Measurement of Autophagy Flux in the Nervous System in Vivo

Supplementary material to

“Measurement of autophagy flux in the nervous system in vivo”

by Karen Castillo, Vicente Valenzuela, Soledad Matus,Melissa Nassif, Maritza Oñate, Yerko Fuentealba,Gonzalo Encina, Tey Irrazabal, Geoffrey Parsons, Felipe A. Court, Bernard Schneider, Donna Armentano, and Claudio Hetz.

Supplementary Figure legends

Supplementary Figure 1In vivo neuronal transduction with AAV2/2-GFP through ICV of newborn mice. Spinal cord images of animals transduced at P0 with AAV2/2_GFP particles showing transversal (i) and longitudinal (ii) sections, co-stained with Hoechst nuclei marker. Longitudinal hemisection of AAV-GFP transduced mice immunolabeled with anti-GFP (iii) and anti-CHAT antibody (iv), a marker of motoneurons. Cells presenting co-expression of GFP and CHAT are indicated with redarrowheads. Scale bars: 150 m (upper) and 30 m (lower).

Supplementary Figure 2mTOR inhibition in nervous system of animals treated with rapamycin. (a) Western blot analysis of animals treated with 2 mg/kg rapamicyn 3 days per week during 2 weeks. Then, animals where sacrificed and the tissue processed for western blot analysis of phosphorylated mTOR (p-mTOR), total mTOR and HSP90 levels (left panel). Quantification of phospho-mTOR levels relative to total mTOR was determined.Mean and standard error is presented. Non-parametric t-test was used to compare statistical significance between drug treated and non-treated animals. *: p 0.05.(b)Western blot analysis of animals treated as in (a) to detect phosphorylated p70S6K (p-p70S6K) and total p70S6K levels (left panel). Quantification of phospho-p70S6K levels relative to total p70S6K is showed.Data represent mean and standard error. Non-parametric t-test was used to compare statistical significance between rapamycin treated and non-treated animals. *: p 0.05. The antibody also detect p-p85 S6K.

Supplementary Figure 3In vivo neuronal transduction with AAV2/2_mCherry-GFP-LC3 through ICV of newborn mice.(a)A representative image of a sagittal section of brain tissue showing the hippocampus region with cells transduced with the autophagy sensor AAV2/2_mCherry-GFP-LC3. Upper panel: 5X representative picture of whole hippocampus, scale bar: 200 m. Lower panel: superzoom magnification of cells transduced of the pictures showed, scale bar: left 60 m and right 8 m.(b)Reproducibility of AAV2/2-mCherry-GFP-LC3 transduction in the cerebellum. View of the cerebellar crest of three independent animals injected with AAV2/2_mCherry-GFP-LC3. Images show sections of the Purkinje cell layer of cerebellum.

Supplementary Figure 4Immunofluorescence analysis of cerebellum sections of animals injected with AAV2/2-mCherry-GFP-LC3 and treated with rapamycin or trehalose, co-stained with anti-Lamp2 antibody (blue). Imaged for each channel are provided with different merge analysis. Right panel: A zoom is provided to depict the co-localization of the mCherry dots and LAMP-2. Of note, in addition to neurons, glial cells are highly positive for LAMP 2 staining (red arrows as examples). Scale bar: 30 m, and 10 m for zoom.

Supplementary Figure 5 Autophagy enhancement in DRGs axons treated with rapamycin.Electron micrographs of DRG axons exposed to 600 nM rapamycin and processed for EM. Pictures show autophagosomal structures as is evidenced by the presence of multimembrane vesicles containing cargoes. Scale bar: 500 nm.