Figure 6. Behavioral tests and neuropathology in mice with conditional Psmf1 inactivation.
(a) Psmf1fl/fl UBC-Cre-ERT2/+ Ai14/+ brains post perfusion (28 days after injection of tamoxifen or corn oil as a control). Tamoxifen-induced recombination induced tdTomato (red) expression and marked cells in which Psmf1 was inactivated (Supplementary File 8). Successful tamoxifen-induced recombination was macroscopically assessed based on the color of dissected mouse brains, which appeared dark pink in mice with severe motor deficit in the rotarod assay (m6, m9) and light pink in mice with mild to no motor deficit in the rotarod assay (m1, m5) compared to controls (ctrl1, ctrl2), which reflected high (~90%) or low (30–50%) recombination rates, respectively. Psmf1fl/fl Ai14 UBC-Cre-ERT2 mice were injected with either corn oil (control) or tamoxifen to induce Cre recombination of floxed Psmf1 exon 3 for loss of Psmf1 and a floxed stop cassette in Ai14 for expression of tdTomato to evaluate recombination efficiency. Mice were sacrificed 28 days post injection. (b-c) Rotarod assay. In the rotarod assay, mice with high recombination rates (~90%) developed severe motor deficits (light blue line in (b) and light blue bar in (c)), whereas the motor performance of mice with low (30–50%) recombination rates was mildly or not impaired (green line in (b) and green bar in (c)). Panel (b) shows single rotarod trials for individual mice on each day. X-axis indicates days of testing after the initial four-day training period. Day 1 corresponds to day 14 after tamoxifen injection. In panel (c), each bar represents the average of three trials for a mouse on day 25 post tamoxifen injection (i.e., 12 days after the initial four-day training period). Error bars indicate standard deviations. (d-e-f) Open field assay. Mice with high recombination rates (~90%, fourth subgroup) strongly avoided walking in the open field, which reflects severe anxiety-related behavior. In contrast, the motor performance of mice with low recombination rates (30–50%, third subgroup) was comparable to controls. In panels c-d-e-f, error bars are standard deviations, p values derived by two-tailed t test, *p < 0.05, **p value < 0.01, *** p value < 0.001. (g) Sagittal brain sections stained with either Iba1 (green) for microglia or GFAP for glia (astrocytes) and with Hoechst 33342 for nuclei (blue). (i) In the deep cerebellar nuclei (DCN), reactive microglia in tamoxifen-induced mice indicated gliosis induction. Scale bar 100 μm. (ii) Higher magnification in the DCN shows morphologically distinct ramified microglia with more amoeboid appearance upon loss of Psmf1. Scale bar 20 μm. (iii) In the DCN of tamoxifen-induced mice we observe an increase of glia staining strongly for GFAP in the DCN, a marker for astrogliosis (of note, GFAP also stains the Bergmann glia in the molecular layer). Scale bar 100 μm. (iv) Higher magnification image of (C). Scale bar 20 μm.