In summary, the power of α-syn to discriminate between AD and DLB can be considered moderate (Table 2), as previously reported (17-18). However, our study shows that the differences observed between AD and DLB appear from the prodromal stage.
Our study has a limitation in that we do not know the exact concentration of hemoglobin in our samples. Indeed, it has been shown that hemoglobin plays a role in α-syn levels in the CSF (19-22). These studies have shown that beyond 200-500 ng/mL (depending on the study) hemoglobin leads to an artificial increase by interfering with the α-syn assay. However, our samples were visually inspected upon arrival at the laboratory and any samples with pink coloration due to the presence of hemoglobin were rejected. This control is reported to eliminate hemorrhagic samples with more than 500 red cells per µL (24). Furthermore, on arrival at the laboratory, samples were centrifuged at 1700 g for 10 min to eliminate as many blood cells as possible that could have contaminated the CSF, thus limiting hemoglobin levels in our samples.
Early modification of α-syn levels
Regarding the results of the total α-syn assay, we found a significant difference between the DLB group and the AD group. Similar results have previously been highlighted in many publications (17, 18, 23-28), with α-syn levels being higher in AD patients compared to DLB patients. These results have even been confirmed in an autopsy series of patients (29).
The originality of our results is to show that, at the prodromal stage, AD patients had significantly higher α-syn levels than DLB patients. So far, only one recent publication has looked at the prodromal stage and has shown results similar to ours (30); however, in that study there were no patients at the demented stage. Thus we have highlighted more precisely the absence of any change in α-syn levels between the prodromal and dementia stages whatever the pathology (AD or DLB). Thus, total α-syn levels are modified from the prodromal stages (Fig. 2A), suggesting that changes in α-syn levels are implemented early.
Ability of α-syn to discriminate between neurological controls and DLB and AD patients
α-syn levels of our control subjects were not significantly different from the AD and DLB groups, most likely because of the different neurological pathologies in this group, which made it heterogeneous. In the same way in the literature, it is usually the case that DLB patients were not significantly different from controls (17, 18, 24, 26, 28,31-35), but a number of publications showed significantly lower levels of α-syn in DLB patients compared to control patients (27, 29, 36, 37). Garcia-Ayllon et al. even showed that this decrease could take place from the DLB prodromal stage (30).
Interestingly, even if some studies, like ours, showed CSF α-syn levels that were numerically higher, but not significantly so, in AD patients than in CS patients (21), most studies comparing CS patients and AD patients showed that total α-syn levels were significantly higher in AD patients (19, 21, 24, 27, 38), suggesting an α-syn increase in AD patients. On the other hand, by observing the group of patients with AD+DLB co-morbidity, it can be seen that the mean α-syn values were at the same level as those of the pure AD groups. This result reinforces the idea that the change in α-syn levels in the CSF is related to an α-syn increase in AD rather than an α-syn decrease in DLB. There are several possible explanations for this increase in AD patients. First, α-syn could be released from damaged neurons (39, 40), as has been hypothesized for the increased levels of CSF tau in AD. Second, an increase in α-syn production was confirmed by Larson et al, who highlighted a 1.67-fold increase in α-syn mRNA levels in the inferior temporal gyrus of AD patients, when compared to age-matched controls, leading to an increase in α-syn monomers even though these AD patients did not have detectable Lewy bodies (41). Thus, the increase in α-syn production in the brains of AD patients is believed to be responsible for its increase in CSF. In addition, it has been shown that high levels of α-syn may cause cognitive deficits by reducing the release of neurotransmitters by inhibiting the recycling of synaptic vesicles (42). Thus, it is likely that these increases in soluble α-syn (even in monomeric form) in the brains of AD patients are the source of an important correlate of decreased cognitive function in AD.
As DLB patients also have neuronal damage, it may seem surprising that there is no α-syn increase in DLB patients. There are two possible explanations for this. First, the aggregating processes of α-syn present in DLB patients are responsible for the decrease in α-syn levels in the CSF, as observed for Aβ42 in AD. The second explanation is that for the same level of cognitive impairment, DLB patients have less neurodegeneration than AD patients (43, 44), which may explain the lower value in DLB patients.
The different proteinopathies have synergistic adverse effects
Thus, while AD patients have amyloid plaques and DLB patients have Lewy bodies, CSF of AD patients presents an α-syn level increase and CSF of DLB patients an Aβ42 decrease. These results indicate that these pathologies seem to be related in one way or another, which would explain the high frequency of co-morbidities, or at least histological hallmarks commonly found between these 2 pathologies. More than 80% of DLB patients showed moderate or abundant cortical amyloid plaques (45), and α-synuclein pathology is also found in up to 50% of patients with AD (for a review, see (46)), suggesting a close link between amyloidopathy and synucleinopathy. In addition, other publications indicate that Tau protein may also have a negative synergy with amyloidopathy and synucleinopathy (47, 48), reinforcing the close link between these different neurodegenerative diseases.
Ability of the combination of α-syn with standard AD-related biomarkers to discriminate DLB from AD
ROC curves (Table 2) show that even combining α-syn results with Alzheimer biomarkers does not improve the discrimination power compared to the combination of Alzheimer biomarkers alone (t-Tau_phospho-Tau_Aβ42 or Aβ42/Aβ40, AUC=0.95, Alzheimer biomarkers + α-syn AUC=0.95). However, this result needs to be put into perspective given that the CSF's Alzheimer biomarkers are taken into account in the diagnosis, leading to a bias due to an overestimation of the discrimination effectiveness of these Alzheimer biomarkers. Despite taking into account the CSF result, some patients, particularly those clinically considered as Alzheimer's, present an atypical CSF profile. However, we are quite confident in the diagnosis, in fact, some patients have started to be included in the study from 2013 and consequently we have a relatively long follow-up of these patients, which has allowed us to reclassify some of them.
To conclude, the total α-syn assay can participate to discriminate between DLB and AD patients, whatever the stage, but with insufficient specificity and sensitivity. Thus, there is currently a clear lack of new biomarkers specific to DLB for its differential diagnosis. However, other biomarkers are under study. While some are directly related to α-syn, such as the α-syn oligomers, fibrils or phosphorylation on S129 of α-syn, there are other post-translational modifications or even biomarkers which are unrelated to the direct aggregation processes of α-syn, such as YKL-40, neurogranin, VILIP-1 (for review, see (8)); yet these biomarkers suffer from a lack of hindsight to determine if they are actually relevant in the biological diagnosis of DLB. Further studies are therefore needed to confirm these results.