Journal: Cellular and Molecular Life Sciences
Article Title: The role of tyrosine hydroxylase–dopamine pathway in Parkinson’s disease pathogenesis
doi: 10.1007/s00018-022-04574-x
Figure Lengend Snippet: LRRK2 G2019S mutation deregulates the TH–DA pathway, leading to DA neuron vulnerability. A – G LRRK2 enhances TH expression in PC12 cells in an LRRK2 kinase activity-dependent manner. A , C and F Representative Western blot data of up-regulated TH levels in PC12 cells under transient overexpression of WT, mutant G2019S or 3KD LRRK2. A FL LRRK2, C LRRK2 RCK domains and F LRRK2 kinase domain. B , D and G , Densitometric analysis of TH protein bands of Western blot gels in A , C and F , respectively. *, at least P < 0.01, compared with arbitrary value of TH protein bands of GFP-transfected control cells. E Quantitative real-time RT-PCR analysis of TH expression in PC12 cells under overexpression of WT, mutant G2019S or 3KD LRRK2 RCK domains. * P < 0.01, compared with relative TH expression level of GFP-transfected control cells. H Expression of WT and mutant G2019S LRRK2 RCK domains impair PC12 cell viability, which can be partially rescued by 500 µM GSH treatment. *, at least P < 0.01, compared with cell viability of the respective WT or G2019S RCK domains transfected PC12 cells in the absence of GSH treatment. (I–P) Stable transfection of LRRK2 up-regulates TH and DA levels, sensitizing human dopaminergic SH-SY5Y cells to stress challenges. I Representative Western blot TH protein bands in SH-SY5Y cells stably transfected with empty vector, WT or mutant G2019S FL LRRK2. J Densitometric analysis of Western blot TH protein bands in I . * P < 0.001, compared with arbitrary value of TH bands of empty vector transfected control SH-SY5Y cells. K and L Mutant G2019S LRRK2-induced DA content increase in stably transfected SH-SY5Y cells can be abrogated by treatment with 2 mM α-MT, a specific TH inhibitor. K Quantitative analysis of DA content by HPLC, *, at least P < 0.05, compared with DA content of the respective cells in the absence of α-MT. L Representative HPLC chromatography of DA peaks. M Quantitative real-time RT-PCR analysis of TH and LRRK2 expression level in stably transfected SH-SY5Y cells. *, at least P < 0.01, compared with TH and LRRK2 expression levels of empty vector transfected SH-SY5Y cells. N Stable transfection of mutant G2019S LRRK2 sensitizes SH-SY5Y cells to H 2 O 2 challenge. *, at least P < 0.05, compared with cell viability of empty vector transfected SH-SY5Y cells treated with the respective dosage of H 2 O 2 . ( O and P ) 2 mM α-MT treatment alleviates LRRK2-induced SH-SY5Y cell viability impairment under 100 µM Fe 2+ ( O ) or 100 µM Fe 3+ ( P ) challenges. *, at least P < 0.05, compared with cell death rate of the respective stable cells without iron species challenges. #, at least P < 0.05, compared with cell death rate of respective SH-SY5Y cells under the respective iron species challenges
Article Snippet: LRRK2 R1441G TG mice generated from BAC containing the entire human mutant R1441G LRRK2 were also provided by the Jackson Laboratory (#009604) [ ].
Techniques: Mutagenesis, Expressing, Activity Assay, Western Blot, Over Expression, Transfection, Control, Quantitative RT-PCR, Stable Transfection, Plasmid Preparation, Chromatography
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![Comparison of [18F]FDOPA images from four groups of mice, showing significantly decreased uptake of the ligand among Tg mice: A coregistered coronal [ 18 F]FDOPA images of the non-Tg mouse striatum; B coregistered coronal [ 18 F]FDOPA images of the hLRRK2 mouse striatum; C coregistered coronal [ 18 F]FDOPA images of the hLRRK2 R1441G HEM mouse striatum; D coregistered coronal [ 18 F]FDOPA images of the hLRRK2 R1441G HOM mouse striatum; E average [ 18 F]FDOPA uptake in the region of interest (striatum) in various groups. The uptake values are A , B , C , and D for Group 1,2,3, and 4, respectively Data are presented as the mean ± SD (n = 5–6). *Significant effect, p < 0.05; **Significant effect, p < 0.01; ***Significant effect, p < 0.005; ****Significant effect, p < 0.001](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5908/pmc09375908/pmc09375908__12929_2022_844_Fig4_HTML.jpg)
