Fig 2: TRKB localizes to the basal body and axoneme of hTERT-RPE1 cells in the presence of BDNF.(A–M) Immunofluorescent staining of hTERT-RPE1 cells transfected with empty vector (EV), shBBS4 or both 3'UTR shBBS4 and BBS4 expression construct (Rescue). Cells were either cultured in BDNF-deficient (BDNF-) or BDNF-supplemented (BDNF+) media and stained using antibody against TRKB (red) or ciliary markers labeling axoneme (ARL13B, green) and basal body (?-tubulin, green, arrows). Region around cilia denoted by dashed box and magnified inset. Scale bar = 10 µm. Imaged at 100× magnification. (N) Quantification of ciliary localization of TRKB calculated as the proportion of either basal bodies (black bars) or axonemes (striped bars) that co-localize with TRKB. Error bars represent standard deviation. *significant difference (p<0.01, chi-square test).

Fig 3: Reduced TRKB activation with loss of ciliary axoneme.(A–D) Immunofluorescent staining of empty vector (EV) control or shKIF3A-treated hTERT-RPE1 cells cultured in BDNF-supplemented media and stained using antibody against pTRKB (red) or ciliary markers labeling axoneme (ARL13B, green) or basal body (γ-tubulin, green). Region around cilia denoted by dashed box and magnified inset. Scale bar = 10 µm. Imaged at 100× magnification. (E) Quantification of basal body localization of pTRKB and TRKB in control (black) or shKIF3A-treated (striped) cells calculated as the proportion that co-localize with pTRKB or TRKB with or without BDNF. Error bars represent standard deviation. No significant difference between control and shKIF3A. (F) Western blot detection for pTRKB and TRKB in hTERT-RPE1 cells treated with or without BDNF and with or without shKIF3A. (G) Quantification of TRKB activation calculated as the average ratio of pTRKB to TRKB protein, measured by ImageJ densitometry analysis. Error bars depict standard deviation across a minimum of three experiments. *significant change (p<0.01, t-test) from control; **significant change (p<0.01, t-test) from BDNF-treated control cells. (H) Western blot detection in hTERT-RPE1 cells of pTRKB and TRKB, as well as Actin, in the presence or absence of BDNF and the presence or absence of a short hairpin targeting the 3′UTR (shBBS4) or a vector expressing BBS4. (I) Quantification of the average activation of TRKB in hTERT-RPE1 cells quantified as the amount of pTRKB relative to the amount of TRKB for indicated treatments. *significant change (p<0.01, t-test) from control; **significant change (p<0.05, t-test) from BDNF-treated control.

Fig 4: Abnormal pTrkB receptor expression and localization are observed in the early stage of cerebellar development and in vitro GCs of Npc1nmf164 mice. A, B Representative immunoblots of pTrkB protein expression in extracts from wt and Npc1nmf164 mice at different stages of cerebellar development (PN8,11, 15, and 30) and in DIV0 GCs. Western blot quantification of pTrkB in wt (white bars) and Npc1nmf164 (black bars) mice. Histograms indicate the abundance (mean ± SD) of each protein determined by the densitometry of protein bands obtained by taking total TrkB (TrkB) as an internal reference. Arrowhead indicates TrkB truncated form present in GCs. Significance is shown as a p value calculated using an unpaired t test. *p < .05 versus wt. **p < .01 versus wt (n = 5 mice/group). C, D Representative fields of confocal images are shown in the figure. Detection of pTrkB receptor (red) and nuclei (Hoechst 33258, blue) in PN8 cerebellar sections and in DIV3 GCs. The GCs in the EGL are magnified in the boxed regions of panel C. Scale bar: 20 μm. Comparison of the mean fluorescence intensity (y-axis) generated by staining sagittal sections of PN8 cerebellar cortex layers of wt and Npc1nmf164 mice with antibodies against pTrkB receptor. D GCs expressing pTrkB receptors in the soma (arrowheads) and in the leading process (asterisks) are indicated. Data shown represent the means ± SD and were evaluated by unpaired t test. **p < .01 versus wt (n = 3 mice/group). ML, molecular layer; PC, Purkinje cell; IGL, internal granular layer. Quantitative analysis of in vitro GCs with BDNF-dependent pTrkB polarization in wt and Npc1 mutant mice is shown as a p value calculated using an unpaired t test. *p < .05 versus wt