Journal: Journal of Cell Science

Article Title: Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells

doi: 10.1242/jcs.226241

Figure Lengend Snippet: Alpha-synuclein induces autophagy in microglial cells. (A) BV2 microglial cells were left untreated or stimulated at different time points with AS monomers (mAS) or fibrils (fAS) at 1 μM. Cells were then fixed and stained for LC3. (B–H) BV2 GFP–LC3 cells (B,C) or primary microglial cells (F,G) were left untreated or stimulated with Alexa Fluor 647-labelled AS fibrils (1 µM). After 12 h cells were immunostained with anti-LAMP1 (red) antibody and primary microglial cells were also stained for LC3. Images shown are z-stack projections. (D,H) 3D surface-rendered magnifications of the selected area above. (E,I) Mean±s.e.m. LC3-positive vesicles in unstimulated or treated BV2 (E) and primary microglial cells (I) were determined using ImageJ particle counting plugin after cell deconvolution (n=20). (J) Cell lysates from BV2 cells cultured with AS fibrils or monomers (1 µM) were collected at different time points and LC3 and β-actin protein levels were examined using western immunoblotting. Bafilomycin A1 (BAF) was added for the last 3 h. Graph shows quantification of mean±s.e.m. LC3-II expression relative to β-actin using densitometry. Results from at least three independent experiments were analysed by one-way ANOVA followed by post-hoc Dunnet's test; n=3. **P<0.01; ***P<0.001; #P<0.01 when comparing AS at 12 h (AS12) with AS at 12 h in the presence of BAF (AS12+BAF); Nd, no significant difference. pMC, primary microglial cells.

Article Snippet: Spatial deconvolution, 3D surface-rendered images and 3D surface-rendered movies were created with SVI Huygens Software.

Techniques: Staining, Cell Culture, Western Blot, Expressing