Sandhoff disease is a lysosomal storage disease caused by simultaneous deficiencies of beta-hexosaminidase A (HexA; alphabeta) and B (HexB; betabeta), due to a primary defect of the beta-subunit gene (HEXB) associated with excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N-acetylhexosamine residues at their non-reducing termini, and with neurosomatic manifestations. To elucidate the neuroinflammatory mechanisms involved in its pathogenesis, we analyzed the expression of chemokines in Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex beta-subunit gene allele (Hexb-/-). We demonstrated that chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) was induced in brain regions, including the cerebral cortex, brain stem and cerebellum, of SD mice from an early stage of the pathogenesis but not in other systemic organs. On the other hand, little changes in other chemokine mRNAs, including those of RANTES (regulated upon activation, normal T expressed and secreted), MCP-1 (monocyte chemotactic protein-1), SLC (secondary lymphoid-tissue chemokine), fractalkine and SDF-1 (stromal derived factor-1), were detected. Significant up-regulation of MIP-1alpha mRNA and protein in the above-mentioned brain regions was observed in parallel with the accumulation of natural substrates of HexA and HexB. Immunohistochemical analysis revealed that MIP-1alpha-immunoreactivity (IR) in the above-mentioned brain regions of SD mice was co-localized in Iba1-IR-positive microglial cells and partly in glial fibrillary acidic protein (GFAP)-IR-positive astrocytes, in which marked accumulation of N-acetylglucosaminyl (GlcNAc)-oligosaccharides was observed from the presymptomatic stage of the disease. In contrast, little MIP-1alpha-IR was observed in neurons in which GM2 accumulated predominantly. These results suggest that specific induction of MIP-1alpha might coincide with the accumulation of GlcNAc-oligosaccharides due to a HexB deficiency in resident microglia and astrocytes in the brains of SD mice causing their activation and acceleration of the progressive neurodegeneration in SD mice.

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Sandhoff disease is an autosomal recessive lysosomal storage disease caused by a defect of the b-subunit gene (HEXB) associated with simultaneous deficiencies of b-hexosaminidase A (HexA; ab) and B (HexB; bb), and excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N-acetylglucosamine (GlcNAc) residues at their non-reducing termini. Recent studies have shown the involvement of microglial activation in neuroinflammation and neurodegeneration of this disease. We isolated primary microglial cells from the neonatal brains of Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex b-subunit gene allele (Hexb-/-). The cells expressed microglial cell-specific ionized calcium binding adaptor molecule 1 (Iba1)-immunoreactivity (IR) and antigen recognized by Ricinus communis agglutinin lectin-120 (RCA120), but not glial fibrillary acidic protein (GFAP)-IR specific for astrocytes. They also demonstrated significant intracellular accumulation of GM2 and GlcNAc-oligosaccharides. We produced a lentiviral vector encoding for the murine Hex b-subunit and transduced it into the microglia from SD mice with the recombinant lentivirus, causing elimination of the intracellularly accumulated GM2 and GlcNAc-oligosaccharides and secretion of Hex isozyme activities from the transduced SD microglial cells. Recomibinant HexA isozyme isolated from the conditioned medium of a Chinese hamster ovary (CHO) cell line simultaneously expressing the human HEXA (a-subunit) and HEXB genes was also found to be incorporated into the SD microglia via cell surface cation-independent mannose 6-phosphate receptor and mannose receptor to degrade the intracellularly accumulated GM2 and GlcNAc-oligosaccharides. These results suggest the therapeutic potential of recombinant lentivirus encoding the murine Hex b-subunit and the human HexA isozyme (ab heterodimer) for metabolic cross-correction in microglial cells involved in progressive neurodegeneration in SD mice. Keywords: b-hexosmanidase A, enzyme replacement therapy, GM2 ganglioside, lentiviral vector, N-acetylglucosaminyl oligosaccharides, Sandhoff disease. Abbreviations used: BSA, bovine serum albumin; CHO, Chinese hamster ovary; CHO-HEXA/HEXB, a CHO cell line simultaneously expressing the human HEXA and HEXB genes; CIM6P/IGFIIR, cationindependent mannose-6-phosphate receptor/insulin-like growth factor II; CM, conditioned medium; DMEM, Dulbecco's modified Eagle's medium; DNaseI, deoxyribonuclease I; FCS, fetal calf serum; FITC, fluorescein isothiocynate; G418, neomycin sulfate derivative; GalNAc, N-acetylgalactosamine; GFAP, glial fibrillary acidic protein; GlcNAc, N-acetylglucosamine; GlcNAc-oligosacharides, oligosaccharides with GlcNAc residues at their non-reducing termini; GM2, GM2 ganglioside; GMB28, a mouse monoclonal antibody against GM2; HBSS, Hank's balanced salt saline; Hex, b-hexosaminidase; HEXA, human gene encoding Hex a-subunit; HEXB, human gene encoding Hex b-subunit; Hexb, murine gene encoding Hex b-subunit; Iba1, ionized calcium bindin...

Lysosomal b-hexosaminidase (Hex, EC 3.2.1.52) is a glycosidase that catalyzes the hydrolysis of terminal N-acetylhexosamine residues at the non-reducing ends of oligosaccharides of glycoconjugates.1,2) There are two major Hex isozymes in mammals including man, HexA (ab, a heterodimer of a-and b-subunits) and HexB (bb, a homodimer of b-subunit), and a minor unstable isozyme, HexS (aa, a homodimer of a-subunit). All these Hex isozymes can degrade terminal b-1,4 linked N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) residues, while HexA and HexS prefer negatively charged substrates and cleave off the terminal N-acetylglucosamine-6-sulfate (GlcNAc-6-S) residues in keratan sulfate. Only HexA is essential for cleavage of the GalNAc residue from GM2 in co-operation with GM2 activator protein. 1-3)Tay-Sachs disease (TS) and Sandhoff disease (SD) are autosomal recessive GM2 gangliosidoses caused by mutations of HEXA on chromosome 15q23-24, and HEXB on chromosome 5q13, respectively. 1,2) The genes exhibit sequence homology, and the gene products exhibit 57% similarity in amino acid sequence. In TS, the genetic defect of HEXA causes a deficiency of HexA with excessive accumulation of GM2 in the central nervous system (CNS), resulting in neurological disorders. In SD, the inherited defect of HEXB leads to simultaneous deficiencies of HexA and HexB with accumulation of GM2 in the CNS and of GlcNAc-oligosaccharides, resulting in systemic manifestations including hepatosplenomegaly as well as the neurological symptoms. So far no effective treatment has been established.Mice have the same gene organization, i.e. Hexa encoding the a-subunit on chromosome 9 and Hexb encoding the bsubunit on chromosome 13, and have the same Hex isozyme system. 1,4,5) The amino acid sequences deduced from Hexa and Hexb cDNAs are 55% identical, and each exhibits homology to the human counterpart, 84% and 75%, respectively.5) Hex gene-disrupted mice have been established, [6][7][8][9] and Hex gene knock-out mice exhibiting neurosomatic manifestations similar to those in the human deficiency patients have been utilized as disease models for developing therapeutic approaches including bone-marrow transplantation, recombinant enzyme replacement and substrate-depletion. [10][11][12] Gene transfer has been also achieved in fibroblasts from TS and SD patients and their disease models by different kinds of viral vector, including adeno-and retrovirus. [13][14][15][16][17][18] Cotransduction with two vectors encoding the a-and b-subunits caused a high level of HexA synthesis, secretion and corrective effects on the disease models.14,15) On the other hand, recent progress in enzyme replacement therapy (ERT) for lysosomal storage diseases, including Gaucher disease, 19) Fabry disease 20,21) and mucopolysaccharidosis I 22) let us expect its application for GM2 gangliosidoses, although the difficulty in delivery of recombinant enzymes across the blood-brain barrier into the brain parenchyma should be solved.In this study, we examined the therap...