In multiple sclerosis (MS), demyelinated CNS lesions fail to sufficiently remyelinate, despite the presence of oligodendrocyte precursor cells (OPCs) capable of differentiating into mature ...oligodendrocytes. MS lesions contain damaged myelin debris that can inhibit OPC maturation and hinder repair. rHIgM22 is an experimental human recombinant IgM antibody that promotes remyelination in animal models and is being examined in patients with MS. rHIgM22 binds to CNS myelin and partially rescues OPC process outgrowth on myelin. Since rHIgM22 does not affect OPC process outgrowth in vitro on permissive substrate, we examined the possibility that it acts by enhancing phagocytic clearance of myelin debris by microglia. In this study, we tested if rHIgM22 binding could tag myelin for microglial phagocytosis. A mouse microglial cell line and primary rat microglia were treated with myelin and rHIgM22 and assayed for myelin phagocytosis. We found that: 1) rHIgM22 stimulates myelin phagocytosis in a dose-dependent manner; 2) rHIgM22-mediated myelin phagocytosis requires actin polymerization; and 3) rHIgM22-stimulation of myelin phagocytosis requires activity of rHIgM22 Fc domain and activation of Complement Receptor 3. Since myelin inhibits OPC differentiation, stimulation of phagocytic clearance of damaged myelin may be an important means by which rHIgM22 promotes remyelination.
In addition to their well-known functions in cellular energy transduction, mitochondria play an important role in modulating the amplitude and time course of intracellular Ca2+signals. In many cells, ...mitochondria act as Ca2+buffers by taking up and releasing Ca2+, but this simple buffering action by itself often cannot explain the organelle's effects on Ca2+signaling dynamics. Here we describe the functional interaction of mitochondria with store-operated Ca2+channels in T lymphocytes as a mechanism of mitochondrial Ca2+signaling. In Jurkat T cells with functional mitochondria, prolonged depletion of Ca2+stores causes sustained activation of the store-operated Ca2+current, ICRAC(CRAC, Ca2+release-activated Ca2+). Inhibition of mitochondrial Ca2+uptake by compounds that dissipate the intramitochondrial potential unmasks Ca2+-dependent inactivation of ICRAC. Thus, functional mitochondria are required to maintain CRAC-channel activity, most likely by preventing local Ca2+accumulation near sites that govern channel inactivation. In cells stimulated through the T-cell antigen receptor, acute blockade of mitochondrial Ca2+uptake inhibits the nuclear translocation of the transcription factor NFAT in parallel with CRAC channel activity and Ca2+ielevation, indicating a functional link between mitochondrial regulation of ICRACand T-cell activation. These results demonstrate a role for mitochondria in controlling Ca2+channel activity and signal transmission from the plasma membrane to the nucleus.
A porcine model of spinal cord injury (SCI) was used to evaluate the neuroprotective effects of magnesium chloride (MgCl
) within a polyethylene glycol (PEG) formulation, called "AC105" (Acorda ...Therapeutics Inc., Ardsley, NY). Specifically, we tested the hypothesis that AC105 would lead to greater tissue sparing at the injury site and improved behavioral outcome when delivered in a clinically realistic time window post-injury. Four hours after contusion/compression injury, Yucatan minipigs were randomized to receive a 30-min intravenous infusion of AC105, magnesium sulfate (MgSO
), or saline. Animals received 4 additional infusions of the same dose at 6-h intervals. Behavioral recovery was tested for 12 weeks using two-dimensional (2D) kinematics during weight-supported treadmill walking and the Porcine Injury Behavior Scale (PTIBS), a 10-point locomotion scale. Spinal cords were evaluated ex vivo by diffusion-weighted magnetic resonance imaging (MRI) and subjected to histological analysis. Treatment with AC105 or MgSO
did not result in improvements in locomotor recovery on the PTIBS or in 2D kinematics on weight-supported treadmill walking. Diffusion weighted imaging (DWI) showed severe loss of tissue integrity at the impact site, with decreased fractional anisotropy and increased mean diffusivity; this was not improved with AC105 or MgSO
treatment. Histological analysis revealed no significant increase in gray or white matter sparing with AC105 or MgSO
treatment. Finally, AC105 did not result in higher Mg
levels in CSF than with the use of standard MgSO
. In summary, when testing AC105 in a porcine model of SCI, we were unable to reproduce the promising therapeutic benefits observed previously in less-severe rodent models of SCI.
Both preclinical evidence and clinical evidence suggest that α7 nicotinic acetylcholine receptor activation (α7nAChR) improves cognitive function, the decline of which is associated with conditions ...such as Alzheimer's disease and schizophrenia. Moreover, allosteric modulation of α7nAChR is an emerging therapeutic strategy in an attempt to avoid the rapid desensitization properties associated with the α7nAChR after orthosteric activation. We used a calcium assay to screen for positive allosteric modulators (PAMs) of α7nAChR and report on the pharmacologic characterization of the novel compound RO5126946 (5-chloro-N-(1S,3R)-2,2-dimethyl-3-(4-sulfamoyl-phenyl)-cyclopropyl-2-methoxy-benzamide), which allosterically modulates α7nAChR activity. RO5126946 increased acetylcholine-evoked peak current and delayed current decay but did not affect the recovery of α7nAChRs from desensitization. In addition, RO5126946's effects were absent when nicotine-evoked currents were completely blocked by coapplication of the α7nAChR-selective antagonist methyl-lycaconitine. RO5126946 enhanced α7nAChR synaptic transmission and positively modulated GABAergic responses. The absence of RO5126946 effects at human α4β2nAChR and 5-hydroxytryptamine 3 receptors, among others, indicated selectivity for α7nAChRs. In vivo, RO5126946 is orally bioavailable and brain-penetrant and improves associative learning in a scopolamine-induced deficit model of fear conditioning in rats. In addition, procognitive effects of RO5126946 were investigated in the presence of nicotine to address potential pharmacologic interactions on behavior. RO5126946 potentiated nicotine's effects on fear memory when both compounds were administered at subthreshold doses and did not interfere with procognitive effects observed when both compounds were administered at effective doses. Overall, RO5126946 is a novel α7nAChR PAM with cognitive-enhancing properties.
Neuregulins are important growth factors involved in cardiac development and response to stress. Certain isoforms and fragments of neuregulin have been found to be cardioprotective. The effects of a ...full-length neuregulin-1β isoform, glial growth factor 2 (GGF2; USAN/INN; also called cimaglermin) were investigated in vitro. Various dosing regimens were then evaluated for their effects on left ventricular (LV) function in rats with surgically-induced myocardial infarction. In vitro, GGF2 bound with high affinity to erythroblastic leukemia viral oncogene (ErbB) 4 receptors, potently promoted Akt phosphorylation, as well as reduced cell death following doxorubicin exposure in HL1 cells. Daily GGF2 treatment beginning 7–14 days after left anterior descending coronary artery ligation produced improvements in LV ejection fraction and other measures of LV function and morphology. The improvements in LV function (e.g. 10% point increase in absolute LV ejection fraction) with GGF2 were dose-dependent. LV performance was substantially improved when GGF2 treatment was delivered infrequently, despite a serum half-life of less than 2h and could be maintained for more than 10 months with treatment once weekly or once every 2 weeks. These studies confirm previous findings that GGF2 may improve contractile performance in the failing rat heart and that infrequent exposure to GGF2 may improve LV function and impact remodeling in the failing myocardium. GGF2 is now being developed for the treatment of heart failure in humans.
Molecules that inhibit store‐operated calcium entry (SOCE) are potentially useful immunomodulating agents. The identification of proteins involved in this pathway may further enable the ...identification of selective inhibitors. Herein we document some examples of the small‐molecule inhibitors of SOCE that have been reported to date. We also describe methods that were used to characterize the mechanism of action of these inhibitors.
Controlled variation in intracellular calcium concentration is a key component of the immune response signaling pathway in lymphocytes. Store‐operated calcium entry (SOCE) in these cells provides a prolonged increase in cytoplasmic Ca2+ concentrations and ultimately leads to the production of pro‐inflammatory cytokines. Molecules that inhibit SOCE could therefore be useful immunomodulating agents for the treatment of rheumatoid arthritis, psoriasis, inflammatory bowel disease, and other conditions. Although the presence of the SOCE signaling pathway in lymphocytes and other cells involved in the immune response has been known for many years, key proteins involved in SOCE were identified only recently. The identification of these proteins may further enable the identification of agents that inhibit SOCE without affecting other cellular processes. This contribution documents representative examples of the small‐molecule inhibitors of SOCE that have been reported to date. Where possible, methods that were used to characterize the mechanism of action of the inhibitors are also described.
Molecules that inhibit store‐operated calcium entry (SOCE) are potentially useful immunomodulating agents. The identification of proteins involved in this pathway may further enable the identification of selective inhibitors. Herein we document some examples of the small‐molecule inhibitors of SOCE that have been reported to date. We also describe methods that were used to characterize the mechanism of action of these inhibitors.
The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been ...reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e., not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gαq.
Magnesium (Mg
) homeostasis is impaired following spinal cord injury (SCI) and the loss of extracellular Mg
contributes to secondary injury by various mechanisms, including glutamate neurotoxicity. ...The neuroprotective effects of high dose Mg
supplementation have been reported in many animal models. Recent studies found that lower Mg
doses also improved neurologic outcomes when Mg
was formulated with polyethylene glycol (PEG), suggesting that a PEG/ Mg
formulation might increase Mg
delivery to the injured spinal cord, compared with that of MgSO
alone. Here, we assessed spinal extracellular Mg
and glutamate levels following SCI in rats using microdialysis. Basal levels of extracellular Mg
(∼0.5 mM) were significantly reduced to 0.15 mM in the core and 0.12 mM in the rostral peri-lesion area after SCI. A single intravenous infusion of saline or of MgSO
at 192 μmoL/kg did not significantly change extracellular Mg
concentrations. However, a single infusion of AC105 (a MgCl
in PEG) at an equimolar Mg
dose significantly increased the Mg
concentration to 0.3 mM (core area) and 0.25 mM (rostral peri-lesion area). Moreover, multiple AC105 treatments completely restored the depleted extracellular Mg
concentrations after SCI to levels in the uninjured spinal cord. Repeated MgSO
infusions slightly increased the Mg
concentrations while saline infusion had no effect. In addition, AC105 treatment significantly reduced extracellular glutamate levels in the lesion center after SCI. These results indicate that intravenous infusion of PEG-formulated Mg
normalized the Mg
homeostasis following SCI and reduced potentially neurotoxic glutamate levels, consistent with a neuroprotective mechanism of blocking excitotoxicity.
Abstract Neurotensin (NT) is a neuropeptide implicated in the pathophysiology of schizophrenia and in mediating the efficacy of antipsychotic drugs. NT is also involved in the regulation of body ...temperature and pain sensitivity. Using neurotensin receptor 1 (NTR1) knockout (KO) and wild-type (WT) mice, these studies evaluated the involvement of NTR1 in the behavioral responses produced by peripheral administration of NT agonists (NT-2 and NT69L). Animals were characterized in paradigms designed to assess hypothermia, antinociception, and antipsychotic-like effects. Under basal conditions, there were no phenotypic differences between NTR1 KO and WT mice. In WT mice, both NTR1 agonists decreased core body temperature (active doses in mg/kg, i.p., for NT-2 and NT69L, respectively: 1 and 3), increased tail withdrawal latencies (1 and 3), produced decreased spontaneous climbing (0.1, 0.3, 1 and 1, 3, 10) and reversed apomorphine-induced climbing (0.3, 1 and 1, 3). In contrast, none of the effects of either agonist were present in KO mice. These results suggest that NTR1: (1) does not play a major role in the control of basal thermoregulation, nociception or psychomotor stimulation in mice (barring possible developmental plasticity), (2) does mediate these behavioral responses to NT agonists, and (3) may play a role in the potential antipsychotic effects of these agonists.
A number of drugs and drug candidates, including fenfluramine and ergot derivatives, are associated with valvulopathy in humans; however, these responses are poorly predicted from animal studies.
In ...vitro and
in vivo evidence suggests that these compounds exert their pathological effect through activation of serotonin 2B receptor (5HT2BR) signaling. However, the variable effect of fenfluramine and other 5HT2BR agonists in rodents has cast doubt on the relevance of animal findings to predicting human risk. Herein, a candidate compound, RO3013, induced subendocardial cell proliferation in the mitral and tricuspid valves in rats after only 3 days of daily dosing. Additionally, there was a treatment-related increase in immunostaining of the proliferation marker Ki67, and phosphorylated Smad3 in the heart indicative of TGFβ signaling co-localized with 5HT2BR expression. To substantiate the hypothesis that RO3013-induced valvular proliferation is secondary to 5HT2BR activation, the compound was evaluated
in vitro and found to bind to the human 5HT2BR with a
K
i of 3.8
μM; however, it was virtually devoid of agonist activity in a functional assay in human cells. By contrast, RO3013 bound to the rat 5HT2BR with a
K
i of 1.2
μM and activated the receptor with an EC50 of 0.5
μM. This agonist potency estimate is in good agreement with the free plasma concentrations of RO3013 at which valvular proliferation was observed. These results suggest that the rat may be susceptible to 5HT2BR-mediated valvular proliferation similar to humans; yet, the significant differences between binding and functional activities can be a possible explanation for the observed species-selective receptor responses.
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