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Cdk12 and Cdk13 regulate axonal elongation through a common signaling pathway that modulates Cdk5 expression
Experimental Neurology 261 (2014) 10–21
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Regular Article
Cdk12 and Cdk13 regulate axonal elongation through a common signaling pathway that modulates Cdk5 expression Hong-Ru Chen a, Guan-Ting Lin b, Chun-Kai Huang a, Ming-Ji Fann a,c,⁎ a b c
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 11221, Taiwan, ROC Institute of Neuroscience, National Yang-Ming University, Taipei 11221, Taiwan, ROC Brain Research Center, National Yang-Ming University, Taipei 11221, Taiwan, ROC
a r t i c l e
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Article history: Received 25 April 2014 Revised 16 June 2014 Accepted 18 June 2014 Available online 3 July 2014 Keywords: Cyclin-dependent kinase Axonal elongation Gene expression
a b s t r a c t Cdk12 and Cdk13 are Cdc2-related proteins that share 92% identity in their kinase domains. Using in situ hybridization and Western blot analysis, we detected the expression of Cdk12 and Cdk13 mRNAs and their proteins in developing mouse embryos, especially during development of the nervous system. We explored the roles of Cdk12 and Cdk13 in neuronal differentiation using the P19 neuronal differentiation model. Upon knockdown of Cdk12 or Cdk13, no effect on differentiated cell numbers was detected, but a substantial decrease of numbers of neurons with long neurites was identified. Similarly, knockdown of Cdk12 or Cdk13 in primarily cultured cortical neurons shortens the averaged axonal length. A microarray analysis was used to examine changes in gene expression after knockdown or overexpression of Cdk12 and we identified Cdk5 as a molecule potentially involved in mediating the effect of Cdk12 and Cdk13. Depletion of Cdk12 or Cdk13 in P19 cells significantly reduces Cdk5 expression at both the mRNA and protein levels. Expression of Cdk5 protein in the developing mouse brain is also reduced in conditional Cdk12-knockout mice in proportion to the residual amount of Cdk12 protein present. This suggests that the reduced axonal outgrowth after knockdown of Cdk12 or Cdk13 might be due to lower Cdk5 expression. Furthermore, overexpression of Cdk5 protein in P19 cells was able to partially rescue the neurite outgrowth defect observed when Cdk12 or Cdk13 is depleted. Together, these findings suggest that Cdk12 and Cdk13 regulate axonal elongation through a common signaling pathway that modulates Cdk5 expression. © 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Introduction Neurons are highly polarized cells with two functionally distinct domains that emerge from the cell body; these are the single long axon, which transmits signals, and the multiple shorter dendrites, which specialize in receiving signals (Craig and Banker, 1994). Once axon specification occurs, the axonal process begins to grow at a rate approximately 10 times faster than that of the dendritic processes (Dotti et al., 1988); this is due to differences in the patterns of microtubule organization between the dendrites and the axons (Conde and Caceres, 2009; Kuijpers
Abbreviations: Cdk5, cyclin-dependent kinase 5; Cdk12, cyclin-dependent kinase 12; Cdk13, cyclin-dependent kinase 13; CNS, central nervous system; DIV, days in vitro; K-S test, two sample Kolmogorov–Smirnov test; MAP2, microtubule-associated protein 2; SMI312, antibody against axonal phosphorylated neurofilament. ⁎ Corresponding author at: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan, ROC. Fax: +86 2 2823 4898. E-mail address: [email protected] (M.-J. Fann).
and Hoogenraad, 2011; Stiess and Bradke, 2011). Axons have tau-bound microtubules of uniform orientation with their plus end pointing outward, whereas dendrites have microtubule-associated protein 2 (MAP2)-bound microtubules of mixed orientation with many minus ends pointing outward (Baas et al., 1989; Burton, 1988; Conde and Caceres, 2009). Axonal and dendritic microtubule networks furthermore differ in their patterns of post-translational modifications and these result in unique interactions with other microtubule-associated proteins (Hammond et al., 2010; Witte et al., 2008). Several types of microtubule post-translational modifications, including tyrosination/ detyrosination and phosphorylation, are also known to modulate the sorting mechanisms of motor proteins in axons and dendrites and these consequently augment differences between axons and dendrites (Fukushima et al., 2009; Sirajuddin et al., 2014). For example, detyrosination modification of microtubules occurs more frequently in axons, which facilitates the recruitment of kinesin-1 to transport collapsin response mediator protein-2 in axons (Hammond et al., 2010; Kimura et al., 2005). The physiological relevance of microtubule phosphorylation in axons and dendrites is another field of active
http://dx.doi.org/10.1016/j.expneurol.2014.06.024 0014-4886/© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
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research. It has been suggested that phosphorylation of the microtubule networks is able to positively or negatively regulate the polymerization of microtubules (Fourest-Lieuvin et al., 2006; Khawaja et al., 1988). For example, Cdk1/cyclin B-mediated phosphorylation at Ser172 of β-tubulin impairs the incorporation of these modified subunits into polymers (Fourest-Lieuvin et al., 2006). Similarly, phosphorylation of β-tubulin at tyrosine residues prevents the incorporation of these β-tubulin molecules into microtubules (Ley et al., 1994). One of the molecules engaging in neuronal microtubule dynamics is Cdk5, a member of cyclin-dependent kinases (Cdk) (Smith, 2003; Veeranna et al., 2000). Cdk5 phosphorylates a number of neuronal cytoskeletal proteins involved in neuronal migration, neurite growth and synaptogenesis (Cheung and Ip, 2007; Cheung et al., 2006; Cruz and Tsai, 2004; Xie et al., 2006). Furthermore, substrates of Cdk5, such as tau, MAP2, the dynein-associated protein, Nudel, and SIRT2, are known to be involved in cytoskeletal dynamics. This implies that Cdk5 plays a role as a critical signal transmission hub that is positioned between a range of cell surface receptors and various cytoskeletal systems (Pandithage et al., 2008; Smith, 2003; Speranza et al., 2013; Veeranna et al., 2000). Protein kinases are critical regulators that affect the cell cycle, differentiation, metabolism, cell death and various other critical cellular processes (Ickowicz et al., 2012; Malumbres, 2011; Yuan et al., 2013)
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and we previously explored the roles of a number of uncharacterized protein kinases. In this study we have focused on Cdk12 and Cdk13 due to their abundant expression in the nervous system (Lin and Fann, 1998). They share 92% identity in the kinase domain, and are known to interact with cyclin L and cyclin K (Blazek et al., 2011; Bosken et al., 2014; Chen et al., 2006, 2007; Dai et al., 2012). At the molecular level, they are able to facilitate transcription of long genes with many exons, and are involved in mRNA alternative splicing (Blazek et al., 2011; Chen et al., 2006, 2007; Rodrigues et al., 2012). At the cellular level, Cdk12 and Cdk13 are able to maintain self-renewal in embryonic stem cells (Dai et al., 2012). In the present study, we examined additional possible functions of Cdk12 and Cdk13 during neuronal development. Using P19 cells as a neuronal differentiation model, we asked whether changes in Cdk12 and Cdk13 expression affect neuronal differentiation and neurite outgrowth. The P19 cells are mouse embryonic carcinoma cells and are able to differentiate into neurons when there is overexpression of the proneural gene Ascl1 (Farah et al., 2000). We found that reduced expression of Cdk12 or Cdk13 resulted in a shortening of neurite elongation in P19 cells and an inhibition of axonal extension in primary cultured cortical neurons, but reduced Cdk12 or Cdk13 expression had no effects on dendrite extension. We further demonstrated that Cdk12 and Cdk13 promote axonal elongation through a common signaling pathway that involves modulation of Cdk5
Fig. 1. Expression of Cdk12 and Cdk13 in the developing nervous system. (A) Cdk12 and Cdk13 mRNA expression in mouse embryos at various developmental stages was analyzed by whole-mount in situ hybridization. The results from the antisense probes are shown in a–d and e–h, while those from the sense probes are shown in a′–d′ and e′–h′. Cdk12 is expressed in all cells, except in the heart (H), but with higher levels in forebrain (F), midbrain (M), primitive streak (PS) and tail bud mesoderm (TBM). A similar expression patterns is observed for Cdk13 mRNA expression (e–h). The scale bar represents 300 μm. (B, C) Expression of the Cdk12 and Cdk13 proteins in developing brains was analyzed by Western blotting.
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expression at the RNA level. This is a novel finding that couples Cdk12 and Cdk13 functioning with Cdk5 signaling during axonal elongation. Materials and methods Animals Adult C57BL/6J mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). All mice were handled according to the university guidelines and all experiments were approved by the National Yang-Ming University Animal Care and Use Committee. For the timed pregnancies, mice were set up in the late afternoon and when plugs were detected the next morning, this was designated as E0.5. Whole-mount in situ hybridization Mouse embryos were dissected at different developmental stages, fixed in 4% paraformaldehyde, and permeabilized by treatment with proteinase K. After hybridization and washing, embryos were incubated with an alkaline phosphatase-conjugated anti-digoxygenin antibody (Roche), washed again and then stained using a NBT/BCIP mixture (Roche) in the presence of 10% polyvinyl alcohol (31–50 kD; Sigma). After visualization of the alkaline phosphatase activity, embryos were post-fixed and cleared in 100% glycerol for photography. Sense and antisense RNA probes were synthesized using an in vitro transcription kit from Roche, and were purified using a Sephadex G-50 column (Amersham Biosciences). RNA inhibitor (RNase) was purchased from Promega. Vector construction The various Cdk12 and Cdk13 plasmids had been constructed previously (Chen et al., 2006, 2007). Protocols described by Chung et al. (2006) were followed to construct the microRNA mimics. Oligonucleotides against mouse Cdk5, Cdk12 and Cdk13 sequences as well as against their equivalent scramble sequences were synthesized and cloned into the vector UI4-puro-SIBR to obtain pCdk5-s, pCdk5-i1, pCdk5-i2, pCdk12-s, pCdk12-i1, pCdk12-i2, pCdk13-s, pCdk13-i1 and pCdk13-i2, or into the vector UI4-GFP-SIBR to obtain pCdk12-s-e, pCdk12-i1-e, pCdk13-s-e and pCdk13-i1-e. The sequences of the oligonucleotides are listed in Table S1. Mouse Cdk5 activator, p35, was amplified by PCR and cloned into the vectors pUS2. All constructs were verified by restriction enzyme mapping and sequencing. Antibodies Rabbit anti-Cdk12 and mouse anti-Cdk13 polyclonal antibodies had been produced previously (Chen et al., 2006, 2007). Mouse anti-β-
tubulin antibody (E7, 1:10 000) and anti-Myc antibody (9E10, 1:5000) were obtained from the Developmental Studies Hybridoma Bank. Mouse anti-Flag M2 monoclonal antibody (1:1000) was purchased from Sigma. Mouse anti-Cdk5, rabbit anti-MAP2 and mouse anti-tau1 antibodies were obtained from Millipore. Mouse anti-pan-axonal neurofilament marker (SMI312) and mouse anti-neuronal class III βtubulin (TuJ1) were purchased from Covance. TRITC-conjugated goat anti-mouse antiserum was obtained from the Jackson Laboratory. Other secondary antisera were purchased from Chemicon or Bethyl Laboratories. Cell culture and transfection P19 cells were cultured in α-minimal essential medium (α-MEM) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine and incubated in a 5% CO2/95% humidified atmosphere at 37 °C. For neuronal differentiation, P19 cells were transfected with pAscl1 and other plasmids using Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer. The expression plasmid pAscl1 (pUS2-Ascl1) from D. L. Turner was used for neuronal differentiation, which has previously been shown to convert P19 cells into a relatively homogenous population of electrophysiologically differentiated neurons (Farah et al., 2000; Yu et al., 2002). Eight hours after transfection, 8 μg/ml puromycin was applied to select transfected cell. The cells were then replated onto 6-well plates coated with 2 μg/ml laminin (Invitrogen) at 24 h after transfection, and cultured in 1% FBS OPTI-MEM. The medium was changed at two days after transfection. Cells were harvested for immunocytochemistry or protein extraction at three days after transfection. Mouse cortical neurons were prepared from E14.5 mouse embryos. The cortex was mechanically dissociated by passing through a glass pipette for twenty times and then filtered through a 70-μm nylon mesh filter (BD Biosciences). Cells were plated onto plastic culture plates coated with 30 μg/ml poly-L-lysine and 2 μg/ml laminin and cultured in Dulbecco's modified Eagle's medium containing 10% FBS, 100 U/ml penicillin and streptomycin, and N2 supplement and B27 supplement (Invitrogen). The cells were kept at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. To deliver plasmids into cortical neurons, electroporation was performed before plating using the Amaxa Mouse Neuron Nucleofector kit and the Amaxa Nucleofecor II (Lonza). Conditions were optimized for 4 × 107 cells and 10 μg of plasmid per electroporation, which gave a survival and transfection efficiency of 60% and 35% to 40%, respectively. Immunofluorescence microscopy and neurite counting Cultured neurons were rinsed once with PBS and fixed in 4% paraformaldehyde for 1 h. Samples were stained with the appropriate primary and secondary antibodies and then finally stained with DAPI (1 μg/ml) for 15 min. Samples were then examined using a fluorescence microscope (Zeiss). Numbers of TuJ1-positive cells and DAPI-positive cells
Fig. 2. Knockdown of Cdk12 and Cdk13 decreases neurite elongation in P19 cells. (A) A schematic depiction of the experimental procedure. (B) Representative images of P19 cells transfected with the indicated Cdk12 constructs. (C) Differentiation rates of P19 cells transfected with the indicated constructs. Differentiated neuronal cells were detected by TuJ1 staining. The number of TuJ1-positive cells was divided by the number of DAPI positive cells in 30 randomly selected fields. (D) Average neurite length of P19 cells transfected with the indicated constructs. The lengths of neurites marked by TuJ1 staining were manually quantified for each selected neuron using Neuron J software. In each experiment, 300 neurons from randomly selected fields for each transfected condition were evaluated. Data from three independent experiments were analyzed by Student's t-test and are presented as mean ± SEM. **, p b 0.01; ***, p b 0.001. (E) The distribution of neurite lengths using P19 cells transfected with the indicated constructs. Data from three independent experiments are presented as mean ± SEM. Statistical analysis using two sample Kolmogorov–Smirnov test (K–S test) shows p N 0.05 for pCdk12 when compared to pEF, and p N 0.05, p b 0.01, and p b 0.001 for pCdk12-s, pCdk12-i1, and pCdk12-i2, respectively, when compared to pUI4. (F) Representative images of P19 cells transfected with the indicated Cdk13 constructs. (G) Differentiation rates of P19 cells transfected with the indicated constructs. (H) Average neurite length of P19 cells transfected with the indicated constructs. Data from three independent experiments are presented as mean ± SEM. ***, p b 0.001 by Student's t-test. (I) Distribution of neurite lengths of P19 cells transfected with the indicated constructs. Data from three independent experiments are presented as mean ± SEM. The two sample K–S test shows p N 0.05 for pCdk13 when compared to pEF, and p N 0.05, p b 0.001, and p b 0.001 for pCdk13-s, pCdk13-i1, and pCdk13-i2, respectively, when compared to pUI4. Scale bars 100 μm.
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were counted using the Image J plugin, NeurphologyJ (Ho et al., 2011). The differentiation rate was calculated by dividing TuJ1-positive cell numbers by DAPI-positive cell numbers. The tracking of P19 cell
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neurites and cortical neurons neurites was performed manually, and the length of each neurite was quantified using the Image J plugin, ImageJ. In each experiment, 300 neurons from randomly selected fields
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for each condition were evaluated. All experiments were repeated at least three times. Targeted disruption of the mouse Cdk12 gene To generate Cdk12 conditional knockout mice, a targeting vector containing Cdk12 sequences between exon 3 and exon 4 flanked by loxP, and FRT-flanked Neo cassette was prepared from a BAC clone using the recombineering approach (Copeland et al., 2001; Liu et al., 2003). The targeting vector was linearized and electroporated into embryonic stem (ES) cells, which was performed by Transgenic Mouse Models Core Facility, Taiwan. After selection with antibiotics, cell clones with correct homologous recombination were identified by Southern blot hybridization. After Neo pop-out, a correctly targeted ES clone was injected into blastocysts and the resultant chimeras were bred with C57BL/6J mice. Genotyping was performed by Southern blotting and/or PCR using the probe and primers listed in Table S1. To conditionally delete Cdk12 in the nervous system, NestinCreER T2 mice were used for Cre-mediated recombination after injecting Tamoxifen. Most mice used for this study were on a C57BL/6-129Sv mixed background. Protein extraction and Western blotting Previously described procedures were followed during these experiments (Wong et al., 2010). Quantitative real-time PCR assay Total RNA was extracted using a RNeasy Mini kit (Qiagen) following the manufacturer's suggestions. Reverse transcription was performed with 1.5 μg of total RNA using SuperScript III reverse transcriptase (Invitrogen). The real-time quantitative PCR was performed on a LightCycler 480 using the Universal Probe Library (UPL) (Roche). A web service (http://qpcr.probefinder.com/roche3. html) was used to select targeted input sequences, UPL probes, and primer pairs. The sequences of the primers are listed in Table S1. Negative controls containing no cDNA template were included in each experiment. The results were analyzed by LightCycler software version 3.5 (Roche) to determine the threshold cycle (Cp) for each reaction. Results Expression of Cdk12 and Cdk13 in the developing nervous system We used whole-mount in situ hybridization and Western blotting to detect the spatial and temporal expression patterns of Cdk12 and Cdk13 at both the mRNA and protein levels. Cdk12 mRNA is present in E6.5 embryo proper and trophoblasts (Fig. 1Aa). During E7–E7.5, Cdk12 is expressed in all cells, but with stronger expression in the primitive streak (PS) (Fig. 1Ab). In E8.5 and E9.5 embryos, stronger signals are detected in the forebrain, midbrain and tail bud mesoderm (TBM), but not in the heart (Figs. 1Ac and Ad). Cdk13 is also ubiquitously expressed but not in the heart at the various developmental stages examined (Figs. 1Ae–Ah). We further analyzed
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expression of Cdk12 and Cdk13 at the protein level in the developing brain at later developmental stages and found that Cdk12 and Cdk13 proteins were consistently expressed in the brain at all embryonic stages (Figs. 1B and C), which suggests that Cdk12 and Cdk13 may play a general role in the development of the nervous system. Knockdown of Cdk12 or Cdk13 leads to an axonal extension defect through a common signaling pathway Initially, we examined whether Cdk12 and Cdk13 participate in neuronal differentiation using the P19 neuronal differentiation model. P19 is a mouse embryonal carcinoma cell line that is able to be induced to differentiate into neuronal cells by ectopic expression of proneural gene Ascl1 and gives rise to unipolar or bipolar neurites. Overexpression constructs that increase Cdk12 or Cdk13 protein expression more than nine folds in P19 cells, and two RNA interference constructs for each gene that decrease Cdk12 or Cdk13 protein expression to less than 40% of the control level were constructed (Figs. S1A and C). The Cdk12 interference constructs do not inhibit Cdk13 protein expression, and vice versa (Figs. S1B and D). Differentiated P19 cells exhibit unipolar or bipolar neurites whose lengths can be revealed by staining with TuJ1 antibody, which recognizes a neuronal specific beta-III-tubulin expressed in early postmitotic neurons. Overexpression of either Cdk12 or Cdk13 in P19 cells was found not to affect differentiation of P19 cells cultured for 3 days after transfection, either in terms of the numbers of differentiated TuJ1-positive cells or in terms of the average length of outgrown neurites (Figs. 2Bb, C, D, Fb, G, and H). Surprisingly, however, knockdown of Cdk12 or Cdk13 decreased the average lengths of neurites in differentiated P19 neurons (312.66 ± 0.34 μm, mean ± SEM, for the pUI4 control, 227.17 ± 7.27 μm for pCdk12-i1, Fig. 2D; and 306.95 ± 4.3 μm for pUI4 control, 202.52 ± 5.75 μm for pCdk13-i1, Fig. 2H), although there was no change in the numbers of differentiated cells (Figs. 2C and G). Detailed analysis of the neurite length distribution revealed that cells with a deficiency in either Cdk12 or Cdk13 were able to send out processes but had reduced capability to extend these processes to long distances (N 300 μm) (Figs. 2E and I). Thus, distribution of the neurite length was skewed to the right in Cdk12-depleted and Cdk13depleted cells, suggesting that Cdk12 and Cdk13 are critical for neurite elongation in P19 cells. Next, we analyzed whether Cdk12 and Cdk13 affect axonal elongation through a common pathway. Interestingly, overexpression of Cdk13 in Cdk12-depleted cells reduced the percentage of short neurite (100–200 μm) from 38% to 27%, and increased the percentage of longer neurite (300–400 μm) from 16% to 23% (Figs. 3B and F). Overexpression of Cdk12 in Cdk13-depleted cells was also able to decrease the percentage of short neurite (100–200 μm) from 44% to 33%, and to raise percentage of the longer neurite (300– 400 μm) from 8% to 13% (Figs. 3D and H). Furthermore, the average neurite lengths after overexpression approached those of the control cells, when compared to those of the Cdk12-depleted or Cdk13depleted P19 cells (Figs. 3E and G). These results suggest that the same underlying signal pathway, at least in part, may mediate action of both Cdk12 and Cdk13 (Fig. 3).
Fig. 3. Cdk12 and Cdk13 regulate neurite elongation through a common signaling pathway in differentiated P19 cells. (A, C) The expression levels of Cdk12, Cdk13, and Cdk5 proteins in P19 cells transfected with the indicated constructed. Ectopically expressed Cdk12 and Cdk13 were detected by anti-myc antibody. (B, D) Representative images of P19 cells transfected with the indicated constructs are shown. (E, F) The average neurite lengths and the neurite length distribution of P19 cells transfected with the indicated Cdk13 and Cdk12 constructs are shown. *, p b 0.05. There is a significant difference between the pCdk12-i1 and pCdk12-i1 + pCdk13 groups by the two sample K–S test (p b 0.01). (G, H) The average neurite lengths and neurite length distribution in P19 cells transfected with the indicated Cdk12 and Cdk13 constructs are shown. *, p b 0.05. There is a significant difference between pCdk13-i1 and pCdk13-i1 + pCdk12 groups by the two sample K–S test (p b 0.001). Scale bars 100 μm.
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As Cdk12 and Cdk13 proteins are detected in mouse E14.5 cortical neurons (Fig. S2), we examined whether Cdk12 and Cdk13 are able to modulate neurite extension in cultured cortical neurons. Dissociated cortical neurons derived from mouse E14.5 embryos were electroporated with the various constructs. We used antiMAP2 antibody to demarkate dendrites, and anti-phosphorylated neurofilament antibody (SMI312) and anti-tau1 antibody to identify axons. After 3 days of culture in vitro (DIV), depletion of either Cdk12 or Cdk13 did not affect the neuronal polarity in cultured cortical neurons (Fig. S3). These neurons exhibited several MAP2positive processes, and one long SMI312-positive process. In Cdk12-depleted and Cdk13-depleted cortical neurons, the average length of dendrites was not affected (12.67 ± 1.19 μm in control cells vs. 12.33 ± 1.44 μm and 12.36 ± 0.72 μm in Cdk12-depleted and Cdk13-depleted neurons, respectively, Figs. 4A, B); however, the average length of axons was decreased (101 ± 5.25 μm in control cells vs. 42 ± 3.3 μm and 41 ± 2.05 μm in Cdk12-depleted and Cdk13-depleted neurons, respectively, Figs. 4C, D). At 5 DIV, dendrites and axons of the control neurons had grown further (Figs. 4E–H). Dendrite growth had taken place in the Cdk12depleted and Cdk13-depleted neurons (compare Figs. 4F with B); however, the growth of the axons of Cdk12-depleted and Cdk13depleted neurons was arrested during these two days (compare Figs. 4H with D). Staining with tau1 gave similar results at 3 DIV (Fig. S4). Together, these findings support the idea that Cdk12 and Cdk13 are essential for axonal extension. Expression of Cdk5 is regulated by Cdk12 and Cdk13 To dissect the underlying mechanisms by which Cdk12 and Cdk13 regulate axonal elongation, we performed microarray analysis and compared the global gene expression profiles of the control, Cdk12-overexpressing and Cdk12-knockdown P19 cells. Using these findings, we then selected candidate genes whose expression was decreased in the Cdk12-knockdown P19 cells but did not change in the Cdk12-overexpressing P19 cells, both in comparison to cells transfected with the control vector. Among these candidates identified, Cdk5 mRNA was found to be down-regulated in Cdk12knockdown P19 cells to 27% of the control using the microarray analysis. As Cdk5 is an important molecule in terms of neurite outgrowth during neural development, we then investigated whether Cdk12 and Cdk13 were indeed able to regulate Cdk5 mRNA expression using quantitative RT-PCR analysis. Knockdown of either Cdk12 or Cdk13 was found to reduce Cdk5 mRNA level to ~ 40% of the control level (Fig. 5A), which is a level compatible with the decreased levels of Cdk12 or Cdk13 protein when their corresponding RNA interference constructs are transfected into P19 cells. Cdk5 protein expression is also reduced to ~ 50% of the control (Fig. 5B, C), which suggests that regulation of Cdk5 by Cdk12 and Cdk13 is primarily at the RNA level. Previously we have generated a Cdk12 conditional knockout mouse by inserting loxP sites into intron 2 and intron 4 (Fig. S5A). We took advantage of variable efficiency of the Cre recombinase in embryos after injecting Tamoxifen into pregnant mice, and asked whether there is a dose–response relationship between the in vivo expression of Cdk12 and the expression of Cdk5. We crossed Cdk12 f/f mice with Nestin-CreER T2 ; Cdk12 del/+ mice, and injected
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Tamoxifen at E10.5 into pregnant mice. Whole brain was then dissected from the E14.5 embryos and protein extracts from different embryos were subjected to Western analysis using anti-Cdk12 antibodies and anti-Cdk5 antibodies (Fig. 5D). A linear correlation between Cdk12 expression and Cdk5 expression with a value for r of 0.98 was found (Fig. 5E). Overexpression of Cdk5 partially rescues effects of Cdk12 or Cdk13 knockdown To examine whether Cdk5 is the downstream gene that is controlled by Cdk12 and Cdk13 in P19 cells, we performed RNA interference experiments to knockdown Cdk5 mRNA and analyzed the effect on neurite extension in P19 cell model. Transfection of two RNA interference constructs (pCdk5-i1 and pCdk5-i2) was found to reduce Cdk5 protein expression to 50% of the control (Fig. S1E). Upon knockdown of Cdk5, the number of differentiated P19 cells does not change (Fig. 5G), but it was found that the neurite length distribution was skewed to the right in a pattern that is similar to what we observed for Cdk12-depleted or Cdk13-depleted cells (Figs. 5H, I). It should be noted that the decrease in average neurite length and change in skewness after knockdown of Cdk5 were found to be less than the changes induced by Cdk12 and Cdk13 depletion. We next examine whether overexpression of Cdk5 was able to rescue the defects found in Cdk12-depleted and Cdk13-depleted P19 cells. When Cdk5 was ectopically expressed in Cdk12-depleted and Cdk13-depleted P19 cells with Cdk5 activator, p35, the neurites grew to a longer length; furthermore, the average neurite length and the distribution of the neurite length approached that of the control cells, when compared to the Cdk12-depleted or Cdk13-depleted P19 cells (Fig. 6). Thus, the defect of neurite extension due to knockdown of Cdk12 or Cdk13 was found to be partially rescued by Cdk5 overexpression. However, overexpression of Cdk12 or Cdk13 in Cdk5-depleted P19 cells had no effect on neurite outgrowth in comparison to knockdown of Cdk5 alone (Fig. S6). These findings suggest that the Cdk5 is one of the downstream mediators for Cdk12 and Cdk13 when axonal elongation occurs. Discussion Cdk12 and Cdk13 are essential for axonal elongation Our investigation provides evidence that Cdk12 and Cdk13 contribute to axonal growth. Upon depletion of Cdk12 or Cdk13, cultured P19 cells and primary cortical neurons lose the ability to extend their axons to greater distances, although they are still able to initiate neurites (Figs. 2 and 4). Overexpression of Cdk13 is able to partially rescue the neurite outgrowth defect in Cdk12-depleted P19 cells, and vice versa, which suggests that they act through common downstream effector(s) to promote axonal elongation (Fig. 3). One protein affected by both Cdk12 and Cdk13 is Cdk5, and both are able to modulate Cdk5 protein expression in vitro; furthermore, overexpression of Cdk5 in Cdk12-depleted and Cdk13-depleted P19 cells is able to partially rescue the neurite outgrowth defect (Fig. 6). Interestingly, overexpression of Cdk13 increases the endogenous Cdk5 protein expression level and is able to rescue partially the defects in Cdk12-depleted P19 cells, also
Fig. 4. Cdk12 and Cdk13 enhance axonal elongation in primary cultured cortical neurons. Cortical neurons from the E14.5 mouse cortex were transfected with the indicated constructs. The cells were cultured, fixed, and immunostained with anti-MAP2 (A, E) or anti-SMI312 (C, G) antibodies at 3 DIV (A–D) and 5 DIV (E–H). The arrows indicate MAP2-positive and SMI312positive neurites. (B, D, F, H) Quantification of the length of MAP2-positive dendrites and the SMI312-positive axons as shown in A, C, E, and G was performed. For each condition, the data were collected from 300 randomly selected cells. Data from three independent experiments were analyzed by Student's t-test and are presented as mean ± SEM. **, p b 0.01; ***, p b 0.001. Scale bars 50 μm.
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showing causality relationship. Overexpression of Cdk12 in Cdk13depleted P19 cells gives the same results (Figs. 3A, C). The findings from Cdk12 knockout mice further confirm that expression of Cdk5 is indeed regulated by the level of Cdk12 proteins in vivo. However, the observation that Cdk5 overexpression in Cdk12-depleted and Cdk13depleted P19 cells brings about only a partial rescue of the neurite elongation defect, which implies that there are other effectors involved in Cdk12-dependent and Cdk13-dependent axonal elongation (Fig. 7). Cdk12 and Cdk13 have overlapping, but not identical, functions Cdk12 and Cdk13 have similar protein sequences and structural motifs. They are known to be engaged in a number of similar biological processes (Chen et al., 2006, 2007; Dai et al., 2012; Kohoutek and Blazek, 2012). For example, both Cdk12 and Cdk13 play an indispensable role in maintaining the pluripotency of mouse embryonic stem cells (Dai et al., 2012). Furthermore, both Cdk12 and Cdk13 take part in RNA splicing regulation in a similar manner (Chen et al., 2006, 2007). In this study, we have shown that their expression patterns seem also to be very similar (Fig. 1). Expression of Cdk12 and Cdk13 is initiated quite early during development, beginning as early as the E6.5 mouse embryo. Both are expressed ubiquitously, except that they are both absent from the developing heart. This similarity in expression pattern and function between Cdk12 and Cdk13 suggests that deficiency in either protein is unlikely to generate an obvious phenotype in mouse embryos. It is thus surprising that Cdk12 knockout mice die at post-implantation stage at E5.5 (data not shown) and this suggests that Cdk13 is not a redundant version of Cdk12 and is not able to completely complement Cdk12 deficiency. Furthermore, it has been shown that Cdk12, rather than Cdk13, activates transcription via the phosphorylation of Ser2 at the carboxylterminal domain of RNAPII and this then facilitates transcription of long exons during elongation (Bartkowiak et al., 2010; Blazek et al., 2011; Cheng et al., 2012; Ghamari et al., 2013). It is likely that these two genes are derived from an ancestor gene but have then evolved divergently and are now an evolutionary distinct far part in vertebrates (Malumbres et al., 2009). This may have resulted in subtle differences in terms of cellular localization as well as changes in their preference in terms of substrates and cyclins. A more thorough study is needed in the future in order to explore these fascinating possibilities. Axons, but not dendrites, are affected by the activity of Cdk12 and Cdk13 Axonal outgrowth involves many steps. The roles of Cdk12 and Cdk13 seem to be limited to axonal elongation. They are not required for specification and maintenance of neuronal polarity, since knockdown of Cdk12 and Cdk13 does not affect the numbers of SMI312positive cortical neurons or MAP2-positive cortical neurons at 3 DIV and 5 DIV (Figs. 4 and S4). However, knockdown of Cdk12 and Cdk13 dose affect axonal elongation and this effect occurs without affecting dendrite extension. This suggests that both Cdk12 and
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Cdk13 are able to regulate the axonal specific microtubule organization but not dendritic cytoskeletal organization, although expression of Cdk12 and Cdk13 is distributed evenly across both dendrites and axons (data not shown). Currently, the underlying mechanisms by which Cdk12 and Cdk13 are able to generate this differential effect remain unclear. It seems likely that Cdk12 and Cdk13 regulate tubulin polymerization and microtubule stability, which is highly critical in axons. Silencing of Cdk12 and/or Cdk13 may also bring abnormal microtubule remodeling and a change in microtubule dynamics in axons. Details of the downstream Cdk12 and Cdk13 substrate proteins and how they contribute to this differential effect on axons and dendrites remain obscure. Future investigation in this area will further strengthen our understanding of the development of axons and dendrites. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.expneurol.2014.06.024. Author contribution H.-R. Chen, G.-T. Ling, and M.-J. Fann conceived and designed the experiments. H.-R. Chen, G.-T. Ling, and C.-K. Huang conducted experiments. H.-R. Chen and M.-J. Fann wrote the manuscript. Conflicts of interest Authors declare no conflict of interest. Acknowledgments We thank L.-S. Kao, J.-Y. Yu, Y.-C. Chern, E. Hwang, and M.-m. Poo for their discussion and critical reading of the manuscript, E. Hwang is thanked for providing NeurphologyJ software. We would also like to thank the Developmental Studies Hybridoma Bank for providing monoclonal antibodies. This research was supported by the National Science Council (NSC101-2320-B-010-064) and the Ministry of Education (Aim for the Top University Plan) to M.-J. Fann. References Baas, P.W., Black, M.M., Banker, G.A., 1989. Changes in microtubule polarity orientation during the development of hippocampal neurons in culture. J. Cell Biol. 109, 3085–3094. Bartkowiak, B., Liu, P., Phatnani, H.P., Fuda, N.J., Cooper, J.J., Price, D.H., Adelman, K., Lis, J.T., Greenleaf, A.L., 2010. CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1. Genes Dev. 24, 2303–2316. Blazek, D., Kohoutek, J., Bartholomeeusen, K., Johansen, E., Hulinkova, P., Luo, Z., Cimermancic, P., Ule, J., Peterlin, B.M., 2011. The cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes. Genes Dev. 25, 2158–2172. Bosken, C.A., Farnung, L., Hintermair, C., Merzel Schachter, M., Vogel-Bachmayr, K., Blazek, D., Anand, K., Fisher, R.P., Eick, D., Geyer, M., 2014. The structure and substrate specificity of human Cdk12/Cyclin K. Nat. Commun. 5, 3505. Burton, P.R., 1988. Dendrites of mitral cell neurons contain microtubules of opposite polarity. Brain Res. 473, 107–115. Chen, H.H., Wang, Y.C., Fann, M.J., 2006. Identification and characterization of the CDK12/ cyclin L1 complex involved in alternative splicing regulation. Mol. Cell. Biol. 26, 2736–2745.
Fig. 5. Depletion of Cdk12 or Cdk13 leads to down-regulation of Cdk5 and depletion of Cdk5 causes a neurite elongation defect in P19 cells. (A) Quantitative PCR was performed to measure Cdk5 expression in P19 cells transfected with various Cdk12 or Cdk13 constructs. Data from three independent experiments were analyzed by Student's t-test and are presented as mean ± SEM. **, p b 0.01. (B) Knockdown of Cdk12 or Cdk13 in P19 cells reduces Cdk5 protein expression as revealed by Western blotting. (C) Quantification of (B). The data from three independent experiments were analyzed by Student's t-test and are presented as mean ± SEM. **, p b 0.01. (D) Western blot analysis of Cdk12, Cdk13, and Cdk5 protein expression in embryonic brains with different Cdk12 knockout backgrounds. (E) The amount of Cdk5 protein present was plotted against the amount of Cdk12 proteins present using mouse embryos that had different genotypes and different levels of Cre activities. Expression levels of Cdk12 and Cdk5 in a randomly selected Cdk12f/+mouse (marked with *) were defined as having an arbitrary unit (A.U.) value of one. (F) Representative images of P19 cells transfected with the indicated Cdk5 constructs. (G) Differentiation rates of P19 cells transfected with the indicated constructs. (H) The average neurite lengths of P19 cells transfected with the indicated constructs. Data from three independent experiments are presented as mean ± SEM. **, p b 0.01; ***, p b 0.001 by Student's t-test. (I) The distribution of neurite lengths of P19 cells transfected with the indicated constructs. Data from three independent experiments are presented as mean ± SEM. The two sample K–S test shows p N 0.05, p b 0.05, and p b 0.05 for pCdk5-s, pCdk5-i1, and pCdk5-i2, respectively, when compared to pUI4. Scale bars 100 μm.
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Fig. 7. A proposed model for the action of Cdk12 and Cdk13 in differentiated neurons. Cdk12 and Cdk13 independently regulate axonal elongation in differentiated neurons, in part, through modulation of Cdk5 expression.
Chen, H.H., Wong, Y.H., Geneviere, A.M., Fann, M.J., 2007. CDK13/CDC2L5 interacts with L-type cyclins and regulates alternative splicing. Biochem. Biophys. Res. Commun. 354, 735–740. Cheng, S.W., Kuzyk, M.A., Moradian, A., Ichu, T.A., Chang, V.C., Tien, J.F., Vollett, S.E., Griffith, M., Marra, M.A., Morin, G.B., 2012. Interaction of cyclin-dependent kinase 12/CrkRS with cyclin K1 is required for the phosphorylation of the C-terminal domain of RNA polymerase II. Mol. Cell. Biol. 32, 4691–4704. Cheung, Z.H., Ip, N.Y., 2007. The roles of cyclin-dependent kinase 5 in dendrite and synapse development. Biotechnol. J. 2, 949–957. Cheung, Z.H., Fu, A.K., Ip, N.Y., 2006. Synaptic roles of Cdk5: implications in higher cognitive functions and neurodegenerative diseases. Neuron 50, 13–18. Chung, K.H., Hart, C.C., Al-Bassam, S., Avery, A., Taylor, J., Patel, P.D., Vojtek, A.B., Turner, D. L., 2006. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. Nucleic Acids Res. 34, e53. Conde, C., Caceres, A., 2009. Microtubule assembly, organization and dynamics in axons and dendrites. Nat. Rev. Neurosci. 10, 319–332. Copeland, N.G., Jenkins, N.A., Court, D.L., 2001. Recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2, 769–779. Craig, A.M., Banker, G., 1994. Neuronal polarity. Annu. Rev. Neurosci. 17, 267–310. Cruz, J.C., Tsai, L.H., 2004. A Jekyll and Hyde kinase: roles for Cdk5 in brain development and disease. Curr. Opin. Neurobiol. 14, 390–394. Dai, Q., Lei, T., Zhao, C., Zhong, J., Tang, Y.Z., Chen, B., Yang, J., Li, C., Wang, S., Song, X., Li, L., Li, Q., 2012. Cyclin K-containing kinase complexes maintain self-renewal in murine embryonic stem cells. J. Biol. Chem. 287, 25344–25352. Dotti, C.G., Sullivan, C.A., Banker, G.A., 1988. The establishment of polarity by hippocampal neurons in culture. J. Neurosci. 8, 1454–1468. Farah, M.H., Olson, J.M., Sucic, H.B., Hume, R.I., Tapscott, S.J., Turner, D.L., 2000. Generation of neurons by transient expression of neural bHLH proteins in mammalian cells. Development 127, 693–702. Fourest-Lieuvin, A., Peris, L., Gache, V., Garcia-Saez, I., Juillan-Binard, C., Lantez, V., Job, D., 2006. Microtubule regulation in mitosis: tubulin phosphorylation by the cyclindependent kinase Cdk1. Mol. Biol. Cell 17, 1041–1050. Fukushima, N., Furuta, D., Hidaka, Y., Moriyama, R., Tsujiuchi, T., 2009. Post-translational modifications of tubulin in the nervous system. J. Neurochem. 109, 683–693.
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Ghamari, A., van de Corput, M.P., Thongjuea, S., van Cappellen, W.A., van Ijcken, W., van Haren, J., Soler, E., Eick, D., Lenhard, B., Grosveld, F.G., 2013. In vivo live imaging of RNA polymerase II transcription factories in primary cells. Genes Dev. 27, 767–777. Hammond, J.W., Huang, C.F., Kaech, S., Jacobson, C., Banker, G., Verhey, K.J., 2010. Posttranslational modifications of tubulin and the polarized transport of kinesin-1 in neurons. Mol. Biol. Cell 21, 572–583. Ho, S.Y., Chao, C.Y., Huang, H.L., Chiu, T.W., Charoenkwan, P., Hwang, E., 2011. NeurphologyJ: an automatic neuronal morphology quantification method and its application in pharmacological discovery. BMC Bioinforma. 12, 230. Ickowicz, D., Finkelstein, M., Breitbart, H., 2012. Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases. Asian J. Androl. 14, 816–821. Khawaja, S., Gundersen, G.G., Bulinski, J.C., 1988. Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level. J. Cell Biol. 106, 141–149. Kimura, T., Watanabe, H., Iwamatsu, A., Kaibuchi, K., 2005. Tubulin and CRMP-2 complex is transported via Kinesin-1. J. Neurochem. 93, 1371–1382. Kohoutek, J., Blazek, D., 2012. Cyclin K goes with Cdk12 and Cdk13. Cell Div. 7, 12. Kuijpers, M., Hoogenraad, C.C., 2011. Centrosomes, microtubules and neuronal development. Mol. Cell. Neurosci. 48, 349–358. Ley, S.C., Verbi, W., Pappin, D.J., Druker, B., Davies, A.A., Crumpton, M.J., 1994. Tyrosine phosphorylation of alpha tubulin in human T lymphocytes. Eur. J. Immunol. 24, 99–106. Lin, S.D., Fann, M.J., 1998. Differential expression of protein kinases in cultured primary neurons derived from the cerebral cortex, hippocampus, and sympathetic ganglia. J. Biomed. Sci. 5, 111–119. Liu, P., Jenkins, N.A., Copeland, N.G., 2003. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 13, 476–484. Malumbres, M., 2011. Physiological relevance of cell cycle kinases. Physiol. Rev. 91, 973–1007. Malumbres, M., Harlow, E., Hunt, T., Hunter, T., Lahti, J.M., Manning, G., Morgan, D.O., Tsai, L.H., Wolgemuth, D.J., 2009. Cyclin-dependent kinases: a family portrait. Nat. Cell Biol. 11, 1275–1276. Pandithage, R., Lilischkis, R., Harting, K., Wolf, A., Jedamzik, B., Luscher-Firzlaff, J., Vervoorts, J., Lasonder, E., Kremmer, E., Knoll, B., Luscher, B., 2008. The regulation of SIRT2 function by cyclin-dependent kinases affects cell motility. J. Cell Biol. 180, 915–929. Rodrigues, F., Thuma, L., Klambt, C., 2012. The regulation of glial-specific splicing of Neurexin IV requires HOW and Cdk12 activity. Development 139, 1765–1776. Sirajuddin, M., Rice, L.M., Vale, R.D., 2014. Regulation of microtubule motors by tubulin isotypes and post-translational modifications. Nat. Cell Biol. 16, 335–344. Smith, D., 2003. Cdk5 in neuroskeletal dynamics. Neurosignals 12, 239–251. Speranza, L., Chambery, A., Di Domenico, M., Crispino, M., Severino, V., Volpicelli, F., Leopoldo, M., Bellenchi, G.C., di Porzio, U., Perrone-Capano, C., 2013. The serotonin receptor 7 promotes neurite outgrowth via ERK and Cdk5 signaling pathways. Neuropharmacology 67, 155–167. Stiess, M., Bradke, F., 2011. Neuronal polarization: the cytoskeleton leads the way. Dev. Neurobiol. 71, 430–444. Veeranna, Shetty, K.T., Takahashi, M., Grant, P., Pant, H.C., 2000. Cdk5 and MAPK are associated with complexes of cytoskeletal proteins in rat brain. Brain Res. Mol. Brain Res. 76, 229–236. Witte, H., Neukirchen, D., Bradke, F., 2008. Microtubule stabilization specifies initial neuronal polarization. J. Cell Biol. 180, 619–632. Wong, Y.H., Lu, A.C., Wang, Y.C., Cheng, H.C., Chang, C., Chen, P.H., Yu, J.Y., Fann, M.J., 2010. Protogenin defines a transition stage during embryonic neurogenesis and prevents precocious neuronal differentiation. J. Neurosci. 30, 4428–4439. Xie, Z., Samuels, B.A., Tsai, L.H., 2006. Cyclin-dependent kinase 5 permits efficient cytoskeletal remodeling–a hypothesis on neuronal migration. Cereb. Cortex 16, i64–i68. Yu, J.Y., DeRuiter, S.L., Turner, D.L., 2002. RNA interference by expression of shortinterfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. 99, 6047–6052. Yuan, H.X., Xiong, Y., Guan, K.L., 2013. Nutrient sensing, metabolism, and cell growth control. Mol. Cell 49, 379–387.
Fig. 6. Overexpression of Cdk5 partially rescues neurite defects in Cdk12-depleted and Cdk13-depleted P19 cells. (A, C) The expression levels of Cdk12, Cdk13, and Cdk5 proteins in P19 cells transfected with the indicated constructed. (B, D) Representative images of P19 cells transfected with the indicated constructs and pp35 are shown. (E, F) The average neurite lengths and the neurite length distribution of P19 cells transfected with the indicated Cdk5 and Cdk12 constructs are shown. **, p b 0.01. There is a significant difference between the pCdk12-i1 + pp35 and pCdk12-i1 + pCdk5 + pp35 groups by the two sample K–S test (p b 0.01). (G, H) The average neurite lengths and neurite length distribution in P19 cells transfected with the indicated Cdk5 and Cdk13 constructs are shown. **, p b 0.01. There is a significant difference between pCdk13i1 + pp35 and pCdk13-i1 + pCdk5 + pp35 groups by the two sample K–S test (p b 0.001). Scale bars 100 μm.
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Regular Article
Cdk12 and Cdk13 regulate axonal elongation through a common signaling pathway that modulates Cdk5 expression Hong-Ru Chen a, Guan-Ting Lin b, Chun-Kai Huang a, Ming-Ji Fann a,c,⁎ a b c
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 11221, Taiwan, ROC Institute of Neuroscience, National Yang-Ming University, Taipei 11221, Taiwan, ROC Brain Research Center, National Yang-Ming University, Taipei 11221, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 25 April 2014 Revised 16 June 2014 Accepted 18 June 2014 Available online 3 July 2014 Keywords: Cyclin-dependent kinase Axonal elongation Gene expression
a b s t r a c t Cdk12 and Cdk13 are Cdc2-related proteins that share 92% identity in their kinase domains. Using in situ hybridization and Western blot analysis, we detected the expression of Cdk12 and Cdk13 mRNAs and their proteins in developing mouse embryos, especially during development of the nervous system. We explored the roles of Cdk12 and Cdk13 in neuronal differentiation using the P19 neuronal differentiation model. Upon knockdown of Cdk12 or Cdk13, no effect on differentiated cell numbers was detected, but a substantial decrease of numbers of neurons with long neurites was identified. Similarly, knockdown of Cdk12 or Cdk13 in primarily cultured cortical neurons shortens the averaged axonal length. A microarray analysis was used to examine changes in gene expression after knockdown or overexpression of Cdk12 and we identified Cdk5 as a molecule potentially involved in mediating the effect of Cdk12 and Cdk13. Depletion of Cdk12 or Cdk13 in P19 cells significantly reduces Cdk5 expression at both the mRNA and protein levels. Expression of Cdk5 protein in the developing mouse brain is also reduced in conditional Cdk12-knockout mice in proportion to the residual amount of Cdk12 protein present. This suggests that the reduced axonal outgrowth after knockdown of Cdk12 or Cdk13 might be due to lower Cdk5 expression. Furthermore, overexpression of Cdk5 protein in P19 cells was able to partially rescue the neurite outgrowth defect observed when Cdk12 or Cdk13 is depleted. Together, these findings suggest that Cdk12 and Cdk13 regulate axonal elongation through a common signaling pathway that modulates Cdk5 expression. © 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Introduction Neurons are highly polarized cells with two functionally distinct domains that emerge from the cell body; these are the single long axon, which transmits signals, and the multiple shorter dendrites, which specialize in receiving signals (Craig and Banker, 1994). Once axon specification occurs, the axonal process begins to grow at a rate approximately 10 times faster than that of the dendritic processes (Dotti et al., 1988); this is due to differences in the patterns of microtubule organization between the dendrites and the axons (Conde and Caceres, 2009; Kuijpers
Abbreviations: Cdk5, cyclin-dependent kinase 5; Cdk12, cyclin-dependent kinase 12; Cdk13, cyclin-dependent kinase 13; CNS, central nervous system; DIV, days in vitro; K-S test, two sample Kolmogorov–Smirnov test; MAP2, microtubule-associated protein 2; SMI312, antibody against axonal phosphorylated neurofilament. ⁎ Corresponding author at: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan, ROC. Fax: +86 2 2823 4898. E-mail address: [email protected] (M.-J. Fann).
and Hoogenraad, 2011; Stiess and Bradke, 2011). Axons have tau-bound microtubules of uniform orientation with their plus end pointing outward, whereas dendrites have microtubule-associated protein 2 (MAP2)-bound microtubules of mixed orientation with many minus ends pointing outward (Baas et al., 1989; Burton, 1988; Conde and Caceres, 2009). Axonal and dendritic microtubule networks furthermore differ in their patterns of post-translational modifications and these result in unique interactions with other microtubule-associated proteins (Hammond et al., 2010; Witte et al., 2008). Several types of microtubule post-translational modifications, including tyrosination/ detyrosination and phosphorylation, are also known to modulate the sorting mechanisms of motor proteins in axons and dendrites and these consequently augment differences between axons and dendrites (Fukushima et al., 2009; Sirajuddin et al., 2014). For example, detyrosination modification of microtubules occurs more frequently in axons, which facilitates the recruitment of kinesin-1 to transport collapsin response mediator protein-2 in axons (Hammond et al., 2010; Kimura et al., 2005). The physiological relevance of microtubule phosphorylation in axons and dendrites is another field of active
http://dx.doi.org/10.1016/j.expneurol.2014.06.024 0014-4886/© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
H.-R. Chen et al. / Experimental Neurology 261 (2014) 10–21
research. It has been suggested that phosphorylation of the microtubule networks is able to positively or negatively regulate the polymerization of microtubules (Fourest-Lieuvin et al., 2006; Khawaja et al., 1988). For example, Cdk1/cyclin B-mediated phosphorylation at Ser172 of β-tubulin impairs the incorporation of these modified subunits into polymers (Fourest-Lieuvin et al., 2006). Similarly, phosphorylation of β-tubulin at tyrosine residues prevents the incorporation of these β-tubulin molecules into microtubules (Ley et al., 1994). One of the molecules engaging in neuronal microtubule dynamics is Cdk5, a member of cyclin-dependent kinases (Cdk) (Smith, 2003; Veeranna et al., 2000). Cdk5 phosphorylates a number of neuronal cytoskeletal proteins involved in neuronal migration, neurite growth and synaptogenesis (Cheung and Ip, 2007; Cheung et al., 2006; Cruz and Tsai, 2004; Xie et al., 2006). Furthermore, substrates of Cdk5, such as tau, MAP2, the dynein-associated protein, Nudel, and SIRT2, are known to be involved in cytoskeletal dynamics. This implies that Cdk5 plays a role as a critical signal transmission hub that is positioned between a range of cell surface receptors and various cytoskeletal systems (Pandithage et al., 2008; Smith, 2003; Speranza et al., 2013; Veeranna et al., 2000). Protein kinases are critical regulators that affect the cell cycle, differentiation, metabolism, cell death and various other critical cellular processes (Ickowicz et al., 2012; Malumbres, 2011; Yuan et al., 2013)
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and we previously explored the roles of a number of uncharacterized protein kinases. In this study we have focused on Cdk12 and Cdk13 due to their abundant expression in the nervous system (Lin and Fann, 1998). They share 92% identity in the kinase domain, and are known to interact with cyclin L and cyclin K (Blazek et al., 2011; Bosken et al., 2014; Chen et al., 2006, 2007; Dai et al., 2012). At the molecular level, they are able to facilitate transcription of long genes with many exons, and are involved in mRNA alternative splicing (Blazek et al., 2011; Chen et al., 2006, 2007; Rodrigues et al., 2012). At the cellular level, Cdk12 and Cdk13 are able to maintain self-renewal in embryonic stem cells (Dai et al., 2012). In the present study, we examined additional possible functions of Cdk12 and Cdk13 during neuronal development. Using P19 cells as a neuronal differentiation model, we asked whether changes in Cdk12 and Cdk13 expression affect neuronal differentiation and neurite outgrowth. The P19 cells are mouse embryonic carcinoma cells and are able to differentiate into neurons when there is overexpression of the proneural gene Ascl1 (Farah et al., 2000). We found that reduced expression of Cdk12 or Cdk13 resulted in a shortening of neurite elongation in P19 cells and an inhibition of axonal extension in primary cultured cortical neurons, but reduced Cdk12 or Cdk13 expression had no effects on dendrite extension. We further demonstrated that Cdk12 and Cdk13 promote axonal elongation through a common signaling pathway that involves modulation of Cdk5
Fig. 1. Expression of Cdk12 and Cdk13 in the developing nervous system. (A) Cdk12 and Cdk13 mRNA expression in mouse embryos at various developmental stages was analyzed by whole-mount in situ hybridization. The results from the antisense probes are shown in a–d and e–h, while those from the sense probes are shown in a′–d′ and e′–h′. Cdk12 is expressed in all cells, except in the heart (H), but with higher levels in forebrain (F), midbrain (M), primitive streak (PS) and tail bud mesoderm (TBM). A similar expression patterns is observed for Cdk13 mRNA expression (e–h). The scale bar represents 300 μm. (B, C) Expression of the Cdk12 and Cdk13 proteins in developing brains was analyzed by Western blotting.
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expression at the RNA level. This is a novel finding that couples Cdk12 and Cdk13 functioning with Cdk5 signaling during axonal elongation. Materials and methods Animals Adult C57BL/6J mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). All mice were handled according to the university guidelines and all experiments were approved by the National Yang-Ming University Animal Care and Use Committee. For the timed pregnancies, mice were set up in the late afternoon and when plugs were detected the next morning, this was designated as E0.5. Whole-mount in situ hybridization Mouse embryos were dissected at different developmental stages, fixed in 4% paraformaldehyde, and permeabilized by treatment with proteinase K. After hybridization and washing, embryos were incubated with an alkaline phosphatase-conjugated anti-digoxygenin antibody (Roche), washed again and then stained using a NBT/BCIP mixture (Roche) in the presence of 10% polyvinyl alcohol (31–50 kD; Sigma). After visualization of the alkaline phosphatase activity, embryos were post-fixed and cleared in 100% glycerol for photography. Sense and antisense RNA probes were synthesized using an in vitro transcription kit from Roche, and were purified using a Sephadex G-50 column (Amersham Biosciences). RNA inhibitor (RNase) was purchased from Promega. Vector construction The various Cdk12 and Cdk13 plasmids had been constructed previously (Chen et al., 2006, 2007). Protocols described by Chung et al. (2006) were followed to construct the microRNA mimics. Oligonucleotides against mouse Cdk5, Cdk12 and Cdk13 sequences as well as against their equivalent scramble sequences were synthesized and cloned into the vector UI4-puro-SIBR to obtain pCdk5-s, pCdk5-i1, pCdk5-i2, pCdk12-s, pCdk12-i1, pCdk12-i2, pCdk13-s, pCdk13-i1 and pCdk13-i2, or into the vector UI4-GFP-SIBR to obtain pCdk12-s-e, pCdk12-i1-e, pCdk13-s-e and pCdk13-i1-e. The sequences of the oligonucleotides are listed in Table S1. Mouse Cdk5 activator, p35, was amplified by PCR and cloned into the vectors pUS2. All constructs were verified by restriction enzyme mapping and sequencing. Antibodies Rabbit anti-Cdk12 and mouse anti-Cdk13 polyclonal antibodies had been produced previously (Chen et al., 2006, 2007). Mouse anti-β-
tubulin antibody (E7, 1:10 000) and anti-Myc antibody (9E10, 1:5000) were obtained from the Developmental Studies Hybridoma Bank. Mouse anti-Flag M2 monoclonal antibody (1:1000) was purchased from Sigma. Mouse anti-Cdk5, rabbit anti-MAP2 and mouse anti-tau1 antibodies were obtained from Millipore. Mouse anti-pan-axonal neurofilament marker (SMI312) and mouse anti-neuronal class III βtubulin (TuJ1) were purchased from Covance. TRITC-conjugated goat anti-mouse antiserum was obtained from the Jackson Laboratory. Other secondary antisera were purchased from Chemicon or Bethyl Laboratories. Cell culture and transfection P19 cells were cultured in α-minimal essential medium (α-MEM) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine and incubated in a 5% CO2/95% humidified atmosphere at 37 °C. For neuronal differentiation, P19 cells were transfected with pAscl1 and other plasmids using Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer. The expression plasmid pAscl1 (pUS2-Ascl1) from D. L. Turner was used for neuronal differentiation, which has previously been shown to convert P19 cells into a relatively homogenous population of electrophysiologically differentiated neurons (Farah et al., 2000; Yu et al., 2002). Eight hours after transfection, 8 μg/ml puromycin was applied to select transfected cell. The cells were then replated onto 6-well plates coated with 2 μg/ml laminin (Invitrogen) at 24 h after transfection, and cultured in 1% FBS OPTI-MEM. The medium was changed at two days after transfection. Cells were harvested for immunocytochemistry or protein extraction at three days after transfection. Mouse cortical neurons were prepared from E14.5 mouse embryos. The cortex was mechanically dissociated by passing through a glass pipette for twenty times and then filtered through a 70-μm nylon mesh filter (BD Biosciences). Cells were plated onto plastic culture plates coated with 30 μg/ml poly-L-lysine and 2 μg/ml laminin and cultured in Dulbecco's modified Eagle's medium containing 10% FBS, 100 U/ml penicillin and streptomycin, and N2 supplement and B27 supplement (Invitrogen). The cells were kept at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. To deliver plasmids into cortical neurons, electroporation was performed before plating using the Amaxa Mouse Neuron Nucleofector kit and the Amaxa Nucleofecor II (Lonza). Conditions were optimized for 4 × 107 cells and 10 μg of plasmid per electroporation, which gave a survival and transfection efficiency of 60% and 35% to 40%, respectively. Immunofluorescence microscopy and neurite counting Cultured neurons were rinsed once with PBS and fixed in 4% paraformaldehyde for 1 h. Samples were stained with the appropriate primary and secondary antibodies and then finally stained with DAPI (1 μg/ml) for 15 min. Samples were then examined using a fluorescence microscope (Zeiss). Numbers of TuJ1-positive cells and DAPI-positive cells
Fig. 2. Knockdown of Cdk12 and Cdk13 decreases neurite elongation in P19 cells. (A) A schematic depiction of the experimental procedure. (B) Representative images of P19 cells transfected with the indicated Cdk12 constructs. (C) Differentiation rates of P19 cells transfected with the indicated constructs. Differentiated neuronal cells were detected by TuJ1 staining. The number of TuJ1-positive cells was divided by the number of DAPI positive cells in 30 randomly selected fields. (D) Average neurite length of P19 cells transfected with the indicated constructs. The lengths of neurites marked by TuJ1 staining were manually quantified for each selected neuron using Neuron J software. In each experiment, 300 neurons from randomly selected fields for each transfected condition were evaluated. Data from three independent experiments were analyzed by Student's t-test and are presented as mean ± SEM. **, p b 0.01; ***, p b 0.001. (E) The distribution of neurite lengths using P19 cells transfected with the indicated constructs. Data from three independent experiments are presented as mean ± SEM. Statistical analysis using two sample Kolmogorov–Smirnov test (K–S test) shows p N 0.05 for pCdk12 when compared to pEF, and p N 0.05, p b 0.01, and p b 0.001 for pCdk12-s, pCdk12-i1, and pCdk12-i2, respectively, when compared to pUI4. (F) Representative images of P19 cells transfected with the indicated Cdk13 constructs. (G) Differentiation rates of P19 cells transfected with the indicated constructs. (H) Average neurite length of P19 cells transfected with the indicated constructs. Data from three independent experiments are presented as mean ± SEM. ***, p b 0.001 by Student's t-test. (I) Distribution of neurite lengths of P19 cells transfected with the indicated constructs. Data from three independent experiments are presented as mean ± SEM. The two sample K–S test shows p N 0.05 for pCdk13 when compared to pEF, and p N 0.05, p b 0.001, and p b 0.001 for pCdk13-s, pCdk13-i1, and pCdk13-i2, respectively, when compared to pUI4. Scale bars 100 μm.
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were counted using the Image J plugin, NeurphologyJ (Ho et al., 2011). The differentiation rate was calculated by dividing TuJ1-positive cell numbers by DAPI-positive cell numbers. The tracking of P19 cell
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neurites and cortical neurons neurites was performed manually, and the length of each neurite was quantified using the Image J plugin, ImageJ. In each experiment, 300 neurons from randomly selected fields
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for each condition were evaluated. All experiments were repeated at least three times. Targeted disruption of the mouse Cdk12 gene To generate Cdk12 conditional knockout mice, a targeting vector containing Cdk12 sequences between exon 3 and exon 4 flanked by loxP, and FRT-flanked Neo cassette was prepared from a BAC clone using the recombineering approach (Copeland et al., 2001; Liu et al., 2003). The targeting vector was linearized and electroporated into embryonic stem (ES) cells, which was performed by Transgenic Mouse Models Core Facility, Taiwan. After selection with antibiotics, cell clones with correct homologous recombination were identified by Southern blot hybridization. After Neo pop-out, a correctly targeted ES clone was injected into blastocysts and the resultant chimeras were bred with C57BL/6J mice. Genotyping was performed by Southern blotting and/or PCR using the probe and primers listed in Table S1. To conditionally delete Cdk12 in the nervous system, NestinCreER T2 mice were used for Cre-mediated recombination after injecting Tamoxifen. Most mice used for this study were on a C57BL/6-129Sv mixed background. Protein extraction and Western blotting Previously described procedures were followed during these experiments (Wong et al., 2010). Quantitative real-time PCR assay Total RNA was extracted using a RNeasy Mini kit (Qiagen) following the manufacturer's suggestions. Reverse transcription was performed with 1.5 μg of total RNA using SuperScript III reverse transcriptase (Invitrogen). The real-time quantitative PCR was performed on a LightCycler 480 using the Universal Probe Library (UPL) (Roche). A web service (http://qpcr.probefinder.com/roche3. html) was used to select targeted input sequences, UPL probes, and primer pairs. The sequences of the primers are listed in Table S1. Negative controls containing no cDNA template were included in each experiment. The results were analyzed by LightCycler software version 3.5 (Roche) to determine the threshold cycle (Cp) for each reaction. Results Expression of Cdk12 and Cdk13 in the developing nervous system We used whole-mount in situ hybridization and Western blotting to detect the spatial and temporal expression patterns of Cdk12 and Cdk13 at both the mRNA and protein levels. Cdk12 mRNA is present in E6.5 embryo proper and trophoblasts (Fig. 1Aa). During E7–E7.5, Cdk12 is expressed in all cells, but with stronger expression in the primitive streak (PS) (Fig. 1Ab). In E8.5 and E9.5 embryos, stronger signals are detected in the forebrain, midbrain and tail bud mesoderm (TBM), but not in the heart (Figs. 1Ac and Ad). Cdk13 is also ubiquitously expressed but not in the heart at the various developmental stages examined (Figs. 1Ae–Ah). We further analyzed
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expression of Cdk12 and Cdk13 at the protein level in the developing brain at later developmental stages and found that Cdk12 and Cdk13 proteins were consistently expressed in the brain at all embryonic stages (Figs. 1B and C), which suggests that Cdk12 and Cdk13 may play a general role in the development of the nervous system. Knockdown of Cdk12 or Cdk13 leads to an axonal extension defect through a common signaling pathway Initially, we examined whether Cdk12 and Cdk13 participate in neuronal differentiation using the P19 neuronal differentiation model. P19 is a mouse embryonal carcinoma cell line that is able to be induced to differentiate into neuronal cells by ectopic expression of proneural gene Ascl1 and gives rise to unipolar or bipolar neurites. Overexpression constructs that increase Cdk12 or Cdk13 protein expression more than nine folds in P19 cells, and two RNA interference constructs for each gene that decrease Cdk12 or Cdk13 protein expression to less than 40% of the control level were constructed (Figs. S1A and C). The Cdk12 interference constructs do not inhibit Cdk13 protein expression, and vice versa (Figs. S1B and D). Differentiated P19 cells exhibit unipolar or bipolar neurites whose lengths can be revealed by staining with TuJ1 antibody, which recognizes a neuronal specific beta-III-tubulin expressed in early postmitotic neurons. Overexpression of either Cdk12 or Cdk13 in P19 cells was found not to affect differentiation of P19 cells cultured for 3 days after transfection, either in terms of the numbers of differentiated TuJ1-positive cells or in terms of the average length of outgrown neurites (Figs. 2Bb, C, D, Fb, G, and H). Surprisingly, however, knockdown of Cdk12 or Cdk13 decreased the average lengths of neurites in differentiated P19 neurons (312.66 ± 0.34 μm, mean ± SEM, for the pUI4 control, 227.17 ± 7.27 μm for pCdk12-i1, Fig. 2D; and 306.95 ± 4.3 μm for pUI4 control, 202.52 ± 5.75 μm for pCdk13-i1, Fig. 2H), although there was no change in the numbers of differentiated cells (Figs. 2C and G). Detailed analysis of the neurite length distribution revealed that cells with a deficiency in either Cdk12 or Cdk13 were able to send out processes but had reduced capability to extend these processes to long distances (N 300 μm) (Figs. 2E and I). Thus, distribution of the neurite length was skewed to the right in Cdk12-depleted and Cdk13depleted cells, suggesting that Cdk12 and Cdk13 are critical for neurite elongation in P19 cells. Next, we analyzed whether Cdk12 and Cdk13 affect axonal elongation through a common pathway. Interestingly, overexpression of Cdk13 in Cdk12-depleted cells reduced the percentage of short neurite (100–200 μm) from 38% to 27%, and increased the percentage of longer neurite (300–400 μm) from 16% to 23% (Figs. 3B and F). Overexpression of Cdk12 in Cdk13-depleted cells was also able to decrease the percentage of short neurite (100–200 μm) from 44% to 33%, and to raise percentage of the longer neurite (300– 400 μm) from 8% to 13% (Figs. 3D and H). Furthermore, the average neurite lengths after overexpression approached those of the control cells, when compared to those of the Cdk12-depleted or Cdk13depleted P19 cells (Figs. 3E and G). These results suggest that the same underlying signal pathway, at least in part, may mediate action of both Cdk12 and Cdk13 (Fig. 3).
Fig. 3. Cdk12 and Cdk13 regulate neurite elongation through a common signaling pathway in differentiated P19 cells. (A, C) The expression levels of Cdk12, Cdk13, and Cdk5 proteins in P19 cells transfected with the indicated constructed. Ectopically expressed Cdk12 and Cdk13 were detected by anti-myc antibody. (B, D) Representative images of P19 cells transfected with the indicated constructs are shown. (E, F) The average neurite lengths and the neurite length distribution of P19 cells transfected with the indicated Cdk13 and Cdk12 constructs are shown. *, p b 0.05. There is a significant difference between the pCdk12-i1 and pCdk12-i1 + pCdk13 groups by the two sample K–S test (p b 0.01). (G, H) The average neurite lengths and neurite length distribution in P19 cells transfected with the indicated Cdk12 and Cdk13 constructs are shown. *, p b 0.05. There is a significant difference between pCdk13-i1 and pCdk13-i1 + pCdk12 groups by the two sample K–S test (p b 0.001). Scale bars 100 μm.
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As Cdk12 and Cdk13 proteins are detected in mouse E14.5 cortical neurons (Fig. S2), we examined whether Cdk12 and Cdk13 are able to modulate neurite extension in cultured cortical neurons. Dissociated cortical neurons derived from mouse E14.5 embryos were electroporated with the various constructs. We used antiMAP2 antibody to demarkate dendrites, and anti-phosphorylated neurofilament antibody (SMI312) and anti-tau1 antibody to identify axons. After 3 days of culture in vitro (DIV), depletion of either Cdk12 or Cdk13 did not affect the neuronal polarity in cultured cortical neurons (Fig. S3). These neurons exhibited several MAP2positive processes, and one long SMI312-positive process. In Cdk12-depleted and Cdk13-depleted cortical neurons, the average length of dendrites was not affected (12.67 ± 1.19 μm in control cells vs. 12.33 ± 1.44 μm and 12.36 ± 0.72 μm in Cdk12-depleted and Cdk13-depleted neurons, respectively, Figs. 4A, B); however, the average length of axons was decreased (101 ± 5.25 μm in control cells vs. 42 ± 3.3 μm and 41 ± 2.05 μm in Cdk12-depleted and Cdk13-depleted neurons, respectively, Figs. 4C, D). At 5 DIV, dendrites and axons of the control neurons had grown further (Figs. 4E–H). Dendrite growth had taken place in the Cdk12depleted and Cdk13-depleted neurons (compare Figs. 4F with B); however, the growth of the axons of Cdk12-depleted and Cdk13depleted neurons was arrested during these two days (compare Figs. 4H with D). Staining with tau1 gave similar results at 3 DIV (Fig. S4). Together, these findings support the idea that Cdk12 and Cdk13 are essential for axonal extension. Expression of Cdk5 is regulated by Cdk12 and Cdk13 To dissect the underlying mechanisms by which Cdk12 and Cdk13 regulate axonal elongation, we performed microarray analysis and compared the global gene expression profiles of the control, Cdk12-overexpressing and Cdk12-knockdown P19 cells. Using these findings, we then selected candidate genes whose expression was decreased in the Cdk12-knockdown P19 cells but did not change in the Cdk12-overexpressing P19 cells, both in comparison to cells transfected with the control vector. Among these candidates identified, Cdk5 mRNA was found to be down-regulated in Cdk12knockdown P19 cells to 27% of the control using the microarray analysis. As Cdk5 is an important molecule in terms of neurite outgrowth during neural development, we then investigated whether Cdk12 and Cdk13 were indeed able to regulate Cdk5 mRNA expression using quantitative RT-PCR analysis. Knockdown of either Cdk12 or Cdk13 was found to reduce Cdk5 mRNA level to ~ 40% of the control level (Fig. 5A), which is a level compatible with the decreased levels of Cdk12 or Cdk13 protein when their corresponding RNA interference constructs are transfected into P19 cells. Cdk5 protein expression is also reduced to ~ 50% of the control (Fig. 5B, C), which suggests that regulation of Cdk5 by Cdk12 and Cdk13 is primarily at the RNA level. Previously we have generated a Cdk12 conditional knockout mouse by inserting loxP sites into intron 2 and intron 4 (Fig. S5A). We took advantage of variable efficiency of the Cre recombinase in embryos after injecting Tamoxifen into pregnant mice, and asked whether there is a dose–response relationship between the in vivo expression of Cdk12 and the expression of Cdk5. We crossed Cdk12 f/f mice with Nestin-CreER T2 ; Cdk12 del/+ mice, and injected
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Tamoxifen at E10.5 into pregnant mice. Whole brain was then dissected from the E14.5 embryos and protein extracts from different embryos were subjected to Western analysis using anti-Cdk12 antibodies and anti-Cdk5 antibodies (Fig. 5D). A linear correlation between Cdk12 expression and Cdk5 expression with a value for r of 0.98 was found (Fig. 5E). Overexpression of Cdk5 partially rescues effects of Cdk12 or Cdk13 knockdown To examine whether Cdk5 is the downstream gene that is controlled by Cdk12 and Cdk13 in P19 cells, we performed RNA interference experiments to knockdown Cdk5 mRNA and analyzed the effect on neurite extension in P19 cell model. Transfection of two RNA interference constructs (pCdk5-i1 and pCdk5-i2) was found to reduce Cdk5 protein expression to 50% of the control (Fig. S1E). Upon knockdown of Cdk5, the number of differentiated P19 cells does not change (Fig. 5G), but it was found that the neurite length distribution was skewed to the right in a pattern that is similar to what we observed for Cdk12-depleted or Cdk13-depleted cells (Figs. 5H, I). It should be noted that the decrease in average neurite length and change in skewness after knockdown of Cdk5 were found to be less than the changes induced by Cdk12 and Cdk13 depletion. We next examine whether overexpression of Cdk5 was able to rescue the defects found in Cdk12-depleted and Cdk13-depleted P19 cells. When Cdk5 was ectopically expressed in Cdk12-depleted and Cdk13-depleted P19 cells with Cdk5 activator, p35, the neurites grew to a longer length; furthermore, the average neurite length and the distribution of the neurite length approached that of the control cells, when compared to the Cdk12-depleted or Cdk13-depleted P19 cells (Fig. 6). Thus, the defect of neurite extension due to knockdown of Cdk12 or Cdk13 was found to be partially rescued by Cdk5 overexpression. However, overexpression of Cdk12 or Cdk13 in Cdk5-depleted P19 cells had no effect on neurite outgrowth in comparison to knockdown of Cdk5 alone (Fig. S6). These findings suggest that the Cdk5 is one of the downstream mediators for Cdk12 and Cdk13 when axonal elongation occurs. Discussion Cdk12 and Cdk13 are essential for axonal elongation Our investigation provides evidence that Cdk12 and Cdk13 contribute to axonal growth. Upon depletion of Cdk12 or Cdk13, cultured P19 cells and primary cortical neurons lose the ability to extend their axons to greater distances, although they are still able to initiate neurites (Figs. 2 and 4). Overexpression of Cdk13 is able to partially rescue the neurite outgrowth defect in Cdk12-depleted P19 cells, and vice versa, which suggests that they act through common downstream effector(s) to promote axonal elongation (Fig. 3). One protein affected by both Cdk12 and Cdk13 is Cdk5, and both are able to modulate Cdk5 protein expression in vitro; furthermore, overexpression of Cdk5 in Cdk12-depleted and Cdk13-depleted P19 cells is able to partially rescue the neurite outgrowth defect (Fig. 6). Interestingly, overexpression of Cdk13 increases the endogenous Cdk5 protein expression level and is able to rescue partially the defects in Cdk12-depleted P19 cells, also
Fig. 4. Cdk12 and Cdk13 enhance axonal elongation in primary cultured cortical neurons. Cortical neurons from the E14.5 mouse cortex were transfected with the indicated constructs. The cells were cultured, fixed, and immunostained with anti-MAP2 (A, E) or anti-SMI312 (C, G) antibodies at 3 DIV (A–D) and 5 DIV (E–H). The arrows indicate MAP2-positive and SMI312positive neurites. (B, D, F, H) Quantification of the length of MAP2-positive dendrites and the SMI312-positive axons as shown in A, C, E, and G was performed. For each condition, the data were collected from 300 randomly selected cells. Data from three independent experiments were analyzed by Student's t-test and are presented as mean ± SEM. **, p b 0.01; ***, p b 0.001. Scale bars 50 μm.
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showing causality relationship. Overexpression of Cdk12 in Cdk13depleted P19 cells gives the same results (Figs. 3A, C). The findings from Cdk12 knockout mice further confirm that expression of Cdk5 is indeed regulated by the level of Cdk12 proteins in vivo. However, the observation that Cdk5 overexpression in Cdk12-depleted and Cdk13depleted P19 cells brings about only a partial rescue of the neurite elongation defect, which implies that there are other effectors involved in Cdk12-dependent and Cdk13-dependent axonal elongation (Fig. 7). Cdk12 and Cdk13 have overlapping, but not identical, functions Cdk12 and Cdk13 have similar protein sequences and structural motifs. They are known to be engaged in a number of similar biological processes (Chen et al., 2006, 2007; Dai et al., 2012; Kohoutek and Blazek, 2012). For example, both Cdk12 and Cdk13 play an indispensable role in maintaining the pluripotency of mouse embryonic stem cells (Dai et al., 2012). Furthermore, both Cdk12 and Cdk13 take part in RNA splicing regulation in a similar manner (Chen et al., 2006, 2007). In this study, we have shown that their expression patterns seem also to be very similar (Fig. 1). Expression of Cdk12 and Cdk13 is initiated quite early during development, beginning as early as the E6.5 mouse embryo. Both are expressed ubiquitously, except that they are both absent from the developing heart. This similarity in expression pattern and function between Cdk12 and Cdk13 suggests that deficiency in either protein is unlikely to generate an obvious phenotype in mouse embryos. It is thus surprising that Cdk12 knockout mice die at post-implantation stage at E5.5 (data not shown) and this suggests that Cdk13 is not a redundant version of Cdk12 and is not able to completely complement Cdk12 deficiency. Furthermore, it has been shown that Cdk12, rather than Cdk13, activates transcription via the phosphorylation of Ser2 at the carboxylterminal domain of RNAPII and this then facilitates transcription of long exons during elongation (Bartkowiak et al., 2010; Blazek et al., 2011; Cheng et al., 2012; Ghamari et al., 2013). It is likely that these two genes are derived from an ancestor gene but have then evolved divergently and are now an evolutionary distinct far part in vertebrates (Malumbres et al., 2009). This may have resulted in subtle differences in terms of cellular localization as well as changes in their preference in terms of substrates and cyclins. A more thorough study is needed in the future in order to explore these fascinating possibilities. Axons, but not dendrites, are affected by the activity of Cdk12 and Cdk13 Axonal outgrowth involves many steps. The roles of Cdk12 and Cdk13 seem to be limited to axonal elongation. They are not required for specification and maintenance of neuronal polarity, since knockdown of Cdk12 and Cdk13 does not affect the numbers of SMI312positive cortical neurons or MAP2-positive cortical neurons at 3 DIV and 5 DIV (Figs. 4 and S4). However, knockdown of Cdk12 and Cdk13 dose affect axonal elongation and this effect occurs without affecting dendrite extension. This suggests that both Cdk12 and
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Cdk13 are able to regulate the axonal specific microtubule organization but not dendritic cytoskeletal organization, although expression of Cdk12 and Cdk13 is distributed evenly across both dendrites and axons (data not shown). Currently, the underlying mechanisms by which Cdk12 and Cdk13 are able to generate this differential effect remain unclear. It seems likely that Cdk12 and Cdk13 regulate tubulin polymerization and microtubule stability, which is highly critical in axons. Silencing of Cdk12 and/or Cdk13 may also bring abnormal microtubule remodeling and a change in microtubule dynamics in axons. Details of the downstream Cdk12 and Cdk13 substrate proteins and how they contribute to this differential effect on axons and dendrites remain obscure. Future investigation in this area will further strengthen our understanding of the development of axons and dendrites. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.expneurol.2014.06.024. Author contribution H.-R. Chen, G.-T. Ling, and M.-J. Fann conceived and designed the experiments. H.-R. Chen, G.-T. Ling, and C.-K. Huang conducted experiments. H.-R. Chen and M.-J. Fann wrote the manuscript. Conflicts of interest Authors declare no conflict of interest. Acknowledgments We thank L.-S. Kao, J.-Y. Yu, Y.-C. Chern, E. Hwang, and M.-m. Poo for their discussion and critical reading of the manuscript, E. Hwang is thanked for providing NeurphologyJ software. We would also like to thank the Developmental Studies Hybridoma Bank for providing monoclonal antibodies. This research was supported by the National Science Council (NSC101-2320-B-010-064) and the Ministry of Education (Aim for the Top University Plan) to M.-J. Fann. References Baas, P.W., Black, M.M., Banker, G.A., 1989. Changes in microtubule polarity orientation during the development of hippocampal neurons in culture. J. Cell Biol. 109, 3085–3094. Bartkowiak, B., Liu, P., Phatnani, H.P., Fuda, N.J., Cooper, J.J., Price, D.H., Adelman, K., Lis, J.T., Greenleaf, A.L., 2010. CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1. Genes Dev. 24, 2303–2316. Blazek, D., Kohoutek, J., Bartholomeeusen, K., Johansen, E., Hulinkova, P., Luo, Z., Cimermancic, P., Ule, J., Peterlin, B.M., 2011. The cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes. Genes Dev. 25, 2158–2172. Bosken, C.A., Farnung, L., Hintermair, C., Merzel Schachter, M., Vogel-Bachmayr, K., Blazek, D., Anand, K., Fisher, R.P., Eick, D., Geyer, M., 2014. The structure and substrate specificity of human Cdk12/Cyclin K. Nat. Commun. 5, 3505. Burton, P.R., 1988. Dendrites of mitral cell neurons contain microtubules of opposite polarity. Brain Res. 473, 107–115. Chen, H.H., Wang, Y.C., Fann, M.J., 2006. Identification and characterization of the CDK12/ cyclin L1 complex involved in alternative splicing regulation. Mol. Cell. Biol. 26, 2736–2745.
Fig. 5. Depletion of Cdk12 or Cdk13 leads to down-regulation of Cdk5 and depletion of Cdk5 causes a neurite elongation defect in P19 cells. (A) Quantitative PCR was performed to measure Cdk5 expression in P19 cells transfected with various Cdk12 or Cdk13 constructs. Data from three independent experiments were analyzed by Student's t-test and are presented as mean ± SEM. **, p b 0.01. (B) Knockdown of Cdk12 or Cdk13 in P19 cells reduces Cdk5 protein expression as revealed by Western blotting. (C) Quantification of (B). The data from three independent experiments were analyzed by Student's t-test and are presented as mean ± SEM. **, p b 0.01. (D) Western blot analysis of Cdk12, Cdk13, and Cdk5 protein expression in embryonic brains with different Cdk12 knockout backgrounds. (E) The amount of Cdk5 protein present was plotted against the amount of Cdk12 proteins present using mouse embryos that had different genotypes and different levels of Cre activities. Expression levels of Cdk12 and Cdk5 in a randomly selected Cdk12f/+mouse (marked with *) were defined as having an arbitrary unit (A.U.) value of one. (F) Representative images of P19 cells transfected with the indicated Cdk5 constructs. (G) Differentiation rates of P19 cells transfected with the indicated constructs. (H) The average neurite lengths of P19 cells transfected with the indicated constructs. Data from three independent experiments are presented as mean ± SEM. **, p b 0.01; ***, p b 0.001 by Student's t-test. (I) The distribution of neurite lengths of P19 cells transfected with the indicated constructs. Data from three independent experiments are presented as mean ± SEM. The two sample K–S test shows p N 0.05, p b 0.05, and p b 0.05 for pCdk5-s, pCdk5-i1, and pCdk5-i2, respectively, when compared to pUI4. Scale bars 100 μm.
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Fig. 7. A proposed model for the action of Cdk12 and Cdk13 in differentiated neurons. Cdk12 and Cdk13 independently regulate axonal elongation in differentiated neurons, in part, through modulation of Cdk5 expression.
Chen, H.H., Wong, Y.H., Geneviere, A.M., Fann, M.J., 2007. CDK13/CDC2L5 interacts with L-type cyclins and regulates alternative splicing. Biochem. Biophys. Res. Commun. 354, 735–740. Cheng, S.W., Kuzyk, M.A., Moradian, A., Ichu, T.A., Chang, V.C., Tien, J.F., Vollett, S.E., Griffith, M., Marra, M.A., Morin, G.B., 2012. Interaction of cyclin-dependent kinase 12/CrkRS with cyclin K1 is required for the phosphorylation of the C-terminal domain of RNA polymerase II. Mol. Cell. Biol. 32, 4691–4704. Cheung, Z.H., Ip, N.Y., 2007. The roles of cyclin-dependent kinase 5 in dendrite and synapse development. Biotechnol. J. 2, 949–957. Cheung, Z.H., Fu, A.K., Ip, N.Y., 2006. Synaptic roles of Cdk5: implications in higher cognitive functions and neurodegenerative diseases. Neuron 50, 13–18. Chung, K.H., Hart, C.C., Al-Bassam, S., Avery, A., Taylor, J., Patel, P.D., Vojtek, A.B., Turner, D. L., 2006. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. Nucleic Acids Res. 34, e53. Conde, C., Caceres, A., 2009. Microtubule assembly, organization and dynamics in axons and dendrites. Nat. Rev. Neurosci. 10, 319–332. Copeland, N.G., Jenkins, N.A., Court, D.L., 2001. Recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2, 769–779. Craig, A.M., Banker, G., 1994. Neuronal polarity. Annu. Rev. Neurosci. 17, 267–310. Cruz, J.C., Tsai, L.H., 2004. A Jekyll and Hyde kinase: roles for Cdk5 in brain development and disease. Curr. Opin. Neurobiol. 14, 390–394. Dai, Q., Lei, T., Zhao, C., Zhong, J., Tang, Y.Z., Chen, B., Yang, J., Li, C., Wang, S., Song, X., Li, L., Li, Q., 2012. Cyclin K-containing kinase complexes maintain self-renewal in murine embryonic stem cells. J. Biol. Chem. 287, 25344–25352. Dotti, C.G., Sullivan, C.A., Banker, G.A., 1988. The establishment of polarity by hippocampal neurons in culture. J. Neurosci. 8, 1454–1468. Farah, M.H., Olson, J.M., Sucic, H.B., Hume, R.I., Tapscott, S.J., Turner, D.L., 2000. Generation of neurons by transient expression of neural bHLH proteins in mammalian cells. Development 127, 693–702. Fourest-Lieuvin, A., Peris, L., Gache, V., Garcia-Saez, I., Juillan-Binard, C., Lantez, V., Job, D., 2006. Microtubule regulation in mitosis: tubulin phosphorylation by the cyclindependent kinase Cdk1. Mol. Biol. Cell 17, 1041–1050. Fukushima, N., Furuta, D., Hidaka, Y., Moriyama, R., Tsujiuchi, T., 2009. Post-translational modifications of tubulin in the nervous system. J. Neurochem. 109, 683–693.
21
Ghamari, A., van de Corput, M.P., Thongjuea, S., van Cappellen, W.A., van Ijcken, W., van Haren, J., Soler, E., Eick, D., Lenhard, B., Grosveld, F.G., 2013. In vivo live imaging of RNA polymerase II transcription factories in primary cells. Genes Dev. 27, 767–777. Hammond, J.W., Huang, C.F., Kaech, S., Jacobson, C., Banker, G., Verhey, K.J., 2010. Posttranslational modifications of tubulin and the polarized transport of kinesin-1 in neurons. Mol. Biol. Cell 21, 572–583. Ho, S.Y., Chao, C.Y., Huang, H.L., Chiu, T.W., Charoenkwan, P., Hwang, E., 2011. NeurphologyJ: an automatic neuronal morphology quantification method and its application in pharmacological discovery. BMC Bioinforma. 12, 230. Ickowicz, D., Finkelstein, M., Breitbart, H., 2012. Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases. Asian J. Androl. 14, 816–821. Khawaja, S., Gundersen, G.G., Bulinski, J.C., 1988. Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level. J. Cell Biol. 106, 141–149. Kimura, T., Watanabe, H., Iwamatsu, A., Kaibuchi, K., 2005. Tubulin and CRMP-2 complex is transported via Kinesin-1. J. Neurochem. 93, 1371–1382. Kohoutek, J., Blazek, D., 2012. Cyclin K goes with Cdk12 and Cdk13. Cell Div. 7, 12. Kuijpers, M., Hoogenraad, C.C., 2011. Centrosomes, microtubules and neuronal development. Mol. Cell. Neurosci. 48, 349–358. Ley, S.C., Verbi, W., Pappin, D.J., Druker, B., Davies, A.A., Crumpton, M.J., 1994. Tyrosine phosphorylation of alpha tubulin in human T lymphocytes. Eur. J. Immunol. 24, 99–106. Lin, S.D., Fann, M.J., 1998. Differential expression of protein kinases in cultured primary neurons derived from the cerebral cortex, hippocampus, and sympathetic ganglia. J. Biomed. Sci. 5, 111–119. Liu, P., Jenkins, N.A., Copeland, N.G., 2003. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 13, 476–484. Malumbres, M., 2011. Physiological relevance of cell cycle kinases. Physiol. Rev. 91, 973–1007. Malumbres, M., Harlow, E., Hunt, T., Hunter, T., Lahti, J.M., Manning, G., Morgan, D.O., Tsai, L.H., Wolgemuth, D.J., 2009. Cyclin-dependent kinases: a family portrait. Nat. Cell Biol. 11, 1275–1276. Pandithage, R., Lilischkis, R., Harting, K., Wolf, A., Jedamzik, B., Luscher-Firzlaff, J., Vervoorts, J., Lasonder, E., Kremmer, E., Knoll, B., Luscher, B., 2008. The regulation of SIRT2 function by cyclin-dependent kinases affects cell motility. J. Cell Biol. 180, 915–929. Rodrigues, F., Thuma, L., Klambt, C., 2012. The regulation of glial-specific splicing of Neurexin IV requires HOW and Cdk12 activity. Development 139, 1765–1776. Sirajuddin, M., Rice, L.M., Vale, R.D., 2014. Regulation of microtubule motors by tubulin isotypes and post-translational modifications. Nat. Cell Biol. 16, 335–344. Smith, D., 2003. Cdk5 in neuroskeletal dynamics. Neurosignals 12, 239–251. Speranza, L., Chambery, A., Di Domenico, M., Crispino, M., Severino, V., Volpicelli, F., Leopoldo, M., Bellenchi, G.C., di Porzio, U., Perrone-Capano, C., 2013. The serotonin receptor 7 promotes neurite outgrowth via ERK and Cdk5 signaling pathways. Neuropharmacology 67, 155–167. Stiess, M., Bradke, F., 2011. Neuronal polarization: the cytoskeleton leads the way. Dev. Neurobiol. 71, 430–444. Veeranna, Shetty, K.T., Takahashi, M., Grant, P., Pant, H.C., 2000. Cdk5 and MAPK are associated with complexes of cytoskeletal proteins in rat brain. Brain Res. Mol. Brain Res. 76, 229–236. Witte, H., Neukirchen, D., Bradke, F., 2008. Microtubule stabilization specifies initial neuronal polarization. J. Cell Biol. 180, 619–632. Wong, Y.H., Lu, A.C., Wang, Y.C., Cheng, H.C., Chang, C., Chen, P.H., Yu, J.Y., Fann, M.J., 2010. Protogenin defines a transition stage during embryonic neurogenesis and prevents precocious neuronal differentiation. J. Neurosci. 30, 4428–4439. Xie, Z., Samuels, B.A., Tsai, L.H., 2006. Cyclin-dependent kinase 5 permits efficient cytoskeletal remodeling–a hypothesis on neuronal migration. Cereb. Cortex 16, i64–i68. Yu, J.Y., DeRuiter, S.L., Turner, D.L., 2002. RNA interference by expression of shortinterfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. 99, 6047–6052. Yuan, H.X., Xiong, Y., Guan, K.L., 2013. Nutrient sensing, metabolism, and cell growth control. Mol. Cell 49, 379–387.
Fig. 6. Overexpression of Cdk5 partially rescues neurite defects in Cdk12-depleted and Cdk13-depleted P19 cells. (A, C) The expression levels of Cdk12, Cdk13, and Cdk5 proteins in P19 cells transfected with the indicated constructed. (B, D) Representative images of P19 cells transfected with the indicated constructs and pp35 are shown. (E, F) The average neurite lengths and the neurite length distribution of P19 cells transfected with the indicated Cdk5 and Cdk12 constructs are shown. **, p b 0.01. There is a significant difference between the pCdk12-i1 + pp35 and pCdk12-i1 + pCdk5 + pp35 groups by the two sample K–S test (p b 0.01). (G, H) The average neurite lengths and neurite length distribution in P19 cells transfected with the indicated Cdk5 and Cdk13 constructs are shown. **, p b 0.01. There is a significant difference between pCdk13i1 + pp35 and pCdk13-i1 + pCdk5 + pp35 groups by the two sample K–S test (p b 0.001). Scale bars 100 μm.