Cdk5 - Wiley Online Library

Journal of Neurochemistry, 2005, 93, 502–512

doi:10.1111/j.1471-4159.2005.03058.x

Control of cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability Fan-Yan Wei,* Kazuhito Tomizawa, Toshio Ohshima,à Akiko Asada,* Taro Saito,* Chan Nguyen,§ James A. Bibb,§ Koichi Ishiguro,¶ Ashok B. Kulkarni,** Harish C. Pant, Katsuhiko Mikoshiba,à Hideki Matsui and Shin-ichi Hisanaga* *Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minami-osawa, Hachiohji, Tokyo, Japan Department of Physiology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan àLaboratory for Developmental Neurobiology, Brain Science Institute, Wako, Saitama, Japan §Department of Psychiatry, The University of Texas South-western Medical Center, Dallas, Texas, USA ¶Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo, Japan **Functional Genomics Unit, National Institute of Dental & Craniofacial Research and Laboratory of Neurochemistry, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland, USA

Abstract Although the roles of cyclin-dependent kinase 5 (Cdk5) in neurodevelopment and neurodegeneration have been studied extensively, regulation of Cdk5 activity has remained largely unexplored. We report here that glutamate, acting via NMDA or kainate receptors, can induce a transient Ca2+/ calmodulin-dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome-dependent degradation of a Cdk5 activator p35, and thus ultimately down-regulation of Cdk5 activity. The relevance of this

regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation. p35–/– mice exhibited a lower threshold for induction of long-term potentiation. Thus excitatory glutamatergic neurotransmission regulates Cdk5 activity through p35 degradation, and this pathway may contribute to plasticity. Keywords: calmodulin, cyclin-dependent kinase 5, proteasome, glutamate, N-methyl-D-aspartate, long-term potentiation. J. Neurochem. (2005) 93, 502–512.

Cyclin-dependent kinase 5 (Cdk5) is a cdc2-like kinase that requires the neuronal-specific activator p35 or its homologue p39 for activity (Tang and Wang 1996; Dhavan and Tsai 2001; Hisanaga and Saito 2003). The essential role of Cdk5 in neuronal migration during brain development has been documented in Cdk5- and p35-deficient mice (Ohshima et al. 1996, 1999; Chae et al. 1997). Furthermore, deregulation of Cdk5 by proteolytic conversion of p35 to p25 is suggested to be involved in neuronal cell death (Kusakawa et al. 2000; Lee et al. 2000; Nath et al. 2000) and some neurodegenerative diseases (Patrick et al. 1999; Nguyen et al. 2001; Bu et al. 2002). Emerging evidence suggests a role for Cdk5 in the synaptic activity of mature neurons. In the presynaptic compartment, Cdk5 may regulate neurotransmitter release through phosphorylation of synapsin I (Matsubara et al. 1996) and the

a-subunit of the P/Q type voltage-dependent Ca2+ channel (Tomizawa et al. 2002). Cdk5 also regulates synaptic vesicle endocytosis through phosphorylation of dynamin I and

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Received November 21, 2004; revised manuscript received December 21, 2004; accepted December 22, 2004. Address correspondence and reprint requests to Shin-ichi Hisanaga, Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minami-osawa, Hachiohji, Tokyo 192–0397, Japan. E-mail: [email protected] Abbreviations used: APDC, 4-aminopyrolidine-2,4-dicarbonate; CaMKII, Ca2+-calmodulin-dependent protein kinase II; Cdk5, cyclindependent kinase 5; CHX, cycloheminide; CKI, casein kinase I; DHPG, dihydroxyphenylglycine; EPSP, excitatory postsynaptic potential; L-AP4, L-(+)-2-amino-4-phosphonobutyric acid; LTP, long-term potentiation; PKC, protein kinase C; PP2B, protein phosphatase 2B; TBS, theta burst stimulation; ZLLH, carbobenzoxy-L-leucyl-L-leucinal.

2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 502–512

Glutamatergic regulation of Cdk5–p35 activity 503

amphiphysin I (Floyd et al. 2001; Nguyen and Bibb 2003; Tan et al. 2003; Tomizawa et al. 2003). In the postsynaptic compartment, Cdk5 has been shown to phosphorylate the NR2A subunit of the NMDA receptor (Li et al. 2001; Wang et al. 2003), PSD95 (Morabito et al. 2004), and protein phosphatase inhibitor-1 (Bibb et al. 2001), as well as its striatal homologue, DARPP-32 (Bibb et al. 1999). Furthermore, NMDA stimulation increases the association of p35 with Ca2+-calmodulin-dependent protein kinase IIa (CaMKIIa), a major component of the postsynaptic density (Dhavan et al. 2002). Despite this impressive list of downstream effectors, the identities of upstream regulators of Cdk5 have remained unknown. We report here that ionotropic glutamate receptors regulate Cdk5 activity through the modulation of p35 stability. Evidence that this novel signal transduction pathway may contribute to synaptic plasticity is also presented.

Materials and methods Materials Glutamate, NMDA, BAPTA-AM, calphostin C, KN-62, A23187, and W7 were purchased from Sigma (St. Louis, MO, USA). Kainate was obtained from Tocris (Avonmouth Bristol, UK). Lactacystin, roscovitine, Suc-LLL-MCA and anti-Cdk5 antibody DC-17 were from Calbiochem (San Diego, CA, USA). Carbobenzoxy-L-leucylL-leucinal (ZLLH) was obtained from the Peptide Institute (Osaka, Japan). Antibodies against p35 (C-19 and N-20), Cdk5 (C-8), and inhibitor-1 (N-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-Ser67 inhibitor-1 antibody was provided by Paul Greengard (The Rockefeller University, NY, USA). Cyclosporine A was provided by Kazuo Nagai (Tokyo Institute of Technology, Yokohama, Japan). Calmodulin was purified from porcine brains by the method of Yazawa et al. (1980). Culture and metabolic labeling of primary cortical neurons Neurons from embryonic day 15–16 mouse (ICR, SLC, Tokyo, Japan) brain cortices were plated at a density of 2 · 105 cells/cm2 in polyethyleneimine-coated dishes in Dulbecco’s modified Eagle’s medium and Ham’s F-12 (1 : 1) supplemented with 5% fetal bovine serum and 5% horse serum (Saito et al. 1998). Cytosine arabinoside (20 lM) was added to the culture medium 3 days after plating to inhibit the proliferation of glial cells. All experiments were performed on day 7 of culture. When treating neurons with various inhibitors, the inhibitors were added to the culture medium 30 min before the neurons were stimulated with glutamate, kainate, or NMDA. For metabolic phosphorylation of p35, neurons were cultured in the presence of [32P]orthophosphate in phosphate-free Dulbecco’s modified Eagle’s medium for 3 h (Wada et al. 1998). Preparation of cell extracts and immunoprecipitation Neurons were lysed by freezing and thawing in lysis buffer (10 mM MOPS, pH 7.2, 1 mM MgCl2, 1 mM EGTA, 0.1 mM EDTA, 0.3 M NaCl, 0.5% Nonidet P-40 (NP-40), 1 lg/mL leupeptin, 1 mM dithiothreitol), and the supernatant (cell extract) was collected after centrifugation at 10 000 g for 30 min. Anti-Cdk5 antibody C-8 (2 lL) was added to 100 lL of extract (corresponding to 50 lg

protein). After a 1-h incubation at 4C, 20 lL protein A–Sepharose CL-4B beads (50% slurry in lysis buffer; Amersham Pharmacia Biotech., Tokyo, Japan) was added, and the sample was further incubated for 1 h at 4C. The beads were washed four times with 10 mM MOPS, pH 7.2, 1 mM EGTA, 0.1 mM EDTA, and 1 mM MgCl2, then used for the histone H1 kinase assay (Kusakawa et al. 2000). For the detection of 32P-labeled p35, neurons were suspended in RIPA buffer [10 mM Tris-HCl, pH 7.5, 1 mM EGTA, 0.15 M NaCl, 1% NP-40, 0.1% sodium dodecylsulfate, 10 mM b-glycerophosphate, 5 mM NaF, 1 mM p-nitrophosphate, 0.2 mM Pefabloc SC (Merck, Darmstadt, Germany), 1 lg/mL leupeptin, 1 mM dithiothreitol] and lysed by freezing and thawing. The cell extract (supernatant) was collected after centrifugation at 10 000 g for 30 min. p35 was isolated from the extract by immunoprecipitation with the anti-p35 antibody, C-19, as described previously (Kusakawa et al. 2000). 32P-labeled p35 prepared by immunoprecipitation was separated by sodium dodecyl sulfate–gel electrophoresis on a 12.5% polyacrylamide gel, and the 32P incorporated into the p35 was detected by a BAS 2000 Image Analyzer (Fuji film, Tokyo, Japan). To detect p35 and Cdk5 in whole-cell extracts, neurons were collected by centrifugation at 300 g for 3 min, immediately frozen in liquid nitrogen, and lysed in 100 lL sodium dodecyl sulfate–gel electrophoresis sample buffer (31.25 mM Tris-HCl, pH 6.8, 5% glycerol, 1% sodium dodecyl sulfate, 2.5% b-mercaptoethanol) by sonication and boiling for 5 min. Detection of histone H1 kinase activity in Cdk5–p35 and the phosphorylation of p35 The kinase reaction of immunoprecipitated Cdk5–p35 was initiated by adding 0.1 mM [c-32P]ATP to a reaction mixture containing 10 mM MOPS, pH 7.2, 1 mM MgCl2, and 0.3 mg/mL histone H1. After 30-min incubation at 35C, the reaction was stopped by the addition of sodium dodecyl sulfate–gel electrophoresis sample buffer, and the mixture was immediately boiled for 5 min. Samples were separated by sodium dodecyl sulfate–gel electrophoresis on a 15% polyacrylamide gel, and the radioactivity associated with histone H1 was quantified using a BAS2000 Image Analyzer. 32 P-Labeled p35 prepared by immunoprecipitation was separated by sodium dodecyl sulfate–gel electrophoresis on a 12.5% polyacrylamide gel, and the 32P incorporated into the p35 was detected by a BAS2000 Image Analyzer. Preparation of mouse brain extract and incubation with Ca2+-calmodulin Brains of 7–8-week-old mice (ICR) were homogenized in 10 vol. of 20 mM MOPS, pH 7.4, 1 mM MgCl2, 0.1 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5% NP-40, 1 mM dithiothreitol, 0.1 mM Pefabloc, and 10 lg/mL leupeptin at 4C with a Teflon-pestle glass homogenizer. The brain extract was obtained by centrifugation at 10 000 g for 15 min. The brain extract was incubated with 0.5 mM CaCl2 and 50 lg/mL calmodulin at 35C for 30 min. In some case, 0.1 mM W7 was added to inhibit calmodulin activity. Cdk5–p35 was immunoprecipitated with anti-Cdk5 antibody (C8) or anti-p35 antibody (C19). The kinase activity was measured as described above. p35, p25 and Cdk5 were detected by immunoblotting as described below.


504 F.-Y. Wei et al.

Fluorogenic peptide substrate assay for proteasome activity For the measurement of the proteasome activity, neurons were lysed in ice-cold 20 mM HEPES, pH 7.2, 0.1 mM EDTA, 1 mM ATP, 20% glycerol, and 0.04% NP-40. The cell extract was collected after centrifugation at 10 000 g for 15 min. The extract was incubated at 37C with Suc-LLL-MCA (100 lM) in reaction buffer (50 mM TrisHCl, pH 8.0, 5 mM EGTA) for 30 min. The reaction was stopped by adding 1% sodium dodecyl sulfate. Cleavage of peptides was measured by excitation at 380 nm and emission at 460 nm. Hippocampal slice pharmacology Mouse hippocampal slices were acutely prepared essentially as previously described (Caporaso et al. 2000). Microdissected hippocampal slices (400 lm thick) were treated with 100 lM NMDA for the indicated times. All slices were incubated in Kreb’s buffer for equal amounts of time (60 min). Homogenates were prepared by sonication in boiling 1% sodium dodecyl sulfate with 50 mM NaF. Equal amounts of protein as determined by BCA assay were loaded in each lane. For the histone H1 kinase assay, slices were lysed by sonication in lysis buffer, and the supernatant was collected after centrifugation at 10 000 g for 30 min. Immunoprecipitation of Cdk5/p35 was carried out using anti-p35 antibody (N-20) according to the method described above. Injection of kainate into mice and preparation of the brain extract Seven-week-old mice (ICR) were injected subcutaneously with 50 mg kainate per kg body weight, or the same volume of saline. Mice were killed 30 min after injection, and the cerebral cortices were dissected and immediately frozen in liquid nitrogen. The cerebral cortices were homogenized with a Teflon homogenizer in lysis buffer. The brain extract was collected as the supernatant after centrifugation at 10 000 g for 30 min. Long-term potentiation induction and electrophysiological recording in hippocampus slices p35 knockout mice were generated, maintained in 129/ Sv · C57BL/6J backgrounds, and genotyped as described previously (Ohshima et al. 2001). All mice were handled in accordance with institutional guidelines and housed in as pathogen-free environment on a 12 : 12 h light : dark cycle. Electric stimulation was carried out in 6–8-week-old mice as described previously (Lu et al. 1999; Tomizawa et al. 2003). Briefly, a glass micropipette filled with artificial CSF (aCSF, 1– 5 MW resistance) was placed in the stratum radiatum of the CA1 region to record the field excitatory postsynaptic potentials (fEPSPs), and a bipolar stimulating electrode was placed along the Schaffer collateral fibers. The intensity of the stimulation was adjusted to produce an EPSP with a slope between 35% and 50% of the maximum. The test stimulation was delivered once per minute (0.017 Hz). Long-term potentiation (LTP) was induced by thetaburst stimulation (TBS), a strong stimulation paradigm. TBS protocols consisted of four pulses at 100 Hz repeated for 15 times with an interval of 200 ms between each burst. Slices were lysed by sample buffer 15 min after stimulation. Data are shown as mean (± SEM) percentage of the intensity of p35–/– signal in unstimulated control slices.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting Protein extracts (10 lg) were separated by 12.5% polyacrylamide gel sodium dodecyl sulfate–gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After probing with primary antibodies and then anti-rabbit or anti-mouse IgG secondary antibodies (DAKO, Glostrup Denmark), the reaction was detected with either the BCIP/NBT phosphatase substrate system (KLP, Gaithersburg, MD, USA) or the enhanced chemiluminescence system (ECL; Amersham Pharmacia Biotech). The amount of p35 was quantified using NIH Image Analyzer after incorporation of the blot images into Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA) using a scanner. All experiments in this study were performed multiple times with similar results, and representative results are shown in the figures. Data were analyzed using the Student t-test to compare the two conditions and p < 0.05 was considered to be significant.

Results

Stimulation of NMDA or kainate receptors reduces p35 levels in primary cortical neurons Long-term stimulation with excitotoxic glutamate has been shown to induce cleavage of p35 to p25 by calpain during neuronal cell death (Fig. 1a) (Lee et al. 2000). In characterizing this effect, it was observed that exposure of primary cortical cultured neurons to glutamate for shorter periods (up to 1 h) decreased levels of p35 to 25% that of untreated neurons, but without the generation of p25, as assessed by immunoblot analysis (Fig. 1b). Cortical neurons treated with glutamate for 1 h did not show any signs of cell death and were able to survive for, at least, several days after the removal of glutamate, indicating that this decrease in p35 is a process occurring in living neurons. To determine which glutamate receptor classes were involved in this novel effect, neurons were treated with various specific agonists. Two ionotropic glutamate receptor agonists, NMDA and kainate, induced a dose-dependent decrease in p35 levels in a time course similar to that of glutamate (Figs 1c and d). This effect was similarly observed by immunfluorescent analysis of neuronal cultures (data not shown). Furthermore, an NMDA receptor antagonist (MK801) and a kainate/AMPA receptor antagonist (CNQX) suppressed the effects of kainate and NMDA on p35 levels (Fig. 1e). In contrast, group I, II and III metabotropic glutamate receptors agonists (DHPG, dihydroxyphenylglycine; APDC, 4-aminopyrolidine-2,4-dicarbonate; L-AP4, L-(x)-2-amino-4-phosphonobutyric acid, respectively) did not decrease the levels of p35, but some of them rather increased slightly (Fig. 1f). A small amount of p25 was detected in some cultures, but the short-term treatment with NMDA or kainite resulted in no further increase. These results indicate that the glutamate-induced reduction in p35 levels occurred via specific activation of ionotropic NMDA and kainate receptors.



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Fig. 1 Ionotropic glutamate receptors regulate p35 levels in cultured mouse cortical neurons. Cultured cortical neurons were treated with 1 mM glutamate for 0, 1, 6, 12 h (a), with 0.1 mM glutamate for 0, 1, 5, 15, 30, 60 min (upper panel of b), or at indicated concentrations for 1 h (lower panel of b). p35 and cyclin-dependent kinase 5 (Cdk5) were detected by immunoblotting whole-cell lysates. Quantification of the amount of p35 in the upper panel is shown in the middle panel of (b). Cultured neurons were similarly treated with 0.1 mM NMDA (c) or 0.1 mM kainate (d). Black arrowheads and white arrows indicate p35 and Cdk5, respectively. White arrowheads indicate p25 or the position of p25. (e) Cortical neurons were treated with 0.1 mM NMDA or kainate in the presence of 2 lM MK801 or 20 lM CNQX for 1 h. (f) p35 levels in cortical neurons treated with 0–100 lM DHPG, APDC, or L-AP4 for 1 h.

2000; Kerokoski et al. 2002), whereas the turnover of p35 in healthy neurons is dependent on the ubiquitin–proteasome system (Patrick et al. 1998; Saito et al. 1998). To examine whether the decrease in p35 resulted from proteasomedependent degradation, neurons were treated with NMDA or kainate in the presence of a proteasome inhibitor, lactacystin, or a calpain inhibitor, ZLLH (Fig. 2b). Lactacystin completely inhibited the decrease in p35, indicating the reduction in p35 was due to proteasomal activity. To ensure that the enhanced p35 degradation was specific and not due to an overall increase in proteasomal activity after NMDA or kainate treatment, proteasomal activity was measured using a fluorogenic peptide (Fig. 2c). The proteasome inhibitor MG132 was included as a control (MG in Fig. 2c). Overall proteasomal activity was not enhanced after stimulation with NMDA or kainate, indicating that these agents cause a reduction in p35 by a process of degradation signal tagging, such as ubiquitination. Cyclin-dependent kinase 5 phosphorylates p35 in response to NMDA or kainate receptor activation NMDA and kainate receptors are Ca2+-permeable cation channels (Ozawa et al. 1998; Craig and Boudin 2001). The Ca2+-activated enzymes, protein kinase C (PKC), Ca2+-

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Fig. 2 NMDA and kainate stimulate the proteasome-dependent degradation of p35. (a) Cultured neurons were treated with 10 lg/mL cycloheximide (CHX, squares in lower graph) or 0.1 mM NMDA (black circles) or kainite (white circles). p35 levels were detected by immunoblotting with anti-p35 antibody (upper panels) and quantitated by densitometric analysis (lower graph). This is one of two independent experiments with the similar results. (b) Neurons were treated with 0.1 mM NMDA or 0.1 mM kainate for 1 h in the presence of 10 lM ZLLH, 20 lM lactacystin (Lacta), or no inhibitors (–). The lysates were probed by immunoblotting with anti-p35 antibody. (c) Proteasome activity in neurons treated with 0.1 mM NMDA (N) or 0.1 mM kainate (K) for 30 min and 60 min. The neuron extracts were incubated with the specific proteasome substrate (Z-LLL-MCA), together with 5 lM MG132 (MG) in one case, for 30 min. Cleavage activity is expressed as a percentage of the activity of control neurons.



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Fig. 3 The degradation of p35 is mediated by cyclin-dependent kinase 5 (Cdk5) but not Ca2+-calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), protein phosphatase 2B (PP2B), or casein kinase I (CKI). Cultured neurons were treated with 0.1 mM NMDA or 0.1 mM kainate in the presence of 50 lM KN-62, 1 lM calphostin C (CalC), 2 lM cyclosporine A (CsA), 20 lM casein kinase inhibitor (CKI-7) or 50 lM roscovitine for 1 h. p35 was detected by immunoblotting with anti-p35 antibody (upper panel) and quantitated by densitometric analysis (black bars for NMDA and white bars for kainate in lower panel). *p < 0.001.

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calmodulin-dependent protein kinase II (CaMKII), and protein phosphatase 2B (PP2B), are known to mediate signals originating from NMDA or kainate receptor activation. To examine the role of these enzymes in mediating p35 degradation, a CaMKII inhibitor (KN-62), a PKC inhibitor (calphostin C), and a PP2B inhibitor (cyclosporine A) were employed (Fig. 3). However, when added to the culture medium, none of these inhibitors suppressed the NMDA or kainate-induced degradation of p35, suggesting the existence of a novel signaling from NMDA and kainite receptors. Phosphorylation of p35 is required for degradation of p35 via the proteasome pathway (Patrick et al. 1998; Saito et al. 1998), and it has been shown that p35 can be phosphorylated by casein kinase I (CKI) and Cdk5 in vitro (Tsai et al. 1994; Patrick et al. 1999; Liu et al. 2001; Saito et al. 2003). The possible involvement of these kinases was examined by using inhibitors specific for each (Fig. 3). Neurons were treated with NMDA or kainate in the presence of either a CKI inhibitor (CKI-7) or a Cdk5 inhibitor (roscovitine). Roscovitine, but not CKI-7, suppressed the degradation of p35. Furthermore, roscovitine alone did not change the level of p35 during this incubation period (data not shown). To further evaluate the role that Cdk5 plays in the degradation of p35, kinase activity was determined using Cdk5 immunoprecipitated from cell culture lysates before and after stimulation with NMDA or kainate (Figs 4a and b). The kinase activity of Cdk5/p35, as assessed by phosphorylation of histone H1, increased transiently 1–2 min after the addition of NMDA or kainate, and then decreased gradually over the next hour. To examine whether this curious biphasic regulation of Cdk5 activity was reflected in levels of p35 phosphorylation, cortical neurons were cultured in the

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Fig. 4 NMDA or kainate transiently activates cyclin-dependent kinase 5 (Cdk5) kinase activity and stimulates phosphorylation of p35. (a) and (b) cyclin-dependent kinase 5 (Cdk5)–p35 was isolated by immunoprecipitation from cultured neurons treated with 0.1 mM NMDA (a) or 0.1 mM kainate (b) for 0, 1, 5, 15, 30, or 60 min. The kinase activity was measured using histone H1 as the substrate (H1 in upper panels of a and b). Quantification of kinase activity is shown in the middle panels of (a) and (b) (n ¼ 3). An inset in (a) represents the activation of Cdk5 at 1 and 2 min after NMDA application. Black and white arrowheads in lower panel indicate p35 and the position of p25, respectively. (c) and (d) Cortical neurons cultured in the presence of [32P]orthophosphate were stimulated by 0.1 mM NMDA or 0.1 mM kainate for 1 min in the presence or absence of 50 lM roscovitine (Ros in c). p35 was immunoprecipitated and its phosphorylation status was detected by autoradiography (c) and quantified (d). *p < 0.05.

presence of [32P]orthophosphate. p35 was isolated from neuronal extracts before and after stimulation with NMDA or kainate, and its phosphorylation was detected by autoradiography. Indeed, phosphorylation of p35 increased at 1 min after the addition of NMDA or kainate (Figs 4c and d) and this effect was obliterated by roscovitine (Fig. 4c). Thus, transient activation of Cdk5 by NMDA or kainate results in autophosphorylation and then degradation of p35. The activation of cyclin-dependent kinase 5 and subsequent degradation of p35 is Ca2+-calmodulin dependent There are three known mechanisms by which the kinase activity of Cdk5 can be stimulated: an increase in the level of p35, the conversion of p35 to p25, and the phosphorylation of Cdk5 (Patrick et al. 1999; Sharma et al. 1999; Zukerberg


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et al. 2000; Hashiguchi et al. 2002). We examined these as possible causes of the immediate activation of Cdk5. However, there was neither an increase in the amount of p35 nor in its conversion to p25 in the first few minutes after stimulation with NMDA or kainate (Figs 4a and b, lower panels). In addition, we did not detect phosphorylation of Cdk5 when the Cdk5–p35 complex was prepared from NMDA- or kainate-treated cortical neurons cultured in the presence of 32P (data not shown). In an effort to identify the novel signaling mechanism responsible for the observed up-regulation, focus was given to the ionotropic glutamate receptors. First, the transient upregulation of Cdk5 activity by NMDA and kainate receptors was confirmed using specific receptor antagonists (Figs 5a and b). MK801 and CNQX inhibited both the transient upregulation and the subsequent down-regulation of the Cdk5 kinase activity. A major function of these receptors is the conductance of Ca2+ current into neurons. Although no Ca2+dependent downstream kinases and phosphatases were found to mediate NMDA- or kainate-induced p35 degradation (Fig. 3), Ca2+ entry could still be required for transient Cdk5 activation and subsequent p35 degradation. Indeed, both were suppressed when neurons were treated with BAPTAAM, a membrane-permeable Ca2+ chelator (Figs 5a–c). In contrast, when intracellular Ca2+ levels were increased with the Ca2+ ionophore A23187, p35 was cleaved to p25

Fig. 5 The activation of cyclin-dependent kinase 5 (Cdk5) and subsequent degradation of p35 is Ca2+-calmodulin-dependent. (a) and (b) Neurons were stimulated with 0.1 mM NMDA (a) or 0.1 mM kainate (b) for 1 min and 60 min in the presence of the antagonists MK801 and CNQX, respectively, or 0.2 mM BAPTA-AM (BAPTA). Histone H1 phosphorylation by immunoprecipitated Cdk5/p35 is shown by autoradiograph in upper panel. The kinase activity was measured and expressed in relation to the activity just before NMDA/kainate addition (0 min) in lower panel. *p < 0.05 and **p < 0.001. (c) Cultured neurons were treated with 0.1 mM NMDA or 0.1 mM kainate in the presence of 0.2 mM BAPTA-AM (BAPTA) for 1 h. p35 was detected by immunoblotting with anti-p35 antibody. (d) Cultured neurons were treated with 0.1 mM NMDA, 0.1 mM kainate or 5 lM A23187 for 1 h. The lysates were probed by immunoblotting with anti-p35 antibody. Black and white arrowheads indicate p35 and p25, respectively. (e) The histone H1 kinase activity of Cdk5 immunoprecipitated from brain extract preincubated with 0.05 mg/mL calmodulin (CaM) or 0.1 mM W-7 in the presence (+) or absence (–) of 0.5 mM CaCl2 for 60 min on ice. In one case, 50 lM roscovitine was included in the assay to show that histone H1 phosphorylation was due to Cdk5 activity. (f) Inhibition of Ca2+calmodulin-activated histone H1 kinase activity by roscovitine. Histone H1 kinase activity of immunoprecipitates prepared as described in (e) was measured in the presence or absence of 50 lM roscovitine. (g) Immunoblotting of anti-p35 immunoprecipitates with anti-Cdk5 antibody. (h) Immunoblottings of the brain extract incubated with Ca2+ or Ca2+-calmodulin with anti-p35 antibody (upper panel) and anti-Cdk5 antibody (lower panel). (i) Cortical neurons cultured in the presence of [32P]orthophosphate were stimulated by 0.1 mM NMDA for 1 min in the presence or absence of 0.2 mM W-7. p35 was immunoprecipitated and its phosphorylation status was detected by autoradiography and quantified. *p < 0.05. (j) Primary cultured neurons were treated with 0.1 mM NMDA or 0.1 mM kainate in the presence or absence of 0.2 mM W-7 for 1 h. The level of p35 was determined by immunoblotting.

(Fig. 5d). These results suggest that the transient activation of Cdk5 leading to the subsequent degradation of p35 occurs at physiological Ca2+ concentrations induced by treatment with NMDA or kainate. At higher concentrations of Ca2+, calpain cleavage of p35 to p25 predominates. The effect of Ca2+ on the histone H1 kinase activity of Cdk5/p35 purified from Sf9 cells or immunoprecipitated from mouse brain extracts was next assessed, but addition of Ca2+ had no effect (data not shown), ruling out a direct activation of Cdk5 by Ca2+. The possibility of a Ca2+dependent Cdk5 activator was also investigated by measuring Cdk5 kinase activity immunoprecipitated from brain extract preincubated with Ca2+ (Fig. 5e). Preincubation with Ca2+ stimulated the histone H1 kinase activity of Cdk5. Furthermore, when calmodulin was added to the brain extract during preincubation, Cdk5 displayed even higher kinase activity. Moreover, the addition of a calmodulin inhibitor (W-7) negated the activation of Cdk5 by Ca2+. To ensure that the observed histone H1 kinase activity in the immunoprecipitates was due to Cdk5–p35 and not another Ca2+- or Ca2+-calmodulin-activated protein kinase, roscovi-



In vivo stimulation of NMDA and kainate receptors reduces p35 levels, ultimately leading to a decrease in cyclin-dependent kinase 5 activity The physiologic relevance of this novel NMDA/kainate signaling pathway was next assessed by theta-burst stimulation (TBS), a strong stimulation paradigm, of the Schaffer collateral/CA1 pathway in hippocampal slices followed by immunoblot analysis to determine the level of p35 after 30 min of stimulation (Fig. 6a). The level of p35 in TBS slices was decreased to 74.2 ± 10% (n ¼ 7, p < 0.05) of the controls (lower panel of Fig. 6a). Furthermore, in three of nine slices, an upward mobility shift suggestive of phosphorylation of p35 was observed when sodium dodecyl sulfate–gel electrophoresis was run for longer length (for example, see lane 2 of TBS in Fig. 6a). The upward shift of p35 by autophosphorylation with Cdk5 was observed in the process of p35 degradation in brain extracts and cultured neurons (Saito et al. 2003), but it is unclear why only one-third of slices displayed this upward shift. In another in vivo system, seven-week-old mice were subcutaneously administered 50 mg of kainate/kg body weight. As kainate is a known epileptogenic agent (Bortolotto et al. 1999), the mice exhibited mild seizures 30 min after injection as expected. At this point, the mice were killed and levels of p35 in the cerebral cortex were compared with those of saline-injected control mice by immunoblotting (Fig. 6b). p35 was reduced to 75.5 ± 6.88% (n ¼ 10, p < 0.05) of control levels in kainate-injected mice. We did not observe p25 in a brain of mice treated with kainite for 30 min. Finally, to confirm the ultimate inactivation of Cdk5 by NMDA in intact neuronal tissue, NMDA modulation of a Cdk5-specific site (Ser67) (Bibb et al. 2001) on protein phosphatase inhibitor-1 was examined in hippocampal slices (Fig. 6c, P-Ser67 Inhibitor-1 in upper panel and black circles in lower graph). Phosphorylation of Ser67 was greatly reduced in NMDA-treated slices, in which the Cdk5 activity (H1 in upper panel and white circles in lower graph) and the

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tine was used as a control. Roscovitine inhibited both the basal and Ca2+-calmodulin-activated H1 kinase activity of Cdk5/p35 (Figs 5e and f). Further, the increased kinase activity of Cdk5 was neither due to the increased immunoprecipitation of Cdk5–p35 after incubation (Fig. 5g) nor due to the cleavage of p35 to p25 during the incubation with Ca2+ or Ca2+-calmodulin (Fig. 5h, white arrowhead). Inclusion of protease inhibitors suppressed the p25 generation. These biochemical studies were extended to primary cortical neurons (Figs 5i and j). W-7 inhibited NMDA/ kainate-induced transient phosphorylation (Fig. 5i) and subsequent degradation (Fig. 5j) of p35, supporting the hypothesis that calmodulin activates Cdk5 leading to p35 degradation.

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Fig. 6 In vivo stimulation of NMDA and kainate receptors reduces p35 levels, ultimately leading to a decrease in cyclin-dependent kinase 5 (Cdk5) activity. (a) Hippocampal slices were treated with theta burst stimulation (TBS), and after 30 min p35 was analyzed by immunoblotting (TBS in upper panel). p35 of unstimulated slices is shown in control (Ctrl) of upper panel. Levels of p35 were quantified and expressed in relation to those of unstimulated (Ctrl) slices (lower panel). Values represent the mean ± SEM for seven slices after the largest and smallest data were removed. *p < 0.05. (b) Mice were killed 30 min after the subcutaneous injection of kainate (50 mg/kg) or saline. Levels of p35 in the cerebral cortex were determined by immunoblotting. Values represent the mean ± SEM of 10 mice. *p < 0.05. (c) Hippocampal slices were treated with 0.1 mM NMDA for 0, 5 10, 20, 40 and 60 min. Cdk5, p35, inhibitor-1 and phosphorylated inhibitor-1 were examined by immunoblotting with anti-Cdk5, anti-p35, anti-inhibitor-1 and anti-phospho-Ser67 inhibitor-1 antibodies (upper panel). The kinase activity of Cdk5–p35 was measured with immunoprecipitated Cdk5–p35 using histone H1 as substrate. Autoradiograph is shown in upper panel (H1) and quantification is white circles in lower panel. The level of phosphorylated inhibitor-1 normalized to total inhibitor-1 protein was quantified (black circles in lower panel, n ¼ 4).

protein amount of p35 (p35 in upper panel) were also decreased, though a little bit delayed after the dephosphorylation of Ser67. p35–/– mice have a lower threshold for long term potentiation Down-regulation of Cdk5/p35 activity by NMDA may have important consequences for synaptic plasticity. This possibility was investigated using the Schaffer collateral/CA1 pathway of p35–/– mice, which possess the ultimate downregulation of p35. In this paradigm, a difference might be expected to be observed between wild-type and p35–/– mice under a weak stimulation protocol. As induction of LTP by weak stimulation does not result in p35 degradation, a



difference in the level of LTP induction between wild type and p35–/– mice would represent the contribution of p35 down-regulation of LTP. Stronger stimulation protocols would be expected to negate this result, because the wildtype mice would degrade their p35 to a level closer to that of the p35–/– mice. Indeed, LTP induction by a weak TBS protocol consisting of two bursts of four pulses (2 · 4) was significantly different between wild-type and p35–/– mice (Fig. 7). This weak stimulation failed to induce LTP in wildtype mice (109 ± 6.2%, n ¼ 7), whereas it induced stable LTP in p35–/– mice (138 ± 8.5%, n ¼ 8). However, the effects of stronger TBS protocols consisting of four (4 · 4), six (4 · 6), or eight bursts (4 · 8) of four pulses at theta rhythm (Fig. 7b) and tetanic stimulation (data not shown)

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Fig. 7 p35–/– mice exhibit a lower threshold for long-term potentiation (LTP) induction. (a) LTP induction by theta burst stimulation (TBS), two bursts of four pulses at 100 Hz. After a stable baseline had been recorded for 30 min, TBS was delivered (arrow). Data are the mean ± SEM as percentages of the average baseline excitatory postsynaptic potential (EPSP) slope. Insets: representative tracings from wild-type and p35–/– mice of baseline and 60 min post-TBS. Calibration bars are 1.0 mV and 5.0 ms. (b) Comparison of LTP magnitude induced by different TBS protocols. Data are the mean ± SEM at 60 min post-TBS as percentages of the average baseline EPSP slope. White bar, wild-type; gray bar, p35–/–. *p < 0.01.

were indistinguishable between the groups. These results indicate that the threshold for LTP induction is lowered in the hippocampus of p35–/– mice. Discussion

Cdk5 is active only when associated with an activation subunit (Tang et al. 1997; Amin et al. 2002). Thus, the level of p35 is a primary determinant of Cdk5 kinase activity, although phosphorylation of Cdk5 has been suggested to modulate its kinase activity as well (Sharma et al. 1999; Zukerberg et al. 2000; Sasaki et al. 2002). Consequently, one approach to the function of Cdk5/p35 is to determine what factors control the synthesis and degradation of p35. The synthesis of p35 can be stimulated by neurotrophic factors (Tokuoka et al. 2000; Harada et al. 2001) or extracellular matrix components (Paglini et al. 1998; Li et al. 2000). We show here that glutamate, the major excitatory neurotransmitter in the central nervous system (Ozawa et al. 1998; Craig and Boudin 2001), induces p35 degradation. This antithetical regulation of Cdk5 activity by neurotrophins and a neurotransmitter via p35 synthesis and degradation may prove to be important in a variety of processes. Glutamate receptors consist of two major superfamilies of receptors, usually referred to as ionotropic and metabotropic glutamate receptors, both of which are further classified into several subgroups (Ozawa et al. 1998; Craig and Boudin 2001). Each glutamate receptor has respective functions in various neuronal processes that depend on the stage of development and brain regions involved. In these studies we have demonstrated that degradation of p35 was mediated by NMDA and kainate ionotropic glutamate receptors, but not by metabotropic glutamate receptors. After 1 h of treatment, p35 was decreased to 25% of control levels. Although prolonged exposure to glutamate can result in cytotoxic effects, our cortical neurons did not display any evidences for neuronal dell death after the 1 h treatment, indicating that the p35 degradation occurred in viable neurons. Although treatment of neurons for 1 h resulted into degradation of the majority of p35 in neurons, its decrease was detectable even after 5 min treatment (Fig. 1). Because Cdk5–p35 is localized throughout in neurons including cell body, axon and dendrites (Nikolic et al. 1996), it is not unexpected that treatments of 1 h would be required in order to observe a decrease of p35. However, clearly the shorter exposure was sufficient to cause degradation, possibly by initially targeting p35 localized in postsynaptic region where glutamate ionotropic receptors are concentrated. Activation of ionotropic glutamate receptors stimulated the proteasomal degradation of p35 unassociated with an increase in p25. Proteolysis of p35 to p25 was observed only after extended treatment (6–12 h) with glutamate, consistent with the results of Lee et al. (2000). In contrast,



Kerokoski et al. (2002) reported that p35 is cleaved to p25 within 30 min of glutamate or NMDA administration. This discrepancy may be due to differences in the experimental conditions. Kerokoski et al. (2002) treated hippocampal neurons with glutamate or NMDA in the presence of 2.5 mM CaCl2, whereas we treated cortical neurons with glutamate or NMDA without further addition of Ca2+ to the medium (Saito et al. 1998). A larger Ca2+ influx due to higher concentrations of Ca2+ in the medium may induce more activation of calpain, resulting in the cleavage of p35 to p25, as was shown with the Ca2+ ionophore-treated neurons. In contrast, the novel signaling pathway discussed here functions under physiological Ca2+ concentrations. In this novel pathway, glutamate, acting via ionotropic NMDA or kainate receptors, leads to the inactivation of Cdk5 by a pathway involving the proteasomal degradation of p35. Ca2+ entry induced by NMDA or kainate triggers the degradation of p35 through a transient activation of Cdk5/ p35 that results in phosphorylation of p35. Thus, transient activation of Cdk5 by NMDA ultimately results in inhibition of Cdk5 activity, which would result in decreased level of phosph-Ser67 inhibitor-1. NMDA-induced reduction in the level of phospho-Ser67 inhibitor-1 in striatal slices was previously attributed to stimulation of calcineurin (PP2B), the phosphatase responsible for dephosphorylation of this site (Bibb et al. 2001). The present results suggest that the decrease in Ser67 phosphorylation may be the result of coordinated action between decreased kinase activity and enhanced phosphatase activity. Although metabotropic glutamate receptor agonists did not induce the inactivation of Cdk5/p35 in cortical neurons, one report claims that DHPG, a group I mGluR agonist, increases Cdk5 activity in neostriatal slices via casein kinase I-dependent phosphorylation of p35 (Liu et al. 2001). Glutamate may differentially regulate Cdk5 activity depending on the expression pattern of the various types of glutamate receptors in neurons. NMDA- and kainate-induced Ca2+ signaling is known to be mediated by the Ca2+-activated enzymes PKC, CaMKII, and PP2B (Platenik et al. 2000). However, none of these enzymes was found to be involved in the transient activation of Cdk5 or the degradation of p35. Instead, the Ca2+-binding protein calmodulin was implicated in this process. The regulation of Cdk5 activity by Ca2+ is very complex, as p35 was recently found to bind in a Ca2+-dependent manner to Ca2+-CaMKII and a-actinin, both of which are enriched in the postsynaptic region (Dhavan et al. 2002). It will be interesting to investigate the relationship between the regulation of Cdk5–p35 kinase activity by Ca2+ and the Ca2+-dependent interaction of Cdk5–p35 with Ca2+-CaMKII and a-actinin. Cdk5 activity may be regulated by these binding partners. The importance of the regulation of Cdk5 activity by Ca2+ and glutamate may lie in the relationship between Cdk5 and synaptic plasticity. Indeed, Cdk5 has been

implicated in associative and aversive learning by animal behavioral studies (Fischer et al. 2003). Support for the physiological relevance of the novel signaling described herein is provided by several in vivo models ranging from TBS or NMDA treatment of hippocampal slices to kainate treatment of whole animals. A critical observation further supporting the physiological relevance of this pathway is that p35–/– mice exhibit a lower threshold for LTP than wild-type mice. However, this finding must be considered carefully, because the Cdk5 signaling system could be reorganized compensatory in p35–/– mice or neuronal cell connections could potentially be disorganized in p35–/– mice as was implicated by difference in baseline fEPSP between p35–/– and wild-type mice. In actuality, neuronal cell layers were only mildly or marginally affected in the CA1 region of the hippocampus (Chae et al. 1997 and Toshio Ohshima. et al., in preparation). All together, our data strongly implicate the down-regulation of Cdk5 in NMDA-dependent LTP induction. Acknowledgements We thank Dr Satoru Tahakashi at the National Institute of Dental and Craniofacial Research, National Institutes of Health, for the critical reading, Dr Atsuko Uchida for helpful advice on experiments, Dr Kazuo Nagai for cyclosporine A, and Dr Paul Greengard at The Rockefeller University for the phospho-Ser67 inhibitor-1 antibody. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (C), Advanced Brain Science Project from the Ministry of Education, Culture, Sports, and Science and Technology, of Japan (SH) and and the National Institute of Drug Abuse (JAB).

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