After injection of AAV-hSNCA, a dose dependent level of expressio

After injection of AAV-hSNCA, a dose dependent level of expression of hSNCA-IR was observed in soma and fibers in ipsilateral SN and ventral tegmental area (VTA) and in fibers in ipsilateral striatum (ST) (Fig. 1a). A dose dependent significant loss of TH-IR neurons in these rats was also observed (Table S1). Reduced contralateral forelimb use was observed at the lowest dose (0.6×1010 vg) of AAV-hSNCA (Fig. 1b). When different ratios of mir30-SNCA were examined, hSNCA-IR was found to be reduced in rats that received GSK2118436 price the lowest dose of mir30-SNCA (1:3 ratio), although hSNCA expression was still detectable

in cell bodies in the SN and in fibers in both SN and ST. At the highest dose of mir30-SNCA (1:55 ratio), hSNCA-IR was not detected in Obeticholic Acid supplier ST and only rare hSNCA-IR cells or fibers were detected in the SN, although a diffuse background of hSNCA-IR was observed in the SN (Fig. 1a). A statistically significant protection from the AAV-hSNCA-induced deficit in contralateral forelimb use

was observed at a hSNCA to mir30-SNCA ratio of 1:55, but not at a ratio of 1:29 or 1:3 in this pilot study with n=3 (contra: F5,12=3.8, p=0.0275; ipsi: F5,12=6.2, p=0.0046; Fig. 1b). However, no significant differences in numbers of TH-IR neurons between control and injected SN at any ratio of AAV-hSNCA to AAV-mir30-SNCA were found ( Table S1). Because TH neuron counts do not differ between injected and control SN at any ratio of hSNCA to mir30-SNCA ( Table S1), the optimal ratio was determined by the efficiency of hSNCA-IR silencing and the protection against the deficit in forelimb motor behavior, which differs among hSNCA to mir30-SNCA ratios. Based on the results of this pilot study, the subsequent efficacy experiments were carried out using the 1:55 hSNCA to mir30-SNCA ratio. To confirm that rats in each treatment

group were transduced with the vectors to the same extent, DNA levels of hSNCA and turbo GFP (representing either mir30-SNCA or a control, non-silencing, Janus kinase (JAK) silencing vector containing a scrambled target sequence (NS)) were determined by quantitative real time QPCR at 10d (Fig. S2a and b) and 2 months (Fig. 2a) survival in the ventral midbrain. All groups received similar levels of hSNCA vector DNA (Fig. S2b and Fig. 2a). Groups injected with AAV-hSNCA and AAV-mir30-SNCA, or AAV-hSNCA and AAV-NS, received similar levels of silencing vector DNA, as measured by turbo GFP (Fig. S2a). hSNCA DNA also was detected in the ST of rats that received AAV-hSNCA alone, but not in ST from other treatment groups (Fig. S3). hSNCA expression levels were examined at the mRNA level in the ventral midbrain and ST at 10d (Fig. S2c) and 2 months (Fig. 2b and c) using qRT-PCR to confirm hSNCA expression and silencing.

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