Fig. 6
Intranuclear inclusions in neocortical astrocytes and Bergmann glia in Gfa2-CGG99 mice. a-b EGFP histofluorescence (green) in neocortical astrocytes from a (a) Gfa2-CGG99, and a (b) Gfa2-CGG11 transgenic mouse. The majority of astrocytes in the Gfa2-CGG99 mouse cortex expressed eGFP, while fewer astrocytes showed expression in the Gfa2-CGG11 mouse. Sections were immunoreacted against ubiquitin (red) and nuclei stained with DAPI (blue). Red/yellow fluorescent puncta are due to autofluorescence of lipofuscin granules in neurons and microglia associated with normal aging in the mouse cortex. Scale bars: 50 μm. c-d Intranuclear inclusion (c) in an eGFP histofluorescent Gfa2-CGG99 mouse astrocyte immunostained for ubiquitin (arrow, orange/yellow). d Ubiquitin-stained intranuclear inclusion (arrow, cyan) in a Gfa2-CGG99 astrocyte verified as an astrocyte by staining with anti-GFAP (red fluorescence). Scale bars: 5 μm. e-h Typically appearing protoplasmic astrocyte from a WT mouse immunostained for Gfap (e, green). Shown for comparison with histofluorescent (green) Gfa2-CGG99 astrocytes in f-h). f Green histofluorescence in an astrocyte from a Gfa2-CGG11 transgenic mouse, and from a (g) Gfa2-CGG99 transgenic mouse. Both show a cloud-like histofluorescence emanating from their astrocytic processes, and neither contained a ubiquitin-positive intranuclear inclusion. h Intranuclear inclusion (arrow, yellow) in a green histofluorescence astrocyte from a Gfa2-CGG99 mouse. Although not quantified, astrocytes in Gfa2-CGG99 mice with inclusions appeared to show less green eGFP histofluorescence compared to astrocytes without inclusions (e.g., compare green histofluorescence in g and h). Scale bars 10 μm. I-L EGFP histofluorescence (green) in cerebellar Bergmann glia from a Gfa2-CGG11 (i) and Gfa2-CGG99 (j) transgenic mouse. In panel, i note the green histofluorescence in the soma and radial glial processes of the Bergmann glia in the Gfa2-CGG11 mouse (small arrows). Ubiquitin-stained intranuclear inclusions (arrows, red fluorescence) in Bergmann glia (Figs. j and l, arrows) and in a protoplasmic astroglia in the granule cell layer of the cerebellum (Fig. 6k, arrow) from a Gfa2-CGG99 mouse. Microglia immunolabeled with Iba1 (red fluorescence) did not show eGFP histofluorescence and did not have ubiquitin-stained intranuclear inclusions (l, small arrow). Scale bars 10 μm. Figs. a-c,e-l immunostained ubiquitin visualized with Alexa 568 2o antibody (red). Fig. d, immunostained ubiquitin visualized with Alexa 488 fluorescent 2o antibody (green), astrocytes immunostained for GFAP and visualized with Alexa 568 (red). Fig. e, astrocyte immunostained for GFAP and visualized with Alexa 488 (green). Fig. L, microglia immunostained for Iba1 and visualized with Alexa 568 (red). Figs. a-l nuclei stained with DAPI (blue)