Introduction 20 30 14 15 27 2 21 34 17 19 25 28 31 36 1 18 25 31 41 3 9 26 32 33 38 40 42 3 42 26 9 10 12 7 23 In the present study, we silenced the expression of α-synuclein using vector mediated RNAi in dopaminergic MN9D cells and examined the effect of down-regulation of α-synuclein on TH and DA biosynthesis. Materials and Methods Construction of shRNA Expression Plasmids ® Escherichia coli Cell Culture 8 2 Transfection and Selection l + RNA Isolation, RT-PCR and Real-time RT-PCR + 2 2 Immunofluorescence Staining TM Cell Viability Assay 4 Western Blot Analysis 39 l l g 3 2 l l n 2 2 Statistical Analysis p Results Construction of the shRNA Expression Plasmids After synthesis, each pair of the ss oligos was annealed to generate ds oligos. The annealing efficiency was analyzed by 4% agarose gel electrophoresis. Each ds oligo annealing reactant showed a clearly detectable molecular weight band around 50 bp, as expected for the length of the designed ds oligos. The 50 bp bands were brighter than the corresponding lower molecular weight band, which represented the unannealed ss oligos in the annealing reactant. These data indicate that each pair of ss oligos annealed effectively to form ds oligos. The ds oligos were ligated with linearized pENTR™/H1/TO vectors and the positive clones were expanded and verified by PCR. Bands near 350 bp were found in all PCR reactants, indicating that the ds oligos were ligated into the pENTR/H1/TO vectors. Further analysis by sequencing confirmed that the orientation and sequence of the inserted ds oligos were correct. α-Synuclein Expression in MN9D Transfected Cells 1 1 1 1 1 - Fig. 1 a Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 b n F p p c Bar d Lane 1 Lane 2 Lane 3 − e − − n F p p  Effects of Silencing α-Synuclein Expression on Cell Viability 2 − n F p p − 2 Fig. 2 − n F p p  TH Expression and Activity in MN9D Transfected Cells 3 42 − − + 3 − + 3 − + 3 l l − 4 l + l l − Fig. 3 a n b Lane 1 Lane 2 Lane 3 − Lane 4 + c − + n d − + − + n F p − p − p + p +  Fig. 4 l l l − + n F p − p − p + p +  Effects of Silencing α-Synuclein Expression on DA Levels 5 − − 5 Fig. 5 − n F p p  Discussion E. coli TM − − − 16 35 5 13 4 − 42 3 26 − l − 24 26 − − 29 37 37 24 6 22 26 In conclusion, our data demonstrate that in PD pathology, silencing of α-synuclein expression results in enhanced TH activity and increased DA levels in neurons. We propose that in cells that unable to remove excess DA from the cytosol, DA itself and its oxidized products including reactive oxygen species and reactive DA quinones may lead to cell injury or even death. Further understanding of the normal function of α-synuclein in maintaining DA homeostasis may help to identify novel preventative or therapeutic strategies for PD.