Yang Z. Huang

Signal Transduction in Neuronal Migration: Roles of GTPase Activating Proteins and the Small GTPase Cdc42 in the Slit-Robo Pathway

The Slit protein guides neuronal and leukocyte migration through the transmembrane receptor Roundabout (Robo). We report here that the intracellular domain of Robo interacts with a novel family of Rho GTPase activating proteins (GAPs). Two of the Slit-Robo GAPs (srGAPs) are expressed in regions responsive to Slit. Slit increased srGAP1-Robo1 interaction and inactivated Cdc42. A dominant negative srGAP1 blocked Slit inactivation of Cdc42 and Slit repulsion of migratory cells from the anterior subventricular zone (SVZa) of the forebrain. A constitutively active Cdc42 blocked the repulsive effect of Slit. These results have demonstrated important roles for GAPs and Cdc42 in neuronal migration. We propose a signal transduction pathway from the extracellular guidance cue to intracellular actin polymerization.

Regulation of Neuregulin Signaling by PSD-95 Interacting with ErbB4 at CNS Synapses

Neuregulins (NRGs) and their receptors, the ErbB protein tyrosine kinases, are essential for neuronal development, but their functions in the adult CNS are unknown. We report that ErbB4 is enriched in the postsynaptic density (PSD) and associates with PSD-95. Heterologous expression of PSD-95 enhanced NRG activation of ErbB4 and MAP kinase. Conversely, inhibiting expression of PSD-95 in neurons attenuated NRG-mediated activation of MAP kinase. PSD-95 formed a ternary complex with two molecules of ErbB4, suggesting that PSD-95 facilitates ErbB4 dimerization. Finally, NRG suppressed induction of long-term potentiation in the hippocampal CA1 region without affecting basal synaptic transmission. Thus, NRG signaling may be synaptic and regulated by PSD-95. A role of NRG signaling in the adult CNS may be modulation of synaptic plasticity.

Neuregulin-1 Enhances Depolarization-Induced GABA Release

Neuregulin-1 (NRG1), a regulator of neural development, has been shown to regulate neurotransmission at excitatory synapses. Although ErbB4, a key NRG1 receptor, is expressed in glutamic acid decarboxylase (GAD)-positive neurons, little is known about its role in GABAergic transmission. We show that ErbB4 is localized at GABAergic terminals of the prefrontal cortex. Our data indicate a role of NRG1, both endogenous and exogenous, in regulation of GABAergic transmission. This effect was blocked by inhibition or mutation of ErbB4, suggesting the involvement of ErbB4. Together, these results indicate that NRG1 regulates GABAergic transmission via presynaptic ErbB4 receptors, identifying a novel function of NRG1. Because both NRG1 and ErbB4 have emerged as susceptibility genes of schizophrenia, these observations may suggest a mechanism for abnormal GABAergic neurotransmission in this disorder.

Zinc-Mediated Transactivation of TrkB Potentiates the Hippocampal Mossy Fiber-CA3 Pyramid Synapse

The receptor tyrosine kinase, TrkB, is critical to diverse functions of the mammalian nervous system in health and disease. Evidence of TrkB activation during epileptogenesis in vivo despite genetic deletion of its prototypic neurotrophin ligands led us to hypothesize that a non-neurotrophin, the divalent cation zinc, can transactivate TrkB. We found that zinc activates TrkB through increasing Src family kinase activity by an activity-regulated mechanism independent of neurotrophins. One subcellular locale at which zinc activates TrkB is the postsynaptic density of excitatory synapses. Exogenous zinc potentiates the efficacy of the hippocampal mossy fiber (mf)-CA3 pyramid synapse by a TrkB-requiring mechanism. Long-term potentiation of this synapse is impaired by deletion of TrkB, inhibition of TrkB kinase activity, and by CaEDTA, a selective chelator of zinc. The activity-dependent activation of synaptic TrkB in a neurotrophin-independent manner provides a mechanism by which this receptor can regulate synaptic plasticity.

Molecular Signaling Mechanisms Underlying Epileptogenesis

Epilepsy, a disorder of recurrent seizures, is a common and frequently devastating neurological condition. Available therapy is only symptomatic and often ineffective. Understanding epileptogenesis, the process by which a normal brain becomes epileptic, may help identify molecular targets for drugs that could prevent epilepsy. A number of acquired and genetic causes of this disorder have been identified, and various in vivo and in vitro models of epileptogenesis have been established. Here, we review current insights into the molecular signaling mechanisms underlying epileptogenesis, focusing on limbic epileptogenesis. Study of different models reveals that activation of various receptors on the surface of neurons can promote epileptogenesis; these receptors include ionotropic and metabotropic glutamate receptors as well as the TrkB neurotrophin receptor. These receptors are all found in the membrane of a discrete signaling domain within a particular type of cortical neuron—the dendritic spine of principal neurons. Activation of any of these receptors results in an increase Ca2+concentration within the spine. Various Ca2+-regulated enzymes found in spines have been implicated in epileptogenesis; these include the nonreceptor protein tyrosine kinases Src and Fyn and a serine-threonine kinase [Ca2+-calmodulin–dependent protein kinase II (CaMKII)] and phosphatase (calcineurin). Cross-talk between astrocytes and neurons promotes increased dendritic Ca2+ and synchronous firing of neurons, a hallmark of epileptiform activity. The hypothesis is proposed that limbic epilepsy is a maladaptive consequence of homeostatic responses to increases of Ca2+concentration within dendritic spines induced by abnormal neuronal activity. The epilepsies, disorders of recurrent seizures, affect about 1% of the population worldwide. Available therapy is symptomatic in that drugs inhibit seizures but are not disease-modifying; that is, no effective pharmacological prevention or cure has been identified. The term epileptogenesis refers to the process by which a normal brain becomes epileptic. Understanding the cellular mechanisms of epileptogenesis in molecular terms may help identify molecular targets for which small-molecule therapeutics can be developed to prevent epileptogenesis in individuals at high risk. A number of acquired and genetic causes of this disorder have been identified, and various in vivo and in vitro models of epileptogenesis have been established. The objective of this STKE Review, with 5 figures, 2 tables, and 199 references, is to review current insights into the molecular signaling mechanisms underlying epileptogenesis, focusing on limbic epileptogenesis in particular.

Expression of PSD-95/SAP90 is critical for N-methyl-d-aspartate receptor-mediated thermal hyperalgesia in the spinal cord

PSD-95/SAP90, a molecular scaffold protein, attaches the N-methyl-D-aspartate receptor to cellular signaling pathways through PSD-95/DLG/Z0-1 domain interactions at neuronal synapses.(5,9) This suggests that PSD-95/SAP90 might be involved in many physiological and pathophysiological actions triggered via the N-methyl-D-aspartate receptor in the central nervous system. Here, we present evidence that suppression of the expression of PSD-95/SAP90 in the spinal cord significantly attenuated facilitation of the tail-flick reflex triggered through N-methyl-D-aspartate receptor activation but not baseline tail-flick reflex latency. Moreover, PSD-95/SAP90s messenger RNA and protein were enriched in the spinal cord and selectively distributed in the superficial dorsal horn, where PSD-95/SAP90 overlapped with the N-methyl-D-aspartate receptor. In spinal cord neurons, PSD-95/SAP90 interacted with the N-methyl-D-aspartate receptor subunits 2A/2B. It is indicated that activation of the N-methyl-D-aspartate receptor in spinal hyperalgesia results in association of the N-methyl-D-aspartate receptor with PSD-95/SAP90 and that PSD-95/SAP90 is required for noxious thermal hyperalgesia triggered via the N-methyl-D-aspartate receptor at the spinal cord level. The present findings may provide novel insights into the mechanisms for persistent sensitization of the somatosensory system.

Human homologue of Drosophila CNK interacts with Ras effector proteins Raf and Rlf

Connector enhancer of KSR (CNK) is a multidomain protein that participates in Ras signaling in Drosophila eye development. In this report we identify the human homologue of CNK, termed CNK2A, and a truncated alternatively spliced variant, CNK2B. We characterize CNK2 phosphorylation, membrane localization, and interaction with Ras effector molecules. Our results show that MAPK signaling appears to play a role in the phosphorylation of CNK2 in vivo. CNK2 is found in both membrane and cytoplasmic fractions of the cell. In MDCK cells, full‐length CNK2 is localized to the lateral plasma membrane. Consistent with previous reports, we show CNK2 interacts with Raf. CNK2 interaction was mapped to the regulatory and kinase domains of Raf, as well as to the carboxyl‐terminal half of CNK2. CNK2 also interacts with the Ral signaling components, Ral GTPase, and the Ral‐GDS family member Rlf. CNK2 interaction was mapped to the GEF domain of Rlf. The ability of CNK2 to interact with both Ras effector proteins Raf and Rlf suggests that CNK2 may integrate signals between MAPK and Ral pathways through a complex interplay of components.—Lanigan T. M., Liu A., Huang Y. Z., Mei L., Margolis B., Guan K.‐L. Human homologue of Drosophila CNK interacts with Ras effector proteins Raf and Rlf. FASEB J. 17, 2048–2060 (2003)

Mutual regulation of Src family kinases and the neurotrophin receptor TrkB.

The neurotrophin receptor tyrosine kinase TrkB is critical to diverse biological processes. We investigated the interplay of Src family kinases (SFKs) and TrkB to better understand mechanisms of TrkB signaling in physiological and pathological conditions. We compared and contrasted the role of SFKs in TrkB signaling following activation of TrkB by two mechanisms, its transactivation by zinc, and its activation by its prototypic neurotrophin ligand, brain-derived neurotrophic factor (BDNF). Using biochemical, pharmacological, and chemical genetic studies of cultured rodent neurons, we found that zinc promotes preferential phosphorylation of Tyr-705/Tyr-706 of TrkB by a SFK-dependent but TrkB kinase-independent mechanism, a signaling event critical for transactivation of TrkB by zinc. By contrast, SFK activity is not essential for BDNF-mediated activation of TrkB, yet SFK activity is increased as a consequence of TrkB activation by BDNF. Moreover, BDNF-induced phosphorylation of Tyr-705/Tyr-706 of TrkB was inhibited by SFK inhibitors, implicating a regulatory role of SFKs in TrkB activation by BDNF. In sum, SFKs are activated by TrkB and, in turn, SFKs can promote TrkB activation. We propose models depicting the mutual regulation of SFKs and TrkB following activation of TrkB by zinc and BDNF.

Neuregulin-induced expression of the acetylcholine receptor requires endocytosis of ErbB receptors.

Neuregulin-induced expression of the acetylcholine receptor (AChR) contributes to high concentration of the receptor at the neuromuscular junction (NMJ). Neuregulin-1 activates ErbB tyrosine kinases and subsequently intracellular kinases including Erk that is required for induced AChR expression. Recent studies demonstrate that ligand-induced internalization may regulate signaling of various receptor tyrosine kinases. However, the role of induced ErbB endocytosis in regulating AChR expression was unclear. Here we provide evidence that ErbB tyrosine kinases became rapidly internalized in response to neuregulin. The internalization required the kinase activity of ErbB proteins and involved a clathrin-dependent endocytic pathway. Moreover, neuregulin-induced Erk activation and AChR expression were attenuated when ErbB endocytosis was blocked. These results indicate that ErbB proteins undergo endocytosis in response to neuregulin, and this process is required for neuregulin signaling and induced AChR expression.

A Peptide Uncoupling BDNF Receptor TrkB from Phospholipase Cγ1 Prevents Epilepsy Induced by Status Epilepticus.

The BDNF receptor tyrosine kinase, TrkB, underlies nervous system function in both health and disease. Excessive activation of TrkB caused by status epilepticus promotes development of temporal lobe epilepsy (TLE), revealing TrkB as a therapeutic target for prevention of TLE. To circumvent undesirable consequences of global inhibition of TrkB signaling, we implemented a novel strategy aimed at selective inhibition of the TrkB-activated signaling pathway responsible for TLE. Our studies of a mouse model reveal that phospholipase Cγ1 (PLCγ1) is the dominant signaling effector by which excessive activation of TrkB promotes epilepsy. We designed a novel peptide (pY816) that uncouples TrkB from PLCγ1. Treatment with pY816 following status epilepticus inhibited TLE and prevented anxiety-like disorder yet preserved neuroprotective effects of endogenous TrkB signaling. We provide proof-of-concept evidence for a novel strategy targeting receptor tyrosine signaling and identify a therapeutic with promise for prevention of TLE caused by status epilepticus in humans.