Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Caveolin-3 is a muscle-specific protein integrated in the caveolae, which are small invaginations of the plasma membrane. Mutations of the caveolin-3 gene, localized at 3p25, have been reported to be involved in the pathogenesis of limb-girdle muscular dystrophy (LGMD1C or caveolinopathy) with mild clinical symptoms, inherited through an autosomal dominant form of genetic transmission. To elucidate the pathogenetic mechanism, we developed caveolin-3-deficient mice for use as animal models of caveolinopathy. Caveolin-3 mRNA and its protein were absent in homozygous mutant mice. In heterozygous mutant mice, both the mRNA and its protein were normal in size, but their amounts were reduced by about half. The density of caveolae in skeletal muscle plasma membrane was roughly proportional to the amount of caveolin-3. In homozygous mutant mice, muscle degeneration was recognized in soleus muscle at 8 weeks of age and in the diaphragm from 8 to 30 weeks, although there was no difference in growth and movement between wild-type and mutant mice. No apparent muscle degeneration was observed in heterozygous mutant mice, indicating that pathological changes caused by caveolin-3 gene disruption were inherited through the recessive form of genetic transmission.

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The dopamine D2 receptor (D2) system has been implicated in several neurological and psychiatric disorders, such as schizophrenia and Parkinson's disease. There are two isoforms of the D2 receptor: the long form (D2L) and the short form (D2S). The two isoforms are generated by alternative splicing of the same gene and differ only by 29 amino acids in their protein structures. Little is known about the distinct functions of either D2 isoform, primarily because selective pharmacological agents are not available. We generated D2L receptor-deficient (D2L-/-) mice by making a subtle mutation in the D2 gene. D2L-/- mice (which still express functional D2S) displayed reduced levels of locomotion and rearing behavior. Interestingly, haloperidol produced significantly less catalepsy and inhibition of locomotor activity in D2L-/- mice. These findings suggest that D2L and D2S may contribute differentially to the regulation of certain motor functions and to the induction of the extrapyramidal side effects associated with the use of typical antipsychotic drugs (e.g., haloperidol). Quinpirole induced a similar initial suppression of locomotor activity in both D2L-/- and wild-type mice. In ad

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beta-Sarcoglycan, one of the subunits of the sarcoglycan complex, is a transmembranous glycoprotein which associates with dystrophin and is the molecule responsible for beta-sarcoglycanopathy, a Duchenne-like autosomal recessive muscular dystrophy. To develop an animal model of beta-sarcoglycanopathy and to clarify the role of beta-sarcoglycan in the pathogenesis of the muscle degeneration in vivo, we developed beta-sarcoglycan-deficient mice using a gene targeting technique. beta-Sarcoglycan-deficient mice (BSG(-/-) mice) exhibited progressive muscular dystrophy with extensive degeneration and regeneration. The BSG(-/-) mice also exhibited muscular hypertrophy characteristic of beta-sarcoglycanopathy. Immunohistochemical and immunoblot analyses of BSG(-/-) mice demonstrated that deficiency of beta-sarcoglycan also caused loss of all of the other sarcoglycans as well as of sarcospan in the sarcolemma. On the other hand, laminin-alpha 2, alpha- and beta-dystroglycan and dystrophin were still present in the sarcolemma, However, the dystrophin-dystroglycan complex in BSG(-/-) mice was unstable compared with that in the wild-type mice. Our data suggest that loss of the sarcoglycan complex and sarcospan alone is sufficient to cause muscular dystrophy, that beta-sarcoglycan is an important protein for formation of the sarcoglycan complex associated with sarcospan and that the role of the sarcoglycan complex and sarcospan may be to strengthen the dystrophin axis connecting the basement membrane with the cytoskeleton.

DOI： 10.1093/hmg/8.9.1589

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

To investigate cis-elements responsible for catecholaminergic (CAnergic) neuron-specific expression of the tyrosine hydroxylase (TH) gene, we produced lines of transgenic mice carrying 5.0-kb, 2.5-kb and 0.2-kb fragments from the 5'-flanking region of the human TH gene fused to a reporter gene. chloramphenicol acetyltransferase (CAT), and designated them as TC 50. TC 25, and TC 02, respectively. and reporter gene expression in transgenic mice was analyzed by CAT assay by immunocytochemistry with anti-CAT antibody. High-level CAT expression was observed in the brain and adrenal gland using the 5.0-kb promoter of the TC 50 mice, but ectopic expression was consistently observed in several somatic tissues, e.g. thymus, colon, and testis. In brain, expression was achieved in CAnergic neurons with the largest construct (5.0 kb), but not with 2.5 kb or 0.2 kb of 5' flanking sequence. However, TC 50 mice also expressed CAT immunoreactivity in non-CAnergic neurons. In the TC 25 line CAT immunoreactivity was detected only in some non-CAnergic neurons. In the TC 02 line no CAT immunoreactivity was detected in any of the tissues examined. These results indicate that the 5.0-kb DNA fragment of the TH gene upstream region contains activity to express CAT in CAnergic neurons and surprisingly, lacks some regulatory elements attenuating ectopic expression, and that the 2.5-kb and 0.2-kb fragment are not sufficient for the proper expression. We discuss the presence of the tissue-specific regulatory elements in the structure portion of the TH gene and/or 3'-flanking region.

DOI： 10.1016/0169-328X(92)90236-5

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Authorship：Lead author   Language：English   Publishing type：Doctoral thesis   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The chromosomal gene for human phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) was isolated from a human genomic library using a cloned human PNMT cDNA as a probe, and the nucleotide sequence was determined. PNMT is encoded in a single gene which consists of three exons. We observed newly the presence of minor PNMT mRNA (type B) besides the major mRNA (type A) as reported previously (Kaneda et al., J. Biol. Chem. 263, 7672-7677, 1988) by Northern hybridization. Type B mRNA carries an approximately 700 nucleotide-long untranslated region in the 5? terminus. This suggests that two types of mRNA are produced from a single gene through the use of two alternative promoters. A TATA-like sequence locates 30 base pair upstream from the cap site of type A mRNA. Upstream of the cap site, there are several sequences resembling Spl binding sites and glucocorticoid responsive elements, with the latter also found in the first intron.

DOI： 10.1016/0197-0186(89)90176-9

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Abstract

The common marmoset (Callithrix jacchus) has attracted attention as a valuable primate model for the analysis of human diseases. Despite the potential for primate genetic modification, however, its widespread lab usage has been limited due to the requirement for a large number of eggs. To make up for traditional oocyte retrieval methods such as hormone administration and surgical techniques, we carried out an alternative approach by utilizing ovarian tissue from deceased marmosets that had been disposed of. This ovarian tissue contains oocytes and can be used as a valuable source of follicles and oocytes. In this approach, the ovarian tissue sections were transplanted under the renal capsules of immunodeficient mice first. Subsequent steps consist of development of follicles by hormone administrations, induction of oocyte maturation and fertilization, and culture of the embryo. This method was first established with rat ovaries, then applied to marmoset ovaries, ultimately resulting in the successful acquisition of the late-stage marmoset embryos. This approach has the potential to contribute to advancements in genetic modification research and disease modeling through the use of primate models, promoting biotechnology with non-human primates and the 3Rs principle in animal experimentation.

DOI： 10.1038/s41598-023-45224-x

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.celrep.2023.112398

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We previously demonstrated that fatty acid-binding protein 3 null (FABP3-/-) mice exhibit resistance to nicotine-induced conditioned place preference (CPP). Here, we confirm that the FABP3 inhibitor, MF1 ((4-(2-(1-(2-chlorophenyl)-5-phenyl-1H-pyrazol-3-yl)phenoxy) butanoic acid), successfully reduces nicotine-induced CPP scores in mice. MF1 (0.3 or 1.0 mg/kg) was orally administered 30 min before nicotine, and CPP scores were assessed in the conditioning, withdrawal, and relapse phases. MF1 treatment decreased CPP scores in a dose-dependent manner. Failure of CPP induction by MF1 (1.0 mg/kg, p.o.) was associated with the inhibition of both CaMKII and ERK activation in the nucleus accumbens (NAc) and hippocampal CA1 regions. MF1 treatment reduced nicotine-induced increases in phosphorylated CaMKII and cAMP-response element-binding protein (CREB)-positive cells. Importantly, the increase in dopamine D2 receptor (D2R) levels following chronic nicotine exposure was inhibited by MF1 treatment. Moreover, the quinpirole (QNP)-induced increase in the level of CaMKII and ERK phosphorylation was significantly inhibited by MF1 treatment of cultured NAc slices from wild type (WT) mice; however, QNP treatment had no effect on CaMKII and ERK phosphorylation levels in the NAc of D2R null mice. Taken together, these results show that MF1 treatment suppressed D2R/FABP3 signaling, thereby preventing nicotine-induced CPP induction. Hence, MF1 can be used as a novel drug to block addiction to nicotine and other drugs by inhibiting the dopaminergic system.

DOI： 10.3390/ijms24076644

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NMDA-type glutamate receptors (NMDARs) are tetrameric channel complex composed of two subunits of GluN1, which is encoded by a single gene and diversified by alternative splicing, and two subunits from four subtypes of GluN2, leading to various combinations of subunits and channel specificities. However, there is no comprehensive quantitative analysis of GluN subunit proteins for relative comparison, and their compositional ratios at various regions and developmental stages have not been clarified. Here we prepared six chimeric subunits, by fusing an N-terminal side of the GluA1 subunit with a C-terminal side of each of two splicing isoforms of GluN1 subunit and four GluN2 subunits, with which titers of respective NMDAR subunit antibodies could be standardized using common GluA1 antibody, thus enabling quantification of relative protein levels of each NMDAR subunit by western blotting. We determined relative protein amounts of NMDAR subunits in crude, membrane (P2) and microsomal fractions prepared from the cerebral cortex, hippocampus and cerebellum in adult mice. We also examined amount changes in the three brain regions during developmental stages. Their relative amounts in the cortical crude fraction were almost parallel to those of mRNA expression, except for some subunits. Interestingly, a considerable amount of GluN2D protein existed in adult brains, although its transcription level declines after early postnatal stages. GluN1 was larger in quantity than GluN2 in the crude fraction, whereas GluN2 increased in the membrane component-enriched P2 fraction, except in the cerebellum. These data will provide the basic spatio-temporal information on the amount and composition of NMDARs.

DOI： 10.1016/j.neuint.2023.105517

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Extended pluripotent stem cells (EPSCs) derived from mice and humans showed an enhanced potential for chimeric formation. By exploiting transcriptomic approaches, we assessed the differences in gene expression profile between extended EPSCs derived from mice and humans, and those newly derived from the common marmoset (marmoset; Callithrix jacchus). Although the marmoset EPSC-like cells displayed a unique colony morphology distinct from murine and human EPSCs, they displayed a pluripotent state akin to embryonic stem cells (ESCs), as confirmed by gene expression and immunocytochemical analyses of pluripotency markers and three-germ-layer differentiation assay. Importantly, the marmoset EPSC-like cells showed interspecies chimeric contribution to mouse embryos, such as E6.5 blastocysts in vitro and E6.5 epiblasts in vivo in mouse development. Also, we discovered that the perturbation of gene expression of the marmoset EPSC-like cells from the original ESCs resembled that of human EPSCs. Taken together, our multiple analyses evaluated the efficacy of the method for the derivation of marmoset EPSCs.

DOI： 10.1111/gtc.13000

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Posture and gait are maintained by sensory inputs from the vestibular, visual, and somatosensory systems and motor outputs. Upon vestibular damage, the visual and/or somatosensory systems functionally substitute by cortical mechanisms called "sensory reweighting". We investigated the cerebrocortical mechanisms underlying sensory reweighting after unilateral labyrinthectomy (UL) in mice. Arc-dVenus transgenic mice, in which the gene encoding the fluorescent protein dVenus is transcribed under the control of the promoter of the immediate early gene Arc, were used in combination with whole-brain three-dimensional (3D) imaging. Performance on the rotarod was measured as a behavioral correlate of sensory reweighting. Following left UL, all mice showed the head roll-tilt until UL10, indicating the vestibular periphery damage. The rotarod performance worsened in the UL mice from UL1 to UL3, which rapidly recovered. Whole-brain 3D imaging revealed that the number of activated neurons in S1, but not in V1, in UL7 was higher than that in sham-treated mice. At UL7, medial prefrontal cortex (mPFC) and agranular insular cortex (AIC) activation was also observed. Therefore, sensory reweighting to the somatosensory system could compensate for vestibular dysfunction following UL; further, mPFC and AIC contribute to the integration of sensory and motor functions to restore balance.

DOI： 10.1038/s41598-022-19678-4

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.11249/jsbpjjpp.33.3_100

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When writing an object's name, humans mentally construct its spelling. This capacity critically depends on use of the dual-structured linguistic system, in which meaningful words are represented by combinations of meaningless letters. Here we search for the evolutionary origin of this capacity in primates by designing dual-structured bigram symbol systems where different combinations of meaningless elements represent different objects. Initially, we trained Japanese macaques (Macaca fuscata) in an object-bigram symbolization task and in a visually-guided bigram construction task. Subsequently, we conducted a probe test using a symbolic bigram construction task. From the initial trial of the probe test, the Japanese macaques could sequentially choose the two elements of a bigram that was not actually seen but signified by a visually presented object. Moreover, the animals' spontaneous choice order bias, developed through the visually-guided bigram construction learning, was immediately generalized to the symbolic bigram construction test. Learning of dual-structured symbols by the macaques possibly indicates pre-linguistic adaptations for the ability of mentally constructing symbols in the common ancestors of humans and Old World monkeys.

DOI： 10.1038/s41598-022-07563-z

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Dopaminergic neurotransmission via dopamine D1 receptors (D1Rs) is considered to play an important role not only in reward-based learning but also in aversive learning. The contextual and auditory cued fear conditioning tests involve the processing of classical fear conditioning and evaluates aversive learning memory. It is possible to evaluate aversive learning memory in two different types of neural transmission circuits. In addition, when evaluating the role of dopaminergic neurotransmission via D1R, to avoid the effects in D1R-mediated neural circuitry alterations during development, it is important to examine using mice who D1R expression in the mature stage is suppressed. Herein, we investigated the role of dopaminergic neurotransmission via D1Rs in aversive memory formation in contextual and auditory cued fear conditioning tests using D1R knockdown (KD) mice, in which the expression of D1Rs could be conditionally and reversibly controlled with doxycycline (Dox) treatment. For aversive memory, we examined memory formation using recent memory 1 day after conditioning, and remote memory 4 weeks after conditioning. Furthermore, immunostaining of the brain tissues of D1RKD mice was performed after aversive footshock stimulation to investigate the distribution of activated c-Fos, an immediate-early gene, in the hippocampus (CA1, CA3, dentate gyrus), striatum, amygdala, and prefrontal cortex during aversive memory formation. After aversive footshock stimulation, immunoblotting was performed using hippocampal, striatal, and amygdalar samples from D1RKD mice to investigate the increase in the amount of c-Fos and phosphorylated SNAP-25 at Ser187 residue. When D1R expression was suppressed using Dox, behavioral experiments revealed impaired contextual fear learning in remote aversion memory following footshock stimulation. Furthermore, expression analysis showed a slight increase in the post-stimulation amount of c-Fos in the hippocampus and striatum, and a significant increase in the amount of phosphorylated SNAP-25 in the hippocampus, striatum, and prefrontal cortex before and after stimulation. These findings indicate that deficiency in D1R-mediated dopaminergic neurotransmission is an important factor in impairing contextual fear memory formation for remote memory.

DOI： 10.3389/fnbeh.2022.751053

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Dopamine in the prefrontal cortex is essential for the regulation of social behavior. However, stress-causing social withdrawal also promotes dopamine release in the prefrontal cortex. Thus, this evidence suggests opposite functions of dopamine in the prefrontal cortex. However, the influence of dopamine on prefrontal functions is yet to be fully understood. Here, we show that dopamine differentially modulated the neuronal activity triggered by social stimuli in the prefrontal cortex, depending on the duration of the dopamine activation (transient or sustained activation). Using chemogenetic techniques, we have found that social behavior was negatively regulated by a sustained increase in dopamine neuronal activity in the ventral tegmental area, while it was positively regulated by an acute increase. The duration of social interactions was positively correlated with the transient dopamine release triggered by social stimuli in the prefrontal cortex and negatively correlated with the sustained increase in prefrontal dopamine levels. Furthermore, the elevation of neural calcium signal, triggered by social stimuli, in the prefrontal cortex was attenuated by the persistent elevation of prefrontal dopamine levels, whereas an acute increase in dopamine levels enhanced it. Additionally, the chronic excess of dopamine suppressed c-Fos induction triggered by social stimuli in prefrontal neurons expressing dopamine D1 receptors, but not D2 receptors. These results suggest that sustained activation of prefrontal dopamine, at the opposite of its transient activation, can reduce prefrontal activity associated with social behavior, even for identical dopamine concentrations. Thus, dopamine plays opposite roles in modulating prefrontal activity depending on the duration of its action.

DOI： 10.1096/fj.202101637R

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BACKGROUND: Evaluation of an experimental and preclinical islet transplantation (IsletTx) model to elucidate associated clinical problems is vital. This study aimed to introduce a simple methodology for producing a swine autologous IsletTx model as a preliminary step in an allogeneic transplant experiment. METHODS AND MATERIALS: Twenty-seven pigs were included in the study. Total pancreatectomy (TP) was performed in 8 pigs (TP group), TP with autologous IsletTx in 9 (TP + IsletTx group), and distal pancreatectomy (DP) with autologous IsletTx in 10 (DP + IsletTx group). An open biopsy was performed on all pigs during postoperative day 14 using an infrared imaging (IRI) system. Laboratory data and postoperative survival were analyzed and compared according to the procedures done. RESULTS: Postoperative survival rate was significantly higher in the pigs with autologous IsletTx than in those without (P = .026). There were no significant differences in survival between the TP + IsletTx and DP + IsletTx groups (P = .746). Significant hyperglycemia was not observed in both groups, but the DP + IsletTx group remained relatively stable throughout the postoperative course. There were no differences in serum creatinine, aspartate aminotransferase, and alanine aminotransferase levels between the 2 groups. By selective liver lobe transplantation and administration of the IRI system, localization of the transplanted islets via open biopsy was achieved. CONCLUSIONS: We successfully developed an autologous IsletTx model and an open biopsy system using a swine model. This study will aid in the development of an allogeneic IsletTx experiment that may improve transplantation outcomes.

DOI： 10.1016/j.transproceed.2021.10.029

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α-synuclein accumulation into dopaminergic neurons is a pathological hallmark of Parkinson's disease. We previously demonstrated that fatty acid-binding protein 3 (FABP3) is critical for α-synuclein uptake and propagation to accumulate in dopaminergic neurons. FABP3 is abundant in dopaminergic neurons and interacts with dopamine D2 receptors, specifically the long type (D2L). Here, we investigated the importance of dopamine D2L receptors in the uptake of α-synuclein monomers and their fibrils. We employed mesencephalic neurons derived from dopamine D2L
-/-, dopamine D2 receptor null (D2 null), FABP3-/-, and wild type C57BL6 mice, and analyzed the uptake ability of fluorescence-conjugated α-synuclein monomers and fibrils. We found that D2L receptors are co-localized with FABP3. Immunocytochemistry revealed that TH+ D2L-/- or D2 null neurons do not take up α-synuclein monomers. The deletion of α-synuclein C-terminus completely abolished the uptake to dopamine neurons. Likewise, dynasore, a dynamin inhibitor, and caveolin-1 knockdown also abolished the uptake. D2L and FABP3 were also critical for α-synuclein fibrils uptake. D2L and accumulated α-synuclein fibrils were well co-localized. These data indicate that dopamine D2L with a caveola structure coupled with FABP3 is critical for α-synuclein uptake by dopaminergic neurons, suggesting a novel pathogenic mechanism of synucleinopathies, including Parkinson's disease.

DOI： 10.3390/biomedicines9010049

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Dopamine (DA) exerts well-known functions in the brain as a neurotransmitter. In addition, it plays important physiological roles in peripheral organs, but it is largely unknown how and where peripheral DA is synthesized and regulated. Catecholamines in peripheral tissues are either produced within the tissue itself and/or derived from sympathetic neurons, which release neurotransmitters for uptake by peripheral tissues. To evaluate DA-producing ability of each peripheral tissue, we generated conditional KO mice (cKO mice) in which the tyrosine hydroxylase (TH) gene is ablated in the sympathoadrenal system, thus eliminating sympathetic neurons as a DA source. We then examined the alterations in the noradrenaline (NA), DA, and 3,4-dihydroxyphenylalanine (DOPA) contents in peripheral organs and performed immunohistochemical analyses of TH-expressing cells. In the heart and pancreas of cKO mice, both the TH protein and NA levels were significantly decreased, and the DA contents were decreased in parallel with NA contents, indicating that the DA supply originated from sympathetic neurons. We found TH-immunoreactive cells in the stomach and lung, where the TH protein showed a decreasing trend, but the DA levels were not decreased in cKO mice. Moreover, we found a significant correlation between the DA content in the kidney and the plasma DOPA concentration, suggesting that the kidney takes up DOPA from blood to make DA. The aforementioned data unravel differences in the DA biosynthetic pathway among tissues and support the role of sympathetic neurons as a DA supplier.

DOI： 10.1016/j.jbc.2021.100544

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Synapse formation and regulation require signaling interactions between pre- and postsynaptic proteins, notably cell adhesion molecules (CAMs). It has been proposed that the functions of neuroligins (Nlgns), postsynaptic CAMs, rely on the formation of trans-synaptic complexes with neurexins (Nrxns), presynaptic CAMs. Nlgn3 is a unique Nlgn isoform that localizes at both excitatory and inhibitory synapses. However, Nlgn3 function mediated via Nrxn interactions is unknown. Here we demonstrate that Nlgn3 localizes at postsynaptic sites apposing vesicular glutamate transporter 3-expressing (VGT3+) inhibitory terminals and regulates VGT3+ inhibitory interneuron-mediated synaptic transmission in mouse organotypic slice cultures. Gene expression analysis of interneurons revealed that the αNrxn1+AS4 splice isoform is highly expressed in VGT3+ interneurons as compared with other interneurons. Most importantly, postsynaptic Nlgn3 requires presynaptic αNrxn1+AS4 expressed in VGT3+ interneurons to regulate inhibitory synaptic transmission. Our results indicate that specific Nlgn-Nrxn signaling generates distinct functional properties at synapses.

DOI： 10.7554/eLife.59545

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Dopamine (DA) plays an important role in the basal ganglia (BG) for motor control, and DA deficiency as seen in Parkinson's disease, causes movement disorders. DA activates the direct pathway nerve via the D1 receptor (D1R) and inhibits the indirect pathway nerve via the D2 receptor (D2R). To understand the role of DA signaling, we review recent studies of the roles of D1R and D2R with respect to motor control, neural activity and memory learning using genetically engineered mice, and investigate their involvement in the BG oscillation phenomenon.

DOI： 10.11477/mf.1416201666

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Pathophysiological roles of cardiac dopamine system remain unknown. Here, we show the role of dopamine D1 receptor (D1R)-expressing cardiomyocytes (CMs) in triggering heart failure-associated ventricular arrhythmia. Comprehensive single-cell resolution analysis identifies the presence of D1R-expressing CMs in both heart failure model mice and in heart failure patients with sustained ventricular tachycardia. Overexpression of D1R in CMs disturbs normal calcium handling while CM-specific deletion of D1R ameliorates heart failure-associated ventricular arrhythmia. Thus, cardiac D1R has the potential to become a therapeutic target for preventing heart failure-associated ventricular arrhythmia.

DOI： 10.1038/s41467-020-18128-x

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Dopamine (DA) signaling in the basal ganglia plays important roles in motor control. Motor deficiencies were previously reported in dopamine receptor D1 (D1R) and D2 (D2R) knockout mice. While these results indicate the involvement of DA receptors in motor execution, the null knockout (KO) mouse lacks the specificity necessary to determine when and where in the brain D1R and D2R function in motor execution. To address these questions, we restricted the loss of function temporally and spatially by using D1R conditional knockdown (cKD) mice and mice injected with antagonists against DA receptors directly into the dorsal striatum. In addition, we address the DA and acetylcholine (ACh) balance hypothesis by using antagonists against ACh receptors. We tested the motor ability of the mice with a newly devised task named the accelerating step-wheel. In this task, the maximum running speed was measured in a situation where the wheel rotation speed was gradually accelerated in one trial. We found significant decreases in the maximum running speed of D1R cKD mice and the mice injected with the antagonist against D1R or muscarinic ACh receptor. These results indicated that D1R and muscarinic ACh receptor in the dorsal striatum play pivotal roles in the execution of walking/running.

DOI： 10.1016/j.neures.2019.12.001

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The shortage of donor lungs hinders lung transplantation, the only definitive option for patients with end-stage lung disease. Blastocyst complementation enables the generation of transplantable organs from pluripotent stem cells (PSCs) in animal models. Pancreases and kidneys have been generated from PSCs by blastocyst complementation in rodent models. Here, we report the generation of lungs using mouse embryonic stem cells (ESCs) in apneumic Fgf10 Ex1mut/Ex3mutmice by blastocyst complementation. Complementation with ESCs enables Fgf10-deficient mice to survive to adulthood without abnormalities. Both the generated lung alveolar parenchyma and the interstitial portions, including vascular endothelial cells, vascular and parabronchial smooth muscle cells, and connective tissue, largely originate from the injected ESCs. These data suggest that Fgf10 Ex1mut/Ex3mutblastocysts provide an organ niche for lung generation and that blastocyst complementation could be a viable approach for generating whole lungs.

DOI： 10.1016/j.celrep.2020.107626

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The generation of mature, functional, thyroid follicular cells from pluripotent stem cells would potentially provide a therapeutic benefit for patients with hypothyroidism, but in vitro differentiation remains difficult. We earlier reported the in vivo generation of lung organs via blastocyst complementation in fibroblast growth factor 10 (Fgf10), compound, heterozygous mutant (Fgf10 Ex1mut/Ex3mut) mice. Fgf10 also plays an essential role in thyroid development and branching morphogenesis, but any role thereof in thyroid organogenesis remains unclear. Here, we report that the thyroids of Fgf10 Ex1mut/Ex3mut mice exhibit severe hypoplasia, and we generate thyroid tissues from mouse embryonic stem cells (ESCs) in Fgf10 Ex1mut/Ex3mut mice via blastocyst complementation. The tissues were morphologically normal and physiologically functional. The thyroid follicular cells of Fgf10 Ex1mut/Ex3mut chimeric mice were derived largely from GFP-positive mouse ESCs although the recipient cells were mixed. Thyroid generation in vivo via blastocyst complementation will aid functional thyroid regeneration.

DOI： 10.3389/fendo.2020.609697

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Nicotine in tobacco causes psychological dependence through its rewarding effect in the central nervous system (CNS). Although nicotine dependence is explained by dopamine receptor (DR) signaling together with nicotinic acetylcholine receptors (nAChRs), the synaptic molecular mechanism underlying the interaction between dopamine receptor and nAChRs remains unclear. Since reward signaling is mediated by dopamine receptors, we hypothesized that the dopamine D2 receptor (D2R), in part, mediates the synaptic modulation of nicotine-induced conditioned place preference (CPP) in addition to dopamine D1 receptor. To investigate the involvement of D2R, wild-type (WT) and dopamine D2 receptor knockout (D2RKO) mice were assessed using the CPP task after induction of nicotine-induced CPP. As expected, D2RKO mice failed to induce CPP behaviors after repeated nicotine administration (0.5 mg/kg). When kinase signaling was assessed in the nucleus accumbens and hippocampal CA1 region after repeated nicotine administration, both Ca2+/calmodulin-dependent protein kinase (CaMKII) and extracellular signal-regulated kinase (ERK) were upregulated in WT mice but not in D2RKO mice. Likewise, nicotine-induced CPP was associated with elevation of pro- brain-derived neurotropic factor (BDNF) and BDNF protein levels in WT mice, but not in D2RKO mice. Taken together, in addition to dopamine D1 receptor signaling, dopamine D2 receptor signaling is critical for induction of nicotine-induced CPP in mice.

DOI： 10.1007/s12035-019-1635-x

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Other Link： http://link.springer.com/article/10.1007/s12035-019-1635-x/fulltext.html

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Spinocerebellar ataxia 42 (SCA42) is a neurodegenerative disorder recently shown to be caused by c.5144G > A (p.Arg1715His) mutation in CACNA1G, which encodes the T-type voltage-gated calcium channel CaV3.1. Here, we describe a large Japanese family with SCA42. Postmortem pathological examination revealed severe cerebellar degeneration with prominent Purkinje cell loss without ubiquitin accumulation in an SCA42 patient. To determine whether this mutation causes ataxic symptoms and neurodegeneration, we generated knock-in mice harboring c.5168G > A (p.Arg1723His) mutation in Cacna1g, corresponding to the mutation identified in the SCA42 family. Both heterozygous and homozygous mutants developed an ataxic phenotype from the age of 11-20 weeks and showed Purkinje cell loss at 50 weeks old. Degenerative change of Purkinje cells and atrophic thinning of the molecular layer were conspicuous in homozygous knock-in mice. Electrophysiological analysis of Purkinje cells using acute cerebellar slices from young mice showed that the point mutation altered the voltage dependence of CaV3.1 channel activation and reduced the rebound action potentials after hyperpolarization, although it did not significantly affect the basic properties of synaptic transmission onto Purkinje cells. Finally, we revealed that the resonance of membrane potential of neurons in the inferior olivary nucleus was decreased in knock-in mice, which indicates that p.Arg1723His CaV3.1 mutation affects climbing fiber signaling to Purkinje cells. Altogether, our study shows not only that a point mutation in CACNA1G causes an ataxic phenotype and Purkinje cell degeneration in a mouse model, but also that the electrophysiological abnormalities at an early stage of SCA42 precede Purkinje cell loss.

DOI： 10.1016/j.nbd.2019.104516

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Mental disorders are caused by genetic and environmental factors. We here show that deficiency of an isoform of dopamine D2 receptor (D2R), D2LR, causes stress vulnerability in mouse. This occurs through dysfunction of serotonin [5-hydroxytryptamine (5-HT)] 1A receptor (5-HT1AR) on serotonergic neurons in the mouse brain. Exposure to forced swim stress significantly increased anxiety- and depressive-like behaviors in D2LR knock-out (KO) male mice compared with wild-type mice. Treatment with 8-OH-DPAT, a 5-HT1AR agonist, failed to alleviate the stress-induced behaviors in D2LR-KO mice. In forced swim-stressed D2LR-KO mice, 5-HT efflux in the medial prefrontal cortex was elevated and the expression of genes related to 5-HT levels was upregulated by the transcription factor PET1 in the dorsal raphe nucleus. Notably, D2LR formed a heteromer with 5-HT1AR in serotonergic neurons, thereby suppressing 5-HT1AR-activated G-protein-activated inwardly rectifying potassium conductance in D2LR-KO serotonergic neurons. Finally, D2LR overexpression in serotonergic neurons in the dorsal raphe nucleus alleviated stress vulnerability observed in D2LR-KO mice. Together, we conclude that disruption of the negative feedback regulation by the D2LR/5-HT1A heteromer causes stress vulnerability.SIGNIFICANCE STATEMENT Etiologies of mental disorders are multifactorial, e.g., interactions between genetic and environmental factors. In this study, using a mouse model, we showed that genetic depletion of an isoform of dopamine D2 receptor, D2LR, causes stress vulnerability associated with dysfunction of serotonin 1A receptor, 5-HT1AR in serotonergic neurons. The D2LR/5-HT1AR inhibitory G-protein-coupled heteromer may function as a negative feedback regulator to suppress psychosocial stress.

DOI： 10.1523/JNEUROSCI.0079-19.2019

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cell Press  

We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.

DOI： 10.1016/j.celrep.2018.07.056.

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Language：Japanese   Publisher：(一社)日本外科学会  

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Language：English   Publisher：生命科学系学会合同年次大会運営事務局  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

BACKGROUND: The dystroglycan complex consists of two subunits: extracellular α-dystroglycan and membrane-spanning β-dystroglycan, which provide a tight link between the extracellular matrix and the intracellular cytoskeleton. Previous studies showed that 43 kDa β-dystroglycan is proteolytically cleaved into the 30 kDa fragment by matrix metalloproteinases (MMPs) in various non-muscle tissues, whereas it is protected from cleavage in muscles by the sarcoglycan complex which resides close to the dystroglycan complex. It is noteworthy that cleaved β-dystroglycan is detected in muscles from patients with sarcoglycanopathy, sarcoglycan-deficient muscular dystrophy. In vitro assays using protease inhibitors suggest that both MMP-2 and MMP-9 contribute to the cleavage of β-dystroglycan. However, this has remained uninvestigated in vivo. METHODS: We generated triple-knockout (TKO) mice targeting MMP-2, MMP-9 and γ-sarcoglycan to examine the status of β-dystroglycan cleavage in the absence of the candidate matrix metalloproteinases in sarcoglycan-deficient muscles. RESULTS: Unexpectedly, β-dystroglycan was cleaved in muscles from TKO mice. Muscle pathology was not ameliorated but worsened in TKO mice compared with γ-sarcoglycan single-knockout mice. The gene expression of MMP-14 was up-regulated in TKO mice as well as in γ-sarcoglycan knockout mice. In vitro assay showed MMP-14 is capable to cleave β-dystroglycan. CONCLUSIONS: Double-targeting of MMP-2 and MMP-9 cannot prevent cleavage of β-dystroglycan in sarcoglycanopathy. Thus, matrix metalloproteinases contributing to β-dystroglycan cleavage are redundant, and MMP-14 could participate in the pathogenesis of sarcoglycanopathy.

DOI： 10.1016/j.bbrc.2017.08.048

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press  

DOI： 10.1016/j.bbrc.2017.08.048.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Aberrant dopamine D-2 receptor (D2R) activity is associated with neuropsychiatric disorders, making those receptors targets for antipsychotic drugs. Here, we report that novel signaling through the intracellularly localized D2R long isoform (D2LR) elicits extracellular signal-regulated kinase (ERK) activation and dendritic spine formation through Rabex-5/ platelet-derived growth factor receptor-beta (PDGFR beta)-mediated endocytosis in mouse striatum. We found that D2LR directly binds to and activates Rabex-5, promoting early-endosome formation. Endosomes containing D2LR and PDGFR beta are then transported to the Golgi apparatus, where those complexes trigger Gai3-mediated ERK signaling. Loss of intracellular D2LR-mediated ERK activation decreased neuronal activity and dendritic spine density in striatopallidal medium spiny neurons (MSNs). In addition, dendritic spine density in striatopallidal MSNs significantly increased following treatment of striatal slices from wild-type mice with quinpirole, a D2R agonist, but those changes were lacking in D2LR knockout mice. Moreover, intracellular D2LR signaling mediated effects of a typical antipsychotic drug, haloperidol, in inducing catalepsy behavior. Taken together, intracellular D2LR signaling through Rabex-5/PDGFR beta is critical for ERK activation, dendritic spine formation and neuronal activity in striatopallidal MSNs of mice.

DOI： 10.1038/mp.2016.200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Glial cell line-derived neurotrophic factor (GDNF) positively regulates the development and maintenance of in vitro dopaminergic neurons. However, the in vivo influences of GDNF signals on the brain dopamine system are controversial and not fully defined. To address this question, we analyzed dopaminergic phenotypes of the transgenic mice that overexpress GDNF under the control of the glial Gfap promoter. Compared with wild-type, the GDNF transgenic mice contained higher levels of GDNF protein and phosphorylated RET receptors in the brain. However, there were reductions in the levels of tyrosine hydroxylase (TH), dopamine, and its metabolite homovanillic acid in the striatum of transgenic mice. The TH reduction appeared to occur during postnatal development. Immunohistochemistry revealed that striatal TH density was reduced in transgenic mice with no apparent signs of neurodegeneration. In agreement with these neurochemical traits, basal levels of extracellular dopamine and high K+-induced dopamine efflux were decreased in the striatum of transgenic mice. We also explored the influences of GDNF overexpression on lomomotor behavior. GDNF transgenic mice exhibited lower stereotypy and rearing in a novel environment compared with wild-type mice. These results suggest that chronic overexpression of GDNF in brain astrocytes exerts an opposing influence on nigrostriatal dopamine metabolism and neurotransmission.

DOI： 10.1016/j.neulet.2017.06.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ASSOC RESEARCH VISION OPHTHALMOLOGY INC  

Purpose: Mutations in genes encoding the dystrophin-associated glycoprotein complex (DGC) can cause muscular dystrophy and disturb synaptic transmission in the photoreceptor ribbon synapse. However, the molecular composition and specific functions of the photoreceptor DGC remain unknown. Brefeldin A-resistant Arf-GEF 2 (BRAG2), also known as IQSEC1, is a guanine nucleotide exchange factor for ADP-ribosylation factor 6 (Arf6), a critical GTPase that regulates endosomal trafficking and actin cytoskeleton remodeling. In the present study, we characterized the expression of BRAG2a, an alternative splicing isoform of BRAG2, in the adult mouse photoreceptor. Methods: Immunofluorescence and immunoelectron microscopic analyses of adult mouse retinas were performed using a novel anti-BRAG2a antibody. Pull-down, immunoprecipitation, and in situ proximity ligation assays were performed to examine the interaction between BRAG2a and the DGC in vivo. Results: Immunofluorescence demonstrated punctate colocalization of BRAG2a with β-dystroglycan in the outer plexiform layer. Immunoelectron microscopy revealed the localization of BRAG2a at the plasma membrane of lateral walls and processes of photoreceptor terminals within the synaptic cavity. Pull-down and immunoprecipitation assays using retinal lysates demonstrated the protein complex formation between BRAG2a with the DGC. In situ proximity ligation assays further detected a close spatial relationship between BRAG2a and β-dystroglycan in the outer plexiform layer. Conclusions: The present study provided evidence that BRAG2a is a novel component of the photoreceptor DGC, suggesting functional involvement of the BRAG2a-Arf6 pathway downstream of the DGC.

DOI： 10.1167/iovs.17-21746

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

BACKGROUND: α-Synuclein (αSYN) has been genetically implicated in familial and sporadic Parkinson's disease (PD), and is associated with disease susceptibility, progression and pathology. Excess amounts of αSYN are toxic to neurons. In the brain, microglial αSYN clearance is closely related to neuronal survival. Leucine-rich repeat kinase 2 (LRRK2) is the one of the other genes implicated in familial and sporadic PD. While LRRK2 is known to be expressed in microglia, its true function remains to be elucidated. In this study, we investigated αSYN clearance by microglia isolated from LRRK2-knockout (KO) mice. RESULTS: In LRRK2-KO microglia, αSYN was taken up in larger amounts and cleared from the supernatant more effectively than for microglia isolated from wild-type (WT) mice. This higher clearance ability of LRRK2-KO microglia was thought to be due to an increase of Rab5-positive endosomes, but not Rab7- or Rab11-positive endosomes. Increased engagement between Rab5 and dynamin 1 was also observed in LRRK2-KO microglia. CONCLUSION: LRRK2 negatively regulates the clearance of αSYN accompanied by down-regulation of the endocytosis pathway. Our findings reveal a new functional role of LRRK2 in microglia and offer a new insight into the mechanism of PD pathogenesis.

DOI： 10.1186/s12868-016-0315-2

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The corticostriatothalamic circuit regulates learning behaviors via dopamine neurotransmission. D2 long (D2L) receptors are an isoform of dopamine D2 receptors (D2Rs) and may act mainly at postsynaptic sites. It is well known that D2Rs influence high brain functions, but the roles of individual D2R isoforms are still unclear. To assess the influence of D2L receptors in visual discrimination learning, we performed visual discrimination and reversal tasks with D2L knockout mice using a touchscreen operant system. There were no significant differences in an operant conditioning task between genotypes. However, D2L knockout mice were impaired in both visual discrimination and reversal learning tasks. D2L knockout mice were also significantly slower than wild-type mice in collecting the reward in the visual discrimination task. These results indicate that D2L receptors play an important role in visual discrimination and reversal learning.

DOI： 10.1159/000447970

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CSHL Press  

Considerable evidence has demonstrated a critical role for the nucleus accumbens (NAc) in the acquisition and flexibility of behavioral strategies. These processes are guided by the activity of two discrete neuron types, dopamine D1- or D2-receptor expressing medium spiny neurons (D1-/D2-MSNs). Here we used the IntelliCage, an automated group-housing experimental cage apparatus, in combination with a reversible neurotransmission blocking technique to examine the role of NAc D1- and D2-MSNs in the acquisition and reversal learning of a place discrimination task. We demonstrated that NAc D1- and D2-MSNs do not mediate the acquisition of the task, but that suppression of activity in D2-MSNs impairs reversal learning and increased perseverative errors. Additionally, global knockout of the dopamine D2L receptor isoform produced a similar behavioral phenotype to D2-MSN-blocked mice. These results suggest that D2L receptors and NAc D2-MSNs act to suppress the influence of previously correct behavioral strategies allowing transfer of behavioral control to new strategies.

DOI： 10.1101/lm.042507.116

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

LRRK2 is the causal molecule of autosomal dominant familial Parkinson's disease. B2 cells express a much higher LRRK2 mRNA level than B1 cells. To reveal the function of LRRK2 in B cells, we analyzed B cell functions in LRRK2-knockout (LRRK2(-/-)) mice. LRRK2(-/-) mice had significantly higher counts of peritoneal B1 cells than wild-type mice. After BCR stimulation, phosphor-Erk1/2 of splenic B2 cells was enhanced to a higher degree in LRRK2(-/-) mice. LRRK2(-/-) mice had a significantly higher serum IgA level, and TNP-Ficoll immunization increased the titer of serum anti-TNP IgM antibody. LRRK2 may play important roles in B cells.

DOI： 10.1016/j.jneuroim.2016.01.005

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Language：English   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

DOI： 10.1038/ncomms8295

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Language：Japanese   Publisher：日本生物学的精神医学会  

統合失調症の疾患モデル動物の評価には、行動の変化を解析することが多いことから、行動解析法の妥当性の検討が必要である。疾患モデルとして遺伝子操作マウスは有用であるが、異なる研究室からの解析結果の再現性が課題であり、その解決は容易でない。例えばモデル動物の運動量評価のためには遺伝背景等を同一条件とし、かつ実験方法が同一であることが望ましい。本稿ではコンジェニック系統のドーパミン受容体遺伝子変異マウスを用いて、ホームケージにおける運動量・摂食量・飲水量の解析方法について我々が開発した実験方法を紹介する。今後、統合失調症の疾患モデル動物の新たな行動解析の方法として評価することを考えている。(著者抄録)

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2015&ichushi_jid=J05574&link_issn=&doc_id=20150812470003&doc_link_id=%2Fcy8jbiop%2F2015%2F002602%2F004%2F0087-0094%26dl%3D0&url=http%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fcy8jbiop%2F2015%2F002602%2F004%2F0087-0094%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Duchenne muscular dystrophy (DMD) is a chronic and life-threatening disease that is initially supported by muscle regeneration but eventually shows satellite cell exhaustion and muscular dysfunction. The life-long maintenance of skeletal muscle homoeostasis requires the satellite stem cell pool to be preserved. Asymmetric cell division plays a pivotal role in the maintenance of the satellite cell pool. Here we show that granulocyte colony-stimulating factor receptor (G-CSFR) is asymmetrically expressed in activated satellite cells. G-CSF positively affects the satellite cell population during multiple stages of differentiation in ex vivo cultured fibres. G-CSF could be important in developing an effective therapy for DMD based on its potential to modulate the supply of multiple stages of regenerated myocytes. This study shows that the G-CSF-G-CSFR axis is fundamentally important for long-term muscle regeneration, functional maintenance and lifespan extension in mouse models of DMD with varying severities.

DOI： 10.1038/ncomms7745

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We previously reported the different motor abilities of D1R knockout (KO), D2R KO and wild-type (WT) mice. To understand the interaction between the cerebellum and the striatal direct and indirect pathways, we examined the expression patterns of immediate early genes (IEG) in the cerebellum of these three genotypes of mice. In the WT naive mice, there was little IEG expression. However, we observed a robust expression of c-fos mRNA in the vermis and hemisphere after running rota-rod tasks. In the vermis, c-fos was expressed throughout the lobules except lobule 7, and also in crus 1 of the ansiform lobule (Crus1), copula of the pyramis (Cop) and most significantly in the flocculus in the hemisphere. jun-B was much less expressed but more preferentially expressed in Purkinje cells. In addition, we observed significant levels of c-fos and jun-B expressions after handling mice, and after the stationary rota-rod task in naive mice. Surprisingly, we observed significant expression of c-fos and jun-B even 30 min after single weighing. Nonetheless, certain additional c-fos and jun-B expressions were observed in three genotypes of the mice that experienced several sessions of motor tasks 24 h after stationary rota-rod task and on days 1 and 5 after rota-rod tasks, but no significant differences in expressions after the running rota-rod tasks were observed among the three genotypes. In addition, there may be some differences 24 h after the stationary rota-rod task between the naive mice and the mice that experienced several sessions of motor tasks.

DOI： 10.3389/fcell.2015.00038

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

BACKGROUND: Voltage-dependent block of the NMDA receptor by Mg2+ is thought to be central to the unique involvement of this receptor in higher brain functions. However, the in vivo role of the Mg2+ block in the mammalian brain has not yet been investigated, because brain-wide loss of the Mg2+ block causes perinatal lethality. In this study, we used a brain-region specific knock-in mouse expressing an NMDA receptor that is defective for the Mg2+ block in order to test its role in neural information processing. RESULTS: We devised a method to induce a single amino acid substitution (N595Q) in the GluN2A subunit of the NMDA receptor, specifically in the hippocampal dentate gyrus in mice. This mutation reduced the Mg2+ block at the medial perforant path-granule cell synapse and facilitated synaptic potentiation induced by high-frequency stimulation. The mutants had more stable hippocampal place fields in the CA1 than the controls did, and place representation showed lower sensitivity to visual differences. In addition, behavioral tests revealed that the mutants had a spatial working memory deficit. CONCLUSIONS: These results suggest that the Mg2+ block in the dentate gyrus regulates hippocampal spatial information processing by attenuating activity-dependent synaptic potentiation in the dentate gyrus.

DOI： 10.1186/1756-6606-7-44

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Whole-cell or cell-attached analysis was carried out in dopamine (DA) D2 receptor (D2R) knock-out (KO) mice to elucidate the function of this receptor in the regulation of GABAergic synaptic transmission onto striatal cholinergic interneurons as well as their spontaneous firing. In slice preparation obtained from wild-type mice, evoked GABAergic inhibitory postsynaptic currents (IPSCs) showed frequency-dependent suppression, and this suppression significantly decreased in the presence of a D2-like receptor antagonist or in D2R KO mice. Contribution of N-type calcium channel was also significantly reduced in the striatal cholinergic interneurons of the D2R KO mice compared with that in the wild-type mice. Spontaneous firing of striatal cholinergic interneurons was inhibited by 5- or 10-Hz stimulation, and the suppression was decreased in the presence of a D2-like receptor antagonist or in D2R KO mice. These findings substantiate the physiological role of D2R in the regulation of GABAergic synaptic transmission onto striatal cholinergic interneurons as well as their excitability, confirming the tight coupling between D2R and N-type calcium channels in the regulation of GABA release. © 2014 IBRO.

DOI： 10.1016/j.neuroscience.2014.01.010

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In the rodent brain, certain G protein-coupled receptors and adenylyl cyclase type 3 are known to localize to the neuronal primary cilium, a primitive sensory organelle protruding singly from almost all neurons. A recent chemical screening study demonstrated that many compounds targeting dopamine receptors regulate the assembly of Chlamydomonas reinhardtii flagella, structures which are analogous to vertebrate cilia. Here we investigated the effects of dopaminergic inputs loss on the architecture of neuronal primary cilia in the rodent striatum, a brain region that receives major dopaminergic projections from the midbrain. We first analyzed the lengths of neuronal cilia in the dorsolateral striatum of hemi-parkinsonian rats with unilateral lesions of the nigrostriatal dopamine pathway. In these rats, the striatal neuronal cilia were significantly longer on the lesioned side than on the non-lesioned side. In mice, the repeated injection of reserpine, a dopamine-depleting agent, elongated neuronal cilia in the striatum. The combined administration of agonists for dopamine receptor type 2 (D2) with reserpine attenuated the elongation of striatal neuronal cilia. Repeated treatment with an antagonist of D2, but not of dopamine receptor type 1 (D1), elongated the striatal neuronal cilia. In addition, D2-null mice displayed longer neuronal cilia in the striatum compared to wild-type controls. Reserpine treatment elongated the striatal neuronal cilia in D1-null mice but not in D2-null mice. Repeated treatment with a D2 agonist suppressed the elongation of striatal neuronal cilia on the lesioned side of hemi-parkinsonian rats. These results suggest that the elongation of striatal neuronal cilia following the lack of dopaminergic inputs is attributable to the absence of dopaminergic transmission via D2 receptors. Our results provide the first evidence that the length of neuronal cilia can be modified by the lack of a neurotransmitter's input.

DOI： 10.1371/journal.pone.0097918

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Polypyrimidine tract-binding protein (PTB) is a well-characterized RNA-binding protein and known to be preferentially expressed in neural stem cells (NSCs) in the central nervous system; however, its role in NSCs in the developing brain remains unclear. To explore the role of PTB in embryonic NSCs in vivo, Nestin-Cremediated conditional Ptb knockout mice were generated for this study. In the mutant forebrain, despite the depletion of PTB protein, neither abnormal neurogenesis nor flagrant morphological abnormalities were observed at embryonic day 14.5 (E14.5). Nevertheless, by 10 weeks, nearly all mutant mice succumbed to hydrocephalus (HC), which was caused by a lack of the ependymal cell layer in the dorsal cortex. Upon further analysis, a gradual loss of adherens junctions (AJs) was observed in the ventricular zone (VZ) of the dorsal telencephalon in the mutant brains, beginning at E14.5. In the AJs-deficient VZ, impaired interkinetic nuclear migration and precocious differentiation of NSCs were observed after E14.5. These findings demonstrated that PTB depletion in the dorsal telencephalon is causally involved in the development of HC and that PTB is important for the maintenance of AJs in the NSCs of the dorsal telencephalon.

DOI： 10.1093/cercor/bhs161

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Polypyrimidine tract-binding protein (PTB) is a well-characterized RNA-binding protein and known to be preferentially expressed in neural stem cells (NSCs) in the central nervous system; however, its role in NSCs in the developing brain remains unclear. To explore the role of PTB in embryonic NSCs in vivo, Nestin-Cre-mediated conditional Ptb knockout mice were generated for this study. In the mutant forebrain, despite the depletion of PTB protein, neither abnormal neurogenesis nor flagrant morphological abnormalities were observed at embryonic day 14.5 (E14.5). Nevertheless, by 10 weeks, nearly all mutant mice succumbed to hydrocephalus (HC), which was caused by a lack of the ependymal cell layer in the dorsal cortex. Upon further analysis, a gradual loss of adherens junctions (AJs) was observed in the ventricular zone (VZ) of the dorsal telencephalon in the mutant brains, beginning at E14.5. In the AJs-deficient VZ, impaired interkinetic nuclear migration and precocious differentiation of NSCs were observed after E14.5. These findings demonstrated that PTB depletion in the dorsal telencephalon is causally involved in the development of HC and that PTB is important for the maintenance of AJs in the NSCs of the dorsal telencephalon.

DOI： 10.1093/cercor/bhs161

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The basal ganglia-thalamocortical circuitry plays a central role in selecting actions that achieve reward-seeking outcomes and avoid aversive ones. Inputs of the nucleus accumbens (NAc) in this circuitry are transmitted through two parallel pathways: the striatonigral direct pathway and the striatopallidal indirect pathway. In the NAc, dopaminergic (DA) modulation of the direct and the indirect pathways is critical in reward-based and aversive learning and cocaine addiction. To explore how DA modulation regulates the associative learning behavior, we developed an asymmetric reversible neurotransmission-blocking technique in which transmission of each pathway was unilaterally blocked by transmission-blocking tetanus toxin and the transmission on the intact side was pharmacologically manipulated by local infusion of a receptor-specific agonist or antagonist. This approach revealed that the activation of D1 receptors and the inactivation of D2 receptors postsynaptically control reward learning/cocaine addiction and aversive learning in a direct pathway-specific and indirect pathway-specific manner, respectively. Furthermore, this study demonstrated that aversive learning is elicited by elaborate actions of NMDA receptors, adenosine A2a receptors, and endocannabinoid CB1 receptors, which serve as key neurotransmitter receptors in inducing long-term potentiation in the indirect pathway. Thus, reward and aversive learning is regulated by pathway-specific neural plasticity via selective transmitter receptors in the NAc circuit.

DOI： 10.1073/pnas.1220358110

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DOI： 10.1073/pnas.1220358110

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MicroRNAs (miRNAs) are endogenous small RNAs of 18-23 nucleotides that regulate gene expression. Recently, plasma miRNAs have been investigated as biomarkers for various diseases. In the present study, we explored whether miRNA expression profiling of various muscle cells may be useful for the diagnosis of various diseases involving muscle necrosis. miRNA expression profiling was assessed by miRNA array and real-time reverse-transcriptase polymerase chain reaction by using a reverse primer of a stem loop structure. Profiling of various muscle cells of mouse, including cardiac muscles, skeletal muscles, and vascular and visceral smooth muscles, indicated that profiling of miR-1, miR-133a, miR-133b, miR-145, miR-206, miR-208a, miR-208b, and miR499 were adequate to discriminate muscle cells. miR-145 was remarkably highly expressed in smooth muscles. miR-208a and miR-499 were highly expressed in cardiomyocytes. miR-133a was highly expressed in fast-twitch skeletal muscles. miR-206 and miR-208b were expressed in the slow-twitch skeletal muscles, and they can likely discriminate fast- and slow-twitch types of skeletal muscle cells. We observed that brown fat adipose cells had an miRNA expression profile very similar to those of skeletal muscle cells in the mouse. Plasma concentrations of miR-133a and miR-145 were extremely useful in diagnosing skeletal muscle necrosis in a mouse model of Duchenne muscular dystrophy and colon smooth muscle necrosis in a rat ischemic colitis model, respectively. In the present study, we investigated the miRNA expression profiles of various muscular tissues. Our results suggest that expression profiling would be useful for the diagnosis of various diseases such as muscular necrosis.

DOI： 10.2220/biomedres.34.289

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Alterations in the activity of the dopamine D2 receptor (D2R) have been implicated in several neurological and psychiatric disorders, including schizophrenia, Parkinson's disease, Huntington's disease, Tourette syndrome, attention-deficit hyperactivity disorder (ADHD), and drug addiction. Two isoforms of D2R, long form (D2LR) and short form (D2SR), have been identified. The specific function of each D2R isoform is poorly understood, primarily because isoform-selective pharmacological agents are not available. Using homologous recombination, we have generated D2LR knockout (KO) mice. D2LR KO mice are completely deficient in D2LR, but still express functional D2SR at a level similar to the total D2R level in wild-type (WT) mice. D2LR is generally the predominant isoform expressed in WT mice. We showed that D2LR KO mice displayed a number of robust behavioral phenotypes distinct from WT mice, indicating that D2LR and D2SR have differential functions. In this chapter we describe the generation and characterization of the D2LR KO mouse. This genetic approach provides a valuable research tool to investigate the functional role of individual D2R isoforms in the mammalian central nervous system (CNS).

DOI： 10.1007/978-1-62703-251-3_11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Humana Press Inc.  

Alterations in the activity of the dopamine D2 receptor (D2R) have been implicated in several neurological and psychiatric disorders, including schizophrenia, Parkinson's disease, Huntington's disease, Tourette syndrome, attention-deficit hyperactivity disorder (ADHD), and drug addiction. Two isoforms of D2R, long form (D2LR) and short form (D2SR), have been identified. The specific function of each D2R isoform is poorly understood, primarily because isoform-selective pharmacological agents are not available. Using homologous recombination, we have generated D2LR knockout (KO) mice. D2LR KO mice are completely deficient in D2LR, but still express functional D2SR at a level similar to the total D2R level in wild-type (WT) mice. D2LR is generally the predominant isoform expressed in WT mice. We showed that D2LR KO mice displayed a number of robust behavioral phenotypes distinct from WT mice, indicating that D2LR and D2SR have differential functions. In this chapter we describe the generation and characterization of the D2LR KO mouse. This genetic approach provides a valuable research tool to investigate the functional role of individual D2R isoforms in the mammalian central nervous system (CNS). © Springer Science+Business Media, LLC 2013.

DOI： 10.1007/978-1-62703-251-3_11

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The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1(+/-) mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.

DOI： 10.1371/journal.pone.0080737

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DOI： 10.1111/j.1440-1681.2012.05723.x

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Methyl CpG-binding protein 2 gene (MeCP2) mutations are implicated in Rett syndrome (RTT), one of the common causes of female mental retardation. Two MeCP2 isoforms have been reported: MeCP2_e2 (splicing of all four exons) and MeCP2_e1 (alternative splicing of exons 1, 3, and 4). Their relative expression levels vary among tissues, with MeCP2_e1 being more dominant in adult brain, whereas MeCP2_e2 is expressed more abundantly in placenta, liver, and skeletal muscle. In this study, we performed specific disruption of the MeCP2_e2-defining exon 2 using the Cre-loxP system and examined the consequences of selective loss of MeCP2_e2 function in vivo. We performed behavior evaluation, gene expression analysis, using RT-PCR and real-time quantitative PCR, and histological analysis. We demonstrate that selective deletion of MeCP2_e2 does not result in RTT-associated neurological phenotypes but confers a survival disadvantage to embryos carrying a MeCP2_e2 null allele of maternal origin. In addition, we reveal a specific requirement for MeCP2_e2 function in extraembryonic tissue, where selective loss of MeCP2_e2 results in placenta defects and up-regulation of peg-1, as determined by the parental origin of the mutant allele. Taken together, our findings suggest a novel role for MeCP2 in normal placenta development and illustrate how paternal X chromosome inactivation in extraembryonic tissues confers a survival disadvantage for carriers of a mutant maternal MeCP2_e2 allele. Moreover, our findings provide an explanation for the absence of reports on MeCP2_e2-specific exon 2 mutations in RTT. MeCP2_e2 mutations in humans may result in a phenotype that evades a diagnosis of RTT.

DOI： 10.1074/jbc.M111.309864

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The gonadal primordium first emerges as a thickening of the embryonic coelomic epithelium, which has been thought to migrate mediodorsally to form the primitive gonad. However, the early gonadal development remains poorly understood. Mice lacking the paired-like homeobox gene Emx2 display gonadal dysgenesis. Interestingly, the knockout (KO) embryonic gonads develop an unusual surface accompanied by aberrant tight junction assembly. Morphological and in vitro cell fate mapping studies showed an apparent decrease in the number of the gonadal epithelial cells migrated to mesenchymal compartment in the KO, suggesting that polarized cell division and subsequent cell migration are affected. Microarray analyses of the epithelial cells revealed significant up-regulation of Egfr in the KO, indicating that Emx2 suppresses Egfr gene expression. This genetic correlation between the two genes was reproduced with cultured M15 cells derived from mesonephric epithelial cells. Epidermal growth factor receptor signaling was recently shown to regulate tight junction assembly through sarcoma viral oncogene homolog tyrosine phosphorylation. We show through Emx2 KO analyses that sarcoma viral oncogene homolog tyrosine phosphorylation, epidermal growth factor receptor tyrosine phosphorylation, and Egfr expression are up-regulated in the embryonic gonad. Our results strongly suggest that Emx2 is required for regulation of tight junction assembly and allowing migration of the gonadal epithelia to the mesenchyme, which are possibly mediated by suppression of Egfr expression. Copyright © 2010 by The Endocrine Society.

DOI： 10.1210/en.2010-0915

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The gonadal primordium first emerges as a thickening of the embryonic coelomic epithelium, which has been thought to migrate mediodorsally to form the primitive gonad. However, the early gonadal development remains poorly understood. Mice lacking the paired-like homeobox gene Emx2 display gonadal dysgenesis. Interestingly, the knockout (KO) embryonic gonads develop an unusual surface accompanied by aberrant tight junction assembly. Morphological and in vitro cell fate mapping studies showed an apparent decrease in the number of the gonadal epithelial cells migrated to mesenchymal compartment in the KO, suggesting that polarized cell division and subsequent cell migration are affected. Microarray analyses of the epithelial cells revealed significant up-regulation of Egfr in the KO, indicating that Emx2 suppresses Egfr gene expression. This genetic correlation between the two genes was reproduced with cultured M15 cells derived from mesonephric epithelial cells. Epidermal growth factor receptor signaling was recently shown to regulate tight junction assembly through sarcoma viral oncogene homolog tyrosine phosphorylation. We show through Emx2 KO analyses that sarcoma viral oncogene homolog tyrosine phosphorylation, epidermal growth factor receptor tyrosine phosphorylation, and Egfr expression are up-regulated in the embryonic gonad. Our results strongly suggest that Emx2 is required for regulation of tight junction assembly and allowing migration of the gonadal epithelia to the mesenchyme, which are possibly mediated by suppression of Egfr expression.

DOI： 10.1210/en.2010-0915

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Planar cell polarity (PCP) genes are essential for establishing planar cell polarity in both invertebrate and vertebrate tissues and are known to regulate cellular morphogenesis and cell movements during development. We focused on Prickle, one of the core components of the PCP pathway, and deleted one of two mouse prickle homologous genes, mpk1. We found that the deletion of mpk1 gene resulted in early embryonic lethality, between embryonic day (E)5.5 and E6.5, associated with failure of distal visceral endoderm migration and primitive streak formation. The mpk1(-/-) epiblast tissue was disorganized, and analyses at the cellular level revealed abnormal cell shapes, mislocalized extracellular matrix (ECM) proteins, and disrupted orientation of mitotic spindles, from which loss of apico-basal (AB) polarity of epiblast cells are suspected. Furthermore, we show mpk1 genetically interacts with another core PCP gene Vangl2/stbm in the epiblast formation, suggesting that PCP components are commonly required for the establishment and/or the maintenance of epiblast AB polarity. This was further supported by our finding that overexpression of DeltaPET/LIM (DeltaP/L), a dominant-negative Pk construct, in Xenopus embryo disrupted uniform localization of an apical marker PKCzeta, and expanded the apical domain of ectoderm cells. Our results demonstrate a role for mpk1 in AB polarity formation rather than expected role as a PCP gene.

DOI： 10.1073/pnas.0901332106

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Planar cell polarity (PCP) genes are essential for establishing planar cell polarity in both invertebrate and vertebrate tissues and are known to regulate cellular morphogenesis and cell movements during development. We focused on Prickle, one of the core components of the PCP pathway, and deleted one of two mouse prickle homologous genes, mpk1. We found that the deletion of mpk1 gene resulted in early embryonic lethality, between embryonic day (E)5.5 and E6.5, associated with failure of distal visceral endoderm migration and primitive streak formation. The mpk1(-/-) epiblast tissue was disorganized, and analyses at the cellular level revealed abnormal cell shapes, mislocalized extracellular matrix (ECM) proteins, and disrupted orientation of mitotic spindles, from which loss of apico-basal (AB) polarity of epiblast cells are suspected. Furthermore, we show mpk1 genetically interacts with another core PCP gene Vangl2/stbm in the epiblast formation, suggesting that PCP components are commonly required for the establishment and/or the maintenance of epiblast AB polarity. This was further supported by our finding that overexpression of Delta PET/LIM (Delta P/L), a dominant-negative Pk construct, in Xenopus embryo disrupted uniform localization of an apical marker PKC zeta, and expanded the apical domain of ectoderm cells. Our results demonstrate a role for mpk1 in AB polarity formation rather than expected role as a PCP gene.

DOI： 10.1073/pnas.0901332106

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Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell-derived VEGFR2(+) mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)-BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A-induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras(-/-) mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2(+) progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2(+) progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6-9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras-Erk signaling to direct endothelial specification of VEGFR2(+) vascular progenitor cells.

DOI： 10.1083/jcb.200709127

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell-derived VEGFR2(+) mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)-BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A-induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras(-/-) mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2(+) progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2(+) progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6-9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras-Erk signaling to direct endothelial specification of VEGFR2(+) vascular progenitor cells.

DOI： 10.1083/jcb.200709127

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Although platelet-derived growth factor (PDGF)-BB activates PDGF receptor-beta (PDGFR-beta) and, in turn, inhibits the glutamate N-methyl-D-aspartate (NMDA) receptor function, whether PDGF-BB modulates the CNS function mediated by another glutamate receptors, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, remains poorly understood. Here we now report the inhibitory effect of PDGF-BB on the AMPA receptor function in the nucleus tractus solitarius (NTS) by using slice patch-clamp techniques. Excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the tractus solitarius in mouse NTS second-order neurons. EPSCs were nearly completely eliminated by CNQX but not by MK-801, implying mediation through non-NMDA receptors. PDGF-BB significantly decreased the amplitude of EPSCs without affecting the mean decay time constant. This inhibitory effect was transient and reversible after removing PDGF-BB. Furthermore, PDGF-BB significantly reduced the amplitude of AMPA-induced currents in NTS neurons, which showed that PDGF-BB could suppress the AMPA receptor-mediated excitatory input via the postsynaptic mechanism. The inhibitory effect of PDGF-BB on EPSCs was not observed in mutant mice with conditional deletion of the PDGFR-beta gene in neurons. Together, these studies suggest that the PDGF-B/PDGFR-beta axis inhibits the AMPA receptor-mediated synaptic transmission that comprises the major part of the primary afferent to the NTS second-order neuron. The detected inhibitory action may be involved in the CNS regulation of the respiratory response.

DOI： 10.1016/j.brainres.2007.05.037

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Dystroglycan (DG) is a widely expressed, transmembrane glycoprotein complex that plays important roles by connecting the extracellular matrix to the cytoskeleton. The alpha- and beta-DG subunits are produced by the cleavage of residues 653 and 654 of the precursor. To clarify the mechanisms involved in cleavage and subunit association, we performed a series of mutation analyses and made the following discoveries: (i) Disruption of the intramolecular disulfide bridge between Cys669 and Cys713 in beta-DG completely abolishes the cleavage, (ii) deletions in the loop region (669-713) and in the C-terminal region of alpha-DG (550-645) abolish the cleavage, (iii) disruption of the disulfide bridge and deletions in the loop region deteriorate the alpha- and beta-DG subunit association, and (iv) at the cleavage site, especially, positions P1' (Ser654) and P6' (Trp659) are critical. Thus, the critical role of the Cys669-Cys713 disulfide bridge formation is, most likely, to form a specific tertiary structure, in which the alpha- and beta-DG domains interact and the cleavage site becomes susceptible to proteolytic reactions. The Cys669 and Cys713 pair is broadly conserved in vertebrates and in some invertebrates, suggesting that the disulfide bridge formation was established early in the evolution of DG.

DOI： 10.1111/j.1365-2443.2006.01033.x

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Caveolin-3 is a muscle-specific membrane protein that serves as a scaffold of various molecules. As previously reported, caveolin-3 deficiency causes muscle degeneration in mice. In the present study, gene expression profiles, analyzed in the skeletal muscles of caveolin-3 deficient mice using the DNA microarray technique, showed that the gene of osteopontin, a versatile regulator of inflammation and tissue repair, was significantly down-regulated. This is in contrast to mdx mice showing a markedly up-regulated osteopontin gene in their skeletal muscles. Recently, osteopontin has been reported to be important in the pathogenesis of muscular dystrophy. We examined whether up-regulated osteopontin gene expression in mdx muscles is altered by the deficiency of caveolin-3. To this end, we developed caveolin-3 and dystrophin double-deficient mice and used them for the analysis. Levels of osteopontin mRNA and protein in the double-deficient mice clearly decreased compared with those in mdx mice.

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Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) are widely expressed in the mammalian CNS, though their functional significance remains unclear. The corresponding null-knockout mutations are lethal. Here, we developed novel mutant mice in which the gene encoding the beta subunit of PDGFR (PDGFR-beta) was genetically deleted in CNS neurons to elucidate the role of PDGFR-beta, particularly in the post-natal stage. Our mutant mice reached adulthood without apparent anatomical defects. In the mutant brain, immunohistochemical analyses showed that PDGFR-beta detected in neurons and in the cells in the subventricular zone of the lateral ventricle in wild-type mice was depleted, but PDGFR-beta detected in blood vessels remained unaffected. The cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with controls. Furthermore, TdT-mediated dUTP-biotin nick end labeling (TUNEL)-positive neuronal cell death and lesion formation in the cerebral hemisphere were extensively exacerbated in our mutant mice after direct injection of NMDA without altered NMDA receptor expression. Our results clearly demonstrate that PDGFR-beta expressed in neurons protects them from cryogenic injury and NMDA-induced excitotoxicity.

DOI： 10.1111/j.1471-4159.2006.03922.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

In developing mammalian telencephalon, the loss of adherens junctions and cell cycle exit represent crucial steps in the differentiation of neuroepithelial cells into neurons, but the relationship between these cellular events remains obscure. Atypical protein kinase C (aPKC) is known to contribute to junction formation in epithelial cells and to cell fate determination for Drosophila neuroblasts. To elucidate the functions of aPKClambda, one out of two aPKC members, in mouse neocortical neurogenesis, a Nestin-Cre mediated conditional gene targeting system was employed. In conditional aPKClambda knockout mice, neuroepithelial cells of the neocortical region lost aPKClambda protein at embryonic day 15 and demonstrated a loss of adherens junctions, retraction of apical processes and impaired interkinetic nuclear migration that resulted in disordered neuroepithelial tissue architecture. These results are evidence that aPKClambda is indispensable for the maintenance of adherens junctions and may function in the regulation of adherens junction integrity upon differentiation of neuroepithelial cells into neurons. In spite of the loss of adherens junctions in the neuroepithelium of conditional aPKClambda knockout mice, neurons were produced at a normal rate. Therefore, we concluded that, at least in the later stages of neurogenesis, regulation of cell cycle exit is independent of adherens junctions.

DOI： 10.1242/dev.02389

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The original sarcoglycan (SG) complex has four subunits and comprises a subcomplex of the dystrophin-dystrophin-associated protein complex. Each SG gene has been shown to be responsible for limb-girdle muscular dystrophy, called sarcoglycanopathy (SGP). In this review, we detail the characteristics of the SG subunits, and the mechanism of the formation of the SG complex and various molecules associated with this complex. We discuss the molecular mechanisms of SGP based on studies mostly using SGP animal models. In addition, we describe other SG molecules, epsilon- and zeta-SGs, with special reference to their expression and roles in vascular smooth muscle, which are currently in dispute. We further consider the maternally imprinted nature of the epsilon-SG gene. Finally, we stress that the SG complex cannot work by itself and works in a larger complex system, called the transverse fixation system, which forms an array of molecules responsible for various muscular dystrophies.

DOI： 10.1002/mus.20349

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Synemin is an intermediate filament protein shown previously to interact with alpha-dystrobrevin and desmin. Immunoblot analysis detects a beta-synemin protein of 170 kDa in human skeletal muscle and an alpha-synemin protein of 225 kDa in monkey brain. Low-resolution immunohistochemical analysis localizes beta-synemin within muscle along the sarcolemma, whereas confocal microscopic analysis further refines localization to the costamere and muscle Z-lines. In addition to these locations, beta-synemin is also enriched at the neuromuscular and myotendinous junctions, other regions that undergo high stress during myofiber contraction. Based on its localization and its expression pattern, it is proposed that beta-synemin functions as a structural protein involved in maintaining muscle integrity through its interactions with alpha-dystrobrevin, desmin, and other structural proteins.

DOI： 10.1002/mus.20111

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC PUBL  

The sarcoglycanopathies are a group of four autosomal recessive limb girdle muscular dystrophies (LGMD 2D, 2E, 2C, and 2F), caused by mutations of the alpha-, beta-, gamma-, or delta-sarcoglycan genes, respectively. The delta-sarcoglycan-deficient hamster has been the most utilized model for gene delivery to muscle by recombinant adeno-associated virus (AAV) vectors; however, human patients with delta-sarcoglycan deficiency are exceedingly rare, with only two patients described in the United States. Here, we report construction and use of AAV vectors expressing either alpha- or beta-sarcoglycan, the genes responsible for the most common forms of the human sarcoglycanopathies. Both vectors showed successful short-term genetic, biochemical, and histological rescue of both alpha- and beta-sarcoglycan-deficient mouse muscle. However, comparison of persistence of expression in 51 injected mice showed substantial differences between AAV alpha-sarcoglycan (alpha-SG) and beta-sarcoglycan (beta-SG) vectors. AAV-beta-SG showed long-term expression with no decrease in expression for more than 21 months after injection, whereas AAV-alpha-SG showed a dramatic loss of positive fibers between 28 and 41 days post-injection (p = 0.006). Loss of immunopositive myofibers was correlated with significant inflammatory cell infiltrate, primarily macrophages. To determine whether the loss of alpha-sarcoglycan-positive fibers was due to an immune response or cytotoxic effect of alpha-sarcoglycan overexpression, severe combined immunodeficient (SCID) mouse muscle was assayed for cytotoxicity after injection with AAV-alpha-SG, AAV-beta-SG, or phosphate-buffered saline. The results were consistent with overexpression of a- sarcoglycan causing significant cytotoxicity. The cytotoxicity of alpha-sarcoglycan, and not beta- or delta-sarcoglycan overexpression, was consistent with biochemical studies of the hierarchical order of assembly of the sarcoglycan complex. Our data suggest that even closely related proteins might require different levels of expression to avoid toxicity and achieve long-term tissue rescue.

DOI： 10.1089/10430340260201725

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To evaluate the promoter function of the 5 ' -flanking sequence of mouse gamma -sarcoglycan (gamma -SG) gene in vivo, H e generated transgenic mice harboring this sequence fused with enhanced green fluorescent protein reporter gene, The reporter expression was restricted in striated muscles and particularly strong in all myofibers in skeletal muscles. Using these mice, a e examine the spatial and temporal transcriptional patterns of the gamma -SG gene during mouse skeletal muscle development. The expression of basic helix loop helix transcriptional factors preceded that of the reporter. Differences between the expression of reporter and endogenous gamma -SG genes in non-muscle tissues suggested the existence of additional promoter elements in the endogenous gene, and the analysis of endogenous mRNAs demonstrated the existence of a novel upstream exon and promoter active in non-muscle tissues. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V, All rights reserved.

DOI： 10.1016/S0014-5793(01)02368-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Catecholamine neurotransmitters are synthesized by hydroxylation of tyrosine to L-dihydroxyphenylalanine (L-Dopa) by tyrosine hydroxylase (TH). The elimination of TH in both pigmented and albino mice described here, like pigmented TH-null mice reported previously (Kobayashi et al., 1995; Zhou et al., 1995), demonstrates the unequivocal requirement for catecholamines during embryonic development. Although the lack of TH is fatal, TH-null embryos can be rescued by administration of catecholamine precursors to pregnant darns. Once born, TH-null pups can survive without further treatment until weaning. Given the relatively rapid half-life of catecholamines, we expected to find none in postnatal TH-null pups. Despite the fact that the TH-null pups lack TH and have not been supplemented with catecholamine precursers, catecholamines are readily detected in our pigmented line of TH-null mice by glyoxylic acid-induced histofluorescence at postnatal day 7 (P7) and P15 and quantitatively at P15 in sympathetically innervated peripheral organs, in sympathetic ganglia, in adrenal glands, and in brains. Between 2 and 22% of wild-type catecholamine concentrations are found in these tissues in mutant pigmented mice. To ascertain the source of the catecholamine, we examined postnatal TH-null albino mice that lack tyrosinase, another enzyme that converts tyrosine to L-Dopa but does so during melanin synthesis. In contrast to the pigmented TH-null mice, catecholamine histofluorescence is undetectable in postnatal albino mutants, and the catecholamine content of TH-null pups lacking tyrosinase is 18% or less than that of TH-null mice with tyrosinase. Thus, these extraordinary circumstances reveal that tyrosinase serves as an alternative pathway to supply catecholamines.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

We have examined the role of NMDA receptor-mediated neural activity in the formation of periphery-related somatosensory patterns, using genetically engineered mice. We demonstrate that ectopic expression of a transgene of an NMDAR1 splice variant rescues neonatally fatal NMDAR1 knockout (KO) mice, although the average life span varies depending on the level of the transgene expression. In NMDAR1 KO mice with "high" levels of the transgene expression, sensory periphery-related patterns were normal along both the trigeminal and dorsal column pathways. In the KO mice with "low" levels of the transgene expression, the patterns were absent in the trigeminal pathway. Our results indicate that NMDA receptor-mediated neural activity plays a critical role in pattern formation; along the ascending somatosensory pathways.

DOI： 10.1016/S0896-6273(00)80412-2

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F52 is a myristoylated, alanine-rich substrate for protein kinase C, We have generated F52-deficient mice by the gene targeting technique. These mutant mice manifest severe neural tube defects that are not associated with other complex malformations, a phenotype reminiscent of common human neural tube defects. The neural tube defects observed include both exencephaly and spina hifida, and the phenotype exhibits partial penetrance with about 60% of homozygous embryos developing neural tube defects, Exencephaly is the prominent type of defect and leads to high prenatal lethality, Neural tube defects are observed in a smaller percentage of heterozygous embryos (about 10%). Abnormal brain development and tail formation occur in homozygous mutants and are likely to be secondary to the neural tube defects, Disruption of F52 in mice therefore identifies a gene whose mutation results in isolated neural tube defects and may provide an animal model for common human neural tube defects.

DOI： 10.1073/pnas.93.5.2110

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Two steps of gene targeting were used to replace the p53 gene with the E.coli beta-galactosidase (lacZ) gene in mouse embryonic stem (ES) cells. The first targeting vector consisted of neo and herpes simplex virus thymidine kinase (HSV-tk) genes as a neo-tk cassette in the middle of the targeting vector. At the first targeting, the homologous recombinants became G418 resistant and ganciclovir (GANC) sensitive and were selected by G418 alone. At the second targeting, homologous recombination reciprocally exchanged the neo-tk cassette in the ES cell chromosome with the lacZ fragment in the second targeting vector and thus made the ES cells GANC resistant. We obtained two ES cell clones, in which the p53 gene for both had been replaced with a totally non-homologous sequence of the lacZ gene. The germ-line transmission of the manipulated ES cells also demonstrated that the entire procedure had no detrimental effects on ES cells at all. (C) 1994 Academic Press, Inc.

DOI： 10.1006/bbrc.1994.2005

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Using a microdialysis method, we observed a similar steady-state L-3,4-dihydroxyphenylalanine accumulation in the striatum of transgenic mice carrying the human tyrosine hydroxylase (TH) gene after NSD-1015 perfusion (10(-4) M) as compared to nontransgenic mice. Basal extracellular levels of 3,4-dihydroxyphenylacetic acid before the perfusion of NSD-1015 were also comparable in both transgenic and non-transgenic striata. The results suggest that the in vivo activity of TH in the striatum of transgenic mice was retained to the normal level by some regulatory mechanism(s) in spite of the increased expression of the enzyme protein.

DOI： 10.1016/0304-3940(93)90608-N

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We investigated the regulatory mechanism of dopamine biosynthesis in the striatum of transgenic mice carrying multiple copies of human tyrosine hydroxylase (TH). The in vitro TH activity of transgenic striatum at pH 7.0 was approximately 2.8-fold higher than that of non-transgenic striatum. This augmentation is similar to that of the in vitro TH activity at pH 6.0, indicating that the expression of human TH in transgenic striatum induced little change in the phosphorylation level of TH. L-3,4-Dihydroxyphenylalanine (DOPA) formation in striatal slices of transgenic mice was approximately 2.7-fold higher than that of non-transgenic mice. The addition of 0.5 mM (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4) to the incubation medium brought a negligible increase in DOPA formation in both cases. These results suggest that 6R-BH4 is not the limiting factor of TH in situ both in the transgenic and non-transgenic mice.

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Epinephrine-producing cells are characterized by the presence of phenylethanolamine N-methyltransferase (PNMT), which catalyzes the formation of epinephrine from norepinephrine. We generated a line of transgenic mice carrying a chimeric gene containing human PNMT cDNA fused to the 4-kilobase fragment of the human dopamine beta-hydroxylase (DBH) gene promoter, to switch catecholamine phenotype in the nervous and endocrine systems. Human PNMT transcripts and immunoreactivity were mainly detected, in norepinephrine neurons in brain and sympathetic ganglion as well as in norepinephrine-producing cells in adrenal medulla of transgenic mice, indicating that the human DBH gene promoter of 4 kilobases is sufficient to direct expression of the gene in norepinephrine-producing cells. Analysis of catecholamines in the various tissues showed that the expression of human PNMT in transgenic mice induced the appearance of epinephrine in sympathetic ganglion and dramatic changes in norepinephrine and epinephrine levels in brain, adrenal gland, and blood. These results indicate that the additional PNMT expression in norepinephrine-producing cells can convert these cells to the epinephrine phenotype, and suggest that norepinephrine-producing cells normally possess the basic machinery required for the synthesis of epinephrine except for PNMT. Thus it appears that the only major difference between norepinephrine- and epinephrine-producing cells is the expression of PNMT. Our transgenic animals provide an experimental model to investigate the functional differences between norepinephrine and epinephrine.

DOI： 10.1073/pnas.89.5.1631

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

The mouse tyrosine hydroxylase (TH) gene was isolated from a genomic library by cross-hybridization with human TH cDNA probe. Nucleotide sequence analysis of two overlapping genomic clones showed that this gene is split into 13 exons distributed about 7.5 kb in length. The transcription initiation site was determined by primer extension analysis with mouse adrenal gland poly(A)+RNA. The structure of the mouse TH gene was similar to that of the human TH gene, but it contained neither the alternative splice donor site around the 3'-end of the first exon nor an independent exon corresponding to the second exon of the human TH gene. There were the canonical TATA and GC boxes, cyclic AMP responsive element (CRE), and AP1 binding site in the 5'-flanking region of the mouse TH gene.

DOI： 10.1016/S0006-291X(05)80151-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

We have previously reported the distribution of human tyrosine hydroxylase (TH) transgene expression in dopaminergic neurons (ventral tegmental area and substantia nigra), adrenal gland, and non-catecholaminergic neurons in the forebrain of transgenic (Tg) mice. In this paper, we analysed the transgene expression in catecholaminergic (CAergic) neurons in the lower brainstem of Tg mice, by in situ hybridization and immunocytochemistry at the light and electron microscopic levels. High-level hybridization signals of the human TH mRNA were observed in the locus ceruleus and nucleus tractus solitarii of the Tg brain. Intense TH immunoreactivity was expressed specifically in the Tg brainstem, as was observed in non-Tg mice. These results reveal that the human TH transgene contains the regulatory elements responsible for the expression in three kinds of CAergic (dopaminergic, noradrenergic and adrenergic) neurons of the mouse brain.

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We have recently reported the production of transgenic (Tg) mice carrying the human tyrosine hydroxylase (TH) gene, and have described tissue-specific expression of the transgene in catecholaminergic (CAergic) neurons and adrenal glands. This paper describes the transgene expression in non-catecholaminergic (nCAergic) neurons in the brain of Tg mice by immunocytochemistry and in situ hybridization. In adult Tg mice, human TH was atypically expressed in the olfactory (typically, the anterior olfactory nucleus and pyriform cortex) and visual (typically, n. suprachiasmaticus and n. parabigeminalis) systems, in addition to typical CAergic neuron-rich nuclei in the brain. These results suggest the possibility that TH plays some novel roles in sensory systems.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

The cDNAs for tyrosine hydroxylase were cloned from a mouse brain cDNA library by plaque hybridization. Since the longest cDNA clone lacked approximately 150 bp sequence of its N-terminal region, additional 5' region was obtained using polymerase chain reaction. Nucleotide sequence determination of cDNAs revealed that mouse tyrosine hydroxylase m-RNA encodes 498 amino acids with a calculated molecular mass of 55,990. The amino acid sequence of mouse tyrosine hydroxylase is highly homologous to rat (97%) and human (92%) enzymes.

DOI： 10.1016/0006-291X(91)90472-J

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH)gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：JAPAN SCIENTIFIC SOC PRESS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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Publishing type：Research paper (scientific journal)   Publisher：American Physiological Society  

The purpose of this study was to provide new evidence favoring the hypothesis that cardiovascular information from arterial baroreceptors is integrated with the nigrostriatal system that contributes to regulation of motor activity. Samples of extracellular striatal dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were collected by the technique of in vivo microdialysis and analyzed by high-performance liquid chromatography-electron capture detection. Rats were prepared with a guide tube placed in the caudate-putamen for subsequent insertion of microdialysis probes. During the 1st wk after sinoaortic denervation (SAD) or sham operation (SO), a microdialysis probe was inserted and perfused with Ringer solution at the rate of 2 microliter/min in the freely moving rats. Samples were collected every 20 min before and after injection of pargyline, 100 mg/kg ip. The results showed that SAD rats have approximately 50% less extracellular striatal DA, DOPAC, and HVA than SO rats (P less than 0.01). After blockade of monoamine oxidase activity with pargyline, striatal DA accumulated three times faster in SO than SAD rats suggesting DA synthesis is reduced in SAD rats. These data provide further evidence that the arterial baroreceptor system affects dopaminergic metabolism in the nigrostriatal system possibly as a means for integration of cardiovascular and motor activity.

DOI： 10.1152/ajpregu.1988.254.2.r396

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

The purpose of this study was to provide new evidence favoring the hypothesis that cardiovascular information from arterial baroreceptors is integrated with the nigrostriatal system that contributes to regulation of motor activity. Samples of extracellular striatal dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were collected by the technique of in vivo microdialysis and analyzed by high-performance liquid chromatography-electron capture detection. Rats were prepared with a guide tube placed in the caudate-putamen for subsequent insertion of microdialysis probes. During the 1st wk after sinoaortic denervation (SAD) or sham operation (SO), a microdialysis probe was inserted and perfused with Ringer solution at the rate of 2 microliter/min in the freely moving rats. Samples were collected every 20 min before and after injection of pargyline, 100 mg/kg ip. The results showed that SAD rats have approximately 50% less extracellular striatal DA, DOPAC, and HVA than SO rats (P less than 0.01). After blockade of monoamine oxidase activity with pargyline, striatal DA accumulated three times faster in SO than SAD rats suggesting DA synthesi

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CAMBRIDGE UNIV PRESS  

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Language：Japanese  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2011.07.371

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2010.07.2219

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2010.07.1652

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2009.09.284

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SPRINGER TOKYO  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

DOI： 10.1016/j.nmd.2006.05.197

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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The sarcoglycan complex is composed of four membrane-spanning dystrophin-associated proteins (DAPs) and is essential for skeletal muscle survival, since the absence or markedly reduced expression of this complex due to mutation of any one of the sarcoglycan genes causes a group of muscular dystrophies, collectively termed sarcoglycanopathy. Although one of the putative functions of the sarcoglycan complex is its participation in signaling processes, detailed studies have been scarce. Very recently, it was shown that gene knockout mice for a DAP, α-dystrobrevin, exhibit a dystrophic phenotype, possibly due to defects in muscle cell signaling. To clarify the putative function of the sarcoglycan complex, it is essential to determine whether or not there is a link between it and the intracellular signaling molecules. To elucidate this, we developed new methods for preparing various DAP complexes containing the sarcoglycan complex from the purified dystrophin-DAP complex. It was suggested from one of the complexes prepared that the sarcoglycan-sarcospan complex (the sarcoglycan complex associated with sarcospan) is associated with syntrophin and/or dystrobrevin. Further analysis of this complex revealed that the N-terminal half of dystrobrevin participates in this association. It is thus considered that the sarcoglycan-sarcospan complex is linked to the signaling protein neuronal nitric oxide synthase via α-syntrophin associated with dystrobrevin.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：UNIV CHICAGO PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：UNIV CHICAGO PRESS  

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Language：Japanese  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-LISS  

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