Tsuji Daisuke, Kuroki Aya, Ishibashi Yasuhiro, Itakura Tomohiro, Itoh Kohji
Department of Medicinal Biotechnology, Institute for Medicinal Resources, Graduate School of Pharmaceutical Sciences, The University of Tokushima, 178 Sho-machi, Tokushima, Japan.
J Neurochem. 2005 Sep;94(6):1631-8. doi: 10.1111/j.1471-4159.2005.03317.x. Epub 2005 Aug 10.
Sandhoff disease is an autosomal recessive lysosomal storage disease caused by a defect of the beta-subunit gene (HEXB) associated with simultaneous deficiencies of beta-hexosaminidase A (HexA; alphabeta) and B (HexB; betabeta), 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 beta-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 beta-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 (alpha-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 beta-subunit and the human HexA isozyme (alphabeta heterodimer) for metabolic cross-correction in microglial cells involved in progressive neurodegeneration in SD mice.
桑德霍夫病是一种常染色体隐性溶酶体贮积病，由β亚基基因（HEXB）缺陷引起，该缺陷导致β-己糖胺酶A（HexA；αβ）和B（HexB；ββ）同时缺乏，以及GM2神经节苷脂（GM2）和在其非还原末端带有N-乙酰葡糖胺（GlcNAc）残基的寡糖过度蓄积。最近的研究表明，小胶质细胞活化参与了该疾病的神经炎症和神经退行性变。我们从通过破坏小鼠Hexβ亚基基因等位基因（Hexb-/-）产生的桑德霍夫病模型小鼠（SD小鼠）的新生大脑中分离出原代小胶质细胞。这些细胞表达小胶质细胞特异性的离子钙结合衔接分子1（Iba1）免疫反应性（IR）和蓖麻凝集素-120（RCA120）识别的抗原，但不表达星形胶质细胞特异性的胶质纤维酸性蛋白（GFAP）-IR。它们还表现出GM2和GlcNAc-寡糖在细胞内的显著蓄积。我们构建了一种编码小鼠Hexβ亚基的慢病毒载体，并用重组慢病毒将其转导到SD小鼠的小胶质细胞中，导致细胞内蓄积的GM2和GlcNAc-寡糖被清除，转导后的SD小胶质细胞分泌Hex同工酶活性。从同时表达人HEXA（α亚基）和HEXB基因的中国仓鼠卵巢（CHO）细胞系的条件培养基中分离出的重组HexA同工酶，也被发现通过细胞表面非依赖阳离子的甘露糖6-磷酸受体和甘露糖受体被摄取到SD小胶质细胞中，以降解细胞内蓄积的GM2和GlcNAc-寡糖。这些结果表明，编码小鼠Hexβ亚基和人HexA同工酶（αβ异二聚体）的重组慢病毒对参与SD小鼠进行性神经退行性变的小胶质细胞进行代谢交叉校正具有治疗潜力。
