Synthetic Multivalent Ligands as Probes of Signal Transduction

Cell-surface receptors acquire information from the extracellular environment and coordinate intracellular responses. Many receptors do not operate as individual entities, but rather as part of dimeric or oligomeric complexes. Coupling the functions of multiple receptors may endow signaling pathways with the sensitivity and malleability required to govern cellular responses. Moreover, multireceptor signaling complexes may provide a means of spatially segregating otherwise degenerate signaling cascades. Understanding the mechanisms, extent, and consequences of receptor co-localization and interreceptor communication is critical; chemical synthesis can provide compounds to address the role of receptor assembly in signal transduction. Multivalent ligands can be generated that possess a variety of sizes, shapes, valencies, orientations, and densities of binding elements. This Review focuses on the use of synthetic multivalent ligands to characterize receptor function.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50669/1/2348_ftp.pd

Effects of maternal enflurane exposure on NR2B expression in the hippocampus of their offspring

This work aims to study the pathogenesis of learning and memory impairment in offspring rats resulting from maternal enflurane anesthesia by focusing on the expression of the N-methyl-d-aspartic acid receptor subunit 2B (NR2B) in the hippocampus of the offspring. Thirty female Sprague-Dawley rats were randomly divided into three groups: control (C group), 4 h enflurane exposure (E1 group), and 8 h enflurane exposure (E2 group) groups. Eight to ten days after the initiation of pregnancy, rats from the E1 and E2 groups were allowed to inhale 1.7% enflurane in 2 L/min oxygen for 4 h and 8 h, respectively. Rats from the C group were allowed to inhale 2 L/min of oxygen only. The Morris water maze was used to assay the learning and memory function of the offspring on postnatal days 20 and 30. RT-PCR and immunohistochemistry assays were then used to measure the mRNA levels and protein expression of NR2B, respectively. Relative to offspring rats from the C group, those from the E1 and E2 groups exhibited longer escape latencies, lesser number of crossings over the platform, and less time spent in the target quadrant in the spatial exploration test (P 0.05) in terms of mRNA levels and protein expression of NR2B. The cognitive function of the offspring is impaired when maternal rats are exposed to enflurane during early pregnancy. A possible mechanism of this effect is related to the down-regulation of NR2B expression

Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance

Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non-Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2-5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load synaptic protein turnover places on individual neurons is very substantial

The Regulation of Dendritic Spine Plasticity by EphB and N-methyl-D-aspartate Receptors Through Spatial Control Over Cofilin Activity in Mature Hippocampal Neurons

Dendritic spines are the post-synaptic sites of most excitatory synapses in the brain, and changes in their morphology are implicated in synaptic plasticity and long-term memory. F-actin dynamics are thought to be a basis for both the formation of dendritic spines during development and their structural plasticity (Ethell and Pasquale, 2005; Pontrello and Ethell, 2009). We have shown that the F-actin-severing protein cofilin, which is regulated by phosphorylation, can induce remodeling of mature dendritic spines in hippocampal neurons (Shi et al., 2009). We also demonstrate that β-Arrestins play an important role in spatial control over cofilin activity in dendritic spines, which underlies NMDA-mediated dendritic spine remodeling. Cofilin activity in dendritic spines is regulated through CaMKII-mediated suppression of cofilin activity by phosphorylation, and calcineurin-dependent cofilin activation through its dephosphorylation. In addition, while the EphB receptor promotes spine stabilization through cofilin inactivation, the NMDA receptor prevents EphB-mediated cofilin inactivation through calcineurin. NMDAR activation also promotes the translocation of cofilin to dendritic spines, an event that requires cofilin dephosphorylation and is also dependent on β-Arrestins, which have recently been shown to scaffold cofilin with its regulators, LIM kinase and slingshot phosphatase (Zoudilova et al., 2010). Our studies demonstrate that cofilin clustering in the spines is affected in both β-Arrestin1- and β-Arrestin2- deficient neurons under normal synaptic activity, and a constitutively-active cofilinS3A mutant fails to translocate to spines in response to NMDA in β-Arrestin2 KO neurons. Moreover, while wt neurons display dendritic spine remodeling in response to NMDA or with over-expression of cofilinS3A, β-Arrestin2-deficient neurons are resistant to both NMDA-induced and cofilinS3A-induced spine remodeling. In contrast, dominant-negative cofilinS3D prevents NMDA-induced dendritic spine remodeling in wt neurons, and also rescues a mature spine phenotype that is lost in β-Arrestin1 KO neurons. In addition, over-expression of β-Arrestin in the KO neurons rescues spine abnormalities. β-Arrestin1-deficient neurons also develop immature spines in vivo, whereas hippocampal neurons lacking β-Arrestin2 develop normal mature spines, but fail to remodel in response to NMDA. Our studies demonstrate novel functions of β-Arrestin1 in the development of mature dendritic spines, and β-Arrestin2 in NMDAR-mediated dendritic spine plasticity through spatial control over cofilin activity

From clinic to classroom: A case study of a literacy specialist examining teacher learning and instructional decision making

The purpose of this case study was to explore how the specific content knowledge developed in a newly certified literacy specialist’s practicum experiences are taken up in her practice as she supports the learning of her struggling literacy learners in the situated context of the middle school. Focused on her perspective, the present study explored the behaviors, beliefs, and stances of a literacy specialist cultured in diagnostic decision making and reflective processes and what her practices may represent as she attempts to meet the needs of her struggling seventh grade literacy learners. The present study examined the way a literacy specialist assesses her students’ areas of strength and development, confronts specific difficulties experienced by her students and the process of planning an effective program of re-mediation for the improvement of comprehension. Conversely, this study explores the way a literacy specialist responds to the professional challenges she faced in her professional role. In concert with the literacy specialist’s perspective of her role, teacher learning, and the role reflective practice as it relates to instructional decision making in a re-medial classroom context was central to the investigation of this process. Findings relative to a newly literacy specialist’s practicum experiences lead to positive implications for literacy education. The first implication suggests providing for the development of research-based pedagogical content knowledge and curricular knowledge situated within authentic contexts to inform practice supported teacher learning and instructional decision making. The second implication for literacy education suggests a literacy specialist’s efficient decision making involves the adoption of guiding principles. The third implication suggests the adoption of guiding principles occurs through the effective development of reflective practice

Signaling Mode of the Broad-Spectrum Conserved CO2 Receptor Is One of the Important Determinants of Odor Valence in Drosophila

Odor detection involves hundreds of olfactory receptors from diverse families, making modeling of hedonic valence of an odorant difficult, even in Drosophila melanogaster where most receptors have been deorphanised. We demonstrate that a broadly tuned heteromeric receptor that detects CO2 (Gr21a, Gr63a) and other odorants is a key determinant of valence along with a few members of the Odorant receptor family in a T-maze, but not in a trap assay. Gr21a and Gr63a have atypically high amino acid conservation in Dipteran insects, and they use both inhibition and activation to convey positive or negative valence for numerous odorants. Inhibitors elicit a robust Gr63a-dependent attraction, while activators, strong aversion. The attractiveness of inhibitory odorants increases with increasing background CO2 levels, providing a mechanism for behavior modulation in odor blends. In mosquitoes, valence is switched and activation of the orthologous receptor conveys attraction. Reverse chemical ecology enables the identification of inhibitory odorants to reduce attraction of mosquitoes to skin

Age-Related Oxidative Redox and Metabolic Changes Precede Intraneuronal Amyloid-β Accumulation and Plaque Deposition in a Transgenic Alzheimer’s Disease Mouse Model

BackgroundMany identified mechanisms could be upstream of the prominent amyloid-β (Aβ) plaques in Alzheimer's disease (AD).ObjectiveTo profile the progression of pathology in AD.MethodsWe monitored metabolic signaling, redox stress, intraneuronal amyloid-β (iAβ) accumulation, and extracellular plaque deposition in the brains of 3xTg-AD mice across the lifespan.ResultsIntracellular accumulation of aggregated Aβ in the CA1 pyramidal cells at 9 months preceded extracellular plaques that first presented in the CA1 at 16 months of age. In biochemical assays, brain glutathione (GSH) declined with age in both 3xTg-AD and non-transgenic controls, but the decline was accelerated in 3xTg-AD brains from 2 to 4 months. The decline in GSH correlated exponentially with the rise in iAβ. Integrated metabolic signaling as the ratio of phospho-Akt (pAkt) to total Akt (tAkt) in the PI3kinase and mTOR pathway declined at 6, 9, and 12 months, before rising at 16 and 20 months. These pAkt/tAkt ratios correlated with both iAβ and GSH levels in a U-shaped relationship. Selective vulnerability of age-related AD-genotype-specific pAkt changes was greatest in the CA1 pyramidal cell layer. To demonstrate redox causation, iAβ accumulation was lowered in cultured middle-age adult 3xTg-AD neurons by treatment of the oxidized redox state in the neurons with exogenous cysteine.ConclusionThe order of pathologic progression in the 3xTg-AD mouse was loss of GSH (oxidative redox shift) followed by a pAkt/tAkt metabolic shift in CA1, iAβ accumulation in CA1, and extracellular Aβ deposition. Upstream targets may prove strategically more effective for therapy before irreversible changes

Coadaptation of the chemosensory system with voluntary exercise behavior in mice.

Ethologically relevant chemical senses and behavioral habits are likely to coadapt in response to selection. As olfaction is involved in intrinsically motivated behaviors in mice, we hypothesized that selective breeding for a voluntary behavior would enable us to identify novel roles of the chemosensory system. Voluntary wheel running (VWR) is an intrinsically motivated and naturally rewarding behavior, and even wild mice run on a wheel placed in nature. We have established 4 independent, artificially evolved mouse lines by selectively breeding individuals showing high VWR activity (High Runners; HRs), together with 4 non-selected Control lines, over 88 generations. We found that several sensory receptors in specific receptor clusters were differentially expressed between the vomeronasal organ (VNO) of HRs and Controls. Moreover, one of those clusters contains multiple single-nucleotide polymorphism loci for which the allele frequencies were significantly divergent between the HR and Control lines, i.e., loci that were affected by the selective breeding protocol. These results indicate that the VNO has become genetically differentiated between HR and Control lines during the selective breeding process. Although the role of the vomeronasal chemosensory receptors in VWR activity remains to be determined, the current results suggest that these vomeronasal chemosensory receptors are important quantitative trait loci for voluntary exercise in mice. We propose that olfaction may play an important role in motivation for voluntary exercise in mammals

Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation

CD22 is an inhibitory coreceptor on the surface of B cells that attenuates B cell antigen receptor (BCR) signaling and, therefore, B cell activation. Elucidating the molecular mechanisms underlying the inhibitory activity of CD22 is complicated by the ubiquity of CD22 ligands. Although antigens can display CD22 ligands, the receptor is known to bind to sialylated glycoproteins on the cell surface. The propinquity of CD22 and cell-surface glycoprotein ligands has led to the conclusion that the inhibitory properties of the receptor are due to cis interactions. Here, we examine the functional consequences of trans interactions by employing sialylated multivalent antigens that can engage both CD22 and the BCR. Exposure of B cells to sialylated antigens results in the inhibition of key steps in BCR signaling. These results reveal that antigens bearing CD22 ligands are powerful suppressors of B cell activation. The ability of sialylated antigens to inhibit BCR signaling through trans CD22 interactions reveals a previously unrecognized role for the Siglec-family of receptors as modulators of immune signaling