Fig. 3: Aggregation propensity and neurotoxicity of synthetic and brain-amplified α-synuclein fibrils. | Nature Communications

Fig. 3: Aggregation propensity and neurotoxicity of synthetic and brain-amplified α-synuclein fibrils.

From: Phenotypic manifestation of α-synuclein strains derived from Parkinson’s disease and multiple system atrophy in human dopaminergic neurons

Fig. 3

Similar levels of pSYN were detected by HTRF in neuronal lysates seeded with a 0.1 μM or b 1 μM fibrils when comparing de novo fibrils to each case of PD- or MSA-amplified strains (n = 9 for panels a and b). c Confocal images of SNCATRIP dopaminergic neurons treated with de novo or brain-amplified fibrils. pSYN (upper), tyrosine hydroxylase (TH, lower). Images are representative of three independent differentiations (scale bar: 50 µm) and quantified in d, pSYN-positive aggregates induced by MSA-amplified fibrils were shorter and more speckled, whereas pSYN-positive aggregates induced by PD-amplified fibrils were neuritic and longer in length compared to ones induced by de novo-generated fibrils (n = 9). e Proteinase K digestion of cell lysates followed by immunoblotting with a mixture of anti-α-synuclein antibodies (ASyM, 4B12 and 10D2) revealed different fragments and resistance to proteolysis in PD-seeded versus MSA-seeded neuronal aggregates (immunoblot representative of n = 2). f Representative confocal images from three independent differentiations depicting nuclear fragmentation (DAPI) and axonal degeneration (TUJ1), which were especially prominent in SNCATRIP neurons treated with PD- or MSA-amplified fibrils. Healthy control (upper) and SNCATRIP (lower) lines were treated with de novo fibrils, PD- or MSA-amplified fibrils (scale bar: 30 µm). g Viability was similar across conditions at 2 weeks post seeding (n = 9). h Increased nuclear fragmentation was detected in SNCATRIP lines seeded with MSA-amplified fibrils followed by PD-amplified fibrils at 3 weeks post seeding (n = 9). Healthy control lines did not exhibit a similarly progressive phenotype (n = 9). i α-Syn monomer and homogenates (hm) from each PD and MSA cases did not cause toxicity in contrast to corresponding amplified fibrils (n = 3). NSC = non-seeded control, which means untreated neurons, Mono = neurons treated with monomeric α-Syn. Each dot corresponds to one clone differentiated once and data are mean ± s.e.m from at least n = 3 differentiations per clone. In panels d, g, h, i *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test. Source data for a, b, d, e, g, h, i are provided as a Source Data file.

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