We explore the novel mechanism of neural cell survival by BAFF-R signals using a murine model, in which ablation of the BAFF-R in vivo is combined with the acceleration of neurodegeneration by overexpressing mSOD1. Our findings establish BAFF as a critical member of neurotrophic factors thatdirects neural cell survival independent of the action for lymphoid cells.Materials and Methods Ethics StatementThis study was carried out in strict accordance with both the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science and the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Research Institute for Microbial Diseases and Immunology Frontier Research Center of Osaka University, who specifically approved this study 16574785 (Permit number: Biken-AP-H21-28-0). AllFigure 1. BAFF-R expression in mouse primary cultured neurons, on AKT inhibitor 2 cost spinal cord neurons and on Neuro2a cells. (A ) Primary cultured mouse neurons (A and B), sections of a mouse spinal cord (C and D) and Neuro2a neuroblastoma cells (E and F) were co-stained with Cy5-conjugated anti AFF-R antibodies (A, C and E) or control antibodies (B, D and F) and an Alexa488-conjugated anti-microtubule-associated protein 2 (Map2) antibody (A, B, E and F) or anti-Neurofilament H Non-Phosphorylated (SMI-32) antibody (C and D). (G and H) Sections of a mouse spinal cord were costained with Cy5-conjugated anti AFF-R antibodies and an Alexa488 conjugated anti-GFAP antibody (G) or FITC-conjugated tomato lectin (H). 49, 6diamidino-2-phenylindole (DAPI) was also used to stain nuclei. Scale bars represent 100 mm (for panel A, B, E and F), 50 mm (for panel C and D), 25 mm (for panel G), and 10 mm (for panel H) respectively. (I) BAFF-R mRNA expression in 6? microglia cells, Neuro2a cells, and primary cultured neurons was examined by quantitative RT-PCR. The data are presented as the mean 6 s.d. of samples examined in triplicate. doi:10.1371/journal.pone.0070924.gNeuroprotection by B Cell Activating Factor (BAFF)Figure 2. BAFF expression in mouse primary cultured neurons and on mouse spinal cord neurons. (A ) Primary cultured mouse neurons (A and B) and sections of a mouse spinal cord (C and D) were co-stained with Cy5-conjugated anti-BAFF antibodies (A and C) or control antibodies (B and D) and an Alexa488-conjugated anti-Map2 antibody (A and B) or anti-SMI-32 antibody (C and D). DAPI was also used to stain nuclei. Scale bars represent 20 mm (calculated for each panel). (E) BAFF mRNA expression in 6? microglia cells and primary cultured neurons was examined by quantitative RT-PCR. The data are presented as the mean 6 s.d. of samples examined in triplicate. doi:10.1371/journal.pone.0070924.gsurgery was performed under sodium 58-49-1 site pentobarbital anesthesia, and all necessary steps were taken to ameliorate suffering to animals involved in the study.Cell cultureNeurons were prepared from cerebral cortices of mouse embryos (E13.5) as previously described [13]. Neurons were maintained in Neurobasal medium (Gibco, MD, USA) containing 2 B27 supplements (Gibco) for 4? days before experimentation. Astrocytes were prepared as previously described [13]. Briefly, cells collected from cerebral cortices of newborn mice were plated onto a flask coated with poly-L-lysine (PLL; Sigma) and incubated in media consisting of Dulbecco’s mo.We explore the novel mechanism of neural cell survival by BAFF-R signals using a murine model, in which ablation of the BAFF-R in vivo is combined with the acceleration of neurodegeneration by overexpressing mSOD1. Our findings establish BAFF as a critical member of neurotrophic factors thatdirects neural cell survival independent of the action for lymphoid cells.Materials and Methods Ethics StatementThis study was carried out in strict accordance with both the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science and the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Research Institute for Microbial Diseases and Immunology Frontier Research Center of Osaka University, who specifically approved this study 16574785 (Permit number: Biken-AP-H21-28-0). AllFigure 1. BAFF-R expression in mouse primary cultured neurons, on spinal cord neurons and on Neuro2a cells. (A ) Primary cultured mouse neurons (A and B), sections of a mouse spinal cord (C and D) and Neuro2a neuroblastoma cells (E and F) were co-stained with Cy5-conjugated anti AFF-R antibodies (A, C and E) or control antibodies (B, D and F) and an Alexa488-conjugated anti-microtubule-associated protein 2 (Map2) antibody (A, B, E and F) or anti-Neurofilament H Non-Phosphorylated (SMI-32) antibody (C and D). (G and H) Sections of a mouse spinal cord were costained with Cy5-conjugated anti AFF-R antibodies and an Alexa488 conjugated anti-GFAP antibody (G) or FITC-conjugated tomato lectin (H). 49, 6diamidino-2-phenylindole (DAPI) was also used to stain nuclei. Scale bars represent 100 mm (for panel A, B, E and F), 50 mm (for panel C and D), 25 mm (for panel G), and 10 mm (for panel H) respectively. (I) BAFF-R mRNA expression in 6? microglia cells, Neuro2a cells, and primary cultured neurons was examined by quantitative RT-PCR. The data are presented as the mean 6 s.d. of samples examined in triplicate. doi:10.1371/journal.pone.0070924.gNeuroprotection by B Cell Activating Factor (BAFF)Figure 2. BAFF expression in mouse primary cultured neurons and on mouse spinal cord neurons. (A ) Primary cultured mouse neurons (A and B) and sections of a mouse spinal cord (C and D) were co-stained with Cy5-conjugated anti-BAFF antibodies (A and C) or control antibodies (B and D) and an Alexa488-conjugated anti-Map2 antibody (A and B) or anti-SMI-32 antibody (C and D). DAPI was also used to stain nuclei. Scale bars represent 20 mm (calculated for each panel). (E) BAFF mRNA expression in 6? microglia cells and primary cultured neurons was examined by quantitative RT-PCR. The data are presented as the mean 6 s.d. of samples examined in triplicate. doi:10.1371/journal.pone.0070924.gsurgery was performed under sodium pentobarbital anesthesia, and all necessary steps were taken to ameliorate suffering to animals involved in the study.Cell cultureNeurons were prepared from cerebral cortices of mouse embryos (E13.5) as previously described [13]. Neurons were maintained in Neurobasal medium (Gibco, MD, USA) containing 2 B27 supplements (Gibco) for 4? days before experimentation. Astrocytes were prepared as previously described [13]. Briefly, cells collected from cerebral cortices of newborn mice were plated onto a flask coated with poly-L-lysine (PLL; Sigma) and incubated in media consisting of Dulbecco’s mo.
