However, TH-IR cell counts are not statistically different in injected SNs of all treatment groups. At 2 months, TH-IR neuron numbers also are reduced (p≤0.001) in hSNCA-expressing SN (i.e. hSNCA: 8518±586, n=6, and hSNCA and NS: 6466±264, n=5) compared to respective control click here SN (hSNCA: 12,145±204, n=6, and hSNCA and NS: 12,254±262, n=5). SNCA gene silencing ameliorates this deficit in TH-IR neurons

because rats where hSNCA was silenced with mir30-SNCA have a less severe reduction (p≤0.05) in the number of TH-IR neurons in the injected SN (10,355±732, n=6) compared to the respective control SN (12,633±213), and this reduction is not significant in comparison to the control SNs from the hSNCA-expressing groups. Injection of AAV-hSNCA and AAV-NS exacerbates the deficit in TH-IR neurons in that SNs injected with AAV-hSNCA and AAV-NS have reduced TH-IR neurons compared to SNs injected with AAV-hSNCA and AAV-mir30-SNCA, as well as those injected with AAV-hSNCA alone (p≤0.05 compared to hSNCA, p≤0.001 compared INK 128 to hSNCA and mir30-SNCA; F5,28=28.90, p<0.0001). Note that although significant

differences were observed between treated SNs at 2 months, and not at 1 month, the pattern and magnitude of effects at 1 and 2 months are very similar and do not differ significantly between time points, which was verified by a lack of significant effect of time or interaction of time and treatment by 2-way ANOVA. To further examine effects of hSNCA expression and silencing on DA neurons in the SN, the ventral midbrain was dissected from rats injected with AAV-hSNCA, or AAV-hSNCA and either AAV-mir30-SNCA or AAV-NS silencing vector and endogenous rat DA phenotypic DNA Damage inhibitor markers were examined at the mRNA and protein levels (Fig. 5). TH mRNA levels (Fig. 5a) are reduced in ventral midbrain injected with either AAV-mir30-SNCA or AAV-NS silencing vector compared to AAV-hSNCA-injected or control ventral midbrain, and this reduction is greatest in ventral midbrain injected with AAV-hSNCA and AAV-NS, which have reduced TH mRNA levels compared to all control ventral midbrains (F5,24=15.66,

p<0.0001). Protein levels follow this same trend in that ventral midbrain injected with either AAV-mir30-SNCA or AAV-NS silencing vector exhibit reduced TH protein using a pan TH antibody (F5,24=6.148, p=0.0008; Fig. 5c), as well as Ser40 phosphorylated (P-Ser40) TH antibody ( Fig. 5d), an activated form of TH, compared to AAV-hSNCA-injected and control ventral midbrain. However, control ventral midbrains from rats that received either silencing vector also show reduced P-Ser40 TH protein expression (F5,24=8.421, p=0.0007). Interestingly, protein levels of vesicular monoamine transporter 2 (VMAT2, Fig. 5e) are not significantly affected by treatment, suggesting that expression of TH is selectively affected by silencing vector in DA neurons.