Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration
doi: 10.1073/pnas.1605195113
Figure Lengend Snippet: ATROSAB is neuroprotective via enhanced TNFR2 signaling. (A) Primary neurons, isolated from hu/mTNFR1-k/i mice, were stimulated with or without ATROSAB (100 µg/mL) for 30 min followed by addition of wild-type EHD2-sc–mTNF (10 ng/mL). Then cells were incubated for 24 h and lyzed, and phosphorylation of PKB/Akt was quantified by Western blot (n = 4, ±SEM). (B) Primary neurons, isolated from hu/mTNFR1-k/i mice, were stimulated with or without ATROSAB (100 µg/mL) for 30 min followed by addition of non–TNFR-selective EHD2-sc–mTNF or mouse TNFR2-selective EHD2-sc–mTNFR2. After 24 h, glutamate (5 µM) was added and cells were incubated for an additional hour. Then medium was exchanged to remove glutamate and cells were incubated for 23 h. Cell viability was determined by MTT assay. Data are shown as percentage of MTT signal of untreated control cells (n = 3; ±SEM). Representative images show (C) ChAT-positive cholinergic innervations in the somatosensory cortex or (E) CD11B-positive activated macrophage/microglia in magnocellular nucleus basalis. NMDA injected into the NBM induced an extensive cholinergic fiber loss in the layer V of somatosensory cortex and a massive volume of macrophage/microglial activation compared with the control group. However, ATROSAB treatment attenuated fiber loss and significantly reduced macrophage/microglial activation induced by NMDA. ATROSAB neuroprotection against fiber loss was prevented by TNFR2 antagonistic MAB426. However, MAB426 alone did not significantly alter NMDA-induced NBM lesion. Parallel bars indicated the layer V of the somatosensory cortex in which quantitative measurements were performed. (D) Quantification of cholinergic fiber density in layer V of the somatosensory cortex. Fiber density was measured in eight sections per mouse. (F) Quantification of total extent of activated macrophage/microglia around the injections. Macrophage/microglial activation was measured in a series of sections with macrophage/microglial activation. n = 7 mice/group. All data in bar charts represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001, one-way ANOVA with post hoc comparisons Tukey.
Article Snippet: Then, 0.5 × 10 6 cells were incubated with antibodies against mouse TNFR1 (HP8002; Hycult Biotec), human TNFR1 (HP9002; Hycult Biotec), mouse TNFR2 (HP8003; Hycult Biotec), and human TNFR2 (HP9003; Hycult Biotec) for 45 min on ice.
Techniques: Isolation, Incubation, Western Blot, MTT Assay, Injection, Activation Assay