Human Remyelination Promoting Antibody Stimulates Astrocytes Proliferation Through Modulation of the Sphingolipid Rheostat in Primary Rat Mixed Glial Cultures.

Remyelination promoting human IgMs effectively increase the number of myelinated axons in animal models of multiple sclerosis. Hence, they ultimately stimulate myelin production by oligodendrocytes (OLs); however, their exact mechanism of action remains to be elucidated, and in particular, it remains unclear whether they are directly targeting OLs, or their action is mediated by effects on other cell types. We assessed the effect of remyelination promoting antibody rHIgM22 on the proliferative response and on the ceramide/sphingosine 1-phosphate rheostat in mixed glial cell cultures (MGCs). rHIgM22 treatment caused a time-dependent increase in PDGFαR protein in MGCs. Forty-eight hours of treatment with rHIgM22 induced a dose-dependent proliferative response (evaluated as total cell number and as EdU(+) cell number) in MGCs. When the proliferation response of MGCs to rHIgM22 was analyzed as a function of the cell types, the most significant proliferative response was associated with GLAST(+) cells, i.e., astrocytes. In many cell types, the balance between different sphingolipid mediators (the "sphingolipid rheostat"), in particular ceramide and sphingosine 1-phosphate, is critical in determining the cell fate. rHIgM22 treatment in MGCs induced a moderate but significant inhibition of total acidic sphingomyelinase activity (measured in vitro on cell lysates), the main enzyme responsible for the stimulus-mediated production of ceramide, when treatment was performed in serum containing medium, but no significant differences were observed when antibody treatment was performed in the absence of serum. Moreover, rHIgM22 treatment, either in the presence or in absence of serum, had no effects on ceramide levels. On the other hand, rHIgM22 treatment for 24 h induced increased production and release of sphingosine 1-phosphate in the extracellular milieu of MGC. Release of sphingosine 1-phosphate upon rHIgM22 treatment was strongly reduced by a selective inhibitor of PDGFαR. Increased sphingosine 1-phosphate production does not seem to be mediated by regulation of the biosynthetic enzymes, sphingosine kinase 1 and 2, since protein levels of these enzymes and phosphorylation of sphingosine kinase 1 were unchanged upon rHIgM22 treatment. Instead, we observed a significant reduction in the levels of sphingosine 1-phosphate lyase 1, one of the key catabolic enzymes. Remarkably, rHIgM22 treatment under the same experimental conditions did not induce changes in the production and/or release of sphingosine 1-phosphate in pure astrocyte cultures. Taken together, these data suggest that rHIgM22 indirectly influences the proliferation of astrocytes in MGCs, by affecting the ceramide/sphingosine 1-phosphate balance. The specific cell population directly targeted by rHIgM22 remains to be identified, however our study unveils another aspect of the complexity of rHIgM22-induced remyelinating effect.

Human IgM antibody rHIgM22 promotes phagocytic clearance of myelin debris by microglia.

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.

Transient Changes in Hepatic Physiology That Alter Bilirubin and Bile Acid Transport May Explain Elevations in Liver Chemistries Observed in Clinical Trials of GGF2 (Cimaglermin Alfa).

GGF2 is a recombinant human neuregulin-1ß in development for chronic heart failure. Phase 1 clinical trials of GGF2 were put on hold when transient elevations in serum aminotransferases and total bilirubin were observed in 2 of 43 subjects who received single doses of GGF2 at 1.5 or 0.378 mg/kg. However, aminotransferase elevations were modest and not typical of liver injury sufficient to result in elevated serum bilirubin. Cynomolgus monkeys administered a single 15 mg/kg dose of GGF2 had similar transient elevations in serum aminotransferases and bilirubin as well as transient elevations in serum bile acids. However, no hepatocellular necrosis was observed in liver biopsies obtained during peak elevations. When sandwich-cultured human hepatocytes were treated with GGF2 for up to 72 h at concentrations approximately 0.8-fold average plasma Cmax for the 0.378 mg/kg dose, no cytotoxicity was observed. Gene expression profiling identified approximately 50% reductions in mRNAs coding for bilirubin transporters and bile acid conjugating enzymes, as well as changes in expression of additional genes mimicking the interleukin-6-mediated acute phase response. Similar gene expression changes were observed in GGF2-treated HepG2 cells and primary monkey hepatocytes. Additional studies conducted in sandwich-cultured human hepatocytes revealed a transient and GGF2 concentration-dependent decrease in hepatocyte bile acid content and biliary clearance of taurocholate without affecting biliary taurocholate efflux. Taken together, these data suggest that GGF2 does not cause significant hepatocellular death, but transiently modifies hepatic handling of bilirubin and bile acids, effects that may account for the elevations in serum bilirubin observed in the clinical trial subjects.

Effects of neuregulin GGF2 (cimaglermin alfa) dose and treatment frequency on left ventricular function in rats following myocardial infarction.

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-1ß 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.

AC105 Increases Extracellular Magnesium Delivery and Reduces Excitotoxic Glutamate Exposure within Injured Spinal Cords in Rats.

Magnesium (Mg2+) homeostasis is impaired following spinal cord injury (SCI) and the loss of extracellular Mg2+ contributes to secondary injury by various mechanisms, including glutamate neurotoxicity. The neuroprotective effects of high dose Mg2+ supplementation have been reported in many animal models. Recent studies found that lower Mg2+ doses also improved neurologic outcomes when Mg2+ was formulated with polyethylene glycol (PEG), suggesting that a PEG/ Mg2+ formulation might increase Mg2+ delivery to the injured spinal cord, compared with that of MgSO4 alone. Here, we assessed spinal extracellular Mg2+ and glutamate levels following SCI in rats using microdialysis. Basal levels of extracellular Mg2+ (∼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 MgSO4 at 192 µmoL/kg did not significantly change extracellular Mg2+ concentrations. However, a single infusion of AC105 (a MgCl2 in PEG) at an equimolar Mg2+ dose significantly increased the Mg2+ concentration to 0.3 mM (core area) and 0.25 mM (rostral peri-lesion area). Moreover, multiple AC105 treatments completely restored the depleted extracellular Mg2+ concentrations after SCI to levels in the uninjured spinal cord. Repeated MgSO4 infusions slightly increased the Mg2+ 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 Mg2+ normalized the Mg2+ homeostasis following SCI and reduced potentially neurotoxic glutamate levels, consistent with a neuroprotective mechanism of blocking excitotoxicity.

The Evaluation of Magnesium Chloride within a Polyethylene Glycol Formulation in a Porcine Model of Acute Spinal Cord Injury.

A porcine model of spinal cord injury (SCI) was used to evaluate the neuroprotective effects of magnesium chloride (MgCl2) 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 (MgSO4), 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 MgSO4 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 MgSO4 treatment. Histological analysis revealed no significant increase in gray or white matter sparing with AC105 or MgSO4 treatment. Finally, AC105 did not result in higher Mg2+ levels in CSF than with the use of standard MgSO4. 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.

Isoproterenol acts as a biased agonist of the alpha-1A-adrenoceptor that selectively activates the MAPK/ERK pathway.

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 ß2-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 ß-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.

Characterization of RO5126946, a Novel α7 nicotinic acetylcholine receptor-positive allosteric modulator.

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 α4ß2nAChR 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.

Effects of neurotensin-2 receptor deletion on sensorimotor gating and locomotor activity.

Endogenous neurotensin (NT) has been implicated in brain processes relevant to schizophrenia as well as the therapeutic effects of antipsychotic drugs (APDs) used to treat this disorder. Converging evidence suggests that NT1 receptors mediate the antipsychotic-like effects of NT, such as prepulse inhibition (PPI) elevation. However, the role of NT2 receptors in these effects is not known. To investigate the contribution of NT2 receptors to the regulation of PPI, we measured baseline PPI and acoustic startle response (ASR), in male and female wild type (WT) and NT2 knockout (KO) mice. For comparison, we also measured locomotor activity. Baseline PPI was significantly elevated in both male (P<0.01) and female (P<0.01) NT2 KO compared to WT mice, while ASR was significantly decreased in KO mice of both genders (P<0.01). In contrast, female but not male KO mice exhibited significantly less baseline ambulations (P<0.05). These data support the regulation of baseline PPI, ASR and locomotor activity by endogenous NT acting at the NT2 receptor. Further studies investigating the role of NT2 receptors in the modulation of APD-like effects are warranted.

Mechanism of subendocardial cell proliferation in the rat and relevance for understanding drug-induced valvular heart disease in humans.

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 TGFß 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 3.8µM; 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 1.2µM and activated the receptor with an EC50 of 0.5µM. 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.

Small-molecule inhibitors of store-operated calcium entry.

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 Ca(2+) 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.

Involvement of the neurotensin receptor 1 in the behavioral effects of two neurotensin agonists, NT-2 and NT69L: lack of hypothermic, antinociceptive and antipsychotic actions in receptor knockout mice.

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.

Facilitatory interplay in alpha 1a and beta 2 adrenoceptor function reveals a non-Gq signaling mode: implications for diversification of intracellular signal transduction.

Agonist occupied alpha(1)-adrenoceptors (alpha(1)-ARs) engage several signaling pathways, including phosphatidylinositol hydrolysis, calcium mobilization, arachidonic acid release, mitogen-activated protein (MAP) kinase activation, and cAMP accumulation. The natural agonist norepinephrine (NE) activates with variable affinity and intrinsic efficacy all adrenoceptors, and in cells that coexpress alpha(1)- and beta-AR subtypes, such as cardiomyocytes, this leads to coactivation of multiple downstream pathways. This may result in pathway cross-talk with significant consequences to heart physiology and pathologic state. To dissect signaling components involved specifically in alpha(1A)- and beta(2)-AR signal interplay, we have developed a recombinant model system that mimics the levels of receptor expression observed in native cells. We followed intracellular Ca(2+) mobilization to monitor in real time the activation of both G(q) and G(s) pathways. We found that coactivation of alpha(1A)- and beta(2)-AR by the nonselective agonist NE or via a combination of the highly selective alpha(1A)-AR agonist A61603 and the beta-selective agonist isoproterenol led to increases in Ca(2+) influx from the extracellular compartment relative to stimulation with A61603 alone, with no effect on the associated transient release of Ca(2+) from intracellular stores. This effect became more evident upon examination of an alpha(1A)-AR variant exhibiting a partial defect in coupling to G(q), and we attribute it to potentiation of a non G(q)-pathway, uncovered by application of a combination of xestospongin C, an endoplasmic reticulum inositol 1,4,5-triphosphate receptor blocker, and 2-aminoethoxydiphenyl borate, a nonselective storeoperated Ca(2+) entry channel blocker. We also found that stimulation with A61603 of a second alpha(1A)-AR variant entirely unable to signal induced no Ca(2+) unless beta(2)-AR was concomitantly activated. These results may be accounted for by the presence of alpha(1A)/beta(2)-AR heterodimers or alternatively by specific adrenoceptor signal cross-talk resulting in distinct pharmacological behavior. Finally, our findings provide a new conceptual framework to rationalize outcomes from clinical studies targeting alpha- and beta-adrenoceptors.