-PM fusion events in response to a high-frequency stimulus. Proteomic analysis of neuronal EVs by Liquid Chromatography Mass Spectrometry (LC-MS) demonstrated bFGF to increase the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs, whereas VAMP2 was decreased. Conversely, knocking-down VAMP3 in cultured neurons abolished the effect of bFGF on EV release. Similar to bFGF, the classical neurotrophins BDNF and NGF increased neuronal EV release in a VAMP3-dependent manner. In summary, our results thus reveal a new function for neurotrophic factor signalling in controlling neuronal exosome release and support the investigation of growth factor-mediated signal transduction via EVs in the healthy and diseased CNS.

Project description:Background : In mucopolysaccharidosis type III (MPS III, also known as Sanfilippo syndrome), a pediatric neurodegenerative disorder, accumulation of abnormal glycosaminoglycans (GAGs) induces severe neuroinflammation by triggering the microglial pro-inflammatory cytokines production via a TLR4-dependent pathway. But the extent of the microglia contribution to the MPS III neuropathology remains unclear. Extracellular vesicles (EVs) mediate intercellular communication and are known to participate in the pathogenesis of adult neurodegenerative diseases. However, characterization of the molecular profiles of EVs released by MPS III microglia and their effects on neuronal functions have not been described. Methods : Here, we isolated EVs secreted by the microglial BV-2 cells after treatment with GAGs purified from urines of Sanfilippo patients (MPS-GAGs EVs) or from age-matched healthy subjects (WT-GAGs EVs) to explore the EVs’ proteins and small RNA profiles using LC–MS/MS and RNA sequencing. We next performed a functional assay by immunofluorescence following WT-GAGs- or MPS-GAGs-EVs uptake by WT primary cortical neurons and analyzed their extensions metrics after staining of βIII-tubulin and MAP2 by confocal microscopy. Results : Functional enrichment analysis for both proteomics and RNA sequencing data from MPS-GAGs-EVs revealed a specific content involved in neuroinflammation and neurodevelopment pathways. Treatment of cortical neurons with MPS-GAGs-EVs induced a disease-associated phenotype demonstrated by a lower total neurite surface area, an impaired somatodendritic compartment, and a higher number of immature dendritic spines. Conclusions : This study shows, for the first time, that GAGs from patients with Sanfilippo syndrome can induce microglial secretion of EVs that deliver a specific molecular message to recipient naive neurons, while promoting the neuroinflammation, and depriving neurons of neurodevelopmental factors. This work provides a framework for further studies of biomarkers to evaluate efficiency of emerging therapies.

Project description:Toxoplasma gondii (T. gondii) cyst formation in the central nervous system only occurs in neurons allowing the parasite to remain latent 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 including neurons and play an important role in neuronal-astrocyte interactions including the regulation of glutamate receptors on astrocytes. Previous work has demonstrated 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), CD63 ELISA, 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. EVs from infected neurons contained secreted Toxoplasma proteins GRA1, GRA2, GRA7, MAG1 and MAG2 associated with cyst formation. Following incubation of neuronal EVs with primary astrocytes, a proportion of EVs colocalize to the nucleus. EVs from infected neurons altered gene expression of astrocytes leading to a downregulation of GLT-1 protein expression and an increase in pro-inflammatory transcriptional signatures. These results demonstrate the ability of a parasitic infection in the brain to alter EV production and the fundamental communication between neurons and astrocytes.

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.

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	240926_AG_Glatzel_Shafiq_Lombino_Cells_1.pdResult	Other
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	240926_AG_Glatzel_Shafiq_Lombino_Cells_2.raw	Raw
	240926_AG_Glatzel_Shafiq_Lombino_Cells_3.raw	Raw

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Extracellular vesicles released from cortical neurons influence spontaneous activity of recipient neurons.

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Extracellular Vesicles Released From Cortical Neurons Influence Spontaneous Activity of Recipient Neurons.

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