Context-dependent perturbation of neural systems in transgenic mice expressing a cytosolic prion protein

Abstract

We analyzed the relationship between pathogenic protein expression and perturbations to brain anatomy and physiology in a genetic model of prion disease. In this model, the mouse line 1D4, neuropathology is promoted by accumulation of a cytosolic form of the prion protein (cyPrP). CyPrP distribution was determined and compared with anatomical magnetic resonance imaging (MRI) data, a form of functional MRI based on manganese labeling, and immediate early gene mapping with an antibody to c-Fos. Significant discrepancies between 1D4 and control mice became apparent well in advance of overt behavioral pathology in the mutant mice. Alterations to brain structure and function in the mutants varied among brain regions, however, and differed strikingly even among regions with the highest levels of cyPrP expression. In the cerebellum, gross neurodegeneration was accompanied by increased Mn(2+)-enhanced MRI signal, raising the possibility that compensatory mechanisms act to preserve cerebellar function in the face of massive atrophy. In the hippocampus of 1D4 mice, no significant structural alterations were observed, but both Mn(2+)-enhanced MRI and c-Fos data indicated perturbations to neurophysiology. In the neocortex, there were no clear neural activity differences between 1D4 and control animals, but mutant mice showed significant reduction in cortical thickness. Our finding that distinct combinations of anatomical and functional abnormalities accompanied cyPrP overexpression in different parts of the brain indicates the importance of context in conditioning effects of protein pathogens, and exemplifies the notion that neurodegenerative phenotypes extend beyond cell death and the immediate consequences of atrophy for particular neural systems.

Fig. 1.

Expression pattern of cyPrP in transgenic mice. Immunohistochemistry with an antibody against PrP was performed on sagittal brain sections from mice expressing cyPrP on a PrP^C knock-out background. No counter stain was used. Mutant protein expression varied strongly among brain regions (A), and could be visualized with multiple exposure times (see Supplementary Fig. S1); scale bar=1 mm. Knock-out mice without the transgene showed no staining (not shown). Close-up views are shown for regions of interest denoted by black boxes in panel A. (B) In the neocortex, high cyPrP expression was observed, particularly in layer 2 (layers 1–4 numbered, scale bar=100 μm for panels B–D). (C) The hippocampus showed high expression (dentate gyrus, DG, and CA3 subfields labeled). (D) Prominent cyPrP expression was also observed in the cerebellum; here, levels were highest in the granule cell layer (G), but sporadic expression was also observed in the Purkinje (P) and molecular (M) layers.

Fig. 2.

Comparison of brain anatomy between 1D4 and WT animals in two age groups. Macroscopic anatomical differences between 1D4 mutants and wild-type controls were quantified using MRI data from anesthetized animals. Characteristic landmarks were identified in brain images, and distances between pairs of landmarks were measured. Landmark positions are shown here superimposed on horizontal, coronal, and sagittal sections (top to bottom) through the Mn²⁺-enhanced T₁-weighted MRI scan of a representative WT animal (A); image resolution 125 μm (isotropic), scale bar=1 mm. Distances were quantified from the brains of 2–5 mo animals (B) and 7–12 mo animals (C). Bars denote mean±S.E.M. for distance measurements from WT (black) and 1D4 (gray) animals, using landmarks in the cerebellum (Cb), neocortex (Cx), and hippocampus (H). Asterisks indicate comparisons judged to be significant after Bonferroni correction for multiple tests (Mann–Whitney U test, p<0.005).

Fig. 3.

Brain region-specific differences in manganese-enhanced MRI signal between mutant and control mice. T₁-weighted MRI scans were obtained 24 h after intraperitoneal injection of the contrast agent MnCl₂ (37.7 mg/kg). During the labeling period, mice were maintained in their home cages, without additional stimulation. Images from 1D4 and WT animals were spatially normalized and used to compute a three-dimensional t-value map indicating signal differences between the two sets of animals (A). The top and bottom panels show sagittal and horizontal sections through the t-value map, respectively; relative positions of the sections are denoted by dotted lines. “Hot” colors indicate areas where mutant animals had significantly greater MRI signal than controls; “cold” colors indicate areas where mutant animals displayed hypointense signal. Both positive and negative t-values were thresholded using p < 0.0054 (|t|≥3.19, false discovery rate 10%) as a criterion for statistical significance. Independent analysis of manganese-enhanced MRI signal changes was performed by defining regions of interest (ROIs) in individual brain images, and averaging ROI-specific signal from multiple animals of each strain and age group. Results of this comparison are shown for ROIs in the cerebellum (Cb), the neocortex (Cx), and the hippocampus (H), both in 2–5mo animals (B) and in 7–12 mo animals (C). Insets in the bar graphs show average signal in the hippocampus, broken down by subfield (CA1, CA3, and DG). Black and gray bars correspond to WT and 1D4 animals, respectively, with error bars denoting S.E.M.

Fig. 4.

High resolution analysis of Mn²⁺-enhanced MRI intensity in the cerebellum. Representative T₁-weighted images (TE/TR=6/50 ms) acquired on a 9.4 T MRI scanner, with 50 μm in-plane and 100 μm out of plane resolution. MRI signal from three adjacent sagittal slices around the midline was averaged to produce the images shown for individual WT (left) and 1D4 cyPrP transgenic (right) animals (both 12 mo). Both images show a narrow lamina of hyperintense signal winding through the cerebellar cortex, probably corresponding to the Purkinje cell layer (P). Inner and outer gray matter regions correspond to the granular (G) and molecular (M) layers of the cerebellum, respectively. White matter (wm) appears hypointense. Scale bars are divided into 1 mm increments; comparison of the scale bars with WT and 1D4 sagittal cross sections indicates the pronounced reduction in cerebellar size observed in cyPrP-expressing mutants.

Fig. 5.

c-Fos expression after exposure to a novel environment. Expression of c-Fos was visualized using standard immunohistochemical methods. Foci of c-Fos accumulation were apparent in the hippocampus of a representative WT animal (A). The numbers of c-Fos positive nuclei per unit area were determined for WT (black) and 1D4 (gray) animals for ROIs in the neocortex (Cx) and hippocampus (H), including hippocampal subfields (inset). Error bars denote the S.E.M. for each measurement, and asterisks identify comparisons for which differences between WT and 1D4 mice were statistically significant after Bonferroni correction.