Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

Abstract

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction.

Figure 1

SNCA_Tri line overexpresses aSyn after differentiation. (a) aSyn immunofluorescence during NiPSCs differentiation (DNA counterstaining in blue). (b) Quantification of aSyn expression by total aSyn brightness (mean background fluorescence subtracted). The 30* time point corresponds only to cells with upregulated aSyn expression selected by image segmentation. All reported values are normalized to the number of analyzed cells. (c) SNCA mRNA abundance quantified by real-time PCR showing an increased transcription with neuronal differentiation and higher aSyn expression in the triplication line after DA2 differentiation (n=3). (d) Representative immunoblot showing greater abundance of the 15 kDa monomeric aSyn in SNCA_Tri NiPSCs and differentiated cells compared with control 1. (e) aSyn quantification analysis from immunoblots (n=4). Statistical differences were determined by P-values of *P<0.05, **P<0.01 and ***P<0.001

Figure 2

SNCA triplication impairs neuronal stem cell differentiation. (a) aSyn concentration determined by ELISA in protein extracts from NiPSCs. (b) Expression of tyrosine hydroxylase (TH) determined by immunoblot after DA2 differentiation. (c) TH immunofluorescence (green) in differentiated DAn neurons. DNA counterstained with DRAQ5 (blue); Tuj1 is represented in red (representative images from control 2, SNCA_Tri-C1-Scr, SNCA_Tri-C2 and SNCA_Tri-C2_KD lines are given in Supplementary Figure S4). (d) Abundance of TH⁺ neurons expressed as a percentage of the total number of cells. Neurons were obtained from three independent differentiations and imaged at a magnification of × 20. More than 15 000 cells were analyzed in each cell line. (e) Abundance of GABA⁺ neurons expressed as a percentage of the total number of cells. Results were acquired and analyzed as described for TH⁺ cells. (f) Immunoblot showing aSyn and TH expression in LUHMES cells flow-sorted for expression of aSyn-IRES-GFP (aSyn o/e) or GFP only (GFP Control). (g) TH quantification analysis of LUHMES immunoblots (n=3). Statistical differences were determined by P-values of *P<0.05 and ***P<0.001

Figure 3

Electrophysiological characterization of differentiated NiPSCs. (a) Examples of three distinctive changes in membrane potential elicited by current injection. AP-firing cells were distinguished from Spikelet-firing cells by two criteria: (i) the slower kinetics of their APs measured under current clamp and (ii) the larger peak amplitudes of their I_Na(V) measured under voltage clamp. Maximum rates of rise of APs, which were obtained from the differentiated membrane potential, were generally ≥5 mV/ms for AP-firing but lower for Spikelet-firing cells. Peak amplitudes of I_Na(V) were generally ≥200 pA in AP-firing but lower in Spikelet-firing cells. In passive cells I_Na(V) was undetectable. (b) Relative fraction of AP-firing, Spikelet-firing and Passive cells. (c) Characterization of I_Na(V) and I_K(V) in control, SNCA_Tri and SNCA_Tri_KD cells. The mean I–V relationships of I_Na(V) and I_K(V) are shown in the left panels. Representative recordings are shown in the right panels for control and triplication lines. See text for details

Figure 4

SNCA triplication reduces neurite outgrowth. (a) Neurite morphology of dopaminergic neurons from control 1, SNCA_Tri-C1, SNCA_Tri-C1_KD, SNCA_Tri_C2, and SNCA_Tri-C2_KD differentiated NiPSCs, identified by TH immunocytochemistry. (b) High magnification images of neurite morphology in the patient and control lines: MAP2 (green) and Lmx1A (red) (c–f) Quantification of dopaminergic neurite morphology per cell in terms total neurite length (c), mean length of the longest neurite (d), number of neurites (e) and Schoenen Ramification Index (f). The data are shown in scatter box plots representing the median and interquartile range of each group and explicitly explained in the Results. Statistical differences were determined by P-values of *P<0.05, **P<0.01 and ***P<0.001

Figure 5

SNCA triplication increases macroautophagy in differentiated neurons. NiPSCs and differentiated neurons pretreated with vehicle or chloroquine (5 μM) for 18 h prior to fixation and immunofluorescence analysis. (a) Macroautophagy flux determined by LC3 immunostaining (green) in the presence (+) or absence (−) of chloroquine. DNA was counterstained with DRAQ5 (blue). (b) Quantification of the intraneuronal LC3+ puncta per cell (>50 cells per line analyzed in the NiPSCs stage and >100 cells in the DA2 stage). Blue arrows designate the differences in LC3+ autophagosomes between the cells pretreated with chloroquine and vehicle, and are used as an estimation of the macroautophagic flux. Statistical differences were determined by P-values of *P<0.05, **P<0.01 and ***P<0.001