Graphene quantum dots prevent α-synucleinopathy in Parkinson's disease

Abstract

Though emerging evidence indicates that the pathogenesis of Parkinson's disease is strongly correlated to the accumulation^1,2 and transmission^3,4 of α-synuclein (α-syn) aggregates in the midbrain, no anti-aggregation agents have been successful at treating the disease in the clinic. Here, we show that graphene quantum dots (GQDs) inhibit fibrillization of α-syn and interact directly with mature fibrils, triggering their disaggregation. Moreover, GQDs can rescue neuronal death and synaptic loss, reduce Lewy body and Lewy neurite formation, ameliorate mitochondrial dysfunctions, and prevent neuron-to-neuron transmission of α-syn pathology provoked by α-syn preformed fibrils^5,6. We observe, in vivo, that GQDs penetrate the blood-brain barrier and protect against dopamine neuron loss induced by α-syn preformed fibrils, Lewy body/Lewy neurite pathology and behavioural deficits.

Fig. 1 |. Effect of GQDs on α-syn fibrillization and fibril disaggregation.

a, Schematic representation of α-syn fibrillization (5 mg ml⁻¹ α-syn monomers) and disaggregation (5 mgml⁻¹ α-syn fibrils) in the presence and absence of GQDs (5 mg ml⁻¹). b,c, Kinetics of α-syn fibrillization using aliquots of the reaction monitored by ThT fluorescence (b) and turbidity assays (c) (n = 4 biologically independent samples; two-way analysis of variance (ANOVA) with a post hoc Bonferroni test, ***P<0.001; error bars are standard deviation, s.d.). Mean values and P values are provided in Supplementary Table 1. d, TEM images of α-syn after fibrillization in the absence (left) and presence (right) of GQDs. e,f, Kinetics of preformed α-syn fibrils after incubation with GQDs using aliquots of the reaction monitored by ThT fluorescence (e) and turbidity assays at various time points (f) (n = 4 biologically independent samples; two-way ANOVA with a post hoc Bonferroni test, ***P<0.001; error bars are s.d.). Mean values and P values are provided in Supplementary Table 1. g, Quantifications of the end-to-end length and number of α-syn fibrils per μm2. Mean values of end-to-end length are 937.84, 245.52, 123.13, 66.27 and 51.02 nm at 0, 6, 12, 24 and 72 h (n = 50 fibrils at each time point; one-way ANOVA with a post hoc Bonferroni test, P< 0.0001 at 6, 12, 24 and 72 h; error bars are s.d.). Mean values of number are 15.53, 37.01, 63.83, 97.52 and 44.38 per μm² at 0, 6, 12, 24 and 72 h (n = 6, biologically independent samples; one-way ANOVA with a post hoc Bonferroni test, P = 0.0483, P<0.0001, P<0.0001 and P = 0.0050 at 6, 12, 24 and 72 h, respectively; error bars are s.d.). h, Amount of remaining α-syn fibrils (as % of number at 0 h), determined by multiplying the end-to-end length and number of α-syn fibrils at the same time points (0, 6, 12, 24 and 72 h) in the presence of GQDs. Mean values are 100.00, 47.33, 42.00, 36.67 and 13.83 at 0, 6, 12, 24 and 72 h (n = 6, biologically independent samples; one-way ANOVA with a post hoc Bonferroni test, error bars are s.d.). i, TEM images of preformed α-syn fibrils at various time points (6 and 12 h, 1, 3 and 7 days) in the absence (top) and presence (bottom) of GQDs. j, Representative image of α-syn fibrils dot-blot assay at various time points (0 and 12 h, 1, 3 and 7days) with α-syn filament specific antibody. These experiments were independently repeated three times with similar results. k, BN–PAGE analysis of α-syn, prepared with aliquots of reaction run at various time points (0, 3, 6 and 12 h, 1, 3 and 7days). These experiments were independently repeated three times with similar results.

Fig. 2 |. Detailed analysis of the interaction between GQDs and mature α-syn fibrils during the dissociation process.

a, TEM images for binding between biotinylated GQDs and α-syn fibrils with low and high magnifications. Arrowheads indicate biotinylated GQDs enhanced with ultra-small gold-streptavidin nanoparticles. b, Quantification of the average width of α-syn fibrils during the disaggregation process after 1 h of incubation (mean±s.d.): 14.74±0.70 nm and 17.42± 0.83 nm for fibrils and fibrils + GQDs-biotin (n = 20 for each group, two-tailed Student’s t-test). c, NMR chemical shift difference obtained from full ¹H–¹⁵N HSQC spectra. The decreased intensity ratio of NMR chemical shifts is presented for each residue after binding with GQDs. d, Time course simulation dynamics of the interaction between GQDs and mature α-syn fibrils, and 65 ns snapshot image (bottom right) of the interaction between GQDs and mature α-syn fibrils with designated sidechains attributed for the major binding force. e, Time-dependent plots for r.m.s.d. of atomic positions, SASA for the α-syn fibril only group and α-syn fibril and GQDs group, total potential energy (ΔU_tot), electrostatic energy (ΔE_elec) and van der Waals energy (ΔE_van) for α-syn fibril and GQDs group, respectively (from the top). f, Time-dependent secondary structure plot calculated by the DSSP algorithm. g, CD spectra of α-syn monomers and α-syn fibrils without and with GQDs after 7 days of incubation. h, Fractional secondary structure contents ratio of α-syn fibrils and α-syn fibrils disaggregated by GQDs calculated using the algorithm CONTIN/LL (n = 5 biologically independent samples). Data presented as mean± s.d. (fibrils versus fibrils + GQDs (% of content); coil: 20.13± 5.74 versus 24.58±3.64; β-turn: 22.39± 4.62 versus 25.89±1.34; β-sheet: 53.30± 3.53 versus 29.76±3.35; α-helix: 4.17± 1.25 versus 19.74± 1.52).

Fig. 3 |. Effect of GQDs on α-syn PFF-induced neuronal death, pathology and transmission in vitro.

a–c, Neuronal death assessed by TUNEL (a), alamarBlue (b) and LDH assays (c) treated with α-syn PFFs (1 μg ml⁻¹) in the absence and presence of GQDs (1 μg ml⁻¹) in 10 DIV mouse cortical neurons for 7 days. Mean values of TUNEL (% of TUNEL+ cells) are 18.94, 15.86, 61.39 and 30.45 for PBS, GQDs, PFFs and PFFs + GQDs; mean values of alamarBlue (% of control) are 100.00, 99.16, 49.33 and 78.86 for PBS, GQDs, PFFs and PFFs + GQDs; mean values of LDH assay (% of control) are 100.00, 95.43, 154.58 and 113.33 for PBS, GQDs, PFFs and PFFs + GQDs (n = 6 biologically independent samples; two-way ANOVA with post hoc Bonferroni test; NS, not significant; error bars are s.d.). d, Representative immunoblot levels with p-α-syn antibody. e, Quantifications of the SDS-insoluble fraction normalized to the levels of β-actin. Mean values are 1.00, 1.49, 16.35 and 6.86 for PBS, GQDs, PFFs and PFFs + GQDs (n = 6 biologically independent samples; two-way ANOVA with post hoc Bonferroni test; NS, not significant; error bars are s.d.). f, Representative p-α-syn immunostaining micrographs with p-α-syn antibody. g, Quantifications of p-α-syn immunofluorescence intensities normalized to PBS control. Mean values are 1.00, 0.99, 13.62 and 4.69 for PBS, GQDs, PFFs and PFFs + GQDs (n = 6 biologically independent samples; two-way ANOVA with post hoc Bonferroni test; NS, not significant; error bars are s.d.). h, Schematic representation of the microfluidic device for the transmission of pathologic α-syn, composed of three connected chambers. i, Representative images of p-α-syn immunostained neurons in the microfluidic device 14days post α-syn PFFs addition. j, Quantifications of p-α-syn immunofluorescence intensities. Areas occupied by p-α-syn were measured in each chamber. Mean values of chamber 1 are 15.67, 3.12 and 4.52 for PFFs, PFFs + GQDs (C1) and PFFs + GQDs (C2); mean values of chamber 2 are 8.52, 2.56 and 3.90 for PFFs, PFFs + GQDs (C1) and PFFs + GQDs (C2); mean values of chamber 3 are 6.04, 1.72 and 1.67 for PFFs, PFFs + GQDs (C1) and PFFs + GQDs (C2) (n = 6 biologically independent samples; two-way ANOVA with post hoc Bonferroni test; NS, not significant; error bars are s.d.).

Fig. 4 |. Effect of GQDs on α-syn-induced pathologies in vivo.

a, Schematic illustration of injection coordinates of α-syn PFFs (5μg) for stereotaxic intrastriatal injection in C57BL/6 mice. As a treatment, 50 μg of GQDs or PBS were i.p. injected biweekly for 6 months. AP, anteroposterior; ML, mediolateral; DV, dorsoventral; Ctx, cortex; STR, striatum; IHC, immunohistochemistry. b, Representative TH immunohistochemistry images in the substantia nigra of α-syn PFF-injected hemisphere in the absence (top) and presence (bottom) of GQDs. c, Stereological counting of the number of TH- and Nissl-positive neurons in the substantia nigra via unbiased stereological analysis after 6 months of α-syn PFF injection with and without GQDs injection. Mean values are 5,061, 5,096, 3,155 and 4,080 Nissl-positive neurons; 4,039, 4,068, 2,327 and 3,221 TH-positive neurons (n = 6 biologically independent animals; two-way ANOVA with a post hoc Bonferroni test; NS, not significant; error bars are s.d.). d, Representative TH immunohistochemistry images in the striatum of α-syn PFF-injected hemisphere. e, Quantifications of TH-immunopositive fibre densities in the striatum. Mean values for relative optical densities of TH+ signals are 1.00, 1.03, 0.42 and 0.90 for PBS + PBS, PBS + GQDs, PFFs+ PBS and PFFs + GQDs (n = 6 biologically independent animals; two-way ANOVA with a post hoc Bonferroni test; NS, not significant; error bars are s.d.). f, Assessments of the behavioural deficits measured by the use of forepaws in the cylinder test. Mean values (% of ticks) are 51.00, 51.67, 29.83 and 44.17 for PBS+ PBS, PBS + GQDs, PFFs + PBS, and PFFs + GQDs (n = 6 biologically independent animals; two-way ANOVA with a post hoc Bonferroni test; NS, not significant; error bars are s.d.). g, Assessments of the behavioural deficits measured by the ability to grasp and descend from a pole. Mean values are 10.25, 11.38, 22.73 and 14.43 s for PBS + PBS, PBS + GQDs, PFFs + PBS and PFFs + GQDs (n = 6 biologically independent animals; two-way ANOVA with a post hoc Bonferroni test; NS, not significant; error bars are s.d.). h, Representative p-α-syn immunostaining images in the striatum (STR) and substantia nigra (SN) of α-syn PFF-injected hemisphere. i, Quantifications of p-α-syn immunoreactive neurons in the striatum and substantia nigra. Mean values (number of inclusions) are 43.33 and 12.50 for PFFs+ PBS and PFFs + GQDs of striatum (STR); 17.67 and 8.00 for PFFs+ PBS and PFFs + GQDs of substantia nigra (n = 6 biologically independent animals; two-tailed Student’s t-test; error bars are s.d.). j, Distribution of LB/LN-like pathology in the CNS of α-syn PFF-injected hemisphere (p-α-syn positive neurons, red dots; p-α-syn positive neurites, red lines).