D to neuronal cultures. Blocking BAFF-R ligation with TACI-Ig inhibited wild-type, but not Title Loaded From File Baffrm/m, neuronal survival in a dose-dependent manner (Fig. 3 C). However, TACI-Ig had no effect on the survival of 6? microglial cells or primary cultured murine astrocytes (Figure S1). Collectively, these results indicate that the functional interaction between BAFF and BAFF-R on neuronal cells contributes to their survival. Finally, to determine whether BAFF-R has a neuroprotective role in vivo, we first evaluated the BAFF and BAFF-R expression in the spinal cord of transgenic mice overexpressing the mutant human SOD1 gene (mSOD1 mice), which show not only ALS-like symptoms but also the degeneration of both upper and lower motor neurons [16]. At the age of 70 days, relative expression level of BAFF and BAFF-R in the spinal cords of mSOD1 mice was 0.9860.19 and 1.7260.14 respectively. At the age of 130 days, BAFF and BAFF-R expression increased up to 1.8360.32 and 2.2060.30 respectively (Figure S2), suggesting that BAFF-BAFFR axis may be involved in the regulation of neuronal survival in mSOD1 mice. Next, we introduced a transgene encoding a mutated human SOD1 into Baffrm/m mice. The resulting mSOD1/Baffrm/m mice displayed significantly accelerated weight loss around 17 weeks of age compared to mSOD1/Baffr+/+ mice (Fig. 4 A). In addition, mSOD1/Baffrm/m mice exhibited significant muscle weakness compared to control mice in a hanging-wire test (Fig. 4 B). Furthermore, mSOD1/Baffrm/m mice had shorter life spans compared to control mSOD1 mice (mSOD1/Baffr+/+ mice: 152.361.3 days (n = 35); mSOD1/ Baffrm/m mice: 141.662.3 days (n = 27)) (Fig. 4 C). Consistent with these clinical manifestations, mSOD1/Baffrm/m mice had significantly fewer myelinated axons in sciatic nerves than mSOD1/Baffr+/+ mice at 75 days of age (Fig. 4 D), although Nissl staining showed no significant difference in the number of motor neurons in the anterior horns (data not shown). On the other hand, there were no differences in the numbers of microglia and astrocytes between mSOD1/Baffrm/m and control mice,although neurodegeneration in ALS often involves neuroinflammation mediated by these cells (Fig. 4 E and F) [17,18,19,20]. Other groups previously showed that disease is exacerbated in mSOD1 mice that are deficient for RAG2 or CD4+ T cells [21,22], suggesting that immune cells contribute to the pathogenesis of ALS. Thus, it is possible that the accelerated disease in mSOD1/ Baffrm/m mice is due to defective mature B cells or a deficiency in BAFF-R on bone marrow-derived cells. However, a recent study showed that B cell-deficient mSOD1 mice (mSOD1/mMT mice) did not show altered mortality or rotarod performance compared with control mice [23]. Indeed, we confirmed that disease progression and severity in mSOD1/mMT mice were indistinguishable from those in control mSOD1 mice (mean survival time of mSOD1/mMT mice: 157.361.5 days (n = 20); mean survival time of control mSOD1 mice: 152.361.3 days (n = 35)) (Fig. 5 A?C), indicating that B cells do not play a protective role in ALS. To further determine the neuroprotective role of BAFF-R in bone marrow-derived 23977191 cells, we then transferred bone L optical density of 561024 at 600 nm (OD600 = 561024), and the suspension was marrow cells from Baffrm/m or wild-type mice into mSOD1/Baffrm/m mice. The disease progression of mSOD1/Baffrm/m mice reconstituted with bone marrow cells from Baffrm/m mice was comparable to that of mSOD1/Baffrm/m mice reconstituted with wild-type bone marrow cells (mean survival time of mSOD1/Ba.D to neuronal cultures. Blocking BAFF-R ligation with TACI-Ig inhibited wild-type, but not Baffrm/m, neuronal survival in a dose-dependent manner (Fig. 3 C). However, TACI-Ig had no effect on the survival of 6? microglial cells or primary cultured murine astrocytes (Figure S1). Collectively, these results indicate that the functional interaction between BAFF and BAFF-R on neuronal cells contributes to their survival. Finally, to determine whether BAFF-R has a neuroprotective role in vivo, we first evaluated the BAFF and BAFF-R expression in the spinal cord of transgenic mice overexpressing the mutant human SOD1 gene (mSOD1 mice), which show not only ALS-like symptoms but also the degeneration of both upper and lower motor neurons [16]. At the age of 70 days, relative expression level of BAFF and BAFF-R in the spinal cords of mSOD1 mice was 0.9860.19 and 1.7260.14 respectively. At the age of 130 days, BAFF and BAFF-R expression increased up to 1.8360.32 and 2.2060.30 respectively (Figure S2), suggesting that BAFF-BAFFR axis may be involved in the regulation of neuronal survival in mSOD1 mice. Next, we introduced a transgene encoding a mutated human SOD1 into Baffrm/m mice. The resulting mSOD1/Baffrm/m mice displayed significantly accelerated weight loss around 17 weeks of age compared to mSOD1/Baffr+/+ mice (Fig. 4 A). In addition, mSOD1/Baffrm/m mice exhibited significant muscle weakness compared to control mice in a hanging-wire test (Fig. 4 B). Furthermore, mSOD1/Baffrm/m mice had shorter life spans compared to control mSOD1 mice (mSOD1/Baffr+/+ mice: 152.361.3 days (n = 35); mSOD1/ Baffrm/m mice: 141.662.3 days (n = 27)) (Fig. 4 C). Consistent with these clinical manifestations, mSOD1/Baffrm/m mice had significantly fewer myelinated axons in sciatic nerves than mSOD1/Baffr+/+ mice at 75 days of age (Fig. 4 D), although Nissl staining showed no significant difference in the number of motor neurons in the anterior horns (data not shown). On the other hand, there were no differences in the numbers of microglia and astrocytes between mSOD1/Baffrm/m and control mice,although neurodegeneration in ALS often involves neuroinflammation mediated by these cells (Fig. 4 E and F) [17,18,19,20]. Other groups previously showed that disease is exacerbated in mSOD1 mice that are deficient for RAG2 or CD4+ T cells [21,22], suggesting that immune cells contribute to the pathogenesis of ALS. Thus, it is possible that the accelerated disease in mSOD1/ Baffrm/m mice is due to defective mature B cells or a deficiency in BAFF-R on bone marrow-derived cells. However, a recent study showed that B cell-deficient mSOD1 mice (mSOD1/mMT mice) did not show altered mortality or rotarod performance compared with control mice [23]. Indeed, we confirmed that disease progression and severity in mSOD1/mMT mice were indistinguishable from those in control mSOD1 mice (mean survival time of mSOD1/mMT mice: 157.361.5 days (n = 20); mean survival time of control mSOD1 mice: 152.361.3 days (n = 35)) (Fig. 5 A?C), indicating that B cells do not play a protective role in ALS. To further determine the neuroprotective role of BAFF-R in bone marrow-derived 23977191 cells, we then transferred bone marrow cells from Baffrm/m or wild-type mice into mSOD1/Baffrm/m mice. The disease progression of mSOD1/Baffrm/m mice reconstituted with bone marrow cells from Baffrm/m mice was comparable to that of mSOD1/Baffrm/m mice reconstituted with wild-type bone marrow cells (mean survival time of mSOD1/Ba.