Lysosomal storage and pathologenesis in a novel in vitro cellular model of Sandhoff disease

Sandhoff disease is a devastating autosomal recessive GM2 gangliosidosis in which a deficiency of β-hexosaminidase results in lysosomal storage of the enzyme's substrates, including GM2 and GA2 glycolipid as well as glycoprotein-derived oligosaccharides (OS). The effect of individual storage products on the induction of neurodegeneration in the gangliosidoses has not been well defined.
Currently there is no valid in vitro cellular model for studying this disease so the aim of this work was to chemically induce the disease phenotype using a potent, reversible β- hexosaminidase inhibitor, N-benzyl-2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol (NBnLABNAc, SRI). Multiple cell lines, both human and mouse-derived were studied, and following SRI treatment, murine RAW264.7 macrophage-like cells exhibited the Sandhoff phenotype, in agreement with glycoconjugate storage found in mouse models, and were used for further study.
The structures of the glycosphingolipids (GSLs) and OS, stored due to β-hexosaminidase inhibition, as well as the cellular localization were determined by normal phase HPLC and mass spectrometry, and subcellular fractionation and chemical extraction of the cytosol and lysosome, respectively.
These stored lysosomal β-hexosaminidase substrates resulted in a tightly regulated inflammatory response where caspase-dependent apoptosis was prevented.
It remained unclear which of the glycoconjugates were responsible for triggering this cytokine-mediated state, so substrate (glycolipid) reduction therapeutics Nbutyldeoxynojirimycin (NB-DNJ) and N-butyldeoxygalactonojirimycin (NB-DGJ) were administered to SRI-induced storage cells. Reduced GSL storage levels were observed but OS accumulation, although changed in structural composition, did not decrease, as predicted from the mechanism of action of each of the imino sugar inhibitors used.
Both imino sugars restored GM2 levels whereas the GA2 glycolipid and OS levels remained elevated but the inflammatory response was normalized, implicating the sole contributor to the pathogenesis of Sandhoff disease is GM2 ganglioside. However, NBDNJ treatment resulted in an inflammatory response at high concentrations, presumably due to α-glucosidase-mediated inhibition of protein folding, demonstrating the greater potential of NB-DGJ in treating Sandhoff disease and possibly other related disorders.
Besides substrate reduction therapy, chaperone-mediated therapy (CMT) has promise in treating lysosomal storage disorders such as Sandhoff disease. Following administration of an inhibitor of the deficient enzyme to act as a molecular chaperone that stabilises the conformation of the mutant enzyme, ER associated degradation is avoided, improving traffic to the lysosome. The partial increase in enzyme activity using CMT may be sufficient to adjust the critical threshold enzyme activity to levels where GSL storage in the lysosome is reduced to non-pathological concentrations.
Cells derived from human Sandhoff and Tay-Sachs patients were assayed for β-hexosaminidase activity, GSL and OS storage levels. However, despite the lack of β-hexosaminidase activity, GM2 and GA2 glycolipid levels were negligible, possibly due to the cellular origin. Following treatment with SRI, elevated activity of β-hexosaminidase did not result in significant amelioration of the GSL storage levels. In terms of OS storage and enzyme activity, SRI induced differential effects depending on the residual β-hexosaminidase activity present, demonstrating the limitations of this compound in enhancing the total activity of β-hexosaminidase in these cells exhibiting GM2 gangliosidosis.