Perinatal abrogation of Cdk5 expression in brain results in neuronal migration defects

Cyclin-dependent kinase 5 (Cdk5) is essential for the proper development of the CNS, as is evident from the perinatal lethality of conventional Cdk5 knockout (Cdk5-/-) mice. Cdk5 is also implicated in numerous complex functions of the adult CNS such as synaptic transmission, synaptic plasticity, and neuronal signaling. To elucidate the molecular roles of Cdk5 in the adult CNS, we have abrogated neuronal expression of Cdk5 in perinatal mice by using a cre-loxP system. The Cdk5-loxP flanked mice were crossed with the cre-transgenic mice in which the cre expression is driven by the murine neurofilament-heavy chain promoter, resulting in generation of viable Cdk5 conditional knockout mice with the restricted deletion of the Cdk5 gene in specific neurons beginning around embryonic day 16.5. Twenty-five percent of the Cdk5 conditional knockout mice carrying the heterozygous cre allele had neuronal migration defects confined to brain areas where neuronal migration continues through the perinatal period. These results indicate that abrogation of Cdk5 expression in mature neurons results in a viable mouse model that offers further opportunities to investigate the molecular roles of Cdk5 in the adult CNS.     

The brain-specific activator p35 allows Cdk5 to escape inhibition by p27Kip1 in neurons.

Cell cycle withdrawal in postmitotic cells involves cyclin-dependent kinase (Cdk) inhibitors that repress cell cycle Cdk activity. During mouse neurogenesis, cortical postmitotic neurons are shown here to accumulate high levels of the p27 Cdk inhibitor compared with their progenitor neuroblasts. Elevated p27 levels in staged embryo brain extracts correlate with p27 binding to Cdk2, and Cdk inactivation. Yet, Cdk5, which is associated with the noncyclin activator p35 in neurons, remains active in the presence of high p27 levels. Both in vitro and in vivo, p27 and related inhibitors can recognize a cyclin D-Cdk5 complex but not a p35-Cdk5 complex. The results indicate that the choice of activator determines the susceptibility of Cdk5 to p27 and related Cdk inhibitors, and thus its ability to act in postmitotic cells.     

Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.

Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in developing brain. Cdk5 purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. We report here generation of Cdk5(-/-) mice through gene targeting and their phenotypic analysis. Cdk5(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of Cdk5(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization.     

PKCδ regulates cortical radial migration by stabilizing the Cdk5 activator p35

Cyclin-dependent kinase 5 (Cdk5) and its activator p35 are critical for radial migration and lamination of cortical neurons. However, how this kinase is regulated by extracellular and intracellular signals during cortical morphogenesis remains unclear. Here, we show that PKCδ, a member of novel PKC expressing in cortical neurons, could stabilize p35 by direct phosphorylation. PKCδ attenuated the degradation of p35 but not its mutant derivative, which could not be phosphorylated by PKCδ. Down-regulation of PKCδ by in utero electroporation of specific small interference RNA (siRNA) severely impaired the radial migration of cortical neurons. This migration defect was similar to that caused by down-regulation of p35 and could be prevented by cotransfection with the wild-type but not the mutant p35. Furthermore, PKCδ could be activated by the promigratory factor brain-derived neurotrophic factor (BDNF) and was required for the activation of Cdk5 by BDNF. Both PKCδ and p35 were required for the promigratory effect of BDNF on cultured newborn neurons. Thus, PKCδ may promote cortical radial migration through maintaining the proper level of p35 in newborn neurons.     

ApoER2/VLDL receptor and Dab1 in the rostral migratory stream function in postnatal neuronal migration independently of Reelin

Postnatal migration of interneuron precursors from the subventricular zone to the olfactory bulb occurs in chains that form the substrate for the rostral migratory stream. Reelin is suggested to induce detachment of neuroblasts from the chains when they arrive at the olfactory bulb. Here we show that ApoER2 and possibly very-low-density lipoprotein receptor (VLDLR) and their intracellular adapter protein Dab1 are involved in chain formation most likely independent of Reelin. F-spondin, which is present in the stream, may act as ligand for ApoER2 and VLDLR. In mice lacking either both receptors or Dab1 chain formation is severely compromised, and as a consequence the rostral migratory stream is virtually absent and neuroblasts accumulate in the subventricular zone. The mutant animals exhibit severe neuroanatomical defects in the subventricular zone and in the olfactory bulb. These data demonstrate a cell-autonomous function of ApoER2, and most likely VLDLR and Dab1, in postnatal migration of neuroblasts in the forebrain, which is suggested to depend on ligands other than Reelin.     

Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase

Mammalian Cdk5 is a member of the cyclin-dependent kinase family that is activated by a neuron-specific regulator, p35, to regulate neuronal migration and neurite outgrowth. p35/Cdk5 kinase colocalizes with and regulates the activity of the Pak1 kinase in neuronal growth cones and likely impacts on actin cytoskeletal dynamics through Pak1. Here, we describe a functional homologue of Cdk5 in budding yeast, Pho85. Like Cdk5, Pho85 has been implicated in actin cytoskeleton regulation through phosphorylation of an actin-regulatory protein. Overexpression of CDK5 in yeast cells complemented most phenotypes associated with pho85Delta, including defects in the repression of acid phosphatase expression, sensitivity to salt, and a G(1) progression defect. Consistent with the functional complementation, Cdk5 associated with and was activated by the Pho85 cyclins Pho80 and Pcl2 in yeast cells. In a reciprocal series of experiments, we found that Pho85 associated with the Cdk5 activators p35 and p25 to form an active kinase complex in mammalian and insect cells, supporting our hypothesis that Pho85 and Cdk5 are functionally related. Our results suggest the existence of a functionally conserved pathway involving Cdks and actin-regulatory proteins that promotes reorganization of the actin cytoskeleton in response to regulatory signals.     

Cyclin-dependent kinase 5 activity regulates pain signaling

Several molecules and cellular pathways have been implicated in nociceptive signaling, but their precise molecular mechanisms have not been clearly defined. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase implicated in the development and disease of the mammalian nervous system. The precise role of this kinase in sensory pathways has not been well characterized. Here we report a molecular role for Cdk5 in nociception. We identified the expression of Cdk5 and its activator p35 in nociceptive neurons, which is modulated during a peripheral inflammatory response. Increased calpain activity in sensory neurons after inflammation resulted in the cleavage of p35 to p25, which forms a more stable complex with Cdk5 and, consequently, leads to elevation of Cdk5 activity. p35 knockout mice (p35(-/-)), which exhibit significantly decreased Cdk5 activity, showed delayed responses to painful thermal stimulation compared with WT controls. In contrast, mice overexpressing p35, which exhibit elevated levels of Cdk5 activity, were more sensitive to painful thermal stimuli than were controls. In conclusion, our data demonstrate a role for Cdk5/p35 activity in primary afferent nociceptive signaling, suggesting that Cdk5/p35 may be a target for the development of analgesic drugs.     

Increased activity of cyclin-dependent kinase 5 leads to attenuation of cocaine-mediated dopamine signaling

Cocaine, a drug of abuse, increases synaptic dopamine levels in the striatum by blocking dopamine reuptake at axon terminals. Cyclin-dependent kinase 5 (Cdk5) and its activator p35, proteins involved in phosphorylation of substrates in postmitotic neurons, have been found to be up-regulated after chronic exposure to cocaine. To further examine the effects of Cdk5 and p35 induction on striatal dopamine signaling, we generated two independent transgenic mouse lines in which Cdk5 or p35 was overexpressed specifically in neurons. We report here that increased Cdk5 activity, as a result of p35 but not of Cdk5 overexpression, leads to attenuation of cocaine-mediated dopamine signaling. Increased Cdk5-mediated phosphorylation of dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa (DARPP-32) at Thr-75, was accompanied by decreased phosphorylation of DARPP-32 at Thr-34. Increased Cdk5-mediated phosphorylation of extracellular signal-regulated kinase kinase 1 at Thr-286 was accompanied by decreased activation of extracellular signal-regulated kinase 1/2. These effects contributed to attenuation of cocaine-induced phosphorylation of cAMP response element-binding protein as well as a lesser induction of c-fos in the striatum. These results support the idea that Cdk5 activity is involved in altered gene expression after chronic exposure to cocaine and hence impacts the long-lasting changes in neuronal function underlying cocaine addiction.     

Reelin,Disabled 1, and beta 1 integrins are required for the formation of the radial glial scaffold in the hippocampus

The extracellular matrix molecule Reelin is required for the correct positioning of neurons during the development of the forebrain. However, the mechanism of Reelin action on neuronal migration is poorly understood. Reelin is assumed to act on neurons directly, but it may also affect the differentiation of glial cells necessary for neuronal migration. Here we show that a regular glial scaffold fails to form in vivo in the dentate gyrus of mice deficient of Reelin or Disabled 1, a neuronal adaptor protein in the Reelin signaling pathway. A subset of these defects is observed in mice that lack beta(1)-class integrins, known to bind Reelin. Moreover, recombinant Reelin induced branching of glial processes in vitro. Our data suggest that Reelin affects glial differentiation via Disabled 1 and beta(1)-class integrin-dependent signaling pathways.     

Nuclear localization of Cdk5 is a key determinant in the postmitotic state of neurons

Cyclin-dependent kinase 5 (Cdk5) is a nontraditional Cdk that is primarily active in postmitotic neurons. Its best known substrates are cytoskeletal proteins. Less appreciated is its role in the maintenance of a postmitotic state. We show here that in cycling cells (NIH 3T3), the localization of Cdk5 changes from predominantly nuclear to cytoplasmic as cells reenter a cell cycle after serum starvation. Similarly, when beta-amyloid peptide is used to stimulate cultured primary neurons to reenter a cell cycle, they too show a loss of nuclear Cdk5. Blocking nuclear export pharmacologically abolishes cell cycle reentry in wild-type but not Cdk5(-/-) neurons, suggesting a Cdk5-specific effect. Cdk5 overexpression targeted to the nucleus of Cdk5(-/-) neurons effectively blocks the cell cycle, but cytoplasmic targeting is ineffective. Further, in both human Alzheimer's disease as well as in the R1.40 mouse Alzheimer's model and the E2f1(-/-) mouse, neurons expressing cell cycle markers consistently show reduced nuclear Cdk5. Thus, both in vivo and in vitro, neurons that reenter a cell cycle lose nuclear Cdk5. We propose that the nuclear Cdk5 plays an active role in allowing neurons to remain postmitotic as they mature and that loss of nuclear Cdk5 leads to cell cycle entry.     

Cyclin-dependent kinase 5 regulates steroidogenic acute regulatory protein and androgen production in mouse Leydig cells

The roles of cyclin-dependent kinase 5 (Cdk5) in central nervous system and neurodegenerative diseases have been intensely investigated in recent decades. Because protein expressions of Cdk5 and its regulator, p35, have been identified in Leydig cells, it is informative to further explore the novel function of Cdk5/p35 in male reproduction. Here we show that Cdk5/p35 protein expression and kinase activity in mouse Leydig cells are regulated by human chorionic gonadotrophin (hCG) in both dose- and time-dependent manners. Blocking of Cdk5 by molecular inhibitors or small interfering RNA resulted in reduction of testosterone production by Leydig cells. cAMP, a second messenger in LH signaling, was identified as a factor in hCG-dependent regulation of Cdk5/p35. Importantly, Cdk5 protein and kinase activity could support accumulation of steroidogenic acute regulatory (StAR) protein, a crucial component of steroidogenesis. We additionally addressed the protein interaction between Cdk5/p35 and StAR. The Cdk5-dependent serine phosphorylation of StAR indicated a possible mechanism by which Cdk5 induced accumulation of StAR protein. In conclusion, Cdk5 modulates hCG-induced androgen production in mouse Leydig cells, possibly through regulation of StAR protein levels. These results indicate that Cdk5 may play an important role in male reproductive endocrinology and is a potential therapeutic target in androgen-related diseases.     

Reelin is preferentially expressed in neurons synthesizing γ-aminobutyric acid in cortex and hippocampus of adult rats

During embryonic development of brain laminated structures, the protein Reelin, secreted into the extracellular matrix of the cortex and hippocampus by Cajal–Retzius (CR) cells located in the marginal zone, contributes to the regulation of migration and positioning of cortical and hippocampal neurons that do not synthesize Reelin. Soon after birth, the CR cells decrease, and they virtually disappear during the following 3 weeks. Despite their disappearance, we can quantify Reelin mRNA (approximately 200 amol/μg of total RNA) and visualize it by in situ hybridization, and we detect the translated product of this mRNA by immunocytochemistry preferentially in γ-aminobutyric acid (GABA)ergic neurons of adult rat cortex and hippocampus. In adult rat cerebellum, Reelin is expressed in glutamatergic neurons (granule cells). The translated product of this mRNA is readily exported from the granule cell somata to the parallel fibers, where it has been detected by electron microscopy in axon terminals located presynaptically to Purkinje cell dendrites.     

Cdk5 is essential for adult hippocampal neurogenesis

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.     

Estrogen receptor (ER)beta knockout mice reveal a role for ERbeta in migration of cortical neurons in the developing brain

The present study stems from our previous observations that the brains of adult estrogen receptor beta knockout (ERbeta-/-) mice show regional neuronal hypocellularity especially in the cerebral cortex. We now show that ERbeta is necessary for late embryonic development of the brain and is involved in both neuronal migration and apoptosis. At embryonic day (E)18.5, ERbeta-/- mouse brains were smaller than those of the wild-type (WT) littermates, and there were fewer neurons in the cortex. There were no differences in size or cellularity at E14.5. When proliferating cells were labeled with 5'-bromodeoxyuridine (BrdUrd) on E12.5, a time when cortical neurogenesis in mice begins, and examined on E14.5, there was no difference between WT and ERbeta-/- mice in the number of labeled cells in the cortex. However, when BrdUrd was administered between E14.5 and E16.5, a time when postmitotic neurons migrate to layers of the cortex, there were fewer BrdUrd-labeled cells in the superficial cortical layers by E18.5 and postnatal day 14 in mice lacking ERbeta. At E18.5, there were more apoptotic cells in the ventricular zone of mice lacking ERbeta. In addition, the processes of the cortical radial glia, which are essential for guiding the migrating neurons, were fragmented. These findings suggest that by influencing migration and neuronal survival, ERbeta has an important role in brain development.     

Regulation of cyclin-dependent kinase 5 and casein kinase 1 by metabotropic glutamate receptors

Cyclin-dependent kinase 5 (Cdk5) is a multifunctional neuronal protein kinase that is required for neurite outgrowth and cortical lamination and that plays an important role in dopaminergic signaling in the neostriatum through phosphorylation of Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa). Casein kinase 1 (CK1) has been implicated in a variety of cellular functions such as DNA repair, circadian rhythm, and intracellular trafficking. In the neostriatum, CK1 has been found to phosphorylate Ser-137 of DARPP-32. However, first messengers for the regulation of Cdk5 or CK1 have remained unknown. Here we report that both Cdk5 and CK1 are regulated by metabotropic glutamate receptors (mGluRs) in neostriatal neurons. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist for group I mGluRs, increased Cdk5 and CK1 activities in neostriatal slices, leading to the enhanced phosphorylation of Thr-75 and Ser-137 of DARPP-32, respectively. The effect of DHPG on Thr-75, but not on Ser-137, was blocked by a Cdk5-specific inhibitor, butyrolactone. In contrast, the effects of DHPG on both Thr-75 and Ser-137 were blocked by CK1-7 and IC261, specific inhibitors of CK1, suggesting that activation of Cdk5 by mGluRs requires CK1 activity. In support of this possibility, the DHPG-induced increase in Cdk5 activity, measured in extracts of neostriatal slices, was abolished by CK1-7 and IC261. Treatment of acutely dissociated neurons with DHPG enhanced voltage-dependent Ca(2+) currents. This enhancement was eliminated by either butyrolactone or CK1-7 and was absent in DARPP-32 knockout mice. Together these results indicate that a CK1-Cdk5-DARPP-32 cascade may be involved in the regulation by mGluR agonists of Ca(2+) channels.     

Aberrant motor axon projection, acetylcholine receptor clustering, and neurotransmission in cyclin-dependent kinase 5 null mice

Cyclin-dependent kinase (Cdk)5 is a key regulator of neural development. We have previously demonstrated that Cdk5/p35 are localized to the postsynaptic muscle and are implicated in the regulation of neuregulin/ErbB signaling in myotube culture. To further elucidate whether Cdk5 activity contributes to neuromuscular junction (NMJ) development in vivo, the NMJ of Cdk5-/- mice was examined. Consistent with our previous demonstration that Cdk5 phosphorylates ErbB2/3 to regulate its tyrosine phosphorylation, we report here that the phosphorylation of ErbB2 and ErbB3 and the ErbB2 kinase activity are reduced in Cdk5-deficient muscle. In addition, Cdk5-/- mice also display morphological abnormalities at the NMJ pre- and postsynaptically. Whereas the outgrowth of the main nerve trunk is grossly normal, the intramuscular nerve projections exhibit profuse and anomalous branching patterns in the Cdk5-/- embryos. The central band of acetylcholine receptor (AChR) clusters is also wider in Cdk5-/- diaphragms, together with the absence of S100 immunoreactivity along the phrenic nerve during late embryonic stages. Moreover, we unexpectedly discovered that the agrin-induced formation of large AChR clusters is significantly increased in primary muscle cultures prepared from Cdk5-null mice and in C2C12 myotubes when Cdk5 activity was suppressed. These abnormalities are accompanied by elevated frequency of miniature endplate potentials in Cdk5-null diaphragm. Taken together, our findings reveal the essential role of Cdk5 in regulating the development of motor axons and neuromuscular synapses in vivo.     

A reelin-integrin receptor interaction regulates Arc mRNA translation in synaptoneurosomes

Reelin is synthesized and secreted into extracellular matrix by cortical gamma-aminobutyric acid (GABA)ergic interneurons and binds with high affinity to the extracellular domain of integrin receptors expressed in dendritic shaft and spine postsynaptic densities (DSPSD) of pyramidal neurons. In heterozygous reeler mice, reelin bound to DSPSD, and the expression of Arc (activity-regulated cytoskeletal protein) is lower than in wild-type mice. We studied the effect of reelin on Arc and total protein synthesis in synaptoneurosomes (SNSs) prepared from mouse neocortex. Recombinant full-length mouse reelin displaces the high affinity (K(D) = 60 fM) binding of [(125)I]echistatin (a competitive integrin receptor antagonist) to integrin receptors with a K(i) of 22 pM and with a Hill slope close to 1. Echistatin (50-100 nM) competitively antagonizes and abates reelin binding. The addition of reelin (2-40 pM) to SNSs enhances the incorporation of [(35)S]methionine into Arc and other rapidly translated proteins in a concentration-dependent manner. This incorporation is virtually abolished by 50-100 nM echistatin or by 5-10 nM rapamycin, a blocker of the mammalian target of rapamycin kinase. We conclude that reelin binds with high affinity to integrin receptors expressed in SNSs and thereby activates Arc protein synthesis.     

Neuronal migration is retarded in mice lacking the tissue plasminogen activator gene

Neuronal migration is a critical phase of brain development, where defects can lead to severe ataxia, mental retardation, and seizures. In the developing cerebellum, granule neurons turn on the gene for tissue plasminogen activator (tPA) as they begin their migration into the cerebellar molecular layer. Granule neurons both secrete tPA, an extracellular serine protease that converts the proenzyme plasminogen into the active protease plasmin, and bind tPA to their cell surface. In the nervous system, tPA activity is correlated with neurite outgrowth, neuronal migration, learning, and excitotoxic death. Here we show that compared with their normal counterparts, mice lacking the tPA gene (tPA(-/-)) have greater than 2-fold more migrating granule neurons in the cerebellar molecular layer during the most active phase of granule cell migration. A real-time analysis of granule cell migration in cerebellar slices of tPA(-/-) mice shows that granule neurons are migrating 51% as fast as granule neurons in slices from wild-type mice. These findings establish a direct role for tPA in facilitating neuronal migration, and they raise the possibility that late arriving neurons may have altered synaptic interactions.     

AMP-activated protein kinase (AMPK) activity is not required for neuronal development but regulates axogenesis during metabolic stress

Mammalian brain connectivity requires the coordinated production and migration of billions of neurons and the formation of axons and dendrites. The LKB1/Par4 kinase is required for axon formation during cortical development in vivo partially through its ability to activate SAD-A/B kinases. LKB1 is a master kinase phosphorylating and activating at least 11 other serine/threonine kinases including the metabolic sensor AMP-activated protein kinase (AMPK), which defines this branch of the kinome. A recent study using a gene-trap allele of the β1 regulatory subunit of AMPK suggested that AMPK catalytic activity is required for proper brain development including neurogenesis and neuronal survival. We used a genetic loss-of-function approach producing AMPKα1/α2-null cortical neurons to demonstrate that AMPK catalytic activity is not required for cortical neurogenesis, neuronal migration, polarization, or survival. However, we found that application of metformin or AICAR, potent AMPK activators, inhibit axogenesis and axon growth in an AMPK-dependent manner. We show that inhibition of axon growth mediated by AMPK overactivation requires TSC1/2-mediated inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. Our results demonstrate that AMPK catalytic activity is not required for early neural development in vivo but its overactivation during metabolic stress impairs neuronal polarization in a mTOR-dependent manner.