Figure 6

Modulating HDAC4 expression enhances AAV9 transduction. (A) Western blot analysis of pulldowns between whole brain lysates and AAV9 (4 × 10¹¹ vg for either virus) with streptavidin-agarose corroborates data from LC/MS. Note that endogenous SRSF1 expression was not detectable by conventional western blot, and so SRSF1-flag was overexpressed in HEK cells and incubated with the AAVs. Images shown are from multiple blots taken from a single pulldown probed for different interactors. Pulldowns were repeated at least three times for all targets shown. (B) Both AAV9 and AAV9-138 can bind to HDAC4 indicating that the HDAC4-AAV interaction does not arise purely from the capsid modification. Images shown are the same pulldown probed on different blots. (C) Two different miRNA sequences for HDAC4 demonstrate at least a 50% knockdown in HEK293T cells normalized to the control. (D) Western blot analysis of protein lysates taken 48 hrs after AAV-mcherry was added at 50,000 vg/cell show that the miHDAC4 #2 sequence augmented mcherry expression compared to a control miRNA scramble sequence. Tubulin immunoreactivity measured after stripping blot of mcherry signal. (E) Densitometry of western blots show that pretreating cells with the miHDAC4 #2 sequence yielded a 1.9 ± 0.48 (n = 5) fold increase in mcherry expression. (F) HDAC4 knockdown significantly (Student’s unpaired t-test used for each gene) reduces SF3B1, SRSF1, and SRSF5 levels in HEK293T cells stably expressing either a lentiviral miR-RNAi control or a miR-RNAi-HDAC4. (G,H) HDAC4 knockdown HEK cells show a significant increase in AAV9 particles in the nucleus compared to miR-RNAi controls at 20 minutes (Mann-Whitney test, n = 7, *p = 0.026) but not 60 minutes (Mann-Whitney test, miControl: n = 9; miHDAC4: n = 5, p = 0.15). Corrected total cell fluorescence (CTCF) was used to quantify the AAV9-138 particles as described in methods. Arrows indicate regions where viral particles are clearly co-localizing with nucleolin. Error bars represent mean ± SEM. The AAV9-138 particles are stained red and nucleolin is stained in green. Scale bar 10 µm.