Increased CSF α-synuclein levels in Alzheimer's disease: Correlation with tau levels

Alzheimer’s & Dementia - (2014) 1–9

Research Article

Increased CSF a-synuclein levels in Alzheimer’s disease: Correlation with tau levels Sylvie Slaetsa,y, Eugeen Vanmechelenb,y, Nathalie Le Bastarda, Hilde Decraemerb, Manu Vandijckb, Jean-Jacques Martinc, Peter Paul De Deyna,c,d,e, Sebastiaan Engelborghsa,d,* a

Reference Center for Biological Markers of Dementia (BIODEM), Laboratory of Neurochemistry and Behavior, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium b Innogenetics NV (Miraca/Fujirebio Group), Ghent, Belgium c Biobank, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium d Department of Neurology and Memory Clinic, Hospital Network Antwerp (ZNA) Middelheim and Hoge Beuken, Antwerp, Belgium e Department of Neurology and Alzheimer Research Center, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands

Abstract

Background: Given the difficult clinical differential diagnosis between Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB), growing interest resulted in research on a-synuclein as a potential cerebrospinal fluid biomarker (CSF) for synucleinopathies. Methods: CSF a-synuclein-140 concentrations were determined by a prototype xMAPÔ bead-based assay (Innogenetics NV, Belgium). In addition, CSF amyloid b1-42 (Ab1-42), total tau (T-tau), and phosphorylated tau (P-tau181P) levels were determined. Results: CSF a-synuclein levels were higher in AD patients as compared with cognitively healthy controls (P 5 .019) and patients with synucleinopathies (P , .001). CSF a-synuclein levels were correlated with T-tau (P , .001) and P-tau181P (P , .001) levels in autopsy-confirmed AD patients. A diagnostic algorithm using a-synuclein and P-tau181P discriminated neuropathologically confirmed AD from DLB patients, resulting in sensitivity and specificity values of 85% and 81%, respectively. Conclusion: Because CSF a-synuclein levels were significantly higher in AD as compared with synucleinopathies, a-synuclein might have a value as a biomarker for differential dementia diagnosis. Ó 2014 The Alzheimer’s Association. All rights reserved.

Keywords:

Biomarker; Dementia; a-Synuclein; Cerebrospinal fluid; Alzheimer’s disease; Dementia with Lewy bodies; tau

1. Background Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB) are the two most common causes of neurodegenerative dementia in the elderly. On the basis of a routine clinical dementia workup, it is difficult to discriminate between AD and DLB because of overlapping symptoms between both conditions [1]. However, given the differences in pharmacological treatment options (e.g., choice of cholinesterase inhibitor, severe adverse effects to antipsychotic drugs in y

Sylvie Slaets and Eugeen Vanmechelen contributed equally to this article. *Corresponding author. Tel.: 132-32652394; Fax: 132-32652618. E-mail address: [email protected]

DLB patients), discriminating between DLB and AD is clinically relevant. Moreover, an early and accurate (differential) dementia diagnosis will be indispensable once diseasemodifying drugs for AD or other neurodegenerative brain diseases become available because these (potentially toxic) drugs will probably be pathology-specific. Although the cerebrospinal fluid (CSF) biomarkers amyloid-b1-42 (Ab1-42), total tau (T-tau), and phosphorylated tau (P-tau181P) have an added diagnostic value for the differential dementia diagnosis [2], concomitant amyloid pathology in DLB limits the use of CSF Ab1-42 for the differential AD versus DLB diagnosis [3]. DLB and other synucleinopathies, including Parkinson’s disease (PD), Parkinson’s disease dementia (PDD), and multiple system atrophy (MSA), are characterized by the

1552-5260/$ - see front matter Ó 2014 The Alzheimer’s Association. All rights reserved. http://dx.doi.org/10.1016/j.jalz.2013.10.004

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accumulation of the protein a-synuclein in Lewy bodies or glial cytoplasmic inclusions [4]. There are already several studies that evaluated the potential role of a-synuclein as a diagnostic biomarker for synucleinopathies, but results were conflicting. Some studies found decreased a-synuclein levels in patients with a synucleinopathy as compared with controls or AD patients [5–12], whereas others could not find significant differences between different diagnostic groups [13–16]. These conflicting results may be due to the examination of splicing variants of a-synuclein and the paucity of studies including autopsy-confirmed dementia patients [9]. In this study, we will focus on the diagnostic value of full-length asynuclein for the AD versus DLB differential diagnosis in a population of well-characterized patients of who a substantial proportion have autopsy-confirmed dementia diagnoses. 2. Methods 2.1. Study population CSF samples of patients with a synucleinopathy, AD patients, and cognitively healthy controls (CHCs) were selected from the Biobank of the Institute Born-Bunge (Antwerp, Belgium). The study population consisted of 105 patients with a synucleinopathy, 128 AD patients, and 29 CHC subjects. An overview of the population is given in Figure 1. Data on age at sampling, gender, and MiniMental State Examination (MMSE) scores within 3 months of lumbar puncture (LP) were available. For a subset of the AD and DLB samples, neuropathological confirmation of clinical diagnosis was available. 2.2. Clinical diagnostic criteria Probable DLB was diagnosed according to the criteria of McKeith and colleagues [17]. The criteria for PD were based on the presence of at least two of the four motor deficiencies that characterize the disease (resting tremor, bradykinesia, rigidity, and postural instability) [18]. Patients with PD were responsive to levodopa. The diagnosis of PDD was

based on the presence of PD and dementia with onset at least 1 year after the onset of PD. The diagnosis of dementia was based on the Diagnostic and Statistical Manual of Mental Disorders, fourth edition [19] criteria after routine blood sampling, an extensive neuropsychological examination, and brain magnetic resonance imaging (MRI; or computed tomography scan in the case of contraindications for MRI). MSA was diagnosed according to the consensus criteria and consisted of three patients with MSA with predominant parkinsonism features (MSA-P) and four patients with MSA with predominant cerebellar features (MSA-C) [20]. The diagnosis of probable AD was made according to the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS)–Alzheimer’s Disease and Related Disorders Association (ADRDA) criteria [21]. In addition to nondiseased controls (n 5 2), the CHC group consisted of patients with cervicalgia (n 5 1), radiculopathy (n 5 3), peripheral cranial nerve paralysis (n 5 2), disturbance of equilibrium (n 5 1), polyneuropathy (n 5 6), vertigo (n 5 5), atypical pain syndrome (n 5 1), medication-induced confusion (n 5 1), headache (n 5 3), discrete syringomyelia at the mid-dorsal level and without involvement of the cervical spinal cord (n 5 1), urinary incontinence (n 5 1), urosepsis (n 5 1), and low back pain (n 5 1).The CHC group was recruited among hospitalized patients at the Neurology Department. In all of these patients, central nervous system pathology that could explain the patients’ clinical symptoms was ruled out by means of an extensive neurological workup. 2.3. Neuropathological criteria As described previously [22], all pathological diagnoses were established by the same neuropathologist (J.J.M.), who was blinded for the CSF results. For the diagnosis of AD, the neuropathological criteria of Braak and Braak [23] and Braak and colleagues [24] were applied. The pathological criteria of McKeith and colleagues [17] were applied to make the diagnosis of DLB. MSA was neuropathologically diagnosed according to the criteria of Gilman and colleagues [20]. 2.4. CSF sampling and storage CSF was obtained by LP at the L3/L4 or L4/L5 interspace. Most samples (206 of 262) were centrifuged (3000g; 10 minutes) immediately after the LP to avoid blood contamination of our samples. CSF samples were immediately frozen in liquid nitrogen and stored in polypropylene vials at 280 C until analysis. The samples were collected at the Hospital Network Antwerp (ZNA) Middelheim and Hoge Beuken according to a standard protocol [2,25].

Fig.1. Overview of the study population. np, neuropathologically confirmed. AD, Alzheimer’s disease; CHC, cognitively health control; DLB, dementia with Lewy bodies; PD, Parkinson’s disease; PDD, Parkinson’s disease with dementia; MSA, multiple system atrophy.

2.5. CSF analysis CSF levels of Ab1-42, T-tau, and P-tau181P were determined with commercially available single-analyte enzyme-linked

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immunoabsorbent assays (ELISAs; INNOTEST b-AMYLOID(1-42), INNOTEST hTAU-Ag, and INNOTEST PHOSPHO-TAU(181P), CE marked, Innogenetics, Ghent, Belgium). For each assay, the clinical samples, together with a blank (sample diluent), the calibrator series, and the appropriate controls, were tested strictly following the test instructions provided in the kit insert. All samples were run in duplicate. If the replicate difference was greater than 20% (calculated as [(value 1 – value 2) ! 100]/average), then the samples were retested. If concentrations were out of range, then the value was set equal to the highest/lowest concentration increased or decreased with the difference between two highest/lowest calibration points of the calibration curve divided by 2.

with an arbitrary cutoff of median fluorescence intensity of 150 [30]. Hemoglobin levels were determined (Human Hemoglobin ELISA Kit, Bethyl Laboratories, Inc., Montgomery, TX) for all samples of neuropathologically confirmed patients for whom sufficient CSF was available (49 of 50). Hemoglobin levels were also determined for four samples with a-synuclein levels greater than 1000 pg/mL. In addition, 26 samples were also selected at random from the whole population to determine the hemoglobin levels. Samples with hemoglobin levels above 3000 ng/mL were excluded from statistical analysis (AD: n 5 3; DLB: n 5 3). Also, one AD sample with an extreme a-synuclein level of greater than 1000 pg/mL was excluded from the analysis.

2.6. A bead-based xMAPÔ a-synuclein assay

2.7. Statistical analyses

The bead-based xMAPÔ a-synuclein assay has been described in the work of Hall and colleagues [26]. In short, the antibody Syn1a/9B6, IgG1, recognizing the C-terminal L113-Y126 epitope [27], was covalently coupled to the carboxymethylated beads (region 126). The biotinylated detector monoclonal antibody Syn3 b/4D8, IgG1 was mapped to the epitope K43-K60 in exon 3 (Figure 2). These beads were also combined with sandwich assays for Ab1-42 (21F12/ 82E1bio), T-tau (BT2/42F8bio), and P-tau (AT270/ 42F8bio); however, because the levels correlated very well with the respective INNOTEST ELISAs, and all data for INNOTEST were available, only INNOTEST results for Ab1-42, T-tau, and P-tau were further analyzed. Heterophilic or human anti-mouse antibodies (HAMAs) are a common problem in high-sensitive immunoassays [28], which has also been acknowledged in CSF [29]. Therefore, a bead controlling for heterophilic antibody interference was included in the multianalyte assay. In none of the 262 CSF samples analyzed in this study did we observe heterophilic antibodies

To normalize the biomarker data, concentrations were log10 transformed. For the comparison of the demographic variables among the synucleinopathies, AD, and CHC groups, an analysis of variance (ANOVA) test with Bonferroni correction was used. A c2 test was used to compare gender distribution between the different groups. To confirm the results in a neuropathologically confirmed data set, nonparametric statistics were used because of the small number of subjects. A Mann–Whitney U test was used to evaluate the difference between a-synuclein levels between AD and DLB patients. To determine the relationship between the different biomarkers in neuropathological confirmed AD and DLB patients, a Spearman rank correlation test was executed. A decision tree was made using the Chi-squared automatic interaction detection method (CHAID). The maximum tree depth was two levels, the significance (Pearson) for splitting nodes and merging categories was set to 0.05, the maximum number of iterations was 100, and the

Fig. 2. Schematic representation of human a-synuclein and the epitopes of monoclonal antibodies Syn1a/9B6 and Syn3 b/4D8. The N-terminal region (light blue) of human a-synuclein contains four repeats (dark blue) and an alternatively spliced exon, exon 3. The middle part (orange) contains two repeats and the nonamyloid component region. The C-terminal region (red) has one alternatively spliced exon, exon 5. Four different splicing variants of a-synuclein have been described: (1) the largest or a-syn140 contains exon 3 and exon 5, (2) a-syn126 misses exon 3 whereas (3) a-syn112 misses exon 5, and (4) the shortest a-syn98 misses both exons and is the shortest form. As shown in the figure, a sandwich immunoassay with Syn1a/9B6 and Syn3 b/4D8 will predominantly measure a-syn140. The a-syn peptides identified in CSF-a-syn by mass spectrometry as described in Mollenhauer and colleagues [6] are shown in green bars. CSF, cerebrospinal fluid.

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minimum number of cases in parent nodes was 10 and for a child node was 5. The minimum change in expected cell frequencies was 0.001, and the weight of DLB cases was increased by two. A significance level less than .05 was considered significant. Statistical analyses were performed using SPSS 20 (SPSS Inc., Chicago, IL).

biomarkers was examined in the neuropathologically confirmed study population. A significant and moderately positive correlation was found between T-tau and a-synuclein (n 5 32; r 5 0.655; n 5 32; P , .001) and P-tau181P and a-synuclein (n 5 32; r 5 0.776; P , .001) in AD patients but not in DLB patients (n 5 13; P 5 .448; P 5 .972; Figure 4). These correlations between a-synuclein and T-tau and P-tau181P were also present in the clinical cohort (AD: P , .001, r 5 0.536; synucleinopathies: P , .001, r 5 0.344; and AD: P , .001, r 5 0.620; synucleinopathies: P 5 .001, r 5 0.311). The CSF biomarkers Ab1-42, T-tau, P-tau181P, and a-synuclein data from the neuropathologically confirmed AD and DLB patients were entered in a decision tree model to optimally discriminate between AD and DLB patients. The final decision tree retained P-tau181P and a-synuclein (Figure 5). In a first step, dementia patients were subdivided according to a-synuclein levels. In the subgroup with high CSF a-synuclein levels, P-tau181P levels were used to subdivide this group. A sensitivity of 85% (11 of 13) and specificity of 81% (26 of 32) was reached using this model, and the accuracy of this tree was 82% (37of 45) for differentiating AD from DLB.

3. Results The demographic and clinical data of the patients with synucleinopathies, AD, and CHC groups are summarized in Table 1. For a subset of AD (n 5 34) and DLB (n 5 15) patients, autopsy confirmation of clinical diagnosis was available, of which some samples were excluded because of hemoglobin levels above 3000 ng/mL (AD: n 5 2; DLB: n 5 2). A significant difference was found in CSF a-synuclein concentrations among the three diagnostic groups (P , .001). The AD group presented with higher levels of a-synuclein in CSF when compared with synucleinopathies (P ,.001) and CHC (P 5.019; Figure 3). In AD patients, the levels of T-tau and P-tau181P were increased whereas Ab1-42 was decreased when compared with CHC and patients with synucleinopathies (for all comparisons: P , .001). To confirm the results in neuropathologically confirmed patients, 32 AD and 13 DLB patients were selected from the database. Also in this subpopulation, higher a-synuclein levels were found in AD patients as compared with DLB patients (P 5 .001; Table 1). In the neuropathologically confirmed subpopulation, no difference was found in CSF Ab1-42 values between AD and DLB patients. Definite AD patients had increased T-tau and P-tau181P levels as compared with definite DLB patients (Table 1). No significant correlation could be found between a-synuclein levels and neuropathology in DLB patients (senile plaques: P 5 .345; neurofibrillary tangles: P 5 .549; Lewy bodies: P 5 .142). The correlation between the different

4. Discussion The purpose of this study was to determine the performance of a-synuclein-140 as a CSF biomarker for the AD versus DLB differential diagnosis. 4.1. a-Synuclein-140 It is well established in brain studies that alternative splicing of synuclein transcripts is closely associated with Lewy body pathology [31,32]. To what extent this alternative splicing specifically affects protein biomarker analysis is not known. Most of the in-house developed

Table 1 Demographic, clinical, and biomarker data Synucleinopathy Gender (male/female) Age (years) MMSE score (/30) Ab1-42 (pg/mL) T-tau (pg/mL) P-tau181P (pg/mL) a-synuclein (pg/mL)

68/34*

z

73 6 10* (n 5 102) 20 6 6*z (n 5 69) 608 6 236* (n 5 102) 289 6 174* (n 5 102) 48 6 24* (n 5 102) 104 6 50* (n 5 102)

AD

CHC

P z

56/68*

12/17

78 6 9* (n 5 124) 17 6 7*y (n 5 112) 476 6 159*y (n 5 124) 586 6 359*y (n 5 124) 79 6 41*y (n 5 124) 147 6 74*y (n 5 124)

74 6 8 (n 5 29) 28 6 2yz (n 5 11) 653 6 216y (n 5 29) 286 6 138y (n 5 29) 51 6 20y (n 5 29) 111 6 53y (n 5 29)

.002 .001 ,.001 ,.001 ,.001 ,.001 ,.001

DLB (np)

AD (np)

P

11/2

24/8

.482

77 6 8 (n 5 13) 18 6 1 (n 5 8) 421 6 172 (n 5 13) 364 6 252 (n 5 13) 53 6 36 (n 5 13) 111 6 61 (n 5 13)

79 6 9 (n 5 32) 13 6 7 (n 5 24) 443 6 126 (n 5 32) 731 6 513 (n 5 32) 97 6 61 (n 5 32) 192 6 91 (n 5 32)

.483 .034 .271 .004 .003 .001

Abbreviations: AD, Alzheimer’s disease; ANOVA, analysis of variance; CHC, cognitively healthy control; DLB, dementia with Lewy bodies; MMSE, MiniMental State Examination; np, neuropathologically confirmed; T-tau, total tau; P-tau181P, phosphorylated tau; Ab1-42, amyloid-b. NOTE. Data are shown as mean 6 SD. An ANOVA with Bonferroni correction was used to compare the synucleinopathy, AD, and CHC groups with the exception of the gender distribution (c2 with Bonferroni correction). A Mann–Whitney U test was used to evaluate the difference between np DLB and AD patients with exception of the gender distribution (c2). Significant differences are indicated with the following symbols: *Synucleinopathy vs AD. y AD vs CHC. z Synucleinopathy vs CHC.

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[27]. Thus, assays using this antibody preferentially quantify full-size a-synuclein or synuclein-140 and synuclein-126 but not synuclein-112 and synuclein-98. Furthermore, Syn3 b/4D8 [27], used in this study, has its epitope in exon 3. Thus, a sandwich assay based on Syn1a/9B6 and Syn3 b/4D8 as described in this study specifically quantifies synuclein-140 (Figure 2). Comparative studies using different assay formats combined with high-sensitive characterization via two-dimensional and/or mass spectrometry as described in Wang and colleagues [34] will help to address its relevance toward the development of an optimal a-synuclein biomarker assay. 4.2. CSF a-synuclein in synucleinopathies

Fig. 3. Boxplot of a-synuclein levels in the total population and the definite AD and DLB subgroup. AD, Alzheimer’s disease; CHC, cognitively healthy control; DLB, dementia with Lewy bodies.

sandwich immunoassays use one monoclonal and polyclonal antibody. The a-synuclein-specific monoclonal antibody, 211, maps to a C-terminal epitope within exon 5 [33], as well as the Syn1a/9B6 antibody used in this study

In this study, significantly higher CSF a-synuclein levels were found in AD patients when compared with patients with a synucleinopathy and CHC. This increase in AD of a-synuclein is congruent with the results of several other studies [6,7,12,35,36] (Table 2). In the current study, no difference could be found between CHC and synucleinopathies, which is in contrast with other studies [5,6,8,9,11,37]. When interpreting these results, it should be taken into account that all of these studies examined different isoforms of a-synuclein (Table 2). Also, our population was not age- and gender-matched. Patients with a synucleinopathy were younger than AD patients, and there were more males in the synucleinopathy population when compared with the AD and CHC populations. Although differences

Fig. 4. The correlation between CSF a-synuclein and T-tau (r 5 0.655; P , .001) as well as P-tau181P (r 5 0.776; P , .001) levels in AD patients are shown in panels A and C. These correlations could not be found in the DLB population (B and D). The dotted line is the 95% prediction interval for the calculated regression line in a solid blue line. If the correlation was not statistically significant, then no regression line is shown. T-tau, total tau; P-tau181P, phosphorylated tau.

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AD as a result of the pathophysiological processes related to AD because this correlation could not be found in neuropathologically confirmed DLB patients. Because cognitive decline in AD is related to synapse loss and a-synuclein is a synaptic protein, a-synuclein might be a marker for synaptic loss in AD. Indeed, a study of Korff and colleagues [36] demonstrated a correlation between a-synuclein and cognition. The increase of a-synuclein CSF levels in AD might also be a result of a higher expression of a-synuclein in the brains of AD patients as demonstrated in Larson and colleagues [43]. In the latter study, an approximate 2-fold increase of soluble a-synuclein in brains of AD patients was found when compared with CHCs. 4.4. CSF a-synuclein for the AD versus DLB differential diagnosis Fig. 5. Classification tree based on neuropathologically confirmed AD and DLB patients. AD, Alzheimer’s disease; DLB, dementia with Lewy bodies; P-tau181P, phosphorylated tau.

in gender and age were to be expected because AD is more frequent amongst elderly and women [38], these age and gender differences could have influenced our results because age and gender have previously been shown to influence asynuclein levels [5,37,39]. Nevertheless, our results indicate that in synucleinopathies, the accumulation of a-synuclein in Lewy bodies does not influence the CSF a-synuclein levels compared with CHCs. 4.3. CSF a-synuclein in AD patients In the current study, we further examined the ability of a-synuclein as a biomarker in a neuropathologically confirmed AD and DLB population. Higher CSF a-synuclein levels were found in definite AD patients. These results are in line with several other studies [12,26,37], but not all [10,14]. When only considering the neuropathologically confirmed patients in Mollenhauer and colleagues [9], a clear increase in CSF a-synuclein levels in AD patients was found as well. Subsequently investigating the increase of a-synuclein in AD patients, we found a moderately positive correlation between a-synuclein and T-tau and P-tau181P levels in CSF. Again, this positive correlation is in line with Wennstrom and colleagues [35,36,39], but contrasting results have been reported by Parnetti and colleagues [11]; therefore, further research is required. CSF T-tau levels are probably related to the neurodegenerative process that occurs in AD [40]. Moreover, increased CSF a-synuclein levels have been observed in Creutzfeldt-Jakob disease [6], traumatic brain injury [41], multiple sclerosis, and neuromyelitis optica [42]. Our findings suggest that a-synuclein is also raised in

In the current study population, no significant difference in CSF Ab1-42 levels was found between AD and DLB patients. This might be the result of concomitant AD pathology in 73% (11 of 15) of neuropathologically confirmed DLB patients in the current study, as was recently demonstrated [3]. Therefore, CSF Ab1-42 levels alone cannot be used to discriminate between AD and DLB patients. Because CSF a-synuclein levels are significantly increased in AD when compared with DLB patients and might contribute to the discrimination between these two dementia forms, a diagnostic algorithm was constructed using the CHAID method to optimally discriminate the neuropathologically confirmed AD from DLB patients. The decision tree used CSF a-synuclein and P-tau181P levels, reaching sensitivity and specificity values of 85% and 81%, respectively. The sensitivity of this decision tree is better than the sensitivity that is reached when using clinical diagnostic criteria (32.1%) [44]. This diagnostic algorithm achieves the threshold of 80% sensitivity and specificity values as set forward by the consensus criteria for diagnostic markers of AD [45]. 4.5. Limitations and future directions Because different isoforms were used in different studies with a-synuclein, it is difficult to compare our results with those of the other studies. In addition, when determining a-synuclein in CSF, it is important to avoid blood contamination because erythrocytes are a major source of a-synuclein. In the current study, most samples (206 of 262) were centrifuged (3000g, 10 minutes) immediately after the LP, and the supernatant was aliquoted and immediately frozen in liquid nitrogen and stored in polypropylene vials at 280 C until analysis. For the neuropathologically confirmed population we also determined CSF hemoglobin levels. Samples with increased hemoglobin levels were excluded (AD: n 5 3; DLB: n 5 3) from statistical analysis. For CSF a-synuclein research it will

PD: P , .001 DLB: P , .001

NS

NS

P , .001

DLB: NS

PD: P , .01 DLB: NS PD: P , .01 DLB: NS

DLB: NS

1190 6 810 n 5 51 55 (40-70)* n 5 90

38,000 6 29,000 n 5 40 1420 6 1260 n 5 55 59 (49-68)* n 5 70 ,1000 ng/L

,500 erythrocytes/mL

Mollenhauer et al. [9] Training cohort Hall et al. [26]

Wang et al. [34] Validation cohort Spies et al. [14]

–

Luminex 211/FL-140 ELISA 211/FL-140 ELISA mSA-1/Syn-1 Luminex Syn1a/Syn3 b

Shi et al. [46]

,200 ng/mL

Luminex211/FL-140

Study

,200 ng/mL

490 6 170 n 5 137 487 6 181 n 5 128 30,400 6 19,100 n 5 57 1730 6 1830 n 5 76 67 (51-83)* n 5 107

380 6 100 n 5 126 399 6 137 n 5 116

DLB PD Controls

a-synuclein levels

Exclusion criteria for blood contamination Assay (Technology 1 antibodies)

Table 2 Overview of the literature

Abbreviations: AD, Alzheimer’s disease; DLB, dementia with Lewy bodies; ELISA, enzyme-linked immunoabsorbent assay; NS, not significant. NOTE. An overview of research papers examining CSF a-synuclein levels in at least 40 AD, 40 control individuals, and synucleinopathies. Papers that did not fulfill these criteria were included in the discussion. Confounding factors like age, gender, blood contamination, etc that influence CSF alpha-synuclein levels might have contributed to the differences that were found between these studies. For every paper the method of a-synuclein detection is indicated. The a-synuclein levels are shown as mean 6 SD except for the concentrations indicated with *, these results are shown as median (interquartile range).

DLB: P 5 .091

NS PD: P , .05

PD: P , .01

P . .100 PD: P , .001

550 6 150 n 5 50 556 6 155 n 5 50 37,000 6 36,100 n 5 131 1850 6 1470 n 5 62 94 (76-121)* n 5 48

PD: P , .001

Controls vs AD Controls vs PD-DLB AD

AD vs PD-DLB

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always be important to consider possible contamination with blood. Because low diagnostic accuracy of the clinical differential diagnosis hampers the interpretation of biomarker studies dealing with AD and DLB patients, a subset of neuropathologically confirmed patients was included. A limitation of using neuropathologically confirmed patients is the relatively small number of subjects. Therefore, our diagnostic paradigm should be confirmed in an independent study that includes autopsyconfirmed patients. An independent study is also necessary to assess sensitivity and specificity because in the current study the validation and the training cohorts were identical, which could have led to an overestimation of sensitivity and specificity. 5. Conclusion When compared with synucleinopathies and CHCs, CSF a-synuclein levels are higher in AD. Given the significant correlations among T-tau, P-tau181P, and a-synuclein in neuropathologically confirmed AD patients, the increase of CSF a-synuclein is probably related to the pathophysiological processes in AD. A biomarker paradigm with a-synuclein and P-tau181P could discriminate between neuropathologically confirmed AD and DLB patients, resulting in sensitivity and specificity values of 85% and 81%, respectively. Acknowledgments This work was supported by the University Research Fund of the University of Antwerp; the Foundation for Alzheimer Research (SAO-FRA); the Institute Born-Bunge; the agreement between the Institute Born-Bunge and the University of Antwerp; the Central Biobank Facility of the Institute Born-Bunge/University Antwerp; an unrestricted educational grant of Janssen, Belgium; Neurosearch Antwerp; the Research Foundation– Flanders (FWO-Vlaanderen); the Interuniversity Attraction Poles (IAP) program P7/16 of the Belgian Science Policy Office; the Methusalem Excellence Grant of the Flemish Government, Belgium; and the Medical Research Foundation Antwerp. This work is part of the BIOMARKAPD project within the European Union Joint Programme for Neurodegenerative Disease Research. The authors acknowledge the administrative assistance and the clinical staff of the Department of Neurology and Memory Clinic of Hospital Network Antwerp (ZNA), Middelheim and Hoge Beuken, Antwerp, Belgium. S.S. and E.V. serve as joint first authors. E.V. has been with ADx NeuroSciences, Ghent, Belgium, since September 2011. N.L.B. has been with Immunogenetics NV (Miraca/ Fujirebio Group), Ghent, Belgium since September 2012. Conflict of Interest: E.G. was an employee of Innogenetics NV (Miraca/Fujirebio Group), Ghent, Belgium until September 2011. N.L.B., H.D., and M.V. are employees of Innogenetics

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NV (Miraca/Fujirebio Group), Ghent, Belgium. S.E. was a member of the Innogenetics Advisory Board Dementia. [8]

RESEARCH IN CONTEXT

1. Systematic review: The literature was reviewed by using Pubmed with following search string: “dementia with Lewy bodies” or “Parkinson” or “Alzheimer’s disease” or “synucleinopathy” and “cerebrospinal fluid” and “alpha-synuclein”. When reviewing published research involving CSF a-synuclein as a biomarker, we found conflicting results and only one study with neuropathologically confirmed dementia diagnoses. 2. Interpretation: Our findings suggested that increased CSF levels of a-synuclein in AD are probably related to the pathophysiological processes in AD. The classification tree based on our population confirms that a-synuclein is a possible biomarker for discriminating AD and DLB. 3. Future directions: The proposed classification tree should be tested in an independent study population, preferably with neuropathologically confirmed dementia diagnoses. In future research, more attention should be directed toward the different splicing variants of a-synuclein. The combination of CSF a-synuclein with other biological markers such as imaging should be considered.

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

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