Multi-Stage Modeling of the Kinetics of Activation of CaMKII

Ca 2+ /calmodulin-dependent protein kinase 2 (CaMKII) plays an important role in induction of long-term potentiation and formation of memory. It is abundant in dendritic spines, and is activated when Ca 2+ flows into the postsynaptic cytosol through open NMDA-type glutamate receptors. Its function is fine-tuned through interaction with other proteins as well as through subunit interactions and regulatory autophosphorylation. We have undertaken a multi-stage project to study the critical kinetics of activation of CaMKII in the spine by combining modeling and experimental studies. We are using computational modeling and simulations on various platforms, coupled with biochemical experiments in vitro, and eventually in vivo, to understand CaMKII regulation. The project includes the following steps: 1. Determining the parameters governing activation of a monomeric subunit. The CaMKII holoenzyme is a large dodecamer of similar, homologous subunits held together by interactions between the association domains located at the carboxyl end of each subunit. Individual, monomeric subunits can be expressed recombinantly by removing the association domain. Computer simulations of activation of monomeric CaMKII by Ca 2+ /calmodulin at both saturating and non-saturating concentrations in a test tube have helped to identify the binding parameters that are most crucial for modeling of regulation of CaMKII and thus have indicated the most useful biochemical assays to measure those parameters (Pepke et al., 2010). We are using these measurements to fine-tune our model of activation of individual catalytic subunits. 2. Building a model of the holoenzyme. Because a CaMKII holoenzyme contains 12 similar subunits, each of which can exist in several states, the holoenzyme can have a large number of state combinations. Thus, modeling the entire holoenzyme requires a computational framework that avoids the ensuing combinatorial complexity. The stochastic simulator MCell provides an elegant, rule-based way of modeling state changes in the CaMKII holoenzyme. 3. Modeling cooperativity that arises from the dodecameric structure of CaMKII. Autophosphorylation at threonine-286, which activates CaMKII subunits, is an inter-subunit event. Thus, it is greatly facilitated by the close proximity of subunits in the holoenzyme. In addition, the subunits within the holoenzyme are arranged as dimers which appears to result in cooperativity in the binding of Ca 2+ /CaM to individual subunits of the dimer (Chao et al., 2010). An accurate model of activation of subunits in the holoenzyme and their autophosphorylation will allow us to explore the effects of cooperativity on CaMKII activation on various time scales. 4. Modeling CaMKII within the context of a postsynaptic spine CaMKII interacts with a variety of other proteins, both in the postsynaptic density (PSD), close to major sources of Ca 2+ influx, and in other parts of the spine. In the fourth stage of this project we plan to implement kinetic models of activation of CaMKII in the context of an MCell model of Ca 2+ influx into a spine upon activation of NMDA-type glutamate receptors (Keller et al., 2008; Keller et al., 2011, submitted). We will explore the effects of different localization and numbers of CaMKII holoenzymes in the spine on CaMKII activation. References: Pepke, S., Kinzer-Ursem, T., Mihalas, S., and Kennedy, M.B. (2010). A dynamic model of interactions of Ca 2+ , calmodulin, and catalytic subunits of Ca 2+ /calmodulin-dependent protein kinase II. PLoS Comput Biol 6, e1000675. Chao, L.H., Pellicena, P., Deindl, S., Barclay, L.A., Schulman, H., and Kuriyan, J. (2010). Intersubunit capture of regulatory segments is a component of cooperative CaMKII activation. Nat Struct Mol Biol 17, 264-272. Keller, D.X., Franks, K.M., Bartol, T.M., Jr., and Sejnowski, T.J. (2008). Calmodulin activation by calcium transients in the postsynaptic density of dendritic spines. PLoS ONE 3, e2045. Keller, D.X., Bartol, T.M., Kinney, J.P, Kennedy, M.B., Bajaj, C., Harris, K.M., and Sejnowski, T.J. Regulation of synaptic calcium transients in reconstructed dendritic spines of hippocampal CA1 pyramidal neurons, submitted

Choroidal thickness measured using swept-source optical coherence tomography is reduced in patients with type 2 diabetes

Objective To compare choroidal thickness between patients with type 2 diabetes (T2D) and healthy controls measured using swept-source optical coherence tomography (SS-OCT). Methods The sample comprised 157 eyes of 94 T2D patients, 48 eyes of which had diabetic macular edema (DME), and 71 normal eyes of 38 healthy patients. Subfoveal (SF) choroidal thickness, and choroidal thickness at 500-µm intervals up to 2500 µm nasal and temporal from the fovea were measured using the SS-OCT. Choroidal thicknesses were compared between groups using Student’s t-test. Additionally, Pearson correlations were calculated between diabetes duration, glycosylated hemoglobin (HbA1c) levels, and choroidal thickness. Results Mean diabetes duration was 16.6±9.5 years, while mean glycosylated hemoglobin was 7.7 ±1.3%. Overall, the choroid was significantly thinner in T2D patients. Individuals with DME had reduced choroidal thickness in all measurements, except at 2000 and 2500-µm nasal positions, compared to healthy controls. There was a moderate correlation between choroidal thickness and HbA1c levels in DME patients (SF: r = 0.342; p = 0.017). Diabetes duration did not correlate significantly with choroidal thickness. Conclusion SS-OCT measurements revealed that the choroid was significantly thinner in T2D patients, moderate non-proliferative diabetic retinopathy patients, and DME patients than in healthy individuals. Further studies are needed to clarify the effect of diabetes on this layer and the relationship between choroidal thickness and DME

Repeatability of choroidal thickness measurements assessed with swept-source optical coherence tomography in healthy and diabetic individuals

PURPOSE: To assess the intrasession repeatability of choroidal thickness measurements obtained using swept-source optical coherence tomography in Type 2 diabetic (T2D) patients and healthy controls. METHODS: This was a single-center, prospective, observational, cross-sectional study with consecutive inclusion of 33 healthy subjects and 43 T2D patients. Subjects underwent three consecutive swept-source optical coherence tomography scans in a single session. After automatic delineation of the choroid, subfoveal choroidal thickness, and thickness at 500-µm intervals up to 2, 500 µm nasal and temporal from the fovea were measured using the software caliper by the same operator. Intraclass correlation coefficients (ICCs), coefficients of variation, and test-retest variability were calculated. RESULTS: Mean subfoveal choroidal thickness in healthy subjects and in T2D patients was 229.97 ± 79.9 and 192.67 ± 74.3 µm, respectively (P = 0.013). All intrasession intraclass correlation coefficients were higher than 0.95 and 0.99, respectively. Coefficients of variations were less than 4.4% and 1.8%, respectively. Test-retest variability ranged from 0.76 µm to 11.12 µm and 0.64 µm to 6.29 µm, respectively. No significant differences were found in the intrasession repeatability of any choroidal measurement between healthy subjects and T2D patients. CONCLUSION: Swept-source optical coherence tomography provided excellent intrasession repeatability of choroidal thickness measurements in healthy subjects and T2D patients

A Multi-State Model of the CaMKII Dodecamer Suggests a Role for Calmodulin in Maintenance of Autophosphorylation

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) accounts for up to 2 percent of all brain protein and is essential to memory function. CaMKII activity is known to regulate dynamic shifts in the size and signaling strength of neuronal connections, a process known as synaptic plasticity. Increasingly, computational models are used to explore synaptic plasticity and the mechanisms regulating CaMKII activity. Conventional modeling approaches may exclude biophysical detail due to the impractical number of state combinations that arise when explicitly monitoring the conformational changes, ligand binding, and phosphorylation events that occur on each of the CaMKII holoenzyme’s subunits. To manage the combinatorial explosion without necessitating bias or loss in biological accuracy, we use a specialized syntax in the software MCell to create a rule-based model of a twelve-subunit CaMKII holoenzyme. Here we validate the rule-based model against previous experimental measures of CaMKII activity and investigate molecular mechanisms of CaMKII regulation. Specifically, we explore how Ca²⁺/CaM-binding may both stabilize CaMKII subunit activation and regulate maintenance of CaMKII autophosphorylation. Noting that Ca²⁺/CaM and protein phosphatases bind CaMKII at nearby or overlapping sites, we compare model scenarios in which Ca²⁺/CaM and protein phosphatase do or do not structurally exclude each other’s binding to CaMKII. Our results suggest a functional mechanism for the so-called “CaM trapping” phenomenon, wherein Ca²⁺/CaM may structurally exclude phosphatase binding and thereby prolong CaMKII autophosphorylation. We conclude that structural protection of autophosphorylated CaMKII by Ca²⁺/CaM may be an important mechanism for regulation of synaptic plasticity

Computational reconstitution of spine calcium transients from individual proteins

We have built a stochastic model in the program MCell that simulates Ca^(2+) transients in spines from the principal molecular components believed to control Ca^(2+) entry and exit. Proteins, with their kinetic models, are located within two segments of dendrites containing 88 intact spines, centered in a fully reconstructed 6 × 6 × 5 μm^3 cube of hippocampal neuropil. Protein components include AMPA- and NMDA-type glutamate receptors, L- and R-type voltage-dependent Ca^(2+) channels, Na^+/Ca^(2+) exchangers, plasma membrane Ca^(2+) ATPases, smooth endoplasmic reticulum Ca^(2+) ATPases, immobile Ca2+ buffers, and calbindin. Kinetic models for each protein were taken from published studies of the isolated proteins in vitro. For simulation of electrical stimuli, the time course of voltage changes in the dendritic spine was generated with the desired stimulus in the program NEURON. Voltage-dependent parameters were then continuously re-adjusted during simulations in MCell to reproduce the effects of the stimulus. Nine parameters of the model were optimized within realistic experimental limits by a process that compared results of simulations to published data. We find that simulations in the optimized model reproduce the timing and amplitude of Ca^(2+) transients measured experimentally in intact neurons. Thus, we demonstrate that the characteristics of individual isolated proteins determined in vitro can accurately reproduce the dynamics of experimentally measured Ca^(2+) transients in spines. The model will provide a test bed for exploring the roles of additional proteins that regulate Ca^(2+) influx into spines and for studying the behavior of protein targets in the spine that are regulated by Ca^(2+) influx

Cardioprotective Effects of Cell Permeable NADPH oxidase inhibitors in Myocardial Ischemia/Reperfusion Injury

During myocardial ischemia/reperfusion (I/R), the generation of reactive oxygen species (ROS) contributes to post-reperfused cardiac injury and contractile dysfunction. Activation of NADPH oxidase (NOX) during reperfusion generates ROS, and exacerbates I/R injury. We hypothesize that reducing ROS formation through inhibition of NOX will attenuate myocardial I/R injury in isolated perfused rat hearts subjected to I(30min)/R(45min) compared to untreated I/R hearts. The cell-permeable NOX inhibiting peptide, gp91 ds/tat (RKKRRQRRR-CSTRIRRQL-Amide, MW=2452 g/mol, 20μM, n=5), significantly improved post-reperfused cardiac function compared to controls (n=15,

Espon-Interstrat. Espon in Integrated Territorial Strategies.

The INTERSTRAT project’s overall aim is “to encourage and facilitate the use of ESPON 2013 Programme findings in the creation and monitoring of Integrated Territorial Development Strategies (ITDS) and to support transnational learning about the actual and potential contribution of ESPON to integrated policy-making.” We defined integrated territorial development as ‘the process of shaping economic, social and environmental change through spatially sensitive policies and programmes’

B-cell dysregulation in Crohn's disease is partially restored with infliximab therapy

Background: B-cell depletion can improve a variety of chronic inflammatory diseases, but does not appear beneficial for patients with Crohn's disease. Objective: To elucidate the involvement of B cells in Crohn's disease, we here performed an 'in depth' analysis of intestinal and blood B-cells in this chronic inflammatory disease. Methods: Patients with Crohn's disease were recruited to study B-cell infiltrates in intestinal biopsies (n = 5), serum immunoglobulin levels and the phenotype and molecular characteristics of blood B-cell subsets (n = 21). The effects of infliximab treatment were studied in 9 patients. Results: Granulomatous tissue showed infiltrates of B lymphocytes rather than Ig-secreting plasma cells. Circulating transitional B cells and CD21low B cells were elevated. IgM memory B cells were reduced and natural effector cells showed decreased replication histories and somatic hypermutation (SHM) levels. In contrast, IgG and IgA memory B cells were normally present and their Ig gene transcripts carried increased SHM levels. The numbers of transitional and natural effector cells were normal in patients who responded clinically well to infliximab. Conclusions: B cells in patients with Crohn's disease showed signs of chronic stimulation with localization to granulomatous tissue and increased molecular maturation of IgA and IgG. Therapy with TNFα-blockers restored the defect in IgM memory B-cell generation and normalized transitional B-cell levels, making these subsets candidate markers for treatment monitoring. Together, these results suggest a chronic, aberrant B-cell response in patients with Crohn's disease, which could be targeted with new therapeutics that specifically regulate B-cell function

Signaling in Secret: Pay-for-Performance and the Incentive and Sorting Effects of Pay Secrecy

Key Findings: Pay secrecy adversely impacts individual task performance because it weakens the perception that an increase in performance will be accompanied by increase in pay; Pay secrecy is associated with a decrease in employee performance and retention in pay-for-performance systems, which measure performance using relative (i.e., peer-ranked) criteria rather than an absolute scale (see Figure 2 on page 5); High performing employees tend to be most sensitive to negative pay-for- performance perceptions; There are many signals embedded within HR policies and practices, which can influence employees’ perception of workplace uncertainty/inequity and impact their performance and turnover intentions; and When pay transparency is impractical, organizations may benefit from introducing partial pay openness to mitigate these effects on employee performance and retention