ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo

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1 Supplementary Information ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo Carlos Barcia* 1,2, Carmen M Ros 1,2, Valentina Annese 1,2, Maria Angeles Carrillo-de Sauvage 1,2, Francisco Ros-Bernal 1,2, Aurora Gómez 1,2, José Enrique Yuste 1,2, Carmen María Campuzano 1, Vicente de Pablos 1,2, Emiliano Fernandez-Villalba 1,2, Maria Trinidad Herrero 1,2 1 Clinical and Experimental Neuroscience, 2 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), School of Medicine, University of Murcia, Campus de Espinardo, 30071, Murcia, Spain. *Corresponding Author: Carlos Barcia Clinical and Experimental Neuroscience and Centro de Investigación Biomedica en Red de Enfermedades Neurodegenerativas (CIBERNED), School of Medicine, University of Murcia, Campus de Espinardo, Murcia, Spain. Tel Fax barcia@um.es This document contains the following supplementary information: 1. Supplementary Figures S1 to S8 2. Supplementary Figure legends S1 to S8 3. Legends to Supplementary Movies 1 to 7 1

2 Supplementary Figure S1 Supplementary Figure S1. Specific activation of microglia can be clearly identified in the SNpc 24 h after MPTP. (A) Diagram of a representative section of the mouse mesencephalon at the level of the SNpc, ventral and lateral to the Red Nucleus (RN). (B) At 24 h after MPTP injection, an area of activated microglia can be observed locally at the SNpc in mouse brain. Pictures of the sections of the SNpc of a control mouse (upper row) and an MPTP-injected mouse (bottom row) stained for TH and Iba-1. The arrow indicates the specific area of microglial activation. Scale bar: 500µm. 2

3 Supplementary Figure S2 Supplementary Figure S2. Quantification of F4/80 + cells in the SNpc. The ROCK inhibitor HA-1077 does not affect the increase of F4/80 + cells after MPTP treatment. *p<0.05; **p<

4 Supplementary Figure S3 Supplementary Figure S3. (A) F-actin staining is ubiquitous and can be observed in the dopaminergic and non-dopaminegic cells of the SNpc. Confocal sections (0.5 µm) show the phalloidin staining (Magenta) for F-actin, combined with TH-immunostaining (red) to label dopaminergic neurons in untreated mouse SNpc. A merge of both channels and DAPI is shown in the merged image. (B) The intensity of relative fluorescence of F-actin cluster in microglia is higher than in the neighboring cells. The confocal image on the left, obtained from an MPTP-treated mouse, shows the merged 4

5 channels of F-actin, Iba-1 and DAPI. The picture on the right shows the F-actin staining alone. The yellow broken arrow indicates the segment of relative fluorescence measurement. The graph on the right shows the intensity [I] of F-actin relative fluorescence measured along the broken line. Note that the highest level of fluorescence was obtained in the microglial F-actin cluster (1), compared with the other cell (2). (C) The relative fluorescence of Cdc42 cluster in microglia is higher than in the surrounding cells. The confocal image on the left, obtained from an MPTP-treated mouse, shows the merged channels of Cdc42, Iba-1 and DAPI. The image on the right shows the Cdc42 immuno-staining alone. The yellow broken arrow indicates the segment of relative fluorescence measurement. The graph on the right shows the intensity [I] of Cdc42 fluorescence measured along the broken line. Note that the highest level was obtained in the microglial Cdc42 cluster (1), compared with the other cells (2). Scale bar in A: 20 µm. 5

6 Supplementary Figure S4 Supplementary Figure S4. Confocal sections of Cdc42 clustering in two microglial cells in the nigrostriatal pathway after MPTP treatment (1, 2). Confocal images of the immunostaining for Iba-1 (green) and Cdc42 (red), combined with DAPI counterstaining (blue) are shown. Clustering of Cdc42 is clearly observed in microglial cells compared with the neighboring cells. This Cdc42 cluster is located at the protrusion. Descriptive diagrams of the images are illustrated at the bottom. 6

7 Supplementary Figure S5 Supplementary Figure S5. (A) Quantification of the thionine + neurons in the SNpc of all experimental groups. HA-1077 treatment prevents MPTP-induced neuron elimination. (B) The levels of MPP + in the striatum measured using HPLC show that HA-1077 does not block the entrance of MPP + into the striatum. 7

8 Supplementary Figure S6 Supplementary Figure S6. (A) Confocal images of the immunofluorescence of GM130 (magenta), TH (red) and Iba-1 (green) combined with DAPI as a counterstain in the SNpc of mice. The size of the Golgi apparatus in microglial cells is notably bigger in MPTP-treated animals and it is reduced in HA-1077-treated mice. The insert shows the magnification of a representative microglial cell in each experimental group. (B) Quantification of the area of microglial Golgi for each experimental group. The area of the microglial Golgi is significantly bigger in MPTP-treated animals, and this condition is reversed with HA-1077 treatment. *p<

9 Supplementary Figure S7 Supplementary Figure S7. (A) Confocal images of the three different channels of the engulfing gliapse shown in Figure 6A 2. The white arrow indicates the engulfed dopaminergic neuron, which displays a pycnotic nucleus and low levels of TH fluorescence. (B) Analysis of the relative fluorescence of the engulfed neuron and other spots of TH in the same field. The white broken lines indicate the segment analyzed and their relative fluorescence is represented in the specific graphs on the right. The numbers indicates the specific points of fluorescence measured. Note that the spots of 9

10 the engulfed neuron (2, 3, 4 and 5) show lower red fluorescence than the other spots not involved in engulfing events (1 and 6). (C) Size comparison between a healthy dopaminergic neuron in the SNpc of a control mouse and an engulfed dopaminergic neuron in the SNpc of an MPTP-treated mouse. Note the differences between the nuclei. 10

11 Supplementary Figure S8 Supplementary Figure S8. Four examples of engulfing gliapses in the SNpc of mice injected with MPTP. In the four cases the engulfing pycnotic nucleus can be observed inside the Iba-1+ microglial cell. Lateral x-z and y-z views are also shown at the level of the engulfed pycnotic nucleus for each case. Iba-1 appears completely surrounding the engulfed nucleus in all dimensions. Scale bar: 15 µm. 11

12 Supplementary Movie Legends Supplementary Movie 1. Slide show showing an example of a typical confocal image of the SNpc of a control mouse compared with the same anatomical structure after MPTP. The arrows indicate the activated microglia cells after MPTP treatment (Iba-1 + cells). Some of the microglial cells are shown in intimate contact with dopaminergic neurons. Supplementary Movie 2. Three-dimensional reconstruction of phagocytosing microglia (green) engulfing a dopaminergic neuron (red) in the SNpc of a mouse treated with MPTP. The nuclei are stained with DAPI (blue). The red channel has been removed in the middle clip highlight the pycnotic nucleus of the neuron inside the phagosome. Supplementary Movie 3a and 3b. A series of confocal optical xy sections through the z-axis in the SNpc of a mouse treated with MPTP. The series captures several phagocytosing microglia containing pycnotic nuclei. The movie shows the different scanned 0.5-µm optical layers along the z-axis. Iba-1 + microglial cells are shown in green, TH + dopaminergic neurons are shown in red and nuclei are stained with DAPI (blue). Multiple engulfing processes are observed along the z-axis of the scanned images where Iba-1 + cells phagocytose pycnotic extra-nuclei. Movie 3b shows the same anatomical location but with higher magnification and resolution. Supplementary Movie 4. Three-dimensional reconstruction of an engulfing microglial cell in the SNpc of a mouse treated with MPTP. An Iba-1 + (green) microglial cell in the 12

13 vicinity of the dopaminergic neurons (red) of the SNpc, containing a pycnotic extranucleus marked with DAPI (blue) is shown at the center of the image. Supplementary Movie 5a and 5b. Three-dimensional reconstruction of an engulfing Iba-1 + microglial cell (green) in the SNpc of an MPTP-treated mouse. A microglial cell (green) in the vicinity of dopaminergic neurons (red). A clipping plane, limited with yellow lines, is positioned toward the phagosome containinig the pycnotic nucleus in the center of the image. Movie 5b shows the same anatomical location but with higher magnification and resolution. Supplementary Movie 6. Example of phagocytosing microglia containing a pycnotic extra-nucleus. A clipping plane, limited with yellow lines, is positioned toward the phagosome containing the pycnotic nucleus in the center of the image. Supplementary Movie 7. Three-dimensional reconstruction of an engulfing microglia containing a pycnotic nucleus showing clustering of F-actin in the protrusion. F-actin staining is observed in magenta. 13