Chemical chaperone therapy for brain pathology in G(M1)-gangliosidosis

Abstract

We synthesized a galactose derivative, N-octyl-4-epi-beta-valienamine (NOEV), for a molecular therapy (chemical chaperone therapy) of a human neurogenetic disease, beta-galactosidosis (GM1-gangliosidosis and Morquio B disease). It is a potent inhibitor of lysosomal beta-galactosidase in vitro. Addition of NOEV in the culture medium restored mutant enzyme activity in cultured human or murine fibroblasts at low intracellular concentrations, resulting in a marked decrease of intracellular substrate storage. Short-term oral administration of NOEV to a model mouse of juvenile GM1-gangliosidosis, expressing a mutant enzyme protein R201C, resulted in significant enhancement of the enzyme activity in the brain and other tissues. Immunohistochemical stain revealed a decrease in the amount of GM1 and GA1 in neuronal cells in the fronto-temporal cerebral cortex and brainstem. However, mass biochemical analysis did not show the substrate reduction observed histochemically in these limited areas in the brain probably because of the brief duration of this investigation. Chemical chaperone therapy may be useful for certain patients with beta-galactosidosis and potentially other lysosomal storage diseases with central nervous system involvement.

Fig. 1.

Structure of inhibitors for β-glucosidase and β-galactosidase. (A) Synthetic derivatives of glucosylceramide (1) and galactosylceramide (2). (B) Structure of N-octyl-4-epi-β-valienamine (NOEV).

Fig. 2.

Spectral data of NOEV. Mass spectrum data of NOEV. (A)¹H-NMR. δ 0.900 (t, 3 H, J_8′7′ = 6.8 Hz, H-8′), 1.290-1.370 (m, 10 H, H-3′, 4′, 5′, 6′, and 7′), 1.490-1.540 (m, 2 H, H-2′), 2.555 (ddd, 1 H, J_gem = 11.2 Hz, J_1′b,2′ = 7.4 Hz, H-1′b), 2.739 (ddd, 1 H, J_gem = 11.2 Hz, J_1′a,2′ = 7.4 Hz, H-1′a), 3.101 (dd, 1 H, J_1,2 = 8.1 Hz, J_1,5a = 2.2 Hz, H-1), 3.435 (dd, 1 H, J_3,2 = 10.0 Hz, J_3,4 = 4.2 Hz, H-3), 3.696 (dd, 1 H, J_2,1 = 8.1 Hz, J_2,3 = 10.0 Hz, H-2), 4.121 (t, 2 H, J_6a,6b = 1.7 Hz, H-6a and 6b), 4.154 (d, 1H, J_4,3 = 4.2 Hz, H-4), 5.718 (d, 1H, J_5a,1 = 2.2 Hz, H-5a). (B)¹³C-NMR, δ 14.466 (C-8′), 23.766, 28.480, 30.448, 30.666, 30.983, 33.050 (C-2′≈7′), 46.925 (C-1′), 61.848 (C-1), 63.958 (C-6), 68.192 (C-4), 70.862 (C-2), 73.929 (C-3), 125.326 (C-5a), 140.716 (C-5). (C) Electrospray ionization mass. [M + H]⁺, [M + Na]⁺: m/z 288.04, 310.04.

Fig. 3.

Effect of NOEV after ganglioside loading on G_M1 storage in cultured mouse fibroblasts expressing human β-galactosidase gene. The cells expressing mutant (R201C) or wild-type (GP8) human β-galactosidase were loaded with ganglioside mixture, and then cultured with additional 0.2 μM NOEV (4 days). Shown is staining with FITC-labeled cholera toxin B. (A and C) R201C cells. (B and D) GP8 cells. (A and B) After NOEV treatment. (C and D) Before NOEV treatment. G_M1 storage was reduced to the control (GP8) level after NOEV treatment. There was no change in GP8 cells.

Fig. 4.

β-Galactosidase activity in mouse tissues after NOEV treatment. Results are shown for oral administration of NOEV water solution (0.1-1 mM) ad libitum for 1 week to R201C mice (3 months old). β-Galactosidase activity is expressed as nmol/mg of protein per 30 min (mean ± SD; n = 3).

Fig. 5.

X-Gal stain of mouse tissues. The R201C mouse, 3 months old, was given orally a water solution of 1 mM NOEV ad libitum for 1 week. The mouse tissues after NOEV treatment are more intensely stained than those without treatment. (A and D) Fronto-temporal cerebral cortex. (B and E) Trigeminal nucleus of brainstem. (C and F) Liver. (A, B, and C) After NOEV treatment. (D, E, and F) Before NOEV treatment. All tissues showed a remarkable increase of X-Gal stain.

Fig. 6.

Immunostaining of G_M1 and G_A1 in the mouse brain. The R201C mouse, 3 months old, was given orally a water solution of 1 mM NOEV ad libitum for 1 week, and the fronto-temporal cortex and brainstem were stained as described in Materials and Methods. (A and C) Fronto-temporal cerebral cortex. (B and D) Trigeminal nucleus of brainstem. (A and B) After NOEV treatment. (C and D) Before NOEV treatment. A remarkable decrease of both G_M1 and G_A1 is observed in the brain after the NOEV treatment.