Proteomic profiling of circulating plasma exosomes reveals novel biomarkers of Alzheimer's disease

Abstract

Background: Neuronal- and astrocyte-derived exosomes have been identified as an optimal source for screening biomarkers for Alzheimer's disease (AD). However, few studies focus on the bulk exosome population isolated from plasma of AD. This study investigated whether proteins in bulk exosomes can aid in the diagnosis of AD.

Methods: The plasma exosomes were collected by ultracentrifuge. Protein samples were extracted from exosomes. Cerebrospinal fluid levels of amyloid β (Aβ)42 and phosphorylated tau (P-tau)181 were measured for diagnostic purposes. A pilot study (controls, 20; AD, 20) followed by a second dataset (controls, 56; AD, 58) was used to establish a diagnostic model of AD. Mass spectrometry-based proteomics was performed to profile the plasma exosomal proteome. Parallel reaction monitoring was used to further confirm the differentially expressed proteins.

Results: In total, 328 proteins in plasma exosomes were quantified. Among them, 31 proteins were altered in AD patients, and 12 were validated. The receiver operating characteristic curve analysis revealed a combination of six proteins (upregulated: Ig-like domain-containing protein (A0A0G2JRQ6), complement C1q subcomponent subunit C (C1QC), complement component C9 (CO9), platelet glycoprotein Ib beta chain (GP1BB), Ras suppressor protein 1 (RSU1); downregulated: disintegrin and metalloproteinase domain 10 (ADA10)) has the capacity to differentiate AD patients from healthy controls with high accuracy. Linear correlation analysis showed that the combination was significantly correlated with cognitive performance.

Conclusions: The combination of plasma exosomal proteins A0A0G2JRQ6, C1QC, CO9, GP1BB, RSU1, and ADA10 acts as a novel candidate biomarker to differentiate AD patients from healthy individuals.

Keywords: Alzheimer’s disease; Biomarker; Diagnosis; Exosome; Proteomics.

Fig. 1

Confirmation of exosomes by transmission electron microscopy (TEM), Western blot, CD9, CD63, and CD81. A Plasma samples contained vesicles of typical exosomes morphology (black arrow) under TEM from an AD patient. Scale bar = 100 nm. B Western blot analysis of Alix, a common exosome surface marker, expression in the exosome samples. Supernates served as the negative control. C–E Measurement of CD9 (C), CD63 (D), and CD81 (E) in exosomes. AD, Alzheimer’s disease; NS, no significance

Fig. 2

Heat map of 15 upregulated and 16 downregulated proteins in the pilot study (dataset 1). The heatmap was generated by log₂ transformation of fold changes, with positive values representing upregulation (depicted in orange), and negative values representing downregulation (depicted in blue). AD, Alzheimer’s disease; A0A075B6R9, probable non-functional immunoglobulin kappa variable 2D-24; B1AHL2, fibulin-1; CC110, coiled-coil domain-containing protein 110; FCN2, ficolin-2;APOL1, apolipoprotein L1; HPTR, haptoglobin-related protein; KVD30, immunoglobulin kappa variable 2D-30; RSU1, Ras suppressor protein 1; GP1BB, platelet glycoprotein Ib beta chain; ALAT2, alanine aminotransferase 2; A0A0G2JRQ6, Ig-like domain-containing protein; CO9, complement component C9; C1QC, complement C1q subcomponent subunit C; CO7, complement component C7; CFAH, complement factor H; ADA10, disintegrin and metalloproteinase domain 10 (ADAM10); IGHA2, immunoglobulin heavy constant alpha 2; KT33A, keratin, type I cuticular Ha3-I; HV428, immunoglobulin heavy variable 4-28; HBB, hemoglobin subunit beta; A2MG, alpha-2-macroglobulin; A1AG2, alpha-1-acid glycoprotein 2; FA12, coagulation factor XII; IGHA1, immunoglobulin heavy constant alpha 1; KV315, immunoglobulin kappa variable 3-15; A0A087X1J7, glutathione peroxidase; AACT, alpha-1-antichymotrypsin; A0A1W2PR11, HLA class I histocompatibility antigen, C alpha chain; LV321, immunoglobulin lambda variable 3-21; HYDIN, hydrocephalus-inducing protein homolog; A0A087WXI2, IgGFc-binding protein

Fig. 3

The measurements of exosomal proteins in dataset 2. C1QC (A), CO9 (B), CFAH (C), KVD30 (D), GP1BB (E), RSU1 (F), and A0A0G2JRQ6 (G) were increased in Alzheimer’s disease (AD) patients, and A2MG (H), ADA10 (I), A1AG2 (J), IGHA1 (K), and HV428 (L) were decreased in AD patients. AD, Alzheimer’s disease; FC, fold change. C1QC, complement C1q subcomponent subunit C; CO9, complement component C9; CFAH, complement factor H; KVD30, immunoglobulin kappa variable 2D-30; GP1BB, platelet glycoprotein Ib beta chain; RSU1, Ras suppressor protein 1; A0A0G2JRQ6, Ig-like domain-containing protein; A2MG, alpha-2-macroglobulin; ADA10, disintegrin and metalloproteinase domain 10 (ADAM10); A1AG2, alpha-1-acid glycoprotein 2; IGHA1, immunoglobulin heavy constant alpha 1; HV428, immunoglobulin heavy variable 4-28

Fig. 4

Establishment of diagnostic panel for Alzheimer’s disease. A Receiver operating characteristic (ROC) curve analyses of the six-protein panel. B ROC analyses of 12 individual exosomal protein. The six proteins included in the panel are in bold. AUC, area under the curve; A0A0G2JRQ6, Ig-like domain-containing protein; C1QC, complement C1q subcomponent subunit C; CO9, complement component C9; GP1BB, platelet glycoprotein Ib beta chain; RSU1, Ras suppressor protein 1; ADA10, disintegrin and metalloproteinase domain 10 (ADAM10); CFAH, complement factor H; KVD30, immunoglobulin kappa variable 2D-30; A2MG, alpha-2-macroglobulin; A1AG2, alpha-1-acid glycoprotein 2; IGHA1, immunoglobulin heavy constant alpha 1; HV428, immunoglobulin heavy variable 4-28. P < 0.001 (A) or 0.01 (B)