Design, synthesis and biological evaluation of 2-pyrrolone derivatives as radioprotectors.

It is known that p53 is an important transcription factor and plays a central role in ionizing radiation (IR)-induced DNA damage responses such as cell cycle arrest, DNA repair and apoptosis. We previously reported that regulating p53 protein is an effective strategy for modulating cell fate by reducing the acute side effects of radiation therapy. Herein, we report on the discovery of STK160830 as a new radioprotector from a chemical library at The University of Tokyo and the design, synthesis and biological evaluation of its derivatives. The radioprotective activity of STK160830 itself and its derivatives that were synthesized in this work was evaluated using a leukemia cell line, MOLT-4 cells as a model of normal cells that express the p53 protein in a structure-activity relationships (SAR) study. The experimental results suggest that a direct relationship exists between the inhibitory effect of these STK160830 derivatives on the expression level of p53 and their radioprotective activity and that the suppression of p53 by STK160830 derivatives contribute to protecting MOLT-4 cells from apoptosis that is induced by exposure to radiation.

Prothymosin alpha-deficiency enhances anxiety-like behaviors and impairs learning/memory functions and neurogenesis.

Prothymosin alpha (ProTα) is expressed in various mammalian organs including the neuronal nuclei in the brain, and is involved in multiple functions, such as chromatin remodeling, transcriptional regulation, cell proliferation, and survival. ProTα has beneficial actions against ischemia-induced necrosis and apoptosis in the brain and retina. However, characterizing the physiological roles of endogenous ProTα in the brain without stress remains elusive. Here, we generated ProTα-deficiency mice to explore whether endogenous ProTα is involved in normal brain functions. We successfully generated heterozygous ProTα knockout (ProTα+/- ) mice, while all homozygous ProTα knockout (ProTα-/- ) offspring died at early embryonic stage, suggesting that ProTα has crucial roles in embryonic development. In the evaluation of different behavioral tests, ProTα+/- mice exhibited hypolocomotor activity in the open-field test and enhanced anxiety-like behaviors in the light/dark transition test and the novelty induced hypophagia test. ProTα+/- mice also showed impaired learning and memory in the step-through passive avoidance test and the KUROBOX test. Depression-like behaviors in ProTα+/- mice in the forced swim and tail suspension tests were comparable with that of wild-type mice. Furthermore, adult hippocampal neurogenesis was significantly decreased in ProTα+/- mice. ProTα+/- mice showed an impaired long-term potentiation induction in the evaluation of electrophysiological recordings from acute hippocampal slices. Microarray analysis revealed that the candidate genes related to anxiety, learning/memory-functions, and neurogenesis were down-regulated in ProTα+/- mice. Thus, this study suggests that ProTα has crucial physiological roles in the robustness of brain.

Prostaglandin E2-mediated attenuation of mesocortical dopaminergic pathway is critical for susceptibility to repeated social defeat stress in mice.

Various kinds of stress are thought to precipitate psychiatric disorders, such as major depression. Whereas studies in rodents have suggested a critical role of medial prefrontal cortex (mPFC) in stress susceptibility, the mechanism of how stress susceptibility is determined through mPFC remains unknown. Here we show a critical role of prostaglandin E(2) (PGE(2)), a bioactive lipid derived from arachidonic acid, in repeated social defeat stress in mice. Repeated social defeat increased the PGE(2) level in the subcortical region of the brain, and mice lacking either COX-1, a prostaglandin synthase, or EP1, a PGE receptor, were impaired in induction of social avoidance by repeated social defeat. Given the reported action of EP1 that augments GABAergic inputs to midbrain dopamine neurons, we analyzed dopaminergic response upon social defeat. Analyses of c-Fos expression of VTA dopamine neurons and dopamine turnover in mPFC showed that mesocortical dopaminergic pathway is activated upon social defeat and attenuated with repetition of social defeat in wild-type mice. EP1 deficiency abolished such repeated stress-induced attenuation of mesocortical dopaminergic pathway. Blockade of dopamine D1-like receptor during social defeat restored social avoidance in EP1-deficient mice, suggesting that disinhibited dopaminergic response during social defeat blocks induction of social avoidance. Furthermore, mPFC dopaminergic lesion by local injection of 6-hydroxydopamine, which mimicked the action of EP1 during repeated stress, facilitated induction of social avoidance upon social defeat. Taken together, our data suggest that PGE(2)-EP1 signaling is critical for susceptibility to repeated social defeat stress in mice through attenuation of mesocortical dopaminergic pathway.

Histone Deacetylase 2 Knockdown Ameliorates Morphological Abnormalities of Dendritic Branches and Spines to Improve Synaptic Plasticity in an APP/PS1 Transgenic Mouse Model.

Disease-modifying therapies, such as neuroprotective and neurorestorative interventions, are strongly desired for Alzheimer's disease (AD) treatment. Several studies have suggested that histone deacetylase 2 (HDAC2) inhibition can exhibit disease-modifying effects in AD patients. However, whether HDAC2 inhibition shows neuroprotective and neurorestorative effects under neuropathic conditions, such as amyloid ß (Aß)-elevated states, remains poorly understood. Here, we performed HDAC2-specific knockdown in CA1 pyramidal cells and showed that HDAC2 knockdown increased the length of dendrites and the number of mushroom-like spines of CA1 basal dendrites in APP/PS1 transgenic mouse model. Furthermore, HDAC2 knockdown also ameliorated the deficits in hippocampal CA1 long-term potentiation and memory impairment in contextual fear conditioning tests. Taken together, our results support the notion that specific inhibition of HDAC2 has the potential to slow the disease progression of AD through ameliorating Aß-induced neuronal impairments.

A Novel RNA Synthesis Inhibitor, STK160830, Has Negligible DNA-Intercalating Activity for Triggering A p53 Response, and Can Inhibit p53-Dependent Apoptosis.

RNA synthesis inhibitors and protein synthesis inhibitors are useful for investigating whether biological events with unknown mechanisms require transcription or translation; however, the dependence of RNA synthesis has been difficult to verify because many RNA synthesis inhibitors cause adverse events that trigger a p53 response. In this study, we screened a library containing 9600 core compounds and obtained STK160830 that shows anti-apoptotic effects in irradiated wild-type-p53-bearing human T-cell leukemia MOLT-4 cells and murine thymocytes. In many of the p53-impaired cells and p53-knockdown cells tested, STK160830 did not show a remarkable anti-apoptotic effect, suggesting that the anti-apoptotic activity is p53-dependent. In the expression analysis of p53, p53-target gene products, and reference proteins by immunoblotting, STK160830 down-regulated the expression of many of the proteins examined, and the downregulation correlated strongly with its inhibitory effect on cell death. mRNA expression analyses by qPCR and nascent RNA capture kit revealed that STK160830 showed a decreased mRNA expression, which was similar to that induced by the RNA synthesis inhibitor actinomycin D but differed to some extent. Furthermore, unlike other RNA synthesis inhibitors such as actinomycin D, p53 accumulation by STK160830 alone was negligible, and a DNA melting-curve analysis showed very weak DNA-intercalating activity, indicating that STK160830 is a useful inhibitor for RNA synthesis without triggering p53-mediated damage responses.

Transcriptome profiling of Lotus japonicus roots during arbuscular mycorrhiza development and comparison with that of nodulation.

To better understand the molecular responses of plants to arbuscular mycorrhizal (AM) fungi, we analyzed the differential gene expression patterns of Lotus japonicus, a model legume, with the aid of a large-scale cDNA macroarray. Experiments were carried out considering the effects of contaminating microorganisms in the soil inoculants. When the colonization by AM fungi, i.e. Glomus mosseae and Gigaspora margarita, was well established, four cysteine protease genes were induced. In situ hybridization revealed that these cysteine protease genes were specifically expressed in arbuscule-containing inner cortical cells of AM roots. On the other hand, phenylpropanoid biosynthesis-related genes for phenylalanine ammonia-lyase (PAL), chalcone synthase, etc. were repressed in the later stage, although they were moderately up-regulated on the initial association with the AM fungus. Real-time RT-PCR experiments supported the array experiments. To further confirm the characteristic expression, a PAL promoter was fused with a reporter gene and introduced into L. japonicus, and then the transformants were grown with a commercial inoculum of G. mosseae. The reporter activity was augmented throughout the roots due to the presence of contaminating microorganisms in the inoculum. Interestingly, G. mosseae only colonized where the reporter activity was low. Comparison of the transcriptome profiles of AM roots and nitrogen-fixing root nodules formed with Mesorhizobium loti indicated that the PAL genes and other phenylpropanoid biosynthesis-related genes were similarly repressed in the two organs.

[Olfactory disturbance screening with the odor stick identification test (OSIT-J) in executive checkups].

The odor stick identification test for Japanese (OSIT-J) has been shown to be useful for detecting and evaluating olfactory disturbances in Japanese people. We studied the usefulness of OSIT-J in screening for olfactory disturbances in 83 Japanese participants (49 male, 34 female) participating in an executive checkup at NTT West Kanazawa Hospital in Japan. The olfactory ability was self-reported on a grade scale. Olfactory function was then evaluated with a three-odors OSIT-J (rose, curry and sweaty socks). Participants with low self-reported olfactory ability or less-than-full scores in the three-odor test were evaluated with an additional 10 odors of OSIT-J. Eight or less points are considered to be lower than average in the 13-odor test of OSIT-J (Saito S, et al.). Eleven of the 83 participants had low self-reported olfactory ability. Four participants with a full score in the three odors test with low self-reported olfactory ability scored more than eight points in the 13-odor test. Thirty-eight participants scored less than three points in the three-odor test. Seven of 29 participants with two points in the three-odor test scored eight or less in the 13-odor test. In the 29 participants, subjects with low self-reported olfactory ability scored significantly lower scores than those without a low self-reported olfactory ability in the 13-odor test. The self-reported olfactory ability was not related to the score in the 13-odor test in the nine participants with one point or less in the three-odor test. Males scored significantly lower scores than females in the three-odor test. However, gender was not significantly related to the rate of olfactory disability estimated based on the 13-odor test. Use of a three-odor OSIT-J along with a self-administered questionnaire pertaining to olfactory disability is useful for olfactory disturbance screening during executive health checkups.

mDia and ROCK Mediate Actin-Dependent Presynaptic Remodeling Regulating Synaptic Efficacy and Anxiety.

Here, we show neuronal inactivation-induced presynaptic remodeling and involvement of the mammalian homolog of Diaphanous (mDia) and Rho-associated coiled-coil-containing kinase (ROCK), Rho-regulated modulators of actin and myosin, in this process. We find that social isolation induces inactivation of nucleus accumbens (NAc) neurons associated with elevated anxiety-like behavior, and that mDia in NAc neurons is essential in this process. Upon inactivation of cultured neurons, mDia induces circumferential actin filaments around the edge of the synaptic cleft, which contract the presynaptic terminals in a ROCK-dependent manner. Social isolation induces similar mDia-dependent presynaptic contraction at GABAergic synapses from NAc neurons in the ventral tegmental area (VTA) associated with reduced synaptic efficacy. Optogenetic stimulation of NAc neurons rescues the anxiety phenotype, and injection of a specific ROCK inhibitor, Y-27632, into the VTA reverses both presynaptic contraction and the behavioral phenotype. mDia-ROCK signaling thus mediates actin-dependent presynaptic remodeling in inactivated NAc neurons, which underlies synaptic plasticity in emotional behavioral responses.

[Clinical usefulness of odor stick identification test for patients with olfactory disturbance].

The odor stick identification test for Japanese (OSITJ) is the latest in measuring olfactory identification. It consists of 13 odorants familiar to the Japanese population. We studied the relationship between the Japanese standard olfactory test (T & T olfactometer) and the OSITJ in 182 patients with olfactory disturbance. The identification ratio in 2 of OSITJ tests using 13 odorants was correlated significantly negatively with the detection and recognition threshold measured by the T & T olfactometer. A high correlation between identification ratio and olfactory thresholds was maintained even when the number of odorants in the OSITJ was reduced to 2. For each odorant used, the identification ratio correlated significantly with olfactory thresholds. Results from the OSITJ provide a measure of the degree of olfactory deficit because the ratio of correct answers obtained by the OSITJ decreased gradually with of the severity of olfactory disturbance. Compared to the T & T olfactometer, the OSITJ has several advantages for use in the clinic. These include minimal odor pollution of the test room, simplicity of use, and shorter clinical time needed to administer the test. The OSITJ may be ideal for use in screening due to the minimum number of odorants needed. In conclusion, the OSITJ is useful for detecting and evaluating olfactory disturbance in Japanese people.

[Roles of altered striatal function in major depression].

Major depression, a psychiatric disorder characterized by depressive mood and loss of interest and pleasure, is a leading cause of disability and suicide in developed countries. However, the mechanisms that underlie major depression remain to be elucidated. Clinical studies on patients with major depression have shown abnormalities in multiple brain areas, each of which can account for a distinct symptom or endophenotype. Notably, the striatum in healthy control subjects responds to positive emotional stimuli and to positive feedback signals during cognitive tasks, but these striatal responses are greatly reduced in depressive patients. Given the role of the striatum in behavioral learning with positive reinforcers, abnormalities as such suggest that impairment in reward processing and/or reinforcement learning in major depression is the basis of anhedonia and reduced psychomotor activity. In animal studies, stress -a risk factor for major depression- is frequently used to induce behavioral depression. Repeated social defeat stress increases the excitability of dopamine neurons and subsequent CREB-mediated transcription in the nucleus accumbens shell, leading to behavioral depression. Surprisingly, this pathway seems not to be involved in behavioral depression caused by prolonged social isolation, suggesting distinct mechanisms underlying the two stressful contexts. In contrast, ΔFosB-mediated gene expression in the nucleus accumbens shell confers resilience to stress. Repeated social defeat stress induces accumbal ΔFosB expression in a stress-resilient subset of individuals, whereas prolonged social isolation decreases this expression, leading to stress susceptibility. In addition to emotional changes, chronic stress also alters the mode of instrumental behavior from goal-directed to habitual responding, with consistent morphological changes in striatal subregions responsible for the corresponding behavioral modes. Therefore, these clinical and preclinical findings suggest that striatal abnormalities play a role in emotional and cognitive changes associated with major depression.

Temporally matched subpopulations of selectively interconnected principal neurons in the hippocampus.

The extent to which individual neurons are interconnected selectively within brain circuits is an unresolved problem in neuroscience. Neurons can be organized into preferentially interconnected microcircuits, but whether this reflects genetically defined subpopulations is unclear. We found that the principal neurons in the main subdivisions of the hippocampus consist of distinct subpopulations that are generated during distinct time windows and that interconnect selectively across subdivisions. In two mouse lines in which transgene expression was driven by the neuron-specific Thy1 promoter, transgene expression allowed us to visualize distinct populations of principal neurons with unique and matched patterns of gene expression, shared distinct neurogenesis and synaptogenesis time windows, and selective connectivity at dentate gyrus-CA3 and CA3-CA1 synapses. Matched subpopulation marker genes and neuronal subtype markers mapped near clusters of olfactory receptor genes. The nonoverlapping matched timings of synaptogenesis accounted for the selective connectivities of these neurons in CA3. Therefore, the hippocampus contains parallel connectivity channels assembled from distinct principal neuron subpopulations through matched schedules of synaptogenesis.

Wnt signaling mediates experience-related regulation of synapse numbers and mossy fiber connectivities in the adult hippocampus.

We investigated how experience regulates the structure of a defined neuronal circuit in adult mice. Enriched environment (EE) produced a robust and reversible increase in hippocampal stratum lucidum synapse numbers, mossy fiber terminal (LMT) numbers, and spine plus synapse densities at LMTs, whereas a distinct mechanism depending on Rab3a promoted LMT volume growth. In parallel, EE increased postsynaptic CA3 pyramidal neuron Wnt7a/b levels. Inhibiting Wnt signaling through locally applied sFRP-1 suppressed the effects of EE on synapse numbers and further reduced synapse numbers in control mice. Wnt7 applied to CA3 mimicked the effects of EE on synapse and LMT numbers. CA3 Wnt7a/b levels were enhanced by excitatory activity and reduced by sFRP-1. Synapse numbers and Wnt7a/b levels peaked in mice aged 6-12 months; a decline in aged mice was reversed by EE. Therefore, behavioral experience specifically regulates adult global stratum lucidum synapse numbers and hippocampal network structure through Wnt signaling.

Cross-cultural comparison of data using the odor stick identification test for Japanese (OSIT-J).

A new olfactory test, the odor stick identification test for Japanese (OSIT-J), has been developed in Japan. To determine if the OSIT-J would be effective cross-culturally, we administered the test to 52 US and 50 Japanese subjects reporting normal olfactory function. The average composite OSIT-J test score for US subjects was significantly lower (77%) than that for Japanese subjects (94%, P < 0.0001). Both US and Japanese subjects correctly identified eight of the 13 odorants included in the OSIT-J with scores of 80% or higher. However, for five odorants, the US subjects' scores fell below 80% and were consistently lower than Japanese subjects, presumably reflecting cultural differences in odor experience. Most of the US subjects found the OSIT-J to be easy, interesting, pleasant, and short in duration. Although the 13-odorant OSIT-J was found to be suitable for testing US populations, elimination of five test odorants that were unfamiliar to US subjects significantly enhanced the test's effectiveness. Findings from this study emphasize the importance of identifying test odorants that may have a cultural bias, a crucial issue when comparing data obtained from different smell tests used at smell and taste centers around the world.

Development of a smell identification test using a novel stick-type odor presentation kit.

The odor identification is strongly influenced by the social and cultural factors; therefore, the odorants used in a smell identification test should be familiar to the test population. In addition, the device used in the test is desired to be simply handled and retain odor quality over time. We developed a novel stick-type odor presentation kit that consists of microcapsules of odorant incorporated into stable cream and the smell identification test using it. Thirteen odorants were selected to be familiar to the test population. In the test, we used two identification methods: one was a modified forced-choice paradigm with "detectable but not recognizable" and "no smell detected" added as choices and the other was a two-step identification paradigm where the participant first selected one of eight odor categories and then chose the specific odor name from the selected category. We verified the performance of the odor stick and the test by stability, using a test-retest paradigm, comparing this test with another smell test, and testing Japanese people from a range of age groups. We conclude that this kit is a useful odor presentation device, and the test using it works effectively as a smell identification test.

Knockdown of an arbuscular mycorrhiza-inducible phosphate transporter gene of Lotus japonicus suppresses mutualistic symbiosis.

cDNA for a major arbuscular mycorrhiza (AM)-inducible phosphate (Pi) transporter of Lotus japonicus, LjPT3, was isolated from Glomus mosseae-colonized roots. The LjPT3 transcript was expressed in arbuscule-containing cells of the inner cortex. The transport activity of the gene product was confirmed by the complementation of a yeast mutant that lacks high-affinity Pi transporters. In contrast to most AM-inducible Pi transporters thus far reported, LjPT3 has an amino acid sequence that has much in common with those of other members of the Pht1 family of plant Pi transporters, such as StPT3 of potato. To understand better the physiological role of this AM-inducible Pi transporter, knockdown transformants of the gene were prepared through hairy root transformation and RNA interference. Under Pi-limiting conditions, the transformants showed a reduction of Pi uptake via AM and growth retardation. The transformants also exhibited a decrease in G. mosseae arbuscules. Additionally, when Mesorhizobium loti was inoculated into the knockdown transformants in combination with G. mosseae, necrotic root nodules were observed. Based on these findings, we consider that the genetically engineered host plants had monitored insufficient Pi uptake via AM or low expression of LjPT3, excluding the existing fungi and rhizobia and/or preventing further development of the fungal and nodule structures.

Cloning and expression analysis of a MAPKKK gene and a novel nodulin gene of Lotus japonicus.

We isolated a cDNA encoding mitogen-activated protein kinase kinase kinase alpha, designated LjM3Kalpha, from Lotus japonicus, a model legume. The gene was expressed constitutively in roots, root nodules, and shoots. We also identified a novel nodulin gene, LjNUF, that shows specific expression in nodules. LjNUF resembles the C-terminal half of a hypothetical protein (pir//D85436), the N-terminal half of which is similar to a portion of mitogen-activated protein kinase kinase kinase gamma. Although LjNUF was predicted to be a secreted protein, its function remains to be clarified.