Micro-RNAs secreted through astrocyte-derived extracellular vesicles cause neuronal network degeneration in C9orf72 ALS
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ABSTRACT:
ORGANISM(S): synthetic construct Homo sapiens
PROVIDER: GSE122640 | GEO | 2019/01/08
REPOSITORIES: GEO
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Project description:This datased was used to obtain a genome-wide expression signature for the early response of mouse motor neurons to mutant SOD1 astrocytes conditioned media. Neurons, far from living in isolation, are surrounded by a host of other neuronal and non-neuronal cells, such as astrocytes. The latter entertain complex functional interactions with neighboring neurons, which, under normal conditions, are important for the their well-being. In pathological situations, however, altered astrocyte behavior may contribute to the demise of neighboring neurons. Such non-cell autonomous pathogenic scenario is increasingly considered in a variety of disorders, including amyotrophic lateral sclerosis (ALS), the most frequent adult-onset paralytic disorder. Assembly and interrogation of gene regulatory models has helped elucidate causal mechanisms responsible for the presentation of several tumor-related phenotypes. To systematically elucidate the effectors of neurodegeneration in a model of ALS, we first developed techniques for the efficient purification of motor neurons (MNs), the primary target of ALS neurodegenerative process. We then generated gene expression profiles to fully characterize the critical timepoints associated with initiation and commitment of MN degenerative progression in an in vitro murine mutant SOD1 (mSOD1) model of ALS. ES cells were derived from transgenic Hlxb9-GFP1Tmj mice expressing eGFP and CD2 driven by the mouse HB9 promoter. These cells were then differentiated into motor neurons (ES-MN) as described previously [PMID 12176325] ES-MN were exposed to non-transgenic (NTg), G93A mutant SOD1 (mSOD1) or wtSOD1 over-expression astrocytes conditioned media for 0 days (time zero control), 1 day, and 3 days. Total RNA was extracted and profiled by RNAseq.
2014-12-17 | E-GEOD-49023 | biostudies-arrayexpress
Project description:Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons (MNs). It was shown that human astrocytes with mutations in genes associated with ALS, like C9orf72 (C9) or SOD1, reduce survival of MNs. Astrocyte toxicity may be related to their dysfunction or the release of neurotoxic factors. We used human induced pluripotent stem cell-derived astrocytes from ALS patients carrying C9orf72 mutations and non-affected donors. We utilized these cells to investigate astrocytic induced neuronal toxicity, changes in astrocyte transcription profile as well as changes in secretome profiles. We report that C9-mutated astrocytes are toxic to MNs via soluble factors. The toxic effects of astrocytes are positively correlated with the length of astrocyte propagation in culture, consistent with the age-related nature of ALS. We show that C9-mutated astrocytes downregulate the secretion of several antioxidant proteins. In line with these findings, we show increased astrocytic oxidative stress and senescence. Importantly, media conditioned by C9-astrocytes increased oxidative stress in wild type MNs. Our results suggest that dysfunction of C9-astrocytes leads to oxidative stress of themselves and MNs, which probably contributes to neurodegeneration. Our findings suggest that therapeutic strategies in familial ALS must not only target MNs but also focus on astrocytes to abrogate nervous system injury.
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Project description:Diversified neurons are essential for sensorimotor function, but whether astrocytes become specialized to optimize circuit performance remains unclear. Large fast α-motor neurons (FαMNs) of spinal cord innervate fast-twitch muscles that generate peak strength. We report that ventral horn astrocytes express the inward rectifying K+ channel Kir4.1 (aka, Kcnj10) around MNs in a VGLUT1-dependent manner. Loss-of astrocyte-encoded Kir4.1 selectively altered FαMN size, function and led to reduced peak strength. Overexpression of Kir4.1 in astrocytes was sufficient to increase MN size through activation of the PI3K/mTOR/pS6 pathway. Kir4.1 was downregulated cell-autonomously in astrocytes derived from amyotrophic lateral sclerosis (ALS) patients with SOD1 mutation. However, astrocyte Kir4.1 was dispensable for FαMN survival even in the mutant SOD1 background. These findings show that astrocyte Kir4.1 is essential for maintenance of peak strength and suggest that Kir4.1 downregulation uncouples symptoms of muscle weakness from MN cell death in ALS.
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Project description:As recently reported by our group, we performed miRNA and gene expression profiling of CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 42 PMF patient samples compared with 31 healthy controls. Integrative analysis of these profiles by means of Ingenuity Pathway Analysis (IPA) allowed the identification of several aberrantly regulated miRNA-mRNA target pairs organized in interaction networks. In particular, our results highlighted the up-regulation of miR-494-3p in CD34+ cells from PMF patients (Norfo R et al, Blood, 2014). Interestingly, among the most upregulated miRNAs, miR-494-3p emerges as being associated to the highest number of downregulated target mRNAs. In order to understand the biological role of miR-494-3p during the hematopoietic commitment and differentiation, we overexpressed this miRNA in cord blood (CB) derived-CD34+ cells. Cells were electroporated with either miR-494-3p miRNA mimic (mimic miR-494) or a negative control mimic (mimic Neg CTR). qRT-PCR confirmed miR-494-3p overexpression 24h and 4 days after transfection (RQ ± SEM, 512.60 ± 137.37, p<.01, and 20.63 ± 3.03, p<.01, respectively). Immunophenotypic analysis of CD41 and CD42b megakaryocyte (MK) lineage differentiation markers, performed on serum-free megakaryocytic ultilineage culture at day 3, 5, 8, 10 and 12, demonstrated that miR-494-3p overexpression induces a significant increase in the MK fraction compared to control samples. Accordingly, morphological analysis of May-Grünwald-Giemsa stained cytospins revealed an expansion of megakaryocytic lineage in samples overexpressing miR-494-3p. These data were further confirmed by collagen-based clonogenic assays which showed a significant increase in the percentage of megakaryocytic colonies in miR-494-3p overexpressing samples compared with controls. In order to better characterize the molecular mechanisms underlying the effects of miR-494-3p on HSPCs differentiation, we performed gene expression analysis of miR-494-3p overexpressing cells 24 hours after the last nucleofection.
2017-06-12 | GSE85250 | GEO
Project description:The microenvironment of injured mucosa has important effects on intestinal stem cell self-renewal and reconstruction of epithelial barrier function in inflammatory bowel disease (IBD). However, the precise status of the interactions between intestinal epithelial cell (IEC) injury, particularly intestinal crypt absence, and microenvironment in IBD is not completely understood. We identified miR-494-3p as important for protection of colonic stemness in intestinal inflammation colonic organoid culture. A novel cytokine-cytokine receptor, EDA-A2/EDA2R, could suppress colonic stemness and epithelial repair during IBD. During intestinal inflammation, high level of LP macrophage-derived EDA-A2 inhibited the nuclear β-catenin/c-Myc axis and organoid growth by targeting EDA2R in colonic crypt stem cells. We further demonstrated that the pro-inflammatory cytokines IL-1β and IL-6 are capable of stimulating macrophages to release EDA-A2 during colitis. Secondly, we identified the cross-talk among IECs, colonic crypts, and lamina propria (LP) macrophages in miR-494-3p-mediated colitis. Furthermore, our study showed that miR-494-3p deficiency in IECs promoted LP macrophage recruitment and M1 activation in DSS-induced colitis mice. In addition, we identified miR-494-3p as critical to dampening IEC injury; specifically, miR-494-3p inhibited inflammation-induced IKKβ/NF-κB activation by targeting the IKKβ 3’UTR in IECs. As such, administration of adequate amounts of a miR-494-3p agomire attenuate colitis in vivo. Consistent with this inference, we showed that miR-494-3p levels were decreased in colonic crypts and serum in colitis mice, and loss of miR-494 potentiated the severity of colonic colitis. Our clinical data on the interactions between miR-494-3p levels in serum exosomes & colonic tissues and associated outcomes support the clinical relevance of miR-494-3p in IBD. The miR-494-3p agomir system, which we designed permits local delivery in vivo in this study, significantly ameliorated the severity of colonic colitis. Our findings no only uncover a miR-494-3p-mediated cross-talk mechanism by which inflamed colonic LP macrophages integrate signals from IECs to regulate colonic stemness and colonic epithelial repair/homeostasis. The miR-494-3p agomir may serve as a potential therapeutic approach in IBD.
2019-09-24 | GSE137889 | GEO
Project description:The microenvironment of injured mucosa has important effects on intestinal stem cell self-renewal and reconstruction of epithelial barrier function in inflammatory bowel disease (IBD). However, the precise status of the interactions between intestinal epithelial cell (IEC) injury, particularly intestinal crypt absence, and microenvironment in IBD is not completely understood. We identified miR-494-3p as important for protection of colonic stemness in intestinal inflammation colonic organoid culture. A novel cytokine-cytokine receptor, EDA-A2/EDA2R, could suppress colonic stemness and epithelial repair during IBD. During intestinal inflammation, high level of LP macrophage-derived EDA-A2 inhibited the nuclear β-catenin/c-Myc axis and organoid growth by targeting EDA2R in colonic crypt stem cells. We further demonstrated that the pro-inflammatory cytokines IL-1β and IL-6 are capable of stimulating macrophages to release EDA-A2 during colitis. Secondly, we identified the cross-talk among IECs, colonic crypts, and lamina propria (LP) macrophages in miR-494-3p-mediated colitis. Furthermore, our study showed that miR-494-3p deficiency in IECs promoted LP macrophage recruitment and M1 activation in DSS-induced colitis mice. In addition, we identified miR-494-3p as critical to dampening IEC injury; specifically, miR-494-3p inhibited inflammation-induced IKKβ/NF-κB activation by targeting the IKKβ 3’UTR in IECs. As such, administration of adequate amounts of a miR-494-3p agomire attenuate colitis in vivo. Consistent with this inference, we showed that miR-494-3p levels were decreased in colonic crypts and serum in colitis mice, and loss of miR-494 potentiated the severity of colonic colitis. Our clinical data on the interactions between miR-494-3p levels in serum exosomes & colonic tissues and associated outcomes support the clinical relevance of miR-494-3p in IBD. The miR-494-3p agomir system, which we designed permits local delivery in vivo in this study, significantly ameliorated the severity of colonic colitis. Our findings no only uncover a miR-494-3p-mediated cross-talk mechanism by which inflamed colonic LP macrophages integrate signals from IECs to regulate colonic stemness and colonic epithelial repair/homeostasis. The miR-494-3p agomir may serve as a potential therapeutic approach in IBD.
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Project description:Defects in stress responses are important contributors in many chronic conditions including cancer, cardiovascular disease, diabetes, and obesity-driven pathologies like non-alcoholic steatohepatitis (NASH). Specifically, endoplasmic reticulum (ER) stress is linked with these pathologies and control of ER stress can ameliorate tissue damage. MicroRNAs have a critical role in regulating diverse stress responses including ER stress. Here we show that miR-494-3p plays a functional role during ER stress. ER stress inducers (tunicamycin and thapsigargin) robustly increase the expression of miR-494 in vitro in an ATF6 dependent manner. Surprisingly, miR-494 pretreatment dampens the induction and magnitude of ER stress in response to tunicamycin in endothelial cells. Conversely, inhibition of miR-494 increases ER stress de novo and amplifies the effects of ER stress inducers. Using Mass Spectrometry (TMT-MS) we identified many proteins that are downregulated by both tunicamycin and miR-494 in cultured human umbilical vein endothelial cells (HUVECs). Among these, we found 6 transcripts which harbor a putative miR-494 binding site. Our data indicates that ER stress driven miR-494 may act in a feedback inhibitory loop to dampen downstream ER stress signaling. We propose that RNA-based approaches targeting miR-494 or its targets may be attractive candidates for inhibiting ER stress dependent pathologies in human disease.
2021-09-09 | PXD019992 | Pride
Project description:Neuroinflammation is an important hallmark in amyotrophic lateral sclerosis (ALS). Experimental evidence has highlighted a role of microglia in the modulation of motor neuron degeneration. However, the exact contribution of microglia to both sporadic and genetic forms of ALS is still unclear. We generated single nuclei profiles of spinal cord and motor cortex from sporadic and C9orf72 ALS patients, as well as controls. We particularly focused on the transcriptomic responses of both microglia and astrocytes. We confirmed that C9orf72 is highly expressed in microglia and shows a diminished expression in carriers of the hexanucleotide repeat expansion (HRE). This resulted in an impaired response to disease, with specific deficits in phagocytic and lysosomal transcriptional pathways. Astrocytes also displayed a dysregulated response in C9orf72 ALS patients, remaining in a homeostatic state. This suggests that C9orf72 HRE alters a coordinated glial response, which ultimately would increase the risk for developing ALS. Our results indicate that C9orf72 HRE results in a selective microglial loss-of-function, likely impairing microglial-astrocyte communication and preventing a global glial response. This is relevant as it indicates that sporadic and familial forms of ALS may present a different cellular substrate, which is of great importance for patient stratification and treatment.
| EGAS00001006711 | EGA