Substrate compositional variation with tissue/region and Gba1 mutations in mouse models--implications for Gaucher disease

Abstract

Gaucher disease results from GBA1 mutations that lead to defective acid β-glucosidase (GCase) mediated cleavage of glucosylceramide (GC) and glucosylsphingosine as well as heterogeneous manifestations in the viscera and CNS. The mutation, tissue, and age-dependent accumulations of different GC species were characterized in mice with Gba1 missense mutations alone or in combination with isolated saposin C deficiency (C*). Gba1 heteroallelism for D409V and null alleles (9V/null) led to GC excesses primarily in the visceral tissues with preferential accumulations of lung GC24∶0, but not in liver, spleen, or brain. Age-dependent increases of different GC species were observed. The combined saposin C deficiency (C*) with V394L homozygosity (4L;C*) showed major GC18:0 degradation defects in the brain, whereas the analogous mice with D409H homozygosity and C* (9H;C*) led to all GC species accumulating in visceral tissues. Glucosylsphingosine was poorly degraded in brain by V394L and D409H GCases and in visceral tissues by D409V GCase. The neonatal lethal N370S/N370S genotype had insignificant substrate accumulations in any tissue. These results demonstrate age, organ, and mutation-specific quantitative differences in GC species and glucosylsphingosine accumulations that can have influence in the tissue/regional expression of Gaucher disease phenotypes.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Glucosylceramide (GC) in Gba1 mouse tissues.

Total GC levels in liver, lung, and spleen from 9V/null (red), 9H/9H (violet), 9V/9V (green), and 4L/4L (orange) mice. In 9V/null the GC levels progressively accumulated from 4 to 52 wks and at greater rates in the lung than in liver and spleen. 9H/9H, 9V/9V, and 4L/4L mice had GC increases slightly above WT (black) levels. Data are mean±S.E. (n = 3–4 mice).

Figure 2. GC species in the liver and lung.

Each panel presents GC species levels in mice having the indicated Gba1 mutation: D409H (9H/9H and 9H;C*), V394L (4L/4L and 4L;C*), D409V (9V/9V, 9V/null, and 9V;C*) and N370S (0S/0S and 0S;C*). The panels are grouped to reflect the various mutations to facilitate comparison of the GC levels. The ordinates differ to facilitate visualizing the differing levels of GC. Age-matched WT tissues were controls for each analysis. 4L;C* lung at 6 wks was compared to WT at 9 wks. Each GC species level in the mutant was analyzed relative to age-matched WT using Student’s t-test. *, p<0.05, **, p<0.01, ***, p<0.0001, ****, p<0.00001. The results and error bars are the mean+S.E. (n = 3–4 mice).

Figure 3. GC species in the spleen and brain.

GC species in the spleen (9H/9H, 9H;C*, 4L/4L, 4L;C*, 9V/9V, and 9V/null), whole brain (9H;C*, 4L;C*, 9V;C*, 0S/0S, and 0S;C*) or cerebral cortex (9H/9H, 9V/9V, 4L/4L, and 9V/null) are presented. GCs in the brain extracts were resolved from galactosylceramide by hydrophobic interaction liquid chromatography and quantified by MS. The spleen of ∼1 day-old mice (0S/0S, 9V;C* and 0S;C*) were not determined. 4L;C* spleen at 6 wks was compared to WT at 9 wks. The data analyses were as in Figure 2.

Figure 4. Glucosylsphingosine levels in Gba1 mutant mice.

Each graph depicts glucosylsphingosine levels in four tissues from mice having the same Gba 1 mutation: D409H (9H/9H and 9H;C*), V394L (4L/4L and 4L;C*), D409V (9V/9V, 9V/null and 9V;C*) and N370S (0S/0S and 0S;C*). Age-matched WT tissues were included as control. The data analyses were as in Figure 2.

Figure 5. Analyses of substrate accumulation in Gba 1 mutant mice.

(A) The mean proportion of each GC species relative to total GC (6 GC species) in age-matched Gba 1 mutants and WT (52 wks) visceral tissues were plotted. In the lung, GC24∶0 was significantly increased in 9V/null, whereas other species were reduced. In the liver, proportion of GC16∶0 was significantly increased in 9H/9H, but not in liver from other mutant mice. GC24∶0 had reduced proportions in 9H/9H and 9H;C* livers, whereas GC24∶1 was increased in 9V/null and 9H;C* livers compared to WT and other mutants. In the spleen, the proportion of GC20∶0 was significantly increased in 9H;C*. Reduced GC species were present in spleen of some mutant mice: GC24∶0 in 9H;C*, GC20∶0 in 9V/null, and GC24∶0 in 9V/9V. The black stars indicate that the proportion of a GC species was significantly increased and the pink stars indicate that the proportion of a GC species was reduced relative to WT. The color gradients indicate the proportion of total GC species represented by GCs with a defined FAAC. (B) The pie charts show the proportions of each GC species in 9V/null lung and WT at 52 wks. The GC24∶0 was 63% in 9V/null lung compared with 39% in WT. The proportions of the other GC species were concomitantly reduced in 9V/null lung. (C) In brain extracts, the proportion of GC18∶0 was significantly increased only in 4L;C* (black star) mice. In 9H;C* brain GC18∶0 was reduced (pink star) and long chain GC species, GC24∶0 and GC24∶1, were significantly increased (black stars). The proportion of each GC species in WT brain at 6 wks and 52 wks were included in the analyses. (D) Glusosylsphingosine concentrations in tissues from each mutant were plotted as the mean of concentration. Significant increases of glusosylsphingosine (black stars) were in 4L;C* brain and spleen, 9H;C* brain, and 9V/null lung, liver and spleen. The glucosylsphingosine levels in WT tissues at 6 wks and 52 wks were included in the analyses. The color gradient indicates glucosylsphingosine concentrations (pmol/mg tissues). The data in (A), (C) and (D) were analyzed with Tukey’s HSD test. The samples that showed significant difference from WT and other mutants (p<0.05) were marked with star (*).

Figure 6. Heatmap of GC species in Gba1 mutant mice.

The fold changes of GC species and glucosylsphingosine levels in individual mutant mice were age-grouped for each organ according to genotype using Gene Spring 11.5.1. The fold change was obtained for each condition relative to the average value of the age-matched WT control. The color gradients represent level of fold change. Yellow indicates that the GC species levels in the mutant were at WT levels and the fold change was 1. The gradient from yellow to red indicates GC species levels in the mutants were above WT levels and fold change were >1, and yellow to green represents

Figure 7. Age-dependent changes of GC species levels in WT mouse tissues.

All GC species in WT mice showed age-dependent decrease with age (from