DISC1 causes associative memory and neurodevelopmental defects in fruit flies.

Originally found in a Scottish family with diverse mental disorders, the DISC1 protein has been characterized as an intracellular scaffold protein that associates with diverse binding partners in neural development. To explore its functions in a genetically tractable system, we expressed the human DISC1 in fruit flies (Drosophila melanogaster). As in mammalian neurons, DISC1 is localized to diverse subcellular domains of developing fly neurons including the nuclei, axons and dendrites. Overexpression of DISC1 impairs associative memory. Experiments with deletion/mutation constructs have revealed the importance of amino-terminal domain (46-290) for memory suppression whereas carboxyl domain (598-854) and the amino-terminal residues (1-45) including the nuclear localization signal (NLS1) are dispensable. DISC1 overexpression also causes suppression of axonal and dendritic branching of mushroom body neurons, which mediate a variety of cognitive functions in the fly brain. Analyses with deletion/mutation constructs reveal that protein domains 598-854 and 349-402 are both required for the suppression of axonal branching, while amino-terminal domains including NLS1 are dispensable. In contrast, NLS1 was required for the suppression of dendritic branching, suggesting a mechanism involving gene expression. Moreover, domain 403-596 is also required for the suppression of dendritic branching. We also show that overexpression of DISC1 suppresses glutamatergic synaptogenesis in developing neuromuscular junctions. Deletion/mutation experiments have revealed the importance of protein domains 403-596 and 349-402 for synaptic suppression, while amino-terminal domains including NLS1 are dispensable. Finally, we show that DISC1 functionally interacts with the fly homolog of Dysbindin (DTNBP1) via direct protein-protein interaction in developing synapses.

Early postnatal GABAA receptor modulation reverses deficits in neuronal maturation in a conditional neurodevelopmental mouse model of DISC1.

Exploring drug targets based on disease-associated molecular mechanisms during development is crucial for the generation of novel prevention and treatment strategies for neurodevelopmental psychiatric conditions. We report that prefrontal cortex (PFC)-specific postnatal knockdown of DISC1 via in utero electroporation combined with an inducible knockdown expression system drives deficits in synaptic GABAA function and dendritic development in pyramidal neurons, as well as abnormalities in sensorimotor gating, albeit without profound memory deficits. We show for the first time that DISC1 is specifically involved in regulating cell surface expression of α2 subunit-containing GABAA receptors in immature developing neurons, but not after full maturation. Notably, pharmacological intervention with α2/3 subtype-selective GABAA receptor positive allosteric modulators during the early postnatal period ameliorates dendritic deficits and behavioral abnormalities induced by knockdown of DISC1. These findings highlight a critical role of DISC1-mediated disruption of postnatal GABA signaling in aberrant PFC maturation and function.

Role for neonatal D-serine signaling: prevention of physiological and behavioral deficits in adult Pick1 knockout mice.

NMDA glutamate receptors have key roles in brain development, function and dysfunction. Regulatory roles of D-serine in NMDA receptor-mediated synaptic plasticity have been reported. Nonetheless, it is unclear whether and how neonatal deficits in NMDA-receptor-mediated neurotransmission affect adult brain functions and behavior. Likewise, the role of D-serine during development remains elusive. Here we report behavioral and electrophysiological deficits associated with the frontal cortex in Pick1 knockout mice, which show D-serine deficits in a neonatal- and forebrain-specific manner. The pathological manifestations observed in adult Pick1 mice are rescued by transient neonatal supplementation of D-serine, but not by a similar treatment in adulthood. These results indicate a role for D-serine in neurodevelopment and provide novel insights on how we interpret data of psychiatric genetics, indicating the involvement of genes associated with D-serine synthesis and degradation, as well as how we consider animal models with neonatal application of NMDA receptor antagonists.

Prognostic value of tropomyosin-related kinases A, B, and C in gastric cancer.

PURPOSE: Tropomyosin-related kinase (Trk) receptors play critical roles in tumor development and are considered attractive targets for cancer therapy. We investigated correlations of the expression of TrkA, TrkB, and TrkC with clinicopathological features and outcomes in gastric cancer. METHODS: Tumor samples were obtained from 221 patients with gastric cancer who underwent gastrectomy between 2003 and 2007. The expression of TrkA, TrkB, and TrkC was analyzed using immunohistochemical staining. The relationship of their expression to clinicopathological factors and outcomes was assessed. RESULTS: High expression of TrkA, TrkB, or TrkC was significantly associated with histopathology (p = 0.022, p < 0.001, and p < 0.001). High expression of TrkA was significantly correlated with variables related to tumor progression, including lymph node metastasis (p = 0.024) and distant metastasis or recurrence (p < 0.001). Distant metastasis or recurrence was found in a significantly higher proportion of patients with high expression of TrkC than in those with low expression (p = 0.036). High expression of TrkA was significantly associated with poorer relapse-free survival (RFS) in univariate analysis (p = 0.001). High expression of TrkA or TrkC was significantly associated with poorer disease-specific survival (DSS) in univariate analysis (p < 0.001 and p = 0.008). In multivariate analysis, TrkA was an independent predictor of RFS [hazard ratio (HR), 2.294; 95 % confidence interval (CI), 1.309-4.032; p = 0.004] and DSS (HR, 2.146; 95 % CI, 1.195-3.861; p = 0.011). Expression of TrkB was not associated with RFS or DSS in univariate analysis. CONCLUSIONS: Our results demonstrated that TrkA expression was associated with tumor progression and poor survival, and was an independent predictor of poor outcomes in gastric cancer patients.

DISC1 regulates trafficking and processing of APP and Aß generation.

We report the novel regulation of proteolytic processing of amyloid precursor protein (APP) by DISC1, a major risk factor for psychiatric illnesses, such as depression and schizophrenia. RNAi knockdown of DISC1 in mature primary cortical neurons led to a significant increase in the levels of intracellular α-C-terminal fragment of APP (APP-CTFα) and the corresponding N-terminal-secreted ectodomain product sAPPα. DISC1 knockdown also elicited a significant decrease in the levels of amyloid beta (Aß)42 and Aß40. These aberrant proteolytic events were successfully rescued by co-expression of wild-type DISC1, but not by mutant DISC1 lacking the amino acids required for the interaction with APP, suggesting that APP-DISC1 protein interactions are crucial for the regulation of the C-terminal proteolysis. In a genetically engineered model in which a major full-length DISC1 isoform is depleted, consistent changes in APP processing were seen: an increase in APP-CTFα and decrease in Aß42 and Aß40 levels. Finally, we found that knockdown of DISC1 increased the expression of APP at the cell surface and decreased its internalization. The presented DISC1 mechanism of APP proteolytic processing and Aß peptide generation, which is central to Alzheimer's disease pathology, suggests a novel interface between neurological and psychiatric conditions.

Sympathetic afferent stimulation inhibits central vagal activation induced by intravenous medetomidine in rats.

AIM: To examine whether sympathetic afferent stimulation (SAS) inhibits central vagal activation induced by α2 -adrenergic stimulation. METHODS: In anaesthetized Wistar-Kyoto rats, a cardiac microdialysis technique was applied to the left ventricle, and the effect of α2 -adrenergic stimulation by medetomidine on myocardial interstitial acetylcholine (ACh) levels was examined in the absence (n = 6) or the presence (n = 6) of SAS delivered from the left stellate ganglion. The effect of electrical vagal efferent stimulation on myocardial interstitial ACh release was also examined in the absence or the presence of SAS (n = 6). RESULTS: Intravenous medetomidine (0.1 mg kg(-1) ) significantly increased myocardial interstitial ACh levels in the absence of SAS (from 1.95 ± 0.79 to 3.36 ± 1.61 nM, P < 0.05), but not in the presence of SAS (from 1.67 ± 0.67 to 2.01 ± 0.78 nM). In contrast, electrical vagal nerve stimulation increased myocardial interstitial ACh level to the same degree regardless of SAS (from 1.66 ± 0.16 to 3.93 ± 0.72 nM without SAS vs. 4.05 ± 0.89 nM with SAS). CONCLUSION: Sympathetic afferent stimulation inhibited medetomidine-induced ACh release, but not electrical stimulation-induced ACh release, suggesting that SAS inhibited medetomidine-induced vagal activation via central mechanisms. While central vagal activation by α2 -adrenergic agonists could be an alternative to electrical vagal activation, blocking sympathetic afferent input may be important to increase the efficacy of α2 -adrenergic agonists in enhancing vagal nerve activity.

High-frequency dominant depression of peripheral vagal control of heart rate in rats with chronic heart failure.

AIM: To examine whether dynamic characteristics of the peripheral vagal control of heart rate (HR) are altered in chronic heart failure (CHF). METHODS: The right vagal nerve was electrically stimulated according to a binary white noise signal, and the transfer function from vagal nerve stimulation (VNS) to HR was estimated in the frequency range from 0.01 to 1 Hz in five control rats and five CHF rats under anaesthetized conditions. The rate of VNS was changed among 10, 20 and 40 Hz. RESULTS: A multiple linear regression analysis indicated that the increase in the VNS rate augmented the ratio of the high-frequency (HF) gain to the steady-state gain in the control group but not in the CHF group. As a result, the dynamic gain of the transfer function in the frequencies near 1 Hz decreased more in the CHF group than in the control group. CONCLUSION: Changes in the dynamic characteristics of the peripheral vagal control of HR may contribute to the manifestation of decreased HF components of HR variability observed in CHF.

Highly efficient generation of definitive endoderm lineage from human induced pluripotent stem cells.

BACKGROUND: Although hepatocytes can be an option for liver transplantation, the shortage of donor organs continues to worsen. Since the development of induced pluripotent stem (iPS) cell technology, it is eagerly anticipated to produce functional elements from pluripotent stem cells. These functional cells differentiated from iPS cells could be used for transplantation, drug screening, and in vitro toxicology. METHODS: Human iPS cells are maintained on Mitomycin C-treated mouse embryonic fibroblast layers in DMEM-Ham F12-based medium supplemented with Knockout Serum Replacement, nonessential amino acids, 2-mercaptoethanol, and Glutamax. Differentiation of human iPS cells into a definitive endodermal lineage was induced with PRMI 1640 medium supplemented with B27 and 100 ng/mL human activin A. Two B27 supplements were examined with and without insulin. Furthermore, the PI3 kinase inhibitor LY294002 was used to examine the effect of inhibiting insulin signaling. RESULTS AND DISCUSSION: We established efficient induction of definitive endodermal differentiation from iPS cells. Quantitative analysis revealed efficient (93.03 ± 2.74%) differentiation of human iPS cells into definitive endoderm cells using B27 minus insulin. This protocol may contribute as a fundamental technique to promote human iPS studies to develop cellular sources for transplantation.

Central vagal activation by alpha(2) -adrenergic stimulation is impaired in spontaneously hypertensive rats.

AIM: To elucidate the abnormality of vagal control in spontaneously hypertensive rats (SHR) by measuring left ventricular myocardial interstitial acetylcholine (ACh) release in response to α(2) -adrenergic stimulation as an index of in vivo vagal nerve activity. METHODS: A cardiac microdialysis technique was applied to the rat left ventricle in vivo, and the effect of α(2) -adrenergic stimulation by medetomidine or electrical vagal nerve stimulation on myocardial interstitial ACh levels was examined in normotensive Wistar-Kyoto rats (WKY) and SHR under anaesthetized conditions. RESULTS: Intravenous medetomidine (0.1 mg kg(-1) ) significantly increased the ACh levels in WKY (from 2.4 ± 0.6 to 4.2 ± 1.3 nmol L(-1) , P < 0.05, n = 7) but not in SHR (from 2.5 ± 0.7 to 2.7 ± 0.7 nmol L(-1) , n = 7). In contrast, electrical vagal nerve stimulation increased the ACh levels in both WKY (from 1.0 ± 0.4 to 2.9 ± 0.9 nmol L(-1) , P < 0.001, n = 6) and SHR (from 0.9 ± 0.2 to 2.2 ± 0.4 nmol L(-1) , P < 0.001, n = 6). Intravenous administration of medetomidine (0.1 mg kg(-1) ) did not affect the vagal nerve stimulation-induced ACh release in either WKY or SHR. CONCLUSION: Medetomidine-induced central vagal activation was impaired in SHR, whereas peripheral vagal control of ACh release was preserved. In addition to abnormal sympathetic control, vagal control by the central nervous system may be impaired in SHR.

Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly.

Disrupted-in-schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that colocalizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a corepressor N-CoR, modulating CRE-mediated gene transcription.

Open clinical study of eye-drops containing tetrapeptides derived from substance P and insulin-like growth factor-1 for treatment of persistent corneal epithelial defects associated with neurotrophic keratopathy.

BACKGROUND/AIMS: Loss of corneal sensation results in the development of persistent corneal epithelial defects. The combination of a substance P-derived peptide (FGLM-amide) and an insulin-like growth factor-1 (IGF-1)-derived peptide (SSSR) stimulates rabbit corneal epithelial migration in vitro and rabbit corneal epithelial wound closure in vivo. The clinical efficacy of eye-drops containing FGLM-amide and SSSR for the treatment of persistent corneal epithelial defects in individuals with neurotrophic keratopathy was examined in a prospective open study. METHODS: Twenty-five consecutive patients (26 eyes) with persistent corneal epithelial defects associated with neurotrophic keratopathy were treated by administration of eye-drops containing FGLM-amide and SSSR. The course of epithelial healing was monitored by slit-lamp examination. RESULTS: Epithelial defects resurfaced completely in 19 of the 26 eyes (73%) within 4 weeks after treatment initiation. Complete resurfacing of epithelial defects was apparent in 18 of 22 (82%) or in one of four (25%) eyes without or with limbal stem cell deficiency, respectively. No adverse effects of treatment were observed in any subject. CONCLUSION: Eye-drops containing FGLM-amide and SSSR induced the rapid resurfacing of persistent epithelial defects in stem cell-positive individuals with neurotrophic keratopathy.

Disorder of phase-related modulation of soleus H-reflex during hip movement in stroke patients.

PURPOSE: The soleus H-reflex during passive hip movement was measured to clarify the modulation of excitability of the soleus monosynaptic reflex during locomotion-like movement in spastic stroke patients. METHOD: The experiment was performed in five patients with spastic hemiparesis. The hip joint was moved passively ranging from 0 to 40 degrees. The knee joint was fixed at full extension and the ankle joint was fixed at the mid-position. During the movement, the soleus M-wave and soleus H-reflex were measured. RESULTS: Flexion caused a decrease in the soleus H-reflex, whereas extension caused an increase symmetrically for both the static and dynamic conditions. In addition, the lowest value was observed at the end of the flexion phase during fast movement. CONCLUSION: These findings indicate that the phase-related modulation of soleus H-reflex during hip movement is partially disordered in stroke patients.

Personal recognition rate improvement using head-top image and an application to walking subject.

A personal identification method using head-top image is improved for the smart house. The personal recognition rate was 86.4 percent in the previous report from eleven subjects who stood still. It was improved to 100 percent in this study by using the same image database of the previous report. Moreover, head-top image sequences were acquired when the subjects under the thermo camera started walking, and the personal recognition rate was 52 percent from six subjects.

Passive movement of hip and knee joints decreases the amplitude of soleus H-reflex in stroke patients.

OBJECTIVES: The study aims to investigate the changes of the H-reflex in soleus (Sol-H-reflex) during hip and knee joint movements in stroke patients. METHODS: The experiments were carried out on five stroke patients with spastic hemiplegia (2 males and 3 females, 48 to 71 years old). Sol-H-reflexes were measured 200 times for each joint movement speed Stimulus was given at random intervals (4 to 5 seconds) during the joint movement. Two movement speeds were used to investigate the effects of movement speed. RESULTS: For both fast and slow movements, the amplitude of the Sol-H-reflex decreased in the middle flexion phase. In contrast, the amplitude of the Sol-H-reflex increased in the middle extension phase. For the fast movement, the Sol-H-reflex was smaller in only a small angle range during the flexion and extension phase in comparison to the slow movement. The Sol-H-reflex during the flexion phase was significantly smaller than during the extension phase at almost all angles for both speeds. The Sol-H-reflex for both speeds was smaller than the Sol-H-reflex at rest for the whole angle range. CONCLUSION: These results demonstrate the differential effects of Sol-H-reflex modulation in stroke patients when compared with normal subjects.