Total and Proteinase K-Resistant α-Synuclein Levels in Erythrocytes, Determined by their Ability to Bind Phospholipids, Associate with Parkinson's Disease

Abstract

A marker for diagnosis of Parkinson's disease (PD), which reflects on the occurrence of peripheral pathogenic mechanisms, would potentially improve therapy. The significance of α-Synuclein (α-Syn) expression in red blood cells (RBC) is currently unclear. Here we investigated whether RBC's-expressed α-Syn may associate with PD. To this aim, we determined the levels of total and proteinase K-resistant α-Syn in samples of packed red blood cells (PRBCs). Twenty-one individuals with PD at various disease stages and 15 healthy controls, with similar demographic features, were recruited to this study. α-Syn levels were determined by their biochemical property to bind phospholipids, using a phospholipid-ELISA assay. A significantly lower ratio of total-to-proteinase K-resistant α-Syn levels was detected in PD patients than in the healthy control group. However, there was considerable overlap between the two groups. Suggesting a need for additional markers to be tested in combination with α-Syn levels. To the best of our knowledge, this is the first evidence for an association between RBCs-expressed α-Syn and pathogenic mechanisms involved in PD.

Figure 1. Detection of α-Syn in samples of packed red blood cells (PRBC).

(a) A PRBCs sample (10 μg proteins), separated on a 10%SDS-PAGE, transferred to nitrocellulose membrane and reacted directly with a chemiluminescence (ECL) detection kit (Pierce, CT, USA), without prior incubation in primary or secondary antibodies. (b) Samples of PRBC were heated at 100 °C for 10 minutes, followed by a spin down at 20,000 × g, to generate the soluble (S)-PRBC and pellet (P)-PRBC. Protein samples (at the indicated amounts) were separated on a 10%SDS-PAGE and the blot was reacted directly with an ECL detection kit as in (a). (c) The blot in (b) reacted with anti α-Syn antibody (Syn #10¹⁹) and a secondary donkey anti mouse-HRP conjugated antibody. Arrow, an α-Syn-specific signal. (d) Protein samples of PRBC or S-PRBC, analyzed by Western blotting as in (c). Filled arrow, non specific bands; line arrow, α-Syn-specific bands.

Figure 2. The phospholipid ELISA assay specifically detects α-Syn in S-PRBC samples.

(a) Samples of human S-PRBC (0–4 μg protein) were applied to a 96-well microtiter dish containing the indicated phospholipids (Phosphatidylinositol (PI); phosphatidylserine (PS); phosphatidylethanolamine (PE)) at a final amount of 100 μg/well, or without phospholipids (-PL) and processed for the detection of α-Syn using anti human α-Syn antibody, α-Syn #10¹⁹). Graph illustrates the means ± SD, n = 3 replicates. (b) Samples of mouse PRBC or S-PRBC from wt or α-Syn-/- (1 μg protein), analyzed by Western blotting using anti- α-Syn antibody, α-Syn #3. Arrows indicate non-specific immunoreactive bands. (c) S-PRBC from wt and α-Syn-/- mice were applied in increasing amounts (0–4 μg protein) into wells of a microtiter dish coated with PI-PS-PE phospholipids (as in (a)), using anti α-Syn antibody, α-Syn#3 or without a primary detecting ab (−1° ab). Graph illustrates the means ± SD of n = 3 repeats.

Figure 3. α-Syn levels in S-PRBC, S-plasma and soluble fraction of human brains

(a) Samples of S-PRBC (0–2.5 μg protein); S-plasma (0–25 μg protein) or the soluble fraction of human brain (HB, 0–13 μg protein) were analyzed by phospholipid-ELISA using PI-PS-PE for capture and anti α-Syn ab, α-Syn#10¹⁹, for detection. (b) Samples of S-plasma, S-PRBC and HB (at the indicated protein amounts), analyzed by Western blotting using anti α-Syn ab, α-Syn#10. A representative blot out of n = 3.

Figure 4. Higher levels of proteinase K-resistant α-Syn in PD samples.

Western blot analysis of PRBC samples (20 μg protein) of healthy controls (a) and PD patients (b), treated with the indicated Proteinase K concentrations for 30 minutes at 37 °C and spun at 20,000 g before loading on a 10% SDS-PAGE. A representative blot, reacted with anti α-Syn (α-Syn#10)¹⁹ and anti β-actin abs. Densitometry analysis of Western blots for HC (c) and PD (d). The levels of α-Syn and β-actin are calculated as percent of the signal obtained without proteinase K (set at 100%). Mean ± SD of n = 6 different samples; *, p < 0.01, t-test. Samples of PRBC from healthy (e) and PD (f), analyzed by Phospholipid-ELISA as in Fig. 2a. Graph present mean ± SD of n = 6.

Figure 5. α-Syn resistance to proteinase K is acquired in the presence of phospholipids.

Samples of SPRBC (5 μg protein) pre-incubated with PI/PS/PE (1:1:1) or cholesterol/sphingomyelin (1:1), at a final amount of 100 μg, or without any lipids prior to incubation with proteinase K. Proteinase-K resistant α-Syn levels determined by phospholipid-ELISA and presented as percent of total α-Syn, detected without Proteinase K. Mean ± SD of n = 3.

Figure 6. Total and proteinase K-resistant α-Syn are detected in samples of SPRBC by a standard sandwich-ELISA but with a lower efficacy.

Samples of human S-PRBC (0–2.5 μg protein) were applied to a 96-well microtiter dishes and α-Syn levels were determined by phospholipid (PL)-ELISA (as in Fig. 2a) or a sandwich- ELISA, using anti α-Syn ab, Syn-1 (Transduction laboratories) as a capturing antibody. α-Syn detection was obtained with α-Syn #3 antibody (PL-ELISA, a and c) or C-20 antibody (Santa Cruz) (sandwich ELISA, b and d). Graph illustrates the means ± SD (n = 3 replicates) of total α-Syn (filled line) and proteinase K-resistant α-Syn (dashed line) levels of a representative sample in each group.

Figure 7. α-Syn levels in healthy controls and PD groups

(a) Total α-Syn levels determined by phospholipid ELISA in healthy control (HC; n = 15); and PD (n = 21). (b) Proteinase K- resistant α-Syn determined by phospholipid ELISA. (c) The ratio of total-to-proteinase K-resistant α-Syn. Bar, represents the mean value of the group. *, P < 0.001, ttest. (d) The ratio of total-to-proteinase K-resistant α-Syn as in (c), presented in a whiskers plot. The horizontal bold lines indicate the medians; the lower part of boxes indicate the first quartile and the upper part the third quartile; the vertical lines indicate the range of values.