Generalized expression of TrkB in DCRNs.

<p>After 20 h in vitro, DCRNs were immunolabeled with anti-TrkB antibody (green) and counterstained with bisbenzimide (Bisb.). Bar: 10 µm.</p

TrkB is expressed by a subset of RGCs lacking Rb expression.

<p>Confocal sections from retinas of E6 chick embryos were double labeled with an anti-TrkB specific antiserum (green) and anti-βIII tubulin (βIII Tub.) (<b>A</b>), anti-Rb (<b>B</b>), or anti-p75^NTR (p75) (<b>C</b>) antibodies (red). Nuclear staining with bisbenzimide is shown in blue (Bisb.). (<b>A</b>) TrkB-positive cells colocalize with βIII tubulin in the basal retina, close to the presumptive GCL (arrowhead). (<b>B</b>) TrkB-positive cells do not express Rb (arrowhead). Yellow arrowhead: a cell expressing Rb. (<b>C</b>) TrkB-positive cells colocalize with p75^NTR (arrowhead). Arrow: TrkB-positive cells surrounding the eye. GCL: ganglion cell layer; PE: pigment epithelium; v: vitreous body. Bar: 20 µm (<b>A</b>,<b>B</b>), 40 µm (<b>C</b>).</p

Cdk1 protein kinase activity measured from DCRNs in either the presence or absence of BDNF.

<p>(<b>A</b>) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. (<b>B</b>) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).</p

Tyr15 phosphorylation in cdk1 by BDNF is independent of Wee1.

<p>(<b>A</b>) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). (<b>B</b>) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ***p<0.005 (Student’s t test; n = 3). <b>^#</b>p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).</p

Cdk1 is expressed by p75^NTR- and TrkB-positive DCRNs and colocalizes with basally-located mitosis.

<p>Confocal sections from retinas of E6 chick embryos were double labeled with the anti-cdk1 specific mAbs (red) (B-6) (<b>A</b>), [A17] (<b>B</b>), and (17) (<b>C</b>-<b>E</b>), as well as with anti-p75^NTR (p75) (<b>C</b>), anti-phosphoHistone H3 (pH3) (<b>D</b>), or anti-TrkB (<b>E</b>) antibodies (green). Nuclear staining with bisbenzimide is shown in blue (Bisb.). (<b>A</b>,<b>B</b>) Cdk1 immunostaining is observed in the cytoplasm of a subpopulation of retinal cells and often in nuclei located apically (arrows). (<b>C</b>) A subset of cdk1-positive cells co-localize with p75^NTR (arrowhead), whereas many other p75^NTR-positive cells lack cdk1 immunolabeling (arrow). (<b>D</b>) Cdk1-positive cells co-localize with phosphoHistone H3 in the basal retina, close to the presumptive GCL (arrow). Arrowhead: apically located nucleus with cdk1-specific immunoreactivity. (<b>E</b>) TrkB-positive cells co-localize with cdk1 (arrow). Boxes: high magnification of the dashed areas. GCL: ganglion cell layer; PE: pigment epithelium; v: vitreous body. Bar: 20 µm (<b>A</b>-<b>D</b>), 40 µm (<b>E</b>).</p

A model for the regulation of cdk1 by BDNF in DCRNs.

<p>TrkB activation by BDNF prevents the increase cyclin B and cdk1 protein expression (dashed line) and induces phosphorylation of cdk1 at Tyr15 (thick black arrow), thus inhibiting cdk1 kinase activity. This effect participates in the G2/M arrest (grey line) observed in tetraploid RGCs.</p

Post-transductional effects of BDNF on cdk1 function are specific for Tyr15.

<p>E6 retinal cells electroporated with either EGFP (-) or the Tyr15Phe mutant form of cdk1 plus cyclin B1 (Mut CC) and EGFP (+) were cultured for 20 h under neurogenic conditions in the presence of different combinations of 100 ng/ml NGF and 2 ng/m BDNF. The percentage of pyknotic nuclei was evaluated in EGFP-positive cells. *p<0.05 (Student’s t test; n = 4).</p

Post-transductional effects of BDNF on cdk1 function.

<p>(<b>A</b>) E6 retinal cells electroporated with either EGFP (-) or cdk1 plus cyclin B1 (CC) and EGFP (+) were cultured under neurogenic conditions for 20 h in the presence of different combinations of 100 ng/ml NGF and 2 ng/ml BDNF. The percentage of mitotic figures was evaluated in the EGFP-positive cells. Lower panels show an example of a mitotic figure (pH3; red) in an EGFP-transfected cell (EGFP) (arrow). (<b>B</b>) E6 retinal cells were electroporated and cultured as above. The percentage of pyknotic nuclei was evaluated in the EGFP-positive cells. Lower panels show an example of a pyknotic nucleus in an EGFP-transfected cell (EGFP) (arrow). Bisb.: bisbenzimide. *p<0.05; ***p<0.005 (Student’s t test; n = 4). Bars: 10 µm.</p