Mixed Neurodevelopmental and Neurodegenerative Pathology in Nhe6-Null Mouse Model of Christianson Syndrome

Widespread reduction of neural tissue in aged Nhe6-null mice. A, C, E, G, I, Representative images of 30-μm brain sections from 22-mo-old WT and MUT male littermate mice after Nissl staining. Shown are coronal brain sections of cortex (A), striatum (C), and hippocampus (E) and sagittal sections of cerebellum (G) and spinal cord (I). Rightmost panels depict sections overlaid with anatomic labels for orientation purposes and blue lines indicating regions of measurement for assessment of atrophy. Two different anterior-posterior thicknesses of spinal cord (1 and 2) are shown and were analyzed (I). D, dorsal; L, lateral; V, ventral; M, medial; A, anterior; P, posterior. Scale bars, 200 μm. B, D, F, H, J, Graphs depicting quantification of thicknesses or areas of different brain regions in MUT male mice compared with WT male littermates, with each graph corresponding to the brain region reflected in the images to its left. WT n = 3, MUT n = 3 (B, D, and F); WT n = 2, MUT n = 4 (H); WT n = 2, MUT n = 2 (J). Data are presented as mean ± SEM. Statistical analyses were conducted using two-tailed Student’s t tests. *, p = 0.025 (B); ***, p = 0.00031 (D); **, p = 0.0098 (F); ****, p = 4.6 × 10^–5 (H); #, p = 0.060 (J, Region 1); p = 0.24 (J, Region 2).

Trajectories of brain tissue changes in Nhe6-null mice from 2 mo to 2 yr. A, C, E, G, Representative images of 30-μm brain sections from 2-mo-old WT and MUT male littermate mice after Nissl staining. Coronal sections were used for cortex (A), striatum (C), and hippocampus (E), and sagittal sections were used for cerebellum (G). Measurements were performed as in Fig. 2. Scale bars, 200 μm. B, D, F, H, Graphs depicting quantification of thicknesses or areas of different brain regions in MUT male mice compared with WT male littermates at time points of 2 months (2 m) and 22 months (22 m; from Fig. 2). Each graph corresponds to the brain region reflected in the images to its left. WT n = 3, MUT n = 3 for each age group (B, D, and F); WT n = 3, MUT n = 3 for 2 m and WT n = 2, MUT n = 4 for 22 m (H). Data are presented as mean ± SEM. Statistical analyses were conducted using two-way ANOVA followed by Tukey’s multiple comparison tests to compare the means for each genotype at each time point to one another. See Results for details. *, p = 0.035 (B); **, p = 0.0020 (WT 2 m to 22 m; D); **, p = 0.0021 (22 m; D); *, p = 0.033 (WT 2 m to 22 m; F); **, p = 0.0036 (22 m; F); *, p = 0.034 (2 m; H); ***, p = 0.0002 (MUT 2 m to 22 m; H); ****, p < 0.0001 (22 m; H).

Temporal progression of PC loss in the primary fissure of the vermis in Nhe6-null cerebellum. A, Representative images of hematoxylin and eosin-stained midsagittal sections of the vermal primary fissure from 12-mo-old WT and MUT male mice. PCF, preculminate fissure; PRI, primary fissure; PPF, prepyramidal fissure; ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale bars, 200 μm (left panels) and 50 μm (right panels). B, C, Representative images of midsagittal sections of the vermal primary fissure immunostained with the PC marker calbindin (red) in WT and MUT male mice at ages 5 mo (B) and 11–13 mo (C). Nuclei were stained with DAPI (blue). Vermis folia are labeled with Roman numerals according to the Allen Mouse Brain Atlas (Lein et al., 2007). Scale bars, 50 μm. D, E, Graphs depicting quantification of PC density (D) and overall calbindin signal (E) in WT and MUT male mice 5 months of age (5 m) and 11–13 months of age (11–13 m). Nhe6-null male mice (MUT) display significantly decreased PC density at both 5 m (*, p = 0.03) and 11–13 m (***, p < 0.001), as well as significantly decreased overall calbindin signal at both ages (**, p = 0.002, 5 m; ***, p = 0.001, 11–13 m). Furthermore, Nhe6-null mice exhibit a progressive decrease in PC density (**, p = 0.006, D) and overall calbindin signal (**, p = 0.01, E) over the 5- to 11–13-mo time period, a decrease that was not observed in WT male mice. F, Graph depicting the trajectory of PC density of MUT male mice in comparison to age-matched WT male mice based on data collected from mice 2, 5, or 11–13 mo. WT n = 3, MUT n = 3 for 5 m and WT n = 5, MUT n = 4 for 11–13 m (D and F); WT n = 4, MUT n = 4 for 5 m and WT n = 6, MUT n = 4 for 11–13 m (E); WT n = 2, MUT n = 2 for 2 m (F). Data are presented as mean ± SEM. Statistical analyses were conducted using two-tailed Student’s t tests.

Anatomic patterns of PC loss in Nhe6-null cerebellum. A, C, Representative images of midsagittal sections of the anterior lobe (lobules II and III), vermal primary fissure (lobules V and VI), and flocculonodular lobe (lobules IX and X) immunostained with the PC marker calbindin (red) in WT and MUT male mice at ages 5 mo (A) and 11–13 mo (C). Nuclei were stained with DAPI (blue). ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale bars, 50 μm. B, D, Graphs depicting quantification of PC density across cerebellar regions in WT and MUT male mice 5 mo (B) and 11–13 mo (D). At 5 mo, PC density is significantly decreased in the anterior lobe (**, p = 0.006) and primary fissure (*, p = 0.03, previously shown in Fig. 4B, D), but not the flocculonodular lobe (p = 0.17), in MUT male mice compared with WT male mice (B). Similarly, at 11–13 mo, MUT male mice show significantly decreased PC density in the anterior lobe (***, p < 0.001) and primary fissure (***, p < 0.001, previously shown in Fig. 4C, D), but not the flocculonodular lobe (p = 0.46), relative to WT male mice (D). Also, there is a statistically significant difference between cerebellar regions in Nhe6-null mice 11–13 mo of age [F_(2,7) = 20.11, p = 0.001], but not in Nhe6-null mice 5 mo of age. WT n = 3, MUT n = 3 for vermal primary fissure and flocculonodular lobe (B); WT n = 3, MUT n = 4 for anterior lobe (B); WT n = 4, MUT n = 3 for anterior lobe and flocculonodular lobe (D); WT n = 5, MUT n = 4 for vermal primary fissure (D). Data are presented as mean ± SEM. Statistical analyses were conducted using two-tailed Student’s t tests for group comparisons and one-way ANOVA followed by Tukey’s multiple comparison tests for cerebellar region comparisons. Note that representative vermal primary fissure images from mice at ages 5 mo (A) and 11–13 mo (C) are the same as those for the respective time points in Fig. 4B, C.

PC loss in the cerebellar vermis as opposed to lateral regions. A, B, Representative images of sagittal sections of whole cerebellar periphery immunostained with the PC marker calbindin (red) in WT and MUT male mice at 5 mo of age. Images in B reflect higher magnification of the respective boxed regions in A. Nuclei were stained with DAPI (blue). ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale bars, 500 μm (A) and 50 μm (B). C, Graph depicting quantitative analysis of PC density in the cerebellar vermis (from Fig. 4D) versus the cerebellar periphery in WT and MUT male mice of 5 mo of age. Differences in PC density in peripheral regions of the cerebellum are not detected in WT versus MUT male mice at this time point (p = 0.16). However, the peripheral cerebellum shows a significantly greater PC density compared with the cerebellar vermis at 5 mo in both WT mice (*, p = 0.02) and MUT mice (**, p = 0.006). As shown in Figs. 4 and 5, Nhe6-null male mice (MUT) at 5 mo of age display significantly decreased PC density in the primary fissure of the vermis (*, p = 0.03). WT n = 3, MUT n = 3 for each cerebellar region analyzed. Data are presented as mean ± SEM. Statistical analyses were conducted using two-tailed Student’s t tests.

Anatomic patterns of cerebellar PC loss in a new exons 2/3 Nhe6-null mouse model. A, Schematic of the targeting approach and vector for generating the exons 2/3 Nhe6-null mouse model. B, C, Loss of Nhe6 mRNA and NHE6 protein expression in the exons 2/3 Nhe6-null mouse model was confirmed via PCR, using mouse tail clippings as a sample source (B), and via Western blot, using whole-brain lysate as a sample source (C), respectively. For Western blotting, the membrane was first probed to detect NHE6, after which the membrane was stripped and reprobed for α-tubulin as a loading control. WT, wild-type; HET, heterozygous; MUT, exons 2/3 Nhe6-null mutant. D, E, Representative images of midsagittal sections of whole cerebellar vermis immunostained with the PC marker calbindin (red) in WT and exons 2/3 Nhe6-null MUT male mice at 6 mo of age. Lower-magnification images depicting lobules I through X are shown in D, and higher-magnification images depicting specific lobules and regions as indicated are shown in E. Nuclei were stained with DAPI (blue). ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale bars, 500 μm (D) and 50 μm (E). F, Graph depicting quantification of PC density across cerebellar regions in WT and MUT male mice at 6 mo of age. Compared with WT males, exons 2/3 MUT male mice exhibit significantly decreased PC density in the anterior lobe (*, p = 0.03) and primary fissure (*, p = 0.02), yet a statistically significant difference is not observed in the flocculonodular lobe (p = 0.75). Furthermore, MUT male mice exhibit a region-specific decrease in PC density in the primary fissure and anterior lobe, but not in the flocculonodular lobe [F_(2,6) = 6.94, p = 0.03]. WT n = 2, MUT n = 3. Data are presented as mean ± SEM. Statistical analyses were conducted using two-tailed Student’s t tests for group comparisons and one-way ANOVA followed by Tukey’s multiple comparison tests for cerebellar region comparisons.

PC loss in Nhe6-null mouse cerebellum, female and male. A, B, Representative images of midsagittal sections of whole cerebellar vermis and the vermal primary fissure immunostained with the PC marker calbindin (red) in WT and MUT female (A) and male (B) mice at 5 mo of age. Lower-magnification images depicting lobules I through X are shown in the left and middle panels, and higher-magnification images depicting the primary fissure are shown in the rightmost panels. Nuclei were stained with DAPI (blue). ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale bars, 500 μm (left and middle panels) and 50 μm (right panels). C, D, Graphs depicting quantitative analysis of PC density (C) and overall calbindin signal (D) in the vermal primary fissure in WT and MUT female and male (from Fig. 4D, E) mice at 5 mo of age. Nhe6-null female mice display significantly decreased PC density (**, p = 0.007, C) and overall calbindin signal (***, p = 0.001, D) compared with WT female mice. As shown in Fig. 4D, E, Nhe6-null male mice at 5 mo of age display significantly decreased PC density (*, p = 0.03, C) and overall calbindin signal (**, p = 0.002, D), respectively, compared with WT male mice. There are no significant differences between Nhe6-null female and male mice. WT n = 3, MUT n = 4 for female (C and D); WT n = 3, MUT n = 3 for male (C); WT n = 4, MUT n = 4 for male (D). Data are presented as mean ± SEM. Statistical analyses were conducted using two-tailed Student’s t tests.

Microglia in the corpus callosum of aged Nhe6-null mouse brain. Representative images of 30-μm coronal brain sections through the CC from 22-mo-old WT and MUT male littermate mice after triple immunohistochemical staining with antibodies against NeuN (green), a marker for neurons; GFAP (red), a marker for reactive astrocytes; and Iba1 (blue), a marker for microglia. Shown are merged images and images of single channels for NeuN, GFAP, and Iba1, respectively. The images reveal strong reactivity of microglia and astrocytes in the CC of aged (22-mo-old) MUT male mice compared with WT male littermates. Scale bars, 200 μm.

Microglia in the cortex, striatum, and hippocampus of aged Nhe6-null mouse brain. A, B, C, Representative images of 30-μm coronal brain sections from 22-mo-old WT and MUT male littermate mice after triple immunohistochemical staining with antibodies against NeuN (green), a marker for neurons; GFAP (red), a marker for astrocytes; and Iba1 (blue), a marker for microglia. Shown are merged images and images of single channels for GFAP and Iba1, respectively, for sections of cortex (A), striatum (B), and hippocampus (C). The images reveal gliosis, as indicated by Iba1 immunostaining, in the respective brain regions of aged (22-mo-old) MUT male mice compared with WT male littermates. Scale bars, 50 μm (A and B) and 200 μm (C). Di-iii, Graphs depicting quantification of gliosis, based on the number of Iba1-positive cells in a region of defined size, in the cortex (Di), striatum (Dii), and hippocampus (Diii) of WT and MUT male littermate mice at 22 mo of age. Microglia density is significantly increased in MUT male mice compared with WT male littermates in the cortex (*, p = 0.019, Di), striatum (**, p = 0.0054, Dii), and CA region of the hippocampus (**, p = 0.0048, Diii) but not in the DG region of the hippocampus (p = 0.18). CA, cornu Ammonis; DG, dentate gyrus. Images similar to those shown in A, B, and C were used for quantitation, with the number of microglia in 228-μm² regions being counted and summed across the total number of regions analyzed. The number of 228-μm² regions analyzed for each brain region for each mouse brain were as follows: 16 (cortex), 36 (striatum), 4 (hippocampus, DG), and 6 (hippocampus, CA). WT n = 3, MUT n = 4. Data are presented as mean ± SEM. Statistical analyses were conducted using two-tailed Student’s t tests.

Microglia in the cerebellum and spinal cord of aged Nhe6-null mouse. A, B, Representative images of 30-μm sagittal sections from 22-mo-old WT and MUT male littermate mice after triple immunohistochemical staining with antibodies against NeuN (green), a marker for neurons; GFAP (red), a marker for astrocytes; and Iba1 (blue), a marker for microglia. Shown are merged images and images of single channels for GFAP and Iba1, respectively, for sections of cerebellum (A) and spinal cord (B). The images reveal gliosis, as indicated by Iba1 immunostaining, in the respective regions of aged (22-mo-old) MUT male mice compared with WT male littermates. Roman numerals overlaying images in A reflect the respective lobules of the vermis folia. GCL, granule cell layer; ML, molecular layer. Scale bars, 100 μm (A) and 200 μm (B).

Activated microglia in the corpus callosum of aged Nhe6-null mouse brain. Representative images of 30-μm coronal brain sections through the CC of 22-mo-old WT and MUT male littermate mice after triple immunohistochemical staining with antibodies against NeuN (green), a marker for neurons; CD68 (red), a marker for activated microglia; and Iba1 (blue), a marker for microglia. Shown are merged images and images of single channels for NeuN, CD68, and Iba1, respectively. The images reveal a more prominent presence of activated microglia, as indicated by microglia containing large CD68-positive puncta, in the CC of aged (22-mo-old) MUT male mice compared with WT male littermates. Scale bar, 50 μm.

Activated microglia in the cortex, striatum, hippocampus, and cerebellum of aged Nhe6-null mouse brain. A–D, Representative images of 30-μm brain sections from 22-mo-old WT and MUT male littermate mice after triple immunohistochemical staining with antibodies against NeuN (green), a marker for neurons; CD68 (red), a marker for activated microglia; and Iba1 (blue), a marker for microglia. Coronal sections were used for cortex (A), striatum (B), and hippocampus (C), and sagittal sections were used for cerebellum (D). Shown are merged images and single-channel images for CD68. ML, molecular layer; GCL, granule cell layer. Scale bars, 50 μm. E, Graph depicting quantification of microglial activation, based on the size of CD68-positive puncta in Iba1-positive cells. For hippocampus, the analysis was divided into two subregions, CA and DG. No CD68 size analysis was done in CA, since there were few CD68-positive microglia and the CD68-positive puncta that were present were very small in size. The size of CD68-positive puncta is significantly increased in MUT male mice compared with WT male littermates in cortex (*, p = 0.015). Although not reaching statistical significance, an increase in size is also noted in striatum (p = 0.11). For the DG of the hippocampus, the size of CD68-positive puncta is not statistically different between MUT male mice and WT male littermates (p = 0.34). WT n = 3, MUT n = 3. Data are mean ± SEM. Statistical analyses were conducted using two-tailed Student’s t tests.

Gliosis in other axonal tracts in aged Nhe6-null mouse. A–C, Representative images of 30-μm brain sections from 22-mo-old WT and MUT male littermate mice after triple immunohistochemical staining with antibodies against NeuN (green), a marker for neurons; CD68 (red), a marker for activated microglia; and Iba1 (blue), a marker for microglia. Shown are merged images for coronal sections of the anterior commissure (A) and medial septum (B), and sagittal sections of the spinal cord (C). The images reveal the presence of abnormal gliosis, as indicated by the strong immunostaining for CD68 and Iba1, in axonal tracts of various regions of aged (22-mo-old) MUT male mice compared with WT male littermates. Scale bars, 50 μm.