Fig 2: Downregulation of BDNF and TrkB in aged microglial cells. a–b Representative dual immunofluorescent images show the distribution of BDNF (a) and TrkB (c) in the Iba1+ cells in the SN of 6- and 12-month-old mice. Scale bar: 10 µm. The percent areas of BDNF+Iba1+/Iba1+ (b) and TrkB+Iba1+/Iba1+ (d) in the SN of 6- and 12-month-old mice. BDNF+ areas were detected from 31 cells in three 6-month-old mice and 56 cells in three 12-month-old mice; TrkB+ areas were obtained from 35 cells in three 6-month-old mice and 73 cells in three 12-month-old mice. ***p < 0.001 versus 6-month-old mice. e–h Correlations (Pearson) between the levels of BDNF and TrkB, and the area and number of Iba1+ cells (n = 24)

Fig 3: Temporal profiles of BDNF, TrkB, and microglial activation in the substantia nigra during aging. a Representative immunohistochemical images show Iba1+ cells in the SN of 3-, 6-, and 12-month-old mice. Quantitative results of Iba1+ cell areas and numbers (n = 8 in each age group) are shown on the right panels. To control for non-specific binding, Iba1 and MHC class II antibodies were replaced by their respective isotype antibodies. b Intensity of Iba1+ signal in isolated microglia. The distribution of the intensity of Iba1+ cells of 3-month-old mice is shown in red as a reference peak. The intensity of 12-month-old mice was much stronger than that of 3-month-old mice as indicated by the right-shifted distribution of the intensity (n = 3). c Representative confocal micrographs show the Iba1+ cells in the SN of 6- and 12-month-old mice. An enlarged single Iba1+ cell is both shown on the right panel. d Representative immunohistochemical images show MHC class II+ cells in the SN of 3-, 6-, and 12-month-old mice. Quantitative results of MHC class II+ cell areas and numbers (n = 5 in each age group) are shown on the right panels. e–i Levels of p-p65, TNF, IL-6, BDNF, and FL-TrkB (n = 8 in each age group). To control for non-specific binding, Iba1 and MHC class II antibodies were replaced by their respective isotype antibodies. Kruskal-Wallis test: *p < 0.05, **p < 0.01, ***p < 0.001 versus 3-month-old mice. Data are presented as mean ± S.D.

Fig 4: Inhibition of TrkA kinase activity by KRC-108. (A) In vitro TrkA kinase activity was measured using the TR-FRET method. KRC-108 was used at the indicated concentrations. The kinase activity of recombinant TrkA protein was measured and the % inhibition was calculated, compared to DMSO control. Data are presented as the means ± standard error of mean (SEM) of three independent experiments. (B) KM12C cells were treated with KRC-108 at the indicated concentrations and were subjected to western blot analyses. TrkA phosphorylation was measured using antibodies against phosphorylated TrkA (p-TrkA) and TrkA. ß-actin was used as loading control. The intensity of the bands was quantified using densitometry. The relative level of p-TrkA vs TrkA was calculated and indicated in the graph. The symbols * and ** indicate p<0.05 and p<0.01, respectively.

Fig 5: Modulation of APP/TrkA association by NGF and trafficking perturbation. (A) Representative images of PLA performed with APP-CT and Trk B3, antibodies showing the interaction of APP and TrkA in primary septal cultures (12 DIV) untreated (Ctrl) or treated with NGF (100 ng/ml for 1 h). Positive signal of interaction is shown as red dot, nuclei are stained with DAPI. (B) Quantification of the PLA signal in primary neurons untreated and treated with NGF (100 ng/ml for 1 h)). The graph represents quantitative analysis of the number of PLA fluorescent red puncta/cell counted in five different fields and for a total of 20–30 cells examined and are expressed as mean ± SEM **p < 0.01 Student t-test. (C) Co-localization of PLA signal with GM130 and calnexin in untreated and NGF treated cells. A representative z-stack of images was collected from each cell. (D) Confocal microscopy images of immunofluorescence and PLA signal [both performed with anti-APP-CT (red) and anti-Trk B3 (green) antibodies], of untreated (Ctrl) and BFA (5 µg/ml for 3 h), cytochalasin D (5 µM for 3 h) and colchicine (100 µM for 3 h) treated primary septal cultures (12 DIV) are shown. PLA dots in cytocalasin D treated cells are distributed in cell body and also along the neuritis coherently with the co-localization signal detected by immunofluorescence. Nuclei are stained with DAPI (blue). (E) Quantification of the PLA signal of APP/TrkA interaction in primary neurons untreated (Ctrl) and treated with BFA, cytochalasin D and colchicine. A total of 20–30 cells were examined. ***p < 0.001 when compared to Ctrl. Student t-test was used for statistical analysis. (F) Representative Western blot analysis with anti APP-CT (A8717, Sigma Aldrich), anti TrkA-CT (Abcam, ab76291) antibodies and anti ß-actin as loading control under treatments indicated.