Manipulation of Lrrk enhances the excess stabilization of the actin cytoskeleton caused by tau.

(A) ELISA specific for F-actin shows an increase in F-actin levels in tau transgenic flies with further increase when Lrrk levels are increased or decreased or when Lrrk-GS is expressed. n = 3. (B) Western blot demonstrating equivalent levels of total actin and total protein, as illustrated by GAPDH, in the genotypes studied. Quantification of actin intensity from three blots is shown on the right panel. (C) Immunofluorescence microscopy images of sections of the indicated genotypes flies stained for actin illustrating actin rods. Images shown are of cortical Kenyon neurons in the mushroom bodies. Scale bars represent 10 μm. (D) The number of actin-rich rod-like structures (actin rods) present in sections from tau transgenic flies is increased when Lrrk levels are increased or decreased. (E) Western blot showing that Lrrk overexpression in a Lrrk mutant background results in Lrrk levels similar to those in wild-type control animals. The blot is reprobed for GAPDH to illustrate equivalent protein levels. Quantification from three different blots is shown on the right panel. (F) Expression of wild-type Lrrk in tau transgenic flies in the Lrrk mutant background does not increase the number of neurons with activated caspase, indicating no increase of neuronal toxicity. (G) There is no increase in cell cycle activation in postmitotic neurons when Lrrk is expressed with tau in flies in the Lrrk mutant background. (H) There is no incr ease in the number of actin rods when Lrrk is expressed with tau in flies with Lrrk mutant background. n = 6 per genotype and time point for histological analyses. *P elav-GAL4/+; UAS-CD8-PARP-Venus/+ in F, and elav-GAL4/+ in the remainder of the panels. Flies are 10 days old. See S1 Data for individual numerical values underlying the summary data displayed in A, B, and D—H. ELISA, enzyme-linked immunosorbent assays; F-actin, filamentous actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Lrrk, leucine-rich repeat kinase; Lrrk-GS, Lrrk carrying the G1914S mutation.