Fig. 1: Characterisation of α-synuclein seeded aggregation in iPSC-derived dopaminergic neurons. | Nature Communications

Fig. 1: Characterisation of α-synuclein seeded aggregation in iPSC-derived dopaminergic neurons.

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

Fig. 1

a Addition of exogenous α-Syn fibrils (+Fib) seeded the aggregation of endogenous α-Syn as evident by pSYN immunoreactivity in red. Images are representative of three independent differentiations (scale bar: 10 µm). b pSYN-positive aggregates were localised in axons (neuritic) or soma (speckled or Inclusion body) and co-localised with p62. Images are representative of three independent differentiations (scale bar: 10 µm). c RIPA-insoluble fractions from seeded neuronal lysates were immunoreactive for pSYN by immunoblotting (n = 3). d Dopaminergic neurons harbouring either the missense A53T mutation (SNCAA53T) or SNCA gene triplication (SNCATRIP) had increased levels of pSYN compared to healthy controls 2 weeks post seeding, with SNCATRIP neurons exhibiting the highest burden of seeded aggregates as quantified by HTRF (n = 9 for HC, n = 9 for SNCATRIP, n = 6 for SNCAA53T). e A single exposure to fibrils on DIV45 at an increasing dose (baseline, 0.01, 0.1, 1 µM) induced a dose and time-dependent increase in pSYN signal as quantified by HTRF (n = 9 for SNCATRIP and n = 5 for HC). f Endogenous α-Syn expression in neurons increased over time in culture. Increased baseline α-Syn expression by ~2.5-fold in SNCATRIP neurons compared to controls as assessed by immunoblotting (n = 3). g Upregulation of SNCA mRNA was triggered by fibril treatment 2 weeks post seeding at DIV45. Induction of SNCA mRNA was more pronounced in SNCATRIP compared to controls (n = 9). h Ribbons exhibited higher seeding propensity than fibrils 4 weeks post seeding as evident by pSYN levels, quantified by HTRF (n = 9). Each dot corresponds to one clone differentiated once and data are mean ± s.e.m from at least n = 3 differentiations per clone. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test (c, d, h), two-way ANOVA followed by Tukey’s multiple comparison test (e), two-sided unpaired Student’s t test (f, g). Source data for c, d, e, f, g, h are provided as a Source Data file.

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