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Proteomics of TurboID-induced biotinylated proteins from astrocyte and astrocyte endeet after acute peripheral inflammation

ABSTRACT: The brain vasculature is tightly regulated to control the exchange of substances between the periphery and the brain parenchyma at an interface called the blood-brain barrier (BBB). Astrocytes are glial cells that wrap the brain vasculature with a specialised structure called the endfoot, which is an integral component of the BBB. We generated adeno-associated viruses (AAVs) that express a promiscuous biotin ligase, TurboID, specifically in astrocytes, and developed a method that allows us to define the in vivo protein composition of the astrocyte endfoot with negligible contamination of other cells or other compartments of the astrocyte. Here, we isolated the biotinylated proteins from both the whole astrocyte and astrocyte endfeet from mice with acute peripheral inflammation.

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ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Blanca Díaz Castro

LAB HEAD: Blanca Diaz-Castro

PROVIDER: PXD056232 | Pride | 2025-09-11

REPOSITORIES: Pride

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			Action	DRS
	MS_Rawfiles_Part1.zip	Other
	MS_Rawfiles_Part2.zip	Other
	report.pg_matrix.tsv	Tabular

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Project description:Astrocytes are key cells in brain aging, helping neurons to undertake healthy aging or otherwise letting them enter into a spiral of neurodegeneration. We aimed to characterize astrocytes cultured from senescence-accelerated prone 8 (SAMP8) mice, a mouse model of brain pathological aging, along with the effects of caloric restriction, the most effective rejuvenating treatment known so far. Analysis of the transcriptomic profiles of SAMP8 astrocytes cultured in control conditions and treated with caloric restriction serum was performed using mRNA microarrays. A decrease in mitochondrial and ribosome mRNA, which was restored by caloric restriction, confirmed the age-related profile of SAMP8 astrocytes and the benefits of caloric restriction. An amelioration of antioxidant and neurodegeneration-related path- ways confirmed the brain benefits of caloric restriction. Studies of oxidative stress and mitochondrial function demonstrated a reduction of oxidative damage and partial improvement of mito- chondria after caloric restriction. In summary, caloric restriction showed a significant tendency to normalize pathologically aged astrocytes through the activation of pathways that are protective against the age-related deterioration of brain physiology. Key words: astrocytes; caloric restriction; mitochondria; oxidative stress; RNA microarrays; SAMP8. Primary cultures enriched in astrocytes were obtained from cerebral cortical tissue from 2-day-old SAMP8 and SAMR1 mice. Astrocyte cultures were established and experiments were routinely carried out after 21 days in culture. Established astrocyte cultures of both SAMR1 and SAMP8 consisted of 85-90% astrocytes, 10-15% microglia and 0.1-1% oligodendroglia. Sera from rats subjected to ad libitum (AL) diet and to CR were obtained as described for the establishment of the CR in vitro model (de Cabo et al., 2003). Serum was heat inactivated at 56°C prior to use in astrocyte culture experiments. Treatment in vitro was performed by adding 10% volume CR or AL serum onto the astrocyte culture medium for 48 h, the cells were harvested and RNA was extracted for the microarray studies. Three biological replicates for each condition were done and RNA was extracted for the microarray studies. Please note that SAM models were developed from AKR/J by Kyoto University. Five litters with severe senescence were selected to further propagate and examine these characteristics. Litters that showed normal aging were selected as a senescence-resistant series (R-series). The genetic background of the SAM mice became suspect after the pathological findings were different from the AKR/J mouse. Each SAM model is genetically different. Each SAM colony was acquired by Harlan by Takeda Chemical Ltd. in 2002. And here is the link to the company site. http://www.harlan.com/products_and_services/research_models_and_services/research_models/sam_inbred_mice/samp8tahsd.hl

Project description:MartínJiménez2017 - Genome-scale reconstruction of the human astrocyte metabolic network This model is described in the article: Genome-Scale Reconstruction of the Human Astrocyte Metabolic Network. Martín-Jiménez CA, Salazar-Barreto D, Barreto GE, González J. Front Aging Neurosci 2017; 9: 23 Abstract: Astrocytes are the most abundant cells of the central nervous system; they have a predominant role in maintaining brain metabolism. In this sense, abnormal metabolic states have been found in different neuropathological diseases. Determination of metabolic states of astrocytes is difficult to model using current experimental approaches given the high number of reactions and metabolites present. Thus, genome-scale metabolic networks derived from transcriptomic data can be used as a framework to elucidate how astrocytes modulate human brain metabolic states during normal conditions and in neurodegenerative diseases. We performed a Genome-Scale Reconstruction of the Human Astrocyte Metabolic Network with the purpose of elucidating a significant portion of the metabolic map of the astrocyte. This is the first global high-quality, manually curated metabolic reconstruction network of a human astrocyte. It includes 5,007 metabolites and 5,659 reactions distributed among 8 cell compartments, (extracellular, cytoplasm, mitochondria, endoplasmic reticle, Golgi apparatus, lysosome, peroxisome and nucleus). Using the reconstructed network, the metabolic capabilities of human astrocytes were calculated and compared both in normal and ischemic conditions. We identified reactions activated in these two states, which can be useful for understanding the astrocytic pathways that are affected during brain disease. Additionally, we also showed that the obtained flux distributions in the model, are in accordance with literature-based findings. Up to date, this is the most complete representation of the human astrocyte in terms of inclusion of genes, proteins, reactions and metabolic pathways, being a useful guide for in-silico analysis of several metabolic behaviors of the astrocyte during normal and pathologic states. This model is hosted on BioModels Database and identified by: MODEL1608180000. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Research regarding the role of astrocytes as M-NM-2-amyloid (AM-NM-2) degrading cells has broadened our view about the mechanisms how these common glia cells function in AlzheimerM-bM-^@M-^Ys disease (AD). Based on previous studies adult mouse astrocytes are able to degrade AM-NM-2 deposits from AD mouse model and human brain tissue sections ex vivo, contrary to neonatal astrocytes. In this study, we studied the possible altered gene expression profiles of adult and neonatal astrocytes cultured for 22 h on top of the AM-NM-2 burdened tg APdE9 or wild-type mouse brain sections using whole genome microarrays. Quantitative RT-PCR analysis confirmed the significant up-regulation of HtrA serine peptidase 1 (Htra1), metallopeptidase 9 (Mmp9), phosphate regulating gene with homologies to endopeptidases on the X chromosome (Phex) and scavenger receptor class A, member 5 (Scara5) in adult astrocytes, whereas neonatal astrocytes up-regulated Mmp9 and down-regulated genes related to cholesterol transport and synthesis: apolipoprotein E (Apoe), 24-dehydrocholesterol reductase (Dhcr24) and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (Hmgcs1). These findings brought out novel genes which expression is altered during AM-NM-2 clearance in adult and neonatal astrocytes ex vivo. Astrocyte cultures: For the adult astrocyte cultures, hippocampi and cortices were isolated from 6-8-week-old C57Bl/6j wild-type and tg eGFP mice and the tissue was suspended in DMEM/F12 (3:1, Gibco BRL, NY, USA) containing 10 % heat-inactivated fetal bovine serum (Gibco BRL) and 100 U / ml penicillin-streptomycin (Gibco BRL). The cells were treated with Percoll (Sigma-Aldrich, St.Louis, USA) and plated onto poly-L-lysine coated flasks in DMEM/F12 (3:1) containing 10 % heat-inactivated fetal bovine serum, 100 U / ml penicillin-streptomycin and G5 supplement (Gibco BRL). Before the experiments the glial cell cultures were shaken to remove microglia at 200 rpm for 2 h. For the neonatal astrocyte cultures, hippocampi and cortices were isolated from 2-day-old wild-type and eGFP mice according to the protocol described above, except that the culture medium was DMEM (+4500mg/l glucose, + L-glutamine, -pyruvate, Gibco BRL) without G5 supplement and the isolation method included no Percoll treatment. Standard anti-GFAP (1:500; Dako Cytomation, Glostrup, Denmark) immunocytochemistry was used to determine the purity of astrocyte cultures. The cells were incubated with biotinylated goat anti-rabbit IgG (1:200 dilution; Vector Laboratories, CA) secondary antibody and Vectastain ABC Elite kit was used (Vector Laboratories) according to manufacturerM-bM-^@M-^Ys protocol. Hydroxen peroxide (H2O2) and nickel enhanced diaminobenzidine (Ni-DAB) was added to detect the immunoreactivity. Three fields per well and at least three wells per cell type were analyzed for the percentage of Ni-DAB stained cells of all cellular profiles in the field. Adult and neonatal astrocyte cultures both contained on average 99.6 % M-BM-1 0.6 % anti-GFAP immunoreactive astrocytes. Anti-Iba (2 M-BM-5g/ml; Wako, Germany) and anti-CD11b (1:500 dilution; Serotec, UK) antibodies were used to detect possible microglial cells. The cultures contained less than 0.4 % of microglial cells in all experiments. Ex vivo treatments: tg APdE9 / wild-type mouse brain section incubated with neonatal (age 3 d) or adult (age 6-8 wks) mouse eGFP expressing astrocytes (4 x 105 cells / well), n=6. The mouse brain sections used were prepared from tg APdE9 mouse line (created by co-injection of chimeric mouse/human APPSwe and PS1-dE9 vectors, both controlled by their own mouse prion protein promoter element [APdE9 mice (Jankowsky et al. 2004) provided by Prof. Heikki Tanila] and their wild type C57Bl/6j littermates. Before the mRNA isolation process, the samples were pooled (n=3). The purity of the samples was checked with FACS (FACSCalibur, Becton Dickinson, USA) using the analyzing program CellQuestTM version 3.3 (Becton Dickinson, USA). After 22 h incubation the culture medium was removed from the wells and the brain sections with cultured eGFP astrocytes were incubated with 1 x trypsin-EDTA (500 M-BM-5l /well) for up to 15 min at 37M-BM-0 C. 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Project description:MicroRNAs (miRNAs) are an evolutionarily conserved large class of small non-coding RNAs that mediate post-transcriptional silencing of genes and influence a broad spectrum of biological processes ranging from embryonic development to organismal death. Our previous study identified the miR-29 family, three paralogous species of miR-29a/b/c, as the most predominantly expressed small RNA in aged mouse brain compared to neonate one. The mouse brain miR-29 is highly astrocytic. Its expression is quiescent during early brain development, and thereafter steadily increases to a saturating level around the end of the reproductive phase. To explore the functional relevance of miR-29 expression to the neural physiology of mouse brain from the mechanistic perspective of mammalian species-specific lifespan, we here designed a gain-of-function approach through forced expression of miR-29 in astrocyte from mouse fetus and surveyed the resulting alteration in the transcriptional and translational levels. DNA microarray analyses retrieved a total of 5,589 genes showing a temporal significant expression changes in the miR-29-transfected fetal astrocytes, and classified them into two gene groups positively or negatively regulated by miR-29. Mass spectrometry (MS)-based quantification of translational products of miR-29-responsive genes identified 18 species of miR-29 target candidates. We performed functional enrichment analyses using bioinformatics resources to elucidate the biological roles of the gene sets thus identified, and obtained their expression trend that favor the processes for facilitating cell differentiation while supporting normal cell proliferation /survival, which was different from the functional signatures of miR-29 at adult stages, implying the bifunctional property of miR-29 depending on the developmental context. Our present results strongly suggest that miR-29 serves as the central coordinator to shift the global gene expression in the developing mouse brain toward matured phenotype, and through which ensure the programmed transition to the post-developmental stage that is inherently set for the mouse lifespan. DNA microarray experiment: Sample labeling, hybridization and washing were performed following the standard protocol detailed in the Agilent One-Color Microarray-Based Gene Expression Analysis ver.5.7. Briefly, a One-color Spike-Mix was diluted 1000-fold and a 5-μl aliquot of the diluted mix were added to every 0.5 μg of total RNA samples (three each of extraction replicates of miR-29-treated sample and control sample named elsewhere) prior to labeling reaction. The labeling reaction was carried out separately for the RNA samples using a Quick Amp Labeling Kit one-color, in the presence of cyanine 3-CTP. The dye-labeled target (1.6 μg as cRNA) was fragmented and hybridized on an Agilent Whole Mouse Genome 4X44K microarray at 65°C for 17 hr with a Gene Expression Hybridization Kit. The hybridized slide was washed in Gene Expression Wash Buffer 1 at room temperature for 1 min, which was followed by a wash for 1 min in Gene Expression Wash Buffer 2. The processed microarrays were scanned using an Agilent DNA Microarray Scanner. Data extraction from raw image files was done with Agilent Feature Extraction software ver.10.7.

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Proteomics of TurboID-induced biotinylated proteins from astrocyte and astrocyte endeet after acute peripheral inflammation

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