The main result of our study was that, among the proposed biomarkers, only tau, α-syn, and α-syn/tau managed to differentiate cognitively healthy from cognitively impaired subjects, with α-syn/Aβ1−42 that could only discriminate between AD and HC. The most performing marker was α-syn/tau, which managed to separate with good and fair accuracy LBD and AD respectively from controls. Of note, none of them was useful for differential diagnosis between AD and LBD groups.
Emerging evidences suggest that neurodegenerative diseases are not related to the cerebral deposition of single/specific abnormal proteins, but rather to a mixed pattern of these misfolded proteins [20, 24, 35]. Several studies support the notion that Aβ1−42, tau, and α-syn interact in vivo to promote aggregation and accumulation of each other and accelerate cognitive dysfunction . Interestingly, their expression levels and aggregation processes are not restricted to the brain, but reach peripheral compartments, possibly configuring a systemic disease [35, 50]. Among peripheral cells, RBCs were demonstrated to be particularly susceptible to the oxidative stress and accumulation of misfolded proteins [25, 31, 33, 51–54]. Herein, a cohort of AD and LBDs patients and a group of HCs were recruited to quantify the RBCs concentrations of α-syn and its heterocomplexes with tau and Aβ1−42, and to test their potential discriminatory accuracy. Indeed, literature highlights need for additional tools to distinguish LBD from AD, especially for the early-stage diagnosis, and a combination of blood biomarkers may be a more promising approach to differentiate AD and LBD from other conditions than looking for a single molecule.
In the present study, Aβ1−42 concentrations in RBCs did not differ between HC and patients (AD and LBD). Very few data are available on the blood levels of amyloid protein. Previous cross-sectional studies confirmed that plasma Aβ of AD patients is not much different from normal controls , but somewhat promising results have been seen for combinations of Aβ1−42 and Aβ1−40. Of note, recent studies allowed us to measure very low amounts of several Aβ-related peptides in plasma using ultrasensitive assays, supporting the use of plasma Aβ42/40 ratios as surrogate biomarkers of cerebral Aβ deposition [56, 57]. Considering the remarkable role of CSF Aβ1−42 in PD conversion to dementia [58, 59], studies on Aβ1−42 contribution in PD are mandatory. The determination of plasmatic α-syn has yielded conflicting results so far in synucleinopathies; a recent meta-analysis concluded for higher levels in PD patients compared to controls , while opposite results in LBD [61–63].
Recently, we explored RBCs Aβ1−42 levels as a potential biomarker, finding comparable levels between HC and PD subjects , or HC and AD . In contrast, Aβ1−42 fibrils in RBCs have been found to be significantly higher in AD patients when compared to HC . However, in this study, the diagnosis was based on purely clinical criteria  and Aβ1−42 fibrils were quantified by a different detection method . Taken together, these data highlight the need for the investigation on peripheral Aβ1−42, and, most importantly, for more uniformed protocols to improve comparability of results.
Doubtless, the contribution of α-syn to LBD diagnosis is more meaningful than Aβ, primary because α-syn content in blood, particularly that stored in RBCs, is much higher than in CSF [30, 66]. In our study, total α-syn concentrations were lower in AD and LBD patients compared to HC. These data confirm the previous results obtained in a different cohort of AD subjects  and PD subjects [30, 33, 34]. Nonetheless, α-syn concentration was not able to discriminate AD subjects from LBD ones.
RBC tau concentration was reduced in AD and LBD subjects compared to HC, without discriminating the two patient populations. To our knowledge, very few studies have assessed the RBC tau protein concentrations in NDs . In a previous study, tau protein in RBCs has been found to be similar in AD and HC , while higher tau levels have been demonstrated in plasma of AD patients [67–70]. Further investigation on tau isoforms and distribution will certainly be of interest, considering the potential contribution of tau pathology in LBD progression, especially in the case of Aβ1−42 copathology.
Heterocomplexes of α-syn with tau and Aβ1−42 have been proven to occur both in cellular models and in patients’ brains [21–24, 34, 35, 71]. Noteworthy, α-syn forms heterocomplexes with both Aβ1−42 and tau proteins in brain tissues and RBCs of senescence-accelerated mice, similarly with previous data reported in human samples [24, 25, 34, 72]. In our study, both α-syn/Aβ1−42 and α-syn/tau concentrations in RBCs were significantly lower in AD patients than HC, as previously reported . Furthermore, α-syn/tau levels were also reduced in LBD subjects than in HC. By contrast, α-syn/Aβ1−42 heterocomplexes in PD patients were higher than in controls and correlate with the disease’s progression . Overall, these data highlight the potential role of α-syn heteromers as biomarkers in dementia and LBD. Interestingly, both α-syn/tau and α-syn/Aβ1−42 heterodimers in RBCs can fairly discriminate AD from HC, and α-syn/tau heterodimers distinguish LBD from HC with good accuracy.
The strength of our study was that AD patients received a biomarker-based diagnosis and a nigrostriatal degeneration were confirmed in LBD patients. However, some caveats are needed. In particular, our sample size is relatively small hindering further stratifications (e.g. specific investigations regarding PDD and DLB subsets). Moreover, our samples are not homogeneous in terms of age, sex prevalence, and disease stages. In fact, AD group consisted of both prodromal (Mild Cognitive Impairment, MCI) and mild demented patients, whereas LBD group was composed only of demented patients and did not comprehend an MCI PD category. Furthermore, given the cross-sectional nature of our study, and the lack of adequate follow-up, it is impossible to explore the prognostic value of these biomarkers. Another limitation is not having compared biomarkers in RBCs with the relative concentrations in plasma and CSF to clarify their clearance process.
Nevertheless, due to the multifactorial etiology of NDs and the existence of multiple elements involved in NDs pathogenesis, it could be interesting to further evaluate RBCs concentrations of phosphorylated tau (specifically reflecting the presence of neurofibrillary tangles, NFTs), phosphorylated α-syn (since it represents the 90% of insoluble α-syn contained in LBs), Aβ1−42 fibrils and aggregates (which bind RBCs in a sharply larger share of AD patients compared to HC), and oligomeric α-syn, whose dosage in RBCs has already shown significant results in PD .