Calcium-Dependent Interplay of Lithium and Tricyclic Antidepressants, Amitriptyline and Desipramine, on N-methyl-D-aspartate Receptors.

The facilitated activity of N-methyl-D-aspartate receptors (NMDARs) in the central and peripheral nervous systems promotes neuropathic pain. Amitriptyline (ATL) and desipramine (DES) are tricyclic antidepressants (TCAs) whose anti-NMDAR properties contribute to their analgetic effects. At therapeutic concentrations <1 µM, these medicines inhibit NMDARs by enhancing their calcium-dependent desensitization (CDD). Li+, which suppresses the sodium−calcium exchanger (NCX) and enhances NMDAR CDD, also exhibits analgesia. Here, the effects of different [Li+]s on TCA inhibition of currents through native NMDARs in rat cortical neurons recorded by the patch-clamp technique were investigated. We demonstrated that the therapeutic [Li+]s of 0.5−1 mM cause an increase in ATL and DES IC50s of ~10 folds and ~4 folds, respectively, for the Ca2+-dependent NMDAR inhibition. The Ca2+-resistant component of NMDAR inhibition by TCAs, the open-channel block, was not affected by Li+. In agreement, clomipramine providing exclusively the NMDAR open-channel block is not sensitive to Li+. This Ca2+-dependent interplay between Li+, ATL, and DES could be determined by their competition for the same molecular target. Thus, submillimolar [Li+]s may weaken ATL and DES effects during combined therapy. The data suggest that Li+, ATL, and DES can enhance NMDAR CDD through NCX inhibition. This ability implies a drug−drug or ion−drug interaction when these medicines are used together therapeutically.

Prediction of long-term treatment outcome in HCV following 24 day PEG-IFN alpha-2b therapy using population pharmacokinetic-pharmacodynamic mixture modeling and classification analysis.

Mathematical models have been widely used for understanding the dynamics of the hepatitis C virus (HCV). We propose a method to predict final clinical outcome for 24 HIV-HCV - coinfected patients with the help of a mathematical model based on the first two weeks of PEG-IFN therapy. Applying a pharmacokinetic-pharmacodynamic (PKPD) approach, together with mixture models, to the adapted model of viral dynamics developed by Neumann et al., we have analyzed the influence of PEG-IFN on the kinetics and interaction of target cells, infected cells and virus mRNA. It was found that PEG-IFN pharmacokinetic parameters were similar in sustained virological responders and nonresponders, while the plasma PEG-IFN concentration that decreases HCV production by 50% (EC50) and the rate of infected cell death were different. The treatment outcome depended mainly on the initial viral mRNA concentration and the rate of infected cell death. The population PKPD approach with a mixture model enabled the determination of individual PKPD parameters and showed high sensitivity (93.5%) and specificity (97.4%) for the prediction of the treatment outcome.

Quantitative systems pharmacology model of α-synuclein pathology in Parkinson's disease-like mouse for investigation of passive immunotherapy mechanisms.

The main pathophysiological hallmark of Parkinson's disease (PD) is the accumulation of aggregated alpha-synuclein (αSyn). Microglial activation is an early event in PD and may play a key role in pathological αSyn aggregation and transmission, as well as in clearance of αSyn and immunotherapy efficacy. Our aim was to investigate how different proposed mechanisms of anti-αSyn immunotherapy may contribute to pathology reduction in various PD-like mouse models. Our mechanistic model of PD pathology in mouse includes αSyn production, aggregation, degradation and distribution in neurons, secretion into interstitial fluid, internalization, and subsequent clearance by neurons and microglia. It describes the influence of neuroinflammation on PD pathogenesis and dopaminergic neurodegeneration. Multiple data from mouse PD models were used for calibration and validation. Simulations of anti-αSyn passive immunotherapy adequately reproduce preclinical data and suggest that (1) immunotherapy is efficient in the reduction of aggregated αSyn in various models of PD-like pathology; (2) prevention of aSyn spread only does not reduce the pathology; (3) a decrease in microglial inflammatory activation and aSyn aggregation may be alternative therapy approaches in PD-like pathology.

Selective inhibitor of sodium-calcium exchanger, SEA0400, affects NMDA receptor currents and abolishes their calcium-dependent block by tricyclic antidepressants.

The open-channel block of N-methyl-D-aspartate receptors (NMDARs) and their calcium-dependent desensitization (CDD) represent conventional mechanisms of glutamatergic synapse regulation. In neurotrauma, neurodegeneration, and neuropathic pain the clinical benefits of cure with memantine, ketamine, Mg2+, and some tricyclic antidepressants are often attributed to NMDAR open-channel block, while possible involvement of NMDAR CDD in the therapy is not well established. Here the effects of selective high-affinity sodium-calcium exchanger (NCX) isoform 1 inhibitor, SEA0400, on NMDA-activated whole-cell currents and their block by amitriptyline, desipramine and clomipramine recorded by patch-clamp technique in cortical neurons of primary culture were studied. We demonstrated that in the presence of extracellular Ca2+, 50 nM SEA0400 caused a reversible decrease of the steady-state amplitude of NMDAR currents, whereas loading neurons with BAPTA or the removal of extracellular Ca2+ abolished the effect. The decrease did not exceed 30% of the amplitude and did not depend on membrane voltage. The external Mg2+ block and 50 nM SEA0400 inhibition of currents were additive, suggesting their independent modes of action. In the presence of Ca2+ SEA0400 speeded up the decay of NMDAR currents to the steady state determined by CDD. The measured IC50 value of 27 nM for SEA0400-induced inhibition coincides with that for NCX1. Presumably, SEA0400 effects are induced by an enhancement of NMDAR CDD through the inhibition of Ca2+ extrusion by NCX1. SEA0400, in addition, at nanomolar concentrations could interfere with Ca2+-dependent effect of tricyclic antidepressants. In the presence of 50 nM SEA0400, the IC50s for NMDAR inhibition by amitriptyline and desipramine increased by about 20 folds, as the Ca2+-dependent NMDAR inhibition disappeared. This observation highlights NCX1 involvement in amitriptyline and desipramine effects on NMDARs and unmasks competitive relationships between SEA0400 and these antidepressants. Neither amitriptyline nor desipramine could affect NCX3. The open-channel block of NMDARs by these substances was not affected by SEA0400. In agreement, SEA0400 did not change the IC50 for clomipramine, which acts as a pure NMDAR open-channel blocker. Thus, NCX seems to represent a promising molecular target to treat neurological disorders, because of the ability to modulate NMDARs by decreasing the open probability through the enhancement of their CDD.

Unveiling the Role of Cholesterol in Subnanomolar Ouabain Rescue of Cortical Neurons from Calcium Overload Caused by Excitotoxic Insults.

Na/K-ATPase maintains transmembrane ionic gradients and acts as a signal transducer when bound to endogenous cardiotonic steroids. At subnanomolar concentrations, ouabain induces neuroprotection against calcium overload and apoptosis of neurons during excitotoxic stress. Here, the role of lipid rafts in interactions between Na/K-ATPase, sodium-calcium exchanger (NCX), and N-methy-D-aspartate receptors (NMDARs) was investigated. We analyzed 0.5-1-nanometer ouabain's effects on calcium responses and miniature post-synaptic current (mEPSCs) frequencies of cortical neurons during the action of NMDA in rat primary culture and brain slices. In both objects, ouabain attenuated NMDA-evoked calcium responses and prevented an increase in mEPSC frequency, while the cholesterol extraction by methyl-ß-cyclodextrin prevented the effects. The data support the conclusions that (i) ouabain-induced inhibition of NMDA-elicited calcium response involves both pre- and post-synapse, (ii) the presence of astrocytes in the tripartite synapse is not critical for the ouabain effects, which are found to be similar in cell cultures and brain slices, and (iii) ouabain action requires the integrity of cholesterol-rich membrane microdomains in which the colocalization and functional interaction of NMDAR-transferred calcium influx, calcium extrusion by NCX, and Na/K-ATPase modulation of the exchanger occur. This regulation of the molecules by cardiotonic steroids may influence synaptic transmission, prevent excitotoxic neuronal death, and interfere with the pharmacological actions of neurological medicines.

Hallmarks of neurodegenerative disease: A systems pharmacology perspective.

Age-related central neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, are a rising public health concern and have been plagued by repeated drug development failures. The complex nature and poor mechanistic understanding of the etiology of neurodegenerative diseases has hindered the discovery and development of effective disease-modifying therapeutics. Quantitative systems pharmacology models of neurodegeneration diseases may be useful tools to enhance the understanding of pharmacological intervention strategies and to reduce drug attrition rates. Due to the similarities in pathophysiological mechanisms across neurodegenerative diseases, especially at the cellular and molecular levels, we envision the possibility of structural components that are conserved across models of neurodegenerative diseases. Conserved structural submodels can be viewed as building blocks that are pieced together alongside unique disease components to construct quantitative systems pharmacology (QSP) models of neurodegenerative diseases. Model parameterization would likely be different between the different types of neurodegenerative diseases as well as individual patients. Formulating our mechanistic understanding of neurodegenerative pathophysiology as a mathematical model could aid in the identification and prioritization of drug targets and combinatorial treatment strategies, evaluate the role of patient characteristics on disease progression and therapeutic response, and serve as a central repository of knowledge. Here, we provide a background on neurodegenerative diseases, highlight hallmarks of neurodegeneration, and summarize previous QSP models of neurodegenerative diseases.

Monoclonal antibody therapy efficacy can be boosted by combinations with other treatments: Predictions using an integrated Alzheimer's Disease Platform.

For many years, clinical research in Alzheimer's disease (AD) has focused on attempts to identify the most explicit biomarker, namely amyloid beta. Unfortunately, the numerous therapies that have been developed have failed in clinical practice. AD arises as a consequence of multiple factors, and as such it requires a more mechanistic analytical approach than statistical modeling. Quantitative systems pharmacology modeling is a valuable tool for drug development. It utilizes in vitro data for the calibration of parameters, embeds them into physiologically based structures, and explores translation between animals and humans. Such an approach allows for a quantitative study of the dynamics of the interactions between multiple factors or variables. Here, we present an overview of the quantitative translational model in AD, which embraces current preclinical and clinical data. The previously published description of amyloid physiology has been updated and joined with a model for tau pathology and multiple intraneuronal processes responsible for cellular transport, metabolism, or proteostasis. In addition, several hypotheses regarding the best correlates of cognitive deterioration have been validated using clinical data. Here, the amyloid hypothesis was unable to predict the aducanumab clinical trial data, whereas simulations of cognitive impairment coupled with tau seeding or neuronal breakdown (expressed as caspase activity) matched the data. A satisfactory validation of the data from multiple preclinical and clinical studies was followed by an attempt to predict the results of combinatorial treatment with targeted immunotherapy and activation of autophagy using rapamycin. The combination is predicted to yield better efficacy than immunotherapy alone.

Quantitative systems pharmacology in neuroscience: Novel methodologies and technologies.

The development and application of quantitative systems pharmacology models in neuroscience have been modest relative to other fields, such as oncology and immunology, which may reflect the complexity of the brain. Technological and methodological advancements have enhanced the quantitative understanding of brain physiology and pathophysiology and the effects of pharmacological interventions. To maximize the knowledge gained from these novel data types, pharmacometrics modelers may need to expand their toolbox to include additional mathematical and statistical frameworks. A session was held at the 10th annual American Conference on Pharmacometrics (ACoP10) to highlight several recent advancements in quantitative and systems neuroscience. In this mini-review, we provide a brief overview of technological and methodological advancements in the neuroscience therapeutic area that were discussed during the session and how these can be leveraged with quantitative systems pharmacology modeling to enhance our understanding of neurological diseases. Microphysiological systems using human induced pluripotent stem cells (IPSCs), digital biomarkers, and large-scale imaging offer more clinically relevant experimental datasets, enhanced granularity, and a plethora of data to potentially improve the preclinical-to-clinical translation of therapeutics. Network neuroscience methodologies combined with quantitative systems models of neurodegenerative disease could help bridge the gap between cellular and molecular alterations and clinical end points through the integration of information on neural connectomics. Additional topics, such as the neuroimmune system, microbiome, single-cell transcriptomic technologies, and digital device biomarkers, are discussed in brief.

GluN2 Subunit-Dependent Redox Modulation of NMDA Receptor Activation by Homocysteine.

Homocysteine (HCY) molecule combines distinct pharmacological properties as an agonist of N-methyl-d-aspartate receptors (NMDARs) and a reducing agent. Whereas NMDAR activation by HCY was elucidated, whether the redox modulation contributes to its action is unclear. Here, using patch-clamp recording and imaging of intracellular Ca2+, we study dithiothreitol (DTT) effects on currents and Ca2+ responses activated by HCY through native NMDARs and recombinant diheteromeric GluN1/2A, GluN1/2B, and GluN1/2C receptors. Within a wide range (1-800 µM) of [HCY]s, the concentration-activation relationships for recombinant NMDARs revealed a biphasicness. The high-affinity component obtained between 1 and 100 µM [HCY]s corresponding to the NMDAR activation was not affected by 1 mM DTT. The low-affinity phase observed at [HCY]s above 200 µM probably originated from thiol-dependent redox modulation of NMDARs. The reduction of NMDAR disulfide bonds by either 1 mM DTT or 1 mM HCY decreased GluN1/2A currents activated by HCY. In contrast, HCY-elicited GluN1/2B currents were enhanced due to the remarkable weakening of GluN1/2B desensitization. In fact, cleaving NMDAR disulfide bonds in neurons reversed the HCY-induced Ca2+ accumulation, making it dependent on GluN2B- rather than GluN2A-containing NMDARs. Thus, estimated concentrations for the HCY redox effects exceed those in the plasma during intermediate hyperhomocysteinemia but may occur during severe hyperhomocysteinemia.

Calcium Export from Neurons and Multi-Kinase Signaling Cascades Contribute to Ouabain Neuroprotection in Hyperhomocysteinemia.

Pathological homocysteine (HCY) accumulation in the human plasma, known as hyperhomocysteinemia, exacerbates neurodegenerative diseases because, in the brain, this amino acid acts as a persistent N-methyl-d-aspartate receptor agonist. We studied the effects of 0.1-1 nM ouabain on intracellular Ca2+ signaling, mitochondrial inner membrane voltage (φmit), and cell viability in primary cultures of rat cortical neurons in glutamate and HCY neurotoxic insults. In addition, apoptosis-related protein expression and the involvement of some kinases in ouabain-mediated effects were evaluated. In short insults, HCY was less potent than glutamate as a neurotoxic agent and induced a 20% loss of φmit, whereas glutamate caused a 70% decrease of this value. Subnanomolar ouabain exhibited immediate and postponed neuroprotective effects on neurons. (1) Ouabain rapidly reduced the Ca2+ overload of neurons and loss of φmit evoked by glutamate and HCY that rescued neurons in short insults. (2) In prolonged 24 h excitotoxic insults, ouabain prevented neuronal apoptosis, triggering proteinkinase A and proteinkinase C dependent intracellular neuroprotective cascades for HCY, but not for glutamate. We, therefore, demonstrated here the role of PKC and PKA involving pathways in neuronal survival caused by ouabain in hyperhomocysteinemia, which suggests existence of different appropriate pharmacological treatment for hyperhomocysteinemia and glutamate excitotoxicity.

Kinetic modeling as a tool to integrate multilevel dynamic experimental data.

The metabolic networks are the most well-studied biochemical systems, with an abundance of in vitro and in vivo data available for quantitative estimation of its kinetic parameters. In this chapter, we present our approach to developing mathematical description of metabolic pathways. The model-based integration of reaction kinetics and the utilization of different types of experimental data including temporal dependencies have been described in detail. Software package DBSolve7 which allows us to develop kinetic model of the biochemical system and integrate experimental data has been presented.

A mathematical model of multisite phosphorylation of tau protein.

Abnormal tau metabolism followed by formation of tau deposits causes a number of neurodegenerative diseases called tauopathies including Alzheimer's disease. Hyperphosphorylation of tau protein precedes tau aggregation and is a topic of interest for the development of pharmacological interventions to prevent pathology progression at early stages. The development of a mathematical model of multisite phosphorylation of tau would be helpful for searching for the targets of pharmacological interventions and candidates for biomarkers of pathology progression. In the present study, we for the first time developed a model of multisite phosphorylation of tau protein and elucidated the relative contribution of kinases to phosphorylation of distinct sites. The model describes phosphorylation of tau or PKA-prephosphorylated tau by GSK3ß and CDK5 and dephosphorylation by PP2A, accurately reproducing the data for short-term kinetics of tau (de)phosphorylation. Our results suggest that kinase inhibition may more specifically prevent tau hyperphosphorylation, e.g., on PHF sites, which are key biomarkers of pathological changes in Alzheimer's disease. The main features of our model are partial phosphorylation of tau residues and merging of random and sequential mechanisms of multisite phosphorylation within the framework of the probability-based approach assuming independent phosphorylation events.

Correction: A mathematical model of multisite phosphorylation of tau protein.

[This corrects the article DOI: 10.1371/journal.pone.0192519.].

Developmental Changes of Synaptic and Extrasynaptic NMDA Receptor Expression in Rat Cerebellar Neurons In Vitro.

Transient expression of different NMDA receptors (NMDARs) plays a role in development of the cerebellum. Whether similar processes undergo during neuronal differentiation in culture is not clearly understood. We studied NMDARs in cerebellar neurons in cultures of 7 and 21 days in vitro (DIV) using immunocytochemical and electrophysiological approaches. Whereas at 7 DIV, the vast majority of neurons were immunopositive for GluN2 subunits, further synaptoginesis was accompanied by the time-dependent loss of NMDARs. In contrast to GluN2B- and GluN2C-containing NMDARs, which at 7 DIV exhibited homogenous distribution in extrasynaptic regions, GluN2A-containing receptors were aggregated in spots both in cell bodies and dendrites. Double staining for GluN2A subunits and synaptophysin, a widely used marker for presynaptic terminals, revealed their co-localization in about 75% of dendrite GluN2A fluorescent spots, suggesting postsynaptic origin of GluN2A subunits. In agreement, diheteromeric GluN2A-containing NMDARs contributed to postsynaptic currents recorded in neurons throughout the timescale under study. Diheteromeric GluN2B-containing NMDARs escaped postsynaptic regions during differentiation. Finally, the developmental switch favored the expression of triheteromeric NMDARs assembled of 2 GluN1/1 GluN2B/1 GluN2C or GluN2D subunits in extrasynaptic regions. At 21 DIV, these receptors represented over 60% of the NMDAR population. Thus, cerebellar neurons in primary culture undergo transformations with respect to the expression of di- and triheteromeric NMDARs that should be taken into account when studying cellular aspects of their pharmacology and functions.

Studying the Progression of Amyloid Pathology and Its Therapy Using Translational Longitudinal Model of Accumulation and Distribution of Amyloid Beta.

Long-term effects of amyloid targeted therapy can be studied using a mechanistic translational model of amyloid beta (Aß) distribution and aggregation calibrated on published data in mouse and human species. Alzheimer disease (AD) pathology is modeled utilizing age-dependent pathological evolution for rate constants and several variants of explicit functions for Aß toxicity influencing cognitive outcomes (Adas-cog). Preventive Aß targeted therapies were simulated to minimize the Aß difference from healthy physiological levels. Therapeutic targeted simulations provided similar predictions for mouse and human studies. Our model predicts that: (1) at least 1 year (2 years for preclinical AD) of treatment is needed to observe cognitive effects; (2) under the hypothesis with functional importance of Aß, a 15% decrease in Aß (using an imaging biomarker) is related to 15-20% cognition improvement by immunotherapy. Despite negative outcomes in clinical trials, Aß continues to remain a prospective target demanding careful assessment of mechanistic effect and duration of trial design.

Regulation of leukotriene and 5oxoETE synthesis and the effect of 5-lipoxygenase inhibitors: a mathematical modeling approach.

BACKGROUND: 5-lipoxygenase (5-LO) is a key enzyme in the synthesis of leukotrienes and 5-Oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (oxoETE). These inflammatory signaling molecules play a role in the pathology of asthma and so 5-LO inhibition is a promising target for asthma therapy. The 5-LO redox inhibitor zileuton (Zyflo IR/CR(®)) is currently marketed for the treatment of asthma in adults and children, but widespread use of zileuton is limited by its efficacy/safety profile, potentially related to its redox characteristics. Thus, a quantitative, mechanistic description of its functioning may be useful for development of improved anti-inflammatory targeting this mechanism. RESULTS: A mathematical model describing the operation of 5-LO, phospholipase A2, glutathione peroxidase and 5-hydroxyeicosanoid dehydrogenase was developed. The catalytic cycles of the enzymes were reconstructed and kinetic parameters estimated on the basis of available experimental data. The final model describes each stage of cys-leukotriene biosynthesis and the reactions involved in oxoETE production. Regulation of these processes by substrates (phospholipid concentration) and intracellular redox state (concentrations of reduced glutathione, glutathione (GSH), and lipid peroxide) were taken into account. The model enabled us to reveal differences between redox and non-redox 5-LO inhibitors under conditions of oxidative stress. Despite both redox and non-redox inhibitors suppressing leukotriene A4 (LTA4) synthesis, redox inhibitors are predicted to increase oxoETE production, thus compromising efficacy. This phenomena can be explained in terms of the pseudo-peroxidase activity of 5-LO and the ability of lipid peroxides to transform 5-LO into its active form even in the presence of redox inhibitors. CONCLUSIONS: The mathematical model developed described quantitatively different mechanisms of 5-LO inhibition and simulations revealed differences between the potential therapeutic outcomes for these mechanisms.

Substrate binding of gelatinase B induces its enzymatic activity in the presence of intact propeptide.

Expression of gelatinase B (matrix metalloprotease 9) in human placenta is developmentally regulated, presumably to fulfill a proteolytic function. Here we demonstrate that gelatinolytic activity in situ, in tissue sections of term placenta, is co-localized with gelatinase B. Judging by molecular mass, however, all the enzyme extracted from this tissue was found in a proform. To address this apparent incongruity, we examined the activity of gelatinase B bound to either gelatin- or type IV collagen-coated surfaces. Surprisingly, we found that upon binding, the purified proenzyme acquired activity against both the fluorogenic peptide (7-methoxycoumarin-4-yl)-acetic acid (MCA)-Pro-Leu-Gly-Leu-3-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl-Ala-Arg-NH(2) and gelatin substrates, whereas its propeptide remained intact. These results suggest that although activation of all known matrix metalloproteases in vitro is accomplished by proteolytic processing of the propeptide, other mechanisms, such as binding to a ligand or to a substrate, may lead to a disengagement of the propeptide from the active center of the enzyme, causing its activation.