Background
Methods
Cell culture and treatments
Primary microglia extraction
Super-SILAC processing and mass spectrometric analysis
Statistical, pathway, and upstream regulator analysis
RT-PCR
Western blot
Results and discussion
Proteomics-based identification of aging-related proteins
Altered functional processes identified in aged microglia
Upregulated | Downregulated |
---|---|
Oxidative phosphorylation—UQCRC2, NDUFB3, ATP5D, TCIRG1, UQCRC1, COX7A2, NDUFA7, ATP5F1, COX4I1, COX6C, SDHA, ATP6V1C1, ATP6V1A, NDUFS5, COX6B1, ATP6V0D1 | Spliceosome—NCBP1, TCERG1, PRPF8, U2AF2, TRA2B, SNRNP200, PRPF3, HNRNPC, DDX5, HNRNPU, SF3B2, SNRPG, PRPF40A |
Oxidoreductase—HSD17B11, UQCRC2, HSD17B10, CYB5R1, ME2, GLUD1, PGD, OGDH, PRDX1, FTH1, HADHA, ACOX3, IVD, CAT, COX7A2, ACADS, COX4I1, CBR4, TECR, COX6C, SOD2, SDHA, DHRS1, CYBA, BLVRB | Nuclear lumen—LMNB1, MTA2, PRPF3, DDX5, CBX5, HNRNPL, NONO, SET, TCERG1, HNRNPH2, ILF2, FRG1, PRPF8, ANP32A, RRS1, ACTL6A, RUVBL1, HNRNPH1, RBM14, LBR, PRPF40A, SMARCA4 |
Mitochondrion—UQCRC2, ATP5D, UQCRC1, COX7A2, ME2, GATM, ACADS, GLUD1, ATP5F1, ECHS1, COX4I1, OGDH, HADHA, HADHB, SOD2, SDHA, ACOT9, IVD, OXCT1, ATPIF1, HMGCL | Helicase activity—DDX17, SNRNP200, RUVBL1, DDX5, CHD4, SMARCA4 |
Hydrogen ion transmembrane transporter activity—TCIRG1, ATP5D, ATP6V1C1, ATP6V1A, COX7A2, COX6B1, ATP5F1, COX4I1, ATP6V0D1, COX6C | Nucleotide binding—RAB5B, TRA2B, U2AF2, HNRNPL, GPD1L, NONO, HSPH1, DDX17, AGPS, TARDBP, TUBB5, ACTL6A, HNRNPC, CHD4, CSTF2, STK24, ELAVL1, ATP1A1, DDX5, CRYZ, HNRNPR, HNRNPH2, ILF2, MAPK14, PSMC1, SNRNP200, RUVBL1, RBM14, SMC1A, HNRNPH1, SMARCA4, MDH1 |
Regulation of actin cytoskeleton organization—DBNL, CORO1A, ARPC3, ARPC2, CAPG, MYO1F, ARPC5, CAPZB | Chromatin organization—SET, SMARCD2, HIST1H1B, MTA2, SMARCC2, ACTL6A, RUVBL1, RBM14, CHD4, CBX5, SMARCA4 |
Lysosome—LAMP1, NPC1, CTSZ, LAMP2, LIPA, HEXA, HEXB, ARL8A, ACP2, CTSB, ARL8B, SCARB2 | Regulation of transcription—SUB1, MTA2, TERF2IP, DDX5, CBX5, NONO, FUBP3, TCERG1, SMARCD2, ILF2, HNRNPUL1, TARDBP, MAPK14, SMARCC2, ANP32A, ACTL6A, RUVBL1, RBM14, CHD4, SMARCA4 |
Generation of precursor metabolites and energy—UQCRC2, NDUFB3, ATP5D, TCIRG1, UQCRC1, NDUFA7, ATP5F1, HK2, OGDH, SOD2, SDHA, ATP6V1C1, ATP6V1A, CYBA, NDUFS5, GOT1, CAT, ATP6V0D1 | Nucleocytoplasmic transport—NCBP1, KPNA6, NUTF2, IPO9, KPNA3 |
Valine, leucine, and isoleucine degradation—HSD17B10, ACADS, IVD, OXCT1, ECHS1, HMGCL, HADHA, HADHB | Proteasome complex—PSMB4, PSMA6, PSMC1 |
Response to oxidative stress—GATM, APOE, CAT, GCLM, PRDX1, SOD2 | DNA repair—NONO, DDB1, SMC1A, APEX1 |
Immune effector process—PTPRC, CD47, MYO1F, FCER1G, INPP5D, PRDX1 | Coenzyme binding—GPD1L, DBT, AGPS, CRYZ, MDH1 |
Cell chemotaxis—CORO1A, FCER1G, NUP85 | Chromatin remodeling complex—MTA2, ACTL6A, CBX5, SMARCA4 |
Antigen processing and presentation of peptide antigen via MHC class I—H2-D1, FCER1G, B2M, TAPBP | |
Apoptotic mitochondrial changes—SH3GLB1, BAX, SOD2 | |
Cellular lipid catabolic process—HEXA, PLCG2, HEXB, HADHA, ACOX3, HADHB |
Disruptions in chromatin remodeling and transcriptional regulation
Loss of nuclear architecture and impairments in RNA processing
Aged microglia exhibit a bioenergetic shift from glucose to fatty acid utilization
Top 10 upstream regulators by p value |
p value of overlap | Activated upstream regulators by z-score |
z-score | Inhibited upstream regulators by z-score |
z-score |
---|---|---|---|---|---|
RICTOR | 3.55E−12 | INSR | 3.43 | MAP4K4 | −3.00 |
MYC | 3.21E−11 | IFNG | 3.37 | RICTOR | −2.84 |
Nrf1 | 1.42E−10 | IKBKB | 3.14 | VCAN | −2.00 |
NFE2L2 | 1.20E−09 | APP | 3.11 | ||
CD3 | 1.22E−09 | NRF1 | 2.97 | ||
TP53 | 7.91E−09 | TP53 | 2.58 | ||
HNF4A | 1.83E−08 | XBP1 | 2.56 | ||
INSR | 3.49E−08 | Ins1 | 2.53 | ||
TGFB1 | 8.12E−08 | CST5 | 2.5 | ||
CST5 | 1.37E−07 | Cdc42 | 2.45 |