Figure 2

LRRK2 kinase inhibition prevents the formation of Tat-induced LRRK2 positive inclusions and normalizes process length in BV-2 cells. We treated BV-2 cells for 12 hours with saline or Tat ± LRRK2i and immunostained for LRRK2 (green). (A) Saline-only treated cells contain diffuse cytoplasmic LRRK2 expression. (B) Tat-treated cells contain brightly staining LRRK2 positive inclusions (white arrows). (C, D) BV-2 cells treated with LRRK2i and either saline or Tat do not contain the inclusions seen in the Tat alone treated group. (E) Graph showing average length of longest microglia process in various experimental conditions. We averaged the longest processes from 100 microglia in monoculture from each condition. Tat-treated microglia had significantly longer processes than saline-treated microglia. LRRK2i normalized the microglia process length in the presence of Tat and had no effect in the presence of saline (***P <0.001, one way ANOVA, Newman-Keuls post-test). In order to ensure that the changes in BV-2 cell morphology were not accompanied by changes in cell proliferation or cell death, we performed total cell counts and trypan blue staining after 12 hours of Tat treatment. We found no statistical difference in total cell number (F) or cell death (G) after 12 hours of Tat exposure (P = 0.28 and P = 0.24 respectively, Student’s t-test). ANOVA, analysis of variance; LRRK2, leucine-rich repeat kinase 2; LRRK2i, LRRK2 inhibitor; Tat, trans activator of transcription.