Figure 6. Late-Onset Neuroinflammation and Axonopathy in NR1 cKO Mice.

(A) Indirect signs of neurodegeneration in white matter tracts at 1 year of age and overview (left) of sagittal cerebellum-spinal cord section from NR1 cKO mice. Signs of local inflammation (Mac3 immunostaining) were more obvious in the ventral white matter (vWM) compared to spinal cord gray matter (GM). Higher magnification (region marked by a red rectangle) reveals activated microglia (Mac3). Scale bar, 20 μm. See also Figure S7.

(B) Neuroinflammation in the ventral white matter of NR1 cKO mice confirmed by increased density of cell nuclei (p = 0.029, n = 4, Student’s t test). In adjacent GM, nuclear densities are unaltered.

(C) Quantification of the Mac3⁺ immunostained area (microgliosis) in vWM of NR1 mutant mice (p = 0.0004, n = 4, Student’s t test)

(D) Quantification of the GFAP⁺ area (astrogliosis) in vWM of NR1 mutant mice (p = 0.013, Student’s t test).

(E and F) By electron microscopy of ventral cervical spinal cord cross sections (E), ultrastructural features of axonal pathology and degeneration were more frequent in NR1 mutant mice compared to controls (F) (n = 4–5 mice with 12–14 randomly taken images, covering 530 μm² each, p = 0.0014, Student’s t test). Scale bar, 2 μm. In (E): A, axonal degeneration; B, blebbing membranes; D, delamination; N, normal myelin.

(G) Motor deficits of NR1 cKO mutants at 10–11 months of age, demonstrated by Rotarod testing on three consecutive days (repeated the following month). The latency to fall is decreased in NR1 mutant mice (red line) compared to littermate controls (F_genotype (1, 17) = 4.95, p = 0.040, n = 9–10, two-way ANOVA).

(H) At age 19 months, NR1 cKO mice (n = 11) display significant neurological deficits compared to controls (n = 8, p < 0.001 Student’s t test). Clinical scores are 0, asymptomatic; 1, hind limb clasping; 2, additionally, hunchback visible; 3, additionally, frequent slipping while walking on grid; 3.5, additionally, failure to hold on inverted grid; 4, additionally, hind limb ataxia and unable to hold on upright grid; 4.5, spastic hind limbs.

(I) Brain sections of 19-month-old NR1 cKO mice immunostained for Mac3⁺ show widespread signs of neuroinflammation in white matter tracts; corpus callosum and fimbria are magnified (right panel).

(J) Quantification of Mac3⁺ immunostained area in NR1 mutants compared to littermate controls and age-matched Cnp1^Cre/+ mice (n = 4–6 mice, p < 0.001, linear mixed effects analysis with post hoc Tukey correction for multiple comparisons).

(K) Axonopathy in white matter tracts revealed by APP immunolabeling (arrow heads) in brain sections of 19-month-old NR1 cKO mice; corpus callosum, fimbria, and subventricular white matter are shown.

(L) Quantification of APP spheroids in NR1 mutants in comparison to littermate controls and age-matched Cnp1^Cre/+ mice (n = 4–6 mice; control versus NR1 cKO p < 0.001 and Cnp1^Cre/+ versus NR1 cKO p = 0.03, linear mixed effects analysis with post hoc Tukey correction for multiple comparisons).