Spinal cord neurone loss and foot placement changes in a rat knock-in model of amyotrophic lateral sclerosis Type 8

Abstract

Amyotrophic lateral sclerosis is an age-dependent cell type-selective degenerative disease. Genetic studies indicate that amyotrophic lateral sclerosis is part of a spectrum of disorders, ranging from spinal muscular atrophy to frontotemporal dementia that share common pathological mechanisms. Amyotrophic lateral sclerosis Type 8 is a familial disease caused by mis-sense mutations in VAPB. VAPB is localized to the cytoplasmic surface of the endoplasmic reticulum, where it serves as a docking point for cytoplasmic proteins and mediates inter-organelle interactions with the endoplasmic reticulum membrane. A gene knock-in model of amyotrophic lateral sclerosis Type 8 based on the VapB^P56S mutation and VapB gene deletion has been generated in rats. These animals display a range of age-dependent phenotypes distinct from those previously reported in mouse models of amyotrophic lateral sclerosis Type 8. A loss of motor neurones in VapB^P56S/+ and VapB^P56S/P56S animals is indicated by a reduction in the number of large choline acetyl transferase-staining cells in the spinal cord. VapB^-/- animals exhibit a relative increase in cytoplasmic TDP-43 levels compared with the nucleus, but no large protein aggregates. Concomitant with these spinal cord pathologies VapB^P56S/+ , VapB^P56S/P56S and VapB^-/- animals exhibit age-dependent changes in paw placement and exerted pressures when traversing a CatWalk apparatus, consistent with a somatosensory dysfunction. Extramotor dysfunction is reported in half the cases of motor neurone disease, and this is the first indication of an associated sensory dysfunction in a rodent model of amyotrophic lateral sclerosis. Different rodent models may offer complementary experimental platforms with which to understand the human disease.

Keywords: ALS8; VAPB; motor neurone disease.

Graphical Abstract

Figure 1

Genotype frequency and growth rates of VapB mutant rats. (A) Schematic of genotypes used in the study. VapB^P56S/+ and VapB^−/+ colonies were bred as heterozygous crossings. No significant difference was seen in the number of male and females and the relative frequency of homozygous and heterozygous animals followed the expected Mendelian pattern. (B) Immunoblot indicates VapB protein levels are reduced to ∼65% of WT in the brains of VapB^P56S/+ animals (64.9%, SD 6.4%, n = 5), and dramatically reduced in vapB^P56S/P56S [7.1%, SD 2.9%, n = 3 (1 male, 2 females), WT n = 4 (2 males, 2 females)]. No full-length VAPB is detected in VapB^−/− brain. (C). Long exposure of immunoblots, including VapB^−/+ samples, reveals a low level of smaller molecular weight proteins in the vapB^−/− animals that may represent the products of transcripts lacking Exon 2 or other alternatively spliced transcripts. Signal at 45 kDa cross-reacting signal serves as a loading control. Anti-sera #6 and #38 used for (B) and (C), respectively. See Supplementary Fig. 2 for uncropped immunoblots. (D) Body weight of female animals for all VapB genotypes was not significantly different from WT animals up to 18 months of age [Kruskal–Wallis test; H(3) = 3.858, P = 0.2772]. By 18 months of age, genotype significantly affected body weight [Kruskal–Wallis test; H(3) = 12.85, P = 0.005]. Male VapB^P56S/P56S and VapB^−/− animals were significantly heavier than WT animals (Dunn’s multiple comparisons test; VapB^P56S/P56S P < 0.01, VapB^−/− P < 0.05). For all genotypes, n ≥ 4, *P < 0.05, **P < 0.01.

Figure 2

Loss of spinal motor neurones in the ALS8 rat. At 18 months, there is a small reduction in ChAT-positive neurons in the lumbrical spinal cord of VapB^P56S/+ and VapB^P56S/P56S. (A) Ten micrometre sections from L3–L5 spinal cord were immunostained for ChAT. Threshold values selected structures in the ventral horn of between 700–2500 μm². (B) The number of large ChAT immunopositive cells was reduced in VapB^P56S/+ and VapB^P56S/P56S. (One-way ANOVA; F_3,18 = 3.817, P = 0.0281; Dunnett’s multiple comparisons test; VapB^P56S/+ P < 0.05, VapB^P56S/P56S P < 0.05.) Three sections were counted and averaged from each animal, n = 5–6 animals per genotype (WT, 3 males, 2 females. VapB^P56S/+, 3 males, 3 females. VapB^P56S/P56S, 3 males, 3 females. VapB^−/−, 2 males, 3 females) *P < 0.05. Scale bar on image 500 µm. Scale bar on threshold analysis 200 µm.

Figure 3

Increased cytoplasmic TDP-4 3 staining in motor neurones from VapB^−/−. Sections from the lumbar region of the spinal cord of 18-month-old VapB animals were immunostained for TDP-43. Signal intensity in nuclear and perinuclear regions was measured and used to determine the ratio of nucleus to cytoplasmic TDP-43. A relative increase in cytoplasmic TDP-43 was detected in the VapB^−/− animals. No large TDP-43 aggregates were detected in the cytoplasm. One-way ANOVA; F_3,19 = 5.613, P = 0.0063; Dunnett’s multiple comparisons test. Three sections containing at least six cells were analysed per animal. n = 5–7 animals per genotype (WT, 3 males, 3 females. VapB^P56S/+, 4 males, 3 females. VapB^P56S/P56S, 3 males, 2 females. VapB^−/−, 2 males, 3 females). +/−, standard error. **P < 0.01. Scale bar 50 μm.

Figure 4

CatWalk analysis of VapB mutant rats. There was no difference in the average speed at which animals traversed the CatWalk runway. Neither genotype, age nor their interaction had a significant effect upon the average speed at which the animals traversed the CatWalk runway (two-way RM ANOVA; F_3,28 = 2.105, P = 0.12; F_1,28 = 0.8333, P = 0.37; F_3,29 = 0.6027, P = 0.97). VapB animals had normal regularity of gait at 6 months but by 18 months a slight deficit was detected in VapB^P56S/P56S animals. Genotype significantly affected regularity (two-way RM ANOVA; F_3,28 = 6.292, P = 0.002) with 18-month-old VapB^P56S/P56S animals showing significantly reduced regularity compared with WT animals (Dunnett’s multiple comparisons test; P < 0.05).

Figure 5

Paw print size parameters decreased progressively over 18 months. There was a statistically significant interaction between the effects of age and genotype on both front and hind paw print length (two-way RM ANOVA; front F_3,28 = 4.639, P = 0.009 | hind F_3,28 = 6.088, P = 0.003). Main effects analysis reveals that front paw length is significantly reduced in VapB^−/− animals relative to WT at 6 and 18 months (Dunnett’s multiple comparisons test; 6 months P < 0.01, 18 months P < 0.001) with VapB^P56S/P56S animals only differed at 18 months (P < 0.001). Similar main effects analysis reveals that hind paw length is significantly reduced relative to WT in VapB^−/− and VapB^P56S/P56S animals at 6 and 18 months (all P < 0.001), while only significant at 18 months in VapB^P56S/+ animals (P < 0.001). A similar pattern was seen for paw print width and total paw print area.

Figure 6

Intensity reflects the pressure exerted on the CatWalk through the paws. Both the mean and maximum intensity values were reduced in the front and hind paws of VapB animals over 18 months compared with WT animals. There was a statistically significant interaction between the effects of age and genotype on both front and hind paw print mean intensity (two-way RM ANOVA; front F_3,28 = 5.187 P = 0.006 | hind F_2,28 = 5.708 P = 0.004) and maximum intensity (two-way RM ANOVA; front F_3,28 = 6.314 P = 0.002 | hind F_3,28 = 7.405 P < 0.001). Main effect analysis reveals that front paw mean intensity is significantly reduced relative to WT in VapB^−/− animals at 6 and 18 months (Dunnett’s multiple comparison test; 6 months P < 0.01, 18 months P < 0.001) but only by 18 months for VapB^P56S/P56S animals (P < 0.001). Significant differences in hind paw mean intensity relative to WT were only observed at 18 months in VapB animals (P56S/+ P < 0.05, P56S/P56S P < 0.001, −/− P < 0.01). A similar pattern was observed for maximum paw print intensity; font paw maximum intensity is significantly reduced VapB^−/− animals at 6 months (P < 0.001) and 18 months (P < 0.001) but only at 18 months for VapB^P56S/P56S (P < 0.001) and VapB^P56S/+ animals (P = 0.006); hind paw maximum intensity is significantly reduced in VapB^P56S/P56S animals at 6 months (P < 0.05) with all VapB genotypes significantly differing from WT at 18 months (P56S/+ P < 0.05, P56S/P56S P < 0.001, −/− P < 0.01). Ten runs were analysed and average from each animal at each time point, n = 7–10 animals per genotype (WT, 4 males, 3 females. VapB^P56S/+, 4 males, 3 females. VapB^P56S/P56S, 5 males, 5 females. Vap^−/−, 5 males, 4 females). *P < 0.05, **P < 0.01, ***P < 0.001.