Mitotic spindle disassembly in human cells relies on CRIPT having hierarchical redox signals.

Swift and complete spindle disassembly in late mitosis is essential for cell survival, yet how it happens is largely unknown in mammalian cells. Here we used real-time live cell microscopy and biochemical assays to show that the primordial dwarfism (PD)-related cysteine-rich protein CRIPT dictates the spindle disassembly in a redox-dependent manner in human cells. This previously reported cytoplasmic protein was found to have a confined nuclear localization with a nucleolar concentration during interphase but was distributed to spindles and underwent redox modifications to form disulfide bonds in CXXC pairs during mitosis. Then, it directly interacted with, and might transfer a redox response to, tubulin subunits via a putative redox exchange among cysteine residues to induce microtubule depolymerization. Expression of CRIPT proteins with mutations of these cysteine residues blocked spindle disassembly, generating two cell types with long-lasting metaphase spindles or spindle remnants. Live-cell recordings of a disease-relevant mutant (CRIPTC3Y) revealed that microtubule depolymerization at spindle ends during anaphase and the entire spindle dissolution during telophase might share a common CRIPT-bearing redox-controlled mechanism.

Dysregulated Ca2+-Permeable AMPA Receptor Signaling in Neural Progenitors Modeling Fragile X Syndrome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder that represents a common cause of intellectual disability and is a variant of autism spectrum disorder (ASD). Studies that have searched for similarities in syndromic and non-syndromic forms of ASD have paid special attention to alterations of maturation and function of glutamatergic synapses. Copy number variations (CNVs) in the loci containing genes encoding alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) subunits are associated with ASD in genetic studies. In FXS, dysregulated AMPAR subunit expression and trafficking affect neural progenitor differentiation and synapse formation and neuronal plasticity in the mature brain. Decreased expression of GluA2, the AMPAR subunit that critically controls Ca2+-permeability, and a concomitant increase in Ca2+-permeable AMPARs (CP-AMPARs) in human and mouse FXS neural progenitors parallels changes in expression of GluA2-targeting microRNAs (miRNAs). Thus, posttranscriptional regulation of GluA2 by miRNAs and subsequent alterations in calcium signaling may contribute to abnormal synaptic function in FXS and, by implication, in some forms of ASD.

Functional changes of AMPA responses in human induced pluripotent stem cell-derived neural progenitors in fragile X syndrome.

Altered neuronal network formation and function involving dysregulated excitatory and inhibitory circuits are associated with fragile X syndrome (FXS). We examined functional maturation of the excitatory transmission system in FXS by investigating the response of FXS patient-derived neural progenitor cells to the glutamate analog (AMPA). Neural progenitors derived from induced pluripotent stem cell (iPSC) lines generated from boys with FXS had augmented intracellular Ca2+ responses to AMPA and kainate that were mediated by Ca2+-permeable AMPA receptors (CP-AMPARs) lacking the GluA2 subunit. Together with the enhanced differentiation of glutamate-responsive cells, the proportion of CP-AMPAR and N-methyl-d-aspartate (NMDA) receptor-coexpressing cells was increased in human FXS progenitors. Differentiation of cells lacking GluA2 was also increased and paralleled the increased inward rectification in neural progenitors derived from Fmr1-knockout mice (the FXS mouse model). Human FXS progenitors had increased the expression of the precursor and mature forms of miR-181a, a microRNA that represses translation of the transcript encoding GluA2. Blocking GluA2-lacking, CP-AMPARs reduced the neurite length of human iPSC-derived control progenitors and further reduced the shortened length of neurites in human FXS progenitors, supporting the contribution of CP-AMPARs to the regulation of progenitor differentiation. Furthermore, we observed reduced expression of Gria2 (the GluA2-encoding gene) in the frontal lobe of FXS mice, consistent with functional changes of AMPARs in FXS. Increased Ca2+ influx through CP-AMPARs may increase the vulnerability and affect the differentiation and migration of distinct cell populations, which may interfere with normal circuit formation in FXS.

Cysteine 893 is a target of regulatory thiol modifications of GluA1 AMPA receptors.

Recent studies indicate that glutamatergic signaling involves, and is regulated by, thiol modifying and redox-active compounds. In this study, we examined the role of a reactive cysteine residue, Cys-893, in the cytosolic C-terminal tail of GluA1 AMPA receptor as a potential regulatory target. Elimination of the thiol function by substitution of serine for Cys-893 led to increased steady-state expression level and strongly reduced interaction with SAP97, a major cytosolic interaction partner of GluA1 C-terminus. Moreover, we found that of the three cysteine residues in GluA1 C-terminal tail, Cys-893 is the predominant target for S-nitrosylation induced by exogenous nitric oxide donors in cultured cells and lysates. Co-precipitation experiments provided evidence for native association of SAP97 with neuronal nitric oxide synthase (nNOS) and for the potential coupling of Ca2+-permeable GluA1 receptors with nNOS via SAP97. Our results show that Cys-893 can serve as a molecular target for regulatory thiol modifications of GluA1 receptors, including the effects of nitric oxide.

Molecular mechanisms controlling synaptic recruitment of GluA4 subunit-containing AMPA-receptors critical for functional maturation of CA1 glutamatergic synapses.

Synaptic recruitment of AMPA receptors (AMPARs) represents a key postsynaptic mechanism driving functional development and maturation of glutamatergic synapses. At immature hippocampal synapses, PKA-driven synaptic insertion of GluA4 is the predominant mechanism for synaptic reinforcement. However, the physiological significance and molecular determinants of this developmentally restricted form of plasticity are not known. Here we show that PKA activation leads to insertion of GluA4 to synaptic sites with initially weak or silent AMPAR-mediated transmission. This effect depends on a novel mechanism involving the extreme C-terminal end of GluA4, which interacts with the membrane proximal region of the C-terminal domain to control GluA4 trafficking. In the absence of GluA4, strengthening of AMPAR-mediated transmission during postnatal development was significantly delayed. These data suggest that the GluA4-mediated activation of silent synapses is a critical mechanism facilitating the functional maturation of glutamatergic circuitry during the critical period of experience-dependent fine-tuning. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Aggregation Limits Surface Expression of Homomeric GluA3 Receptors.

AMPA receptors are glutamate-gated cation channels assembled from GluA1-4 subunits and have properties that are strongly dependent on the subunit composition. The subunits have different propensities to form homomeric or various heteromeric receptors expressed on cell surface, but the underlying mechanisms are still poorly understood. Here, we examined the biochemical basis for the poor ability of GluA3 subunits to form homomeric receptors, linked previously to two amino acid residues, Tyr-454 and Arg-461, in its ligand binding domain (LBD). Surface expression of GluA3 was improved by co-assembly with GluA2 but not with stargazin, a trafficking chaperone and modulator of AMPA receptors. The secretion efficiency of GluA2 and GluA3 LBDs paralleled the transport difference between the respective full-length receptors and was similarly dependent on Tyr-454/Arg-461 but not on LBD stability. In comparison to GluA2, GluA3 homomeric receptors showed a strong and Tyr-454/Arg-461-dependent tendency to aggregate both in the macroscopic scale measured as lower solubility in nonionic detergent and in the microscopic scale evident as the preponderance of hydrodynamically large structures in density gradient centrifugation and native gel electrophoresis. We conclude that the impaired surface expression of homomeric GluA3 receptors is caused by nonproductive assembly and aggregation to which LBD residues Tyr-454 and Arg-461 strongly contribute. This aggregation inhibits the entry of newly synthesized GluA3 receptors to the secretory pathway.

Developmental switch in the kinase dependency of long-term potentiation depends on expression of GluA4 subunit-containing AMPA receptors.

The AMPA-receptor subunit GluA4 is expressed transiently in CA1 pyramidal neurons at the time synaptic connectivity is forming, but its physiological significance is unknown. Here we show that GluA4 expression is sufficient to alter the signaling requirements of long-term potentiation (LTP) and can fully explain the switch in the LTP kinase dependency from PKA to Ca2(+)/calmodulin-dependent protein kinase II during synapse maturation. At immature synapses, activation of PKA leads to a robust potentiation of AMPA-receptor function via the mobilization of GluA4. Analysis of GluA4-deficient mice indicates that this mechanism is critical for neonatal PKA-dependent LTP. Furthermore, lentiviral expression of GluA4 in CA1 neurons conferred a PKA-dependent synaptic potentiation and LTP regardless of the developmental stage. Thus, GluA4 defines the signaling requirements for LTP and silent synapse activation during a critical period of synapse development.

The N-terminal domain modulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization.

AMPA receptors are tetrameric glutamate-gated ion channels that mediate fast synaptic neurotransmission in mammalian brain. Their subunits contain a two-lobed N-terminal domain (NTD) that comprises over 40% of the mature polypeptide. The NTD is not obligatory for the assembly of tetrameric receptors, and its functional role is still unclear. By analyzing full-length and NTD-deleted GluA1-4 AMPA receptors expressed in HEK 293 cells, we found that the removal of the NTD leads to a significant reduction in receptor transport to the plasma membrane, a higher steady state-to-peak current ratio of glutamate responses, and strongly increased sensitivity to glutamate toxicity in cell culture. Further analyses showed that NTD-deleted receptors display both a slower onset of desensitization and a faster recovery from desensitization of agonist responses. Our results indicate that the NTD promotes the biosynthetic maturation of AMPA receptors and, for membrane-expressed channels, enhances the stability of the desensitized state. Moreover, these findings suggest that interactions of the NTD with extracellular/synaptic ligands may be able to fine-tune AMPA receptor-mediated responses, in analogy with the allosteric regulatory role demonstrated for the NTD of NMDA receptors.

Autoinactivation of the stargazin-AMPA receptor complex: subunit-dependency and independence from physical dissociation.

Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This "autoinactivation" has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1-A4 AMPA receptors (all flip isoform) expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.

Post-synapse model cell for synaptic glutamate receptor (GluR)-based biosensing: strategy and engineering to maximize ligand-gated ion-flux achieving high signal-to-noise ratio.

Cell-based biosensing is a "smart" way to obtain efficacy-information on the effect of applied chemical on cellular biological cascade. We have proposed an engineered post-synapse model cell-based biosensors to investigate the effects of chemicals on ionotropic glutamate receptor (GluR), which is a focus of attention as a molecular target for clinical neural drug discovery. The engineered model cell has several advantages over native cells, including improved ease of handling and better reproducibility in the application of cell-based biosensors. However, in general, cell-based biosensors often have low signal-to-noise (S/N) ratios due to the low level of cellular responses. In order to obtain a higher S/N ratio in model cells, we have attempted to design a tactic model cell with elevated cellular response. We have revealed that the increase GluR expression level is not directly connected to the amplification of cellular responses because the saturation of surface expression of GluR, leading to a limit on the total ion influx. Furthermore, coexpression of GluR with a voltage-gated potassium channel increased Ca(2+) ion influx beyond levels obtained with saturating amounts of GluR alone. The construction of model cells based on strategy of amplifying ion flux per individual receptors can be used to perform smart cell-based biosensing with an improved S/N ratio.

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors.

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105(+) endothelial cells, human CD133(+) hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133(+) cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.

Ligand-binding domain determines endoplasmic reticulum exit of AMPA receptors.

AMPA receptors (AMPARs) are tetrameric ion channels that mediate rapid glutamate signaling in neurons and many non-neuronal cell types. Endoplasmic reticulum (ER) quality control mechanisms permit only correctly folded functional receptors to be delivered to the cell surface. We analyzed the biosynthetic maturation and transport of all 12 GluA1-4 subunit splice variants as homomeric receptors and observed robust isoform-dependent differences in ER exit competence and surface expression. In contrast to inefficient ER exit of both GluA3 splice forms and the flop variants of GluA1 and GluA4, prominent plasma membrane expression was observed for the other AMPAR isoforms. Surprisingly, deletion of the entire N-terminal domain did not alter the transport phenotype, nor did the different cytosolic C-terminal tail splice variants. Detailed analysis of mutant receptors led to the identification of distinct residues in the ligand-binding domain as primary determinants for isoform-specific maturation. Considered together with the essential role of bound agonist, our findings reveal the ligand-binding domain as the critical quality control target in AMPAR biogenesis.

Analysis of the potential role of GluA4 carboxyl-terminus in PDZ interactions.

BACKGROUND: Specific delivery to synapses of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors with long-tailed subunits is believed to be a key event in many forms of activity-dependent changes in synaptic strength. GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97). In GluA4, another long-tailed subunit implicated in synaptic plasticity, the PDZ motif is blocked by a single proline residue. This feature is highly conserved in vertebrates, whereas the closest invertebrate homologs of GluA4 have a canonical class I PDZ binding motif. In this work, we have examined the role of GluA4 in PDZ interactions. METHODOLOGY/PRINCIPAL FINDINGS: Deletion of the carboxy-terminal proline residue of recombinant GluA4 conferred avid binding to SAP97 in cultured cells as shown by coimmunoprecipitation, whereas wild-type GluA4 did not associate with SAP97. Native GluA4 and SAP97 coimmunoprecipitated from mouse brain independently of the GluA1 subunit, supporting the possibility of in vivo PDZ interaction. To obtain evidence for or against the exposure of the PDZ motif by carboxyterminal processing of native GluA4 receptors, we generated an antibody reagent specific for proline-deleted GluA4 C-terminus. Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent. CONCLUSION/SIGNIFICANCE: We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.

Engineered synapse model cell: genetic construction and chemical evaluation for reproducible high-throughput analysis.

Bioassay models of neural functions must lend themselves to high-throughput analysis in neural drug discovery. However, smart analysis methods for these functions have not yet been fully established. Here, we describe the development of a synapse model for cell-based biosensing. The engineered synapse model cell expresses ionotropic glutamate receptor on its surface, like the neural postsynaptic membrane. The advantages of the model cell are the ease of handling and reproducibility as compared with the cultured neural cell, and it can be employed to evaluate receptor function through ion flux analysis. The agonist-induced sodium influx was monitored as an agonist concentration-dependent increase in the observed fluorescence signal. Furthermore, we found that our model cell enables the correction of uneven cellular signal levels using a reporter system. Our engineered synapse model cell can be employed as a powerful tool for the screening of lead substances in pharmaceutical high-throughput analysis.

Dynamic visualization of membrane-inserted fraction of pHluorin-tagged channels using repetitive acidification technique.

BACKGROUND: Changes in neuronal excitability, synaptic efficacy and generally in cell signaling often result from insertion of key molecules into plasma membrane (PM). Many of the techniques used for monitoring PM insertion lack either spatial or temporal resolution. RESULTS: We improved the imaging method based on time-lapse total internal reflection fluorescence (TIRF) microscopy and pHluorin tagging by supplementing it with a repetitive extracellular acidification protocol. We illustrate the applicability of this method by showing that brief activation of NMDA receptors ("chemical LTP") in cultured hippocampal neurons induced a persistent PM insertion of glutamate receptors containing the pHluorin-tagged GluR-A(flip) subunits. CONCLUSION: The repetitive acidification technique provides a more accurate way of monitoring the PM-inserted fraction of fluorescently tagged molecules and offers a good temporal and spatial resolution.

Interaction of the RhoA exchange factor Net1 with discs large homolog 1 protects it from proteasome-mediated degradation and potentiates Net1 activity.

Net1 is a nuclear Rho guanine nucleotide exchange factor that is specific for the RhoA subfamily of small G proteins. Truncated forms of Net1 are transforming in NIH3T3 cells, and this activity requires cytoplasmic localization of Net1 as well as the presence of a COOH-terminal PDZ binding site. We have previously shown that Net1 interacts with PDZ domain-containing proteins within the Discs Large (Dlg) family and relocalizes them to the nucleus. In the present work, we demonstrate that Net1 binds directly to the first two PDZ domains of Dlg1 and that both PDZ domains are required for maximal interaction in cells. Furthermore, we show that Net1 is an unstable protein in MCF7 breast epithelial cells and that interaction with Dlg1 significantly enhances Net1 stability. Stabilization by Dlg1 significantly increases the ability of Net1 to stimulate RhoA activation in cells. The stability of endogenous Net1 is strongly enhanced by cell-cell contact, and this correlates with a dramatic increase in the interaction between Net1 and Dlg1. Importantly, disruption of E-cadherin-mediated cell contacts, either by depletion of external calcium or by treatment with transforming growth factor beta, leads to a rapid loss of the interaction between Net1 and Dlg1 and a subsequent increase in the ubiquitylation of Net1. These results indicate that Net1 requires interaction with PDZ domain proteins, such as Dlg1, to protect it from proteasome-mediated degradation and to maximally stimulate RhoA and that this interaction is regulated by cell-cell contact.

Ethanol increases desensitization of recombinant GluR-D AMPA receptor and TARP combinations.

Glutamate receptors are important target molecules of the acute effect of ethanol. We studied ethanol sensitivity of homomeric GluR-D receptors expressed in human embryonic kidney 293 cells and examined whether recently discovered transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory proteins (TARPs) affect ethanol sensitivity. Coexpression of the TARPs, stargazin, and gamma4 increased the time constant (tau-value) of current decay in the presence of agonist, thus slowing the onset of desensitization and increasing the steady-state current. Ethanol produced less inhibition of the peak current than the steady-state current for all types of the GluR-D receptors. In addition, ethanol concentration-dependently accelerated the rate of desensitization, measured as the tau-value of fast decay of peak current. This effect was enhanced with coexpression of TARPs. The recovery from desensitization was slowed down by coexpression of gamma4 but ethanol did not affect this process in any GluR-D combination. The results support the idea that increased desensitization is an important mechanism in the ethanol inhibition of AMPA receptors and indicate that coexpression of TARPs can alter this effect of ethanol.

Agonist occupancy is essential for forward trafficking of AMPA receptors.

Regulated trafficking of AMPA receptors to cell surface and to synapses is an important determinant of neuronal excitability. In the present study, we have addressed the role of agonist binding and desensitization in the early trafficking of glutamate receptor-D (GluR-D) AMPA receptors. Analysis of point-mutated GluR-D receptors, via electrophysiology and immunofluorescence, revealed that agonist-binding activity is essential for efficient delivery to cell surface in transfected cell lines and in neurons. Cotransfection with stargazin could fully rescue the surface expression of nonbinding mutant receptors in cell lines, indicating that stargazin is able to interact with and promote exit of AMPA receptors from endoplasmic reticulum (ER) independently of agonist binding. Secretion of separately expressed ligand-binding domain constructs showed a similar dependency of agonist binding to that observed with full-length GluR-D, supporting the idea that glutamate-induced closure of the binding site cleft is registered by ER quality control as a necessary priming step for transport competence. In contrast to agonist binding, the ability of the receptor to undergo desensitization had only a minor influence on trafficking. Our results are consistent with the hypothesis that AMPA receptors are synthesized as intrinsically unstable molecules, which require glutamate binding for structural stability and for transport-competence.

KCC2 interacts with the dendritic cytoskeleton to promote spine development.

The neuron-specific K-Cl cotransporter, KCC2, induces a developmental shift to render GABAergic transmission from depolarizing to hyperpolarizing. Now we demonstrate that KCC2, independently of its Cl(-) transport function, is a key factor in the maturation of dendritic spines. This morphogenic role of KCC2 in the development of excitatory synapses is mediated by structural interactions between KCC2 and the spine cytoskeleton. Here, the binding of KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N may play an important role. A more general conclusion based on our data is that KCC2 acts as a synchronizing factor in the functional development of glutamatergic and GABAergic synapses in cortical neurons and networks.

Isoform-specific early trafficking of AMPA receptor flip and flop variants.

Flip and flop splice variants of AMPA receptor subunits are expressed in distinct but partly overlapping patterns and impart different desensitization kinetics to cognate receptor channels. In the absence of specific antibodies, isoform-specific differences in trafficking or localization of native flip and flop subunits remain uncharacterized. We report that in several transfected cell lines, transport of homomeric glutamate receptor (GluR)-D(flop) receptors is largely blocked at the endoplasmic reticulum (ER) exit, whereas GluR-D(flip) undergoes complex glycosylation and reaches the plasma membrane at >10x higher levels than GluR-D(flop), as determined by immunofluorescence, patch-clamp recordings and biochemical assays. The transport difference between flip and flop is independent of activity, is primarily determined by amino acid residue 780 (Leu in flop, Val in flip), and is manifested even in the secretion of the soluble ligand-binding domain, suggesting it is independent of oligomerization. Coexpression with stargazin or with the flip isoform rescues the surface expression of GluR-D(flop) near to the level exhibited by GluR-D(flip). Our results demonstrate that the extracellular flip/flop region, via interactions with ER luminal splice form-specific protein(s), plays a hitherto unappreciated and important role in AMPA-receptor trafficking.