Proneurotrophin-3 promotes cell cycle withdrawal of developing cerebellar granule cell progenitors via the p75 neurotrophin receptor
- Rutgers University, United States
Figures
Figure 1 with 1 supplement
Development of p75NTR in the rat cerebellum.
(a) Low magnification images of sagittal sections through the entire cerebellum from postnatal day (P) 2 through P21 showing the abundant immunolabeling for p75NTR in the EGL, which decreases by …
Figure 1—figure supplement 1
No differences in TUNEL labeling in the EGL between Ngfr-/- and WT mice.
Sections from WT or Ngfr-/- mouse P7 cerebellum were processed for TUNEL labeling. Few labeled cells were detected and no difference between genotypes was observed.
Figure 2
Expression of p75NTR, Ki67, and DCX in the P7 rat cerebellum.
(a) Confocal image of p75NTR and the proliferation marker Ki67 showing colocalization in the external EGL. Note that p75NTR is also expressed in developing Purkinje cells. (b) p75NTR is …
Figure 3
Cell cycle withdrawal of GCPs in the EGL is delayed in the Ngfr-/- mice compared to wild type mice.
(a) Low magnification images of the entire cerebellum showing incorporation of EdU at postnatal ages from P5 to P14. GCPs in WT mice began to decrease EdU incorporation at P7, which continued …
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Figure 3—source data 1
Mean number of labeled cells of 3 slides for each animal, and statistical analysis for the graphs shown in 3c, 3e, and 3g.
- https://doi.org/10.7554/eLife.16654.007
Figure 4
Mature neurotrophins have no effect on proliferation of cultured P7 rat GCPs in the absence or presence of Shh.
GCPs from P7 rat were cultured with BrdU for 48 hr in the absence or presence of Shh with or without the different mature neurotrophins. BrdU labeling was analyzed by in-cell Western on the LiCor …
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Figure 4—source data 1
Mean values for each experiment and statistical analysis for all graphs.
- https://doi.org/10.7554/eLife.16654.009
Figure 5 with 2 supplements
ProNT3, but not proNGF or proBDNF, prevented Shh-induced proliferation of cultured GCPs.
(a) GCPs were cultured from P7 rat cerebella with BrdU in the absence or presence of Shh with or without 4 or 8 ng/ml of proNGF, proBDNF or proNT3 for 48 hr. Cells were analyzed by in-cell Western …
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Figure 5—source data 1
Mean values for each experiment and statistical analysis for all graphs in Figure 5.
- https://doi.org/10.7554/eLife.16654.011
Figure 5—figure supplement 1
ProNT3 does not induce apoptosis of cerebellar neurons.
Cerebellar neurons were cultured from P7 rat and treated overnight with proNT3. Cells were labeled with TUNEL and Dapi, and expressed as the ratio of TUNEL-positive cells to total cell number.
Figure 5—figure supplement 2
Proneurotrophins induced death of hippocampal neurons.
Hippocampal neurons were cultured from E18 rat embryos and treated overnight with 4 ng/ml of the indicated proneurotrophin, eliciting loss of approximately 30% of the cells, consistent with previous …
Figure 6
ProNT3 requires p75NTR and SorCS2 to block Shh-induced GCP proliferation.
(a) GCPs from wild type or Ngfr-/- mice were cultured with Shh, proNT3 or Shh+proNT3 for 48 hr in the presence of BrdU and analyzed by in-cell Western analysis of anti-BrdU. (b) Immunostaining for …
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Figure 6—source data 1
Mean values for each experiment and statistical analysis for graphs in 6A and 6c.
- https://doi.org/10.7554/eLife.16654.015
Figure 7 with 1 supplement
Expression and secretion of proNT3 in cerebellum.
(a) Immunostaining of P7 and P14 rat cerebellum with an antibody to the pro domain of proNT3 shows the presence of proNT3 in Purkinje cells, labeled with calbindin. Note the abundant proNT3 labeling …
Figure 7—figure supplement 1
Validation of the proNT3 antibody.
Total brain lysates from Ntf3+/+, Ntf3+/-, and Ntf3-/- mice were immunoprecipitated with goat anti-proNT3 or goat control IgG, and probed with anti-proNT3 on a Western blot. ProNT3 produced from HEK …
Figure 8
HDAC1 expression and regulation by Shh and proNT3.
(a) Immunostaining for HDAC1 in WT and Ngfr-/- mice at P14. Size bar is 20 µm. (b) High magnification images of HDAC1 staining in the EGL in WT and Ngfr-/- mice at P14. Size bar is 10 µm. (c) …
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Figure 8—source data 1
Mean values for each experiment and statistical analysis for all graphs in Figure 8.
- https://doi.org/10.7554/eLife.16654.019
Figure 9
Specific deletion of p75NTR from the EGL elicits increased GCP proliferation.
(a) Ngfrfl/fl mice crossed with the Atoh1-Cre show lack of p75NTR specifically in the EGL while retaining p75NTR expression in Purkinje cells and meninges. Size bar indicates 20 µm. (b) Both Ngfr-/- …
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Figure 9—source data 1
Mean number of labeled cells of 3 slides for each animal, and statistical analysis for all graphs in Figure 9.
- https://doi.org/10.7554/eLife.16654.021
Figure 10
The absence of p75NTR during EGL development has persistent effects in the adult.
(a) Cerebellar size of both Ngfr-/- and Ngfrfl/fl-Atoh1-Cre was increased in the adult compared to WT mice. * indicates significance at p=0.0003. (b) Motor balance on the rotarod was impaired in …
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Figure 10—source data 1
Mean values for each experiment and statistical analysis for all graphs in Figure 10.
- https://doi.org/10.7554/eLife.16654.023
