Fig 2: Knockout of BDNF Activates JAK2/STAT3 Pathway and Elicits Synaptic Plasticity Impairment and Cognitive Deficits(A) BDNF knockout activates JAK2/STAT3 and increases the expression of C/EBPβ and δ-secretase. Immunoblotting was conducted from 16-month-old BDNFf/f mice hippocampus injected with AAV-Cre or AAV-GFP. Data are representative of four mice.(B) Quantification of western blotting (n = 4, *p < 0.05, **p < 0.01, unpaired t test with Welch’s correction).(C) qRT-PCR analysis. Data represent mean ± SEM of 4 mice (**p < 0.01, unpaired t test with Welch’s correction).(D) δ-secretase enzymatic activities. Data represent mean ± SEM (n = 5, **p < 0.01, unpaired t test with Welch’s correction).(E) BDNF knockout stimulates inflammatory cytokine production. Data represent mean ± SEM (n = 6, *p < 0.05, **p < 0.01, unpaired t test with Welch’s correction).(F) Quantification of Aβ levels by ELISA represents mean ± SEM (n = 5,*p < 0.05, **p < 0.01, unpaired t test with Welch’s correction).(G) IHC. Scale bar, 50 μm. Data shown as mean ± SEM of 9 sections from 3 mice in each group (*p < 0.05, unpaired t test with Welch’s correction).(H) Golgi staining was conducted on brain sections from apical dendritic layer of the CA1 region. Scale bar, 5 μm. Data represent mean ± SEM of 10 sections from 3 mice in each group. (*p < 0.05, **p < 0.01, unpaired t test with Welch’s correction).(I) Electronic microscopic (EM) analysis. Scale bar, 1 μm. Data represent mean ± SEM of 10 sections from 3 mice in each group. (*p < 0.05, unpaired t test with Welch’s correction).(J) Electrophysiology analysis (mean ± SEM; n = 6 in each group; *p < 0.05, **p < 0.01, two-way ANOVA and Bonferroni’s post hoc test). The ratio of paired pulses in different groups (left). Input-output curve (middle). LTP (right). Shown traces were representative field excitatory post-synaptic potentials (fEPSPs) recorded before (black) and 60 min after (red) Tris-buffered saline (TBS).(K) Morris Water Maze analysis (mean ± SEM; n = 8-9 mice per group; **p < 0.01, two-way ANOVA and Bonferroni’s post hoc test).See also Figures S4 and S5.

Fig 3: Deprivation of BDNF Increases Cytokines and Activates the JAK2/STAT3 Pathway in Primary Neurons(A) BDNF deprivation activates JAK2/STAT3 and increases the expressions of C/EBPβ and δ-secretase. Immunoblotting was conducted from primary neurons after incubation with anti-BDNF or anti-IgG antibody. Data are representative of three independent experiments.(B) Quantification of western blotting (n = 6 per group, *p < 0.05, **p < 0.01, unpaired t test with Welch’s correction).(C) qRT-PCR analysis of C/EBPβ, LGMN, APP, and MAPT mRNA levels in neurons treated with anti-BDNF or anti-IgG antibody. Data represent mean ± SEM of 5 independent experiments (**p < 0.01, unpaired t test with Welch’s correction).(D) ELISA of whole-cell lysates. Data represent mean ± SEM of 6 samples per group from 3 independent experiments (*p < 0.05, **p < 0.01, unpaired t test with Welch’s correction).(E) BDNF deprivation increases δ-secretase enzymatic activity. Data represent mean ± SEM (n = 5, *p < 0.05, unpaired t test with Welch’s correction).(F) BDNF deprivation stimulates Aβ production. Quantification of Aβ40 and Aβ42 levels by ELISA represents mean ± SEM (n = 5,*p < 0.05, **p < 0.01, unpaired t test with Welch’s correction).(G) LDH assay showing that BDNF deprivation promotes cell death. Data shown as mean ± SEM of 3 independent experiments (*p < 0.05, unpaired t test with Welch’s correction).(H–J) TUNEL and immunofluorescence (IF) double-labeling assay (H). Scale bar, 10 μm. Quantifications of TUNEL positive cells (I) and Tau N368 level (J) represent mean ± SEM (n = 6, *p < 0.05, unpaired t test with Welch’s correction). Scale bars, 10 μm.(K) A schematic diagram showing directionality of gene and encoded protein changes.See also Figure S1.