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.     

谷氨酸棒杆菌的乙醛酸循环与谷氨酸合成

为阐明谷氨酸棒杆菌的乙醛酸循环与菌体的生长以及谷氨酸合成之间的关系 ,以谷氨酸棒杆菌基因组测序用典型菌株Corynebacteriumglutamicum ATCC 130 32为出发菌株 ,构建了乙醛酸循环途径缺失的谷氨酸棒杆菌突变株Corynebacteriumglutamicum WTΔA。该菌株没有异柠檬酸裂解酶活性 ,不能在以乙酸盐为唯一碳源的基本培养基上生长。与出发菌株ATCC13032相比 ,WTΔA在以葡萄糖为唯一碳源的培养基上生长时不受影响 ,说明谷氨酸棒杆菌并不需要乙醛酸循环途径提供菌体生长所需的能量和生物合成反应所需的中间产物。但是 ,与出发菌株ATCC13032相比 ,WTΔA的谷氨酸合成能力大幅下降。     

Ca2+ Permeable AMPA Receptor Induced Long-Term Potentiation Requires PI3/MAP Kinases but Not Ca/CaM-Dependent Kinase II

Ca²⁺ influx via GluR2-lacking Ca²⁺-permeable AMPA glutamate receptors (CP-AMPARs) can trigger changes in synaptic efficacy in both interneurons and principle neurons, but the underlying mechanisms remain unknown. We took advantage of genetically altered mice with no or reduced GluR2, thus allowing the expression of synaptic CP-AMPARs, to investigate the molecular signaling process during CP-AMPAR-induced synaptic plasticity at CA1 synapses in the hippocampus. Utilizing electrophysiological techniques, we demonstrated that these receptors were capable of inducing numerous forms of long-term potentiation (referred to as CP-AMPAR dependent LTP) through a number of different induction protocols, including high-frequency stimulation (HFS) and theta-burst stimulation (TBS). This included a previously undemonstrated form of protein-synthesis dependent late-LTP (L-LTP) at CA1 synapses that is NMDA-receptor independent. This form of plasticity was completely blocked by the selective CP-AMPAR inhibitor IEM-1460, and found to be dependent on postsynaptic Ca²⁺ ions through calcium chelator (BAPTA) studies. Surprisingly, Ca/CaM-dependent kinase II (CaMKII), the key protein kinase that is indispensable for NMDA-receptor dependent LTP at CA1 synapses appeared to be not required for the induction of CP-AMPAR dependent LTP due to the lack of effect of two separate pharmacological inhibitors (KN-62 and staurosporine) on this form of potentiation. Both KN-62 and staurosporine strongly inhibited NMDA-receptor dependent LTP in control studies. In contrast, inhibitors for PI3-kinase ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin) or the MAPK cascade (PD98059 and U0126) significantly attenuated this CP-AMPAR-dependent LTP. Similarly, postsynaptic infusion of tetanus toxin (TeTx) light chain, an inhibitor of exocytosis, also had a significant inhibitory effect on this form of LTP. These results suggest that distinct synaptic signaling underlies GluR2-lacking CP-AMPAR-dependent LTP, and reinforces the recent notions that CP-AMPARs are important facilitators of synaptic plasticity in the brain.     

synaptojanin1 Is Required for Temporal Fidelity of Synaptic Transmission in Hair Cells

To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses.     

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.     

A Functional GTPase Domain,but not its Transmembrane Domain,is Required for Function of the SRP Receptor β-subunit

The signal recognition particle and its receptor (SR) target nascent secretory proteins to the ER. SR is a heterodimeric ER membrane protein whose subunits, SRα and SRβ, are both members of the GTPase superfamily. Here we characterize a 27-kD protein in Saccharomyces cerevisiae (encoded by SRP102) as a homologue of mammalian SRβ. This notion is supported (a) by Srp102p''s sequence similarity to SRβ; (b) by its disposition as an ER membrane protein; (c) by its interaction with Srp101p, the yeast SRα homologue; and (d) by its role in SRP-dependent protein targeting in vivo. The GTP-binding site in Srp102p is surprisingly insensitive to single amino acid substitutions that inactivate other GTPases. Multiple mutations in the GTP-binding site, however, inactivate Srp102p. Loss of activity parallels a loss of affinity between Srp102p and Srp101p, indicating that the interaction between SR subunits is important for function. Deleting the transmembrane domain of Srp102p, the only known membrane anchor in SR, renders SR soluble in the cytosol, which unexpectedly does not significantly impair SR function. This result suggests that SR functions as a regulatory switch that needs to associate with the ER membrane only transiently through interactions with other components.     

The Novel Caspase-3 Substrate Gap43 is Involved in AMPA Receptor Endocytosis and Long-Term Depression

The cysteine protease caspase-3, best known as an executioner of cell death in apoptosis, also plays a non-apoptotic role in N-methyl-d-aspartate receptor-dependent long-term depression of synaptic transmission (NMDAR-LTD) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor endocytosis in neurons. The mechanism by which caspase-3 regulates LTD and AMPA receptor endocytosis, however, remains unclear. Here, we addressed this question by using an enzymatic N-terminal peptide enrichment method and mass spectrometry to identify caspase-3 substrates in neurons. Of the many candidates revealed by this proteomic study, we have confirmed BASP1, Dbn1, and Gap43 as true caspase-3 substrates. Moreover, in hippocampal neurons, Gap43 mutants deficient in caspase-3 cleavage inhibit AMPA receptor endocytosis and LTD. We further demonstrated that Gap43, a protein well-known for its functions in axons, is also localized at postsynaptic sites. Our study has identified Gap43 as a key caspase-3 substrate involved in LTD and AMPA receptor endocytosis, uncovered a novel postsynaptic function for Gap43 and provided new insights into how long-term synaptic depression is induced.Synaptic plasticity (the ability of synapses to change in strength) plays an important role in brain development and cognitive function, including learning and memory. N-methyl-d-aspartate receptor (NMDAR)¹-dependent long-term depression of synaptic transmission (LTD) is a major form of synaptic plasticity that leads to long-lasting decreases in synaptic strength. In NMDAR-LTD, synaptic depression is mainly mediated by removal of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors from the postsynaptic membrane through endocytosis (1–3).Caspases are cysteine-dependent proteases that cleave after an aspartate residue (4–6). Primary specificity for aspartate at the cleavage site is so rare in mammalian proteases that only granzyme B, a serine protease derived from lymphocytes, is known to also have such a property (7, 8). Caspases are best known for their pro-apoptotic function in programmed cell death, or apoptosis (9). Caspases activated at the end of a caspase cascade are “effector” caspases, and among them caspase-3 is the dominant one (10). In addition to apoptosis, caspases also play non-apoptotic roles such as in cell differentiation, dendritic development, and memory consolidation (11–14). For instance, our earlier studies show that NMDAR-LTD requires moderate and transient caspase-3 activation, which does not induce cell death (15, 16).In LTD, caspase-3 is activated by the mitochondrial pathway (15, 16). With the opening of NMDA receptors, calcineurin and protein phosphatase 1 are activated to dephosphorylate the Bcl-2 family protein BAD. Dephosphorylated BAD then translocates to the mitochondria, activating BAX, which is also a member of the Bcl-2 family protein. The subsequent release of cytochrome-c from mitochondria leads to the sequential activation of caspase-9 and caspase-3. Active caspase-3 induces AMPA receptor endocytosis, and therefore depression of synaptic strength. The mechanism by which caspase-3 promotes AMPA receptor endocytosis, however, remains to be determined.Caspases'' cellular functions are primarily mediated by the proteolysis of caspase substrates, resulting in change of their functions. Identification of specific proteins cleaved by caspases is therefore key to understanding the mechanisms mediating their biological functions. Several proteomic approaches, developed specifically for this purpose, have led to the identification of more than 2000 caspase cleavage sites (17–22). In particular, the recently developed subtiligase-based method for isolating proteolytic products has led to the identification of >1,000 putative caspase substrates in human samples (21, 23, 24). Subtiligase is an engineered peptide ligase that ligates esterified peptides onto the N termini of proteins or peptides through free α-amines (25). Because the majority of eukaryotic proteins are N-terminally acetylated—and therefore blocked from subtiligase labeling (26)—subtiligase can couple synthetic tagged peptides selectively to the free N-terminal α-amines of proteins derived from proteolysis. These peptide-conjugated proteolytic products can then be affinity-purified, digested with trypsin and sequenced by mass spectrometry. Identification of peptides ligated to the tagged peptide by subtiligase allows researchers to determine cleavage sites within the substrates.In this study, the subtiligase-based proteomic method was used to find capase-3 substrates in rat neurons, resulting in the identification of 81 putative aspartate cleavage sites in 56 proteins. Of these, 37 proteins (human and a single rat orthologs) were not previously reported in the CASBAH database (20), and 13 (human orthologs) are not included in the DegraBase data set compiled using the subtiligase methodology in non-neuronal tissue (21). Using complimentary methods, we further confirmed that, both in vivo and in vitro, caspase-3 cleaves three of these candidate substrates: growth associated protein 43 (Gap43), drebrin (Dbn1), and brain acid soluble protein 1 (BASP1). Surprisingly, we also found that AMPA receptor endocytosis and LTD induction both require caspase-3 to cleave Gap43, a protein well known for its presynaptic functions, at the sites identified by our study.     

The Interleukin-17 Receptor B Subunit Is Essential for the Th2 Response to Helicobacter pylori,but Not for Control of Bacterial Burden

Helicobacter pylori infection leads to an inflammatory response in 100% of infected individuals. The inflammatory cells which are recruited to the gastric mucosa during infection produce several pro- and anti-inflammatory cytokines including several cytokines in the interleukin-17 family. The anti-inflammatory cytokine, interleukin 25 (IL-25, also known as IL-17E), signals through a receptor, which is a heterotrimeric receptor comprised of two IL-17 receptor A subunits and an IL-17 receptor B subunit. Previous studies in our laboratory demonstrated that IL-17RA is required to control infection with Helicobacter pylori in the mouse model. Moreover, the absence of IL-17 receptor A leads to a significant B cell infiltrate and a remarkable increase in lymphoid follicle formation in response to infection compared to infection in wild-type mice. We hypothesized that IL-25, which requires both IL-17 receptor A and IL-17 receptor B for signaling, may play a role in control of inflammation in the mouse model of Helicobacter pylori infection. IL-17 receptor B deficient mice, IL-17 receptor A deficient mice and wild-type mice were infected with Helicobacter pylori (strains SS1 and PMSS1). At several time points H. pylori- infected mice were sacrificed to investigate their ability to control infection and inflammation. Moreover, the effects of IL-17 receptor B deficiency on T helper cytokine expression and H. pylori- specific serum antibody responses were measured. IL-17 receptor B−/− mice (unlike IL-17 receptor A−/− mice) exhibited similar or modest changes in gastric colonization, inflammation, and Th1 and Th17 helper cytokine responses to wild-type mice infected with Helicobacter pylori. However, H. pylori-infected IL-17 receptor B−/− mice have reduced expression of IL-4 and lower serum IgG1 and IgG2a levels compared to infected IL-17 receptor A−/− and wild-type mice. These data indicate that signaling through the IL-17 receptor B subunit is not necessary for control of Helicobacter pylori in our model.     

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.