Fig 1: Silencing of CDKL5 is associated with multipolar spindle formation and chromosome segregation defects. (a) CDKL5 expression in HeLa cells 60 h after transfection with two different siRNAs targeting CDKL5 or a control siRNA. α-tubulin was used as loading control. The asterisk indicates an unspecific band (b,c) HeLa cells treated as in a were stained against α-tubulin and DAPI 60 h post-transfection. In b, the percentage of mitotic multipolar spindles is reported as mean ± S.E.M. (n = 3; *p < 0.05, ANOVA followed by Dunnet’s post hoc analysis). In c, the frequency of the indicated phenotypes is shown as mean ± S.E.M. (n = 2; *p < 0.05; **p < 0.01; unpaired t-test; approximately 1000 counted cells per condition). In b and c, representative images of the indicated cells are shown above the graphs with DAPI in blue and α-tubulin in red; scale bar, 10 µm. In c, arrows indicate a chromosome bridge and micronuclei. A/T = ana-/telo-phase. (d) HeLa cells were treated as in a and analyzed for phosphorylated histone H3 (P-H3) 60 h after silencing. The percentage of P-H3 positive cells was calculated in three independent experiments counting approximately 2000 cells (mean ± S.E.M., unpaired t-test). (e) CDKL5 was expressed in HeLa cells 60 h post-silencing by transfection of a bicistronic vector expressing also GFP. Vertical cropping was performed to show different exposures of the same membrane; full-length blots are shown in Supplementary Figure S7b. (f) Representative images of spindles in GFP-positive cells (green) treated as in e by staining against α-tubulin (red) and with DAPI (blue). Scale bar, 10 µm. (g) Graph showing number of cells with multipolar spindles upon CDKL5 re-expression in silenced cells. ≥ 60 counted cells per condition (n = 3; mean ± S.E.M, *p < 0.05; ANOVA followed by Dunnet’s post hoc analysis). n.s. = not statistically significant.
Fig 2: Validation of phosphospecific antibodies A–CHEK293 cells co-transfected with full-length WT or KD FLAG-CDKL5 and Strep-ARHGEF2 (A), HA-EB2 (B) or HA-MAP1S (C) are probed with their respective phosphospecific antibodies. Phosphospecific antibodies do not detect phosphomutants, indicating their specificity. ARHGEF2 pS122 (A) and MAP1S pS786 (C) are increased when WT CDKL5 is expressed. High levels of endogenous EB2 pS222 (B) and MAP1S pS812 (C) are not altered with WT CDKL5. Total levels of kinase (FLAG) and substrate (Strep/HA) are detected by epitope tags.D, EFull molecular weight range of Western blots with mouse P20 cortical lysates probed for total EB2 and EB2 pS222 (D) and MAP1S light chain and MAP1S pS812 (E).FEfficient shRNA-mediated knockdown of EB2 in rat primary neurons is shown by the specific loss of EB2 staining in transfected cells. Scrambled shRNA was used as a control. EB2 pS222 signal is apparent in dendrites of the control (arrowheads). The remaining nuclear signal of EB2 pS222 after shRNA-mediated knockdown (*) is considered non-specific. Scale bar is 10 µm. Source data are available online for this figure.
Fig 3: Schematic representation of the CDKL5 gene and protein. (A) Schematic representation of the human isoform hCDKL5-1 [18] with its principal functional domains. The catalytic domain is shown in green while the included ATP binding site (ATP), the Ser/Thr kinase active site (S/T) and the Thr-Glu-Tyr (TEY) motif are highlighted in grey. The C-terminus domain is depicted in red and the nuclear localization signals (NLS1 and NLS2) and nuclear export signal (NES) are in blue and violet, respectively. (B) Schematic representation of the CDKL5 gene with introns and exons represented respectively by lines and boxes. Nucleotide variants investigated in this study are reported in the upper part of the figure. Asterisks denote the exons whose presence differs between different transcript isoforms. The bioinformatics prediction of 3′ and 5′ss scores for each exon in normal and pathological condition is reported in the lower part of the panel.
Fig 4: Ex vivo studies indicate that the c.2376+5G>A variant is associated with exon 16 skipping and nonsense-mediated mRNA decay. (A) Electropherograms showing the sequence of CDKL5 exon 16 in a control (Ctrl) or a heterozygous patient harboring the c.2376+5G>A mutation obtained from the genomic DNA of the corresponding lymphoblastoid cell lines. The position of the mutation is indicated by a blue rectangle. (B) Western blotting analysis of CDKL5 expression in the immortalized lymphocytes from the control (lane 1) or the CDKL5-deficiency disorder (CDD) patient (lane 2). Histograms report the amount of CDKL5 protein isoform calculated by densitometric analysis of bands and normalized to GAPDH. (C) Growth rate curve of immortalized lymphocytes from the control donor or the CDKL5 patient. Asterisks indicate a significant difference between the number of control and patient’s cells at the selected time points. * p < 0.05; *** p < 0.001 by two-way repeated measure ANOVA, followed by Sidak’s multiple comparison test. (D) Reverse transcriptase (RT)-PCR evaluating the splicing pattern of CDKL5 exon 16 in the immortalized lymphocytes using the forward primer located in exon 12 and the reverse primer binding the exon 16–18 junction. Levels of 18S rRNA were used as internal standard. Histograms report the relative amount of correctly spliced CDKL5 mRNA in patient’s cells with respect to control, arbitrarily set at 100%. (E) Evaluation of CDKL5 exon 16 skipping transcripts after 4 h of cycloheximide treatment (CHX; 300 mg/mL) by RT-PCR using the forward primer located in exon 12 and the reverse primer binding the exon 15–18 junction. Thus, 18S amplification was used as internal standard.
Fig 5: CDKL5 influences shootin1 phosphorylation in primary cortical neurons.(A) Total cell extracts of DIV7 cortical neurons were treated with or without lambda phosphatase (λ-PPase) and analyzed by two-dimensional gel electrophoresis and immunoblotting with antibodies against shootin1, β-actin and, as control for the λ-PPase treatment, phopho-ERK1/2. (B) Primary cortical neurons were infected with shLacZ- or shCDKL5#1-expressing viral particles at DIV0 and total cell lysates were prepared at DIV7 and subjected to two-dimensional gel electrophoresis. Shootin1 and NFL were detected by immunoblotting; the single NFL-spot was used as internal control for alignment. Silencing of CDKL5 was confirmed by western blot (right panel). (n = 3).
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