Top-Down Proteomics of Human Saliva Highlights Anti-inflammatory, Antioxidant, and Antimicrobial Defense Responses in Alzheimer Disease

Abstract

Alzheimer disease (AD) is the most prevalent neurodegenerative disease in the elderly, characterized by accumulation in the brain of misfolded proteins, inflammation, and oxidative damage leading to neuronal cell death. By considering the viewpoint that AD onset and worsening may be influenced by environmental factors causing infection, oxidative stress, and inflammatory reaction, we investigated the changes of the salivary proteome in a population of patients with respect to that in healthy controls (HCs). Indeed, the possible use of saliva as a diagnostic tool has been explored in several oral and systemic diseases. Moreover, the oral cavity continuously established adaptative and protective processes toward exogenous stimuli. In the present study, qualitative/quantitative variations of 56 salivary proteoforms, including post-translationally modified derivatives, have been analyzed by RP-HPLC-ESI-IT-MS and MS/MS analyses, and immunological methods were applied to validate MS results. The salivary protein profile of AD patients was characterized by significantly higher levels of some multifaceted proteins and peptides that were either specific to the oral cavity or also expressed in other body districts: (i) peptides involved in the homeostasis of the oral cavity; (ii) proteins acting as ROS/RNS scavengers and with a neuroprotective role, such as S100A8, S100A9, and their glutathionylated and nitrosylated proteoforms; cystatin B and glutathionylated and dimeric derivatives; (iii) proteins with antimicrobial activity, such as α-defensins, cystatins A and B, histatin 1, statherin, and thymosin β4, this last with a neuroprotective role at the level of microglia. These results suggested that, in response to injured conditions, Alzheimer patients established defensive mechanisms detectable at the oral level. Data are available via ProteomeXchange with identifier PXD021538.

Antimicrobial Peptides (AMPs) in the Pathogenesis of Alzheimer’s Disease: Implications for Diagnosis and Treatment

“…An increasing line of evidence indicated that α- and β-defensins can be considered as good biomarkers for AD diagnosis. In particular, the levels of α-defensins 1–2 appear to be high in saliva, blood, serum, and CFS of AD patients [ 43 , 94 , 95 ], the levels of α-defensin 3 in saliva, serum, and CFS [ 43 , 94 , 95 ], whereas the level of α-defensin 4 only in saliva [ 43 ]. In the same manner, an increased level of β-defensin 2 in the serum and CFS of AD patients has been reported compared to healthy controls [ 95 ].…”

“…Other findings indicated that both cystatin A and cystatin B are also two regulation factors of inflammation that can inhibit cathepsins [ 104 ]. In particular, cystatin B may play a protective role in AD through the inhibition of cathepsin B, a β-secretase enzyme that cleaved APP to synthesize Aβ fragments [ 43 , 105 ]. Interestingly, cathepsin B is overexpressed following chronic exposure to some bacteria producing an AD-like phenotype [ 106 ].…”

“…Beyond the possible role of cystatins in the pathogenesis of AD, other studies suggest that they could also be considered good diagnostic biomarkers. Indeed, the levels of cystatin C are reduced in the CFS of AD patients [ 107 ], whereas the levels of cystatins A and B are increased in the saliva of AD patients [ 43 ].…”

“…Thymosin β 4 (Tβ 4 ) is a small multifunctional peptide containing 43 amino acids, which protects tissues against damage and promotes their regeneration [ 111 ]. It has been reported that in the central nervous system Tβ4 is mainly released by activated microglia to inhibit neuroinflammation [ 111 ]; thus, exerting antimicrobial activity [ 43 ]. Therefore, it is plausible to hypothesize that in AD Tβ4 may be released by activated microglia—together with other AMPs and other substances—to counteract the inflammation due to the Aβ accumulation.…”

“…It has recently been proposed that Aβ can be considered an AMP; thus, representing a type of innate immune defense protein that protects the host from a variety of pathogens [ 22 , 39 , 40 ] and that other proteins with antimicrobial activity, such as α- and β-defensins, lactoferrin, cystatins A and B, histatin 1, statherin, and thymosin β4 play a key role in AD [ 41 , 42 , 43 , 44 , 45 ].…”