Calcium-permeable AMPA receptor plasticity is mediated by subunit-specific interactions with PICK1 and NSF

A recently described form of synaptic plasticity results in dynamic changes in the calcium permeability of synaptic AMPA receptors. Since the AMPA receptor GluR2 subunit confers calcium permeability, this plasticity is thought to occur through the dynamic exchange of synaptic GluR2-lacking and GluR2-containing receptors. To investigate the molecular mechanisms underlying this calcium-permeable AMPA receptor plasticity (CARP), we examined whether AMPA receptor exchange was mediated by subunit-specific protein-protein interactions. We found that two GluR2-interacting proteins, the PDZ domain-containing Protein interacting with C kinase (PICK1) and N-ethylmaleimide sensitive fusion protein (NSF), are specifically required for CARP. Furthermore, PICK1, but not NSF, regulates the formation of extrasynaptic plasma membrane pools of GluR2-containing receptors that may be laterally mobilized into synapses during CARP. These results demonstrate that PICK1 and NSF dynamically regulate the synaptic delivery of GluR2-containing receptors during CARP and thus regulate the calcium permeability of AMPA receptors at excitatory synapses.     

AMPA receptor mediated D-serine release from retinal glial cells

The N-methyl-D-aspartate receptor (NMDAR) co-agonist D-serine is important in a number of different processes in the CNS, ranging from synaptic plasticity to disease states, including schizophrenia. D-serine appears to be the major co-agonist acting on retinal ganglion cell NMDA receptors, but the cell type from which it originates and whether its release can be modulated by activity are unknown. In this study, we utilized a mutant mouse line with elevated d-serine to investigate this question. Direct measurements of extracellular D-serine using capillary electrophoresis demonstrate that D-serine can be released from the intact mouse retina through an α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) dependent mechanism. α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-evoked D-serine release persisted in the presence of a cocktail of neural inhibitors but was abolished after administration of a glial toxin. These findings provide the first evidence that extracellular D-serine levels in the retina can be modulated, and that such modulation is contingent upon glial cell activity.     

ApoB mRNA editing is mediated by a coordinated modulation of multiple apoB mRNA editing enzyme components

Apolipoprotein (apo)B mRNA editing is accomplished by a large multiprotein complex. How these proteins interact to achieve the precise single-nucleotide change induced by this complex remains unclear. We investigated the relationship between altered apoB mRNA editing and changes in editing enzyme components to evaluate their roles in editing regulation. In the mouse fetal small intestine, we found that the dramatic developmental upregulation of apoB mRNA editing from approximately 3% to 88% begins with decreased levels of inhibitory CUG binding protein 2 (CUGBP2) expression followed by increased levels of apoB mRNA editing enzyme (apobec)-1 and apobec-1 complementation factor (ACF) (4- and 8-fold) and then by decreased levels of the inhibitory components glycine-arginine-tyrosine-rich RNA binding protein (GRY-RBP) and heterogeneous nuclear ribonucleoprotein (hnRNP)-C1 (75% and 56%). In contrast, the expression of KH-type splicing regulatory protein (KSRP), apobec-1 binding protein (ABBP)1, ABBP2, and Bcl-2-associated athanogene 4 (BAG4) were unaltered. In the human intestinal cell line Caco-2, the increase of apoB mRNA editing from approximately 1.7% to approximately 23% was associated with 6- and 3.2-fold increases of apobec-1 and CUGBP2, respectively. In the mouse large intestine, the editing was 48% and had a 2.7-fold relatively greater CUGBP2 level. Caco-2 and the large intestine thus have increased instead of decreased CUGBP2 and a lower level of editing, suggesting that inhibitory CUGBP2 may play a critical role in the magnitude of editing regulation. Short interfering RNA-mediated gene-specific knockdown of CUGBP2, GRY-RBP, and hnRNP-C1 resulted in increased editing in Caco-2 cells, consistent with their known inhibitory function. These data suggest that a coordinated expression of editing components determines the magnitude and specificity of apoB mRNA editing.     

RNA editing at arg607 controls AMPA receptor exit from the endoplasmic reticulum

AMPA-receptor (AMPAR) transport to synapses plays a critical role in the modulation of synaptic strength. We show that the functionally critical GluR2 subunit stably resides in an intracellular pool in the endoplasmic reticulum (ER). GluR2 in this pool is extensively complexed with GluR3 but not with GluR1, which is mainly confined to the cell surface. Mutagenesis revealed that elements in the C terminus including the PDZ motif are required for GluR2 forward-transport from the ER. Surprisingly, ER retention of GluR2 is controlled by Arg607 at the Q/R-editing site. Reversion to Gln (R607Q) resulted in rapid release from the pool and elevated surface expression of GluR2 in neurons. Therefore, Arg607 is a central regulator. In addition to channel gating, it also controls ER exit and may thereby ensure the availability of GluR2 for assembly into AMPARs.     

Pin1 and WWP2 regulate GluR2 Q/R site RNA editing by ADAR2 with opposing effects

ADAR2 catalyses the deamination of adenosine to inosine at the GluR2 Q/R site in the pre-mRNA encoding the critical subunit of AMPA receptors. Among ADAR2 substrates this is the vital one as editing at this position is indispensable for normal brain function. However, the regulation of ADAR2 post-translationally remains to be elucidated. We demonstrate that the phosphorylation-dependent prolyl-isomerase Pin1 interacts with ADAR2 and is a positive regulator required for the nuclear localization and stability of ADAR2. Pin1(-/-) mouse embryonic fibroblasts show mislocalization of ADAR2 in the cytoplasm and reduced editing at the GluR2 Q/R and R/G sites. The E3 ubiquitin ligase WWP2 plays a negative role by binding to ADAR2 and catalysing its ubiquitination and subsequent degradation. Therefore, ADAR2 protein levels and catalytic activity are coordinately regulated in a positive manner by Pin1 and negatively by WWP2 and this may have downstream effects on the function of GluR2. Pin1 and WWP2 also regulate the large subunit of RNA Pol II, so these proteins may also coordinately regulate other key cellular proteins.     

Functional assembly of AMPA and kainate receptors is mediated by several discrete protein-protein interactions

Functional heterogeneity of ionotropic glutamate receptors arises not only from the existence of many subunits and isoforms, but also from combinatorial assembly creating channels with distinct properties. This heteromerization is subtype restricted and thought to be determined exclusively by the proximal extracellular N-terminal domain of the subunits. However, using functional assays for heteromer formation, we show that, besides the N-terminal domain, the membrane sector and the C-terminal part of S2 are critical determinants for the formation of functional channels. Our results are compatible with a model where the N-terminal domain only mediates the initial subunit associations into dimers, whereas for the assembly of the full functional tetramer, compatibility of the other regions is required.     

Suppression of kainate-evoked AMPA receptor mediated responses by lanthanum in rat sacral dorsal commissural neurons

The effect of lanthanum (La) on kainate (KA) responses in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) was investigated using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. The responses to KA were mediated by activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in SDCN neurons. La(3+) reversibly inhibited KA (100 microM) activated currents (I(KA)) in a concentration-dependent manner over the range from 30 microM to 30 mM, with IC(50) values of 0.64 +/- 0.06 mM at a holding potential (V(H)) of -40 mV. Our further study indicated that the effects of La(3+) on I(KA) were voltage independent. Moreover, the inhibition was not use dependent and was not overcome by increasing the concentration of agonist. These findings indicate that La(3+) is an efficacious inhibitor of AMPA receptor mediated responses which may contribute to its cytotoxic effect.     

Oxidized phospholipid-induced inflammation is mediated by Toll-like receptor 2

Oxidative tissue damage is a hallmark of many chronic inflammatory diseases. However, the precise mechanisms linking oxidative changes to inflammatory reactions remain unclear. Herein we show that Toll-like receptor 2 (TLR2) translates oxidative tissue damage into inflammatory responses by mediating the effects of oxidized phospholipids. Intraperitoneal injection of oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycerophosphorylcholine (OxPAPC) resulted in upregulation of inflammatory genes in wild-type, but not in TLR2(-/-) mice. In vitro, OxPAPC induced TLR2 (but not TLR4)-dependent inflammatory gene expression and JNK and p38 signaling in macrophages. Induction of TLR2-dependent gene expression required reducible functional groups on sn-2 acyl chains of oxidized phospholipids, as well as serum cofactors. Finally, TLR2(-/-) mice were protected against carbon tetrachloride-induced oxidative tissue damage and inflammation, which was accompanied by accumulation of oxidized phospholipids in livers. Together, our findings demonstrate that TLR2 mediates cellular responses to oxidative tissue damage and they provide new insights into how oxidative stress is linked to acute and chronic inflammation.     

Interaction between GRIP and liprin-alpha/SYD2 is required for AMPA receptor targeting

Interaction with the multi-PDZ protein GRIP is required for the synaptic targeting of AMPA receptors, but the underlying mechanism is unknown. We show that GRIP binds to the liprin-alpha/SYD2 family of proteins that interact with LAR receptor protein tyrosine phosphatases (LAR-RPTPs) and that are implicated in presynaptic development. In neurons, liprin-alpha and LAR-RPTP are enriched at synapses and coimmunoprecipitate with GRIP and AMPA receptors. Dominant-negative constructs that interfere with the GRIP-liprin interaction disrupt the surface expression and dendritic clustering of AMPA receptors in cultured neurons. Thus, by mediating the targeting of liprin/GRIP-associated proteins, liprin-alpha is important for postsynaptic as well as presynaptic maturation.     

Synaptic excitation mediated by AMPA receptors in rat cerebellar slices is selectively enhanced by aniracetam and cyclothiazide

AMPA receptors mediate fast, glutamatergic synaptic transmission in the central nervous system. The time-course of the associated postsynaptic current has been suggested to be determined principally by the kinetics of glutamate binding and receptor desensitization. Aniracetam and cyclothiazide are drugs capable of selectively preventing desensitization of the AMPA receptor. To investigate the relevance of desensitization to fast synaptic transmission in the cerebellum we have tested these compounds against AMPA-induced depolarizations and postsynaptic potentials using the grease-gap recording technique. Aniracetam (1 M-5 mM) and cyclothiazide (1 M-500 M) both enhanced the depolarising action of AMPA (1 M) on Purkinje cells in a concentration-dependent manner. At the highest concentrations tested, the increases over controls were approximately 600% and 800% respectively. Aniracetam also increased, in a concentration-dependent manner, the amplitude of the evoked synaptic potentials of both parallel fibre-Purkinje cell and mossy fibre-granule cell pathways, with the highest concentrations tested enhancing the potentials by approximately 60% and 75% respectively. These data suggest that, at two different synapses in the cerebellum, AMPA receptor desensitization occurs physiologically and is likely to contribute to the shape of fast synaptic currents.Abbreviations CNQX 6-cyano-7-nitroquinoxaline-2,3-dione - NMDA N-methyl-D-aspartate - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole propionate - AP5 D-2-amino-5-phosphopentanoate - EPSP excitatory postsynaptic potential - EPSC excitatory postsynaptic current - DMSO dimethyl sulphoxide - NBQX 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline Special issue dedicated to Dr. Robert Balázs.     

Cellular prion protein null mice display normal AMPA receptor mediated long term depression

Cellular prion protein (PrPC) appears to be involved in numerous physiological processes. We have recently shown a novel modulation of NMDA receptors by PrPC that results in neuroprotection via silencing of NMDA receptors containing NR2D subunits, whereas no effects on AMPA receptor function could be observed (Khosravani et al. 2008, J Cell Biol. 181, 551). Here we show that PrP-null mice show a normal response to long-term depression stimuli requiring AMPA receptor activity, thus further supporting our previous findings of a selective action on NMDA receptors among ionotropic glutamate receptors.     

Regulation of AMPA receptor trafficking by N-cadherin

Dendritic spines are dynamically regulated, both morphologically and functionally, by neuronal activity. Morphological changes are mediated by a variety of synaptic proteins, whereas functional changes can be dramatically modulated by the regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor trafficking. Although these two forms of plasticity appear to be highly coordinated, the connections between them are not fully understood. In this study the synaptic cell adhesion molecule N-cadherin was found to associate with AMPA receptors and regulate AMPA receptor trafficking in neurons. N-cadherin and beta-catenin formed a protein complex with AMPA receptors in vivo, and this association was regulated by extracellular Ca2+. In addition, these proteins co-clustered at synapses in cultured neurons. In heterologous cells and in cultured neurons, overexpression of wild-type N-cadherin specifically increased the surface expression level of the AMPA receptor subunit glutamate receptor 1 (GluR1) and this effect was reversed by a dominant-negative form of N-cadherin. Finally, GluR1 increased the surface expression of N-cadherin in heterologous cells. Importantly, recent studies suggest that N-cadherin and beta-catenin play key roles in structural plasticity in neurons. Therefore, our data suggest that the association of N-cadherin with AMPA receptors may serve as a biochemical link between structural and functional plasticity of synapses.     

Cellular prion protein null mice display normal AMPA receptor mediated long term depression

Cellular prion protein (PrP^C) appears to be involved in numerous physiological processes. We have recently shown a novel modulation of NMDA receptors by PrP^C that results in neuroprotection via silencing of NMDA receptors containing NR2D subunits, whereas no effects on AMPA receptor function could be observed (Khosravani, et al. J Cell Biol 2008; 181:551). Here we show that PrP-null mice show a normal response to long-term depression stimuli requiring AMPA receptor activity, thus further supporting our previous findings of a selective action on NMDA receptors among ionotropic glutamate receptors.Key words: AMPA receptor, NMDA receptor, PrP, long term depression, LTDThe role of prion proteins in the pathophysiology of transmissible spongiform encephalopathies is well documented.¹ Although there is a growing body of literature associating normal cellular prion protein (PrP^C) with functions such as regulation of cell proliferation and survival, cell signalling and immune function,¹ the spectrum of physiological roles attributable to PrP^C remains to be determined. This may in part be due to the fact that mice lacking PrP^C display a relatively mild phenotype, unless subjected to insults such as ischemia or seizures, where increased mortality of the PrP-null mice has been reported.^2–5 Interestingly, the increased neuronal damage in PrP-null mice following excitotoxicity is alleviated upon treatment with the N-Methyl-D-Aspartate (NMDA) receptor (NMDAR) inhibitor MK-801,⁶ suggesting a neuroprotective role of PrP^C via an action on NMDARs, but the mechanism was unclear.We recently described a novel action of PrP^C on NMDAR function.⁷ By examining the neurophysiological properties of hippocampal neurons isolated from PrP-null mice, we were able to show that PrP-null mouse neurons exhibit enhanced and drastically prolonged NMDA evoked currents due to a functional upregulation of NMDARs containing NR2D subunits. Biochemical analyses suggested that NR2D subunits, but not NR2B subunits, co-immunoprecipitated with PrP^C, indicating that PrP^C and NMDARs form physical signaling complexes in neurons. The increased NMDAR function could be phenocopied by RNA interference and were rescued upon overexpression of exogenous PrP^C. The enhanced NMDAR activity resulted in increased neuronal excitability, as well as enhanced glutamatergic-based excitotoxicity in both in vitro and in vivo experiments were neurons were transiently exposed to the selective agonist NMDA. Hence, native PrP^C appears to mediate an important neuroprotective role by virtue of its ability to silence NR2D containing NMDARs. In contrast, minor effects on amplitude and rise and decay-time kinetics were observed for both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and GABA_A (miniature and evoked) currents in synaptically mature hippocampal cultures.AMPA and NMDA receptors have been linked to synaptic plasticity, in particular long term potentiation (LTP) and long term depression (LTD). LTP is believed to mostly reflect a strengthening of the postsynaptic response, caused by a brief period of hyperexcitability that releases significant amounts of glutamate such as during a brief tetanic stimulation. This is thought to result in the opening of AMPA receptors, which depolarize the postsynaptic membrane. This in turn increases the activity of postsynaptic NMDARs, because magnesium ions that normally inhibit NMDAR activity, are dislodged by the postsynaptic depolarization, thus allowing NMDARs to become active. This functional activation of NMDARs results in the influx of calcium ions, which in turn initiate a signaling cascade that results in the membrane insertion of additional AMPA receptors, thus strengthening the synapse. This process is thought to involve NMDA receptor isoforms that predominantly contain the NR2A subunits.⁸A synaptic depotentiation process also can take place that results in the opposite effects of LTP; this process is known as long-term depression or LTD, which also has an NMDAR-dependent component. In contrast to brief tetanic stimulation, as is used in the induction of LTP, establishing LTD requires low frequency stimulation (e.g., 1 Hz for 15 min). Successful and repeatable induction of LTD depends on the parameters used for the conditioning stimulus and more importantly on the age of the animal. In juvenile animals (P12–P21) a low frequency protocol is effective and the mechanism of LTD is believed to depend on the activity of NMDA receptors containing NR2B subunits.⁸ Although a clear distinction of the roles between NR2A and NR2B containing NMDARs, in LTP and LTD respectively, has remained controversial,⁹ it is clear that both NR2A and NR2B are key mediators of alterations in synaptic plasticity. In older animals, the conditioning protocol is reported to require modification to include paired-pulses. This is thought to be due to the involvement of predominantly AMPA (and perhaps kainate) receptors in addition to mGluRs responsible for the synaptic depotentiation (reviewed in ref. ¹⁰).As mentioned earlier, our data obtained from hippocampal cultures indicated only a minor effect of PrP^C knockout on AMPA receptor function. Hence, we hypothesized that AMPA receptor-mediated LTD should be similar in both wild type and PrP-null mice. We therefore examined the effect of PrP on LTD in hippocampal slices obtained from P30–P45 wild type mice and Zurich 1 PrP^C knockout mice. Extracellular potentials were recorded using a patch pipette filled with 150 mM NaCl. First, 10 minutes of baseline evoked (every 30 sec) potentials were recorded to ensure stability of the preparation. LTD was then evoked by application of conditioning paired pulses (Δt = 60 ms) delivered at 1 Hz for 15 min. Thereafter, the field response was sampled every 30 sec for 40 min. As shown in Figure 1, this protocol evoked reliable LTD in wild type mice that partially recovered over the time course of about 20 minutes (Fig. 1). In the PrP^C-null slices, LTD was indistinguishable from that observed in the wild type slices (Fig. 1).Open in a separate windowFigure 1LTD in the CA1 region of hippocampal slices from adult (P30–P45) WT and PrP-null mice. The conditioning pulse (arrow head) was delivered as paired-pulses (Δt = 60 ms) at 1 Hz for 15 min at the Schaffer collaterals. Analysis of field excitatory postsynaptic potential (fEPSP) slope revealed no statistically significant differences (Student''s t-test, p > 0.05) in the extent of induced LTD or the time course of its recovery to baseline. Numbers in parentheses indicate number of slices.The age of the animals, combined with the paired pulse protocol used in our experiments was designed to isolate AMPA receptor mediated LTD.¹⁰ The notion that LTD was unaltered in PrP-null mice is consistent with the observation that AMPA currents were not altered in these mice, and that AMPA receptor-mediated spontaneous synaptic events showed only minute changes compared with wild type animals. These data are also consistent with the notion that PrP-null mice show only mild phenotypes in spatial learning, with no apparent overall short-term memory deficits. Collectively, these data further support a selective action of PrP on NMDA receptors, rather than overall glutamatergic synaptic transmission.     

Regulation of ion channel/neurotransmitter receptor function by RNA editing

RNA editing by select adenosine deamination (A-to-I editing) alters functional determinants in certain ion channels and neurotransmitter receptors in vertebrates and invertebrates. In most cases, edited and unedited versions of a given receptor/channel co-exist to expand the functional space of the receptor population. Recent studies have characterized K(+) channels in squid that are edited at multiple positions, revealed a role for Q/R site editing in AMPA receptor assembly, and demonstrated a link between serotonin levels and the extent of editing of a mammalian serotonin receptor.     

PICK1 is a calcium-sensor for NMDA-induced AMPA receptor trafficking

Regulation of AMPA receptor (AMPAR) trafficking results in changes in receptor number at the postsynaptic membrane, and hence modifications in synaptic strength, which are proposed to underlie learning and memory. NMDA receptor-mediated postsynaptic Ca2+ influx enhances AMPAR internalisation, but the molecular mechanisms that trigger such trafficking are not well understood. We investigated whether AMPAR-associated protein-protein interactions known to regulate receptor surface expression may be directly regulated by Ca2+. PICK1 binds the AMPAR GluR2 subunit and is involved in AMPAR internalisation and LTD. We show that PICK1 is a Ca2+-binding protein, and that PICK1-GluR2 interactions are enhanced by the presence of 15 muM Ca2+. Deletion of an N-terminal acidic domain in PICK1 reduces its ability to bind Ca2+, and renders the GluR2-PICK1 interaction insensitive to Ca2+. Overexpression of this Ca2+-insensitive mutant occludes NMDA-induced AMPAR internalisation in hippocampal neurons. This work reveals a novel postsynaptic Ca2+-binding protein that provides a direct mechanistic link between NMDAR-mediated Ca2+ influx and AMPAR endocytosis.     

Ionotropic glutamate receptor AMPA 1 is associated with ovulation rate

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1(Asn) has a weaker affinity to glutamate than GRIA1(Ser), both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1(Asn) have a slower luteinizing hormone (LH) surge than cows with GRIA1(Ser). In addition, cows with GRIA1(Asn) possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1(Ser). Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy.     

人工核酸内切酶介导的新一代基因组编辑技术进展

对基因组特定位置进行针对性修饰的实验方法称为基因组编辑技术。近年出现的ZFN、TALEN、CRISPR/Cas等一系列人工核酸内切酶逐渐形成了新一代基因组编辑技术,极大地促进了基因组靶向修饰技术的发展,并在基因功能研究、基因治疗等领域开始发挥巨大作用。文中对这一技术的基本原理、发展历程和应用方式进行了简要总结。