Fig. 5
High levels of EU triggers Purkinje cell degeneration. a, b Examples of Purkinje cells with a large number of cytosolic EU−/p62 + particles. Note (in a) that the cell with EU−/p62 + particles also exhibits EU + /p62 + bodies, and that the Purkinje cell in b has an atrophied appearance and a nucleus with abnormal chromatin characterized by DAPI-dense foci. Both examples are from mice 11 days after injection. c Example of a Purkinje cell with ubiquitin + particles (blue arrow) stained with 2 different antibodies (Ub(m), monoclonal antibody FK2; Ub, polyclonal antibody). Cytosolic EU + particles do not stain for ubiquitin (red arrows). d Triple staining for ubiquitin, homer3 and EU, showed reduced homer3 staining, reflecting Purkinje cell dendritic atrophy, and ubiquitin + particles in the somato-dendritic domain of Purkinje cells in regions with high EU labelling (9d–h). Parts of the cerebellar cortex without EU labelling (9d–n) in the same mouse show normal homer3 staining and no ubiquitin + particles. e Low- and high-magnification confocal images illustrating the disappearance of calbindin-immunoreactivity in the molecular and Purkinje cell layers (blue arrows) indicating Purkinje cell loss 14 days after EU injection. Purkinje cell loss is associated with the appearance of LGALS3 + phagocytosing microglia cells that occasionally engulf a degenerating Purkinje cell (yellow arrow). White arrows point to atrophied Purkinje cells with shrunken strongly EU + nuclei. f Inverse grey scale wide-field images of caudal vermis (lobule 8 and 9) showing that while absent 7 days after EU injection, LGALS3 + microglia cells are present in the molecular and Purkinje cell layers 9 and 11 days after injection. g, h Immunolabelling for the nucleopore protein Nup62 and Sumo1 that in normal Purkinje cell strongly outline the nuclear envelope, shows that a subset of EU + Purkinje cell nuclei are devoid of nuclear envelope staining (arrows in g, h). These nuclei showed high EU levels, and a somewhat shrunken appearance and in all occasions were immuno-negative for the histone marker variant macroH2A (arrow in h) that in normal Purkinje cells co-distributes with heterochromatin. 0, 1.2 and 2.4 (in g) represent the z-position (in μm) of the individual images in the confocal stack. Scale bars: 5 μm (b, c, h); 10 μm (a, g); 20 μm (d, e); 200 μm (f)