Targeting DNA-PKcs by siRNA.

<p>293 cells are transfected with siRNA (100 pmole) targeting DNA-PKcs mRNA and non-specifically control siRNA using Lipofectamine™ 2000. Two days after transfection, cells were harvested. (A) RT-PCR for the detection of DNA-PKcs mRNA. Amplified cDNA fragments were separated on a 1% agarose gel. (B) Western blot for the detection of DNA-PKcs protein using beta-actin protein as an internal control.</p

Targeting DNA-PKcs reduced rAAV replication.

<p>293 cells were transfected with DNA-PKcs specific siRNA (DNA-PKcs siRNA) or control siRNA. Two days after transfection, cells were infected with rAAV-UF5 and rHSV, or transfected with pUF5 and pDG. rAAV replication was evaluated by Southern blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015073#pone-0015073-g001" target="_blank">Figure 1</a>. (A) Effect of DNA-PKcs siRNA on rAAV replication by Southern blot after vector infection. Concentration of DNA-PKcs siRNA and control siRNA was 100 pmole. (B) Densitometry analysis of data from Figure A. **, <i>P<0.001</i> for monomer; *, <i>P<0.05</i> for dimer and concatamers when compared with control siRNA group. (C) Dose dependent effect of DNA-PKcs siRNA on rAAV DNA replication. (D) Effect of DNA-PKcs siRNA on rAAV after vector and helper plasmid transfection. Left panel, without DpnI digestion; Right panel after. Note: DpnI digestion removes transfected plasmid DNA and shows all <i>de novo</i> replicated rAAV forms (monomer, dimer and concatamers).</p

AAT-ITR interacts with Ku proteins.

<p> (A) construction of AAV-ITR on a magnetic particle. (B) ITR on magnetic bead interacts with Rep78 and Ku proteins. AAV-ITR was bound to purified proteins or HeLa nuclear extract (NE) at room temperature or 37°C and then subjected to western blot analysis using antibodies toDNA-PKcs, Ku80 and Ku70. Rep78 are used as a positive control. (C) T-shaped closed ITR interact with Ku proteins in dose-dependent manner. AAV-ITRs on the bead were treated with Exonuclease III and incubated with different amount of HeLa NE (65µg/µl). The ITR binding proteins were subjected to western blot analysis for DNA-PKcs, Ku80 and Ku70. (D) Competition assay (right panel). When AAV-ITR on bead was incubated with HeLa nuclear extract, free AAV-ITR was added (2.5 fold) as a competitor. Streptavidin-coated magnetic beads and the beads with AAT-ITR (left panel) served as a control and showed addition of AAV-ITR increased pull down of Ku proteins.</p

Structure analysis of replicated DNA.

<p>(A) Map of the rAAV-UF5 vector; X (XbaI), S (SacI), and N (NotI), Two bold lines indicate the position of CMV and Neo^R probes. (B) All possible genome sizes generated from different AAV junctions. I. F., internal fragment; H-H, head-to-head; T-T, tail-to-tail; H-T, head-to-tail. (C and D) Southern hybridization probed with CMV (C), and Neo^R (D) after restriction enzyme digestion of Hirt DNA.</p

rAAV replication in MO59K cells and 293 cell treated with wortmannin.

<p>Hirt DNA was purified two days after viral infection and subjected to Southern blot analysis. All samples were triplicated and hybridized with ³²P labeled CMV probe. Replicated forms of rAAV include double-stranded monomer (about 3.4 kb), or dimer (about 6.8 kb), and concatamers DNA (high molecular weight). Notice that treatment of wortmannin reduced rAAV replication in a dose-dependent manner in both MO59K cells (A) and in 293 cells (B).</p

Rescue of TH+ and NeuN+ cells in the ipsilateral SN with intraperitoneal administration of anti-α-Syn antibodies.

<p>Immunohistochemical staining of the SN region with an anti-TH antibody (A) AAV-GFP, (B) AAV-α-Syn + IgG, (C) AAV-α-Syn + AB1, (D) AAV-α-Syn + AB2. (E) Graph of unbiased stereological estimation of TH+ cells in the SN of treated animals. AB1 treated animals showed similar levels of TH+ cells compared to the GFP control and significantly higher number of TH+ cells compared to the IgG treated group. Data are shown as mean ± SEM (n = 13 AAV-GFP control and n = 7 for treatment groups). (F) Graph of NeuN+ cells of the SN. Stereologic analysis shows a significant rescue of NeuN+ cells in SN sections with AB1 compared with IgG treated animals (n = 9 AAV-GFP control and n = 7 for treatment groups). *P< 0.05, **P< 0.01, ***P< 0.001. Scale bar = 100μm.</p

E6-AP protein levels were restored to wildtype levels in the TR2-UBE3A treated AS mice.

<p>(A) Representative coronal slices through the hippocampus from each group stained for E6-AP. (B) Quantitative analysis of the IHC revealed a significant increase in E6-AP expression in the WT and AS-TR2-UBE3A mice compared to the AS TR2-GFP group, while there was no significant change between the WT and AS TR2-UBE3A mice. Results shown represent the mean with standard error.</p

Increasing E6-AP in the AS mouse results in improvements in early phase LTP.

<p>(A) AS TR2-GFP mice have significant deficits in hippocampal synaptic plasticity. LTP was induced following 20 min of baseline recordings. (B) Immediately following TBS, acute hippocampal slices taken from AS TR2-GFP mice had significant deficits in the average PTP (average of first 5 min recordings of fEPSPs slopes). To compare late phase LTP, the last 5 min recordings of fEPSPs slopes were averaged, and there was no significant difference between any of the groups. (C) There were no significant differences between any of the groups in either PPF or (D) PTP, indicating that short term synaptic plasticity mechanisms are unaffected. Results shown represent the mean with standard error.</p

There were no changes in motor coordination, activity levels, or anxiety.

<p>(A) There was no change in latency to fall of the rotorod in either AS group. (B) Total distance travelled during the open field test revealed no significant difference between any of the treatment groups. (C) Time spent in the open arms of the elevated plus maze was used to determine general anxiety. (D) Time spent immobile in the elevated plus maze was not significantly different in any of the groups. Results shown represent the mean with standard error.</p

The effect of intraperitoneal administered anti-α-Syn antibodies on AAV vector mediated α-Syn expression.

<p>Immunostaining of the SN region with an antibody against α-Syn. Administration of AAV-α-Syn into the rat SN caused significant expression of α-Syn in the SN (B) compared to the AAV-GFP control group (A). Intraperitoneal injection of anti-α-Syn antibody AB1 reduced α-Syn level in the SN (C), while injection with antibody AB2 had a reduced effect (D). Quantitative analysis of levels of α-Syn expression is presented as percent positive area (E). Data are presented as the percent positive area of anti-α-Syn staining throughout the SN (n = 8 animals per group). Asterisk denotes significance (*** P<0.001, * P<0.05) with comparison made to the ipsilateral AAV-GFP group by 1-way ANOVA with post-hoc Bonferroni test. ELISA analysis confirmed a significant reduction in α-Syn levels in the SN with antibody AB1 compared to IgG treatment (F). ### P< 0.001, # P< 0.05 vs. Control AAV-α-Syn + IgG. Data are presented as the mean concentration of α-Syn in pg/μg of protein ± SEM (n = 6 per group). Scale bars are 100μm.</p