Fig. 4. Overexpression of α-synuclein increases intracellular and extracellular dopamine levels with concurrent increased tyrosine hydroxylase expression.
a Schematic and representative baseline-subtracted images of GRABDA2M-HEK cells exposed to increasing concentration of dopamine. Scale bar = 20 μm. b A standard curve of GRABDA2M-HEK cells against known extracellular dopamine concentrations (R2 = 0.98). c Constitutive GRABDA2M-HEK cell fluorescence signal in the absence of dopamine neurons (in culture). Scale bar = 10 μm. d Schematic of GRABDA2M-HEK cells seeded into dopaminergic cultures. In the presence of dopamine, GRABDA2M-HEK cells rapidly increase in fluorescence intensity. e Baseline fluorescence levels denote unstimulated and spontaneous dopamine release from the neurons. The average ratio of the fluorescence signal of the cells adjacent to neuron soma and neuronal processes to the average ratio of GRABDA2M-HEK cells (only) were calculated (relative fluorescence = (FGRABDA2M-HEK cells grown with neurons − Fc)/Fc). GRABDA2M-HEK cells cocultured with naive and α-syn-overexpressing neurons. Scale bar = 50 μm. f GRABDA2M-HEK cells cocultured with α-syn-overexpressing neurons show higher basal fluorescence, indicating higher baseline dopamine release (relative fluorescence) compared to naive neurons (n = 10 from three independent replicates; the data are means ± SEM, two-tailed t test, p = 0.0013). g, h HPLC analysis complements the GRABDA2M-HEK results. Extracellular milieu (g) and cell lysate intracellular milieu (h) revealed increased intracellular and extracellular dopamine levels in α-syn-overexpressing neurons compared to naive neurons (n = 8 each, from eight independent replicates, two-tailed t test; intracellular: p = 0.0071; extracellular: p = 0.0139). i Schematic diagram of quantitative ELISA experimental design for TH in dopaminergic neurons. j Standard curve for TH sandwich ELISA shows average absorbance values for each purified TH protein concentration from multiple consecutive experiments (R2 = 0.99). k TH protein levels were detected and quantified in positive control groups, PC12 cells, whereas no protein was detected in the negative control group, HEK293 cells. l α-Syn-overexpressing neurons exhibited increased levels of TH compared to naive (n = 8–10, two-tailed t test, p = 0.0289). These experiments were performed through a double-blinded experimental design. *p < 0.05, **p < 0.01.