After 72?h, cells were fixed for 15?min at room heat with 4% PFA and then blocked and permeabilized for an hour with 5% BSA and 0

After 72?h, cells were fixed for 15?min at room heat with 4% PFA and then blocked and permeabilized for an hour with 5% BSA and 0.3% Triton\X100. validating PSMs ( Sources for other code used in this study are indicated in the Reagents and Tools Table. Detailed protocols for proteomics sample processing, phosphopeptide enrichment, C18 column preparation, and other cellular assays are available online: Abstract Alzheimers disease (AD) is characterized by the appearance of amyloid\ plaques, neurofibrillary tangles, and inflammation in brain regions involved in memory. Using mass spectrometry, we have quantified the phosphoproteome of the CK\p25, 5XFAD, and Tau P301S mouse models of neurodegeneration. We recognized a shared response including Siglec\F which was upregulated on a subset of reactive microglia. The human paralog Siglec\8 was also upregulated on microglia in AD. Siglec\F and Siglec\8 were upregulated following microglial activation with interferon gamma (IFN) in BV\2 cell collection and human stem cell\derived microglia models. Siglec\F overexpression activates an endocytic and pyroptotic inflammatory response in BV\2 cells, dependent on its sialic acid substrates and immunoreceptor tyrosine\based inhibition motif (ITIM) phosphorylation sites. Related human Siglecs induced a similar response in BV\2 cells. Collectively, our results point to an important role for mouse Siglec\F and human Siglec\8 in regulating microglial activation during neurodegeneration. models of Siglec activation on microglia. We found that treatment with interferon gamma (IFN) increased Siglec\F expression using BV\2 cells, an immortalized cell collection model of mouse microglia (Henn and recognized in AD GWAS studies (Karch & Goate, 2014). Due to the underexplored nature of Siglec\F in neurodegeneration, we next investigated its role in microglial activation. 2.4. Siglec\F is usually upregulated on subsets of inflammatory microglia To determine whether increased Siglec\F phosphorylation was associated with increased Siglec\F protein expression in our models, we used immunofluorescence (IF) imaging Mitoxantrone Hydrochloride to locate and quantify Siglec\F expression. We first validated that this antibody E50\2440 was able to label Siglec\F in BV\2 cells with stable viral protein expression of Siglec\F transporting mutated pTyr sites, with minimal immunoreactivity in control cells expressing an empty vector (Fig EV4A). We next stained CK and CK\p25 brain slices from mice DTX3 after 3?weeks of p25 induction. We observed that a subset of Iba1+ microglia in CK\p25 mice stained positive for Siglec\F expression (Fig?2A). In contrast, we did not observe immunoreactivity for Siglec\F on microglia in CK control mice that lack p25 expression. As previously observed, CK\p25 microglia exhibited gliosis (Mathys human induced pluripotent stem cell\derived microglia (iMGL) model. We observed that iMGLs upregulated Siglec\8 expression after 72?h of treatment with 25?ng/ml IFN by IF (Fig?3B). This effect was less effective with IFN or IFN, suggesting type\II interferon signaling dependence (Fig?3C). We observed that co\treatment of iMGLs with IFN and 5?M tofacitinib was able to reverse the upregulation of Siglec\8 by IF and qPCR (Figs ?(Figs3D3D and EV7F and G). We were able to replicate these effects in iMGLs matured in the presence of homeostasis\inducer transforming growth factor beta (TGF; Butovsky normalized relative to GADPH mRNA levels. Bars show mean??95% CI; n?=?6 replicates; *models, we found that IFN activation upregulates Siglec\F and Siglec\8. These results support a model in which the expression of Siglec\F and Siglec\8 changes on microglia during neurodegeneration and aging, potentially altering microglial inflammatory activity. One main phenotype that we found to be associated with Siglec\F expression on BV\2 cells was the induction of cell death. We found that this effect was dependent on Siglec\F substrate\binding and ITIM phosphorylation. Our transcriptomic data suggest that Siglec\F triggers cell death via a pro\inflammatory pathway including activation of the inflammasome and pyroptosis. Previous work on Siglec\F and Siglec\8 has shown that antibody engagement of these receptors causes a pro\apoptotic response in eosinophils, making them Mitoxantrone Hydrochloride a Mitoxantrone Hydrochloride Mitoxantrone Hydrochloride potential drug target for asthma (Zimmermann 2013) RSEM (v1.3.0)(Li & Dewey 2011) DESeq2 (v1.18.1)Bioconductor GSEA (v3.0 beta\2)Mootha (2003), Tamayo (2005) R (v3.4.4)N/A Salmon (v0.9.1)Patro, Duggal, Love, Irizarry, and Kingsford (2017) Python (v3.7.1)N/A MAGIC (v1.5.5)van Dijk (2012) scipy (v1.3.1)Virtanen (2016) goatools (v1.0.3)Klopfenstein (2015), Subramanian (2005) Fiji\ImageJ (v1.52u)National Inst. Of Health ZEN (v2.1 SP3)Zeiss Other Q Exactive PlusThermoIQLAAEGAAPFALGMBDKQ Exactive HF\XThermoIQLAAEGAAPFALGMBFZLTQ OrbitrapThermoN/AVT100S vibratomeLeicaN/ALSM 710ZeissN/ALSM 880ZeissN/ABD FACS Canto Cell SorterBD BiosciencesN/ALi\Cor Odessey CLxLI\COR BiosciencesN/AIncucyte Plate ImagerEssen BioscienceN/AIllumina HiSeq 2000IlluminaN/ACFX384 Touch Real\Time PCRBioradN/A Mitoxantrone Hydrochloride Open in a separate window 4.2. Methods and Protocols 4.2.1. Mouse models All animal work was approved by the Committee for Animal Care of the Division of Comparative Medicine at the Massachusetts Institute of Technology. The mouse models used in this study were: (i) double transgenic CK\p25, CK control (aged 3C4?months, induced for 2C7?weeks by removal of doxycycline from diet); (ii) 5XFAD; 5XFAD p35KI+/+, WT, WT p35KI+/+ (9C11?months); and (iii) Tau P301S Tg and WT (4C9?months). Mice were housed in groups of three to five on a standard 12?h light/12?h dark cycle, and all experiments were performed during the light cycle. Food and water were provided.

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