Rer1-deficiency in the mouse forebrain results in cerebral degeneration.

(A) Abnormal limb-clasping reflexes in forebrain-specific Rer1 cKO mice. Control (Rer1+/inv-trap) and forebrain-specific Rer1 cKO (Rer1inv-trap/inv-trap; Emx-Cre) mice were suspended by the tail. The forebrain-specific Rer1 cKO mice held and tightened their limbs. (B) Rotarod test of control (Rer1+/inv-trap, Rer1inv-trap/inv-trap, Rer1+/inv-trap; Emx-Cre; n = 7) and forebrain-specific Rer1 cKO (Rer1inv-trap/inv-trap; Emx-Cre; n = 5) male mice at 3 months of age. Mice were placed on a rotating rod and the time spent on the rod was measured. Data are shown as the mean ± standard error of the mean (SEM). n.s.: not significant. (C) Abnormal open field behavior in forebrain-specific Rer1 cKO mice. Traces show representative exploratory behavior in an open field for control (Rer1+/inv-trap) and forebrain-specific Rer1 cKO (Rer1inv-trap/inv-trap; Emx-Cre) mice. (D, E) Quantitative analysis of time spent in the center (D) and total distance traveled (E) for 3-month-old control (Rer1+/inv-trap, Rer1inv-trap/inv-trap, Rer1+/inv-trap; Emx-Cre; n = 7) and forebrain-specific Rer1 cKO (Rer1inv-trap/inv-trap; Emx-Cre; n = 5) male mice. *P t-test). n.s.: not significant. (F) Dorsal views of whole-mount brains from 5-month-old male control (Rer1+/inv-trap) and forebrain-specific Rer1 cKO (Rer1inv-trap/inv-trap; Emx-Cre) male mice. (G) The brain weight (left graph) or the length of cortex (right graph) of 5-month-old male control (Rer1+/inv-trap) and forebrain-specific Rer1 cKO (Rer1inv-trap/inv-trap; Emx-Cre) male mice. Error bars indicate the standard error of the mean (SEM) of three mice. Data are shown as the mean ± standard error of the mean (SEM). P*P t-test). (H, I) Hematoxylin and eosin (H&E)-stained sagittal sections from 5-month-old male control (Rer1+/inv-trap) and Rer1cKO (Rer1inv-trap/inv-trap; Emx-Cre) male mice. (H) The black line shows the length of the cerebral cortex. Shortening of the cerebrum was observed in Rer1 cKO mice. Cx: Cortex, Cb: Cerebellum. Scale bar, 1 mm. (I) Enlarged images of H&E-stained sections of mediolateral cerebral cortex. Cortical thinning and hippocampal morphogenesis anomalies were observed in forebrain-specific Rer1 cKO mice. Cx: Cerebral cortex, Hp, Hippocampus. Scale bar, 200 μm. (J) Neuron loss and gliosis occur in forebrain-specific Rer1 cKO mice. Homogenates of dissected cerebral cortex from control or forebrain-specific Rer1 cKO mice at 5 weeks were immunoblotted with anti-NeuN (neuronal marker) and anti-GFAP (astrocyte marker) antibodies. An anti-actin antibody was used as a loading control. An anti-Rer1 antibody was used to confirm the Rer1 level. (K) Quantitative analysis of the levels of NeuN and GFAP shown in panel J. Graphs show fold changes of NeuN or GFAP (signal intensity of targets normalized to that of actin) from forebrain-specific Rer1 cKO (cKO, gray bar) mice relative to control (Ctrl, black bar) mice. Error bars indicate the SEM of three mice. *P P t-test). (L) Gliosis in forebrain-specific Rer1 cKO mice. Immunohistochemistry using an anti-GFAP antibody on cerebral cortex from 5-month-old control (Rer1+/inv-trap) and forebrain-specific Rer1 cKO (Rer1inv-trap/inv-trap; Emx-Cre) male mice. Scale bar, 200 μm. (M) Graph show quantitative analysis of GFAP-positive cells in control (Ctrl, black bar) or forebrain-specific Rer1 cKO (cKO, gray bar) cortex. Error bars indicate the standard error of the mean (SEM) of six mice. **P t-test).