Disinhibition Mediates a Form of Hippocampal Long-Term Potentiation in Area CA1

The hippocampus plays a central role in memory formation in the mammalian brain. Its ability to encode information is thought to depend on the plasticity of synaptic connections between neurons. In the pyramidal neurons constituting the primary hippocampal output to the cortex, located in area CA1, firing of presynaptic CA3 pyramidal neurons produces monosynaptic excitatory postsynaptic potentials (EPSPs) followed rapidly by feedforward (disynaptic) inhibitory postsynaptic potentials (IPSPs). Long-term potentiation (LTP) of the monosynaptic glutamatergic inputs has become the leading model of synaptic plasticity, in part due to its dependence on NMDA receptors (NMDARs), required for spatial and temporal learning in intact animals. Using whole-cell recording in hippocampal slices from adult rats, we find that the efficacy of synaptic transmission from CA3 to CA1 can be enhanced without the induction of classic LTP at the glutamatergic inputs. Taking care not to directly stimulate inhibitory fibers, we show that the induction of GABAergic plasticity at feedforward inhibitory inputs results in the reduced shunting of excitatory currents, producing a long-term increase in the amplitude of Schaffer collateral-mediated postsynaptic potentials. Like classic LTP, disinhibition-mediated LTP requires NMDAR activation, suggesting a role in types of learning and memory attributed primarily to the former and raising the possibility of a previously unrecognized target for therapeutic intervention in disorders linked to memory deficits, as well as a potentially overlooked site of LTP expression in other areas of the brain.     

Effects of Long-Term Hypoxia/Hypoglycemia on Synaptic Transmission between the CA3 and CA1 Zones in Rat Hippocampal Slices

On rat hippocampal slices using a standard patch-clamp technique in the whole-cell configuration, we studied the effects of long-term (40 to 60 min) hypoxia/hypoglycemia (HH) on excitatory postsynaptic currents (EPSC) evoked by stimulation of Schaffer collaterals in the cells of the CA1 zone. In addition to the earlier described effect of an immediate drop in the EPSC amplitude, a significant transient increase in its amplitude 30-50 min after the beginning of HH was observed. A pharmacologically isolated NMDA component of excitatory synaptic events underwent similar changes: 30-50 min after the blockade of NMDA receptor-mediated current, a fast recovery of its amplitude to the control (or even higher) values occurred. A blocker of NMDA/glutamate (Glu) receptors, D-aminophosphonovaleric acid (D-APV), and a competitive nonspecific antagonist of metabotropic Glu receptors, (RS)--methyl-4-carboxyphenylglycine – (RS)-MCPG – did not influence the HH-induced initial suppression of synaptic transmission but completely eliminated its delayed recovery. Our findings allow us to suppose that NMDA receptors, as well as metabotropic Glu receptors, play important roles in the cascade of biochemical reactions resulting in death of hippocampal pyramidal cells in the course of and after long-term ischemia in vivo.     

M1乙酰胆碱受体对AMPA受体GluA1-Ser831 位点的调控作用

目的:从海马神经元谷氨酸离子型受体--AMPA受体亚基GluA1的831位丝氨酸(GluA1Ser831)磷酸化角度,探讨M1乙酰胆碱受体对AMPA受体GluA1亚基的调控作用及作用机制。方法:本研究以成熟的原代海马神经元为实验对象,用不易被降解的卡巴胆碱(Carbachol,CCh)作为胆碱受体激动剂,以免疫印迹法作为蛋白和磷酸化蛋白的主要检测手段,结合不同蛋白抑制剂研究M1受体调控AMPA受体GluA1亚基的关键信号分子及其机制。结果:1与对照组相比,CCh组Ser831的磷酸化水平显著升高。2CCh促进Ser831磷酸化的现象在M1受体选择性拮抗剂哌仑西平(Pirenzepine)+CCh组消失,CCh升高GluA1-Ser831磷酸化水平的作用由M1受体介导。3蛋白激酶C(ProteinkinaseC,PKC)抑制剂白屈菜红碱(Chelerythrinechloride,CHCL)能对抗CCh促进GluA1-Ser831位点磷酸化的作用,而钙/钙调素依赖性蛋白激酶II(Calcium/calmodulin-dependentkinaseII,CaMKII)抑制剂KN62不能对抗CCh的作用。4为检测体内GluA1-Ser831的磷酸化情况,用小鼠海马组织定位注射CCh和CHCL,CCh组小鼠海马组织GluA1-Ser831位点的磷酸化水平升高,CHCL能对抗这种作用,PKC介导了M1受体激活所导致的GluA1-Ser831磷酸化水平的升高。结论:M1受体通过激活PKC促进GluA1-Ser831的磷酸化。     

A Comparison of the Roles of Protein Kinase C in Long-Term Potentiation in Rat Hippocampal Areas CA1 and CA3

1. Using agonists and antagonists with specificity toward various isozymes, we have examined the role of protein kinase C (PKC) in long-term potentiation (LTP) in rat hippocampal areas CA1 and CA3.2. Agonists (indolactum V but not phorbol ester) and antagonists (sphingosine, staurosporine, chelerytherene) acting at all PKC isozymes reduce or block LTP induction at both sites.3. However ingenol, a relatively specific agonist at the δ and ε isozymes, blocks LTP in the MF-CA3 pathway, but not in the SC-CA1 pathway.4. Go6976, a relatively specific antagonist of the α and β isozymes, blocks LTP in the SC-CA1 pathway at both ages tested (30- and 60-day-old animals), but blocks LTP in the MF-CA3 in 60 but not 30-day-old animals.5. Our studies indicate that different PKC isozymes are crucial to LTP induction in these two areas of hippocampus, and that there are development changes in the profile of isozymes.     

SUMOylation Is Required for Glycine-Induced Increases in AMPA Receptor Surface Expression (ChemLTP) in Hippocampal Neurons

Multiple pathways participate in the AMPA receptor trafficking that underlies long-term potentiation (LTP) of synaptic transmission. Here we demonstrate that protein SUMOylation is required for insertion of the GluA1 AMPAR subunit following transient glycine-evoked increase in AMPA receptor surface expression (ChemLTP) in dispersed neuronal cultures. ChemLTP increases co-localisation of SUMO-1 and the SUMO conjugating enzyme Ubc9 and with PSD95 consistent with the recruitment of SUMOylated proteins to dendritic spines. In addition, we show that ChemLTP increases dendritic levels of SUMO-1 and Ubc9 mRNA. Consistent with activity dependent translocation of these mRNAs to sites near synapses, levels of the mRNA binding and dendritic transport protein CPEB are also increased by ChemLTP. Importantly, reducing the extent of substrate protein SUMOylation by overexpressing the deSUMOylating enzyme SENP-1 or inhibiting SUMOylation by expressing dominant negative Ubc9 prevent the ChemLTP-induced increase in both AMPAR surface expression and dendritic SUMO-1 mRNA. Taken together these data demonstrate that SUMOylation of synaptic protein(s) involved in AMPA receptor trafficking is necessary for activity-dependent increases in AMPAR surface expression.     

Genetic Enhancement of Memory and Long-Term Potentiation but Not CA1 Long-Term Depression in NR2B Transgenic Rats

One major theory in learning and memory posits that the NR2B gene is a universal genetic factor that acts as rate-limiting molecule in controlling the optimal NMDA receptor''s coincidence-detection property and subsequent learning and memory function across multiple animal species. If so, can memory function be enhanced via transgenic overexpression of NR2B in another species other than the previously reported mouse species? To examine these crucial issues, we generated transgenic rats in which NR2B is overexpressed in the cortex and hippocampus and investigated the role of NR2B gene in NMDA receptor-mediated synaptic plasticity and memory functions by combining electrophysiological technique with behavioral measurements. We found that overexpression of the NR2B subunit had no effect on CA1-LTD, but rather resulted in enhanced CA1-LTP and improved memory performances in novel object recognition test, spatial water maze, and delayed-to-nonmatch working memory test. Our slices recordings using NR2A- and NR2B-selective antagonists further demonstrate that the larger LTP in transgenic hippocampal slices was due to contribution from the increased NR2B-containing NMDARs. Therefore, our genetic experiments suggest that NR2B at CA1 synapses is not designated as a rate-limiting factor for the induction of long-term synaptic depression, but rather plays a crucial role in initiating the synaptic potentiation. Moreover, our studies provide strong evidence that the NR2B subunit represents a universal rate-limiting molecule for gating NMDA receptor''s optimal coincidence-detection property and for enhancing memory function in adulthood across multiple mammalian species.     

Effects of A1 and A2 Adenosine Receptor Antagonists on the Induction and Reversal of Long-Term Potentiation in Guinea Pig Hippocampal Slices of CA1 Neurons

1. Using simultaneous recordings of the field EPSP and the population spike in the CA1 neurons of guinea pig hippocampal slices, we confirmed that delivery of a high-frequency stimulation (tetanus: 100 pulses at 100 Hz) produced robust long-term potentiation of synaptic efficacy (LTP) in two independent components, a synaptic component that increases field excitatory postsynaptic potentials (EPSPs) and a component that results in a larger population spike amplitude for a given EPSP size (E-S potentiation).2. In the same cells, reversal of LTP (depotentiation; DP) in the field EPSP and in the E-S component is achieved by delivering low-frequency afferent stimulation (LFS:1 Hz, 1000 pulses) 20 min after the tetanus.3. When the tetanus or LFS was applied to CA1 inputs in the presence of an adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (1 M), the field EPSP was enhances in LTP and attenuated in DP, while the E-S relationship was not significantly affected in either LTP or DP.4. When similar experiments were performed using an A₂ receptor antagonist, CP-66713 (10 M), the field EPSP was blocked in LTP but facilitated in DP, while E-S potentiation was enhanced during both LTP and DP.5. The results show that endogenous adenosine, acting via A₁ or A₂ receptors, modulates both the synaptic and the E-S components of the induction and reversal of LTP. Based on the results, we discuss the key issue of the contribution of these receptors to the dynamics of neuronal plasticity modification in hippocampal CA1 neurons.     

Developmental Hypothyroxinemia Caused by Mild Iodine Deficiency Leads to HFS-Induced LTD in Rat Hippocampal CA1 Region: Involvement of AMPA Receptor

Hypothyroidism induced by severe iodine deficiency (ID) during developmental period seriously damages the central nervous system function. In addition to developmental hypothyroidism induced by severe ID, developmental hypothyroxinemia induced by mild ID is potentially damaging for neurodevelopment and learning and memory in children. Wistar rats were treated with iodine-deficient diet or methimazole (MMZ) during pregnancy and lactation to induce developmental hypothyroxinemia or hypothyroidism in the present study. Pups were weaned on postnatal day (PN) 21 and used for electrophysiological recordings on PN80. It is generally accepted that long-term depression (LTD) is induced at low-frequency stimulation (LFS) in hippocampal CA1 region. Surprisingly, we observed developmental hypothyroxinemia as well as developmental hypothyroidism led to high-frequency stimulation (HFS)-induced LTD in hippocampal CA1 region. The abnormal HFS-induced LTD suggests not only developmental hypothyroidism but also developmental hypothyroxinemia impairs learning and memory. To explore the mechanisms responsible for the HFS-induced LTD, the phosphorylation status of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) was investigated. The results showed that developmental hypothyroxinemia as well as developmental hypothyroidism decreased the phosphorylation of AMPAR subunit glutamate receptor 1 (GluR1) at serine 831 and serine 845 in hippocampal CA1 region. Neither developmental hypothyroxinemia nor developmental hypothyroidism altered the phosphorylation of AMPAR subunit glutamate receptor 2 (GluR2) at serine 880. Increased levels of protein phosphatase-1 (PP1) were also observed in hippocampal CA1 regions of pups subjected to developmental hypothyroxinemia or hypothyroidism. Taken together, our results suggest that the increased levels of PP1 caused by developmental hypothyroxinemia or hypothyroidism may account for the dephosphorylation of GluR1 at serine 831 and serine 845, which may contribute to HFS-induced LTD in hippocampal CA1 region.     

The Ocular Albinism Type 1 Gene Product is an N‐Glycoprotein but Glycosylation is not Required for its Subcellular Distribution

The ocular albinism type 1 (OA1) gene product is a membrane glycoprotein that may play a role in controlling melanosome growth and maturation. A number of mutations in the OA1 gene lead to ocular albinism due at least in part to retention of the aberrant protein in the endoplasmic reticulum. To examine whether N‐glycosylation plays a role in the post‐translational trafficking of the Oa1 protein, we constructed a series of mutant mouse Oa1 cDNAs encoding an Oa1‐green fluorescent protein fusion in which some or all of the potential glycosylation sites were eliminated by site‐directed mutagenesis. Biochemical studies in transfected cells treated with tunicamycin and peptide:N‐glycosidase F suggest that asparagine at amino acid 106 is essential for N‐glycosylation of the protein. Mutation at amino acid 106 that eliminated glycosylation did not affect the endo/lysosomal distribution of the Oa1 protein in either COS cells or cultured murine melanocytes.     

chk-1 is an Essential Gene and is Required for an S-M Checkpoint During Early Embryogenesis

The chk1 gene was first discovered in screens for radiation sensitive mutants in S. pombe [1]. Genetic analysis revealed that chk1 is involved in a DNA damage G2-M checkpoint. Chk1 becomes activated in response to DNA damage and prevents entry into mitosis by inhibiting the cell cycle machinery. This checkpoint decreases the risk of defective DNA being inherited by daughter cells, therefore reducing the risk of genetic instability. In higher eukaryotes, chk1 homologues have similar checkpoint functions. For example, an avian B-lymphoma cell line that is defective for Chk1 fails to arrest in G2-M after DNA damage. Nonetheless, these Chk1 defective cells are viable indicating that Chk1 is not essential for normal somatic cells to divide [2]. In spite of this, mouse and Drosophila homozygous Chk1 mutants die during embryogenesis suggesting that this is an essential gene for embryonic cell cycles [3, 4]. What particular role does Chk1 have in directing embryonic cell divisions? Here we used the model organism, C. elegans, to address the role of chk-1 during development. As expected, disruption of chk-1 by RNAi eliminated the DNA damage checkpoint response in C. elegans. In addition, we revealed that chk-1 was predominantly expressed during embryogenesis and in the postembryonic germline. Indeed, we found that chk-1 had an essential role in embryo and germline development. More specifically, disruption of chk-1 expression resulted in embryo lethality, which was attributed to a defect in an intrinsic S-M hence causing premature entry into M-phase.     

The Sphingolipid Receptor S1PR2 Is a Receptor for Nogo-A Repressing Synaptic Plasticity

Nogo-A is a membrane protein of the central nervous system (CNS) restricting neurite growth and synaptic plasticity via two extracellular domains: Nogo-66 and Nogo-A-Δ20. Receptors transducing Nogo-A-Δ20 signaling remained elusive so far. Here we identify the G protein-coupled receptor (GPCR) sphingosine 1-phosphate receptor 2 (S1PR2) as a Nogo-A-Δ20-specific receptor. Nogo-A-Δ20 binds S1PR2 on sites distinct from the pocket of the sphingolipid sphingosine 1-phosphate (S1P) and signals via the G protein G₁₃, the Rho GEF LARG, and RhoA. Deleting or blocking S1PR2 counteracts Nogo-A-Δ20- and myelin-mediated inhibition of neurite outgrowth and cell spreading. Blockade of S1PR2 strongly enhances long-term potentiation (LTP) in the hippocampus of wild-type but not Nogo-A^−/− mice, indicating a repressor function of the Nogo-A/S1PR2 axis in synaptic plasticity. A similar increase in LTP was also observed in the motor cortex after S1PR2 blockade. We propose a novel signaling model in which a GPCR functions as a receptor for two structurally unrelated ligands, a membrane protein and a sphingolipid. Elucidating Nogo-A/S1PR2 signaling platforms will provide new insights into regulation of synaptic plasticity.     

Cyclin A1/Cdk2 is Sufficient but not Required for the Induction of Apoptosis in Early Xenopus laevis Embryos

The role of cyclin-dependent kinases in cell proliferation is well characterized, whereas their somewhat paradoxical role in catalyzing apoptosis is less understood. One Cdk complex implicated in both cell proliferation and cell death is cyclin A/Cdk2. During early embryonic development of Xenopus laevis, distinct isoforms of cyclin A are expressed at different times. From fertilization through gastrulation, cyclin A1 is the predominant isoform. Cyclin A1 dimerizes with Cdk2 but not Cdk1. In contrast, cyclin A2 is expressed at a low level until gastrulation, when it becomes the major A-type cyclin and associates with both Cdk1 and Cdk2. When Xenopus embryos are treated with ionizing radiation (IR) prior to the midblastula transition (MBT), cyclin A1 protein persists beyond the MBT and forms an active complex with Cdk2. During this window of cyclin A1/Cdk2 activity, the embryo undergoes apoptosis. To test the hypothesis that cyclin A1-associated activity is a mediator of apoptosis, cyclin A1 protein level and associated kinase activity were measured in embryos treated with aphidicolin to induce apoptosis. Both cyclin A1 content and associated kinase activity were sustained after the MBT as embryos underwent apoptosis. To determine whether cyclin A1/Cdk2 was sufficient to induce apoptosis, recombinant cyclin A1/Cdk2 complex was injected into single-celled embryos, which induced apoptosis after the MBT. However, morpholinos targeting translation of cyclins A1 and A2 did not block apoptosis in embryos treated with X-rays or aphidicolin. These data indicate that cyclin A1/Cdk2 is sufficient, but not required for apoptosis during early development.     

The b1 Subunit but not the b2 Subunit Colocalizes with the Human Heart Na+ Channel (hH1) already within the Endoplasmic Reticulum

Voltage-dependent Na+ channels are heteromultimers consisting of a pore-forming a subunit and accessory b subunits. In order to provide more insight into the trafficking and assembly of the cardiac Na+ channel complex, we investigated the subcellular localization of the Na+ channel beta1 and beta2 subunits, both in the absence and presence of the human heart Na+ channel (hH1). We fused spectrally distinct variants of the green fluorescent protein (GFP) to hH1 and to the beta1 and beta2 subunit, and expressed the optically labeled b subunits separately or in combination with hH1 in HEK293 cells. In contrast to the predominant localization of hH1 channels within the endoplasmic reticulum (ER), both beta subunits were clearly targeted to the plasma membrane when expressing their cDNAs alone. Upon coexpression of the a subunit, the beta1 subunit was efficiently retained within the ER and found to be colocalized with hH1. In contrast to this, hH1 and the beta2 subunit were not colocalized, i.e., they were detected mainly within the ER and the plasma membrane, respectively. These results indicate that hH1 and the b2 subunit are transported separately to the plasma membrane whereas the hH1/beta1 complex occurs already within the ER, which possibly facilitates trafficking of the channel complex to the plasma membrane.     

The Cdc42-associated kinase ACK1 is not autoinhibited but requires Src for activation

The non-RTK (receptor tyrosine kinase) ACK1 [activated Cdc42 (cell division cycle 42)-associated kinase 1] binds a number of RTKs and is associated with their endocytosis and turnover. Its mode of activation is not well established, but models have suggested that this is an autoinhibited kinase. Point mutations in its SH3 (Src homology 3)- or EGF (epidermal growth factor)-binding domains have been reported to activate ACK1, but we find neither of the corresponding W424K or F820A mutations do so. Indeed, deletion of the various ACK1 domains C-terminal to the catalytic domain are not associated with increased activity. A previous report identified only one major tyrosine phosphorylated protein of 60 kDa co-purified with ACK1. In a screen for new SH3 partners for ACK1 we found multiple Src family kinases; of these c-Src itself binds best. The SH2 and SH3 domains of Src interact with ACK1 Tyr518 and residues 623-652 respectively. Src targets the ACK1 activation loop Tyr284, a poor autophosphorylation site. We propose that ACK1 fails to undergo significant autophosphorylation on Tyr284 in vivo because it is basophilic (whereas Src is acidophilic). Subsequent ACK1 activation downstream of receptors such as EGFR (EGF receptor) (and Src) promotes turnover of ACK1 in vivo, which is blocked by Src inhibitors, and is compromised in the Src-deficient SYF cell line. The results of the present study can explain why ACK1 is responsive to so many external stimuli including RTKs and integrin ligation, since Src kinases are commonly recruited by multiple receptor systems.     

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.Plant vacuoles are vital organelles for maintaining cell volume and cell turgor, regulating ion homeostasis and pH, disposing toxic materials, and storing and degrading unwanted proteins (Marty, 1999). To perform these diverse functions, vacuoles require an array of different and complex proteins. These proteins are synthesized at the endoplasmic reticulum (ER) and are transported to the vacuole through the vacuolar trafficking pathway. Perturbation of the vacuolar trafficking machinery affects many cellular processes, including tropisms, responses to pathogens, cytokinesis, hormone transport, and signal transduction (Surpin and Raikhel, 2004). The vacuolar trafficking system is comprised of several compartments: the ER, the Golgi apparatus, the trans-Golgi network (TGN), the prevacuolar compartment (PVC), and the vacuole. Vacuolar proteins synthesized at the ER are transported to the cis-Golgi via coat protein complex II (COPII) vesicles and are then transported to the TGN through the Golgi apparatus. In the TGN, proteins are sorted for delivery to their respective locations according to their targeting signal. Vacuolar proteins carrying a vacuolar sorting signal are thought to be recognized by vacuolar sorting receptors (VSRs), which are mainly located in the PVC, although sorting of vacuolar proteins may also occur at the ER and VSRs can be recycled from the TGN to the ER (Castelli and Vitale, 2005; Niemes et al., 2010). Multiple studies suggest that plant VSRs serve as sorting receptors both for lytic vacuole proteins (daSilva et al., 2005; Foresti et al., 2006; Kim et al., 2010) and for storage vacuole proteins (Shimada et al., 2003; Fuji et al., 2007; Zouhar et al., 2010).Osmotic stress is commonly associated with many environmental stresses, including drought, cold, and high soil salinity, that have a severe impact on the productivity of agricultural plants worldwide. Therefore, understanding how plants perceive and respond to osmotic stress is critical for improving plant resistance to abiotic stresses (Zhu, 2002; Fujita et al., 2013). It has long been recognized that osmotic stress can activate several signaling pathways that lead to changes in gene expression and metabolism. One important regulator of these signaling pathways is the phytohormone abscisic acid (ABA), which accumulates in response to osmotic stress. ABA regulates many critical processes, such as seed dormancy, stomatal movement, and adaptation to environmental stress (Finkelstein and Gibson, 2002; Xiong and Zhu, 2003; Cutler et al., 2010). De novo synthesis of ABA is of primary importance for increasing ABA levels in response to abiotic stress. ABA is synthesized through the cleavage of a C40 carotenoid originating from the 2-C-methyl-d-erythritol-4-phosphate pathway, followed by a conversion from zeaxanthin to violaxanthin catalyzed by the zeaxanthin epoxidase ABA1 and then to neoxanthin catalyzed by the neoxanthin synthase ABA4. Subsequently, a 9-cis-epoxycarotenoid dioxygenase (NCED) cleaves the violaxanthin and neoxanthin to xanthoxin. Xanthoxin, in turn, is oxidized by a short-chain alcohol dehydrogenase (ABA2) to abscisic aldehyde, which is converted to ABA by abscisic acid aldehyde oxidase3 (AAO3) using a molybdenum cofactor activated by the molybdenum cofactor sulfurase (ABA3; Nambara and Marion-Poll, 2005). In this pathway, it is generally thought that the cleavage step catalyzed by NCED is the rate-limiting step (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). In Arabidopsis (Arabidopsis thaliana), five members of the NCED family (NCED2, NCED3, NCED5, NCED6, and NCED9) have been characterized (Tan et al., 2003). Of those, NCED3 has been suggested to play a crucial role in ABA biosynthesis, and its expression is induced by dehydration and osmotic stress (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). Thus, understanding how the NCED3 gene is activated in response to osmotic stress is important for the elucidation of the mechanisms that govern plant acclimation to abiotic stress.We have used the firefly luciferase reporter gene driven by the stress-responsive NCED3 promoter to enable the genetic dissection of plant responses to osmotic stress (Wang et al., 2011). Here, we report the characterization of a unique regulator of ABA biosynthesis, 9-cis Epoxycarotenoid Dioxygenase Defective2 (CED2). The ced2 mutants are impaired in osmotic stress tolerance and are defective in the expression of genes required for ABA synthesis and consequently osmotic stress-induced ABA accumulation. The CED2 gene encodes VSR1, previously known to be involved in vacuolar trafficking but not known to be critical for osmotic stress induction of ABA biosynthesis and osmotic stress tolerance. Our study further suggests that intracellular pH changes might act as an early stress response signal triggering osmotic stress-activated ABA biosynthesis.     

The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons

Iron efflux in mammalian cells is mediated by the ferrous iron exporter ferroportin (Fpn); Fpn plasma membrane localization and function are supported by a multicopper ferroxidase and/or the soluble amyloid precursor protein (sAPP). Fpn and APP are ubiquitously expressed in all cell types in the central nervous system including neurons. In contrast, neuronal ferroxidase(s) expression has not been well characterized. Using primary cultures of hippocampal neurons, we examined the molecular mechanism of neuronal Fe efflux in detail. Developmental increases of Fpn, APP, and the ferroxidase hephaestin (Hp) were observed in hippocampal neurons. Iron efflux in these neurons depended on the level of Fpn localized at the cell surface; as noted, Fpn stability is supported by ferroxidase activity, an enzymatic activity that is required for Fe efflux. Iron accumulation increases and iron efflux decreases in Hp knockout neurons. In contrast, suppression of endogenous APP by RNAi knockdown does not affect surface Fpn stability or Fe efflux. These data support the model that the neuronal ferroxidase Hp plays a unique role in support of Fpn-mediated Fe efflux in primary hippocampal neurons. Our data also demonstrate that Hp ferroxidase activity relies on copper bioavailability, which suggests neuronal iron homeostasis will be modulated by cellular copper status.