Learning is accompanied by modulation of postsynaptic signal transduction pathways in neurons. Although the neuronal protein kinase cyclin-dependent kinase 5 (Cdk5) has been implicated in cognitive disorders, its role in learning has been obscured by the perinatal lethality of constitutive knockout mice. Here we report that conditional knockout of Cdk5 in the adult mouse brain improved performance in spatial learning tasks and enhanced hippocampal long-term potentiation and NMDA receptor (NMDAR)-mediated excitatory postsynaptic currents. Enhanced synaptic plasticity in Cdk5 knockout mice was attributed to reduced NR2B degradation, which caused elevations in total, surface and synaptic NR2B subunit levels and current through NR2B-containing NMDARs. Cdk5 facilitated the degradation of NR2B by directly interacting with both it and its protease, calpain. These findings reveal a previously unknown mechanism by which Cdk5 facilitates calpain-mediated proteolysis of NR2B and may control synaptic plasticity and learning.

Circular dichroism measurements were used to study the binding of fd gene 5 protein to fd DNA, to six polydeoxynucleotides (poly[d(A)], poly[d(T)], poly[d(I)], poly[d(C)], poly[d(A-T)], and the random copolymer poly[d(A,T)]), and to three oligodeoxynucleotides (d(pA)20, d(pA)7, and d(pT)7). Titrations of these DNAs with fd gene 5 protein were generally done in a low ionic strength buffer (5 mM Tris-HCl, pH 7.0 or 7.8) to insure tight binding, needed to obtain stoichiometric endpoints. By monitoring the CD of the nucleic acids above 250 nm, where the protein has no significant intrinsic optical activity, we found that there were two modes of binding, with the number of nucleotides covered by a gene 5 protein monomer (n) being close to either 4 or 3. These stoichiometries depended upon which polymer was titrated as well as upon the protein concentration. Single endpoints at nucleotide/protein molar ratios close to 3 were found during titrations of poly[d(T)] and fd DNA (giving n = 3.1 and 2.8 +/- 0.2, respectively), while CD changes with two apparent endpoints at nucleotide/protein molar ratios close to 4 and approximately 3 were found during titrations of poly[d(A)], poly[d(I)], poly[d(A-T)], and poly[d(A,T)] (with the first endpoints giving n = 4.1 4.0, 4.0, and 4.1 +/- 0.3, respectively). Calculations showed that the CD changes we observed during these latter titrations were consistent with a switch between two non-interacting binding modes of n = 4 and n = 3. We found no evidence for an n = 5 binding mode. One implication of our results is that the Brayer and McPherson model for the helical gene 5 protein-DNA complex, which has 5 nucleotides bound per protein monomer (G. Brayer and A. McPherson, J. Biomol. Struct. and Dyn. 2, 495-510, 1984), cannot be correct for the detailed solution structure of the complex. We interpreted the CD changes above 250 nm upon binding of the gene 5 protein to single-stranded DNAs to be the result of a slight unstacking of the bases, along with a significant alteration of the CD contributions of the individual nucleotides in the case of A-and/or T-containing DNAs. Interestingly, CD contributions attributed to nearest-neighbor interactions in free poly[d(A-T)], poly[d(A,T)], poly[d(A)], and poly[d(T)] were partially maintained in the CD spectra of the protein-saturated polymers, so that neighboring nucleotides, when bound to the protein at 20 degrees C, appeared to interact with one another in much the same manner as in the free polymers at 50 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclin-dependent kinase 5 (Cdk5) regulates dopamine neurotransmission and has been suggested to serve as a homeostatic target of chronic psychostimulant exposure. To study the role of Cdk5 in the modulation of the cellular and behavioral effects of psychoactive drugs of abuse, we developed Cre/loxP conditional knock-out systems that allow temporal and spatial control of Cdk5 expression in the adult brain. Here, we report the generation of Cdk5 conditional knock-out (cKO) mice using the ␣CaMKII promoter-driven Cre transgenic line (CaMKII-Cre). In this model system, loss of Cdk5 in the adult forebrain increased the psychomotor-activating effects of cocaine. Additionally, these CaMKII-Cre Cdk5 cKO mice show enhanced incentive motivation for food as assessed by instrumental responding on a progressive ratio schedule of reinforcement. Behavioral changes were accompanied by increased excitability of medium spiny neurons in the nucleus accumbens (NAc) in Cdk5 cKO mice. To study NAc-specific effects of Cdk5, another model system was used in which recombinant adeno-associated viruses expressing Cre recombinase caused restricted loss of Cdk5 in NAc neurons. Targeted knock-out of Cdk5 in the NAc facilitated cocaine-induced locomotor sensitization and conditioned place preference for cocaine. These results suggest that Cdk5 acts as a negative regulator of neuronal excitability in the NAc and that Cdk5 may govern the behavioral effects of cocaine and motivation for reinforcement.

The molecular factors regulating adult neurogenesis must be understood to harness the therapeutic potential of neuronal stem cells. Although cyclin-dependent kinase 5 (Cdk5) plays a critical role in embryonic corticogenesis, its function in adult neurogenesis is unknown. Here, we assessed the role of Cdk5 in the generation of dentate gyrus (DG) granule cell neurons in adult mice. Cre recombinase-mediated conditional knockout (KO) of Cdk5 from stem cells and their progeny in the DG subgranular zone (SGZ) prevented maturation of new neurons. In addition, selective KO of Cdk5 from mature neurons throughout the hippocampus reduced the number of immature neurons. Furthermore, Cdk5 gene deletion specifically from DG granule neurons via viral-mediated gene transfer also resulted in fewer immature neurons. In each case, the total number of proliferating cells was unaffected, indicating that Cdk5 is necessary for progression of adult-generated neurons to maturity. This role for Cdk5 in neurogenesis was activating-cofactor specific, as p35 KO but not p39 KO mice also had fewer immature neurons. Thus, Cdk5 has an essential role in the survival, but not proliferation, of adult-generated hippocampal neurons through both cell-intrinsic and cell-extrinsic mechanisms.cyclin-dependent kinase ͉ dentate gyrus granule cell ͉ doublecortin ͉ nestin-CreERT2 ͉ viral-mediated gene transfer I n the adult brain, neural precursors give rise to immature neurons which mature into granule cells in the hippocampal subgranular zone (SGZ) (1, 2). This process of adult neurogenesis is orchestrated by signaling intrinsic to the new cells as well as extrinsic or microenvironmental influences in the SGZ niche (2, 3). For example, the orphan nuclear receptor tailless intrinsically regulates stem cell proliferation (4) and neurogenesin-1 extrinsically regulates fate specification (5). In contrast to our growing understanding of factors that regulate proliferating cells, the identity and mechanisms of factors that regulate immature neurons remain unclear (3). Elucidation of the intrinsic and extrinsic factors that mediate the formation of new neurons is critical to understand normal brain development and to realize the therapeutic potential of adult neurogenesis.Cyclin-dependent kinase 5 (Cdk5) is a proline-directed, serine/threonine cyclin-dependent kinase family member. Its activity is largely restricted to post-mitotic neurons of the central nervous system (6) and depends on association with the noncyclin cofactors p35 and p39 (7,8). Cdk5 plays a critical role in neuronal migration during development and is highly enriched in the hippocampus. Therefore, it might be predicted to be important in the formation of immature neurons in the adult hippocampus. This hypothesis is supported by the importance of Cdk5 substrates, including nestin and doublecortin (DCX), in adult neurogenesis (9)(10)(11). Here, we demonstrate that Cdk5 is critical for adult hippocampal neurogenesis. Using several approaches, we show that ablation of the Cdk5 gene from either SGZ...

Ischemic stroke is one of the leading causes of morbidity and mortality. Treatment options are limited and only a minority of patients receive acute interventions. Understanding the mechanisms that mediate neuronal injury and death may identify targets for neuroprotective treatments. Here we show that the aberrant activity of the protein kinase Cdk5 is a principal cause of neuronal death in rodents during stroke. Ischemia induced either by embolic middle cerebral artery occlusion (MCAO) in vivo or by oxygen and glucose deprivation in brain slices caused calpain-dependent conversion of the Cdk5-activating cofactor p35 to p25. Inhibition of aberrant Cdk5 during ischemia protected dopamine neurotransmission, maintained field potentials, and blocked excitotoxicity. Furthermore, pharmacological inhibition or conditional knock-out (CKO) of Cdk5 prevented neuronal death in response to ischemia. Moreover, Cdk5 CKO dramatically reduced infarctions following MCAO. Thus, targeting aberrant Cdk5 activity may serve as an effective treatment for stroke.

Cyclin-dependent kinase 5 (Cdk5) is emerging as a neuronal protein kinase involved in multiple aspects of neurotransmission in both post-and presynaptic compartments. Within the reward/motor circuitry of the basal ganglia, Cdk5 regulates dopamine neurotransmission via phosphorylation of the postsynaptic signal transduction pathway integrator, DARPP-32 (dopamine-and cyclic AMPregulated phosphoprotein, M r 32 000). Cdk5 has also been implicated in regulating various steps in the presynaptic vesicle cycle. Here we report that Cdk5 phosphorylates tyrosine hydroxylase (TH), the key enzyme for synthesis of dopamine. Using phosphopeptide mapping, site-directed mutagenesis, and phosphorylation state-specific antibodies, the site was identified as Ser31, a previously defined extracellular signal-regulated kinases 1/2 (ERK1/2) site. The phosphorylation of Ser31 by Cdk5 versus ERK1/2 was investigated in intact mouse striatal tissue using a pharmacological approach. The results indicated that Cdk5 phosphorylates TH directly and also regulates ERK1/2-dependent phosphorylation of TH through the phosphorylation of mitogenactivated protein kinase kinase 1 (MEK1). Finally, phospho-Ser31 TH levels were increased in dopaminergic neurons of rats trained to chronically self-administer cocaine. These results demonstrate direct and indirect regulation of the phosphorylation state of a Cdk5/ERK1/2 site on TH and suggest a role for these pathways in the neuroadaptive changes associated with chronic cocaine exposure. KeywordsCdk5; cocaine; dopamine; MAPK; phosphorylation; tyrosine hydroxylase Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, was first shown to be regulated by protein phosphorylation 30 years ago (Morgenroth et al. 1975). It is a tetrameric 56 kDa mixed-function monooxygenase that is phosphorylated at four serine NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript residues in the N-terminus (Fig. 1a) (Zigmond et al. 1989;Kumer and Vrana 1996;Fitzpatrick 1999). TH activity is enhanced upon phosphorylation at Ser40 by cAMP-dependent protein kinase (PKA) as well as cGMP-dependent protein kinase (PKG), protein kinase C (PKC) and Ca 2+ -calmodulin-dependent protein kinase II (CaMKII). Phosphorylation of TH at Ser19 by CaMKII (Campbell et al. 1986) has also been associated with increased TH activity (Haycock et al. 1998). Both Ser40 and Ser19 are phosphorylated by mitogen-activated protein kinaseactivated protein (MAPKAP) kinase 2. TH is also phosphorylated by Cdk1 at Ser8 in cultured cells (Hall et al. 1991).Phosphorylation of Ser31 by ERK1/2 has also been suggested to activate TH . Interestingly, phosphorylation of Ser31 in humans may be affected through alternative splicing that results in differences in adjacent amino acid sequences (Sutherland et al. 1993b;Haycock 2002). Phospho-Ser31 TH levels have been shown to be increased by neuronal depolarization, phorbol esters, and nerve growth factor treatment in rat brain slices (Lindgren et al. 2002). Clear evidence...

Motor learning and neuro-adaptations to drugs of abuse rely upon neuronal signaling in the striatum. Cyclin-dependent kinase 5 (Cdk5) regulates striatal dopamine neurotransmission and behavioral responses to cocaine. Although the role for Cdk5 in neurodegeneration in the cortex and hippocampus and in hippocampaldependent learning has been demonstrated, its dysregulation in the striatum has not been examined. Here we show that strong activation of striatal NMDA receptors produced p25, the truncated form of the Cdk5 co-activator p35. Furthermore, inducible overexpression of p25 in the striatum prevented locomotor sensitization to cocaine and attenuated motor coordination and learning. This corresponded with reduced dendritic spine density, increased neuro-inflammation, altered dopamine signaling, and shifted Cdk5 specificity with regard to physiological and aberrant substrates, but no apparent loss of striatal neurons. Thus, dysregulation of Cdk5 dramatically affects striatal-dependent brain function and may be relevant to non-neurodegenerative disorders involving dopamine neurotransmission.p25 ͉ spines ͉ addiction ͉ dopamine ͉ neurodegeneration N euronal signal transduction mediates the regulation of synaptic strength that underlies learning and memory. Dysregulation of synaptic signaling may result in neuropsychiatric and neurological disorders. Striatal medium spiny neurons (MSNs) receive midbrain dopaminergic and cortical glutamatergic input and mediate reward-based and motor learning. Furthermore, numerous psychiatric and neurodegenerative illnesses target striatal function. Study of the effects of dysregulation of signaling mechanisms that integrate striatal glutamate and dopamine neurotransmission may provide an understanding of the etiology of such disorders.Cdk5 regulates dopamine neurotransmission (1, 2) and striatal neuron excitability (1, 3). Cocaine targets dopamine neurotransmission by elevating synaptic dopamine levels. Cdk5 modulates the effects of cocaine, including its ability to induce neuroadaptations in signaling, behavior, and neuronal morphology (3-7). Cdk5 also functions in synaptic plasticity and learning (8-10) in hippocampus. Striatal synaptic plasticity involves changes in Cdk5-dependent regulation of dopamine signaling (11), and conditional loss of Cdk5 in the striatum lowers the threshold dose required for cocaine to induce a place preference (3). However, dysregulation of Cdk5 in the striatum and its effects on striatal-mediated behavior and learning has remained largely unexplored.Cdk5 activity is dependent on association with its cofactor p35. Cdk5 may be regulated through auto-phosphorylation of p35 (12, 13) and glutamatergic Ca 2ϩ signaling (14, 15). However, the most prominent feature of Cdk5 regulation may be its hyperactivation upon removal of the first 100 amino acids of p35 by the Ca 2ϩ -dependent protease calpain. The resulting Cdk5/p25 complex engenders aberrant activity and is neurotoxic. P25 overexpression in mouse hippocampus and cortex results in dramatic neurodegenera...

Regulation of cytoskeletal dynamics is essential to neuronal plasticity during development and adulthood. Dysregulation of these mechanisms may contribute to neuropsychiatric and neurodegenerative diseases. The neuronal protein kinase, cyclin-dependent kinase 5 (Cdk5), is involved in multiple aspects of neuronal function, including regulation of cytoskeleton. A neuroproteomic search identified the tubulin-binding protein, stathmin, as a novel Cdk5 substrate. Stathmin was phosphorylated by Cdk5 in vitro at Ser25 and Ser38, previously identified as mitogen-activated protein kinase (MAPK) and p38 MAPKd sites. Cdk5 predominantly phosphorylated Ser38, while MAPK and p38 MAPKd predominantly phosphorylated Ser25. Stathmin was phosphorylated at both sites in mouse brain, with higher levels in cortex and striatum. Cdk5 knockout mice exhibited decreased phospho-Ser38 levels.During development, phospho-Ser25 and -Ser38 levels peaked at post-natal day 7, followed by reduction in total stathmin. Inhibition of protein phosphatases in striatal slices caused an increase in phospho-Ser25 and a decrease in total stathmin. Interestingly, the prefrontal cortex of schizophrenic patients had increased phospho-Ser25 levels. In contrast, total and phospho-Ser25 stoichiometries were decreased in the hippocampus of Alzheimer's patients. Thus, microtubule regulatory mechanisms involving the phosphorylation of stathmin may contribute to developmental synaptic pruning and structural plasticity, and may be involved in neuropsychiatric and neurodegenerative disorders. Keywords: cyclin-dependent kinase 5, mitogen-activated protein kinase, p38 MAPKd (stress-activated protein kinase 4), 3 phosphorylation, stathmin, striatum. The number of synapses on neurons in the human forebrain increases dramatically after birth and then decreases with maturity. Children have enhanced plasticity and a higher capacity for learning and memory compared with adults. During postnatal development, synapses are pruned, following over-growth (Luo and O'Leary 2005). Synaptic restructuring continues throughout adulthood and contributes to the process of aging. Strong, frequently activated connections are maintained and strengthened while weaker and less used synaptic contacts are eliminated. Reduced plasticity due to genetic or epigenetic factors can cause cognitive impairment, while excessive plasticity can cause neurological disorders