Inverse synaptic tagging of inactive synapses via dynamic interaction of Arc/Arg3.1 with CaMKIIß.

The Arc/Arg3.1 gene product is rapidly upregulated by strong synaptic activity and critically contributes to weakening synapses by promoting AMPA-R endocytosis. However, how activity-induced Arc is redistributed and determines the synapses to be weakened remains unclear. Here, we show targeting of Arc to inactive synapses via a high-affinity interaction with CaMKIIß that is not bound to calmodulin. Synaptic Arc accumulates in inactive synapses that previously experienced strong activation and correlates with removal of surface GluA1 from individual synapses. A lack of CaMKIIß either in vitro or in vivo resulted in loss of Arc upregulation in the silenced synapses. The discovery of Arc's role in "inverse" synaptic tagging that is specific for weaker synapses and prevents undesired enhancement of weak synapses in potentiated neurons reconciles essential roles of Arc both for the late phase of long-term plasticity and for reduction of surface AMPA-Rs in stimulated neurons.

Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent ß-amyloid generation.

Assemblies of ß-amyloid (Aß) peptides are pathological mediators of Alzheimer's Disease (AD) and are produced by the sequential cleavages of amyloid precursor protein (APP) by ß-secretase (BACE1) and γ-secretase. The generation of Aß is coupled to neuronal activity, but the molecular basis is unknown. Here, we report that the immediate early gene Arc is required for activity-dependent generation of Aß. Arc is a postsynaptic protein that recruits endophilin2/3 and dynamin to early/recycling endosomes that traffic AMPA receptors to reduce synaptic strength in both hebbian and non-hebbian forms of plasticity. The Arc-endosome also traffics APP and BACE1, and Arc physically associates with presenilin1 (PS1) to regulate γ-secretase trafficking and confer activity dependence. Genetic deletion of Arc reduces Aß load in a transgenic mouse model of AD. In concert with the finding that patients with AD can express anomalously high levels of Arc, we hypothesize that Arc participates in the pathogenesis of AD.

The Angelman Syndrome protein Ube3A regulates synapse development by ubiquitinating arc.

Angelman Syndrome is a debilitating neurological disorder caused by mutation of the E3 ubiquitin ligase Ube3A, a gene whose mutation has also recently been associated with autism spectrum disorders (ASDs). The function of Ube3A during nervous system development and how Ube3A mutations give rise to cognitive impairment in individuals with Angleman Syndrome and ASDs are not clear. We report here that experience-driven neuronal activity induces Ube3A transcription and that Ube3A then regulates excitatory synapse development by controlling the degradation of Arc, a synaptic protein that promotes the internalization of the AMPA subtype of glutamate receptors. We find that disruption of Ube3A function in neurons leads to an increase in Arc expression and a concomitant decrease in the number of AMPA receptors at excitatory synapses. We propose that this deregulation of AMPA receptor expression at synapses may contribute to the cognitive dysfunction that occurs in Angelman Syndrome and possibly other ASDs.

Exposure to complex environments results in more sparse representations of space in the hippocampus.

The neural circuitry mediating sensory and motor representations is adaptively tuned by an animal's interaction with its environment. Similarly, higher order representations such as spatial memories can be modified by exposure to a complex environment (CE), but in this case the changes in brain circuitry that mediate the effect are less well understood. Here, we show that prolonged CE exposure was associated with increased selectivity of CA1 "place cells" to a particular recording arena compared to a social control (SC) group. Furthermore, fewer CA1 and DG neurons in the CE group expressed high levels of Arc protein, a marker of recent activation, following brief exposure to a completely novel environment. The reduced Arc expression was not attributable to overall changes in cell density or number. These data indicate that one effect of CE exposure is to modify high-level spatial representations in the brain by increasing the sparsity of population coding within networks of neurons. Greater sparsity could result in a more efficient and compact coding system that might alter behavioural performance on spatial tasks. The results from a behavioural experiment were consistent with this hypothesis, as CE-treated animals habituated more rapidly to a novel environment despite showing equivalent initial responding.

Arc/Arg3.1 interacts with the endocytic machinery to regulate AMPA receptor trafficking.

Arc/Arg3.1 is an immediate-early gene whose mRNA is rapidly transcribed and targeted to dendrites of neurons as they engage in information processing and storage. Moreover, Arc/Arg3.1 is known to be required for durable forms of synaptic plasticity and learning. Despite these intriguing links to plasticity, Arc/Arg3.1's molecular function remains enigmatic. Here, we demonstrate that Arc/Arg3.1 protein interacts with dynamin and specific isoforms of endophilin to enhance receptor endocytosis. Arc/Arg3.1 selectively modulates trafficking of AMPA-type glutamate receptors (AMPARs) in neurons by accelerating endocytosis and reducing surface expression. The Arc/Arg3.1-endocytosis pathway appears to regulate basal AMPAR levels since Arc/Arg3.1 KO neurons exhibit markedly reduced endocytosis and increased steady-state surface levels. These findings reveal a novel molecular pathway that is regulated by Arc/Arg3.1 and likely contributes to late-phase synaptic plasticity and memory consolidation.

Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors.

Homeostatic plasticity may compensate for Hebbian forms of synaptic plasticity, such as long-term potentiation (LTP) and depression (LTD), by scaling neuronal output without changing the relative strength of individual synapses. This delicate balance between neuronal output and distributed synaptic weight may be necessary for maintaining efficient encoding of information across neuronal networks. Here, we demonstrate that Arc/Arg3.1, an immediate-early gene (IEG) that is rapidly induced by neuronal activity associated with information encoding in the brain, mediates homeostatic synaptic scaling of AMPA type glutamate receptors (AMPARs) via its ability to activate a novel and selective AMPAR endocytic pathway. High levels of Arc/Arg3.1 block the homeostatic increases in AMPAR function induced by chronic neuronal inactivity. Conversely, loss of Arc/Arg3.1 results in increased AMPAR function and abolishes homeostatic scaling of AMPARs. These observations, together with evidence that Arc/Arg3.1 is required for memory consolidation, reveal the importance of Arc/Arg3.1's dynamic expression as it exerts continuous and precise control over synaptic strength and cellular excitability.

Sar1-dependent trafficking of the human calcium receptor to the cell surface.

The molecular mechanisms underlying the exit from the endoplasmic reticulum (ER) for cell surface trafficking of the human calcium receptor (hCaR) remain poorly understood. We investigated the role of the Sar1 small GTP-binding protein in cell surface transport of the hCaR. Disruptions of endogenous Sar1 function with the constitutively active Sar1H79G mutant or depletion using small interfering RNA, attenuates cell surface expression of the hCaR. Mutation of several putative di-acidic ER export motifs in the carboxyl-tail of the receptor revealed no apparent defect in cell surface expression. Truncated mutants lacking most of the carboxyl-terminal sequences or all intracellular domains also showed no impairment in cell surface expression at steady state. A truncated receptor containing only the large amino-terminal extracellular ligand-binding domain (ECD) is secreted into the culture medium and Sar1H79G inhibits this secretion. ECD receptor variants with the cysteines essential for intermolecular disulfide-linked dimerization mutated to serine or four of the asparagine sites for N-glycosylation mutated to alanine also disrupt secretion, indicating proper ECD conformation is critical for forward transport of this receptor.

Spatial exploration induces ARC, a plasticity-related immediate-early gene, only in calcium/calmodulin-dependent protein kinase II-positive principal excitatory and inhibitory neurons of the rat forebrain.

Active behavior, such as exploring a novel environment, induces the expression of the immediate-early gene Arc (activity-regulated cytoskeletal associated protein, or Arg 3.1) in many brain regions, including the hippocampus, neocortex, and striatum. Arc messenger ribonucleic acid and protein are localized in activated dendrites, and Arc protein is required for the maintenance of long-term potentiation and memory consolidation. Although previous evidence suggests that Arc is expressed in neurons, there is no direct demonstration that only neurons can express Arc. Furthermore, there is no characterization of the main neuronal types that express Arc. The data reported here show that behavior- or seizure-induced Arc expression in the hippocampus, primary somatosensory cortex, and dorsal striatum of rats colocalizes only with neuronal (NeuN-positive) and not with glial (GFAP-positive) cells. Furthermore, Arc was found exclusively in non-GABAergic alpha-CaMKII-positive hippocampal and neocortical neurons of rats that had explored a novel environment. Some GAD65/67-positive neurons in these regions were observed to express Arc, but only after a very strong stimulus (electroconvulsive seizure). In the dorsal striatum, spatial exploration induced Arc only in GABAergic and alpha-CaMKII-positive neurons. Combined, these results show that although a very strong stimulus (seizure) can induce Arc in a variety of neurons, behavior induces Arc in the CaMKII-positive principal neurons of the hippocampus, neocortex, and dorsal striatum. These results, coupled with recent in vitro findings of interactions between Arc and CaMKII, are consistent with the hypothesis that Arc and CaMKII act as plasticity partners to promote functional and/or structural synaptic modifications that accompany learning.

Rheb is in a high activation state and inhibits B-Raf kinase in mammalian cells.

Rheb (Ras homolog enriched in brain) is a member of the Ras family of proteins, and is in the immediate Ras/Rap/Ral subfamily. We found in three different mammalian cell lines that Rheb was highly activated, to levels much higher than for Ras or Rap 1, and that Rheb's activation state was unaffected by changes in growth conditions. Rheb's high activation was not secondary to unique glycine to arginine, or glycine to serine substitutions at positions 14 and 15, corresponding to Ras residues 12 and 13, since Rheb R14G and R14G, S15G mutants had similarly high activation levels as wild type Rheb. These data are consistent with earlier work which showed that purified Rheb has similar GTPase activity as Ras, and suggest a relative intracellular deficiency of Rheb GTPase activating proteins (GAPs) compared to Rheb activators. Further evidence for relatively low intracellular GAP activity was that increased Rheb expression led to a marked increase in Rheb activation. Rheb, like Ras and Rap1, bound B-Raf kinase, but in contrast to Ras and Rap 1, Rheb inhibited B-Raf kinase activity and prevented B-Raf-dependent activation of the transcription factor Elk-1. Thus, Rheb appears to be a unique member of the Ras/Rap/Ral subfamily, and in mammalian systems may serve to regulate B-Raf kinase activity.

An autoinhibitory helix in the C-terminal region of phospholipase C-ß mediates Gαq activation.

The enzyme phospholipase C-ß (PLCß) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the Gq family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLCß (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCß3 considerably increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCß.

Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD.

Group I metabotropic glutamate receptors (mGluR) induce long-term depression (LTD) that requires protein synthesis. Here, we demonstrate that Arc/Arg3.1 is translationally induced within 5 min of mGluR activation, and this response is essential for mGluR-dependent LTD. The increase in Arc/Arg3.1 translation requires eEF2K, a Ca(2+)/calmodulin-dependent kinase that binds mGluR and dissociates upon mGluR activation, whereupon it phosphorylates eEF2. Phospho-eEF2 acts to slow the elongation step of translation and inhibits general protein synthesis but simultaneously increases Arc/Arg3.1 translation. Genetic deletion of eEF2K results in a selective deficit of rapid mGluR-dependent Arc/Arg3.1 translation and mGluR-LTD. This rapid translational mechanism is disrupted in the fragile X disease mouse (Fmr1 KO) in which mGluR-LTD does not require de novo protein synthesis but does require Arc/Arg3.1. We propose a model in which eEF2K-eEF2 and FMRP coordinately control the dynamic translation of Arc/Arg3.1 mRNA in dendrites that is critical for synapse-specific LTD.

Basiliximab plus low-dose cyclosporin vs. OKT3 for induction immunosuppression following renal transplantation.

BACKGROUND: Current immunosuppressive therapies are very effective in preventing acute rejection (AR) and graft loss following renal transplantation. Newer agents now make it possible to develop equally efficacious but better tolerated and less toxic strategies. This is especially relevant for our ageing recipients. We now compare the efficacy of basiliximab combined with early low-dose cyclosporin therapy to standard OKT3 induction therapy. METHODS: In this single-centre study, 100 consecutive recipients of cadaveric kidney transplants from November 1998 to August 2000 were treated with basiliximab combined with early low-dose cyclosporin, reduced steroids and mycophenolate mofetil (MMF). Clinical outcomes at 100 d and 1 yr were compared with a group of 26 patients transplanted from March 1995 to November 1998 who received OKT3, delayed full-dose cyclosporin, high-dose steroids and MMF. Amongst basiliximab treated patients, we compared clinical outcomes in those older and younger than 60 yr. RESULTS: Both groups were similar except for a shorter cold ischaemic time in the basiliximab group. Length of stay, number of readmissions, total hospitalization days and cytomegalovirus infections were lower in the basiliximab group. Despite a 40% reduction in steroids, basiliximab-treated patients had fewer biopsy-proven episodes of AR (basiliximab 14% vs. OKT3 35%) and required less antilymphocyte antibody therapy. Clinical outcomes including patient and graft survival were no different between groups. Long-term graft survival for patients over 60 yr was limited primarily by mortality. CONCLUSIONS: Compared with OKT3 induction therapy, the combination of early low-dose cyclosporin and basiliximab is steroid sparing, effective, well tolerated and relatively safe.