In the current study, we analyzed two independent datasets to investigate proteomic profiling of circulating plasma exosomes in AD. We found that a panel of six exosomal proteins was associated with AD. To the best of our knowledge, this is the first time that a diagnostic model of AD was established using bulk exosomes of plasma. It is less invasive and antibody-independent and may therefore be extensively used for AD screening in the general population of old adults.
Biomarkers have important roles in AD diagnosis [
3] and research [
18]. The use of serum or plasma in the diagnosis of AD is gaining more and more attention as it has low invasiveness and relatively low cost. Increasing evidence supports multiple promising blood biomarkers, such as Aβ42 [
19], neurofilament light protein [
20], P-tau181, and P-tau217 [
21]. In this study, we generated a diagnostic panel of AD by detecting exosomal proteins in the blood. Label-free proteomics revealed 15 upregulated and 16 downregulated exosomal proteins in AD. Twelve differentially expressed exosomal proteins were further confirmed by PRM, which is a targeted method for the identification and quantification of proteins due to its reproducible and consistent results [
22] and has been extensively used in multiple studies [
23,
24]. Among the 12 proteins, the combination of six proteins was strongly associated with AD and can distinguish AD from healthy controls with high performance. Furthermore, the six-protein panel is significantly positively correlated with cognitive performance, making it an effective predictor for cognitive decline. Our findings provided convenient and antibody free biomarkers for AD.
By proteomic analysis, we identified a six-protein panel in circulating exosomes that may be a novel biomarker for AD. The differentially expressed proteins are associated with several processes: inflammation and immunity, coagulation, and the proteolytic process of amyloid precursor protein (APP). The levels of several complement-related proteins were elevated in our study, which is consistent with earlier findings in astrocyte-derived exosome research [
12]. Compelling evidence revealed that the complement system was essential in AD pathogenesis [
25]. For example, C1QC associates with other subunits to yield C1q, the initiating component of the classical complement pathway. C1q tags the tau-affected synapses by opsonization and induces subsequent microglia phagocytosis [
26]. Furthermore, mouse models showed that dysregulated C1q-mediated synaptic pruning and/or spine density loss was associated with behavioral deficits, implicating its detrimental role in the inflammatory process [
27]. Nevertheless, C1q exerts direct protective effects on primary cultured neurons against amyloid-induced toxicity [
28,
29]. Together, these findings indicate C1q plays dual roles in AD pathogenesis. Another complement, CO9, is a key subcomponent of membrane attack complex (MAC), which plays a significant role in the downstream cascade of complement-meditated pathways. Studies found that MAC co-localized with Aβ plaques and tau tangles in the brains of AD patients, suggesting that CO9 may contribute to AD pathogenesis [
25]. GP1BB is a subunit of the GPIb-V-IX complex, which is a transmembrane protein in platelets and constitutes the receptor for von Willebrand factor (vWF). vWF is abundantly expressed in cerebrovascular endothelium and associated with enhanced inflammatory activity, promoting endothelial dysfunction and cerebral amyloid angiopathy pathology [
30,
31]. Therefore, it is possible that upregulated GP1BB boosts intracellular signaling response to vWF and, ultimately, deteriorates neurotoxicity. To date, no physiological function of RSU1 has been reported in AD. Nonetheless, there exist two intriguing connections between RSU1 and AD pathogenesis. RSU1 inhibits c-Jun N-terminal kinase (JNK) and enhances extracellular signal-regulated kinase (ERK) activation [
32]. JNK activation links to amyloidogenic protein processing, whereas JNK suppression leads to a significant reduction in Aβ42 peptide levels and total plaque burdens as well as to an increased number of neurons and enhanced cognition [
33]. Moreover, the ERK pathway is involved in neuroprotection against oxidative stress and Aβ toxicity [
34]. As a hypothesis, increased RSU1 level measured in AD patients might reflect a compensatory neuroprotection phenomenon in response to excess Aβ burden. The single downregulated exosomal protein ADA10, also known as disintegrin and metalloproteinase domain 10 (ADAM10), is the major α-secretase for APP processing. ADA10 has a role not only in reducing the production of Aβ peptides and relieving the pathologic impairment in AD but may also in reducing tau pathology, preserving synaptic functions, and promoting hippocampal neurogenesis [
35]. In line with our study, ADA10 in the platelets has been proposed as a potential biomarker for early diagnosis of AD [
36]. Taken together, our findings identify a panel of exosomal proteins as an accurate biomarker for diagnosis of AD and extend the understanding of the disease, albeit it remains undetermined how these proteins contribute to the disease.