In vivo demonstration that alpha-synuclein oligomers are toxic

Abstract

The aggregation of proteins into oligomers and amyloid fibrils is characteristic of several neurodegenerative diseases, including Parkinson disease (PD). In PD, the process of aggregation of α-synuclein (α-syn) from monomers, via oligomeric intermediates, into amyloid fibrils is considered the disease-causative toxic mechanism. We developed α-syn mutants that promote oligomer or fibril formation and tested the toxicity of these mutants by using a rat lentivirus system to investigate loss of dopaminergic neurons in the substantia nigra. The most severe dopaminergic loss in the substantia nigra is observed in animals with the α-syn variants that form oligomers (i.e., E57K and E35K), whereas the α-syn variants that form fibrils very quickly are less toxic. We show that α-syn oligomers are toxic in vivo and that α-syn oligomers might interact with and potentially disrupt membranes.

Fig. 1.

In vitro aggregation of hWT α-syn and α-syn variants. (A) EM of aged α-syn and α-syn variants purified without the streptomycin sulfate-precipitation step (protocol B). (Scale bars: 500 nm.) Insets: Ring-forming entities are shown. (Scale bar: 100 nm.) (B) Time-resolved SEC of α-syn variants: 20 mg/mL α-syn variants were solubilized and their size exclusion profiles were measured immediately (day 0). Subsequently, after days 5 and 10, the samples of the corresponding monomer peak were again put on the SEC column, revealing that E57K and E35K had the capacity to form oligomers over time. For both α-syn variants, we show a 10-fold magnification of the oligomer peak at day 5. The decrease of the monomer peak in E35K and E57K is because they aggregate into amyloids. For the other variants and hWT α-syn, no oligomer peak was observed. (C) EM image of the oligomeric species from the experiment shown in B. (Scale bars: 500 nm.)

Fig. 2.

Toxicity of hWT α-syn and α-syn variants in the SN of lentivirus-injected rats. (A) Injection paradigm: rats unilaterally injected with the different lentiviral constructs in the SN. (B) Histograms representing the decrease in numbers of TH-immunoreactive (TH-IR) neurons in the SN relative to the contralateral side, as evaluated by stereology. The nigral dopaminergic neurons were labeled with TH (C and E). Note that the loss of TH-expressing cells caused by α-syn variant expression was more pronounced close to the injection site (C and E). (B) Histogram representing the percentage of TH-positive neurons compared with the noninjected side. The artificial mutants E35K and E57K led to a significant decrease in TH-positive cells (**P < 0.001 vs. GFP and *P < 0.05 vs. hWT α-syn in B and C). In contrast, no significant loss of TH expression was observed with the lentivirus encoding for α-syn(30–110). (Values refer to means ± SD; n = 4 animals per group). (Scale bars: 250 μm in C and E.) (D) Overexpression of the familial variants A30P and E46K significantly decreased the number of TH-immunoreactive neurons. E35K and E57K were significantly decreased compared with A53T (*P < 0.001). (E) Representative brain slices showing the α-syn variants A30P, E46K, A53T, and hWT α-syn. (F–M) Examples of TH/α-syn double-positive cells shown in the hWT α-syn (F–I) and E57K group (J–M) 1 wk after lentiviral injection. Note the different TH morphology (green staining in F and H) in the hWT α-syn group, TH-positive cells with extended processes are visible (F and H: merged images; TH staining, green; α-syn staining, red; DAPI, blue). (G and I) α-Syn staining in red. In the E57K group, only the cytoplasm of TH-positive cells is visible, but their processes are rare. Some α-syn–positive cells colabel with TH. (J, K, L, and M) In addition, a difference in the morphology of the dendrites of α-syn–positive neurons (TH-negative) was noticed, with larger clumping of the dendrites in the E57K group. (Scale bars in F–M: 20 μm.)

Fig. 3.

Immunoblot analysis of aggregated α-syn from recombinant protein and lentivirus-injected brains. Representative Western blots (A) depict the insoluble fraction of microdissected SN for the different groups indicated after ultracentrifugation. Protein samples (20 μg) from two animals per group were run on denaturing polyacrylamide gels along with the indicated amounts (in ng) of purified recombinant hWT α-syn (protocol C). Blots were probed with an anti–α-syn antibody (Upper) and an anti-actin antibody (Lower) as loading control. The position of protein size markers (in kDa) is shown (Left). Note that, in the insoluble fraction, extensive oligomeric bands are present in the E57K mutant (A), whereas mostly monomers are present in the soluble fraction. Quantitative ECL analysis with a Versadoc imaging system that allows acquisition of data within a dynamical linear range (B) revealed a significant increase in the number of trimers in the insoluble fraction of α-syn (B). The ratio is calculated based on the actin loading and the immunoreactivity of the recombinant protein (h-WT α-syn). (C) Structure–toxicity relationship of α-syn: correlation between percent decrease in TH-positive cells in the SN upon lentiviral injection of α-syn variants (x axis) and relative number of in vivo-derived trimers detected by Western blot (y axis). Interestingly, a comparison between the 1-wk (Fig. 3A) and 3-wk time points (Fig. S7D) indicated that the size of the SDS-stable oligomers appeared to be time-sensitive.