Supplementary material:
METHODS:
Patients selection and clinical diagnosis:
Diagnoses are based on clinical examination, using current diagnostic criteria: UK Brain bank society criteria for Parkinson`s Disease (Hughes et al.,1992,J Neurol Neurosurg Psychiatry55(3):181-4); MDS criteria for PD dementia (Emre et al., 2007,Movement Disorder 15;22(12):1689-707) and the McKeith criteria for possible and probable DLB (McKeith et al., 2005, Neurology 65(12):1863-72).
CSF and serum total- and 5G4- α-synuclein measurement
CSF and serum collections were performed according to standardized protocols (for details see Maetzler et al., 2011, Movement Disorder26(1):176-81). Samples were obtained from our local Neuro Biobank (see Acknowledgement Section). Our biobank fulfils highest international standards, with needle- to freezer time of <60 minutes, and repeated clinical characterizations of the included donors performed by a team of neurologists experienced in the field of movement disorders.
Quantitative determination of human total and 5G4α-synuclein protein was done with ELISA following meticulously the kit instruction manual (Analytic Jena Roboscreen GmbH, Leipzig, Germany) for storage time, repeated measurement as well as preanalytical and analytical steps. Total α-synuclein levels were evaluated in duplicate with the MONO-kit from the above company. All 5G4 α-synuclein measurements were performed at least in duplicate, with two different kits: a standard ELISA and an electrochemiluminescence (ECL)-ELISA, both also provided by the above-mentioned company. According to the manufacturer’s manual, intra- and inter-assay variability vary between 3 and 10 percent.
Supplementary Table 1: Demographic, clinical and neurochemical markers of the outlierswith regard to 5G4 α -synuclein serum levels.
PDND / PDD / HCgroup mean / Outliers / group mean / Outlier / groupmean / Outliers
#1 / #2 / #3 / #4 / #5 / #6 / #7 / #8
Serum 5G4 α-synuclein [ng/mL] / 0.012 / 19.1 / 16.2 / 12.7 / 11.2 / 10.6 / 0.007 / 27.5 / 0.014 / 23.1 / 13.4
Gender / - / M / F / F / M / M / - / F / - / F / M
Age [y] / 69 / 56 / 49 / 63 / 61 / 66 / 77 / 69 / 66 / 60 / 64
Age at onset [y] / 58 / 51 / 47 / 57 / 54 / 53 / 64 / 53 / - / - / -
Disease duration [y] / 11 / 5 / 2 / 6 / 8 / 13 / 13 / 13 / - / - / -
H&Y (0-5) / 2 / 2 / 1 / 2 / 3 / 2 / 2.5 / 3 / - / - / -
UPDRS III (0-108) / 25 / 14 / 13 / 15 / 26 / 35 / 33 / 28 / 1 / 0 / 1
MMSE (0-30) / 28 / 30 / 30 / 28 / 27 / 29 / 23 / 23 / 29 / 30 / 30
CSF tau [pg/mL) / 158 / 167 / 133 / 198 / 156 / 182 / 206 / 147 / 172 / 251 / 108
CSF- p-tau (pg/mL) / 42 / 29 / 26 / 44 / 34 / 20 / 51 / 35 / 43 / 49 / 31
CSF Aβ42 (pg/mL) / 792 / 516 / 1053 / 801 / 769 / 1189 / 614 / 913 / 892 / 1275 / 654
Serum total α-synuclein [ng/mL] / 0.39 / 1.17 / UDL / 0.04 / 0.85 / 0.25 / 0.37 / 0.26 / 0.36 / 0.34 / 0.06
Outlier was defined as individual with serum 5G4 α-synuclein levels above 10 ng/mL.CSF, cerebrospinal fluid; DLB, dementia with Lewy bodies; H&Y, Hoehn & Yahr score; MMSE, Minimental State examination; PDD, Parkinson’s disease with dementia; PDND, PD without dementia; UPDRS-III, Unified PD Rating scale motor part.
Supplementary Table 2. Correlationsbetween serum total and 5G4α-synuclein levels, and clinical, demographic as well as biochemical parameters
Total α-synuclein / 5G4 α-synucleinClinical diagnosis / PDND / PDD / DLB / PDND / PDD / DLB
R2 / p-value / R2 / p-value / R2 / p-value / R2 / p-value / R2 / p-value / R2 / p-value
Age / -0.12 / 0.24 / 0.02 / 0.92 / -0.34 / 0.06 / -0.01 / 0.89 / -14 / 0.47 / 0.09 / 0.63
Gender / p=0.72 / p=0.40 / p=0.42 / P=0.77 / p=0.74 / p=0.57
Disease duration / -0.11 / 0.31 / 0.36 / 0.05 / 0.37 / 0.11 / -0.05 / 0.66 / 0.14 / 0.46 / 0.23 / 0.71
UPDRS-III / 0.10 / 0.63 / 0.42 / 0.14 / 0.63 / 0.18 / -0.18 / 0.64 / 0.28 / 0.33 / 0.14 / 0.42
MMSE / 0.21 / 0.13 / -0.21 / 0.26 / 0.00 / 0.99 / -0.24 / 0.08 / -0.15 / 0.42 / -0.02 / 0.95
CSF tau / 0.11 / 0.30 / 0.17 / 0.38 / -0.07 / 0.83 / -0.00 / 0.97 / 0.15 / 0.44 / 0.03 / 0.93
CSF p-tau / 0.08 / 0.50 / 0.23 / 0.26 / 0.05 / 0.86 / 0.12 / 0.34 / -0.08 / 0.70 / 0.27 / 0.38
CSF Aβ42 / -0.03 / 0.79 / 0.36 / 0.07 / 042 / 0.15 / 0.20 / 0.10 / -0.21 / 0.28 / -0.35 / 0.24
Serum total α-synuclein / - / - / - / - / - / - / 0.10 / 0.33 / -0.03 / 0.86 / 0.00 / 0.99
Serum 5G4 α-synuclein / 0.10 / 0.33 / -0.03 / 0.86 / 0.00 / 0.99 / - / - / - / - / - / -
CSF, cerebrospinal fluid; DLB, dementia with Lewy bodies; MMSE, Minimental State examination; PDD, Parkinson’s disease with dementia; PDND, PD without dementia; R2,correlation coefficient;UPDRS-III, Unified PD Rating scale motor part
Supplementary Table 3. Published studies assessing α-synuclein in CSF or serum in PD, PDD and DLB.
α-synuclein form / CSF / SerumParkinson’s Disease
Total α-synuclein / Ref. / Ref.
↓ (n=15)
↔ (n=7) / [14, 16, 17, 24, 25, 30-32, 35, 38-40, 42-44]
[1, 3, 6, 9, 27, 29, 33] / ↑ (n=2)
↓ (n=2)
↔ (n=10) / [5, 21]
[11,22]
[2, 4, 6, 8, 10, 25, 29, 34, 36] + [present study]
↓ t-α-syn/t-prot (n=3) / [24, 25, 42]
↑ t-tau/ t-α-syn (n=1) / [30]
Oligomeric α-synuclein / ↑ (n=5)
↔ (n=2) / [7, 29, 32, 39, 40]
[9, 31] / ↑ (n=1)
↔ (n=2) / [7]
[11, 29]
↑ o / t-α-syn ratio (n=3) / [31, 32, 40] / ↔ o / t-α-syn ratio (n=1) / [11]
Phosphorylated α-synuclein (PS-129) / ↑ (n=1)
↔ (n=1) / [43]
[9] / ↑ (n=1) / [10]
↑ p/t-α-syn ratio (n=1) / [43]
Phosphorylated oligomeric α-synuclein / ↑ (n=1) / [9] / ↔ (n=1) / [8]
5G4-specific α-synuclein / Not detectable (n=1) / [present study] / ↔ (n=1) / [present study]
α-synuclein autoantibodies / - / ↑ (n=3
↓ (n=1)
↔ (n=3) / [12,13, 46]
[2]
[28, 36, 45]
Parkinson’s Disease Dementia
Total α-synuclein / ↓ (n=1)
↔ (n=2) / [44]
[9, 14] / ↔ (n=1) / [present study]
Oligomeric α-synuclein / ↑ (n=1)
↔ (n=1) / [15]
[9] / -
↑ o / t-α-syn ratio (n=1) / [15]
Phosphorylated α-synuclein (PS-129) / ↔ (n=1) / [9] / -
Phosphorylated oligomeric α-synuclein / ↔ (n=1) / [9] / -
5G4-specific α-synuclein / ↑* (n=1)
Not detectable(n=1) / [41]
[present study] / ↔ (n=1) / [present study]
Dementia with Lewy Bodies
Total α-synuclein / ↓ (n=7)
↔ (n=6) / [18, 23, 24, 25, 30, 38, 44]
[14, 15, 26, 27, 33, 37] / ↓ (n=2) / [20] + [present study]
t-α-syn/t-prot ↓ (n=1)
↔ (n=1) / [25]
[24]
↑ t-tau/ t-α-syn (n=1) / [30]
Oligomeric α-synuclein / ↔ (n=2) / [9, 15] / -
Phosphorylated α-synuclein (PS-129) / ↔ (n=1) / [9] / -
Phosphorylated oligomeric α-synuclein / ↑ (n=1) / [9] / -
5G4-specific α-synuclein / ↑* (n=1)
Not detectable(n=1) / [41]
[present study] / ↔ (n=1) / [present study]
α-synuclein autoantibodies / - / ↑ (n=1) / [19]
* Total number (n) for dementia with Lewy bodies (DLB) and Parkinson’s disease dementia (PDD) samples = 7, not differentiated between diagnoses. ↓, ↑ decrease and increase of levels; ↔ no significant difference; CSF, cerebrospinal fluid; o- α-syn: oligomeric α-synuclein; p- α-syn: phosphorylated α-synuclein; Ref: references;t-α-syn: total α-Synuclein; t-tau: total tau protein; t-prot: total CSF protein.
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