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 1,791 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 Aβ plaques in 5xFAD brains. In in-vitro primary microglial studies, Msn silencing by siRNA decreased Aβ 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Sruti Rayaprolu: srayapr{at}emory.edu
Tianwen Gao: 1758643866{at}qq.com
Hailian Xiao: hxiao6{at}emory.edu
Supriya Ramesha: supriya.ramesha{at}emory.edu
Laura D. Weinstock: lweinstock3{at}gatech.edu
Jheel Shah: jheel.shah{at}emory.edu
Duc M. Duong: dduong{at}emory.edu
Eric B. Dammer: edammer{at}emory.edu
James A. Webster, Jr: james.a.webster{at}emory.edu
James J. Lah: jlah{at}emory.edu
Levi B. Wood: levi.wood{at}me.gatech.edu
Ranjita Betarbet: rbetarb{at}emory.edu
Allan I. Levey: alevey{at}emory.edu
Nicholas T. Seyfried: nseyfri{at}emory.edu
Srikant Rangaraju: srikant.rangaraju{at}emory.edu
Figure 2, 3, and 4 revised; Added Figure 5; Supplemental figures updated; Added new data.
Abbreviations
- Aβ
- amyloid-beta
- ACN
- acetonitrile
- AD
- Alzheimer’s disease
- ANOVA
- analysis of variance
- AGC
- automatic gain control
- AsymAD
- asymptomatic Alzheimer’s disease
- BCA
- bicinchoninic acid
- CE
- collision energy
- COBRA
- concurrent brain cell type acquisition
- DAM
- disease-associated-microglia
- DAPI
- diamidino-2-phenylindole
- DTT
- dithiothreitol
- ER
- endoplasmic reticulum
- ERM
- ezrin-radixin-moesin
- FDR
- false discovery rate
- FBS
- fetal bovine serum
- FACS
- fluorescence activated cell sorting
- FA
- formic acid
- fAβ42-488
- fibrillar fluorescent Aβ42 conjugated to HiLyte Fluor 488
- GO
- gene ontology
- GWAS
- genome-wide association studies
- HCD
- higher energy collision dissociation
- IAA
- iodoacetamide
- KD
- knockdown
- LFQ
- label-free quantitation
- LPS
- lipopolysaccharide
- MACS
- magnetic-activated cell sorting
- MS
- mass spectrometry
- MMSE
- mini-mental status examination
- Msn
- moesin
- PBS
- phosphate buffered saline
- PE
- phycoerythrin
- PSM
- peptide spectral match
- qRT-PCR
- quantitative reverse transcriptase pcr
- rpAD
- rapidly progressive ad
- RT
- room temperature
- SNPs
- single-nucleotide polymorphisms
- siRNA
- small interfering ribonucleic acid
- sAD
- sporadic ad
- SIP
- stock isotonic percoll
- SPS
- synchronous precursor selection
- TMT
- tandem mass tag
- TEAB
- triethylammonium bicarbonate
- TFA
- triflouroacetic acid
- TBS
- tris buffered saline
- WGCNA
- weighted correlation network analysis