Fig. 4: Different alpha-synuclein (αSyn) levels alter mitochondria-associated endoplasmic reticulum membrane (MAM) function.
A Phospholipid synthesis in induced pluripotent stem cell (iPSC)-derived neurons. Quantification of de novo phosphatidylserine (PS) synthesis and the ratio phosphatidylethanolamine (PE) to PS (PE/PS) levels in patient-derived neurons after 30 days of directed differentiation, after incubation with 3H-Serine [3H-Ser] for the indicated times. Data were normalized to the Control cell line, and at least 4 independent differentiations were performed. A repeated measures two-way analysis of variance (ANOVA): PS synthesis: Column (Cell line) factor (F(2,10) = 6.777; *p = 0.0138); Row (time) factor (F(2,20) = 10.92; ***p = 0.0006); PE/PS: Cell line factor (F(2,11) = 13.41; **p = 0.0011); time factor (F(2,22) = 1.180; p = 0.3260). For post-hoc analyses a Tukey’s multiple comparison test was used with single pooled variance, *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001. B Protein levels of PS synthase (PSS)1 and PSS2 at the MAM domain. Analysis of MAM fractions from iPSC-derived neurons probed for PSS1 and PSS2, with a representative image shown, adapted with permission according to CC BY 4.0 from ref. 97. Data are presented as the mean ± SD of at least 5 independent biological replicates (n) analyzed by an one-way ANOVA. The variation between the cell lines of the indicated proteins is given as: PSS1 (F(4,28) = 1.259; p = 0.3094); PSS2: (F(4,28) = 7.741; ***p = 0.0002). Tukey’s multiple comparison was used for post hoc analysis with a single pooled variance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C Assessment of PSS2 activity. Quantification of de novo PS synthesis from [14C]-PE in patient-derived neurons after 30 days of directed differentiation. After subcellular fractionation and quantification, 100 µg of protein isolated from the crude mitochondria was incubated with [14C]-PE for 45 minutes. Lipids were immediately extracted using the chloroform/methanol extraction method followed by a modified Bligh and Dyer protocol. A minimum of 3 independent differentiations per cell line were performed, and data were normalized to the αSyn-KO cell line. A repeated measure one-way ANOVA with assumed sphericity was performed with the variation between the cell lines given: (F (4,14) = 7.893; **p = 0.0015). For post-hoc analyses a Tukey’s multiple comparison test was used with single pooled variance, *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001.
