Sustained Alleviation of Autoimmunity by Activating α2B-adrenergic Receptors.

Catecholamines binding to α- and ß-adrenergic receptors on immune cells have recently been shown to play an important role in regulating immune responses. Although α2-adrenergic receptors are known to modulate the immune response in different ways, the therapeutic exploration of their utility has been limited by the lack of agonists selective for the three α2-adrenergic subtypes. We report in this study the identification of the agonist AGN-762, which activates α2B- and α2C-adrenergic subtypes, but not the α2A subtype. We show that AGN-762 reduced clinical disease in an experimental autoimmune encephalitis model of autoimmune disease via direct or indirect effects on T regulatory cells. The activity of AGN-762 was abrogated by depletion of T regulatory cells, which express the α2B-adrenergic receptor. Furthermore, a drug-induced shift to an anti-inflammatory phenotype was demonstrated in immune cells in the spleen of drug-treated experimental autoimmune encephalitis mice. AGN-762 does not display sedative and cardiovascular side effects associated with α2A subtype agonists. Immune modulation by selective α2-adrenergic agonists represents a novel, to our knowledge, approach for treating autoimmune disease.

The NMDA receptor modulator zelquistinel durably relieves behavioral deficits in three mouse models of autism spectrum disorder.

Autism spectrum disorders (ASD) are complex neurodevelopmental disorders characterized by deficient social communication and interaction together with restricted, stereotyped behaviors. Currently approved treatments relieve comorbidities rather than core symptoms. Since excitation/inhibition balance and synaptic plasticity are disrupted in ASD, molecules targeting excitatory synaptic transmission appear as highly promising candidates to treat this pathology. Among glutamatergic receptors, the NMDA receptor has received particular attention through the last decade to develop novel allosteric modulators. Here, we show that positive NMDA receptor modulation by zelquistinel, a spirocyclic ß-lactam platform chemical, relieves core symptoms in two genetic and one environmental mouse models of ASD. A single oral dose of zelquistinel rescued, in a dose-response manner, social deficits and stereotypic behavior in Shank3Δex13-16-/- mice while chronic intraperitoneal administration promoted a long-lasting relief of such autistic-like features in these mice. Subchronic oral mid-dose zelquistinel treatment demonstrated durable effects in Shank3Δex13-16-/-, Fmr1-/- and in utero valproate-exposed mice. Carry-over effects were best maintained in the Fmr1 null mouse model, with social parameters being still fully recovered two weeks after treatment withdrawal. Among recently developed NMDA receptor subunit modulators, zelquistinel displays a promising therapeutic potential to relieve core symptoms in ASD patients, with oral bioavailability and long-lasting effects boding well for clinical applications. Efficacy in three mouse models with different etiologies supports high translational value. Further, this compound represents an innovative pharmacological tool to investigate plasticity mechanisms underlying behavioral deficits in animal models of ASD.

Stress induced aging in mouse eye.

Aging, a universal process that affects all cells in an organism, is a major risk factor for a group of neuropathies called glaucoma, where elevated intraocular pressure is one of the known stresses affecting the tissue. Our understanding of molecular impact of aging on response to stress in retina is very limited; therefore, we developed a new mouse model to approach this question experimentally. Here we show that susceptibility to response to stress increases with age and is primed on chromatin level. We demonstrate that ocular hypertension activates a stress response that is similar to natural aging and involves activation of inflammation and senescence. We show that multiple instances of pressure elevation cause aging of young retina as measured on transcriptional and DNA methylation level and are accompanied by local histone modification changes. Our data show that repeated stress accelerates appearance of aging features in tissues and suggest chromatin modifications as the key molecular components of aging. Lastly, our work further emphasizes the importance of early diagnosis and prevention as well as age-specific management of age-related diseases, including glaucoma.

Zelquistinel Is an Orally Bioavailable Novel NMDA Receptor Allosteric Modulator That Exhibits Rapid and Sustained Antidepressant-Like Effects.

BACKGROUND: The role of glutamatergic receptors in major depressive disorder continues to be of great interest for therapeutic development. Recent studies suggest that both negative and positive modulation of N-methyl-D-aspartate receptors (NMDAR) can produce rapid antidepressant effects. Here we report that zelquistinel, a novel NMDAR allosteric modulator, exhibits high oral bioavailability and dose-proportional exposures in plasma and the central nervous system and produces rapid and sustained antidepressant-like effects in rodents by enhancing activity-dependent, long-term synaptic plasticity. METHODS: NMDAR-mediated functional activity was measured in cultured rat brain cortical neurons (calcium imaging), hNR2A or B subtype-expressing HEK cells, and synaptic plasticity in rat hippocampal and medial prefrontal cortex slices in vitro. Pharmacokinetics were evaluated in rats following oral administration. Antidepressant-like effects were assessed in the rat forced swim test and the chronic social deficit mouse model. Target engagement and the safety/tolerability profile was assessed using phencyclidine-induced hyperlocomotion and rotarod rodent models. RESULTS: Following a single oral dose, zelquistinel (0.1-100 µg/kg) produced rapid and sustained antidepressant-like effects in the rodent depression models. Brain/ cerebrospinal fluid concentrations associated with zelquistinel antidepressant-like activity also increased NMDAR function and rapidly and persistently enhanced activity-dependent synaptic plasticity (long-term potentiation), suggesting that zelquistinel produces antidepressant-like effects by enhancing NMDAR function and synaptic plasticity. Furthermore, Zelquistinel inhibited phencyclidine (an NMDAR antagonist)-induced hyperlocomotion and did not impact rotarod performance. CONCLUSIONS: Zelquistinel produces rapid and sustained antidepressant effects by positively modulating the NMDARs, thereby enhancing long-term potentiation of synaptic transmission.

Extracellular application of the N-methyl-D-aspartate receptor allosteric modulator rapastinel acts remotely to regulate Ca2+ inactivation at an intracellular locus.

BACKGROUND: A novel N-methyl-D-aspartate receptor (NMDAR) allosteric modulator, rapastinel (RAP, formerly GLYX-13), elicits long-lasting antidepressant-like effects by enhancing long-term potentiation (LTP) of synaptic transmission. RAP elicits these effects by binding to a unique site in the extracellular region of the NMDAR complex, transiently enhancing NMDAR-gated current in pyramidal neurons of both hippocampus and medial prefrontal cortex. METHODS: We compared efficacy of RAP in modulating Schaffer collateral-evoked NMDAR-currents as a function of kinetics of the Ca2+ chelator in the intracellular solution, using whole-cell patch-clamp recordings. The intracellular solution contained either the slow Ca2+ chelator EGTA [3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecane-1,14-dioic acid, 0.5 mmol/l] or the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA {2,2',2″,2‴-[ethane-1,2-diylbis(oxy-2,1-phenylenenitrilo)] tetraacetic acid, 5 mmol/l}. NMDAR-gated currents were pharmacologically isolated by bath application of the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptor antagonist 6-nitro-2,3-dioxo-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (10 µmol/l) plus the GABA receptor blocker bicuculline (20 µmol/l). RESULTS: When the slow Ca2+ chelator EGTA was in the intracellular solution, RAP elicited significant enhancement of NMDAR-gated current at a 1 µmol/l concentration, and significantly reduced current at 10 µmol/l. In contrast, when recording with the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA, NMDAR current increased in magnitude by 84% as BAPTA washed into the cell, and the enhancement of NMDAR current by 1 µmol/l RAP was completely blocked. Interestingly, the reduction in NMDAR current from 10 µmol/l RAP was not affected by the presence of BAPTA in the recording pipette, indicating that this effect is mediated by a different mechanism. CONCLUSION: Extracellular binding of RAP to the NMDAR produces a novel, long-range reduction in affinity of the Ca2+ inactivation site on the NMDAR C-terminus accessible to the intracellular space. This action underlies enhancement in NMDAR-gated conductance elicited by RAP.

Rapastinel, an NMDAR positive modulator, produces distinct behavioral, sleep, and EEG profiles compared with ketamine.

Rapastinel, a positive NMDAR modulator, produces rapid-acting and long-lasting antidepressant-like effects; however, unlike ketamine, the abuse potential for rapastinel is minimal. Ketamine has also been shown to induce psychotomimetic/dissociative side effects, aberrant gamma oscillations, and effects similar to sleep deprivation, which may potentially limit its clinical use. In this study, we compared the side effect profile and potential sleep-altering properties of rapastinel (3, 10, and 30 mg/kg) to ketamine (30 mg/kg) in rodents. In addition, we investigated corresponding changes in transcriptomics and proteomics. Rapastinel exhibited no effect on locomotor activity and prepulse inhibition in mice, while ketamine induced a significant increase in locomotor activity and a significant decrease in prepulse inhibition, which are indications of a psychosis-like state. The effects of rapastinel on sleep architecture were minimal, and rapastinel did not alter gamma frequency oscillations. In contrast, ketamine administration resulted in a greater latency to slow wave and REM sleep, disrupted duration of sleep, and affected duration of wakefulness during sleep. Further, ketamine increased cortical oscillations in the gamma frequency range, which is a property associated with psychosis. Rapastinel induced similar plasticity-related changes in transcriptomics to ketamine in rats but differed in several gene ontology classes, some of which may be involved in the regulation of sleep. In conclusion, rapastinel demonstrated a lower propensity than ketamine to induce CNS-related adverse side effects and sleep disturbances.

Positive N-Methyl-D-Aspartate Receptor Modulation by Rapastinel Promotes Rapid and Sustained Antidepressant-Like Effects.

BACKGROUND: Modulation of glutamatergic synaptic transmission by N-methyl-D-aspartate receptors can produce rapid and sustained antidepressant effects. Rapastinel (GLYX-13), initially described as a N-methyl-D-aspartate receptor partial glycine site agonist, exhibits rapid antidepressant effect in rodents without the accompanying dissociative effects of N-methyl-D-aspartate receptor antagonists. METHODS: The relationship between rapastinel's in vitro N-methyl-D-aspartate receptor pharmacology and antidepressant efficacy was determined by brain microdialysis and subsequent pharmacological characterization of therapeutic rapastinel concentrations in N-methyl-D-aspartate receptor-specific radioligand displacement, calcium mobilization, and medial prefrontal cortex electrophysiology assays. RESULTS: Brain rapastinel concentrations of 30 to 100 nM were associated with its antidepressant-like efficacy and enhancement of N-methyl-D-aspartate receptor-dependent neuronal intracellular calcium mobilization. Modulation of N-methyl-D-aspartate receptors by rapastinel was independent of D-serine concentrations, and glycine site antagonists did not block rapastinel's effect. In rat medial prefrontal cortex slices, 100 nM rapastinel increased N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents and enhanced the magnitude of long-term potentiation without any effect on miniature EPSCs or paired-pulse facilitation responses, indicating postsynaptic action of rapastinel. A critical amino acid within the NR2 subunit was identified as necessary for rapastinel's modulatory effect. CONCLUSION: Rapastinel brain concentrations associated with antidepressant-like activity directly enhance medial prefrontal cortex N-methyl-D-aspartate receptor activity and N-methyl-D-aspartate receptor-mediated synaptic plasticity in vitro. At therapeutic concentrations, rapastinel directly enhances N-methyl-D-aspartate receptor activity through a novel site independent of the glycine coagonist site. While both rapastinel and ketamine physically target N-methyl-D-aspartate receptors, the 2 molecules have opposing actions on N-methyl-D-aspartate receptors. Modest positive modulation of N-methyl-D-aspartate receptors by rapastinel represents a novel pharmacological approach to promote well-tolerated, rapid, and sustained improvements in mood disorders.

Function of brain α2B-adrenergic receptor characterized with subtype-selective α2B antagonist and KO mice.

Noradrenergic signaling, through the α2A and α2C adrenergic receptors modulates the cognitive and behavioral symptoms of disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), and addiction. However, it is unknown whether the α2B receptor has any significant role in CNS function. The present study elucidates the potential role of the α2B receptor in CNS function via the discovery and use of the first subtype-selective α2B antagonist (AGN-209419), and behavioral analyses of α-receptor knockout (KO) mice. Using AGN-209419 as radioligand, α2B receptor binding sites were identified within the olfactory bulb, cortex, thalamus, cerebellum, and striatum. Based on the observed expression patterns of α2 subtypes in the brain, we compared α2B KO, α2A KO and α2C KO mice behavioral phenotypes with their respective wild-type lines in anxiety (plus maze), compulsive (marble burying), and sensorimotor (prepulse inhibition) tasks. α2B KO mice exhibited increased marble burying and α2C KO mice exhibited an increased startle response to a pulse stimulus, but otherwise intact prepulse inhibition. To further explore compulsive behavior, we evaluated novelty-induced locomotor hyperactivity and found that α2B KO and α2C KO mice exhibited increased locomotion in the open field. Interestingly, when challenged with amphetamine, α2C KO mice increased activity at lower doses relative to either α2A KO or WT mice. However, α2B KO mice exhibited stereotypy at doses of amphetamine that were only locomotor stimulatory to all other genotypes. Following co-administration of AGN-209419 with low-dose amphetamine in WT mice, stereotypy was observed, mimicking the α2B KO phenotype. These findings suggest that the α2B receptor is involved in CNS behaviors associated with sensorimotor gating and compulsivity, and may be therapeutically relevant for disorders such as schizophrenia, ADHD, post-traumatic stress disorder, addiction, and obsessive compulsive disorder.

A2E and lipofuscin distributions in macaque retinal pigment epithelium are similar to human.

The accumulation of lipofuscin, an autofluorescent aging marker, in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD). Lipofuscin contains several visual cycle byproducts, most notably the bisretinoid N-retinylidene-N-retinylethanolamine (A2E). Previous studies with human donor eyes have shown a significant mismatch between lipofuscin autofluorescence (AF) and A2E distributions. The goal of the current project was to examine this relationship in a primate model with a retinal anatomy similar to that of humans. Ophthalmologically naive young (<10 years., N = 3) and old (>10 years., N = 4) Macaca fascicularis (macaque) eyes, were enucleated, dissected to yield RPE/choroid tissue, and flat-mounted on indium-tin-oxide-coated conductive slides. To compare the spatial distributions of lipofuscin and A2E, fluorescence and mass spectrometric imaging were carried out sequentially on the same samples. The distribution of lipofuscin fluorescence in the primate RPE reflected previously obtained human results, having the highest intensities in a perifoveal ring. Contrarily, A2E levels were consistently highest in the periphery, confirming a lack of correlation between the distributions of lipofuscin and A2E previously described in human donor eyes. We conclude that the mismatch between lipofuscin AF and A2E distributions is related to anatomical features specific to primates, such as the macula, and that this primate model has the potential to fill an important gap in current AMD research.

A peripheral adrenoceptor-mediated sympathetic mechanism can transform stress-induced analgesia into hyperalgesia.

BACKGROUND: Stress has paradoxical effects on pain, causing stress-induced analgesia but also exacerbating pain via poorly understood mechanisms. Adrenergic neurotransmission is integral in pathways that regulate the response to both pain and stress. Hyperalgesia is often associated with enhanced adrenergic sensitivity of primary afferents, but sympathetic nervous system outflow has not been demonstrated to exacerbate pain perception after stress. METHODS: Rats or C57/BL6 wild-type mice treated with α-2 receptor antagonists or α-2A receptor knockout mice were exposed to ultrasonic noise stress or footshock stress and subsequently tested for hotplate paw withdrawal latencies. The sensory sensitivity of α-2A knockout mice to electrical and chemical stimuli was tested neurophysiologically and behaviorally. The effects of sympatholytic treatments were investigated. RESULTS: Noise and footshock stressors induced thermal hyperalgesia in rats pretreated systemically with α-2 antagonists. Wild-type mice pretreated with α-2 antagonists and α-2A knockout mice also exhibited thermal hyperalgesia induced by noise stress. Local spinal or intraplantar injection of an α-2 antagonist counteracted stress-induced analgesia without causing hyperalgesia. The α-2A knockout mice had decreased thresholds for peripheral sensitization with sulprostone and for windup of the dorsal horn neuronal response to repetitive electrical stimuli. Stress-induced hyperalgesia was abolished and the sensitization was attenuated by sympathectomy or systemic administration of an α-1-adrenergic antagonist. CONCLUSIONS: Sympathetic postganglionic nerves can enhance pain sensation via a peripheral α-1-adrenoceptor mechanism when sympathetic outflow is disinhibited. The net effect of stress on pain sensation reflects a balance between descending spinal inhibition and sympathetic outflow that can shift toward pain facilitation when central and peripheral α-2-adrenoceptor inhibitory mechanisms are attenuated.

Differential effects of PPARgamma ligands on oxidative stress-induced death of retinal pigmented epithelial cells.

PURPOSE: To investigate the role of the peroxisome proliferator-activated receptor (PPAR)-γ in modulating retinal pigmented epithelium (RPE) responses to oxidative stress. METHODS: ARPE-19 cells were treated with the oxidant, t-butylhydroperoxide (tBH) to induce apoptosis. Cells pretreated with synthetic PPARγ agonists of the antidiabetic thiazolidinediones class before tBH challenge were assessed for viability and, by microarray analysis, for effects on gene expression. RESULTS: Treatment of ARPE-19 cells with tBH resulted in a loss of viability and global changes in the pattern of gene expression. PPARγ ligands were found to have differential modulatory effects on tBH-induced apoptosis of RPE cells. Whereas rosiglitazone and pioglitazone potentiated cell death, troglitazone acted as a potent cytoprotective agent. Downregulation of PPARγ expression by an siRNA resulted in enhanced cell death in response to tBH treatment and blocked the cytoprotective effect of troglitazone consistent with a role of PPARγ in mediating this response. Microarray analysis revealed that while rosiglitazone and pioglitazone had little effect on gene changes induced by tBH treatment, troglitazone dramatically reduced the number of changes caused by oxidative stress. A unique subset of genes that were deregulated by tBH and selectively normalized by troglitazone were identified. CONCLUSIONS: These findings demonstrate that PPARγ agonists can have differential effects on RPE survival in response to oxidative stress. Oxidative stress leads to deregulation of a large set of genes in ARPE-19 cells. A specific subset of these genes can be selectively modulated by troglitazone and represent potential novel targets for cytoprotective therapies.

Alpha-1-adrenergic receptor agonist activity of clinical alpha-adrenergic receptor agonists interferes with alpha-2-mediated analgesia.

BACKGROUND: The use of alpha-2 adrenergic agonists for analgesia is limited due to a narrow therapeutic window. Definition of the role of alpha receptor subtypes in alpha agonist mediated analgesia may identify strategies to separate the analgesic from sedative and cardiovascular effects. METHODS: Analgesic activity of brimonidine, clonidine, and tizanidine was investigated in wild-type C57B/6, alpha-2A, and alpha-2C knockout mice with allodynia induced by N-methyl-D-aspartate or sulprostone. The alpha receptor selectivity of the alpha agonists was assessed using functional in vitro recombinant assays. RESULTS: Brimonidine, clonidine, and tizanidine reduced N-methyl-D-aspartate- and sulprostone-induced allodynia in wild-type mice, but not alpha-2A knockout mice. In alpha-2C knockout mice, brimonidine and tizanidine reduced allodynia in both models, whereas clonidine only reduced N-methyl-D-aspartate-induced allodynia. In vitro, clonidine and tizanidine activated alpha-1 and alpha-2 receptors with similar potencies, whereas brimonidine was selective for alpha-2 receptors. In alpha-2C knockout mice with sulprostone-induced allodynia, blockade of clonidine's alpha-1 receptor agonist activity restored clonidine's analgesic efficacy. In wild-type mice, the analgesic potency of intrathecal clonidine and tizanidine was increased 3- to 10-fold by coadministration with the alpha-1A-selective antagonist 5-methylurapidil without affecting sedation. Following intraperitoneal administration, the therapeutic window was negligible for clonidine and tizanidine, but greater for brimonidine. 5-Methylurapidil enhanced the therapeutic window of intraperitoneal clonidine and tizanidine approximately 10-fold. CONCLUSIONS: Alpha-1A receptor agonist activity can counterbalance alpha-2 receptor agonist-induced analgesia. Greater alpha-2 selectivity may enhance the therapeutic window of alpha-2 agonists in the treatment of pain.

Broad modulation of neuropathic pain states by a selective estrogen receptor beta agonist.

The effects of estrogens on pain perception remain controversial. In animal models, both beneficial and detrimental effects of non-selective estrogens have been reported. ERb-131 a non-steroidal estrogen receptor beta ligand was evaluated in several pain animal models involving nerve injury or sensitization. Using functional and binding assays, ERb-131 was characterized as a potent and selective estrogen receptor beta agonist. In vivo, ERb-131 was devoid of estrogen receptor alpha activity as assessed in a rat uterotrophic assay. ERb-131 alleviated tactile hyperalgesia induced by capsaicin, and reversed tactile allodynia caused by spinal nerve ligation and various chemical insults. Moreover, ERb-131 did not influence the pain threshold of normal healthy animals. Thus, estrogen receptor beta agonism is a critical effector in attenuating a broad range of anti-nociceptive states.

CRM1-dependent function of a cis-acting RNA export element.

Viruses often contain cis-acting RNA elements, which facilitate the posttranscriptional processing and export of their messages. These elements fall into two classes distinguished by the presence of either viral or cellular RNA binding proteins. To date, studies have indicated that the viral proteins utilize the CRM1-dependent export pathway, while the cellular factors generally function in a CRM1-independent manner. The cis-acting element found in the woodchuck hepatitis virus (WHV) (the WHV posttranscriptional regulatory element [WPRE]) has the ability to posttranscriptionally stimulate transgene expression and requires no viral proteins to function. Conventional wisdom suggests that the WPRE would function in a CRM1-independent manner. However, our studies on this element reveal that its efficient function is sensitive to the overexpression of the C terminus of CAN/Nup214 and treatment with the antimicrobial agent leptomycin B. Furthermore, the overexpression of CRM1 stimulates WPRE activity. These results suggest a direct role for CRM1 in the export function of the WPRE. This observation suggests that the WPRE is directing messages into a CRM1-dependent mRNA export pathway in somatic mammalian cells.