Takayuki Yamaji

Roles of glutamate signaling in preclinical and/or mechanistic models of depression.

Accumulating evidence suggests that the glutamatergic system plays important roles in the pathophysiology and treatment of major depressive disorder (MDD). Abnormalities in the glutamatergic system are definitely observed in this disorder, and certain glutamatergic agents exhibit antidepressant effects in patients with MDD. In this review, we summarize the preclinical findings suggesting the involvement of glutamate signaling in the pathophysiology and treatment of MDD. Preclinical animal models for depression are often characterized by changes in molecules related to glutamatergic signaling. Some antidepressants exert their effects by affecting glutamatergic system components in animals. Animals with genetically modified glutamatergic function exhibit depression-like behaviors or anti-depressive behavior. In addition, several types of glutamatergic agents have shown antidepressant-like effects in preclinical models for depression. Many types of glutamate receptors (NMDA, AMPA, and metabotropic glutamate receptors) or transporters appear to be involved in the etiology of depression or in the mechanisms of action of antidepressants. These functional proteins related to glutamate signal transduction are potential targets for a new generation of antidepressants with fast-onset effects, such as the NMDA antagonist ketamine.

FK506 potentiates NGF‐induced neurite outgrowth via the Ras/Raf/MAP kinase pathway

Nerve growth factor (NGF) and other members of the neurotrophin family are critical for the survival and differentiation of neurons within the peripheral and central nervous systems. Neurophilin ligands, including FK506, potentiate NGF‐induced neurite outgrowth in several experimental models, although the mechanism of this potentiation is unclear. Therefore, we tested which signaling pathways were involved in FK506‐potentiated neurite outgrowth in SH‐SY5Y neuroblastoma cells using specific pharmacological inhibitors of various signaling molecules. Inhibitors of Ras (lovastatin), Raf (GW5074), or MAP kinase (PD98059 and U0126) blocked FK506 activity, as did inhibitors of phospholipase C (U73122) and phosphatidylinositol 3′ kinase (LY294002). Protein kinase C inhibitors (Go6983 and Ro31‐8220) slightly but significantly inhibited neurite outgrowth, whereas inhibitors of p38 MAPK (SB203580) or c‐Jun N‐terminal kinase (SP600125) had no effect. These data suggest that FK506 potentiates neurite outgrowth through the Ras/Raf/MAP kinase signaling pathway downstream of phospholipase C and phosphatidylinositol 3′ kinase.

Pharmacological characterization of standard analgesics on mechanical allodynia in streptozotocin-induced diabetic rats.

The present study was designed to investigate the anti-allodynic effects of current analgesic agents, such as pregabalin, amitriptyline, mexiletine, morphine, and diclofenac, in a rat model of streptozotocin (STZ)-induced diabetic neuropathy. Diabetic rats developed a sustained decrease in withdrawal threshold response to the von Frey test within 8 weeks after a single injection of STZ (45 mg/kg, i.v.). The anti-allodynic effects of analgesic agents were examined after a single oral or subcutaneous administration at 3 and 7 weeks after beginning of STZ-treatment. Pregabalin (3-30 mg/kg, p.o.), an antiepileptic agent, dose-dependently blocked the mechanical allodynia in rats treated both at 3 and 7 weeks. Mexiletine (10-100 mg/kg, p.o.), a sodium channel blocker, dose-dependently ameliorated mechanical allodynia in rats treated at 3 weeks; however, the efficacy was diminished at 7 weeks. Morphine (1-10 mg/kg, s.c.) was effective in rats treated at 3 weeks; however, it was ineffective at 7 weeks. Conversely, an antidepressant amitriptyline (0.3-3 mg/kg, p.o.) improved mechanical allodynia in rats treated at 7 weeks, whereas it was ineffective at 3 weeks. Diclofenac, a non-steroidal anti-inflammatory drug, was ineffective at both time points. These results demonstrate that, except for diclofenac, the standard analgesic agents tested can effectively alleviate the mechanical allodynia seen in STZ-induced diabetic neuropathy. Their efficacies varied depending on the duration of the diabetic condition, suggesting that temporal changes in pharmacodynamic factors could affect the responsiveness of this model to analgesic agents.

Activation of the serotonin 5-HT2C receptor is involved in the enhanced anxiety in rats after single-prolonged stress

We have recently confirmed that exposure of rats to the single-prolonged stress (SPS) paradigm induces enhanced hypothalamic-pituitary-adrenal (HPA) axis negative feedback and enhanced anxiety, and found that these changes develop time-dependently following stress exposure, suggesting that it could model the neuroendocrinological and behavioral abnormalities of the post-traumatic stress disorder (PTSD) patients. In the present study, microarray analysis was performed using RNA from the hippocampus, amygdala and anterior cingulate cortex of SPS rats and unstressed controls to unveil the molecular changes underlying SPS-induced behavioral changes. Thirty-one genes were found whose time course of expression corresponded to that of behavioral changes. One gene, 5-hydroxytryptamine2C (5-HT2C) receptor, was identified as a putative candidate. The overexpression of the gene in the amygdala of SPS rats was confirmed using real-time PCR 7 days after the SPS exposure. This molecule was then pharmacologically validated using FR260010 (N-[3-(4-methyl-1H-imidazol-1-yl)phenyl]-5,6-dihydrobenzo[h]quinazolin-4-amine dimethanesulfonate), a selective 5-HT2C receptor antagonist. FR260010 (1-10 mg/kg, s.c.) significantly inhibited the enhancement of anxiety in SPS rats. These results demonstrate for the first time that activation of the brain 5-HT2C receptor is involved in the development of behavioral abnormality in this model. This suggests that selective 5-HT2C receptor antagonists might provide novel therapeutic avenues for PTSD treatment.

A novel glycine transporter-1 (GlyT1) inhibitor, ASP2535 (4-[3-isopropyl-5-(6-phenyl-3-pyridyl)-4H-1,2,4-triazol-4-yl]-2,1,3-benzoxadiazole), improves cognition in animal models of cognitive impairment in schizophrenia and Alzheimer's disease.

Hypofunction of brain N-methyl-d-aspartate (NMDA) receptors has been implicated in psychiatric disorders such as schizophrenia and Alzheimers disease. Inhibition of glycine transporter-1 (GlyT1) is expected to increase glycine, a co-agonist of the NMDA receptor and, consequently, to facilitate NMDA receptor function. We have identified ASP2535 (4-[3-isopropyl-5-(6-phenyl-3-pyridyl)-4H-1,2,4-triazol-4-yl]-2,1,3-benzoxadiazole) as a novel GlyT1 inhibitor, and here describe our in vitro and in vivo characterization of this compound. ASP2535 potently inhibited rat GlyT1 (IC(50)=92 nM) with 50-fold selectivity over rat glycine transporter-2 (GlyT2). It showed minimal affinity for many other receptors except for μ-opioid receptors (IC(50)=1.83 μM). Oral administration of ASP2535 dose-dependently inhibited ex vivo [(3)H]-glycine uptake in mouse cortical homogenate, suggesting good brain permeability. This profile was confirmed by pharmacokinetic analysis. We then evaluated the effect of ASP2535 on animal models of cognitive impairment in schizophrenia and Alzheimers disease. Working memory deficit in MK-801-treated mice and visual learning deficit in neonatally phencyclidine (PCP)-treated mice were both attenuated by ASP2535 (0.3-3mg/kg, p.o. and 0.3-1mg/kg, p.o., respectively). ASP2535 (1-3mg/kg, p.o.) also improved the PCP-induced deficit in prepulse inhibition in rats. Moreover, the working memory deficit in scopolamine-treated mice and the spatial learning deficit in aged rats were both attenuated by ASP2535 (0.1-3mg/kg, p.o. and 0.1mg/kg, p.o., respectively). These studies provide compelling evidence that ASP2535 is a novel and centrally-active GlyT1 inhibitor that can improve cognitive impairment in animal models of schizophrenia and Alzheimers disease, suggesting that ASP2535 may satisfy currently unmet medical needs for the treatment of these diseases.

A Simple, Flexible, Nonfluorescent System for the Automated Screening of Neurite Outgrowth:

Measurement of neurite outgrowth is a common assay of neurotrophic activity. However, currently available techniques for measuring neurite outgrowth are either time or resource intensive. The authors established a system in which chronic treatment of a subcloned SH-SY5Y cell line with aphidicolin and various concentrations of nerve growth factor (NGF) induced discernable alterations in proliferation and differentiation. Cells were fixed, labeled with a nonfluorescent dye, and evaluated both manually and with an automated analysis system. NGF increased multiple parameters of differentiation, including neurite length, the proportion of cells extending neurites, and branching, as well as promoting cellular survival/proliferation. Interestingly, although NGF treatment increased the total number of branches, it actually decreased the proportion of branches per neurite length. The authors observed no differences in results obtained using the manual and automated systems, but the automated system was orders of magnitude faster. To demonstrate the flexibility of the system, the authors also show that they could measure changes in differentiation induced by a small-molecule Rho kinase inhibitor, as well as by retinoic acid cotreatment with brain-derived neurotrophic factor. In addition to this flexibility, this system does not require specialized equipment or fluorescent antibodies for analysis and therefore provides a less resource-intensive alternative to fluorescence-based systems.

Depressive-like behavior in adrenocorticotropic hormone-treated rats blocked by memantine.

Hyperactivity of the hypothalamic pituitary-adrenal (HPA) axis plays a role in the pathophysiology of major depressive disorder (MDD). Recent studies suggest the role of the glutamatergic system in the pathophysiology of MDD, and N-methyl-D-aspartate (NMDA) receptor antagonists have shown antidepressant effects in both preclinical and clinical studies. However, little is known about the role of adrenocorticotropic hormone (ACTH) specifically in the glutamatergic response to HPA axis activation. Glutamate is an NMDA receptor agonist, and glycine and D-serine act as co-agonists. Here, we measured brain concentrations of these amino acids in rats given repeated administration of ACTH (100 μg/rat/day, sc, for 14 days). Further, we also evaluated behavioral effects of memantine, a non-competitive NMDA antagonist, on immobility time in the forced swimming test and on locomotor activity in ACTH-treated rats. Compared with control rats, glutamine, glycine, L-serine, and D-serine levels were increased in the hippocampus of ACTH-treated rats; glutamate, glutamine, glycine, L-serine, and D-serine were increased in the cerebellum; and glutamine and glycine were increased in the frontal cortex and striatum, all with statistical significance. Remarkably, these increases in agonists and co-agonists might have led to the augmentation of NMDA receptor activity. ACTH treatment increased immobility time in the forced swimming test and decreased locomotor activity in rats. On the contrary, memantine (10 mg/kg, ip) significantly decreased immobility time in the forced swimming test and increased locomotor activity in ACTH-treated rats. Furthermore, imipramine (15 mg/kg, ip) did not alter immobility time in the forced swimming test whereas this drug significantly decreased locomotor activity in ACTH-treated rats. These results suggest that depressive-like behaviors by chronic ACTH treatment could be blocked by memantine.

FK1706, a novel non-immunosuppressant neurophilin ligand, ameliorates motor dysfunction following spinal cord injury through its neuroregenerative action

Injured spinal cord axons fail to regenerate in part due to a lack of trophic support. While various methods for replacing neurotrophins have been pursued, clinical uses of these methods face significant barriers. FK1706, a non-immunosuppressant neurophilin ligand, potentiates nerve growth factor signaling, suggesting therapeutic potential for functional deficits following spinal cord injury. Here, we demonstrate that FK1706 significantly improves behavioral outcomes in animal models of spinal cord hemisection and contusion injuries in rats. Furthermore, we show that FK1706 is effective even if administration is delayed until 1 week after injury, suggesting that FK1706 has a reasonable therapeutic time-window. Morphological analysis of injured axons in the dorsal corticospinal tract showed an increase in the radius and perimeter of stained axons, which were reduced by FK1706 treatment, suggesting that axonal swelling and retraction balls observed in injured spinal cord were improved by the neurotrophic effect of FK1706. Taken together, FK1706 improves both behavioral motor function and the underlying morphological changes, suggesting that FK1706 may have therapeutic potential in meeting the significant unmet needs in spinal cord injury.

FK1706, a novel non-immunosuppressive immunophilin ligand, modifies the course of painful diabetic neuropathy

FK1706, a derivative of FK506, is a non-immunosuppressive immunophilin ligand with significant neurotrophic activity mediated via FKBP-52 and the RAS/RAF/MAPK signaling pathway. Here, we tested the effect of FK1706 on painful diabetic neuropathy in rat model of diabetes induced by streptozotocin (STZ). FK1706 ameliorated mechanical allodynia in this model at doses over 0.32 mg/kg, p.o., even if treatment was initiated after neuropathy was established, and did not affect plasma glucose levels. Furthermore, this improvement continued at least 4 weeks after the last administration. In morphological analysis, FK1706 treatment also restored intraepidermal nerve fiber density in footpad skin to almost normal levels. Gabapentin also improved mechanical allodynia in the same model, but efficacy disappeared the day after administration stopped. Allodynia responses were potentiated by co-administration of both compounds. Thus, FK1706 ameliorated painful diabetic neuropathy via a different mechanism from gabapentin and improved morphological outcomes, indicating that FK1706 improves painful diabetic neuropathy by modifying the underlying disease pathology.

FK1706, a novel non-immunosuppressive immunophilin ligand, modifies gene expression in the dorsal root ganglia during painful diabetic neuropathy

Abstract Objectives: FK1706, a non-immunosuppressive immunophilin ligand, potentiated nerve growth factor-induced neurite outgrowth, putatively mediated via FKBP-52 and the Ras/Raf/MAPK signaling pathway. It also improved mechanical allodynia accompanied by the recovery of intraepidermal nerve fiber density in a painful diabetic neuropathy in rats. The aim of this study was to demonstrate the gene expression profiling in dorsal root ganglion in streptozotocin-induced diabetic rats related to pain and anti-allodynia effects of FK1706 administration to elucidate the putative mechanisms of its neurotrophic activity in vivo. Here, we analyzed gene expression of the dorsal root ganglia using microarray together with behavioral measurement of mechanical allodynia in diabetic rats to try to capture the global fingerprint of changes in gene expression associated with FK1706 administration. Methods: The withdrawal threshold of streptozotocin-induced diabetic rats was measured by an electronic von Frey system. The gene expression of the ganglia from L4 to L6 obtained from streptozotocin-treated rats with or without chronic administration of FK1706 was analyzed using an Affymetrix GeneChip to extract interesting genes in the development of mechanical allodynia in diabetes and anti-allodynia effect of FK1706. Results: Daily oral administration of FK1706 improved mechanical allodynia without decreasing plasma glucose levels. From gene expression analysis, the expression of thioredoxin interacting protein gene was sustained to increased change, whereas those of collagen I alpha1, II alpha1 and IX alpha1 genes were decreased from 2 to 4 weeks after streptozotocin injection. While no changes occurred after 1 week of commencing of FK1706 administration (2 weeks after streptozotocin injection), changes in expression more than 1·5-fold were observed for genes such as Ckm, Actn3, Atp2a1, Bglap, Acta1, Myl1, Tnnc2, and Mylpf at 2 weeks of FK1706 administration (3 weeks after streptozotocin injection). The genes RGD1564519, Hbb, LOC689064, Arpc4 and S100a9 were upregulated in comparison with streptozotocin-injected control group at 3 weeks of FK1706 administration; on the other hand, those of Actn3, Atp2a1 were downregulated by FK1706. Discussion: FK1706 ameliorates mechanical allodynia with accompanying increases in gene expressions possibly related to neurite outgrowth, development, differentiation, and nociceptive sensitivity.