BAFF Controls Neural Cell Survival through BAFF Receptor

<div><p>Various neuroprotective factors have been shown to help prevention of neuronal cell death, which is responsible for the progression of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, most of these therapeutic potentials have been tested by administration of recombinant proteins, transgenic expression or virus vector-mediated gene transfer. Therefore, it remains to be clarified whether any endogenous factors has advantage for neuroprotection in a pathological nervous system. Here we show the role of BAFF-R signaling pathway in the control of neural cell survival. Both B cell–activating factor (BAFF) and its receptor (BAFF-R) are expressed in mouse neurons and BAFF-R deficiency reduces the survival of primary cultured neurons. Although many studies have so far addressed the functional role of BAFF-R on the differentiation of B cells, impaired BAFF-R signaling resulted in accelerated disease progression in an animal model of inherited ALS. We further demonstrate that BAFF-R deficient bone marrow cells or genetic depletion of B cells does not affect the disease progression, indicating that BAFF-mediated signals on neurons, not on B cells, support neural cell survival. These findings suggest opportunities to improve therapeutic outcome for patients with neurodegenerative diseases by synthesized BAFF treatment.</p></div

Neither B lymphocytes nor bone marrow-derived cells affect disease progression or the survival of mSOD1 mice.

<p>(A–C) The absence of B lymphocytes does not affect body weight (A), motor performance (B), or the survival (C) of mSOD1 mice (log rank test for survival, p = 0.051). In panels A and B, n = 15 for mSOD1/μMT mice and n = 19 for control mSOD1 mice. In panel C, n = 20 for mSOD1/μMT mice and n = 35 for control mSOD1 mice. (D–F) BAFF-R–deficient bone marrow cells do not affect body weight (D), motor performance (E), or the survival (F) of mSOD1 mice. (log rank test for survival, p = 0.284) (n = 7 per group) Data are expressed as the mean ± SEM. *:p<0.05, #:p<0.01.</p

BAFF-R expression in mouse primary cultured neurons, on spinal cord neurons and on Neuro2a cells.

<p>(A–F) 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–BAFF-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 co-stained with Cy5-conjugated anti–BAFF-R antibodies and an Alexa488 conjugated anti-GFAP antibody (G) or FITC-conjugated tomato lectin (H). 4′, 6-diamidino-2-phenylindole (DAPI) was also used to stain nuclei. Scale bars represent 100 μm (for panel A, B, E and F), 50 μm (for panel C and D), 25 μm (for panel G), and 10 μm (for panel H) respectively. (I) BAFF-R mRNA expression in 6–3 microglia cells, Neuro2a cells, and primary cultured neurons was examined by quantitative RT-PCR. The data are presented as the mean ± s.d. of samples examined in triplicate.</p

mSOD1/<i>Baffr</i>^m/m mice exhibit accelerated disease progression and reduced survival.

<p>(A) Changes in the mean body weight of mSOD1/<i>Baffr</i>^m/m mice (n = 20) and mSOD1/<i>Baffr</i>^+/+ mice (n = 19). (B) Time course of motor performance using the hanging-wire test in which mSOD1/<i>Baffr</i>^m/m mice performed significantly worse than mSOD1/<i>Baffr</i>^+/+ mice. (n = 20 and n = 19, respectively) (C) Kaplan-Meier survival curve. Defects in BAFF-R signaling shortened the survival of mSOD1 mice (log rank test for survival, p = 0.0000342) (n = 27 for mSOD1/<i>Baffr</i>^m/m mice and n = 35 for mSOD1/<i>Baffr</i>^+/+ mice) (D) mSOD1/<i>Baffr</i>^m/m mice had significantly fewer myelinated axons than control mSOD1/<i>Baffr</i>^+/+ mice (n = 3 per group). Representative pictures are shown. Scale bar  = 20 μm. (E and F) The number of GFAP-positive or tomato lectin-positive cells in the spinal cord does not differ between mSOD1/<i>Baffr</i>^+/+ and mSOD1/<i>Baffr</i>^m/m mice. Lumbar sections of the spinal cord were stained with an anti-GFAP antibody (E) or with tomato lectin (F). Scale bar  = 100 μm. Data are representative of three animals.</p

Role of BAFF-R in neuronal survival in vitro.

<p>(A) The effect of a BAFF-R deficiency on neuronal survival. Neurons from E13.5 embryos of wild-type and A/WySnJ mice (<i>Baffr</i>^m/m mice) were cultured for 7<b> </b>days under nutrient-limiting conditions, and then cell viability was measured by counting Map2⁺ viable neurons. Representative pictures are shown. Scale bar  = 100 μm. (B) Reduced Akt phosphorylation in <i>Baffr</i>^m/m neurons. After 7<b> </b>days of culture, wild-type and <i>Baffr</i>^m/m neurons were assayed for the levels of total and phospho-Akt by immunoblot analysis. β-actin is shown as a loading control. (C) The effects of blocking BAFF-R on neuronal survival. Neurons from E13.5 embryos of wild-type and <i>Baffr</i>^m/m mice were cultured with TACI-Ig or control human-Ig for 7<b> </b>days under nutrient-limiting conditions, and then Map2⁺ viable neurons were counted. Data are representative of three separate experiments. *:p<0.05, #:p<0.01.</p

BAFF expression in mouse primary cultured neurons and on mouse spinal cord neurons.

<p>(A–D) 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 μm (calculated for each panel). (E) BAFF mRNA expression in 6–3 microglia cells and primary cultured neurons was examined by quantitative RT-PCR. The data are presented as the mean ± s.d. of samples examined in triplicate.</p

¹¹C-Acetate PET Imaging in Patients with Multiple Sclerosis

<div><p>Background</p><p>Activation of glial cells is a cardinal feature in multiple sclerosis (MS) pathology, and acetate has been reported to be selectively uptaken by astrocytes in the CNS. The aim of this study was to investigate the efficacy of PET with ¹¹C-acetate for MS diagnosis.</p><p>Materials and Methods</p><p>Six patients with relapsing-remitting MS and 6 healthy volunteers (HV) were enrolled. The ¹¹C-acetate brain uptake on PET was measured in patients with MS and HV. Volume-of-interest analysis of cerebral gray and white matter based on the segmentation technique for co-registered MRI and voxel-based statistical parametric analysis were performed. Correlation between ¹¹C-acetate uptake and the lesion number in T1- and T2- weighted MR images were also assessed.</p><p>Results</p><p>The standardized uptake value (SUV) of ¹¹C-acetate was increased in both white and gray matter in MS patients compared to HV. Voxel-based statistical analysis revealed a significantly increased SUV relative to that in the bilateral thalami (SUVt) in a broad area of white matter, particularly in the subcortical white matter of MS patients. The numbers of T2 lesions and T1 black holes were significantly correlated with SUV of ¹¹C-acetate in white and gray matter.</p><p>Conclusions</p><p>The ¹¹C-acetate uptake significantly increased in MS patients and correlated to the number of MRI lesions. These preliminary data suggest that ¹¹C-acetate PET can be a useful clinical examination for MS patients.</p></div

¹¹C-acetate CNS biodistribution.

<p>(A)Mean standardized uptake value (SUV) of each lesion. (B) Relative SUV compared to that of the thalamus (SUVt). Data are expressed as the mean ± standard error of the mean (SEM) (n = 6). The Mann–Whitney <i>U</i> test showed a significant difference in the median between the HV and MS groups (*:p<0.0055 after Bonferroni correction). HV  =  healthy volunteers, MS  =  multiple sclerosis.</p

Correlation between ¹¹C-acetate SUV and the number of MRI lesions in patients with MS.

<p>Correlation between ¹¹C-acetate SUV in WM or GM and the number of T1 black holes (A, C) or T2 lesions (B, D) in each hemisphere of the six MS patients. SUV: standardized uptake value.</p

¹¹C-acetate uptake distribution and quantification in MS patients.

<p>(A)Spatially normalized group mean images of ¹¹C-acetate SUVt automatically segmented based on MRI. VOI analysis summarizing the mean SUVt in WM (B) and GM (C), and the WM/GM SUV ratio (D) in the HV and MS groups. The identical analysis performed using spill-in-free VOIs are also shown (E–G). The p-value was calculated using the analysis of covariance to adjust the variance of age. (H) The SPM analysis result is overlaid onto the T1-weighted brain MRI template. Colored voxels indicate T-scores representing significantly increased ¹¹C-acetate uptake (SUVt) in patients with MS compared to HV patients. The spatially normalized PET images were smoothed for the analysis using a 12-mm FWHM isotropic Gaussian kernel. The significance thresholds are corrected for multiple comparisons at the cluster level with a p-value of 0.05 (family-wise error correction). SUV: standardized uptake value.</p