DRP1 levels are equivalent in the

total homogenate and cytoplasmic fraction of control and tau flies. However, the mitochondrial fraction shows specific depletion of DRP1 in tau transgenic flies ( Figure 3B). Similarly, fractionation of control and rTg4510 mouse hippocampus reveals reduced DRP1 in mitochondrial fractions from tau transgenic mice compared to controls ( Figure 3C). Immunoblotting for porin, a mitochondrial membrane protein, and GAPDH are used to confirm enrichment of mitochondrial and cytoplasmic proteins, respectively, during the fractionation procedure. We have previously demonstrated that tau binds to and stabilizes actin. Excess actin stabilization by tau is required for neurotoxicity (Fulga et al., 2007). To determine if increasing F-actin level B-Raf inhibitor clinical trial alters DRP1 localization, we overexpressed the actin nucleating factor WASP using a UAS-WASP see more transgene ( Berger et al., 2008). We also increased expression of the actin bundling protein forked. forked gene dosage was increased with a genomic rescue construct ( Grieshaber et al., 2001). We first used the F-actin sensitive dye rhodamine-phalloidin in whole-mount brains to confirm that WASP and forked increase F-actin ( Figure S4A). We then determined

if stabilizing the actin cytoskeleton influences mitochondrial morphology and DRP1 localization to mitochondria. We find that compared to normal control mitochondria, mitochondria in neurons with increased expression of WASP or forked are frequently elongated ( Figure 4A, arrows). Elongated mitochondria often fail to colocalize with DRP1, whereas normal round to tubular mitochondria maintain DRP1 localization ( Figure 4A, arrowheads). Quantitative analysis demonstrates a significant increase in mitochondrial length resulting from overexpression of WASP or forked (

Figure 4A, graph). As would be expected, increasing the expression of WASP or forked together with human tau markedly enhances mitochondrial elongation and neuronal Resminostat toxicity, without altering tau expression ( Figures S2A, S4B, and S4C). Subcellular fractionation confirms reduced localization of DRP1 to mitochondria with increased forked gene dosage ( Figure 4B). To investigate a possible physical interaction between DRP1 and F-actin, we isolated F-actin from head homogenates of control and forked-over-expressing flies by precipitation with biotinylated phalloidin. Western blotting shows that DRP1 coprecipitates with F-actin. Stabilization of actin by forked overexpression substantially increases the amount of DRP1 coprecipitated with biotinylated phalloidin (Figure 4C).