Nancy Y. Ip

ERKs: A family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF

We recently described the purification and cloning of extracellular signal-regulated kinase 1 (ERK1), which appears to play a pivotal role in converting tyrosine phosphorylation into the serine/threonine phosphorylations that regulate downstream events. We now describe cloning and characterization of two ERK1-related kinases, ERK2 and ERK3, and provide evidence suggesting that there are additional ERK family members. At least two of the ERKs are activated in response to growth factors; their activations correlate with tyrosine phophorylation, but also depend on additional modifications. Transcripts corresponding to the three cloned ERKs are distinctly regulated both in vivo and in a differentiating cell line. Thus, this family of kinases may serve as intermediates that depend on tyrosine phosphorylation to activate serine/threonine phosphorylation cascades. Individual family members may mediate responses in different developmental stages, in different cell types, or following exposure to different extracellular signals.

CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130

Ciliary neurotrophic factor (CNTF) has a variety of actions within the nervous system. While some of the actions of leukemia inhibitory factor (LIF) on neurons resemble those of CNTF, LIF also has broad actions outside of the nervous system that in many cases mimic those of interleukin-6 (IL-6). Comparison of the tyrosine phosphorylations and gene activations induced by CNTF and LIF in neuron cell lines reveals that they are indistinguishable and also very similar to signaling events that characterize LIF and IL-6 responses in hematopoietic cells. We provide a basis for the overlapping actions of these three factors by demonstrating that the shared CNTF and LIF signaling pathways involve the IL-6 signal transducing receptor component gp130. Thus, the receptor system for CNTF is surprisingly unlike those used by the nerve growth factor family of neurotrophic factors, but is instead related to those used by a subclass of hematopoietic cytokines.

Human and rat brain-derived neurotrophic factor and neurotrophin-3: gene structures, distributions, and chromosomal localizations.

The development and maintenance of the vertebrate nervous system depends upon neuronal survival proteins known as neurotrophic factors. Nerve growth factor (NGF) remains the best characterized neurotrophic molecule. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are two recently cloned neurotrophic factors that are homologous to NGF. Here we describe the molecular cloning of the human and rat genes encoding BDNF, as well as the isolation of the human NT-3 gene. On the basis of comparison of our genomic and cDNA clones with those of previously isolated BDNF and NT-3 genes and cDNAs, we make inferences about the structures of processed transcripts derived from the neurotrophin genes and the protein precursors they encode. We demonstrate that the mature form of BDNF is identical in all mammals examined, and that the same is true of the mature form of NT-3. Furthermore, the respective tissue-distributions and neuronal specificities of NT-3 and BDNF are also conserved among mammals. Finally, we localize the gene encoding human BDNF (gene symbol designated BDNF) to chromosome 11, band p13, and the gene encoding human NT-3 (gene symbol designated NTF3) to chromosome 12, band p13.

The α component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development

We recently proposed that ciliary neurotrophic factor (CNTF) shares two receptor components with a generally acting cytokine, leukemia inhibitory factor (LIF), but that CNTF also requires a third receptor component (CNTFR alpha) that is mostly restricted to the nervous system in its expression. Here we demonstrate that a transfected CNTFR alpha gene is sufficient to confer CNTF responsiveness upon hemopoietic cells normally responsive only to LIF, providing evidence that CNTFR alpha is a required receptor component that uniquely characterizes CNTF-responding cells. Consistent with this notion, CNTFR alpha expression could be localized to neurons within all known peripheral targets of CNTF. CNTFR alpha was also widely expressed within neurons of the CNS, suggesting that CNTF has broader CNS actions than previously appreciated. However, in vivo localization of CNTFR alpha, as well as of CNTF itself, is consistent with a particularly important role for CNTF in motor function as well as during neuropoiesis.

Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth

Ciliary neurotrophic factor (CNTF) supports motor neuron survival in vitro and in mouse models of motor neuron degeneration and was considered a candidate for the muscle-derived neurotrophic activity that regulates motor neuron survival during development. However, CNTF expression is very low in the embryo, and CNTF gene mutations in mice or human do not result in notable abnormalities of the developing nervous system. We have generated and directly compared mice containing null mutations in the genes encoding CNTF or its receptor (CNTFR alpha). Unlike mice lacking CNTF, mice lacking CNTFR alpha die perinatally and display severe motor neuron deficits. Thus, CNTFR alpha is critical for the developing nervous system, most likely by serving as a receptor for a second, developmentally important, CNTF-like ligand.

Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells in vivo

Adult rat sciatic nerve is known to express high levels of ciliary neurotrophic factor (CNTF) mRNA and protein. Here we examine the cellular localization of CNTF protein and mRNA in peripheral nerve and the regulation of CNTF expression by peripheral axons. In intact nerve, CNTF immunoreactivity is found predominantly in the cytoplasm of myelin-related Schwann cells. After axotomy, CNTF immunoreactivity and mRNA levels fall dramatically and do not recover unless axons regenerate. This behavior is similar to the pattern of myelin gene expression in these nerves. We conclude that the expression of CNTF in Schwann cells depends on axon-Schwann cell interactions.

Retrograde axonal transport of LIF is increased by peripheral nerve injury: Correlation with increased LIF expression in distal nerve

Leukemia inhibitory factor (LIF) is a cytokine that affects the survival and differentiation of certain neuronal populations in vitro. To identify LIF-responsive neurons in the adult rat, we have demonstrated retrograde axonal transport of 125I-LIF to sensory and motor neurons. The accumulation of 125I-LIF by both cell types was significantly increased by prior sciatic nerve crush. Retrograde transport of 125I-LIF was inhibited by excess unlabeled LIF but not by related cytokines, indicating a specific receptor-mediated mechanism. Northern blot analysis revealed LIF expression in peripheral nerve that was increased in distal segments after axotomy. The correlation between LIF expression and increased retrograde transport following injury suggests that LIF plays a role in peripheral nerve regeneration.

Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism.

The development of dendritic spines is thought to be crucial for synaptic plasticity. Dendritic spines are retracted upon Eph receptor A4 (EphA4) activation, but the mechanisms that control this process are not well understood. Here we report an important function of cyclin-dependent kinase 5 (Cdk5) in EphA4-dependent spine retraction in mice. We found that blocking Cdk5 activity inhibits ephrin-A1–triggered spine retraction and reduction of mEPSC frequency at hippocampal synapses. The activation of EphA4 resulted in the recruitment of Cdk5 to EphA4, leading to the tyrosine phosphorylation and activation of Cdk5. EphA4 and Cdk5 then enhanced the activation of ephexin1, a guanine-nucleotide exchange factor that regulates activation of the small Rho GTPase RhoA. The association between EphA4 and ephexin1 was significantly reduced in Cdk5−/− brains and Cdk5-dependent phosphorylation of ephexin1 was required for the ephrin-A1–mediated regulation of spine density. These findings suggest that ephrin-A1 promotes EphA4-dependent spine retraction through the activation of Cdk5 and ephexin1, which in turn modulates actin cytoskeletal dynamics.

Injury‐induced Regulation of Ciliary Neurotrophic Factor mRNA in the Adult Rat Brain

Ciliary neurotrophic factor (CNTF) is a pleiotropic molecule that acts as a neurotrophic factor for a wide range of embryonic neurons as well as a differentiation factor for sympathetic neuroblasts and O2A progenitor cells in culture. CNTF messenger RNA (mRNA) is present at very low levels in the normal adult rat central nervous system (CNS), but is dramatically up‐regulated after an aspiration lesion of dorsal hippocampus and overlying cortex, in the area coincident with glial scar. The increased level of CNTF mRNA in lesioned hippocampus is maximal by 3 days and is sustained for up to 20 days, the longest time point examined. In contrast, mRNA levels for brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) were slightly decreased during the same period. In situ hybridization experiments revealed that cells expressing CNTF mRNA were concentrated at the margin of the wound, and also present within the gelfoam which filled the lesion cavity. This distribution of CNTF‐expressing cells corresponded very closely to that of cells expressing high levels of glial fibrillary acidic protein mRNA at the wound site. Paralleling the observed increase in CNTF mRNA, increased levels of CNTF‐like neurotrophic activity were apparent in soluble extracts of the lesioned tissues. This neurotrophic activity for ciliary ganglion neurons was completely blocked by the addition of neutralizing antiserum against CNTF. Basic fibroblast growth factor, which has been shown by others to increase after a similar lesion paradigm (Frautschy et al., Brain Res., 553, 291–299, 1991), does not contribute appreciably to this trophic activity. We conclude that CNTF is markedly increased as a function of injury to the CNS and that its expression is most likely restricted to reactive astrocytes in the glial scar.

Regulation of NMDA Receptors by Neuregulin Signaling in Prefrontal Cortex

Recent linkage studies have identified a significant association of the neuregulin gene with schizophrenia, but how neuregulin is involved in schizophrenia is primarily unknown. Aberrant NMDA receptor functions have been implicated in the pathophysiology of schizophrenia. Therefore, we hypothesize that neuregulin, which is present in glutamatergic synaptic vesicles, may affect NMDA receptor functions via actions on its ErbB receptors enriched in postsynaptic densities, hence participating in emotional regulation and cognitive processes that are impaired in schizophrenia. To test this, we examined the regulation of NMDA receptor currents by neuregulin signaling pathways in prefrontal cortex (PFC), a prominent area affected in schizophrenia. We found that bath perfusion of neuregulin significantly reduced whole-cell NMDA receptor currents in acutely isolated and cultured PFC pyramidal neurons and decreased NMDA receptor-mediated EPSCs in PFC slices. The effect of neuregulin was mainly blocked by application of the ErbB receptor tyrosine kinase inhibitor, phospholipase C (PLC) inhibitor, IP3 receptor (IP3R) antagonist, or Ca2+ chelators. The neuregulin regulation of NMDA receptor currents was also markedly attenuated in cultured neurons transfected with mutant forms of Ras or a dominant-negative form of MEK1 (mitogen-activated protein kinase kinase 1). Moreover, the neuregulin effect was prevented by agents that stabilize or disrupt actin polymerization but not by agents that interfere with microtubule assembly. Furthermore, neuregulin treatment increased the abundance of internalized NMDA receptors in cultured PFC neurons, which was also sensitive to agents affecting actin cytoskeleton. Together, our study suggests that both PLC/IP3R/Ca2+ and Ras/MEK/ERK (extracellular signal-regulated kinase) signaling pathways are involved in the neuregulin-induced reduction of NMDA receptor currents, which is likely through enhancing NR1 internalization via an actin-dependent mechanism.