An evaluation method for developing abuse-deterrent opioid formulations with agonist and antagonist combinations using conditioned place preference.

Although opioids are useful narcotic analgesics in clinical settings, their misuse and addiction in the United States of America and other countries are rapidly increasing. Therefore, the development of abuse-deterrent formulations is an urgent issue. We herein investigated how to select the ratio of an opioid and the opioid receptor antagonist, naloxone in abuse-deterrent formulations for mice. The conditioned place preference (CPP) test was used to evaluate the rewarding effects of abused drugs. The opioids morphine (30 µmol/kg), oxycodone (3 µmol/kg), fentanyl (0.4 µmol/kg), and buprenorphine (0.5 µmol/kg) significantly induced place preference in mice. We also examined the optimal ratio of naloxone and opioids to inhibit the rewarding effects of the latter. Naloxone (3-5 µmol/kg) effectively inhibited place preference induced by the opioids tested. We calculated theoretical drug doses that exerted the same pharmacodynamic effects based on two parameters: µ-opioid receptor binding affinity and blood-brain barrier (BBB) permeability. Theoretical doses were very close to the drug doses at which mice showed place preference. Therefore, the CPP test is useful as a behavioral method for evaluating abuse-deterrent formulations of opioids mixed with an antagonist. The ratio of naloxone with opioids, at which mice did not show place preference, may be an effective index for developing abuse-deterrent formulations. Ratios may be calculated for other opioids based on µ-opioid receptor binding affinity and BBB permeability.

Phosphoproteomic of the acetylcholine pathway enables discovery of the PKC-ß-PIX-Rac1-PAK cascade as a stimulatory signal for aversive learning.

Acetylcholine is a neuromodulator critical for learning and memory. The cholinesterase inhibitor donepezil increases brain acetylcholine levels and improves Alzheimer's disease (AD)-associated learning disabilities. Acetylcholine activates striatal/nucleus accumbens dopamine receptor D2-expressing medium spiny neurons (D2R-MSNs), which regulate aversive learning through muscarinic receptor M1 (M1R). However, how acetylcholine stimulates learning beyond M1Rs remains unresolved. Here, we found that acetylcholine stimulated protein kinase C (PKC) in mouse striatal/nucleus accumbens. Our original kinase-oriented phosphoproteomic analysis revealed 116 PKC substrate candidates, including Rac1 activator ß-PIX. Acetylcholine induced ß-PIX phosphorylation and activation, thereby stimulating Rac1 effector p21-activated kinase (PAK). Aversive stimulus activated the M1R-PKC-PAK pathway in mouse D2R-MSNs. D2R-MSN-specific expression of PAK mutants by the Cre-Flex system regulated dendritic spine structural plasticity and aversive learning. Donepezil induced PAK activation in both accumbal D2R-MSNs and in the CA1 region of the hippocampus and enhanced D2R-MSN-mediated aversive learning. These findings demonstrate that acetylcholine stimulates M1R-PKC-ß-PIX-Rac1-PAK signaling in D2R-MSNs for aversive learning and imply the cascade's therapeutic potential for AD as aversive learning is used to preliminarily screen AD drugs.

Inhibition of Rho-kinase ameliorates decreased spine density in the medial prefrontal cortex and methamphetamine-induced cognitive dysfunction in mice carrying schizophrenia-associated mutations of the Arhgap10 gene.

Copy-number variations in the ARHGAP10 gene encoding Rho GTPase-activating protein 10 are associated with schizophrenia. Model mice (Arhgap10 S490P/NHEJ mice) that carry "double-hit" mutations in the Arhgap10 gene mimic the schizophrenia in a Japanese patient, exhibiting altered spine density, methamphetamine-induced cognitive dysfunction, and activation of RhoA/Rho-kinase signaling. However, it remains unclear whether the activation of RhoA/Rho-kinase signaling due to schizophrenia-associated Arhgap10 mutations causes the phenotypes of these model mice. Here, we investigated the effects of fasudil, a brain permeable Rho-kinase inhibitor, on altered spine density in the medial prefrontal cortex (mPFC) and on methamphetamine-induced cognitive impairment in a touchscreen­based visual discrimination task in Arhgap10 S490P/NHEJ mice. Fasudil (20 mg/kg, intraperitoneal) suppressed the increased phosphorylation of myosin phosphatase-targeting subunit 1, a substrate of Rho-kinase, in the striatum and mPFC of Arhgap10 S490P/NHEJ mice. In addition, daily oral administration of fasudil (20 mg/kg/day) for 7 days ameliorated the reduced spine density of layer 2/3 pyramidal neurons in the mPFC. Moreover, fasudil (3-20 mg/kg, intraperitoneal) rescued the methamphetamine (0.3 mg/kg)-induced cognitive impairment of visual discrimination in Arhgap10 S490P/NHEJ mice. Our results suggest that Rho-kinase plays significant roles in the neuropathological changes in spine morphology and in the vulnerability of cognition to methamphetamine in mice with schizophrenia-associated Arhgap10 mutations.

Rho kinase inhibitors ameliorate cognitive impairment in a male mouse model of methamphetamine-induced schizophrenia.

Schizophrenia (SCZ) is a severe psychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive deficits. Current antipsychotic treatment in SCZ improves positive symptoms but has major side effects and little impact on negative symptoms and cognitive impairment. The pathoetiology of SCZ remains unclear, but is known to involve small GTPase signaling. Rho kinase, an effector of small GTPase Rho, is highly expressed in the brain and plays a major role in neurite elongation and neuronal architecture. This study used a touchscreen-based visual discrimination (VD) task to investigate the effects of Rho kinase inhibitors on cognitive impairment in a methamphetamine (METH)-treated male mouse model of SCZ. Systemic injection of the Rho kinase inhibitor fasudil dose-dependently ameliorated METH-induced VD impairment. Fasudil also significantly suppressed the increase in the number of c-Fos-positive cells in the infralimbic medial prefrontal cortex (infralimbic mPFC) and dorsomedial striatum (DMS) following METH treatment. Bilateral microinjections of Y-27632, another Rho kinase inhibitor, into the infralimbic mPFC or DMS significantly ameliorated METH-induced VD impairment. Two proteins downstream of Rho kinase, myosin phosphatase-targeting subunit 1 (MYPT1; Thr696) and myosin light chain kinase 2 (MLC2; Thr18/Ser19), exhibited increased phosphorylation in the infralimbic mPFC and DMS, respectively, after METH treatment, and fasudil inhibited these increases. Oral administration of haloperidol and fasudil ameliorated METH-induced VD impairment, while clozapine had little effect. Oral administration of haloperidol and clozapine suppressed METH-induced hyperactivity, but fasudil had no effect. These results suggest that METH activates Rho kinase in the infralimbic mPFC and DMS, which leads to cognitive impairment in male mice. Rho kinase inhibitors ameliorate METH-induced cognitive impairment, perhaps via the cortico-striatal circuit.

Muscarinic signaling regulates voltage-gated potassium channel KCNQ2 phosphorylation in the nucleus accumbens via protein kinase C for aversive learning.

The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217) and its inhibitory effect on channel activity were predicted. However, whether and how muscarinic signaling phosphorylates KCNQ2 in vivo remains unclear. Here, we found that PKC directly phosphorylated KCNQ2 at T217 in vitro. Carbachol and a muscarinic M1 receptor (M1R) agonist facilitated KCNQ2 phosphorylation at T217 in NAc/striatum slices in a PKC-dependent manner. Systemic administration of the cholinesterase inhibitor donepezil, which is commonly used to treat dementia, and electric foot shock to mice induced the phosphorylation of KCNQ2 at T217 in the NAc, whereas phosphorylation was suppressed by an M1R antagonist. Conditional deletion of Kcnq2 in the NAc enhanced electric foot shock induced aversive learning. Our findings indicate that muscarinic signaling induces the phosphorylation of KCNQ2 at T217 via PKC activation for aversive learning.

Phosphorylation Signals Downstream of Dopamine Receptors in Emotional Behaviors: Association with Preference and Avoidance.

Dopamine regulates emotional behaviors, including rewarding and aversive behaviors, through the mesolimbic dopaminergic pathway, which projects dopamine neurons from the ventral tegmental area to the nucleus accumbens (NAc). Protein phosphorylation is critical for intracellular signaling pathways and physiological functions, which are regulated by neurotransmitters in the brain. Previous studies have demonstrated that dopamine stimulated the phosphorylation of intracellular substrates, such as receptors, ion channels, and transcription factors, to regulate neuronal excitability and synaptic plasticity through dopamine receptors. We also established a novel database called KANPHOS that provides information on phosphorylation signals downstream of monoamines identified by our kinase substrate screening methods, including dopamine, in addition to those reported in the literature. Recent advances in proteomics techniques have enabled us to clarify the mechanisms through which dopamine controls rewarding and aversive behaviors through signal pathways in the NAc. In this review, we discuss the intracellular phosphorylation signals regulated by dopamine in these two emotional behaviors.

Dynamic subcellular localization and transcription activity of the SRF cofactor MKL2 in the striatum are regulated by MAPK.

Dopamine type 1 receptor (D1R) signaling activates protein kinase A (PKA), which then activates mitogen-activated protein kinase (MAPK) through Rap1, in striatal medium spiny neurons (MSNs). MAPK plays a pivotal role in reward-related behavior through the activation of certain transcription factors. How D1R signaling regulates behavior through transcription factors remains largely unknown. CREB-binding protein (CBP) promotes transcription through hundreds of different transcription factors and is also important for reward-related behavior. To identify transcription factors regulated by dopamine signaling in MSNs, we performed a phosphoproteomic analysis using affinity beads coated with CBP. We obtained approximately 40 novel candidate proteins in the striatum of the C57BL/6 mouse brain after cocaine administration. Among them, the megakaryoblastic leukemia-2 (MKL2) protein, a transcriptional coactivator of serum response factor (SRF), was our focus. We found that the interaction between CBP and MKL2 was increased by cocaine administration. Additionally, MKL2, CBP and SRF formed a ternary complex in vivo. The C-terminal domain of MKL2 interacted with CBP-KIX and was phosphorylated by MAPK in COS7 cells. The activation of PKA-MAPK signaling induced the nuclear localization of MKL2 and increased SRF-dependent transcriptional activity in neurons. These results demonstrate that dopamine signaling regulates the interaction of MKL2 with CBP in a phosphorylation-dependent manner and thereby controls SRF-dependent gene expression. Cover Image for this issue: https://doi.org/10.1111/jnc.15067.

Shati/Nat8l deficiency disrupts adult neurogenesis and causes attentional impairment through dopaminergic neuronal dysfunction in the dentate gyrus.

Successful completion of daily activities relies on the ability to select the relevant features of the environment for memory and recall. Disruption to these processes can lead to various disorders, such as attention-deficit hyperactivity disorder (ADHD). Dopamine is a neurotransmitter implicated in the regulation of several processes, including attention. In addition to the higher-order brain function, dopamine is implicated in the regulation of adult neurogenesis. Previously, we generated mice lacking Shati, an N-acetyltransferase-8-like protein on a C57BL/6J genetic background (Shati/Nat8l-/- ). These mice showed a series of changes in the dopamine system and ADHD-like behavioral phenotypes. Therefore, we hypothesized that deficiency of Shati/Nat8l would affect neurogenesis and attentional behavior in mice. We found aberrant morphology of neurons and impaired neurogenesis in the dentate gyrus of Shati/Nat8l-/- mice. Additionally, research has suggested that impaired neurogenesis might be because of the reduction of dopamine in the hippocampus. Galantamine (GAL) attenuated the attentional impairment observed in the object-based attention test via increasing the dopamine release in the hippocampus of Shati/Nat8l-/- mice. The α7 nicotinic acetylcholine receptor antagonist, methyllycaconitine, and dopamine D1 receptor antagonist, SCH23390, blocked the ameliorating effect of GAL on attentional impairment in Shati/Nat8l-/- mice. These results suggest that the ameliorating effect of GAL on Shati/Nat8l-/- attentional impairment is associated with activation of D1 receptors following increased dopamine release in the hippocampus via α7 nicotinic acetylcholine receptor. In summary, Shati/Nat8l is important in both morphogenesis and neurogenesis in the dentate gyrus and attention, possible via modulation of dopaminergic transmission. Cover Image for this issue: https://doi.org/10.1111/jnc.15061.

Microinjection of Reelin into the mPFC prevents MK-801-induced recognition memory impairment in mice.

Reelin, a large extracellular matrix protein, helps to regulate neuronal plasticity and cognitive function. Several studies have shown that Reelin dysfunction, resulting from factors such as mutations in gene RELN or low Reelin expression, is associated with schizophrenia (SCZ). We previously reported that microinjection of Reelin into cerebral ventricle prevents phencyclidine-induced cognitive and sensory-motor gating deficits. However, it remains unclear whether and how Reelin ameliorates behavioral abnormalities in the animal model of SCZ. In the present study, we evaluated the effect of recombinant Reelin microinjection into the medial prefrontal cortex (mPFC) on abnormal behaviors induced by MK-801, an N-methyl-D-aspartate receptor antagonist. Microinjection of Reelin into the mPFC prevented impairment of recognition memory of MK-801-treated mice in the novel object recognition test (NORT). On the other hand, the same treatment had no effect on deficits in sensory-motor gating and short-term memory in the pre-pulse inhibition and Y-maze tests, respectively. To establish the neural substrates that respond to Reelin, the number of c-Fos-positive cells in the mPFC was determined. A significant increase in c-Fos-positive cells in the mPFC of MK-801-treated mice was observed when compared with saline-treated mice, and this change was suppressed by microinjection of Reelin into the mPFC. A K2360/2467A Reelin that cannot bind to its receptor failed to ameliorate MK-801-induced cognitive deficits in NORT. These results suggest that Reelin prevents MK-801-induced recognition memory impairment by acting on its receptors to suppress neural activity in the mPFC of mice.

Generation and analysis of novel Reln-deleted mouse model corresponding to exonic Reln deletion in schizophrenia.

AIM: A Japanese individual with schizophrenia harboring a novel exonic deletion in RELN was recently identified by genome-wide copy-number variation analysis. Thus, the present study aimed to generate and analyze a model mouse to clarify whether Reln deficiency is associated with the pathogenesis of schizophrenia. METHODS: A mouse line with a novel RELN exonic deletion (Reln-del) was established using the CRISPR/Cas9 method to elucidate the underlying molecular mechanism. Subsequently, general behavioral tests and histopathological examinations of the model mice were conducted and phenotypic analysis of the cerebellar granule cell migration was performed. RESULTS: The phenotype of homozygous Reln-del mice was similar to that of reeler mice with cerebellar atrophy, dysplasia of the cerebral layers, and abrogated protein levels of cerebral reelin. The expression of reelin in heterozygous Reln-del mice was approximately half of that in wild-type mice. Conversely, behavioral analyses in heterozygous Reln-del mice without cerebellar atrophy or dysplasia showed abnormal social novelty in the three-chamber social interaction test. In vitro reaggregation formation and neuronal migration were severely altered in the cerebellar cultures of homozygous Reln-del mice. CONCLUSION: The present results in novel Reln-del mice modeled after our patient with a novel exonic deletion in RELN are expected to contribute to the development of reelin-based therapies for schizophrenia.

Methylation analysis for postpartum depression: a case control study.

BACKGROUND: Postpartum depression (PPD) is a major depressive disorder that occurs after childbirth. Objective diagnostic and predictive methods for PPD are important for early detection and appropriate intervention. DNA methylation has been recognized as a potential biomarker for major depressive disorder. In this study, we used methylation analysis and peripheral blood to search for biomarkers that could to lead to the development a predictive method for PPD. METHODS: Study participants included 36 pregnant women (18 cases and 18 controls determined after childbirth). Genome-wide DNA methylation profiles were obtained by analysis with an Infinium Human Methylation 450BeadChip. The association of DNA methylation status at each DNA methylation site with PPD was assessed using linear regression analysis. We also conducted functional enrichment analysis of PPD using The Database for Annotation, Visualization and Integrated Discovery 6.8 to explore enriched functional-related gene groups for PPD. RESULTS: In the analysis with postpartum depressed state as an independent variable, the difference in methylation frequency between the postpartum non-depressed group and the postpartum depressed group was small, and sites with genome-wide significant differences were not confirmed. After analysis by The Database for Annotation, Visualization and Integrated Discovery 6.8, we revealed four gene ontology terms, including axon guidance, related to postpartum depression. CONCLUSIONS: These findings may help with the development of an objective predictive method for PPD.

Astroglial major histocompatibility complex class I following immune activation leads to behavioral and neuropathological changes.

In the central nervous system, major histocompatibility complex class I (MHCI) molecules are mainly expressed in neurons, and neuronal MHCI have roles in synapse elimination and plasticity. However, the pathophysiological significance of astroglial MHCI remains unclear. We herein demonstrate that MHCI expression is up-regulated in astrocytes in the medial prefrontal cortex (mPFC) following systemic immune activation by an intraperitoneal injection of polyinosinic-polycytidylic acid (polyI:C) or hydrodynamic interferon (IFN)-γ gene delivery in male C57/BL6J mice. In cultured astrocytes, MHCI/H-2D largely co-localized with exosomes. To investigate the role of astroglial MHCI, H-2D, or sH-2D was expressed in the mPFC of male C57/BL6J mice using an adeno-associated virus vector under the control of a glial fibrillary acidic protein promoter. The expression of astroglial MHCI in the mPFC impaired sociability and recognition memory in mice. Regarding neuropathological changes, MHCI expression in astrocytes significantly activated microglial cells, decreased parvalbumin-positive cell numbers, and reduced dendritic spine density in the mPFC. A treatment with GW4869 that impairs exosome synthesis ameliorated these behavioral and neuropathological changes. These results suggest that the overexpression of MHCI in astrocytes affects microglial proliferation as well as neuronal numbers and spine densities, thereby leading to social and cognitive deficits in mice, possibly via exosomes created by astrocytes.

Neuronal PAS domain protein 4 (Npas4) controls neuronal homeostasis in pentylenetetrazole-induced epilepsy through the induction of Homer1a.

Neuronal intrinsic homeostatic scaling-down of excitatory synapse has been implicated in epilepsy pathogenesis to prevent the neuronal circuits from hyperexcitability. Recent findings suggest a role for neuronal PAS domain protein 4 (Npas4), an activity-dependent neuron-specific transcription factor in epileptogenesis, however, the underlying mechanism by which Npas4 regulates epilepsy remains unclear. We herein propose that limbic seizure activity up-regulates Npas4-homer1a signaling in the hippocampus, thereby contributing to epileptogenesis in mice. The expression level of Npas4mRNA was significantly increased after the pentylenetetrazol (PTZ) treatment. Npas4KO mice developed kindling more rapidly than their wild-type littermates. The expression of Homer1a in the hippocampus increased after seizure activity. Npas4 increased Homer1a promoter activity in COS7 cells. The PTZ-stimulated induction of Homer1a was attenuated in the hippocampus of Npas4KO mice. The combination of fluorescence in situ hybridization and immunohistochemical analyses revealed that Homer1amRNA co-localized with the Npas4 protein after the convulsive seizure response. PTZ reduced excitatory synaptic transmission at the associational/commissural fibers-CA3 synapses through the Npas4-mediated down-regulation of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in hippocampal CA3 neurons. The adeno-associated virus (AAV)-mediated expression of Homer1a resulted in lower α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor GluA1 subunit levels in the hippocampal plasma membrane fraction than in that from AAV-EGFP-transfected Npas4KO mice. The development of kindling was more strongly suppressed in AAV-Homer1a-microinjected Npas4KO mice than in AAV-EGFP-microinjected Npas4KO mice. These results indicate that Npas4 functions as a molecular switch to initiate homeostatic scaling and the targeting of Npas4-Homer1a signaling may provide new approaches for the treatment of epilepsy.

Innate immune activation of astrocytes impairs neurodevelopment via upregulation of follistatin-like 1 and interferon-induced transmembrane protein 3.

BACKGROUND: Polyriboinosinic-polyribocytidylic acid (polyI:C) triggers a strong innate immune response that mimics immune activation by viral infections. Induction of interferon-induced transmembrane protein 3 (Ifitm3) in astrocytes has a crucial role in polyI:C-induced neurodevelopmental abnormalities. Through a quantitative proteomic screen, we previously identified candidate astroglial factors, such as matrix metalloproteinase-3 (Mmp3) and follistatin-like 1 (Fstl1), in polyl:C-induced neurodevelopmental impairment. Here, we characterized the Ifitm3-dependent inflammatory processes focusing on astrocyte-derived Fstl1 following polyI:C treatment to assess the neuropathologic role of Fstl1. METHODS: Astrocytes were treated with PBS (control) or polyI:C (10 µg/mL). The conditioned medium was collected 24 h after the polyI:C treatment and used as astrocyte condition medium (ACM). The expression of Fstl1 mRNA and extracellular Fstl1 protein levels were analyzed by quantitative PCR and western blotting, respectively. For functional studies, neurons were treated with ACM and the effects of ACM on dendritic elongation were assayed. To examine the role of Fstl1, recombinant Fstl1 protein and siRNA for Fstl1 were used. To investigate the expression of Fstl1 in vivo, neonatal mice were treated with vehicle or polyI:C on postnatal day 2 to 6. RESULTS: ACM prepared with polyI:C (polyI:C ACM) contained significantly higher Fstl1 protein than control ACM, but no increase in Fstl1 was observed in polyI:C ACM derived from Ifitm3-deficient astrocytes. We found that the production of Fstl1 involves the inflammatory responsive molecule Ifitm3 in astrocytes and influences neuronal differentiation. In agreement, the levels of Fstl1 increased in the hippocampus of polyI:C-treated neonatal mice. COS7 cells co-transfected with both Fstl1 and Ifitm3 had higher extracellular levels of Fstl1 than the cells transfected with Fstl1 alone. Treatment of primary cultured hippocampal neurons with recombinant Fstl1 impaired dendritic elongation, and the deleterious effect of polyI:C ACM on dendritic elongation was attenuated by knockdown of Fstl1 in astrocytes. CONCLUSIONS: The extracellular level of Fstl1 is regulated by Ifitm3 in astrocytes, which could be involved in polyI:C-induced neurodevelopmental impairment.

Insular neural system controls decision-making in healthy and methamphetamine-treated rats.

Patients suffering from neuropsychiatric disorders such as substance-related and addictive disorders exhibit altered decision-making patterns, which may be associated with their behavioral abnormalities. However, the neuronal mechanisms underlying such impairments are largely unknown. Using a gambling test, we demonstrated that methamphetamine (METH)-treated rats chose a high-risk/high-reward option more frequently and assigned higher value to high returns than control rats, suggestive of changes in decision-making choice strategy. Immunohistochemical analysis following the gambling test revealed aberrant activation of the insular cortex (INS) and nucleus accumbens in METH-treated animals. Pharmacological studies, together with in vivo microdialysis, showed that the insular neural system played a crucial role in decision-making. Moreover, manipulation of INS activation using designer receptor exclusively activated by designer drug technology resulted in alterations to decision-making. Our findings suggest that the INS is a critical region involved in decision-making and that insular neural dysfunction results in risk-taking behaviors associated with altered decision-making.

Valosin-containing protein (VCP) is a novel IQ motif-containing GTPase activating protein 1 (IQGAP1)-interacting protein.

Scaffold proteins play a pivotal role in making protein complexes, and organize binding partners into a functional unit to enhance specific signaling pathways. IQ motif-containing GTPase activating protein 1 (IQGAP1) is an essential protein for spine formation due to its role in scaffolding multiple signal complexes. However, it remains unclear how IQGAP1 interacts within the brain. In the present study, we screened novel IQGAP1-interacting proteins by a proteomic approach. As a novel IQGAP1-interacting protein, we identified valosin-containing protein (VCP) which is a causative gene in patients with inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD). The physiological interaction of IQGAP1 with VCP was confirmed by an immunoprecipitation assay. Both the N-terminal (N-half) and C-terminal (C-half) fragments of IQGAP1 interacted with the N-terminal region of VCP. Co-localization of IQGAP1 and VCP was observed in the growth corn, axonal shaft, cell body, and dendrites in cultured hippocampal neurons at 4 days in vitro (DIV4). In cultured neurons at DIV14, IQGAP1 co-localized with VCP in dendrites. When HEK293T cells were co-transfected with IQGAP1 and VCP, an immunoprecipitation assay revealed that binding of IQGAP1 with disease-related mutant (R155H or A232E) VCP was markedly reduced compared to wild-type (WT) VCP. These results suggest that reduction of IQGAP1 and VCP interaction may be associated with the pathophysiology of IBMPFD.

Incidence of and risk factors associated with nedaplatin-related hypersensitivity reactions.

BACKGROUND: Nedaplatin (NDP)-related hypersensitivity reactions (HSRs) trigger adverse clinical events. Prediction and prevention of NDP-HSRs are thus essential to minimize the risk and maximize the benefit of NDP therapy. However, the incidence of NDP-HSRs and the associated risk factors remain unclear. METHODS: We retrospectively examined patients who received NDP monotherapy between April 2011 and July 2015 in Nagoya University Hospital. HSRs severity was defined according to the Common Terminology Criteria for Adverse Events version 4 (CTCAE ver.4). Risk factors for NDP-HSRs were determined using multivariate logistic regression. RESULTS: Of 111 patients who received NDP monotherapy, 90 (81%) were female; median age was 59 years (range, 29-78 years). Eighty-eight patients had gynecological cancer and 20 suffered from head and neck cancer. Eight of 111 patients (7.2%) experienced NDP-HSRs, six of which developed in the second NDP cycle. However, all patients with NDP-HSRs were treated with carboplatin (CBDCA) for more than three cycles. Grade 3 and 4 HSRs developed in 2 patients. NDP-HSRs were significantly associated with a history of CBDCA-HSRs (odds ratio 37.5, 95% confidence interval 5.38-262, p < 0.001) and with the interval between NDP administration and the previous platinum treatment (odds ratio 13.9, 95% confidence interval 1.23-158, p = 0.034). CONCLUSION: The risk of NDP-HSRs increases in patients with a history of CBDCA-HSRs and in those administered NDP for more than 6 months after previous platinum treatment. Such individuals must be closely monitored if given NDP, even if they are expected to benefit from the treatment.

[Analysis of Nedaplatin Dose in Patients with Impaired Renal Function].

Nedaplatin(NDP)is a platinum derivative anticancer drug.An NDP dose of 100mg/m2 every 4 weeks is recommended in non-elderly Japanese patient because a higher dose may lead to myelosuppression, such as thrombocytopenia.In a pharmacokinetic analysis, thrombocytopenia was significantly correlated with renal function.However, the correct dose in patients with impaired renal function remains unclear.To evaluate the usefulness of dose reduction in patients with renal dysfunction, we conducted a retrospective study.This study included Japanese solid cancer patients who received NDP monotherapy in Nagoya University Hospital between April 2011 and March 2014. Eighty three patients were evaluated and divided into 2 groups based on renal function: a creatinine clearance(Ccr; mL/min)≥60 group and a Ccr<60 group.The frequency of B Grade 3 thrombocytopenia and neutropenia was significantly higher in the Ccr<60 group than that in the Ccr≥60 group (3.4% vs 32.0%; p=0.001 and 6.8% vs 32.0%; p=0.005, respectively).In the Ccr<60 group, the frequency of BGrade 3 thrombocytopenia and neutropenia was lower in the reduced dose group than that in standard dose(100mg/m2)group (41.7% vs 23.1%; p=0.410 and 41.7% vs 23.1%; p=0.410, respectively).A multiple logistic regression analysis revealed that NDP dose and serum creatinine were risk factors for the incidence of BGrade 3 thrombocytopenia and neutropenia.These results suggest that NDP dose should be reduced to achieve safe drug treatment in patients with Ccr<60.

Isoflurane Induces Transient Impairment of Retention of Spatial Working Memory in Rats.

Postoperative cognitive dysfunction (POCD) occurs in nearly one-third of patients after non-cardiac surgery. Many animal behavior studies have investigated the effect of general anesthesia on cognitive function. However, there have been no studies examining the effects on working memory specifically, with a focus on the retention of working memory. We demonstrate here that isoflurane anesthesia induces deficits in the retention of spatial working memory in rats, as revealed by an increase in isoflurane- induced across-phase errors in the delayed spatial win-shift (SWSh) task with a 30-min delay in an 8-arm radial arm maze on post-anesthesia days (PADs) 1,2,4, and 10. A post-hoc analysis revealed a significant increase in across-phase errors on PAD 1 and recovery on PAD 10 in the isoflurane group. In contrast, within-phase errors independent of the retention of working memory were unaffected by isoflurane. These results demonstrate that isoflurane anesthesia transiently impairs the retention of spatial working memory in rats.

Reward signals downstream of dopamine D1 receptors.

It-is well known that dopamine is necessary for motor function, reward-motivated behaviors. The principal target of dopamine is medium spiny neurons (MSNs), which are a special type of GABAergic inhibitory cell that represents,90% of the neurons within the striatum, including the nucleus accumbens (NAc). There is a distinct class of spatially intermixed MSNs that express dopamine type 1 or 2 receptors (DlR-MSNs or D2R-MSNs, respectively). The D1R-MSN and D2R-MSN pathways control the dynamic balance in the basal ganglia circuit. D1R is coupled -to adenylate cyclase through Golf to activate protein kinase A (PKA), whereas D2R inhibits adenylate cyclase through Gi. Although PKA has been implicated in reward signals downstream of D1Rs by pharmacological approaches, there is no direct evidence indicating that PKA in DlR-MSNs regulates neuronal excitability and reward-related behaviors. We have established the system in which enzymatic activity can be manipulated in the NAc under the control of specific promoters for DlR-MSNs using adeno-associated virus (AAV)-mediated conditional'transgenic techniques. We have also found novel PKA substrates in dopamine receptor signaling using phosphoproteomic approach. This review article focuses on the PKA and its substrates to understand dopamine-related signals in the MSNs. We discuss phosphoproteomic approaches for' comprehensively screening of dopamine signaling. Finally, we highlight RapI pathway as a novel reward signal in vivo.