Calmodulin-dependent Kinase IV Research Articles

CaMKIV Signaling Is Not Essential for the Maintenance of Intrinsic or Synaptic Properties in Mouse Visual Cortex.

Pyramidal neurons in rodent visual cortex homeostatically maintain their firing rates in vivo within a target range. In young cultured rat cortical neurons, Ca2+/calmodulin-dependent kinase IV (CaMKIV) signaling jointly regulates excitatory synaptic strength and intrinsic excitability to allow neurons to maintain their target firing rate. However, the role of CaMKIV signaling in regulating synaptic strength and intrinsic excitability in vivo has not been tested. Here, we show that in pyramidal neurons in visual cortex of juvenile male and female mice, CaMKIV signaling is not essential for the maintenance of basal synaptic or intrinsic properties. Neither CaMKIV conditional knock-down nor viral expression of dominant negative CaMKIV (dnCaMKIV) in vivo disrupts the intrinsic excitability or synaptic input strength of pyramidal neurons in primary visual cortex (V1), and CaMKIV signaling is not required for the increase in intrinsic excitability seen following monocular deprivation (MD). Viral expression of constitutively active CaMKIV (caCaMKIV) in vivo causes a complex disruption of the neuronal input/output function but does not affect synaptic input strength. Taken together, these results demonstrate that although augmented in vivo CaMKIV signaling can alter neuronal excitability, either endogenous CaMKIV signaling is dispensable for maintenance of excitability, or impaired CaMKIV signaling is robustly compensated.

ADNOC and Petronas ink deal to explore collaboration opportunities

Currently, an increasing proportion of adolescent ketamine users simultaneously consume alcohol. However, the potential behavioural and neurological alterations induced by such a drug combination and the underlying mechanisms have not been systematically examined. Therefore, in the present study, the behavioural and morphological changes and the underlying mechanisms were studied in adolescent rats after repeated alcohol and/or ketamine treatment. This study provided the first evidence that co-administration of alcohol (2 and 4 g/kg, i.g.) in adolescent rats significantly potentiated the neurotoxic properties of repeated ketamine (30 mg/kg, i.p.) treatments over 14 days, manifesting as increased locomotor activity, stereotypic behaviour, ataxia and morphological changes. This potentiation was associated with the enhancement by alcohol of ketamine-induced glutamate (Glu) and dopamine (DA) release in the cortex and hippocampus. Further mechanistic study demonstrated that alcohol potentiated ketamine-induced neurotoxicity through down-regulation of Akt (a serine/threonine kinase or protein kinase, PKB), protein kinase A (PKA), calmodulin-dependent kinase IV (CaMK-IV)-mediated cyclic AMP-responsive element binding protein (CREB) pathways and induction of neuronal apoptosis in the cortex and hippocampus of the adolescent rats. As this study provides strong evidence that repeated alcohol and ketamine co-exposure may cause serious neurotoxicity, attention needs to be drawn to the potential risk of this consumption behaviour, especially for adolescents.

Enriched gestation activates the IGF pathway to evoke embryo-adult benefits to prevent Alzheimer\u2019s disease

BackgroundBuilding brain reserves before dementia onset could represent a promising strategy to prevent Alzheimer’s disease (AD), while how to initiate early cognitive stimulation is unclear. Given that the immature brain is more sensitive to environmental stimuli and that brain dynamics decrease with ageing, we reasoned that it would be effective to initiate cognitive stimulation against AD as early as the fetal period.MethodsAfter conception, maternal AD transgenic mice (3 × Tg AD) were exposed to gestational environment enrichment (GEE) until the day of delivery. The cognitive capacity of the offspring was assessed by the Morris water maze and contextual fear-conditioning tests when the offspring were raised in a standard environment to 7 months of age. Western blotting, immunohistochemistry, real-time PCR, immunoprecipitation, chromatin immunoprecipitation (ChIP) assay, electrophysiology, Golgi staining, activity assays and sandwich ELISA were employed to gain insight into the mechanisms underlying the beneficial effects of GEE on embryos and 7–10-month-old adult offspring.ResultsWe found that GEE markedly preserved synaptic plasticity and memory capacity with amelioration of hallmark pathologies in 7–10-m-old AD offspring. The beneficial effects of GEE were accompanied by global histone hyperacetylation, including those at bdnf promoter-binding regions, with robust BDNF mRNA and protein expression in both embryo and progeny hippocampus. GEE increased insulin-like growth factor 1 (IGF1) and activated its receptor (IGF1R), which phosphorylates Ca2+/calmodulin-dependent kinase IV (CaMKIV) at tyrosine sites and triggers its nuclear translocation, subsequently upregulating histone acetyltransferase (HAT) and BDNF transcription. The upregulation of IGF1 mimicked the effects of GEE, while IGF1R or HAT inhibition during pregnancy abolished the GEE-induced CaMKIV-dependent histone hyperacetylation and BDNF upregulation.ConclusionsThese findings suggest that activation of IGF1R/CaMKIV/HAT/BDNF signaling by gestational environment enrichment may serve as a promising strategy to delay AD progression.

Alcohol aggravates ketamine-induced behavioral, morphological and neurochemical alterations in adolescent rats: The involvement of CREB-related pathways

Currently, an increasing proportion of adolescent ketamine users simultaneously consume alcohol. However, the potential behavioural and neurological alterations induced by such a drug combination and the underlying mechanisms have not been systematically examined. Therefore, in the present study, the behavioural and morphological changes and the underlying mechanisms were studied in adolescent rats after repeated alcohol and/or ketamine treatment. This study provided the first evidence that co-administration of alcohol (2 and 4 g/kg, i.g.) in adolescent rats significantly potentiated the neurotoxic properties of repeated ketamine (30 mg/kg, i.p.) treatments over 14 days, manifesting as increased locomotor activity, stereotypic behaviour, ataxia and morphological changes. This potentiation was associated with the enhancement by alcohol of ketamine-induced glutamate (Glu) and dopamine (DA) release in the cortex and hippocampus. Further mechanistic study demonstrated that alcohol potentiated ketamine-induced neurotoxicity through down-regulation of Akt (a serine/threonine kinase or protein kinase, PKB), protein kinase A (PKA), calmodulin-dependent kinase IV (CaMK-IV)-mediated cyclic AMP-responsive element binding protein (CREB) pathways and induction of neuronal apoptosis in the cortex and hippocampus of the adolescent rats. As this study provides strong evidence that repeated alcohol and ketamine co-exposure may cause serious neurotoxicity, attention needs to be drawn to the potential risk of this consumption behaviour, especially for adolescents.

Expression of TRPC6 and BDNF in Cortical Lesions From Patients With Focal Cortical Dysplasia.

Focal cortical dysplasia (FCD) likely results from abnormal migration of neural progenitor cells originating from the subventricular zone. To elucidate the roles in molecules that are involved in neural migration pathway abnormalities in FCDs, we investigated the expression patterns of transient receptor potential canonical channel 6 (TRPC6) and brain-derived neurotrophic factor (BDNF) in cortical lesions from FCD patients and in samples of normal control cortex. TRPC6 and BDNF mRNA and protein levels were increased in FCD lesions. By immunohistochemistry, they were strongly expressed in microcolumns, heterotopic neurons, dysmorphic neurons, and balloon cells (BCs). Colocalization assays revealed that most of the misshapen TRPC6-positive or heterotopic cells had a neuronal lineage with the exception of TRPC6-positive FCDiib patient BCs, which had both neuronal and glial features. Most TRPC6-positive cells were glutamatergic neurons. There was also greater expression of calmodulin-dependent kinase IV (CaMKIV), the downstream factor of TRPC6, in FCD lesions, suggesting that TRPC6 expression promoted dendritic growth and the development of dendritic spines and excitatory synapses via the CaMKIV-CREB pathway in FCD. Thus, overexpression of BDNF and TRPC6 and activation of the TRPC6 signal transduction pathway in cortical lesions of FCD patients may contribute to FC pathogenesis and epileptogenesis.

Subcellular relocation of histone deacetylase 4 regulates growth plate chondrocyte differentiation through Ca2+/calmodulin-dependent kinase IV

Regulatory mechanisms of chondrocyte differentiation in the growth plate are incompletely understood. Here, we find that histone deacetylase 4 (HDAC4) is located in the nucleus of chondrocytes in the proliferation zone and relocates to the cytoplasm of chondrocytes in the prehypertrophic zone in vivo. This suggests that the relocation of HDAC4 from the nucleus to the cytoplasm may play a role during chondrocyte differentiation. Expression of active CaMKIV in chondrocytes promotes HDAC4 relocation into cytoplasm in primary chondrocytes. Conversely, HDAC4 relocation is blocked by a Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) inhibitor. This indicates that CaMKIV signaling plays an important role in regulating HDAC4 relocation. In addition, CaMKIV is required for HDAC4 phosphorylation, which is required for HDAC4 association with the cytoplasmic protein 14-3-3. Active CaMKIV also stimulates runt-related transcription factor-2 (RunX2) and type X collagen (Col X) promoter activities and overcomes repression of these promoter activities by HDAC4. Furthermore, CaMKIV increases gene expression of the chondrocyte differentiation markers Ihh and Col X. Our results demonstrate that CaMKIV induces chondrocyte differentiation through regulation of HDAC4 subcellular relocation, from the nucleus to the cytoplasm, which results in increased activity of RunX2 and transition of chondrocytes from the proliferative to the prehypertrophic stage. Thus, CaMKIV plays an important regulatory role during chondrocyte differentiation.

Targeting lymphocyte signaling pathways as a therapeutic approach to systemic lupus erythematosus

Over the past year several key pathways in systemic lupus erythematosus (SLE) lymphocyte signaling have been identified. Pathways that can be exploited for therapy are discussed in this review. Inhibition of SLE T cell activation by blocking spleen tyrosine kinase (Syk) and SLE T cell migration by blocking CD44 or CXCR4 lead to amelioration of lupus in lupus-prone mice. Similar results can be achieved by boosting CD8+ Treg numbers. Small molecules that block the kinases CaMKIV (calcium and calmodulin dependent kinase IV) and Bruton Tyrosine kinase (Btk) and the phosphatase calcineurin were shown to be effective in treating murine lupus. Finally, gene methylation status determines the expression of several key genes in SLE and strategies to correct it have shown promising results in preclinical studies. Molecules that enhance T cell receptor (TCR) signaling or increase lymphocyte migration can be inhibited successfully with significant improvement of disease intensity in lupus-prone mice using small molecules. Manipulation of promoter methylation and histone acetylation represents a novel way to alter gene transcription in SLE.

Gα13 regulates MEF2-dependent gene transcription in endothelial cells: role in angiogenesis

The alpha subunit of heterotrimeric G13 protein is required for the embryonic angiogenesis (Offermanns et al., Science 275:533-536, 1997). However, the molecular mechanism of Galpha13-dependent angiogenesis is not understood. Here, we show that myocyte-specific enhancer factor-2 (MEF2) mediates Galpha13-dependent angiogenesis. Our data showed that constitutively activated Galpha13Q226L stimulated MEF2-dependent gene transcription. In addition, downregulation of endogenous Galpha13 inhibited thrombin-stimulated MEF2-dependent gene transcription in endothelial cells. Both Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) and histone deacetylase 5 (HDAC5) were involved in Galpha13-mediated MEF2-dependent gene transcription. Galpha13Q226L also increased Ca(2+)/calmodulin-independent CaMKIV activity, while dominant negative mutant of CaMKIV inhibited MEF2-dependent gene transcription induced by Galpha13Q226L. Furthermore, Galpha13Q226L was able to derepress HDAC5-mediated repression of gene transcription and induce the translocation of HDAC5 from nucleus to cytoplasm. Finally, downregulation of endogenous Galpha13 and MEF2 proteins in endothelial cells reduced cell proliferation and capillary tube formation. Decrease of endothelial cell proliferation that was caused by the Galpha13 downregulation was partially restored by the constitutively active MEF2-VP16. Our studies suggest that MEF2 proteins are an important component in Galpha13-mediated angiogenesis.

Elevating Calcium in Th2 Cells Activates Multiple Pathways to Induce IL-4 Transcription and mRNA Stabilization

PMA and ionomycin cause T cell cytokine production. We report that ionomycin alone induces IL-4 and IFN-gamma, but not IL-2, from in vivo- and in vitro-generated murine Th2 and Th1 cells. Ionomycin-induced cytokine production requires NFAT, p38, and calmodulin-dependent kinase IV (CaMKIV). Ionomycin induces p38 phosphorylation through a calcium-dependent, cyclosporine A-inhibitable pathway. Knocking down ASK1 inhibits ionomycin-induced p38 phosphorylation and IL-4 production. Ionomycin also activates CaMKIV, which, together with p38, induces AP-1. Cooperation between AP-1 and NFAT leads to Il4 gene transcription. p38 also regulates IL-4 production by mRNA stabilization. TCR stimulation also phosphorylates p38, partially through the calcium-dependent pathway; activated p38 is required for optimal IL-4 and IFN-gamma.

TRPC6 channels promote dendritic growth via the CaMKIV-CREB pathway

The canonical transient receptor potential channels (TRPCs) are Ca(2+)-permeable nonselective cation channels with various physiological functions. Here, we report that TRPC6, a member of the TRPC family, promotes hippocampal neuron dendritic growth. The peak expression of TRPC6 in rat hippocampus was between postnatal day 7 and 14, a period known to be important for maximal dendritic growth. Overexpression of TRPC6 increased phosphorylation of Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) and cAMP-response-element binding protein (CREB) and promoted dendritic growth in hippocampal cultures. Downregulation of TRPC6 by short hairpin RNA interference against TRPC6 suppressed phosphorylation of both CaMKIV and CREB and impaired dendritic growth. Expressing a dominant-negative form of CaMKIV or CREB blocked the TRPC6-induced dendritic growth. Furthermore, inhibition of Ca(2+) influx suppressed the TRPC6 effect on dendritic growth. Finally, in TRPC6 transgenic mice, the phosphorylation of CaMKIV and CREB was enhanced and the dendritic growth was also increased. In conclusion, TRPC6 promoted dendritic growth via the CaMKIV-CREB pathway. Our results thus revealed a novel role of TRPC6 during the development of the central nervous system (CNS).

Neurotrophin 3 induces structural and functional modification of synapses through distinct molecular mechanisms

The mechanisms by which neurotrophins elicit long-term structural and functional changes of synapses are not known. We report the mechanistic separation of functional and structural synaptic regulation by neurotrophin 3 (NT-3), using the neuromuscular synapse as a model. Inhibition of cAMP response element (CRE)–binding protein (CREB)–mediated transcription blocks the enhancement of transmitter release elicited by NT-3, without affecting the synaptic varicosity of the presynaptic terminals. Further analysis indicates that CREB is activated through Ca2+/calmodulin-dependent kinase IV (CaMKIV) pathway, rather than the mitogen-activated protein kinase (MAPK) or cAMP pathway. In contrast, inhibition of MAPK prevents the NT-3–induced structural, but not functional, changes. Genetic and imaging experiments indicate that the small GTPase Rap1, but not Ras, acts upstream of MAPK activation by NT-3. Thus, NT-3 initiates parallel structural and functional modifications of synapses through the Rap1–MAPK and CaMKIV–CREB pathways, respectively. These findings may have implications in the general mechanisms of long-term synaptic modulation by neurotrophins.

Shuttling of HDAC5 in H9C2 cells regulates YY1 function through CaMKIV/PKD and PP2A

YY1 is a transcription factor that can activate or repress transcription of a variety of genes and is involved in several developmental processes. YY1 is a repressor of transcription in differentiated H9C2 cells and in neonatal cardiac myocytes but an activator of transcription in undifferentiated H9C2 cells. We now present a detailed analysis of the functional domains of YY1 when it is acting as a repressor or an activator and identify the mechanism whereby its function is regulated in the differentiation of H9C2 cells. We show that histone deacetylase 5 (HDAC5) is localized to the cytoplasm in undifferentiated H9C2 cells and that this localization is dependent on Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) and/or protein kinase D (PKD). In differentiated cells, HDAC5 is nuclear and interacts with YY1. Finally, we show that HDAC5 localization in differentiated cells is dependent on phosphatase 2A (PP2A). Our results suggest that a signaling mechanism that involves CaMKIV/PKD and PP2A controls YY1 function through regulation of HDAC5 and is important in the maintenance of muscle differentiation.

Calmodulin-dependent protein kinase IV regulates nuclear export of Cabin1 during T-cell activation

Calcium signaling is critical for activation of T lymphocytes and has been proposed to be transduced through multiple calmodulin target proteins. Whereas the calcineurin-NFAT signaling module is critical for all mammalian T cells, the role of calmodulin-dependent kinase IV (CaMKIV) in mouse naïve CD4+ T-cell activation remains enigmatic. We have applied lentivius-mediated RNA interference of CaMKIV to human T cells and found that knockdown of CaMKIV abrogates T-cell receptor-mediated transcription of the IL-2 gene. We demonstrate that CaMKIV directly phosphorylates Cabin1, a transcriptional corepressor for myocyte enhancer factor 2, creating a docking site for 14-3-3, which causes its nuclear export. CaMKIV-mediated nuclear export of Cabin1 is likely to account for a significant part of the requirement of CaMKIV during human T-cell activation.

Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV

Systemic lupus erythematosus (SLE) T cells express high levels of cAMP response element modulator (CREM) that binds to the IL-2 promoter and represses the transcription of the IL-2 gene. This study was designed to identify pathways that lead to increased binding of CREM to the IL-2 promoter in SLE T cells. Ca2+/calmodulin–dependent kinase IV (CaMKIV) was found to be increased in the nucleus of SLE T cells and to be involved in the overexpression of CREM and its binding to the IL-2 promoter. Treatment of normal T cells with SLE serum resulted in increased expression of CREM protein, increased binding of CREM to the IL-2 promoter, and decreased IL-2 promoter activity and IL-2 production. This process was abolished when a dominant inactive form of CaMKIV was expressed in normal T cells. The effect of SLE serum resided within the IgG fraction and was specifically attributed to anti–TCR/CD3 autoantibodies. This study identifies CaMKIV as being responsible for the increased expression of CREM and the decreased production of IL-2 in SLE T cells and demonstrates that anti–TCR/CD3 antibodies present in SLE sera can account for the increased expression of CREM and the suppression of IL-2 production.

Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV

Systemic lupus erythematosus (SLE) T cells express high levels of cAMP response element modulator (CREM) that binds to the IL-2 promoter and represses the transcription of the IL-2 gene. This study was designed to identify pathways that lead to increased binding of CREM to the IL-2 promoter in SLE T cells. Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) was found to be increased in the nucleus of SLE T cells and to be involved in the overexpression of CREM and its binding to the IL-2 promoter. Treatment of normal T cells with SLE serum resulted in increased expression of CREM protein, increased binding of CREM to the IL-2 promoter, and decreased IL-2 promoter activity and IL-2 production. This process was abolished when a dominant inactive form of CaMKIV was expressed in normal T cells. The effect of SLE serum resided within the IgG fraction and was specifically attributed to anti-TCR/CD3 autoantibodies. This study identifies CaMKIV as being responsible for the increased expression of CREM and the decreased production of IL-2 in SLE T cells and demonstrates that anti-TCR/CD3 antibodies present in SLE sera can account for the increased expression of CREM and the suppression of IL-2 production.

Phosphorylation of NF-κB by calmodulin-dependent kinase IV activates anti-apoptotic gene expression

We previously presented that calmodulin-dependent kinase IV (CaMKIV) mutually interacts with NF-κB and phosphorylates it directly, inducing the increased transcriptional regulation dependent on NF-κB target genes [J. Biol. Chem. 276 (2001) 20005]. Here, we show that Ser 535 residue is phosphorylated by CaMKIV. S535A mutant of p65 was specifically defective in transactivation of NF-κB target gene expression induced by CaMKIV. While coexpression of active CaMKIV with wild-type p65 led to a recovery from etoposide-induced apoptosis and an increase of Bcl-2 protein in cells, cells expressing S535A mutant did not. Taken together these results suggest that phosphorylated NF-κB p65 on Ser 535 by CaMKIV increases NF-κB target gene expression, including anti-apoptotic gene, hence leading to inhibition of apoptosis.

A targeted thyroid hormone receptor alpha gene dominant-negative mutation (P398H) selectively impairs gene expression in differentiated embryonic stem cells.

Thyroid hormone and retinoic acid (RA) are essential for normal neural development in vivo, yet all in vitro differentiation strategies of embryonic stem (ES) cells use only RA. We developed a novel differentiation strategy of mouse ES cells using T(3). A dominant-negative knock-in point mutation (P398H) was introduced into the thyroid hormone receptor alpha gene to determine the influence of T(3) on ES cell differentiation. Differentiation promoted by T(3) (1 nM), RA (1 microM), or combined T(3)/RA was assessed in wild-type (wt) and mutant (m) ES cells on the basis of neuronal-specific gene expression and cell cycle. T(3) alone stimulated neural differentiation in a similar fashion as that seen with RA in both wtES and mES cells. Expression of neurogranin and Ca(2+)/calmodulin-dependent kinase IV mRNA (identified in vivo as T(3)-regulated genes), however, was markedly reduced in mES, compared with wtES cells. RA treatment enhanced apoptosis, significantly greater than that seen with T(3) stimulation. T(3) treatment given with RA significantly reduced the apoptotic effects of RA, an effect not seen in mES cells. T(3)-induced ES cell neural differentiation of thyroid hormone alpha mutant and wtES cells provides an in vitro model to study T(3)-dependent gene regulation in neural development. This system could also be used to identify novel T(3)-regulated genes. The modulation of the apoptotic effects of RA by T(3) may have implications for stem cell therapy.

Conditional mutagenesis of CamKIV

Ca /Calmodulin-dependent kinase IV (CamKIV) has been suggested to be involved in multiple physiological processes including male and female fertility, T-cell maturation, and long-term memory (Corcoran and Means, 2001). It is activated in response to Ca /calmodulin and by phosphorylation of Ca /Calmodulin-dependent kinase. CREB (cAMP response element binding protein) is one of the best defined substrates of this kinase. CamKIV activates CREB-dependent transcription via phosphorylation of serine 133 in the CREB protein (Corcoran and Means, 2001). Inactivation of the CamKIV (Camk4) gene has been reported by two independent groups. Interestingly, in one study the inactivation of CamKIV shows a severe phenotype consisting of impaired viability, male and female infertility, and neurological disorders (Ribar et al., 2000; Wu et al., 2000; Wu et al., 2000), whereas only a mild neurological phenotype was observed in an independent study (Ho et al., 2000). The discrepancy in the phenotypes of the two knockouts could be due to the different placement of the PGKneo cassette into the targeting constructs. It has been shown that the presence of the PGKneo cassette can influence the phenotype of knockout mice (Scacheri et al., 2001). Thus, the generation of CamKIV conditional knockout mice using the Cre/loxP system is an attractive alternative since it allows the generation of a null mutation that lacks the PGKneo cassette (Gu et al., 1994). A fragment of DNA containing the ATP binding domain of the CamKIV gene (exon III) was isolated from a 129-PAC genomic library. Since it has previously been shown that mutation of lysine 71 in the ATP binding domain abolishes kinase activity (Chatila et al., 1996), we chose to flank exon III, which contains this residue with two loxP sites. In addition, deletion of this exon should create a frame shift that prevents translation of the rest of the protein. The targeting construct was generated using ET-cloning (Angrand et al., 1999; Zhang et al., 1998). In the first round of ET-cloning, a loxP site was introduced downstream of exon III of the targeting construct (Fig. 1A). In the second round, the loxP●FRT●PGKTn5neo●FRT cassette was introduced upstream of exon III (Fig. 1A). Finally, the TK gene was introduced in the targeting construct by standard cloning procedures to generate a TK/neo fusion gene (PGKTK/neo). A total of 129 ES cells were electroporated with 20 g of linearized construct. ES cell clones were analysed for the correct homologous recombination event by Southern blot analyses using a 5 external probe (Fig. 1B), a 3 external probe (Fig. 1C) and an internal probe (data not shown). The FRT●PGKTn5TK/neo●FRT cassette was removed from correctly targeted ES cell clones by electroporation of the FLPe-expressing plasmid pCAGGS-FLPe (Fig. 1A) (Schaft et al., 2001). From 200 gancyclovirresistant clones, two were positive for excision of the FRT●PGKTn5TK/neo●FRT cassette as determined by Southern blot analysis. Chimeric animals were generated by blastocyst injection of positive ES cell clones and mated to C57BL/6 animals for germ-line transmission. In order to validate the functionality of the loxP sites in vivo, CamKIV flox animals (CamKIV) were mated with transgenic mice expressing the Cre recombinase under the control of a CamKII BAC (CamKII iCre) (Casanova et al., 2001). Double-transgenic animals CamKIV, CamKII iCre (CamKIV ) showed selective loss of the CamKIV protein only in those regions where the Cre recombinase is expressed. Specifically, CamKIV expression was lost from hippocampus, striatum, and cortex but not cerebellum (Fig. 2). In summary, we have generated mice with a conditional CamKIV allele using a combination of ET-cloning and the Cre/loxP and FLP/FRT systems, which allows the rapid generation of targeting constructs. Furthermore, the ET-cloning technique allows the introduction of loxP and FRT sites into DNA which is independent of restric-

Synapse to nucleus signaling: is Ca 2+ all you need?

The control of gene expression in response to specific synaptic activity is widely thought to be a crucial mechanism involved in many brain processes. Activity dependent gene expression mediated by the transcription factor CREB (cAMP-response-element-binding protein) is believed to be important in stabilizing long-term learning and memory. Full CREB activation requires both phosphorylation of serine 133 in addition to activation of the coactivator of CREB, CREB-binding protein (CBP). The nuclear located Ca2+/calmodulin-dependent kinase IV (CaMKIV) is a key mediator of both of these processes. But the question is, how does the signal of electrical activation at the synapse translate into an instruction for CaMKIV in the nucleus to activate the expression of genes? Current opinion maintains that the Ca2+ binding protein, calmodulin, which has been activated by Ca2+ entry following synaptic activation, is translocated into the nucleus and thereby influences gene expression. However, the findings of Hardingham et al. 1xNuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity. Hardingham, G.E. et al. Nat. Neurosci. 2001; 3: 261–267Crossref | Scopus (331)See all References1 provide a new twist to this story, as they report that Ca2+ alone is sufficient to relay synaptic activation to the regulation of CREB in the nucleus, and nuclear import of cytoplasmic signaling proteins is not necessary in this process.Hardingham et al. first demonstrated that in their experimental system of cultured primary hippocampal neurons, cytoplasmically-injected tagged calmodulin accumulated in the nucleus independently of synaptic activity and as such is unlikely to be a useful mediator of electrical activity signaling in these cells. The authors then injected wheat germ agglutinin into the cytoplasm to block nuclear protein import through the nuclear pore complex. Under these conditions electrical activity-dependent CREB activation was still detected, indicating that nuclear import of proteins was not required to transmit synaptically evoked signals to the nucleus via CaMKIV. Furthermore, they showed that elevating Ca2+ in isolated nuclei was sufficient to trigger signaling to CREB. The authors then showed that Ca2+ influx, through NMDA receptors, resulted in Ca2+-induced Ca2+ release from internal stores. This cytoplasmic Ca2+ release is proposed to amplify and relay the transient Ca2+ elevation at the synapse to an increase in Ca2+ concentration in the nucleus. It was observed that the nuclear Ca2+ concentration elevations peaked just after, and were longer lasting than, the corresponding transient dendritic Ca2+ increase. As a corollary to this finding, when bursts of synaptic activation to the hippocampal neurons were induced at higher frequency, nuclear Ca2+ remained at higher levels for a longer time through a progressive accumulation of burst-induced Ca2+ elevations. This was shown to result in a corresponding increase in CREB-mediated transcription.In this study the authors demonstrated that synaptic input frequency plays a significant role in nuclear Ca2+ dynamics and, by extension, to changes in gene expression. This finding suggests a mechanism for the interpretation of frequency information from synaptic inputs to long-term physiological modifications in the neuron. This mechanism is based on the retention of a synaptically induced Ca2+ signal by the nuclear transcription apparatus. The finding that synaptically-evoked Ca2+ increases in the nucleus were sufficient to activate CREB makes Ca2+ levels in the nucleus a strong candidate as a key player in complex neurobiological events, including learning, memory and cognitive processes.

The ROR nuclear orphan receptor subfamily: Critical regulators of multiple biological processes

The nuclear receptor superfamily, a group of structurally related, ligand-dependent transcription factors, includes a large number of orphan receptors for which no ligand has yet been identified. These proteins function as key regulators of many physiological processes that occur during embryonic development and in the adult. The retinoid-related orphan receptors (RORs) alpha, beta, and gamma comprise one nuclear orphan receptor gene subfamily. RORs exhibit a modular structure that is characteristic for nuclear receptors; the DNA-binding domain is highly conserved and the ligand-binding domain is moderately conserved among RORs. By a combination of alternative promoter usage and exon splicing, each ROR gene generates several isoforms that differ only in their amino terminus. RORs bind as monomers to specific ROR response elements (ROREs) consisting of the consensus core motif AGGTCA preceded by a 5-bp A/T-rich sequence. RORE-dependent transcriptional activation by RORs is cell type-specific and mediated through interactions with nuclear cofactors. RORs have been shown to interact with certain corepressors as well as coactivators, suggesting that RORs are not constitutively active but that their activity is under some regulatory control. RORs likely can assume at least two different conformations: a repressive state, which allows interaction with corepressor complexes, and an active state, which promotes binding of coactivator complexes. Whether the transition between these two states is regulated by ligand binding and/or by phosphorylation remains to be determined. Ca2+/calmodulin-dependent kinase IV (CaMKIV) can dramatically enhance ROR-mediated transcriptional activation. This stimulation involves CaMKIV-mediated phosphorylation not of RORs, but likely of specific nuclear cofactors that interact with RORs. RORalpha is widely expressed. In the cerebellum, its expression is limited to the Purkinje cells. RORalpha-/- mice and the natural RORalpha-deficient staggerer mice exhibit severe cerebellar ataxia due to a defect in Purkinje cell development. In addition, these mice have thin long bones, suggesting a role for RORalpha in bone metabolism, and develop severe atherosclerosis when placed on a high-fat diet. Expression of RORbeta is very restricted. RORbeta is highly expressed in different parts of the neurophotoendocrine system, the pineal gland, the retina, and suprachiasmatic nuclei, suggesting a role in the control of circadian rhythm. This is supported by observations showing alterations in circadian behavior in RORbeta-/- mice. RORgamma, which is most highly expressed in the thymus, plays an important role in thymopoiesis. Thymocytes from RORgamma-/- mice undergo accelerated apoptosis. The induction of apoptosis is, at least in part, due to a down-regulation of the expression of the antiapoptotic gene Bcl-XL. In addition to the thynic phenotype, RORgamma-/- mice lack lymph nodes, indicating that RORgamma is essential for lymph node organogenesis. Overexpression of RORgamma has been shown to inhibit T cell receptor-mediated apoptosis in T cell hybridomas and to repress the induction of Fas-ligand and interleukin 2. These studies demonstrate that RORs play critical roles in the regulation of a variety of physiological processes. Further characterization of the mechanisms of action of RORs will not only lead to the identification of ROR target genes and provide additional insight into their normal physiological functions, but will also determine their roles in disease.