Yeast-Produced Human Recombinant Lysosomal β-Hexosaminidase Efficiently Rescues GM2 Ganglioside Accumulation in Tay-Sachs Disease

Abstract

Background: Tay-Sachs disease (TSD) is an autosomal recessive lysosomal storage disorder characterized by the accumulation of GM2 ganglioside due to mutations in the HEXA gene, which encodes the α-subunit of β-Hexosaminidase A. This accumulation leads to significant neuropathological effects and premature death in affected individuals. No effective treatments exist, but enzyme replacement therapies are under investigation. In our previous work, we demonstrated the internalization and efficacy of human recombinant lysosomal β-hexosaminidase A (rhHex-A), produced in the methylotrophic yeast Pichia pastoris, in reducing lipids and lysosomal mass levels in fibroblasts and neural stem cells derived from patient-induced pluripotent stem cells (iPSCs). In this study, we further evaluated the potential of rhHex-A to prevent GM2 accumulation using fibroblast and neuroglia cells from a TSD patient alongside a relevant mouse model. Methods: Fibroblasts and neuroglial cell lines derived from a murine model and TSD patients were treated with 100 nM rhHexA for 72 h. After treatment, cells were stained by anti-GM2 (targeting GM2 ganglioside; KM966) and anti-LAMP1 (lysosomal-associated membrane protein 1) colocalization staining and incubated with 50 nM LysoTracker Red DND-99 to label lysosomes. In addition, GM2AP and HEXB expression were analyzed to assess whether rhHex-A treatment affected the levels of enzymes involved in GM2 ganglioside degradation. Results: Immunofluorescence staining for LysoTracker and colocalization studies of GM2 and Lamp1 indicated reduced lysosomal mass and GM2 levels. Notably, rhHex-A treatment also affected the expression of the HEXB gene, which is involved in GM2 ganglioside metabolism, highlighting a potential regulatory interaction within the metabolic pathway. Conclusions: Here, we report that rhHex-A produced in yeast can efficiently degrade GM2 ganglioside and rescue lysosomal accumulation in TSD cells.

Keywords: GM2; P. pastoris; Tay–Sachs; murine model; rhHex-A.

Figure 1

LysoTracker staining of recombinant human Hexa protein (rhHex-A, 100 nM) in treated fibroblasts (A) and neuroglia (B) derived from wild-type (WT), Hexa-/-, and Hexa-/-Neu3-/- mice. ImageJ v1.54 program measured the total LysoTracker signal, and the histogram for LysoTracker intensity per cell for fibroblasts (C) and neuroglia (D) is represented. Images were taken under the same light intensity at 100× magnification, and the data are presented as the mean ± S.E.M. Two-way ANOVA analysis was used to determine p-values, as indicated by GraphPad (* p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001) (n = 3).

Figure 2

LysoTracker staining of recombinant human Hexa protein (rhHex-A, 100 nM)-treated fibroblasts (A) and neuroglia (B) cells derived from a human TS patient. ImageJ v1.54 program was used to measure the total LysoTracker signal, and a histogram representing the LysoTracker intensity per cell was generated (C,D). Images were taken under the same light intensity at 100× magnification, and the data are presented as the mean ± S.E.M. Two-way ANOVA analysis was used to determine p-values, as indicated by GraphPad (* p < 0.05, *** p < 0.005, and **** p < 0.001) (n = 3).

Figure 3

GM2/Lamp-1 staining of recombinant human Hexa protein (rhHex-A, 100 nM)-treated fibroblasts (A) and neuroglia cells (B) from WT, Hexa-/- and Hexa-/-Neu3-/- mice. Total GM2 signal was measured by ImageJ v1.54 program and histograms for GM2 intensity per cell for fibroblasts (C) and neuroglia (D) are represented. Images were taken under the same light intensity at 100× magnification, and the data are represented as the mean ± S.E.M. Two-way ANOVA analysis was used to determine p-values, as indicated by GraphPad (* p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001) (n = 3).

Figure 4

GM2/Lamp-1 staining of recombinant human Hexa protein (rhHex-A, 100 nM)-treated fibroblasts (A) and neuroglia (B) cells derived from a human TSD patient. ImageJ v1.54 program was used to measure the total GM2 signal, and histograms representing the GM2 intensity per cell for fibroblasts (C) and neuroglia (D) are shown. Images were taken under the same light intensity at 100× magnification, and the data are presented as the mean ± S.E.M. Two-way ANOVA analysis was used to determine p-values, as indicated by GraphPad (** p < 0.01, *** p < 0.005, and **** p < 0.001) (n = 3).

Figure 5

HexB gene expression analysis in mouse fibroblast (A) and neuroglia (B) generated from WT, Hexa-/-, and Hexa-/-Neu3-/- mice as well as human fibroblasts (C) and neuroglia (D) in untreated (UT) and recombinant human HexA protein (rhHex-A, 100 nM for 72 h)-treated conditions. The data are represented as the mean ± S.E.M. Two-way ANOVA analysis was used to determine p-values, as shown in GraphPad (* p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001).

Figure 6

GM2AP gene expression analysis in mouse fibroblasts (A) and neuroglia (B) generated from WT, Hexa-/-, and Hexa-/-Neu3-/- mice as well as human fibroblasts (C) and neuroglia (D) in untreated (UT) and recombinant human Hexa protein (rhHex-A, 100 nM for 72 h)-treated conditions. The data are represented as the mean ± S.E.M. Two-way ANOVA analysis was used to determine p-values, as shown in GraphPad (* p < 0.05, ** p < 0.01, and **** p < 0.001).