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ABSTRACT: The calcium-calmodulin activated kinase CaMKII mediates many forms of learning and memory. Activity-regulated translocation of CaMKII to synapses is important for its functions in synaptic plasticity. Here, we tested the role of the NMDA receptor subunit GluN2B in recruiting CaMKIIα to synapses with different paradigms: global bath stimulation of NMDA receptors, a chemical long term potentiation (cLTP) protocol that selectively activates synaptic NMDA receptors, or local stimulation of NMDA receptors at a contiguous set of ~10-30 synapses that triggers a propagating synaptic accumulation of CaMKII. Global or cLTP-induced synaptic accumulation of CaMKIIα occurred in wild-type but not sister GluN2B -/- cultured mouse hippocampal neurons. Expression of YFP-GluN2B, but not a similar level of YFP-GluN2A, rescued global and cLTP-induced CaMKIIα translocation. Using chimeric constructs, the pore-forming extracellular and membrane domains of GluN2A combined with the cytoplasmic tail of GluN2B were sufficient to rescue CaMKIIα translocation, whereas the reverse chimera was ineffective. Furthermore, the dual point mutation R1300Q,S1303D in GluN2B that blocks interaction of this high affinity site with CaMKII abolished rescue. Thus, CaMKII binding to GluN2B is required for global and cLTP-induced synaptic accumulation of CaMKIIα. However, surprisingly, locally induced propagating synaptic accumulation of CaMKIIα occurred normally in GluN2B -/- neurons, indistinguishably from wild-type. Thus, synaptic trapping of CaMKII during locally induced propagating translocation occurs by different mechanisms and molecular partners compared with global stimulation and cLTP paradigms. These findings underscore the complex regulatory properties and molecular interactions of CaMKIIα, a key player in synaptic plasticity.
Molecular and Cellular Neuroscience 08/2012; 51(3-4):68-78. · 3.66 Impact Factor
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ABSTRACT: Trafficking of NMDA receptors to the surface of neurons and to synapses is critical for proper brain function and activity-dependent plasticity. Recent evidence suggests that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. Here, we show that glutamate binding to GluN2 is required for trafficking of NMDA receptors to the cell surface. We expressed a panel of GluN2B ligand binding mutants in heterologous cells with GluN1 or in rat cultured neurons and found that surface expression correlates with glutamate efficacy. Such a correlation was found even in the presence of dominant negative dynamin to inhibit endocytosis and surface expression correlated with Golgi localization, indicating differences in forward trafficking. Co-expression of wild type GluN2B did not enhance surface expression of the mutants, suggesting that glutamate must bind to both GluN2 subunits in a tetramer and that surface expression is limited by the least avid of the two glutamate binding sites. Surface trafficking of a constitutively closed cleft GluN2B was indistinguishable from that of wild type, suggesting that glutamate concentrations are typically not limiting for forward trafficking. YFP-GluN2B expressed in hippocampal neurons from GluN2B(-/-) mice rescued synaptic accumulation at similar levels to wild type. Under these conditions, surface synaptic accumulation of YFP-GluN2B mutants also correlated with apparent glutamate affinity. Altogether, these results indicate that glutamate controls forward trafficking of NMDA receptors to the cell surface and to synapses and raise the intriguing idea that NMDA receptors may be functional at intracellular sites.
Journal of Biological Chemistry 06/2012; 287(33):27432-45. · 4.77 Impact Factor
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ABSTRACT: NMDA receptors are calcium-permeable ionotropic receptors that detect coincident glutamate binding and membrane depolarization and are essential for many forms of synaptic plasticity in the mammalian brain. The obligatory GluN1 subunit of NMDA receptors is alternatively spliced at multiple sites, generating forms that vary in N-terminal N1 and C-terminal C1, C2, and C2' cassettes. Based on expression of GluN1 constructs in heterologous cells and in wild type neurons, the prevalent view is that the C-terminal cassettes regulate synaptic accumulation and its modulation by homeostatic activity blockade and by protein kinase C (PKC). Here, we tested the role of GluN1 splicing in regulated synaptic accumulation of NMDA receptors by lentiviral expression of individual GluN1 splice variants in hippocampal neurons cultured from GluN1 (-/-) mice. High efficiency transduction of GluN1 at levels similar to endogenous was achieved. Under control conditions, the C2' cassette mediated enhanced synaptic accumulation relative to the alternate C2 cassette, whereas the presence or absence of N1 or C1 had no effect. Surprisingly all GluN1 splice variants showed >2-fold increased synaptic accumulation with chronic blockade of NMDA receptor activity. Furthermore, in this neuronal rescue system, all GluN1 splice variants were equally rapidly dispersed upon activation of PKC. These results indicate that the major mechanisms mediating homeostatic synaptic accumulation and PKC dispersal of NMDA receptors occur independently of GluN1 splice isoform.
Journal of Biological Chemistry 06/2011; 286(32):28331-42. · 4.77 Impact Factor
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ABSTRACT: Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo, where activity of inputs can be differentially regulated, but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here, we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons.
GluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons, both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures, synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures.
The enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde 'reward' signal generated by WT neurons, although in this paradigm there was no 'punishment' signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous system.
PLoS ONE 01/2011; 6(9):e24423. · 4.09 Impact Factor
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ABSTRACT: Cell adhesion molecules are well characterized for mediating synapse initiation, specification, differentiation, and maturation, yet their contribution to directing dendritic arborization during early brain circuit formation remains unclear. Using two-photon time-lapse imaging of growing neurons within intact and awake embryonic Xenopus brain, we examine roles of β-neurexin (NRX) and neuroligin-1 (NLG1) in dendritic arbor development. Using methods of dynamic morphometrics for comprehensive 3D quantification of rapid dendritogenesis, we find initial trans-synaptic NRX-NLG1 adhesions confer transient morphologic stabilization independent of NMDA receptor activity, whereas persistent stabilization requires NMDA receptor-dependent synapse maturation. Disrupting NRX-NLG1 function destabilizes filopodia while reducing synaptic density and AMPA receptor mEPSC frequency. Altered dynamic growth culminates in reduced dendritic arbor complexity as neurons mature over days. These results expand the synaptotropic model of dendritogenesis to incorporate cell adhesion molecule-mediated morphological stabilization necessary for directing normal dendritic arborization, providing a potential morphological substrate for developmental cognitive impairment associated with cell adhesion molecule mutations.
Neuron 09/2010; 67(6):967-83. · 14.74 Impact Factor
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Hisaki Fujii,
Geoff Cuvelier, Kevin She,
Soudabeh Aslanian,
Hiromi Shimizu,
Amina Kariminia,
Mark Krailo,
Zhengjia Chen,
Rob McMaster,
Axel Bergman,
Frederick Goldman,
Stephen A Grupp,
Donna A Wall,
Andrew L Gilman,
Kirk R Schultz
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ABSTRACT: Numerous chronic graft-versus-host disease (cGVHD) biomarkers have been identified in limited, single-institution studies without validation. We hypothesized that plasma-derived biomarkers could diagnose, classify, and evaluate response in children with cGVHD. We performed a concomitant analysis of a number of known and predicted peripheral blood cGVHD biomarkers from a Children's Oncology Group (COG) phase 3 cGVHD therapeutic trial. A total of 52 newly diagnosed patients with extensive cGVHD were compared for time of onset after blood and marrow transplantation (BMT) (early, 3-8 months; late, > or = 9 months) with 28 time-matched controls with no cGVHD (early, 6 months after BMT; late, 12 months after BMT). Soluble B-cell activation factor (sBAFF), anti-dsDNA antibody, soluble IL-2 receptor alpha (sIL-2Ralpha), and soluble CD13 (sCD13) were elevated in patients with early-onset cGVHD compared with controls. sBAFF and anti-dsDNA were elevated in patients with late-onset cGVHD. Some of the biomarkers correlated with specific organ involvement and with therapeutic response. These 4 biomarkers had high specificity with higher sensitivity in combination. Changes in biomarker concentrations with immune reconstitution after transplantation significantly affected interpretation of results. The identified biomarkers have the potential for improved classification, early response evaluation, and direction of cGVHD treatment, but require validation in larger studies. This study is registered at www.cancer.gov/clinicaltrials as no. COG-ASCT0031.
Blood 03/2008; 111(6):3276-85. · 9.90 Impact Factor
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ABSTRACT: B cells appear to play a role in chronic graft-versus-host disease (cGVHD) as shown in murine models and the success of anti-CD20 B cell antibody treatment in humans. Recent studies have shown that immunostimulatory microbial CpG-DNA splenic responses were enhanced in murine GVHD. We hypothesized that CpG-induced B cell responses are increased in human cGVHD. Newly diagnosed cGVHD patients enrolled on the COG protocol ASCT0031 were divided into early (3-8 months postblood and marrow transplant [BMT]) and late (> or =9 months post-BMT) onset groups and compared to time-matched control BMT patients. A significantly greater percentage of phosphorothioate (PS)-modified CpG stimulated B cells from cGVHD patients demonstrated an increased expression of CD86 compared to controls (P = .0004). This response had a significant correlation between B cell TLR9 expression (r(2) = 0.65; P = .002) and CD86 upregulation using the entirely TLR9-dependent native phosphodiester CpG (P = .003). The PS-modified CpG response at 2 months after initiation of cGVHD therapy demonstrated a trend toward predicting therapeutic response at 9 months post-BMT (P = .07). These findings suggest that an increased number of B cells, primed for a TLR9 response, may play a role in the pathophysiology of cGVHD.
Biology of Blood and Marrow Transplantation 05/2007; 13(4):386-97. · 3.87 Impact Factor
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ABSTRACT: Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy and, although current therapy is widely effective, relapse remains a significant clinical problem for which new treatment strategies are required. The ligation of Toll-like receptors (TLR) on antigen-presenting cells stimulates the generation of strong T-cell helper type 1 (Th1) adaptive immune responses. Although TLR9 ligation has been shown to enhance immunogenicity of a number of leukaemia cell types, there have been few reports of the effects mediated through other TLR. In this study we analysed both the expression of TLR by B-cell precursor ALL cell lines and the effects of individual TLR ligation on the ability of ALL cells to stimulate allogeneic T cells. While ligation of TLR2, TLR 7 and TLR9 led to detectable changes in ALL costimulatory molecule expression, only TLR2 and TLR9 stimulation influenced T-cell responses. The TLR2 ligand Pam3CysSerLys4 provoked the most significant changes in T-cell response, dramatically augmenting interferon-gamma production. These results suggest that TLR ligands, in addition to TLR9 agonists, may provide a strategy to enhance the generation of anti-ALL immune activity by skewing responding T cells towards a Th1 response.
British Journal of Haematology 03/2006; 132(4):452-8. · 4.94 Impact Factor
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ABSTRACT: Immunostimulatory DNA containing unmethylated cytosine-phosphate-guanosine (CpG) induces the development of T helper 1 (Th1) immune responses. The response of B cells to CpG stimulation involves increased proliferation, cytokine production, and costimulatory molecule expression. Similar effects have been observed following CpG stimulation of a variety of malignant B cells. Pediatric precursor B acute lymphoblastic leukemia (B-ALL) cells express low levels of costimulatory molecules and are generally poor stimulators of T-cell responses. In this study, we evaluated the impact of CpG stimulation on precursor B-ALL cell lines and pediatric patient-derived samples. The ability to respond to CpG oligodeoxynucleotides was determined by the level of Toll-like receptor 9 (TLR9) expression. In contrast to both nonleukemic B-cell precursors and mature B cells, the response of precursor B-ALL cells was characterized by increased CD40 expression but only small changes in CD86 levels and no induction of CD80 expression. CpG stimulation of ALL blasts produced increased levels of interleukin-6 (IL-6), IL-8, and IL-10 but no detectable IL-12p70 and led to a skewing of allogeneic T cells, with enhanced interferon gamma (IFN-gamma) production and reduced secretion of IL-5. These results demonstrate the functional relevance of CpG stimulation of precursor B-ALL cells and provide a rational basis for study of these agents for use in treatment of this disease.
Blood 06/2005; 105(9):3641-7. · 9.90 Impact Factor
