Fig. 13.1
Extracellular soluble A-syn oligomers play an important role in both the development and progression of PD. (a) The cascade of A-syn aggregation. The A53T mutation promotes the formation of fibrillar A-syn species, but the A30P mutation does not. The A30P mutation slows the rate of fibril accumulation but strongly promotes the formation of oligomeric species. Thus, the common mechanism of the pathological mutations of SNCA (point mutations such as A53T, A30P, E46K, and triplication) is an increase in A-syn oligomers, not an increase in fibril formation (From [63] (Cookson MR. Annu Rev Biochem. 2005;74:29–52)). (b) Illustration of prion-like propagation of A-syn pathologies that spread spatiotemporally in PD brain during disease progression. A-syn pathologies (Lewy neurites and Lewy bodies) are suggested to first appear in the dorsal motor nucleus of the vagal nerve in the brain stem and anterior olfactory structures (darkest green) and then to spread stereotypically to finally occupy large parts of the brain (From [67] (Brundin P, et al. Nat Rev Mol Cell Biol. 2010;11:301–7)). (c) Prion-like propagation of A-syn pathologies to grafted neurons in PD brain. A-syn-positive Lewy bodies were found in grafted dopaminergic neurons in a patient with PD who had undergone implantation of fetal mesencephalic tissue into the putamen 12 years before death. Classic Lewy bodies in the grafts are immunoreactive for A-syn (From [64] (Li JY, et al. Nat Med. 2008;14:501–3)). (d) The proposed molecular basis of prion-like propagation shown in (b) and (c). Intracellular soluble A-syn oligomers/aggregates can be released from neurons by exocytosis or cell death. The aggregates are taken up by adjacent neuronal cell bodies, for example, and are either retained in the cell soma (local spread of pathology) or transported anterogradely by axons. Alternatively, they are taken up by axon terminals and transported retrogradely to the cell soma. The protein aggregates can spread between brain regions by axonal transport. Those soluble aggregates and/or oligomers of A-syn are considered to be the molecular basis of the prion-like propagation observed in (b) and (c) (From [67] (Brundin P, et al. Nat Rev Mol Cell Biol. 2010;11:301–7))
Fig. 13.2
Specific ELISA for α-synuclein (A-syn) oligomers and quantification of A-syn oligomers in PD and controls [70, 71]. (a) The method of single-antibody sandwich ELISA (SAS-ELISA) and usual sandwich ELISA for the quantification of total A-syn and A-syn oligomers, respectively, in human CSF. The SAS-ELISA uses a well-characterized single monoclonal antibody both for capture and detection of antigens. This type of ELISA cannot detect A-syn monomers because the capture antibody occupies the only antibody-binding site available on monomers. Meanwhile, the SAS-ELISA can theoretically detect A-syn oligomers because multiple antibody-binding sites are exposed on the surface of the three-dimensional structure of the oligomers. (b) Individual values of the measured level of total A-syn (left), A-syn oligomers (middle; RLU = relative luminescence units), and the ratio of A-syn oligomers to total A-syn (C; oligomer/total ratio, %) in CSF from patients with PD (solid circles) and controls (open circles). Each bar represents the mean value. Dashed lines in the middle and right subfigures indicate respective cutoff values that yield the most reliable sensitivity and specificity with ROC curves (9950 RLU/s for the levels of CSF A-syn oligomers; 6.165 % for the ratio of A-syn oligomers to total A-syn in CSF). (c) ROC curves for the levels of CSF A-syn oligomers (open squares) and the ratio of A-syn oligomers to total A-syn in CSF (open circles) for discrimination of PD from controls. Over a range of cutoff points, the arrowhead indicates a cutoff value that yields the most appropriate sensitivity and specificity
13.4.3 Other A-syn Species in Human CSF: Phosphorylated A-syn
Most of the A-syn in Lewy bodies is phosphorylated at the serine residue at position 129 (Ser129) (p129-A-syn) [75–79]. The phosphorylation of S129 alters the propensity of A-syn to aggregate [75, 80, 81].
The usefulness of CSF p129-A-syn levels as a diagnostic and/or surrogate biomarker for synucleinopathy has been investigated. In a large cohort (~600 samples) of patients with PD, MSA, and controls, CSF levels of p129-A-syn showed a positive correlation with the severity of PD symptoms, and the combination of CSF total A-syn (lower in PD) and p129-A-syn (higher in PD) improved discrimination between PD and other forms of parkinsonism [82]. However, a large cohort study (more than 300 subjects) that investigated the longitudinal relationship between CSF levels of p129-A-syn and UPDRS scores in PD patients reported discrepant results [83]. This study showed that the relationship between the levels of CSF p129-A-syn and disease severity may depend on the disease stage of PD in which lower p129-A-syn levels were correlated with a worse clinical condition at early stages but with a better condition at later stages. This observation would make it difficult to use CSF p129-A-syn as a surrogate biomarker for monitoring the disease severity of PD.
13.4.4 Studies Evaluating the Usefulness of CSF A-syn for the Diagnosis of DLB
The diagnostic ability of CSF levels of A-syn species, mainly for the differential diagnosis between AD and DLB, has not been fully investigated, but several studies have been reported. Many studies, including one study targeted at autopsy-proven patients with DLB and AD [43], have shown a reduction in CSF total A-syn in patients with DLB compared to AD [41, 84, 85]. One study also demonstrated that the longer duration of illness was correlated with the lower CSF levels of total A-syn in DLB patients but not in AD patients, suggesting that a reduction in CSF A-syn would be associated with increased severity of synucleinopathy in the brain [84]. However, some studies did not find any significant differences in CSF A-syn levels between patients with DLB and AD [50, 86] or found increased CSF levels of A-syn in DLB compared with AD and controls [87].
To determine the diagnostic utility of CSF A-syn levels in distinguishing DLB from other neurodegenerative dementias, a meta-analysis that included a total of 13 studies comprising 2728 patients was carried out. This study reported that the mean CSF A-syn level was significantly lower in DLB patients compared to those with AD [88] (Fig. 13.3). However, ROC analysis could not be performed in this meta-analysis due to insufficient information available in referred reports, and thus, values for the sensitivity and specificity of the assay were not reported. Another meta-analysis reported in 2015 also showed significantly lower CSF levels of total A-syn in DLB patients compared to AD patients but again did not show the sensitivity and specificity of the assay [89].
Fig. 13.3
Forest plot of the included studies in meta-analysis comparing mean CSF total A-syn concentrations of DLB vs. AD patients. A meta-analysis was carried out to determine the diagnostic utility of CSF total A-syn analysis in distinguishing DLB from other neurodegenerative dementias including DLB. A total of 13 studies that included 2728 patients were included in the final review, and 7 out of 13 studies (1301 patients) were included in the meta-analysis. The mean CSF total A-syn concentration was significantly lower in DLB patients compared to those with AD (weighted mean difference −0.24; 95 % confidence interval, −0.45, −0.03; p = 0.02) (From [88] (Lim X, et al. Parkinsonism Relat Disord. 2013;19:851–8))
One caveat in interpreting the results of the studies that compare CSF A-syn levels between patients with DLB and AD cannot be ignored: there is a subgroup of AD patients with additional Lewy body pathology [90–92]. In the ADNI (Alzheimer’s Disease Neuroimaging Initiative) cohort, there is a clear bimodal distribution of CSF A-syn levels in relationship to t-tau; subjects with abnormally increased t-tau values had high levels of total A-syn, and there were no subjects with elevated levels of CSF A-syn among those with normal t-tau values [93]. Furthermore, a positive correlation was generally noted between CSF total A-syn and p-tau181, but there were some cases with high p-tau181 levels accompanied by low A-syn levels in the ADNI cohort. This result suggests that a mismatch between the measured CSF level of A-syn and the expected levels of A-syn from the levels of CSF p-tau181 would be observed in AD cases with concomitant Lewy body pathology [93].
13.4.5 Measurement of A-syn Species in Human Blood (Plasma/Serum) and Confounding Factors
An ideal biomarker should be noninvasive (such as a blood test) or only moderately invasive (such as CSF biomarkers). Thus, blood tests quantifying A-syn have been reported for the diagnosis of DLB and other synucleinopathies, but their results were inconsistent. Several studies showed significantly higher levels of plasma total A-syn [70, 94, 95] or plasma p129-A-syn [96] in patients with PD. By contrast, some studies found that plasma levels of total A-syn were significantly lower in PD compared to controls [97], as in DLB compared to AD and controls [98]. Other studies found comparable levels of plasma total A-syn among controls, PD, and AD [72, 99]. Those discrepant results are considered to be due to certain confounding factors as mentioned above (see Sect. 13.4.1).
Previous studies emphasized that hemolysis is an important confounding factor that provides a strong positive signal in A-syn ELISAs [42, 57] because greater than 99 % of A-syn in blood resides in RBCs [100]. However, a considerable number of samples with high levels of hemoglobin (Hb) in the study showed average or less than average levels of CSF A-syn [42]. Ishii et al. also reported that there was not a significant relationship between the levels of Hb and CSF or plasma A-syn [54]. Instead of hemolysis, it has been reported that interference from HAs is an important and prevailing confounding factor in ELISAs [101–104]. Recently, Ishii et al. demonstrated that plasma A-syn levels were significantly lower in PD than in controls only under the condition in which HA interference was eliminated. HAs are human antibodies capable of binding to animal immunoglobulins and may possibly interfere with the reaction between animal-derived antibodies and the analyte, which is part of all immunoassays [101–104]. HA interference is known to be more prominent in blood samples than in CSF samples; HAs were found in up to 40 % of human serum samples, and assay interference from HAs occurs in as many as 15 % of serum samples despite highly depending on the specific assay setup [102, 104]. There have been two studies describing HA interference in Aβ ELISA; HA generally affects micro-quantitative ELISA more strongly in plasma than in CSF and produces false positive rather than false negative signals [104, 105]. These findings suggest that HA is an important confounding factor that can generally affect ELISAs that measure very small amounts of antigens and is not limited to the A-syn ELISAs. It should be concluded that HA interference, rather than contamination with RBCs and hemolysis, is a major confounder in some A-syn ELISAs. Eliminating HA interference in A-syn ELISAs is indispensable, although none of the previous studies, other than that of Ishii et al. which examined plasma A-syn with ELISAs, were adjusted for HA interference.
13.5 Other Potential Biomarkers for DLB
There are many reports on CSF biomarkers other than A-syn species for the diagnosis of DLB and synucleinopathy [refer to reviews: 106–109].
13.5.1 AD-Related Biomarkers for Diagnosis and Prediction of Prognosis in DLB and PD
As a diagnostic biomarker, CSF levels of Aβ1-42 are usually decreased in patients with DLB compared to non-demented controls [110]. A meta-analysis of 50 studies demonstrated that Aβ1-42 was moderately lower in AD compared to DLB [111]. Calculation of the Aβ1-42/Aβ1-40 ratio could be promising for the differentiation of AD from DLB [112]. Nevertheless, most studies could not determine exact cutoff scores valuable to distinguish AD and DLB in clinical practice [113–115], including a meta-analysis [111]. The oxidized isoform of Aβ1-40 (Aβ1-40ox) has been shown to be increased in DLB patients compared to patients with PD with dementia (PDD) and non-demented disease controls [116] and has recently also been shown in autopsy-proven AD and DLB [117].
One important prospective cohort study demonstrated that lower baseline CSF Aβ1-42 (≤192 pg/mL), but neither t-tau nor p-tau181, was associated with a more rapid cognitive decline within a 2-year period of follow-up in patients with PD [118]. These results are consistent with previous research showing that AD pathology contributes to cognitive impairment in PD [119–122]. The CSF level of Aβ1-42 may provide clinically useful prognostic information as a prognostic biomarker for cognitive impairment in PD.
In DLB, levels of CSF tau protein are lower compared to AD [113] and higher compared to PD and PDD [123]. Some CSF studies have revealed better specificity for the discrimination of AD when using p-tau181 as a diagnostic biomarker rather than total tau protein [124]. A meta-analysis of 16 studies that included 909 AD patients and 265 DLB patients showed that there was a significant ( p < 0.001) difference between CSF p-tau181 levels in AD (71–136 pg/mL) and DLB (34.5–76.6 pg/mL) [125]. From these findings, quantification of p-tau species in CSF may serve as a specific biomarker to discriminate AD from DLB [124, 126].
13.5.2 PD-Related Biomarkers Other Than A-syn Species for the Diagnosis of DLB
Neurosin, a brain-rich serine protease, can cleave A-syn and thereby may play a major role in the pathomechanisms of diseases associated with A-syn pathology [127–129]. Neurosin was decreased in CSF from patients with synucleinopathy compared with healthy controls and patients with AD. The lowest levels have been found in patients with DLB, thereby offering a potential diagnostic biomarker for DLB [130].
13.5.3 Other Potential CSF/Blood Biomarkers for the Diagnosis of DLB and PDD
Reduced CSF levels of metabolites such as homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) have been reported in DLB compared with AD [131]. Especially MHPG, in combination with the levels of t-tau, p-tau, and Aβ1-42, could increase the sensitivity and specificity of discriminating DLB from AD [132].
With regard to blood biomarkers, it has been reported that serum levels of heart-type fatty acid-binding proteins (FABPs) are distinctly elevated in DLB [133–135], despite lower levels of these proteins that were reported in the brains of AD patients [135]. One study using a multiplex immunoassay that can quantify more than 100 plasma proteins simultaneously identified epidermal growth factor (EGF) as a predictive biomarker of cognitive impairment in patients with PD [136]. In this study, low levels of plasma EGF were not only correlated with poor cognitive test scores at baseline but also predicted an eightfold greater risk of cognitive decline to dementia for those with intact baseline cognition during follow-up period with a median time-to-conversion of 14 months.
13.6 Conclusions and Future Prospective
Recent remarkable advance in functional neuroimaging such as dopamine transporter scans and MIBG scintigraphy have brought us clinically useful imaging biomarkers for the diagnosis of DLB and synucleinopathy. However, those imaging biomarkers have limitations, as mentioned above (see Sect. 13.2.1). Consequently, biochemical biomarkers that are useful for early diagnosis and appropriate management of DLB in clinical practice are also urgently needed.
This chapter has introduced the studies reported so far and discussed the current status of biochemical biomarker candidates for DLB (Table 13.1), but the majority of the studies discussed here were cross-sectional, retrospective, and investigated pathologically unproven subjects. In addition, those studies have other considerable caveats including heterogeneity of examined subjects (both patients and controls) as well as a lack of standardization of sample collection and handling, sample processing, and assay procedures, making them impossible for providing reference values and diagnostic cutoff values that can be globally used. After taking those limitations and caveats into consideration, the summary points of this chapter are as follows:
Table 13.1
Summary of reported candidates of biochemical biomarkers for the diagnosis of DLB and differential diagnosis between DLB and AD and surrogate biomarkers for PD
CSF biomarkers | Plasma/serum biomarkers | |
|---|---|---|
DLB | A-syn↓ | Heart-type FABPs↑ |
(A-syn oligomers? ↑in PD) | EGF↓ | |
Neurosin↓ | ||
Oxidized Aβ1-40↑ | ||
HVA, 5-HIAA, MHPG↓ | ||
DLB vs. AD | Aβ1-42 (AD < DLB) | Heart-type FABPs (AD < DLB) |
t-tau, p-tau (AD > DLB) | ||
PD | A-syn (to monitor disease severity) | EGF (to predict cognitive decline) |
Aβ1-42, p-tau (to predict prognosis) | ||
A-syn oligomers (to predict prognosis) |
The purpose of biomarkers can be typically classified as diagnostic, disease-monitoring, predictive, or effect measurement.
There are important requirements for the ideal diagnostic biomarker, including (1) the ability to reflect fundamental disease process, (2) validation in pathologically confirmed patients, and (3) precision with high sensitivity and specificity.
A-syn plays a pivotal role both in the development of the disease and propagation of the pathology in the brain in PD and other synucleinopathies.
CSF levels of total A-syn can discriminate PD from controls or tauopathies as groups. However, it is hard to diagnose an individual patient only with CSF A-syn. The PPMI data set suggested the CSF total A-syn could be useful for monitoring disease severity as a surrogate biomarker for PD.
CSF levels of A-syn oligomers could be a promising biomarker for the diagnosis of PD. The CSF levels of A-syn oligomers are reported to correlate with motor and cognitive scores in PD, suggesting its possibility as a biomarker to monitor disease severity.
With regard to the diagnosis of DLB, some studies including two meta-analyses demonstrated significantly lower CSF levels of total A-syn in DLB patients compared to AD patients. However, the sensitivity and specificity as well as diagnostic ability of CSF total A-syn still remain to be elucidated. Whether CSF A-syn oligomers are useful or not for the diagnosis of DLB has not been examined.
To study plasma/serum levels of total and oligomeric A-syn, it is essential to eliminate interference from heterophilic antibodies, which have a significant impact on the assays of plasma/serum A-syn despite never being considered in any of the studies reported so far that determined plasma/serum levels of A-syn.
The higher levels of Aβ1-42 and Aβ1-40ox as well as lower levels of p-tau181 and neurosin in CSF from patients with DLB compared to those from AD patients would be useful for the differential diagnosis of these two diseases.
For the future perspective of biomarkers for DLB and synucleinopathy, it will be necessary to accomplish the following two objectives: (A) validation of already-reported candidates and translation of them to clinical practice and (B) identification of novel biomarkers that are more closely associated with the specific disease process of the diseases. For objective (A), the candidate CSF biomarkers include total A-syn, A-syn oligomers, p129-A-syn, and AD-related biomarkers, whereas the candidate plasma biomarkers are A-syn oligomers, EGF, and so on. For objective (B), the most important thing is to identify the true culprit species of A-syn, namely, certain oligomers or protofibrils of A-syn that are specifically relevant to the development (neurotoxicity) and/or progress (prion-like propagation in the brain) of an individual synucleinopathy, including DLB, PDD, PD, and MSA. To accomplish these objectives, future studies should ideally fulfill the following conditions [7, 8, 11, 88]:
- 1.
A large, prospective, multicenter, and longitudinal cohort design
- 2.
Inclusion of patients with DLB and other synucleinopathies and at-risk subjects diagnosed with standardized criteria (pathological or neuroimaging criteria)
- 3.
Simultaneous quantification of multiple biomarkers in the CSF and/or blood by using strictly standardized protocols including quality controls of biomarker assays [53]
- 4.
A long-term follow-up period with serial determinations of the biological markers
- 5.
Final pathological confirmation by examination of the brains and bodies of patients
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