Mice doubly-deficient in lysosomal hexosaminidase A and neuraminidase 4 show epileptic crises and rapid neuronal loss

Abstract

Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the α-subunit of lysosomal β-hexosaminidase A, which converts G(M2) to G(M3) ganglioside. Hexa(-/-) mice, depleted of β-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise G(M2) ganglioside via a lysosomal sialidase into glycolipid G(A2), which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4(-/-);Hexa(-/-)) show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa(-/-) or Neu4(-/-) siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating G(M2) ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa(-/-) mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa(-/-) mice.

Figure 1. Representative electroencephalography (EEG) recordings from double deficient Neu4^−/−;Hexa^−/− and single-deficient Hexa ^−/− mice.

EEG of Neu4^−/−;Hexa^−/− mouse (A) shows simultaneously with a myoclonic jerk (indicated as “twitch” on the figure) high amplitude polyspike discharges involving the cortical electrode with no diffusion to the underlying hippocampus. The EEG of Hexa ^−/− mouse (B) is normal. Inset shows the enlargement of the myoclonic event. Each vertical line on the graph is 1 sec, voltage scale is 30 µV/mm.

Figure 2. Light microscopy of neurons in deep cortex of wild type mice, single Neu4^−/− or Hexa^−/− knockouts and in double Neu4^−/−;Hexa^−/− knockouts without and with seizures.

Affected neurons containing vacuolated cytoplasm shown by arrows are present in Hexa^−/− knockouts (C) and in double knockouts without (D) and with (E) seizures, but absent in wild type mice (A) and single Neu4^−/− knockouts (B). Magnification ×1000. Panels represent typical images obtained for 3 mice for each genotype.

Figure 3. Percent of affected neurons in cortex and hippocampus.

Normal and affected (vacuolized) neurons were counted in 9 epon sections from similar cortical and hippocampal regions of each mouse; 3 mice were studied for each genotype. * - significantly different from Hexa ^−/−.

Figure 4. Electron micrographs of mouse brain tissues.

Cortical neurons from wild type (A) and Neu4^−/− mice (B) show normal lysosomes. Cortical neurons from Hexa^−/− (C) knockouts, and double (Neu4^−/−;Hexa^−/−) knockouts without (D) and with seizures (E) show accumulation of whorls of membranes in lysosomes characteristic of neurons of Tay-Sachs patients. Bars range between 1 and 2 µm. Panels represent typical images obtained for 3 mice for each genotype.

Figure 5. Alteration of G_M2 ganglioside catabolism in the brain tissues of Neu4^−/−;Hexa^−/− mice.

Histograms show the ratio of G_M2 and G_M1 gangliosides as measured by TLC in the extracts of brain tissues from wild-type (WT); Neu4^−/−,Hexa^−/− and Neu4^−/−;Hexa^−/− mice. Values represent means ± S.D. of duplicate measurements performed on 3–11 mice of 4–12 month of age. * p<0.001 as compared with the Hexa^−/− and Neu4^−/−;Hexa^−/− animals by the two-tailed nonparametric t-test. Inset: Representative TLC images of orcinol-stained gangliosides from brain of the WT, Neu4^−/−, Hexa^−/− and Neu4^−/−;Hexa^−/− mice at 12 months of age. Arrows indicate the positions of the ganglioside standards.

Figure 6. Increased accumulation of G_M2 ganglioside in brain neurons of double knockout Neu4^−/−; Hexa^−/− mice.

(A) Typical images of coronal sections from wild type (WT) mouse, mouse with HexA deficiency (Hexa^−/−) and from double knockout mice without (Neu4^−/−;Hexa^−/−) and with episodes of seizures (Neu4^−/−;Hexa^−/− S) stained with anti-G_M2 antibodies. H indicates hippocampus, C – cortex. Scale bar: 100 µm. (B) Amount of neurons storing G_M2-ganglioside in cortex from Neu4^−/−;Hexa^−/− mice with and without episodes of seizures and from a Hexa^−/− mice. The cells were counted in 10 sections from similar cortical regions of each mouse. Three mice were studied for each group. * - significantly different from Hexa^−/−.

Figure 7. Numerous neurons in ventral cortex of double knockout, epileptic mouse store G_M2.ganglioside.

This panel shows G_M2 (green, right panels) and NeuN (red, middle panels) immunostaining in dorsal (A) and ventral (B) cortex in a double knockout, epileptic mouse. G_M2 positive neurons (yellow, right panels) are much more numerous in ventral cortex compared to dorsal cortex. Scale bar: 100 µm. (C) High magnification images showing G_M2 (green, right panel) and NeuN (red, middle panel) immunostaining in layer 56 of ventral cortex in a double knockout, epileptic mouse. NeuN staining is stronger in the perinuclear region and weaker in the cytoplasm of neuronal somata. Scale bar: 5 µm. Panels represent typical images; 10 panels were studied.