A dual role for the RhoGEF Ephexin5 in regulation of dendritic spine outgrowth.

The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines.

Sensory experience regulates cortical inhibition by inducing IGF1 in VIP neurons.

Inhibitory neurons regulate the adaptation of neural circuits to sensory experience, but the molecular mechanisms by which experience controls the connectivity between different types of inhibitory neuron to regulate cortical plasticity are largely unknown. Here we show that exposure of dark-housed mice to light induces a gene program in cortical vasoactive intestinal peptide (VIP)-expressing neurons that is markedly distinct from that induced in excitatory neurons and other subtypes of inhibitory neuron. We identify Igf1 as one of several activity-regulated genes that are specific to VIP neurons, and demonstrate that IGF1 functions cell-autonomously in VIP neurons to increase inhibitory synaptic input onto these neurons. Our findings further suggest that in cortical VIP neurons, experience-dependent gene transcription regulates visual acuity by activating the expression of IGF1, thus promoting the inhibition of disinhibitory neurons and affecting inhibition onto cortical pyramidal neurons.

14-3-3 inhibits Bad-induced cell death through interaction with serine-136.

14-3-3 proteins are a family of multifunctional phosphoserine binding molecules that can serve as effectors of survival signaling. Understanding the molecular basis for the prosurvival effect of 14-3-3 may lead to the development of agents useful in the treatment of disorders involving dysregulated apoptosis. One target of 14-3-3 is the proapoptotic Bcl-2 family member Bad. Serine phosphorylation of Bad is associated with 14-3-3 binding and inhibition of Bad-induced cell death, but the relative contributions of the three known phosphorylation sites to 14-3-3 binding have not been established. Here we demonstrate that S136 of Bad is vital for 14-3-3 interaction, but S112 seems to be dispensable. 14-3-3/Bad interaction was strictly dependent on the presence of phosphorylated S136 in vitro, in yeast, and in mammalian cells. However, mutation of S112 did not affect 14-3-3 binding. The death caused by wild-type and S112A Bad, but not that caused by S136A Bad, could be almost completely abrogated by 14-3-3. These data support a critical role for 14-3-3 in regulating Bad proapoptotic activity. The effect of 14-3-3 on Bad is controlled largely by phosphorylation of S136, whereas S112 may represent a 14-3-3-independent pathway.

The E2F1-3 transcription factors are essential for cellular proliferation.

The retinoblastoma tumour suppressor (Rb) pathway is believed to have a critical role in the control of cellular proliferation by regulating E2F activities. E2F1, E2F2 and E2F3 belong to a subclass of E2F factors thought to act as transcriptional activators important for progression through the G1/S transition. Here we show, by taking a conditional gene targeting approach, that the combined loss of these three E2F factors severely affects E2F target expression and completely abolishes the ability of mouse embryonic fibroblasts to enter S phase, progress through mitosis and proliferate. Loss of E2F function results in an elevation of p21Cip1 protein, leading to a decrease in cyclin-dependent kinase activity and Rb phosphorylation. These findings suggest a function for this subclass of E2F transcriptional activators in a positive feedback loop, through down-modulation of p21Cip1, that leads to the inactivation of Rb-dependent repression and S phase entry. By targeting the entire subclass of E2F transcriptional activators we provide direct genetic evidence for their essential role in cell cycle progression, proliferation and development.

Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway.

Increases in the intracellular concentration of calcium ([Ca2+]i) activate various signaling pathways that lead to the expression of genes that are essential for dendritic development, neuronal survival, and synaptic plasticity. The mode of Ca2+ entry into a neuron plays a key role in determining which signaling pathways are activated and thus specifies the cellular response to Ca2+. Ca2+ influx through L-type voltage-activated channels (LTCs) is particularly effective at activating transcription factors such as CREB and MEF-2. We developed a functional knock-in technique to investigate the features of LTCs that specifically couple them to the signaling pathways that regulate gene expression. We found that an isoleucine-glutamine ("IQ") motif in the carboxyl terminus of the LTC that binds Ca2+-calmodulin (CaM) is critical for conveying the Ca2+ signal to the nucleus. Ca2+-CaM binding to the LTC was necessary for activation of the Ras/mitogen-activated protein kinase (MAPK) pathway, which conveys local Ca2+ signals from the mouth of the LTC to the nucleus. CaM functions as a local Ca2+ sensor at the mouth of the LTC that activates the MAPK pathway and leads to the stimulation of genes that are essential for neuronal survival and plasticity.

Calcium regulation of neuronal gene expression.

Plasticity is a remarkable feature of the brain, allowing neuronal structure and function to accommodate to patterns of electrical activity. One component of these long-term changes is the activity-driven induction of new gene expression, which is required for both the long-lasting long-term potentiation of synaptic transmission associated with learning and memory, and the activity dependent survival events that help to shape and wire the brain during development. We have characterized molecular mechanisms by which neuronal membrane depolarization and subsequent calcium influx into the cytoplasm lead to the induction of new gene transcription. We have identified three points within this cascade of events where the specificity of genes induced by different types of stimuli can be regulated. By using the induction of the gene that encodes brain-derived neurotrophic factor (BDNF) as a model, we have found that the ability of a calcium influx to induce transcription of this gene is regulated by the route of calcium entry into the cell, by the pattern of phosphorylation induced on the transcription factor cAMP-response element (CRE) binding protein (CREB), and by the complement of active transcription factors recruited to the BDNF promoter. These results refine and expand the working model of activity-induced gene induction in the brain, and help to explain how different types of neuronal stimuli can activate distinct transcriptional responses.

Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of Fas ligand.

Elevated levels of beta-Amyloid (Abeta) are present in the brains of individuals with either the sporadic or familial form of Alzheimer's disease (AD), and the deposition of Abeta within the senile plaques that are a hallmark of AD is thought to be a primary cause of the cognitive dysfunction that occurs in AD. Recent evidence suggests that Abeta induces neuronal apoptosis in the brain and in primary neuronal cultures, and that this Abeta-induced neuronal death may be responsible in part for the cognitive decline found in AD patients. In this study we have characterized one mechanism by which Abeta induces neuronal death. We found that in cortical neurons exposed to Abeta, activated c-Jun N-terminal kinase (JNK) is required for the phosphorylation and activation of the c-Jun transcription factor, which in turn stimulates the transcription of several key target genes, including the death inducer Fas ligand. The binding of Fas ligand to its receptor Fas then induces a cascade of events that lead to caspase activation and ultimately cell death. By analyzing the effects of mutations in each of the components of the JNK-c-Jun-Fas ligand-Fas pathway, we demonstrate that this pathway plays a critical role in mediating Abeta-induced death of cultured neurons. These findings raise the possibility that the JNK pathway may also contribute to Abeta-dependent death in AD patients.

Myc requires distinct E2F activities to induce S phase and apoptosis.

Previous work has shown that the Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, we now show that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, we propose that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions.

REST acts through multiple deacetylase complexes.

The RE1 binding silencer protein REST represses neuronal-specific gene expression in nonneuronal cell types. In this issue of Neuron, Ballas et al. show that REST inhibits gene expression via the recruitment of multiple histone deacetylase complexes.

Serum- and glucocorticoid-inducible kinase SGK phosphorylates and negatively regulates B-Raf.

Phosphorylation can both positively and negatively regulate activity of the Raf kinases. Akt has been shown to phosphorylate and inhibit C-Raf activity. We have recently reported that Akt negatively regulates B-Raf kinase activation by phosphorylating multiple residues within its amino-terminal regulatory domain. Here we investigated the regulation of B-Raf by serum and glucocorticoid-inducible kinase, SGK, which shares close sequence identity with the catalytic domain of Akt but lacks the pleckstrin homology domain. We observed that SGK inhibits B-Raf activity. A comparison of substrate specificity between SGK and Akt indicates that SGK is a potent negative regulator of B-Raf. In contrast to Akt, SGK negatively regulates B-Raf kinase activity by phosphorylating only a single Akt consensus site, Ser(364). Under similar experimental conditions, SGK displays a measurably stronger inhibitory effect on B-Raf kinase activity than Akt, whereas Akt exhibits a more inhibitory effect on the forkhead transcription factor, FKHR. The selective substrate specificity is correlated with an enhanced association between Akt or SGK and their preferred substrates, FKHR and B-Raf, respectively. These results indicate that B-Raf kinase activity is negatively regulated by Akt and SGK, suggesting that the cross-talk between the B-Raf and other signaling pathways can be mediated by both Akt and SGK.

Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway.

The PI3K-Akt signaling pathway plays a critical role in mediating survival signals in a wide range of neuronal cell types. The recent identification of a number of substrates for the serine/threonine kinase Akt suggests that it blocks cell death by both impinging on the cytoplasmic cell death machinery and by regulating the expression of genes involved in cell death and survival. In addition, recent experiments suggest that Akt may also use metabolic pathways to regulate cell survival.

EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin.

Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. We report the cloning and characterization of ephexin, a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility.

Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms.

The mechanisms by which neural stem cells give rise to neurons, astrocytes, or oligodendrocytes are beginning to be elucidated. However, it is not known how the specification of one cell lineage results in the suppression of alternative fates. We find that in addition to inducing neurogenesis, the bHLH transcription factor neurogenin (Ngn1) inhibits the differentiation of neural stem cells into astrocytes. While Ngn1 promotes neurogenesis by functioning as a transcriptional activator, Ngn1 inhibits astrocyte differentiation by sequestering the CBP-Smad1 transcription complex away from astrocyte differentiation genes, and by inhibiting the activation of STAT transcription factors that are necessary for gliogenesis. Thus, two distinct mechanisms are involved in the activation and suppression of gene expression during cell-fate specification by neurogenin.

Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a).

Serum- and glucocorticoid-inducible kinases (SGKs) form a novel family of serine/threonine kinases that are activated in response to a variety of extracellular stimuli. SGKs are related to Akt (also called PKB), a serine/threonine kinase that plays a crucial role in promoting cell survival. Like Akt, SGKs are activated by the phosphoinositide-3 kinase (PI3K) and translocate to the nucleus upon growth factor stimulation. However the physiological substrates and cellular functions of SGKs remained to be identified. We hypothesized that SGKs regulate cellular functions in concert with Akt by phosphorylating common targets within the nucleus. The best-characterized nuclear substrates of Akt are transcription factors of the Forkhead family. Akt phosphorylates Forkhead transcription factors such as FKHRL1, leading to FKHRL1's exit from the nucleus and the consequent shutoff of FKHRL1 target genes. We show here that SGK1, like Akt, promotes cell survival and that it does so in part by phosphorylating and inactivating FKHRL1. However, SGK and Akt display differences with respect to the efficacy with which they phosphorylate the three regulatory sites on FKHRL1. While both kinases can phosphorylate Thr-32, SGK displays a marked preference for Ser-315 whereas Akt favors Ser-253. These findings suggest that SGK and Akt may coordinately regulate the function of FKHRL1 by phosphorylating this transcription factor at distinct sites. The efficient phosphorylation of these three sites on FKHRL1 by SGK and Akt appears to be critical to the ability of growth factors to suppress FKHRL1-dependent transcription, thereby preventing FKHRL1 from inducing cell cycle arrest and apoptosis. These findings indicate that SGK acts in concert with Akt to propagate the effects of PI3K activation within the nucleus and to mediate the biological outputs of PI3K signaling, including cell survival and cell cycle progression.

Mutation of E2F2 in mice causes enhanced T lymphocyte proliferation, leading to the development of autoimmunity.

E2Fs are important regulators of proliferation, differentiation, and apoptosis. Here we characterize the phenotype of mice deficient in E2F2. We show that E2F2 is required for immunologic self-tolerance. E2F2(-/-) mice develop late-onset autoimmune features, characterized by widespread inflammatory infiltrates, glomerular immunocomplex deposition, and anti-nuclear antibodies. E2F2-deficient T lymphocytes exhibit enhanced TCR-stimulated proliferation and a lower activation threshold, leading to the accumulation of a population of autoreactive effector/memory T lymphocytes, which appear to be responsible for causing autoimmunity in E2F2-deficient mice. Finally, we provide support for a model to explain E2F2's unexpected role as a suppressor of T lymphocyte proliferation. Rather than functioning as a transcriptional activator, E2F2 appears to function as a transcriptional repressor of genes required for normal S phase entry, particularly E2F1.

Calcium phosphate transfection of DNA into neurons in primary culture.

Calcium phosphate/DNA coprecipitation is a widely used method for the introduction of foreign DNA into cells. DNA and calcium phosphate are allowed to form a precipitate that is then added to cells in culture. The cells internalize the DNA, leading to the expression of the transfected genes in the cell. Despite the simplicity of this method, it has not been used very often for primary neurons because of its potential to cause neuronal toxicity. However, low toxicity and reasonably high transfection efficiency (0.5% to 5%) can be achieved by optimization of the transfection parameter, combined in some cases with the use of inhibitors of neuronal activity. This unit describes a very easy and inexpensive method for neuronal gene delivery that can be used with standard eukaryotic expression vectors for the gene of interest.

Magnitude of the CREB-dependent transcriptional response is determined by the strength of the interaction between the kinase-inducible domain of CREB and the KIX domain of CREB-binding protein.

The activity of the transcription factor CREB is regulated by extracellular stimuli that result in its phosphorylation at a critical serine residue, Ser133. Phosphorylation of Ser133 is believed to promote CREB-dependent transcription by allowing CREB to interact with the transcriptional coactivator CREB-binding protein (CBP). Previous studies have established that the domain encompassing Ser133 on CREB, known as the kinase-inducible domain (KID), interacts specifically with a short domain in CBP termed the KIX domain and that this interaction depends on the phosphorylation of Ser133. In this study, we adapted a recently described Escherichia coli-based two-hybrid system for the examination of phosphorylation-dependent protein-protein interactions, and we used this system to study the kinase-induced interaction between the KID and the KIX domain. We identified residues of the KID and the KIX domain that are critical for their interaction as well as two pairs of oppositely charged residues that apparently interact at the KID-KIX interface. We then isolated a mutant form of the KIX domain that interacts more tightly with wild-type and mutant forms of the KID than does the wild-type KIX domain. We show that in the context of full-length CBP, the corresponding amino acid substitution resulted in an enhanced ability of CBP to stimulate CREB-dependent transcription in mammalian cells. Conversely, an amino acid substitution in the KIX domain that weakens its interaction with the KID resulted in a decreased ability of full-length CBP to stimulate CREB-dependent transcription. These findings demonstrate that the magnitude of CREB-dependent transcription in mammalian cells depends on the strength of the KID-KIX interaction and suggest that the level of transcription induced by coactivator-dependent transcriptional activators can be specified by the strength of the activator-coactivator interaction.

Telephone nursing: evidence of client and organizational benefits.

Telephone nursing (TN) or telephone triage (TT) has been identified as part of a successful cost-reduction demand management strategy. The author examines TN utilization and related client satisfaction, client education, reduction in drop-in clinic visits, and unnecessary ER and urgent care visits associated with an outpatient pediatric clinic population. This study examined 25% of the total of an average of TN 120 phone calls processed by this Southwestern clinic in an average summer month and achieved an 87.3% response rate to followup study questions. "Telephone nursing was performed by specially trained and experienced RNs using approved, written, clinic-specific protocols." The primary goals of the TN program was the "efficient use of health care resources" and provision of the "appropriate level of care at the appropriate time." Over 80% of the callers surveyed reported that if they hadn't been able to speak to the nurse they would have sought medical care elsewhere.

14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation.

The Bcl-2 homology 3 (BH3) domain of prodeath Bcl-2 family members mediates their interaction with prosurvival Bcl-2 family members and promotes apoptosis. We report that survival factors trigger the phosphorylation of the proapoptotic Bcl-2 family member BAD at a site (Ser-155) within the BAD BH3 domain. When BAD is bound to prosurvival Bcl-2 family members, BAD Ser-155 phosphorylation requires the prior phosphorylation of Ser-136, which recruits 14-3-3 proteins that then function to increase the accessibility of Ser-155 to survival-promoting kinases. Ser-155 phosphorylation disrupts the binding of BAD to prosurvival Bcl-2 proteins and thereby promotes cell survival. These findings define a mechanism by which survival signals inactivate a proapoptotic Bcl-2 family member, and suggest a role for 14-3-3 proteins as cofactors that regulate sequential protein phosphorylation events.