The NMDAR modulator NYX-2925 alleviates neuropathic pain via a Src-dependent mechanism in the mPFC.

Previous studies have shown that oral administration of the NMDAR modulator NYX-2925 alleviates pain in several animal models of neuropathic pain and this appears to be through mPFC, but not spinal, mediated mechanisms. While much is known about the impact of neuropathic pain on NMDAR-mediated signaling in the spinal cord, limited studies have focused on the brain. In the current study, we assess signaling changes associated with NMDAR-mediated plasticity in the mPFC and the impact of NYX-2925 administration on the normalization of these signaling changes. We found a decrease in activated Src levels in the mPFC of animals with chronic constriction injury (CCI) of the sciatic nerve. While Src mediated activation of NMDARs was also decreased in CCI animals, the main NMDAR phosphorylation site of CAMKII was not affected. This is in opposition to what has been found in the spinal cord, where both Src and CAMKII activation are increased. Oral administration of NYX-2925 restored levels of activated Src and Src phosphorylation sites on GluN2A and GluN2B in the mPFC, with no effect on activated CAMKII levels. The analgesic effect of NYX-2925 appears dependent on this restoration of Src activation in the mPFC, as co-administering Src activation inhibitors prevented the NYX-2925 analgesic effect. Overall, these data suggest that NMDAR-mediated signaling plays a key role in neuropathic pain, albeit in different directions in the spinal cord vs. the mPFC. Furthermore, the analgesic effect of NYX-2925 appears to involve a restoration of NMDAR-mediated signaling in the mPFC.

NYX-458 Improves Cognitive Performance in a Primate Parkinson's Disease Model.

BACKGROUND: NYX-458 is a N-methyl-d-aspartate receptor (NMDAR) modulator that enhances synaptic plasticity. Dopaminergic cell loss in Parkinson's disease (PD) leads to NMDAR dysregulation in the cortico-striato-pallidal-thalmo-cortical network and altered plasticity in brain regions important to cognitive function. We hypothesize that targeting the NMDAR may be an efficacious approach to treating cognitive impairment in PD. OBJECTIVES: NYX-458 was evaluated in 2 nonhuman primate models of PD. The first, a chronic low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-administration model, was used to assess the effects of NYX-458 on cognitive domains impacted early in PD including attention, working memory, executive function, and visuospatial learning. The second, a high-dose MPTP-administration model, was used to assess potential for NYX-458 induced change in motor symptoms. METHODS: NYX-458 was evaluated in the chronic low-dose MPTP model using the variable delayed response measure to assess attention and working memory and simple discrimination reversal to assess executive function. NYX-458 was also assessed in the high-dose MPTP model as a monotherapy and in combination with low-dose or high-dose levodopa to assess potential impact on motor symptoms. RESULTS: NYX-458 administration resulted in rapid and long-lasting improvement in cognitive function across the domains of attention, working memory, and executive function. Dose levels effective in improving cognitive performance had no effect on PD motor symptoms, the antiparkinsonian benefit of levodopa, or dyskinesia. CONCLUSIONS: NYX-458 provides benefit in specific domains known to be impaired in PD in a dopamine depletion model of PD-like cognitive impairment. These data support the continued evaluation of NYX-458 as a potential therapeutic for cognitive decline in PD. © 2020 International Parkinson and Movement Disorder Society.

Rat ultrasonic vocalizations as a measure of the emotional component of chronic pain.

In humans, chronic pain is often expressed as a spontaneous emotional response which can lead to fragmented sleep. Rat 50-kHz and 20-kHz ultrasonic vocalizations are well-established measures of positive and negative emotional states, respectively. The rat chronic constriction injury model was used to induce chronic pain, and ultrasonic vocalizations were measured in both the heterospecific rough-and-tumble play (i.e. tickling) test as well as during 24-hour home cage recordings. Rates of hedonic 50-kHz ultrasonic vocalizations during the non-stimulus periods of the tickling test, as well as the rewarding value of tickling, were reduced in chronic constriction injury rats compared to sham controls. In the 24-hour home cage recording study, chronic constriction injury animals showed a reduced amplitude in circadian activity, as well as reduced hedonic 50-kHz ultrasonic vocalizations and increased evoked and spontaneous aversive 20-kHz ultrasonic vocalizations. These data demonstrate that rat ultrasonic vocalizations can be used to capture core symptoms of chronic pain and may be useful in the elucidation of the neuronal mechanisms that underlie the affective component of pain.

NYX-2925 Is a Novel N-Methyl-d-Aspartate Receptor Modulator that Induces Rapid and Long-Lasting Analgesia in Rat Models of Neuropathic Pain.

NYX-2925 [(2S,3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)butanamide] is a novel N-methyl-d-aspartate (NMDA) receptor modulator that is currently being investigated in phase 2 clinical studies for the treatment of painful diabetic peripheral neuropathy and fibromyalgia. Previous studies demonstrated that NYX-2925 is a member of a novel class of NMDA receptor-specific modulators that affect synaptic plasticity processes associated with learning and memory. Studies here examined NYX-2925 administration in rat peripheral chronic constriction nerve injury (CCI) and streptozotocin-induced diabetic mechanical hypersensitivity. Additionally, NYX-2925 was examined in formalin-induced persistent pain model and the tail flick test of acute nociception. Oral administration of NYX-2925 resulted in rapid and long-lasting analgesia in both of the neuropathic pain models and formalin-induced persistent pain, but was ineffective in the tail flick model. The analgesic effects of NYX-2925 were blocked by the systemic administration of NMDA receptor antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid. Microinjection of NYX-2925 into the medial prefrontal cortex of CCI rats resulted in analgesic effects similar to those observed following systemic administration, whereas intrathecal administration of NYX-2925 was ineffective. In CCI animals, NYX-2925 administration reversed deficits seen in a rat model of rough-and-tumble play. Thus, it appears that NYX-2925 may have therapeutic potential for the treatment of neuropathic pain, and the data presented here support the idea that NYX-2925 may act centrally to ameliorate pain and modulate negative affective states associated with chronic neuropathic pain.

NYX-2925 Is a Novel NMDA Receptor-Specific Spirocyclic-ß-Lactam That Modulates Synaptic Plasticity Processes Associated with Learning and Memory.

Background: N-methyl-D-aspartate receptors are one member of a family of ionotropic glutamate receptors that play a pivotal role in synaptic plasticity processes associated with learning and have become attractive therapeutic targets for diseases such as depression, anxiety, schizophrenia, and neuropathic pain. NYX-2925 ((2S, 3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)butanamide) is one member of a spiro-ß-lactam-based chemical platform that mimics some of the dipyrrolidine structural features of rapastinel (formerly GLYX-13: threonine-proline-proline-threonine) and is distinct from known N-methyl-D-aspartate receptor agonists or antagonists such as D-cycloserine, ketamine, MK-801, kynurenic acid, or ifenprodil. Methods: The in vitro and in vivo pharmacological properties of NYX-2925 were examined. Results: NYX-2925 has a low potential for "off-target" activity, as it did not exhibit any significant affinity for a large panel of neuroactive receptors, including hERG receptors. NYX-2925 increased MK-801 binding to human N-methyl-D-aspartate receptor NR2A-D subtypes expressed in HEK cells and enhanced N-methyl-D-aspartate receptor current and long-term potentiation (LTP) in rat hippocampal slices (100-500 nM). Single dose ex vivo studies showed increased metaplasticity in a hippocampal LTP paradigm and structural plasticity 24 hours after administration (1 mg/kg p.o.). Significant learning enhancement in both novel object recognition and positive emotional learning paradigms were observed (0.01-1 mg/kg p.o.), and these effects were blocked by the N-methyl-D-aspartate receptor antagonist CPP. NYX-2925 does not show any addictive or sedative/ataxic side effects and has a therapeutic index of >1000. NYX-2925 (1 mg/kg p.o.) has a cerebrospinal fluid half-life of 1.2 hours with a Cmax of 44 nM at 1 hour. Conclusions: NYX-2925, like rapastinel, activates an NMDA receptor-mediated synaptic plasticity process and may have therapeutic potential for a variety of NMDA receptor-mediated central nervous system disorders.

Dysregulation of gene expression in a lysosomal storage disease varies between brain regions implicating unexpected mechanisms of neuropathology.

The characteristic neurological feature of many neurogenetic diseases is intellectual disability. Although specific neuropathological features have been described, the mechanisms by which specific gene defects lead to cognitive impairment remain obscure. To gain insight into abnormal functions occurring secondary to a single gene defect, whole transcriptome analysis was used to identify molecular and cellular pathways that are dysregulated in the brain in a mouse model of a lysosomal storage disorder (LSD) (mucopolysaccharidosis [MPS] VII). We assayed multiple anatomical regions separately, in a large cohort of normal and diseased mice, which greatly increased the number of significant changes that could be detected compared to past studies in LSD models. We found that patterns of aberrant gene expression and involvement of multiple molecular and cellular systems varied significantly between brain regions. A number of changes revealed unexpected system and process alterations, such as up-regulation of the immune system with few inflammatory changes (a significant difference from the closely related MPS IIIb model), down-regulation of major oligodendrocyte genes even though white matter changes are not a feature histopathologically, and a plethora of developmental gene changes. The involvement of multiple neural systems indicates that the mechanisms of neuropathology in this type of disease are much broader than previously appreciated. In addition, the variation in gene dysregulation between brain regions indicates that different neuropathologic mechanisms may predominate within different regions of a diseased brain caused by a single gene mutation.

Dosage thresholds for AAV2 and AAV8 photoreceptor gene therapy in monkey.

Gene therapy is emerging as a therapeutic modality for treating disorders of the retina. Photoreceptor cells are the primary cell type affected in many inherited diseases of retinal degeneration. Successfully treating these diseases with gene therapy requires the identification of efficient and safe targeting vectors that can transduce photoreceptor cells. One serotype of adeno-associated virus, AAV2, has been used successfully in clinical trials to treat a form of congenital blindness that requires transduction of the supporting cells of the retina in the retinal pigment epithelium (RPE). Here, we determined the dose required to achieve targeting of AAV2 and AAV8 vectors to photoreceptors in nonhuman primates. Transgene expression in animals injected subretinally with various doses of AAV2 or AAV8 vectors carrying a green fluorescent protein transgene was correlated with surgical, clinical, and immunological observations. Both AAV2 and AAV8 demonstrated efficient transduction of RPE, but AAV8 was markedly better at targeting photoreceptor cells. These preclinical results provide guidance for optimal vector and dose selection in future human gene therapy trials to treat retinal diseases caused by loss of photoreceptors.

Acute cocaine increases interleukin-1ß mRNA and immunoreactive cells in the cortex and nucleus accumbens.

The cytokine, interleukin-1ß (IL1ß) is a sleep regulatory substance whose expression is enhanced in response to neuronal stimulation. In this study, IL1ß mRNA and immunoreactivity (IR) are evaluated after acute cocaine. First, IL1ß mRNA levels were measured at the start or end of the light period after saline or acute exposure to a low dose of cocaine (5 mg/kg, intraperitoneal (ip)). IL1ß mRNA levels after an acute exposure to cocaine (5 mg/kg, ip) at dark onset were significantly higher than those obtained from rats sacrificed after an acute exposure to saline in the piriform and somatosensory cortex, and nucleus accumbens. Acute exposure of cocaine at 5 mg/kg at dark onset also increased the number of IL1ß-immunoreactive astrocytes in layer I-V of the prefrontal cortex, somatosensory cortex and nucleus accumbens. These data suggest that IL1ß mRNA and protein levels in some of the dopaminergically innervated brain regions are responsive to cocaine.

Expanded repertoire of AAV vector serotypes mediate unique patterns of transduction in mouse brain.

A wide diversity of adeno-associated virus (AAV) structural proteins uncovered from latent genomes in primate tissue has expanded the number of AAV vector serotypes, which can potentially confer unique cell tropism to the vector. We evaluated 17 of these vectors in the mouse brain using green fluorescent protein (GFP) as a reporter gene. A rapid initial evaluation was performed by neonatal lateral ventricle injections. Vectors made with capsids hu.32, hu.37, pi.2, hu.11, rh.8, hu.48R3, and AAV9 for comparison were selected for further analysis based on their ability to transduce large numbers of cells and result in novel patterns of cell transduction. These vectors were injected into adult brains in four major structures (cortex, striatum, hippocampus, and thalamus), and all were found to transduce neurons. In addition, hu.32, hu.11, pi.2, hu.48R3, and rh.8 resulted in GFP expression in some astrocytes or oligodendrocytes. AAVs rh.8, pi.2, hu.32, and hu.11 also appeared to result in neuronal transport of the vector genome. Vector transport was studied by a single unilateral injection into the hippocampus and vector genome was found in projection sites of the hippocampus. These unique patterns of cell transduction expand the potential repertoire for targeting AAV vectors to selected subsets of brain cells.

A single injection of an adeno-associated virus vector into nuclei with divergent connections results in widespread vector distribution in the brain and global correction of a neurogenetic disease.

Neurogenetic disorders typically affect cells throughout the brain. Adeno-associated virus (AAV) vector-mediated transfer of a normal cDNA can correct the metabolic defects at the site of injection, but treatment of the entire brain requires widespread delivery of the normal gene and/or protein. Current methods require multiple injections for widespread distribution. However, some AAV vectors can be transported along neuronal pathways associated with the injected region. Thus, targeting widely dispersed systems in the CNS might be a pathway for gene dispersal from a limited number of sites. We tested this hypothesis in the ventral tegmental area (VTA), a region with numerous efferent and afferent projections. A single 1 mul injection resulted in transport of the vector genome to projection sites in distal parts of the brain. When compared with injections into the striatum, the VTA injection resulted in higher enzyme levels in more regions of the brain. The AAV-9 serotype vector was the most widely disseminated, but AAV-Rh.10 and AAV-1 were also transported after VTA injection. The effect on global lesions of a neurogenetic disease was tested in the mouse model of MPS VII (mucopolysaccharidosis VII), a lysosomal storage disorder. Widespread distribution of the vector genome after AAV-9 VTA injection resulted in even further distribution of the enzyme product, by secretion and uptake by surrounding cells, and complete correction of the storage lesions throughout the entire brain. This unprecedented level of correction from a single injection into the developed brain provides a potential strategy to correct a large volume of brain while minimizing the number of injections.

Brain distribution of cytokine mRNA induced by systemic administration of interleukin-1beta or tumor necrosis factor alpha.

Brain cytokine mRNA levels are impacted by systemic cytokines. For example, systemic interleukin-1beta (IL1beta) increases brain IL1beta mRNA; subdiaphragmatic vagotomy blocks this effect. To localize which brain regions respond to intraperitoneal cytokines, we measured mRNA levels in selected brain regions for a variety of cytokines and growth factors, IL1beta, TNFalpha, interleukin-6 (IL-6), interleukin-10 (IL10), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Relative to saline administration, IL1beta increased IL1beta, TNFalpha and IL6 mRNAs in the nucleus tractus solitarius (NTS), hypothalamus, hippocampus and somatosensory cortex (SSctx), but did not induce any changes in IL10. TNFalpha also increased TNFalpha and IL1beta mRNAs in the hypothalamus, hippocampus and SSctx. TNFalpha increased TNFalpha, IL1beta and IL10 mRNAs in the NTS, but did not induce any changes in IL-6 mRNA. In the amygdala, IL1beta enhanced IL6 mRNA and TNFalpha increased IL1beta mRNAs. In the insular cortex, IL1beta enhanced IL6 mRNA and TNFalpha increased IL1beta mRNA. TNFalpha administration increased NGF mRNA in the SSctx but decreased NGF and BDNF mRNA levels in the insular cortex. Both IL1beta and TNFalpha decreased BDNF mRNA in the amygdala. We also verified the IL1beta-induced increases in TNFalpha mRNA within the NTS using in situ hybridization. These results support the hypothesis that somnogenic doses of IL1beta and TNFalpha enhance their own mRNA levels as well as affect mRNA levels for other sleep-promoting substances.

Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain.

Recombinant adeno-associated viral (AAV) vectors can transduce cells of the CNS, resulting in long-term expression. AAV vector transduction varies depending on the serotype used and the region of the brain injected. AAV serotypes 7, 8, 9, and Rh10 have recently become available, but the transduction capabilities of these serotypes within the CNS have not been determined. We show that AAV 7, 8, 9, and Rh10 vectors expressing cDNA for a lysosomal enzyme transduce neurons, but not astrocytes or oligodendrocytes, in the cortex, striatum, hippocampus, and thalamus. Although all of the vectors contained the same genome, there were markedly different transduction patterns that could be due only to the differences in capsid proteins. The AAV 9 vector was found to undergo vector genome transport to distal neuronal cell bodies via known axonal pathways. This facilitated the distribution of enzyme, resulting in correction of lysosomal storage lesions in regions of a diseased brain that would not be corrected if the genome were not transported.

Time of day differences in IL1beta and TNFalpha mRNA levels in specific regions of the rat brain.

Interleukin-1beta (IL1beta) and tumor necrosis factor-alpha (TNFalpha) are involved in several physiological functions regulated by brain. The current studies were performed to determine whether a diurnal rhythm of IL1beta and TNFalpha mRNAs, determined by real time RT-PCR, existed in specific brain regions linked to the functions of these cytokines. Rats were sacrificed 2 h after light onset (AM) and 1 h prior to dark onset (PM). IL1beta mRNA levels in the AM were significantly higher than those obtained from rats sacrificed in the PM in the prefrontal cortex, parietal cortex, amygdala/piriform cortex, ventral hippocampus, hypothalamus, nucleus tractus solitarius, and nucleus accumbens, but not in the dorsal hippocampus. Time-of-day differences in TNFalpha mRNA levels were observed in all these brain regions. These results support the hypothesis that TNFalpha and IL1beta have physiological roles within the brain.