α-Synuclein interacts directly but reversibly with psychosine: implications for α-synucleinopathies

Abstract

Aggregation of α-synuclein, the hallmark of α-synucleinopathies such as Parkinson's disease, occurs in various glycosphingolipidoses. Although α-synuclein aggregation correlates with deficiencies in the lysosomal degradation of glycosphingolipids (GSL), the mechanism(s) involved in this aggregation remains unclear. We previously described the aggregation of α-synuclein in Krabbe's disease (KD), a neurodegenerative glycosphingolipidosis caused by lysosomal deficiency of galactosyl-ceramidase (GALC) and the accumulation of the GSL psychosine. Here, we used a multi-pronged approach including genetic, biophysical and biochemical techniques to determine the pathogenic contribution, reversibility, and molecular mechanism of aggregation of α-synuclein in KD. While genetic knock-out of α-synuclein reduces, but does not completely prevent, neurological signs in a mouse model of KD, genetic correction of GALC deficiency completely prevents α-synuclein aggregation. We show that psychosine forms hydrophilic clusters and binds the C-terminus of α-synuclein through its amino group and sugar moiety, suggesting that psychosine promotes an open/aggregation-prone conformation of α-synuclein. Dopamine and carbidopa reverse the structural changes of psychosine by mediating a closed/aggregation-resistant conformation of α-synuclein. Our results underscore the therapeutic potential of lysosomal correction and small molecules to reduce neuronal burden in α-synucleinopathies, and provide a mechanistic understanding of α-synuclein aggregation in glycosphingolipidoses.

Figure 1

Knock-out of SNCA removes thioflavin-S-positive inclusions in certain brain regions and improves survival and behavior of TWI mice. (A) Brain tissue from TWI/SCNA−/− (A), TWI/SCNA+/− (B) and TWI/SCNA+/+ (C) were stained for thioflavin-S-positive protein aggregates. Thio-S material was visibly reduced in the lower cortical layers and putamen of TWI/SNCA−/− brains (A) and TWI/SNCA+/− brains (B) with respect to TWI/SNCA+/+ (C). Images (A–C) show anatomical regions encompassing deep cerebral cortex and the putamen. cc, corpus callosum. Scale bar = 200 μm. (D) Unbiased stereology quantitatively revealed a significantly reduced abundance of thio-S+ material in TWI/SNCA−/− and TWI/SNCA+/− brains (p = 0.001, ANOVA) within the Dentate Gyrus (DG), Cerebral Cortex (Cortex), and Thalamus. (E) Psychosine levels measured using LC-MS-MS showed non-significant (n/s) changes between the presence or absence of the SCNA allele in TWI brains. (F) A disease severity score (DSS) measured less severe signs in TWI/SNCA+/− and TWI/SNCA−/− mice compared to TWI/SNCA+/+, while changes were significant at only P36 and P38 for TWI/SNCA+/− (*p < 0.05) and P36 for TWI/SNCA−/− (*p < 0.05). (G) Grip strength, as assessed by a latency-to-fall test, was significantly improved in TWI/SNCA−/− compared to TWI/SNCA+/+ at P30 (*p < 0.05). (H) Nesting ability score, assessed at P20 in each genotype group, was significantly improved in both TWI/SNCA+/− and TWI/SNCA−/− compared to TWI mice (*p < 0.05, **p < 0.01, ANOVA). (I) Kaplan-Meier survival curves reveal significantly longer life of TWI/SNCA+/− and TWI/SNCA−/− compared to TWI. N = 10 mice (TWI/SNA^+/− and TWI/SNCA^−/−) N = 15 mice (TWI/SNCA^+/+).

Figure 2

Neuronal thioflavin-S aggregates and high-molecular-weight α-synuclein aggregates are greatly reduced upon AAV9-GALC gene therapy. Thio-S+ protein aggregates in TWI (B), previously shown to be intra-neuronal and partly comprised of α-synuclein, were eliminated at P40 after neonatal gene therapy using AAV9-GALC vectors alone (D) or in combination with bone marrow transplantation (BMT + AAV9-GALC) (E), but not by BMT alone (C). WT comparison in (A). Images show anatomical regions encompassing deep cerebral cortex and the putamen. cc, corpus callosum. Thio-S+ aggregates (fluorescent units per area) remained largely eliminated in AAV9-GALC and BMT + AAV9-GALC brains (F). *p < 0.05, compared to WT, ANOVA; n = 3–4 mice per group). (G,K) Gene therapy with AAV9-GALC greatly normalized α-synuclein levels in the cortex/putamen region (compare J and K with H). Sections were stained with diamino-bencidine for α-synuclein and counterstained with toluidine blue. (L) Total brain lysates from young (P40) and long-surviving (>P200) TWI, AAV9-GALC treated TWI and control mice were immunoblotted for α-synuclein, and high-molecular-weight bands (~30 to ~100 kDa) were quantified by optical densitometry. Results are expressed as fold levels over basal levels in WT brains. *p < 0.05, ANOVA; n = 3–4 mice per group. Scale bar: 200 μm.

Figure 3

Psychosine and α-synuclein subcellular localization in vivo. Thioflavin-S (thio-S)-positive deposits in the P40 TWI brain co-localized with both the lysosomal marker Lamp-1 (A) and the autophagosomal marker LC3-I (B). TWI autophagosomes were enlarged compared to WT (C) and no aggregated thio-S material was detected in the WT brain (D). Anti-α-synuclein antibodies co-localized intracellularly with thio-S and the lysosomal marker LAMP-1 (E–I, arrows). Thio-S-positive α-synuclein was not detected in WT tissue (J). Isolation of lysosomes further confirmed α-synuclein co-localization with enriched lysosomes (K) and autophagosomes (L) while also staining positive for thio-S (K,L white arrows). Not all organelles that co-localized with α-synuclein were positive for thio-S (L, yellow arrows). Psychosine levels measured in total lysate and isolated lysosomes showed an accumulation in TWI that was significantly enriched in the lysosome/autophagosome fraction compared to the total lysate (n = 3, p < 0.05, ANOVA) (M). Fractions enriched in lysosomes and autophagosomes from TWI and WT brains showed an elevated level of Lamp-1 and LC3-I in addition to high-molecular-weight aggregates of α-synuclein in TWI (N). Ultrastructure EM imaging revealed fibrous material enveloped in apparent autophagic vacuoles (O, double membrane organelle marked by clack arrows), and immunoelectron micrography identified α-synuclein deposits within double-membrane (arrowheads, inset in Q) autophagosome structures (P,Q) in neurons from P40 TWI. Non-specific binding of secondary antibody (anti-IgG-Au, 0.8 nm) was not observed (R). Scale bars: 10 μm (A–L), 0.2 μm (O), 1 μm (P), and 100 nm (R). Uncropped blots displayed in Supplementary Fig. 18.

Figure 4

Psychosine interaction with α-synuclein at different concentrations. CSPs in full-length α-synuclein (20 µM) with psychosine indicated no significant effects at the following ratios: α-synuclein (1) psychosine (0.5) (A); α-synuclein (1):psychosine (1) (B); α-synuclein (1) psychosine (2) (C); α-synuclein (1) psychosine (5) (D). Significant CSPs and line-broadening of signals were observed at the following molar ratios: α-synuclein (1) psychosine (10) (E); α-synuclein (1) psychosine (20) (F); α-synuclein (1) psychosine (30) (G); α-synuclein (1) psychosine (50) (H). The black horizontal line represents the CSP mean plus one SD. The orange horizontal line represents the maximum CSP due to spectral variability between different α-synuclein samples. CSPs higher than both the black and orange lines were considered significant and marked in red. The broadened-beyond-detection signals were assigned by arrows marked in pink in the N-terminus, green in the NAC region, and purple in the C-terminus. The number of signals that were broadened are presented in a pie chart. PSY = psychosine; SYN = α-synuclein.

Figure 5

Psychosine interacts with α-synuclein at cytoplasmic (neutral) and lysosomal (acidic) pH. Overlays of ¹H ¹⁵N-HSQC NMR spectra of α-synuclein (blue) with α-synuclein (1) psychosine (30) (red) at pH 7.6 (A) and α-synuclein (1) psychosine (30) (red) at pH 4.7 (B). Examples of chemical shift changes are circled and highlighted in a box. Black arrows indicate areas of signal broadening. The CSPs were measured from the spectra α-synuclein (1) psychosine (30) (red) at pH 7.6 (C), and α-synuclein (1) psychosine (30) (red) at pH 4.7 (D). The black horizontal line represents the mean CSP plus one SD. The orange horizontal line represents the maximum CSP due to spectral variability between different samples of α-synuclein. CSPs higher than both the black and orange lines were considered significant and are marked in red. The broadened- beyond-detection signals were assigned by arrows marked in pink in the N-terminus, green in the NAC region, and purple in the C-terminus. The number of signals that were broadened are presented in a pie chart. PSY = psychosine; SYN = α-synuclein.

Figure 6

Psychosine interacts with α-synuclein at the C-terminus and truncation of or binding to the C-terminus of α-synuclein releases the N-terminus of α-synuclein. Overlays of ¹H ¹⁵N-HSQC NMR spectra of full-length α-synuclein (20 µM, blue) with psychosine (600 µM, red) (A) and C-terminus truncated α-synuclein (20 µM, blue) with psychosine (600 µM, red) (B). Analysis of ¹⁵N line-width differences (ΔLW, Hz) between full-length α-synuclein and truncated α-synuclein (C) or full-length α-synuclein and α-synuclein in the presence of psychosine (10 µM) (D) shows similar line-narrowing in the N-terminus and line-broadening in the C-terminus. Black arrows mark broadening in the signals of full-length α-synuclein in presence of psychosine. Full-length and truncated α-synuclein spectra are shown in boxes separately. Line-narrowing in the N-terminus is marked in red and line-broadening in the C-terminus in purple. SYN = α-synuclein.

Figure 7

Dopamine blocks psychosine interaction with α-synuclein. Overlays of ¹H ¹⁵N-HSQC NMR spectra of full-length α-synuclein (20 µM, black) with psychosine (600 µM, blue) and with psychosine and dopamine (600 µM, magenta) at 15 hours (A) and 96 hours (B). Black boxes highlight chemical shift changes/reversal. Red boxes highlight signal loss/gain changes. Red arrows show the time-dependent effect of dopamine on signal broadening in the presence/absence of psychosine. PSY = psychosine; SYN = α-synuclein.

Figure 8

Dopamine and carbidopa prevent psychosine-induced aggregation of α-synuclein. NSC34 motor-neuron cells expressing GN-linkaSYn and a-Syn-GC plasmids were used to study α-synuclein by bimolecular fluorescence complementation (BiFC). Psychosine (1 μM) was added to NSC34 cell cultures and the formation of fluorescent aggregates of GN-linkaSYn and a-Syn-GC (A,B) over basal levels in vehicle-treated cells (C) was visualized using confocal microscopy. Psychosine facilitated the formation of medium (0.75–1.50 μm, white arrow in A) and large (>1.5 μm, red arrow in A) fluorescent aggregates. Addition of 10 μM dopamine to psychosine-medium visually prevented aggregation of BiFC fluorescent α-synuclein (E). Bar, 20 μm. Dopamine alone showed insignificant changes in aggregation (F). Similar to dopamine, addition of carbidopa in the psychosine-medium blocked the aggregation mediated by psychosine (I), with insignificant changes in control conditions (J). Sphingosine (1 μM) was used as a control lipid with non-significant differences from basal levels of aggregation. Panel G shows background fluorescence in non-transfected NSC34 cells. Confocal imaging permitted assessment of GFP+ aggregates per cell (D) and the distribution of sizes (H). Significance, *p < 0.05, ANOVA; mean ± standard error of the mean from 3 independent experiments done in triplicate.