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Dopamine induces soluble α-synuclein oligomers and nigrostriatal degeneration

Nature Neuroscience volume 20, pages 1560–1568 (2017)Cite this article

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Abstract

Parkinson's disease (PD) is defined by the loss of dopaminergic neurons in the substantia nigra and the formation of Lewy body inclusions containing aggregated α-synuclein. Efforts to explain dopamine neuron vulnerability are hindered by the lack of dopaminergic cell death in α-synuclein transgenic mice. To address this, we manipulated both dopamine levels and α-synuclein expression. Nigrally targeted expression of mutant tyrosine hydroxylase with enhanced catalytic activity increased dopamine levels without damaging neurons in non-transgenic mice. In contrast, raising dopamine levels in mice expressing human A53T mutant α-synuclein induced progressive nigrostriatal degeneration and reduced locomotion. Dopamine elevation in A53T mice increased levels of potentially toxic α-synuclein oligomers, resulting in conformationally and functionally modified species. Moreover, in genetically tractable Caenorhabditis elegans models, expression of α-synuclein mutated at the site of interaction with dopamine prevented dopamine-induced toxicity. These data suggest that a unique mechanism links two cardinal features of PD: dopaminergic cell death and α-synuclein aggregation.

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Figure 1: TH-RREE lentiviral vector increases dopamine levels and causes hyperactivity in NonTg mice.
Figure 2: Dopamine-induced neurodegeneration of the SN is dependent on α-synuclein.
Figure 3: Dopaminergic neurodegeneration in A53T TH-RREE mice is progressive and ultimately leads to locomotor deficit.
Figure 4: Dopamine induces conformationally distinct α-synuclein oligomers in the mouse brain.
Figure 5: C. elegans expressing A53T α-synuclein lacking the site of interaction with dopamine are resistant to dopamine neurotoxicity.

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Acknowledgements

We thank T. Clarke and T. Pierson from the Wolfe lab for technical assistance with animals and for helpful advice on vector production. We thank H. Bennett from the Kalb lab for initial work on the dopamine neuron degeneration assay in C. elegans and input on worm breeding. We thank L. Spruce, H. Fazelinia and S. Seeholzer from the Children's Hospital of Philadelphia Proteomic Core facility for mass spectrometry. We thank V. Lee (University of Pennsylvania) and G. Miller (Emory University) for generously providing α-synuclein and VMAT2 antibodies, respectively, and S. Przedborski (Columbia University) for the use of stereology equipment and helpful feedback on the manuscript. Finally, we thank R. Lightfoot and members of the Ischiropoulos lab for productive discussions and technical support. This work was supported by grants from the US National Institutes of Health: AG013966 (H.I.), NS038690 (J.H.W.) and NS087077 and NS052325 (R.G.K.). D.E.M. was supported by the US National Institutes of Health Ruth L. Kirschstein National Research Service Award Individual Predoctoral Fellowship NS087779. The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

  1. Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Danielle E Mor, Shachee Doshi, Preetika Gupta, Robert G Kalb, John H Wolfe & Harry Ischiropoulos

  2. AC Immune SA, École Polytechnique fédérale de Lausanne Innovation Park, Lausanne, Switzerland

    Elpida Tsika

  3. Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA

    Joseph R Mazzulli

  4. Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, USA

    Neal S Gould, Robert G Kalb, John H Wolfe & Harry Ischiropoulos

  5. Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama, USA

    Hanna Kim, Kim A Caldwell & Guy A Caldwell

  6. Pharmacology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Malcolm J Daniels & Harry Ischiropoulos

  7. State University of New York Downstate College of Medicine, Brooklyn, New York, USA

    Jennifer L Grossman

  8. College of Arts and Sciences, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Victor X Tan

  9. W.F. Goodman Center for Comparative Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    John H Wolfe

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  1. Danielle E Mor
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Contributions

D.E.M., E.T., J.R.M., R.G.K. and H.I. conceived and designed the experiments. D.E.M., E.T., H.K., N.S.G., M.J.D., S.D., P.G., J.L.G. and V.X.T. performed the experiments and analyzed the data. D.E.M. wrote the paper, with important contributions from J.R.M., E.T., J.H.W., R.G.K., K.A.C., G.A.C. and H.I.

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Integrated supplementary information

Supplementary Figure 1 Increased TH expression and steady-state catecholamine concentrations in SH-SY5Y cells transduced with TH-RREE vector.

(a) Schematic representation of the lentiviral vectors used in this study. TH-RREE contained the gene encoding human tyrosine hydroxylase isoform 1 (hTH-1) with mutations R37E, R38E rendering TH insensitive to feedback inhibition by dopamine. The gene was absent from the empty vector control (CtrlVect), while all other elements were retained. (b) Western blot analysis of lysates showed TH protein only in TH-RREE transduced cells. Neural specific enolase (NSE) was used as a loading control. (n = 4 independent cultures per group). (c) TH immunofluorescence confirmed expression in TH-RREE transduced cells. Scale bar, 100 μm. (n = 5 independent cultures per group). (d) Catecholamines were detected in cells treated with TH-RREE lentivirus. ND, not detected. (n = 3 independent cultures per group except n = 6 independent cultures for DA TH-RREE and n = 6 independent cultures for DOPAC TH-RREE). Box plots show median, 25th and 75th percentiles, minimum and maximum values.

Supplementary Figure 2 TH-RREE vector increases TH expression in A53T mice.

(a) TH levels were increased in the striatum in A53T TH-RREE mice compared with A53T CtrlVect at 5 mpi. GAPDH was used as a loading control. (n = 3 mice per group, P = 0.0048, t = 5.657, d.f. = 4; two-tailed unpaired Student’s t test). (b) TH staining was increased in both the SN (left panels) and striatum (right panels) of A53T TH-RREE mice at 5 mpi. Cresyl Violet (Nissl) counterstain. Scale bar, 10 μm. (n = 4 mice per group). Box plots show median, 25th and 75th percentiles, minimum and maximum values. **P < 0.01.

Supplementary Figure 3 Postsynaptic markers remain unchanged by lentiviral vector treatment.

Striatal levels of the D1 postsynaptic receptor were unaltered in TH-RREE injected A53T and NonTg mice compared with CtrlVect-injected mice at 5 mpi (a-b). The postsynaptic signaling molecule DARPP-32 was also unaffected by viral treatment (a,c). All quantification was normalized to actin loading control. (D1R, n = 3 mice per group except n = 4 mice for NonTg CtrlVect, F(3,9) = 0.2561; DARPP-32, n = 3 mice per group, F(3,10) = 0.3835; one-way ANOVA with Tukey’s correction for multiple comparisons). Box plots show median, 25th and 75th percentiles, minimum and maximum values.

Supplementary Figure 4 Loss of striatal dopamine in A53T mice cannot be accounted for by formation of dopamine-protein adducts.

(a) Triton-soluble (S) and insoluble (P) extracts of striatal tissue from mice at 2.5 or 5 mpi were analyzed by SDS-PAGE and near-infrared fluorescence (nIRF). (b) Total levels of dopamine-protein adduct (combined soluble and insoluble nIRF signal) did not change either between injection groups or within groups over time. (n = 3 mice per group, F(3,8) = 1.763; one-way ANOVA with Tukey’s correction for multiple comparisons). Box plots show median, 25th and 75th percentiles, minimum and maximum values.

Supplementary Figure 5 Analysis of inclusion pathology and insoluble α-synuclein in A53T mice.

(a) Regardless of lentiviral treatment, α-synuclein inclusion pathology, as detected by Syn505 staining, was abundant in brainstem and absent in SN of A53T mice at 5 mpi. Scale bar, 20 μm. (n = 3 mice per group). (b-d) At 5 mpi, SN from A53T mice was sequentially extracted with buffers containing 1% Triton followed by 2% SDS. Triton-insoluble/SDS-soluble fractions from A53T TH-RREE and A53T CtrlVect contained similar levels of monomeric α-synuclein as assessed by LB509 (b), Syn211 (c), and SNL-4 (d) antibodies. Vimentin (Vim) was used as a loading control, and appears for Syn211 and SNL-4 as the same blot since the same membrane is shown. (LB509, n = 3 mice per group, P = 0.2605, t = 1.310, d.f. = 4; Syn211, n = 6 mice per group except n = 4 mice for CtrlVect, P = 0.7716, t = 0.3003, d.f. = 8; SNL-4, n = 6 mice per group except n = 4 mice for CtrlVect, P = 0.7355, t = 0.3498, d.f. = 8; two-tailed unpaired Student’s t test). Box plots show median, 25th and 75th percentiles, minimum and maximum values.

Supplementary Figure 6 Further characterization of α-synuclein and dopamine derived from A53T mice.

(a-b) Triton-soluble SN from A53T TH-RREE and A53T CtrlVect mice at 5 mpi was fractionated by size exclusion chromatography, and the resulting fractions were analyzed by western blot using α-synuclein antibodies Syn211 (C-terminal) (a) and SNL-4 (N-terminal) (b). Oligomer bands that uniquely appear in 122 Å fractions from TH-RREE mice are indicated by arrows. Neural specific enolase (NSE) was used as a loading control, and appears for Syn211 and SNL-4 as the same blot since the same membrane is shown. (n = 4 mice per group). (c) The SDS-PAGE gels were scanned for near-infrared fluorescence, which revealed oxidized dopamine (arrow) only in 72-122 Å fractions from A53T TH-RREE mice. (n = 4 mice per group).

Supplementary Figure 7 Biochemical and structural characterization of α-synuclein oligomers resulting from incubation with dopamine in vitro.

(a) Incubation of recombinant wild-type human α-synuclein under standard aggregation conditions (400 μM, 37°C, 1400 rpm) resulted in Thioflavin T (ThioT)-positive fibril formation, whereas ThioT signal was abolished in the presence of equimolar dopamine (DA). rfu, relative fluorescence units. The data are presented as mean ± s.e.m. (n = 3 independent experiments per group, P-DA day 4 versus +DA day 4 = 0.0045, P-DA day 6 versus +DA day 6 = 0.003, F(1,4) = 8.901; repeated measures two-way ANOVA with Bonferroni’s correction for multiple comparisons). (b) α-Synuclein fibrils exhibited characteristic β-sheet secondary structure by circular dichroism, while α-synuclein species resulting from interaction with dopamine maintained random coil structure on day 5 of incubation. Representative traces are shown. (n = 3 independent experiments per group). (c) Sedimentation analysis and coomassie blue staining revealed that over the 6-day incubation, in the absence of dopamine, soluble α-synuclein (supernatant) was lost while insoluble α-synuclein (pellet) accumulated. In the presence of dopamine, α-synuclein was detected as multiple soluble oligomer species. Similar to ex vivo oligomers extracted from A53T TH-RREE mice, in vitro generated oligomers were immunoreactive to Syn505 (N-terminal) and LB509 (C-terminal) α-synuclein antibodies. (n = 4 independent experiments per group). (d) Immunoelectron microscopy on α-synuclein from day 4 of incubation with dopamine confirmed both Syn505 and LB509 labeling indicating that dopamine-induced oligomers generated in vitro share common epitopes and potentially similar conformations to oligomers resulting from dopamine elevation in vivo. Scale bar, 20 nm. (n = 3 independent experiments per group). **P < 0.01.

Supplementary Figure 8 Dopamine-modified oligomers are not detected in motor cortex.

A53T TH-RREE and CtrlVect cortical tissue from 5 mpi was extracted with 1% Triton X-100 and the soluble fraction was further separated by size exclusion chromatography. Pooled fractions were analyzed by Western blotting using α-synuclein antibodies LB509 (a) and Syn211 (b). No differences in α-synuclein species were detected between TH-RREE and CtrlVect mice. Neural specific enolase (NSE) was used as a loading control. (n = 3 mice per group).

Supplementary Figure 9 α-Synuclein oligomers generated in vitro in the presence of dopamine become internalized and are toxic to primary neurons.

Hippocampal neurons at 7 days in vitro were exposed to 0.5 or 1 μM α-synuclein oligomer that had been prepared in the presence of dopamine (Olig), or 1 μM monomer (Mon), 1 μM dopamine that was incubated in parallel to oligomers but without α-synuclein (DA), or an equivalent dose of PBS. (a) Two weeks post-treatment with 1 μM Olig or PBS, cells were labeled with human α-synuclein antibodies in a two-stage protocol. Cells were live-incubated with Syn204 antibody to label extracellular α-synuclein. Following fixation and permeabilization, the cells were incubated with LB509 to label total α-synuclein. Confocal imaging showed puncta that were labeled by LB509 but not Syn204, indicating that exogenous oligomers had been internalized. These puncta were localized to neurites. Scale bar, 10 μm. (n = 3 independent cultures per group). (b-c) At two weeks post-treatments, cell viability was assayed using Calcein AM and propidium iodide (PI) dyes. The number of viable cells, defined as Calcein AM positive and PI negative, was significantly reduced by exposure to 1 μM Olig but was not affected by Mon or DA controls. Treatment with 0.5 μM Olig was insufficient to induce toxicity, indicating that the effect is dependent on dose. Scale bar, 100 μm. (n = 4 independent cultures per group except n = 3 independent cultures for PBS, n = 3 independent cultures for DA, and n = 5 independent cultures for 1 μM Olig, POlig 1 μM versus PBS = 0.0285, POlig 1 μM versus DA = 0.0308, POlig 1 μM versus Mon = 0.0098, F(4,14) = 6.385; one-way ANOVA with Tukey’s correction for multiple comparisons). Box plots show median, 25th and 75th percentiles, minimum and maximum values. *P < 0.05, **P < 0.01.

Supplementary Figure 10 Mass spectrometry-label free quantification of α-synuclein expression in C. elegans strains.

Parallel reaction monitoring mass spectrometry analysis of human A53T α-synuclein. Data depicts similar signal intensity of the primary fragmentation ion from each of the four unique peptides detected in the four C. elegans strains. The four peptides represent coverage of amino acids 11-21 and 33-43 of human α-synuclein. (n = 1 run per genotype).

Supplementary Figure 11 Full-length blots corresponding to cropped blots in the main figures.

(a) Full-length blot correspondng to Fig. 1a. TH (top arrow) and GAPDH (bottom arrow) were imaged in the same channel. (b) Full-length blots correspondng to Fig. 2e. For each genotype (NonTg, A53T), three blots are shown since DAT and Actin were imaged in separate channels. The left blot shows DAT (arrow), the middle shows Actin (arrow), and the right blot shows the merge of the two channels. (c) Full-length blots correspondng to Fig. 4a. Three blots are shown since α-synuclein and NSE were imaged in separate channels. The left blot shows α-synuclein (arrow), the middle shows NSE (arrow), and the right blot shows the merge of the two channels. (d) Full-length blots correspondng to Fig. 4c. Two blots are shown since α-synuclein and NSE were imaged in separate channels. The top blot shows NSE (arrow), and the bottom blot shows the merge of the two channels. The full-length α-synuclein blot appears in Fig. 4c. * denotes non-specific bands, or bands resulting from incubation with other antibodies not pertinent to the present experiment.

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Mor, D., Tsika, E., Mazzulli, J. et al. Dopamine induces soluble α-synuclein oligomers and nigrostriatal degeneration. Nat Neurosci 20, 1560–1568 (2017). https://doi.org/10.1038/nn.4641

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