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Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer's disease.

ABSTRACT: BACKGROUND:Proteomic characterization of microglia provides the most proximate assessment of functionally relevant molecular mechanisms of neuroinflammation. However, microglial proteomics studies have been limited by low cellular yield and contamination by non-microglial proteins using existing enrichment strategies. METHODS:We coupled magnetic-activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) of microglia with tandem mass tag-mass spectrometry (TMT-MS) to obtain a highly-pure microglial proteome and identified a core set of highly-abundant microglial proteins in adult mouse brain. We interrogated existing human proteomic data for Alzheimer's disease (AD) relevance of highly-abundant microglial proteins and performed immuno-histochemical and in-vitro validation studies. RESULTS:Quantitative multiplexed proteomics by TMT-MS of CD11b?+?MACS-enriched (N?=?5 mice) and FACS-isolated (N?=?5 mice), from adult wild-type mice, identified 1791 proteins. A total of 203 proteins were highly abundant in both datasets, representing a core-set of highly abundant microglial proteins. In addition, we found 953 differentially enriched proteins comparing MACS and FACS-based approaches, indicating significant differences between both strategies. The FACS-isolated microglia proteome was enriched with cytosolic, endoplasmic reticulum, and ribosomal proteins involved in protein metabolism and immune system functions, as well as an abundance of canonical microglial proteins. Conversely, the MACS-enriched microglia proteome was enriched with mitochondrial and synaptic proteins and higher abundance of neuronal, oligodendrocytic and astrocytic proteins. From the 203 consensus microglial proteins with high abundance in both datasets, we confirmed microglial expression of moesin (Msn) in wild-type and 5xFAD mouse brains as well as in human AD brains. Msn expression is nearly exclusively found in microglia that surround A? plaques in 5xFAD brains. In in-vitro primary microglial studies, Msn silencing by siRNA decreased A? phagocytosis and increased lipopolysaccharide-induced production of the pro-inflammatory cytokine, tumor necrosis factor (TNF). In network analysis of human brain proteomic data, Msn was a hub protein of an inflammatory co-expression module positively associated with AD neuropathological features and cognitive dysfunction. CONCLUSIONS:Using FACS coupled with TMT-MS as the method of choice for microglial proteomics, we define a core set of highly-abundant adult microglial proteins. Among these, we validate Msn as highly-abundant in plaque-associated microglia with relevance to human AD.

SUBMITTER: Rayaprolu S

PROVIDER: S-EPMC7206797 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer's disease.

Rayaprolu Sruti S Gao Tianwen T Xiao Hailian H Ramesha Supriya S Weinstock Laura D LD Shah Jheel J Duong Duc M DM Dammer Eric B EB Webster James A JA Lah James J JJ Wood Levi B LB Betarbet Ranjita R Levey Allan I AI Seyfried Nicholas T NT Rangaraju Srikant S

Molecular neurodegeneration 20200507 1

<h4>Background</h4>Proteomic characterization of microglia provides the most proximate assessment of functionally relevant molecular mechanisms of neuroinflammation. However, microglial proteomics studies have been limited by low cellular yield and contamination by non-microglial proteins using existing enrichment strategies.<h4>Methods</h4>We coupled magnetic-activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) of microglia with tandem mass tag-mass spectrometry (TMT-MS) ...[more]

PMID: 32381088

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Project description:BackgroundFlow cytometric sorting can be used to separate sperm based on sex chromosome content. Differential fluorescence emitted by stained X- vs. Y-chromosome-bearing sperm enables sorting and collection of samples enriched in either X- or Y-bearing sperm for use to influence the likelihood that the offspring will be a particular sex. Herein we report the effectiveness of flow cytometric sorting of human sperm and its use in human ART procedures.MethodsThis prospective, observational cohort study of the series of subjects treated with flow cytometrically sorted human sperm was conducted at investigational sites at two private reproductive centers. After meeting inclusion criteria, married couples (n = 4993) enrolled to reduce the likelihood of sex-linked or sex-limited disease in future children (n = 383) or to balance the sex ratio of their children (n = 4610). Fresh or frozen-thawed semen was processed and recovered sperm were stained with Hoechst 33342 and sorted by flow cytometry (n = 7718) to increase the percentage of X-bearing sperm (n = 5635) or Y-bearing sperm (n = 2083) in the sorted specimen. Sorted sperm were used for IUI (n = 4448) and IVF/ICSI (n = 2957). Measures of effectiveness were the percentage of X- and Y-bearing sperm in sorted samples, determined by fluorescence in situ hybridization, sex of babies born, IVF/ICSI fertilization- and cleavage rates, and IUI, IVF/ICSI, FET pregnancy rates and miscarriage rates.ResultsSorted specimens averaged 87.7 ± 5.0% X-bearing sperm after sorting for X and 74.3 ± 7.0% Y-bearing sperm after sorting for Y. Seventy-three percent of sorts were for girls. For babies born, 93.5% were females and 85.3% were males after sorting for X- and Y-bearing sperm, respectively. IUI, IVF/ICSI, and FET clinical pregnancy rates were 14.7%, 30.8%, and 32.1%, respectively; clinical miscarriage rates were 15.5%, 10.2%, and 12.7%.ConclusionsFlow cytometric sorting of human sperm shifted the X:Y sperm ratio. IUI, IVF/ICSI and FET outcomes were consistent with unimpaired sperm function. Results provide evidence supporting the effectiveness of flow cytometric sorting of human sperm for use as a preconception method of influencing a baby's sex.Trial registrationNCT00865735 (ClinicalTrials.gov).

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Dataset Information

Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer's disease.

Publications

Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer's disease.

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