Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons from Familial Parkinson's Disease Patients Display α-Synuclein Pathology and Abnormal Mitochondrial Morphology

Abstract

Accumulation of α-synuclein (α-syn) into Lewy bodies (LBs) and mitochondrial abnormalities are the two cardinal pathobiological features of Parkinson's disease (PD), which are associated with the loss of dopaminergic neurons. Although α-syn accumulates in many different cellular and mouse models, these models generally lack LB features. Here, we generated midbrain dopaminergic (mDA) neuronal cultures from induced pluripotent stem cells (iPSCs) derived from familial PD (fPD) patients and healthy controls. We show that mDA neuronal cultures from fPD patients with A53T mutation and α-syn gene (SNCA) triplication display pathological α-syn deposits, which spatially and morphologically resemble LBs. Importantly, we did not find any apparent accumulation of pathological α-syn in mDA neuronal culture derived from a healthy donor. Furthermore, we show that there are morphological abnormalities in the mitochondrial network in mDA neuronal cultures from fPD patients. Consequently, these cells were more susceptible to mitochondrial damage compared with healthy donor-derived mDA neuronal cultures. Our results indicate that the iPSC-derived mDA neuronal culture platform can be used to investigate the spatiotemporal appearance of LBs, as well as their composition, architecture, and relationship with mitochondrial abnormalities.

Keywords: Lewy bodies; Parkinson’s disease; dopaminergic neurons; iPSC; mitochondria; α-synuclein aggregates.

Figure 1

Differentiation of iPSCs to mDA neurons. (A) Experimental paradigm for human iPSC differentiation toward mDA neurons. (B) Morphology of healthy and PD patients’ cell lines at different stages during differentiation from iPSC into mature mDA neurons.

Figure 2

Characterization of iPSC-derived mDA neurons from a healthy subject and fPD patients. (A) We performed immunostaining with stem cell markers Oct4 and E-cadherin at day 0, midbrain neural precursor marker FOXA2 and LMX1A at day 11, and dopaminergic neuronal marker TH for healthy human and fPD patients (A53T mutation and SNCA triplication) at day 30. Next, we quantified the levels of immunostaining for (B) FOXA2, (C) LMXA1A, and (D) TH in healthy and fPD lines using image analysis. Images are representative of at least three independent differentiations. All data in the graphs are presented as mean ± standard error of the mean (SEM) of three images acquired from three different coverslips. Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. * p < 0.05; Scale bar is 100 µm.

Figure 3

α-syn accumulation in mDA neuronal cultures from fPD patients. (A) Seventy-day-old mDA neuronal cultures, from healthy and familial PD lines, were stained with an anti-α-syn antibody. α-syn accumulation around the nucleus in the A53T mutation and SNCA triplication line is indicated by arrows, whereas the homogenous distribution of native α-syn immunostaining in the healthy line is indicated by the arrowhead. (B) The level of α-syn immunostaining was quantified using image analysis. Images are representative of at least three independent differentiations. All data in (B) are presented as mean ± SEM of three images acquired from three different coverslips. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison post hoc test. * p < 0.05. The scale bar is 10 µm.

Figure 4

Accumulation of phosphorylated α-syn in mDA neuronal culture from fPD patient. (A) Seventy-day-old DA neuronal cultures from healthy and fPD lines were immunostained with an antibody against phosphorylated α-syn at serine 129 residue, a marker of LBs. The punctated accumulation of pathological α-syn next to the nucleus, resembling LBs, is indicated by an arrow, and diffuse pS129 immunostaining, resembling the early stage of LBs, is indicated by an arrowhead. (B) Levels of pS129 immunostaining were quantified using image analysis. Images are representative of at least three independent differentiations. All data in (B) are presented as mean ± SEM of three images acquired from three different coverslips. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison post hoc test. * p < 0.05. The scale bar is 10 µm.

Figure 5

fPD-derived mDA neuronal cultures are more vulnerable to mitochondrial damage. (A) In order to assess abnormalities in mitochondrial morphology and its association with α-syn accumulation, we immunostained mDA neuronal cultures derived from healthy and fPD lines with an antibody against TOM20 and α-syn. mDA neuronal culture from a healthy subject presents a tubular mitochondrial network (arrow). In contrast, TOM20 staining in mDA neuronal cultures from fPD patients with A53T mutation and SNCA triplication appeared clumped (arrowhead), particularly in the cells with α-syn accumulations. (B) Abnormal mitochondrial morphology was quantified using image analysis. (C) mDA neuronal cultures from healthy and fPD lines were treated with different concentrations of CCCP, and cell viability was assessed using the MTT assay. All data in (B) are presented as mean ± SEM of three images acquired from three different coverslips. Data in figure (B) were analyzed by one-way ANOVA followed by Tukey’s multiple comparison post hoc test. * p < 0.05. All data in (C) are presented as mean ± SEM of four wells. Data in figure (C)were analyzed by two-way ANOVA followed by Bonferroni post hoc test. ## (Healthy line vs. SNCA triplication line at 0 µM CCCP) p < 0.01; ### (Healthy line vs. SNCA triplication line at 10 µM CCCP) p < 0.001; $$ (Healthy line vs. SNCA triplication line at 100 µM CCCP) p < 0.01; *** (Healthy line vs. A53T mutation line) p < 0.001; $ (A53T mutation line vs. SNCA triplication line at 10 µM CCCP) p < 0.05.The scale bar is 10 µm.