AMBRA1 controls antigen-driven activation and proliferation of naive T cells.

AMBRA1 (activating molecule in Beclin1-regulated autophagy) is a member of the BECN1 (BECLIN1) protein complex, and it plays a role in autophagy, cell death, tumorigenesis and proliferation. We recently reported that on T-cell receptor (TCR) stimulation, AMBRA1 controlled both autophagy and the cell cycle with metabolic regulation. Accumulating evidence has shown that autophagy and metabolic control are pivotal for T-cell activation, clonal expansion and effector/memory cell fate decision. However, it is unknown whether AMBRA1 is involved in T-cell function under physiological conditions. We found that T cells in Ambra1-conditional knockout (cKO) mice induced an exacerbated graft versus host response when they were transplanted into allogeneic BALB/c mice. Furthermore, Ambra1-deficient T cells showed increased proliferation and cytotoxic capability toward specific antigens in response to in vivo stimulation using allogeneic spleen cells. This enhanced immune response mainly contributed to naive T-cell hyperactivity. The T-cell hyperactivity observed in this study was similar to those in some metabolic factor-deficient mice, but not those in other pro-autophagic factor-deficient mice. Under the static condition, however, naive T cells were reduced in Ambra1-cKO mice, the same as in pro-autophagic factor-deficient mice. Collectively, these results suggested that AMBRA1 was involved in regulating T cell-mediated immune responses through autophagy-dependent and -independent mechanisms.

New PCNT candidate missense variant in a patient with oral and maxillofacial osteodysplasia: a case report.

BACKGROUND: Osteodysplasia of the oral and maxillofacial bone is generally accompanied by systemic bone abnormalities (such as short stature, joint contracture) or other systemic abnormalities (such as renal, dermatological, cardiovascular, optic, or hearing disorders). However, it does not always present this way. Recent reports have suggested that genome-wide sequencing is an effective method for identifying rare or new disorders. Here, we performed whole-exome sequencing (WES) in a patient with a unique form of acquired, local osteodysplasia of the oral and maxillofacial region. CASE PRESENTATION: A 46-year-old woman presented to our hospital with the complaint of gradually moving mandibular teeth (for 6 months), changing facial appearance, and acquired osteolysis of the oral and maxillofacial bones, showing mandibular hypoplasia without family history. Upon skeletal examination, there were no abnormal findings outside of the oral and maxillofacial area; the patient had a height of 157 cm and bone mineral density (according to dual energy x-ray absorptiometry) of 90%. Results of blood and urine tests, including evaluation of bone metabolism markers and neurological and cardiovascular examinations, were normal. We performed WES of genomic DNA extracted from the blood of this patient and her mother, who did not have the disease, as a negative control. We identified 83 new missense variants in the patient, not detected in her mother, including a candidate single nucleotide variant in exon 14 of PCNT (pericentrin). Critical homozygous or compound heterozygous variants in PCNT are a known cause of microcephalic osteodysplastic primordial dwarfism type II accompanied by mandibular hypoplasia, which is similar to the maxillofacial phenotype in this patient. CONCLUSIONS: Protein simulations performed using Polymorphism Phenotyping v2 and Combined Annotation Dependent Depletion software indicated that this missense variant is likely to disrupt the PCNT protein structure. These results suggest that this is a new form of osteolysis related to this PCNT variant.

In Vivo Spiking Dynamics of Intra- and Extratelencephalic Projection Neurons in Rat Motor Cortex.

In motor cortex, 2 types of deep layer pyramidal cells send their axons to other areas: intratelencephalic (IT)-type neurons specifically project bilaterally to the cerebral cortex and striatum, whereas neurons of the extratelencephalic (ET)-type, termed conventionally pyramidal tract-type, project ipsilaterally to the thalamus and other areas. Although they have totally different synaptic and membrane potential properties in vitro, little is known about the differences between them in ongoing spiking dynamics in vivo. We identified IT-type and ET-type neurons, as well as fast-spiking-type interneurons, using novel multineuronal analysis based on optogenetically evoked spike collision along their axons in behaving/resting rats expressing channelrhodopsin-2 (Multi-Linc method). We found "postspike suppression" (~100 ms) as a characteristic of ET-type neurons in spike auto-correlograms, and it remained constant independent of behavioral conditions in functionally different ET-type neurons. Postspike suppression followed even solitary spikes, and spike bursts significantly extended its duration. We also observed relatively strong spike synchrony in pairs containing IT-type neurons. Thus, spiking dynamics in IT-type and ET-type neurons may be optimized differently for precise and coordinated motor control.

Roles of centromedian parafascicular nuclei of thalamus and cholinergic interneurons in the dorsal striatum in associative learning of environmental events.

The thalamus provides a massive input to the striatum, but despite accumulating evidence, the functions of this system remain unclear. It is known, however, that the centromedian (CM) and parafascicular (Pf) nuclei of the thalamus can strongly influence particular striatal neuron subtypes, notably including the cholinergic interneurons of the striatum (CINs), key regulators of striatal function. Here, we highlight the thalamostriatal system through the CM-Pf to striatal CINs. We consider how, by virtue of the direct synaptic connections of the CM and PF, their neural activity contributes to the activity of CINs and striatal projection neurons (SPNs). CM-Pf neurons are strongly activated at sudden changes in behavioral context, such as switches in action-outcome contingency or sequence of behavioral requirements, suggesting that their activity may represent change of context operationalized as associability. Striatal CINs, on the other hand, acquire and loose responses to external events associated with particular contexts. In light of this physiological evidence, we propose a hypothesis of the CM-Pf-CINs system, suggesting that it augments associative learning by generating an associability signal and promotes reinforcement learning guided by reward prediction error signals from dopamine-containing neurons. We discuss neuronal circuit and synaptic organizations based on in vivo/in vitro studies that we suppose to underlie our hypothesis. Possible implications of CM-Pf-CINs dysfunction (or degeneration) in brain diseases are also discussed by focusing on Parkinson's disease.

The intrauterine environment affects learning ability of Tokai high avoider rat offspring derived using cryopreservation and embryo transfer-mediated reproduction.

Embryo transfer (ET) to recipient female animals is a useful technique in biological and experimental animal studies. While cryopreservation of two-cell stage rat embryos and ET to recipient rats are currently well-defined, it is unknown whether these artificial reproductive techniques and maternal factors affect offspring phenotype, particularly higher brain functions. Therefore, we assessed the effects of cryopreservation, ET, and maternal care on learning behaviour of the offspring, using Tokai high avoider (THA) rats that have a high learning ability phenotype. We found that the high learning ability of THA rat offspring was not replicated following ET to surrogate Wistar rats with a low-avoidance phenotype. Additionally, the characteristic phenotype of offspring obtained through mating of ET-derived rats was similar to that of THA rats. A postnatal cross-fostering investigation with the offspring of Wistar and THA rats showed that maternal behaviour, including postnatal care and lactation traits, did not differ between the dams of low-avoidance Wistar rats and THA rats; therefore, learning behaviour was retained in both Wistar and THA rat offspring. We conclude that the offspring phenotype, although unchanged, has an imperceptible effect on the learning ability of ET-derived THA rats through the intrauterine environment of the recipient.

AMBRA1 is involved in T cell receptor-mediated metabolic reprogramming through an ATG7-independent pathway.

Metabolic reprogramming contributes to dynamic alteration of cell functions and characteristics. In T cells, TCR-mediated signaling evokes metabolic reprogramming and autophagy. AMBRA1 is known to serve in the facilitation of autophagy and quality control of mitochondria, but the role of AMBRA1 in T cell metabolic alteration is unknown. Here, we show that AMBRA1, but not ATG7, plays a role in TCR-mediated control of glycolytic factors and mitochondrial mass, while both AMBRA1 and ATG7 are required for autolysosome formation. Our results suggested that AMBRA1 is a core factor that controls both autophagy and metabolic regulation.

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT).

BACKGROUND: The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently. RESULTS: Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice. CONCLUSIONS: The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.

Activity in the nucleus accumbens and amygdala underlies individual differences in prosocial and individualistic economic choices.

Much decision-making requires balancing benefits to the self with benefits to the group. There are marked individual differences in this balance such that individualists tend to favor themselves whereas prosocials tend to favor the group. Understanding the mechanisms underlying this difference has important implications for society and its institutions. Using behavioral and fMRI data collected during the performance of the ultimatum game, we show that individual differences in social preferences for resource allocation, so-called "social value orientation," is linked with activity in the nucleus accumbens and amygdala elicited by inequity, rather than activity in insula, ACC, and dorsolateral pFC. Importantly, the presence of cognitive load made prosocials behave more prosocially and individualists more individualistically, suggesting that social value orientation is driven more by intuition than reflection. In parallel, activity in the nucleus accumbens and amygdala, in response to inequity, tracked this behavioral pattern of prosocials and individualists. In addition, we conducted an impunity game experiment with different participants where they could not punish unfair behavior and found that the inequity-correlated activity seen in prosocials during the ultimatum game disappeared. This result suggests that the accumbens and amygdala activity of prosocials encodes "outcome-oriented emotion" designed to change situations (i.e., achieve equity or punish). Together, our results suggest a pivotal contribution of the nucleus accumbens and amygdala to individual differences in sociality.

Frequent mutations in NOTCH1 ligand-binding regions in Japanese oral squamous cell carcinoma.

Recent studies showed that head and neck squamous cell carcinoma (HNSCC) including oral squamous cell carcinoma (OSCC) of Caucasian, Chinese and Indian patients frequently have NOTCH1 mutations. We found eight of 84 OSCC in Japanese patients have point mutations (9.5%) correspond to the ligand binding region of NOTCH1 protein. Two set of them are the same mutations and all mutations are non-synonymous G>A transitions. In addition, median disease-free survival is significantly longer in patients with NOTCH1-mutated tumors as compared to those without the mutation (P<0.05). The protein structure simulation based on X-ray crystallography indicated that new p.A465T mutation leads to a conformational change of NOTCH1 ligand binding domain as well as the p.G481S mutant NOTCH1 with a loss of flexibility around this residue. These results suggest that NOTCH1 mutation occurs frequently in Japanese OSCC in the vicinity of the ligand binding region and, these mutations cause downregulation of the NOTCH1 function.

Enhancement of alcohol drinking in mice depends on alterations in RNA editing of serotonin 2C receptors.

Serotonin 2C receptors (5-HT(2C)R) are G-protein-coupled receptors with various actions, including involvement in drug addiction. 5-HT2CR undergoes mRNA editing, converting genomically encoded adenosine residues to inosines via adenosine deaminases acting on RNA (ADARs). Here we show that enhanced alcohol drinking behaviour in mice is associated with the degree of 5-HT(2C)R mRNA editing in the nucleus accumbens and dorsal raphe nuceus, brain regions important for reward and addiction. Following chronic alcohol vapour exposure, voluntary alcohol intake increased in C57BL/6J mice, but remained unchanged in C3H/HeJ and DBA/2J mice. 5-HT(2C)R mRNA editing frequency in both regions increased significantly in C57BL/6J mice, as did expressions of 5-HT(2C)R, ADAR1 and ADAR2, but not in other strains. Moreover, mice that exclusively express the unedited isoform (INI) of 5-HT(2C)R mRNA on a C57BL/6J background did not exhibit increased alcohol intake compared with wild-type mice. Our results indicate that alterations in 5-HT(2C)R mRNA editing underlie alcohol preference in mice.

Dopamine neurons learn to encode the long-term value of multiple future rewards.

Midbrain dopamine neurons signal reward value, their prediction error, and the salience of events. If they play a critical role in achieving specific distant goals, long-term future rewards should also be encoded as suggested in reinforcement learning theories. Here, we address this experimentally untested issue. We recorded 185 dopamine neurons in three monkeys that performed a multistep choice task in which they explored a reward target among alternatives and then exploited that knowledge to receive one or two additional rewards by choosing the same target in a set of subsequent trials. An analysis of anticipatory licking for reward water indicated that the monkeys did not anticipate an immediately expected reward in individual trials; rather, they anticipated the sum of immediate and multiple future rewards. In accordance with this behavioral observation, the dopamine responses to the start cues and reinforcer beeps reflected the expected values of the multiple future rewards and their errors, respectively. More specifically, when monkeys learned the multistep choice task over the course of several weeks, the responses of dopamine neurons encoded the sum of the immediate and expected multiple future rewards. The dopamine responses were quantitatively predicted by theoretical descriptions of the value function with time discounting in reinforcement learning. These findings demonstrate that dopamine neurons learn to encode the long-term value of multiple future rewards with distant rewards discounted.

Association of sick building syndrome with neuropathy target esterase (NTE) activity in Japanese.

Sick building syndrome (SBS) is a set of several clinically recognizable symptoms reported by occupants of a building without a clear cause. Neuropathy target esterase (NTE) is a membrane bound serine esterase and its reaction with organophosphates (OPs) can lead to OP-induced delayed neuropathy (OPIDN) and nerve axon degeneration. The aim of our study was to determine whether there was a difference in NTE activity in the peripheral blood mononuclear cells (PBMCs) of Japanese patients with SBS and healthy controls and whether PNPLA6 (alias NTE) gene polymorphisms were associated with SBS. We found that the enzymatic activity of NTE was significantly higher (P < 0.0005) in SBS patients compared with controls. Moreover, population with an AA genotype of a single nucleotide polymorphism (SNP), rs480208, in intron 21 of the PNPLA6 gene strongly reduced the activity of NTE. Fifty-eight SNP markers within the PNPLA6 gene were tested for association in a case-control study of 188 affected individuals and 401 age-matched controls. Only one SNP, rs480208, was statistically different in genotype distribution (P = 0.005) and allele frequency (P = 0.006) between the cases and controls (uncorrected for testing multiple SNP sites), but these were not significant by multiple corrections. The findings of the association between the enzymatic activity of NTE and SBS in Japanese show for the first time that NTE activity might be involved with SBS.

The effects of corticotropin-releasing factor on motor learning.

Corticotropin-releasing factor (CRF) is mainly secreted from the hypothalamic paraventricular nuclei and plays a crucial role in stress-related responses. Recent studies have reported that CRF is a neuromodulator in the central nervous system. In the cerebellum, CRF is essential for the induction of long-term depression (LTD) at the parallel fiber-Purkinje cell synapses. Given that LTD is thought to be one of the fundamental mechanisms of motor learning, CRF may affect motor learning. However, the role of CRF in motor learning in vivo remains unclear. In this study, we aimed to examine the role of CRF in motor learning. This was achieved through a series of behavioral experiments involving the in vivo administration of CRF and its antagonists. Rats injected with CRF directly into the cerebellum exhibited superior performance on the rotarod test, especially during initial training phases, compared to control subjects. Conversely, rats receiving a CRF receptor antagonist demonstrated reduced endurance on the rotating rod compared to controls. Notably, CRF mRNA expression levels in the cerebellum did not show significant variance between the CRF-injected and control groups. These findings imply a critical role of endogenous CRF in cerebellar motor learning and suggest that exogenous CRF can augment this process. (199 words).

Coding of the long-term value of multiple future rewards in the primate striatum.

Decisions maximizing benefits involve a tradeoff between the quantity of a reward and the cost of elapsed time until an animal receives it. The estimation of long-term reward values is critical to attain the most desirable outcomes over a certain period of time. Reinforcement learning theories have established algorithms to estimate the long-term reward values of multiple future rewards in which the values of future rewards are discounted as a function of how many steps of choices are necessary to achieve them. Here, we report that presumed striatal projection neurons represent the long-term values of multiple future rewards estimated by a standard reinforcement learning model while monkeys are engaged in a series of trial-and-error choices and adaptive decisions for multiple rewards. We found that the magnitude of activity of a subset of neurons was positively correlated with the long-term reward values, and that of another subset of neurons was negatively correlated throughout the entire decision-making process in individual trials: from the start of the task trial, estimation of the values and their comparison among alternatives, choice execution, and evaluation of the received rewards. An idiosyncratic finding was that neurons showing negative correlations represented reward values in the near future (high discounting), while neurons showing positive correlations represented reward values not only in the near future, but also in the far future (low discounting). These findings provide a new insight that long-term value signals are embedded in two subsets of striatal neurons as high and low discounting of multiple future rewards.

Improved loop-mediated isothermal amplification for HLA-DRB1 genotyping using RecA and a restriction enzyme for enhanced amplification specificity.

Our aim was to test and develop the use of loop-mediated isothermal amplification (LAMP) for HLA-DRB1 genotyping. Initially, we found that the conventional LAMP protocols produced non-specific and variable amplification results depending on the sample DNA conditions. Experiments with different concentrations of DNase in the reaction mixture with and without T4 DNA ligase-treated samples suggested that the strand displacement activity of DNA polymerase in LAMP, at least in part, started from randomly existing nicks because T4 DNA ligase treatment of sample DNA resulted in no amplification. Such non-specific amplification due to the randomly existing nicks was improved specifically by the addition of RecA of Escherichia coli and a restriction enzyme, for example, PvuII, to the reaction mixture. We applied the modified LAMP (mLAMP) (1) to detect specific HLA-DRB1 alleles by using only specific primers for amplification or (2) for genotyping in multiple samples with a multi-probe typing system. In the latter case, HLA-DRB1 genotyping was developed by combining the mLAMP with amplicon capture using polymorphic region-specific probes fixed onto the bottom of the wells of a 96-well plate and the captured amplicons visualized as a black spot at the bottom of the well. The multi-probe human leukocyte antigen (HLA) typing method and the specific HLA allele detection method could be applied for point-of-care testing due to no requirement for specific and expensive instruments.

X-ray microtomographic visualization of Escherichia coli by metalloprotein overexpression.

This paper reports X-ray microtomographic visualization of the microorganism Escherichia coli overexpressing a metalloprotein ferritin. The three-dimensional distribution of linear absorption coefficients determined using a synchrotron radiation microtomograph with a simple projection geometry revealed that the X-ray absorption was homogeneously distributed, suggesting that every E. coli cell was labeled with the ferritin. The ferritin-expressing E. coli exhibited linear absorption coefficients comparable with those of phosphotungstic acid stained cells. The submicrometer structure of the ferritin-expressing E. coli cells was visualized by Zernike phase contrast using an imaging microtomograph equipped with a Fresnel zone plate. The obtained images revealed curved columnar or bunching oval structures corresponding to the E. coli cells. These results indicate that the metalloprotein overexpression facilitates X-ray visualization of three-dimensional cellular structures of biological objects.

Reward expectation enhances action-related activity of nigral dopaminergic and two striatal output pathways.

Neurons comprising nigrostriatal system play important roles in action selection. However, it remains unclear how this system integrates recent outcome information with current action (movement) and outcome (reward or no reward) information to achieve appropriate subsequent action. We examined how neuronal activity of substantia nigra pars compacta (SNc) and dorsal striatum reflects the level of reward expectation from recent outcomes in rats performing a reward-based choice task. Movement-related activity of direct and indirect pathway striatal projection neurons (dSPNs and iSPNs, respectively) were enhanced by reward expectation, similarly to the SNc dopaminergic neurons, in both medial and lateral nigrostriatal projections. Given the classical basal ganglia model wherein dopamine stimulates dSPNs and suppresses iSPNs through distinct dopamine receptors, dopamine might not be the primary driver of iSPN activity increasing following higher reward expectation. In contrast, outcome-related activity was affected by reward expectation in line with the classical model and reinforcement learning theory, suggesting purposive effects of reward expectation.

Thalamic contributions to Basal Ganglia-related behavioral switching and reinforcement.

Although the existence of prominent connections between the intralaminar thalamic nuclei and the basal ganglia has long been established, the limited knowledge of the functional relevance of this network has considerably hampered progress in our understanding of the neural mechanisms by which the thalamostriatal system integrates and regulates the basal ganglia circuitry. In this brief commentary, we will address this gap of knowledge through a discussion of the key points of a symposium entitled "Thalamic Contributions to Basal Ganglia-Related Behavioral Switching and Reinforcement" that will be presented at the 2011 Society for Neuroscience meeting. Recent anatomical and physiological data that support the role of the thalamostriatal system in action selection, attentional shifting, and reinforcement will be discussed. We will also address the possibility that degeneration of the thalamostriatal system could underlie some of the deficits in redirection of attention in response to salient stimuli seen in Parkinson's disease.

Serotonin2C receptors in the nucleus accumbens are involved in enhanced alcohol-drinking behavior.

Dopamine and serotonin (5-HT) in the nucleus accumbens (ACC) and ventral tegmental area of the mesoaccumbens reward pathways have been implicated in the mechanisms underlying development of alcohol dependence. We used a C57BL/6J mouse model with increased voluntary alcohol-drinking behavior by exposing the mice to alcohol vapor for 20 consecutive days. In the alcohol-exposed mice, the expression of 5-HT(2C) receptor mRNA increased in the ACC, caudate nucleus and putamen, dorsal raphe nucleus (DRN), hippocampus and lateral hypothalamus, while the protein level of 5-HT(2C) receptor significantly increased in the ACC. The expression of 5-HT(7) receptor mRNA increased in the ACC and DRN. Contents of 5-HT decreased in the ACC shell (ACC(S) ) and DRN of the alcohol-exposed mice. The basal extracellular releases of dopamine (DA) and 5-HT in the ACC(S) increased more in the alcohol-exposed mice than in alcohol-naïve mice. The magnitude of the alcohol-induced ACC(S) DA and 5-HT release in the alcohol-exposed mice was increased compared with the control mice. Intraperitoneal (i.p.) administration or local injection into ACC(S) of the 5-HT(2C) receptor antagonist, SB-242084, suppressed voluntary alcohol-drinking behavior in the alcohol-exposed mice. But the i.p. administration of the 5-HT(7) receptor antagonist, SB-258719, did not have significant effects on alcohol-drinking behavior in the alcohol-exposed mice. The effects of the 5-HT(2C) receptor antagonist were not observed in the air-exposed control mice. These results suggest that adaptations of the 5-HT system, especially the upregulation of 5-HT(2C) receptors in the ACC(S) , are involved in the development of enhanced voluntary alcohol-drinking behavior.

Fluorescent transgenic mice suitable for multi-color aggregation chimera studies.

We recently reported a novel method of mouse transgenesis called Pronuclear Injection-based Targeted Transgenisis (PITT) using which a series of fluorescent transgenic (Tg) mice lines were generated. These lines, unlike those generated using conventional random integration methods, express the transgenes faithfully and reproducibly generation after generation. Because of this superior nature, these lines are ideal for the generation of multi-colored aggregation chimeras that can be used to study cell-cell interactions and lineage analyses in living embryos/organs, where the transgenes can be detected and the clonal origin of a given cell population easily traced by its distinct fluorescence. In this study, to verify if Tg fluorescent mice generated through PITT were suitable for such applications, we sought to generate chimeric blastocysts and chimeric-Tg mice by aggregating two- or three-colored 8-cell embryos. Our analyses using these models led to the following observations. First, we noticed that cell mixing was infrequent during the stages of morula to early blastocyst. Second, chimeric fetuses obtained after aggregation of the two-colored 8-cell embryos exhibited uniform cell mixing. And third, in the organs of adult chimeric mice, the mode of cell distribution could be either clonal or polyclonal, as previously pointed out by others. Implications of our novel and improved Tg-chimeric mice approach for clonal cell lineage and developmental studies are discussed.