, 2006; Dias-Santagata et al., 2007). To monitor cell cycle activation in the brains of tau transgenic flies, we immunostained for proliferating cell nuclear antigen (PCNA), an S phase marker abnormally re-expressed in human AD tissue (Busser et al., 1998) as well as tau transgenic flies (Khurana et al.,

2006). Overexpression of DRP1 or knockdown of MARF suppresses cell cycle activation (Figure 2D). Conversely, MARF overexpression and DRP1 knockdown increase cell cycle activation in brains of tau transgenic flies. Western blot analysis shows no alteration in levels of tau in genetically modified backgrounds, confirming that manipulation of DRP1 and UMI-77 nmr MARF do not alter toxicity of tau by simply increasing or decreasing the expression of the tau transgene (Figure S2A). We next explored the mechanism by which tau expression promotes mitochondrial elongation. We first evaluated expression of DRP1 and MARF by real-time PCR, but did not observe changes in mRNA levels ( Figure S3A). We then examined the subcellular localization of DRP1. Cytoplasmic DRP1 protein must translocate to the mitochondria to drive fission ( Frank et al., 2001). To visualize DRP1 in Drosophila, we used a transgenic strain carrying a 9.35 kb genomic rescue construct that has an

in-frame FLAG-FIAsH-HA tag after the start BMS354825 codon of DRP1 ( Verstreken et al., 2005). The presence of this genomic rescue construct does not have a statistically significant effect on tau expression, mitochondrial length, or neuronal toxicity ( Figures S2A, S3B, and S2C), consistent with modest expression of DRP1 from its endogenous promoter ( Figure S2D). Visualizing DRP1 by immunostaining for HA, we find that in control neurons DRP1 signal appears in discrete foci, almost exclusively localized to mitochondria ( Figure 3A, control, arrowheads).

Immunostaining for FLAG shows an equivalent DRP1 staining pattern ( Figure S3D). However, in neurons from tau transgenic animals DRP1 foci are infrequent, all and the majority of mitochondria do not colocalize with DRP1 ( Figure 3A, tau, arrowheads). Signal intensity profiles for DRP1 and mitoGFP verify the tau-mediated inhibition of colocalization ( Figure S3E). One possible explanation for the lack of DRP1 localization to mitochondria in tau transgenic neurons is that elongated mitochondria fail to recruit DRP1 normally. To test this idea we expressed MARF in the absence of tau. In neurons overexpressing MARF we observe the expected increase in mitochondrial length. However, in contrast to results from tau transgenic animals, elongated mitochondria in MARF transgenics do colocalize with DRP1 puncta ( Figure 3A, MARF, arrowheads). Thus, failure of DRP1 to localize to mitochondria in tau transgenic neurons is not likely to be a secondary effect of the mitochondrial elongation itself. Mitochondrial localization of DRP1 is also retained following MARF knockdown, as well as increased or decreased expression of DRP1 ( Figure S3F).