Showing papers by "Shin-ichi Muramatsu published in 2016"

An miRNA-mediated therapy for SCA6 blocks IRES-driven translation of the CACNA1A second cistron

Abstract: Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease characterized by slowly progressive ataxia and Purkinje cell degeneration. SCA6 is caused by a polyglutamine repeat expansion within a second CACNA1A gene product, α1ACT. α1ACT expression is under the control of an internal ribosomal entry site (IRES) present within the CACNA1A coding region. Whereas SCA6 allele knock-in mice show indistinguishable phenotypes from wild-type littermates, expression of SCA6-associated α1ACT (α1ACTSCA6) driven by a Purkinje cell-specific promoter in mice produces slowly progressive ataxia and cerebellar atrophy. We developed an early-onset SCA6 mouse model using an adeno-associated virus (AAV)-based gene delivery system to ectopically express CACNA1A IRES-driven α1ACTSCA6 to test the potential of CACNA1A IRES-targeting therapies. Mice expressing AAV9-mediated CACNA1A IRES-driven α1ACTSCA6 exhibited early-onset ataxia, motor deficits, and Purkinje cell degeneration. We identified miR-3191-5p as a microRNA (miRNA) that targeted CACNA1A IRES and preferentially inhibited the CACNA1A IRES-driven translation of α1ACT in an Argonaute 4 (Ago4)-dependent manner. We found that eukaryotic initiation factors (eIFs), eIF4AII and eIF4GII, interacted with the CACNA1A IRES to enhance α1ACT translation. Ago4-bound miR-3191-5p blocked the interaction of eIF4AII and eIF4GII with the CACNA1A IRES, attenuating IRES-driven α1ACT translation. Furthermore, AAV9-mediated delivery of miR-3191-5p protected mice from the ataxia, motor deficits, and Purkinje cell degeneration caused by CACNA1A IRES-driven α1ACTSCA6 We have established proof of principle that viral delivery of an miRNA can rescue a disease phenotype through modulation of cellular IRES activity in a mouse model.

Freezing of Gait in Parkinson’s Disease Is Associated with Reduced 6-[18F]Fluoro-L-m-tyrosine Uptake in the Locus Coeruleus

Abstract: Freezing of gait (FOG) is a common disorder in Parkinson's disease (PD) and could be attributed to a reduction in brain noradrenaline. The aim of this study was to determine the relationship between aromatic l-amino acid decarboxylase (AADC) activity in the locus coeruleus (LC) and FOG in PD using high-resolution positron emission tomography with an AADC tracer, 6-[(18)F]fluoro-l-m-tyrosine (FMT). We assessed 40 patients with PD and 11 age-matched healthy individuals. PD was diagnosed based on the UK Brain Bank criteria by two movement disorder experts. FOG was directly observed by the clinician and assessed using a patient questionnaire. FMT uptake in the LC, caudate, and putamen was analyzed using PMOD software on coregistered magnetic resonance images. FOG was present in 30 patients. The severity of FOG correlated with the decrease of FMT uptake in the LC regardless of disease duration and the severity of other motor impairments, indicating dysfunction of the noradrenergic network in FOG.

A novel reporter rat strain that conditionally expresses the bright red fluorescent protein tdTomato

Abstract: Despite the strength of the Cre/loxP recombination system in animal models, its application in rats trails that in mice because of the lack of relevant reporter strains. Here, we generated a floxed STOP tdTomato rat that conditionally expresses a red fluorescent protein variant (tdTomato) in the presence of exogenous Cre recombinase. The tdTomato signal vividly visualizes neurons including their projection fibers and spines without any histological enhancement. In addition, a transgenic rat line (FLAME) that ubiquitously expresses tdTomato was successfully established by injecting intracytoplasmic Cre mRNA into fertilized ova. Our rat reporter system will facilitate connectome studies as well as the visualization of the fine structures of genetically identified cells for long periods both in vivo and ex vivo. Furthermore, FLAME is an ideal model for organ transplantation research owing to improved traceability of cells/tissues.

RpA1 ameliorates symptoms of mutant ataxin-1 knock-in mice and enhances DNA damage repair

Abstract: DNA damage and repair is a critical domain of many neurodegenerative diseases. In this study, we focused on RpA1, a candidate key molecule in polyQ disease pathologies, and tested the therapeutic effect of adeno-associated virus (AAV) vector expressing RpA1 on mutant Ataxin-1 knock-in (Atxn1-KI) mice. We found significant effects on motor functions, normalized DNA damage markers (γH2AX and 53BP1), and improved Purkinje cell morphology; effects that lasted for 50 weeks following AAV-RpA1 infection. In addition, we confirmed that AAV-RpA1 indirectly recovered multiple cellular functions such as RNA splicing, transcription and cell cycle as well as abnormal morphology of dendrite and dendritic spine of Purkinje cells in Atxn1-KI mice. All these results suggested a possibility of gene therapy with RpA1 for SCA1.

Evaluation of 6-11C-Methyl-m-Tyrosine as a PET Probe for Presynaptic Dopaminergic Activity: A Comparison PET Study with β-11C-l-DOPA and 18F-FDOPA in Parkinson Disease Monkeys

Abstract: 303 Objectives 90Y microspheres are increasingly used for the treatment of inoperable hepatocellular carcinoma (HCC). Currently, the confirmation of 90Y biodistribution after treatment is assessed indirectly by Bremsstrahlung-SPECT, which is known to have poor statistical counting and inaccurate quantification of actual delivered dose to the tumor (TD) and nontumorous liver (NTD). TD and NTD are important prognostic and radiobiological factors in HCC patients; therefore, we aim to evaluate the efficacy of 90Y PET/CT post radioembolization for dosimetry measurement and for quantitative optimization of injected activity (IA) of 90Y glass microspheres individualized to HCC patients. Methods From April 2014 to Oct 2015, 34 patients (M: 28, F: 6; age range: 34~82y, mean=60.1±13.9y; HBV: 24, HCV: 1) treated by 90Y glass microspheres for intermediate or advanced stage HCC were recruited. All patients underwent pretreatment 99mTc-MAA planar and SPECT/CT to estimate the lung shunting (S:% of IA) and tumor-to-nontumorous liver ratio (TNR), respectively. The dose to the injected liver (ILD) was determined by adjustment of the fixed radiation dose (120Gy recommended by pharmaceutical company) according to patient’s tumor extent, liver size and liver function. The IA was then calculated by a simplified MIRD equation: IA(GBq)= ILD(Gy)/[(1-S)×49.67/W(kg)] (W=ILvolume×1.03, where 1.03kg/L is the liver density). Regional 90Y PET/CT (Biograph mCT) was performed 17~22 hours post radioembolization with 15-min acquisition/bed and “time-of-flight” reconstruction (2 iterations, 21 subsets, 10-mm at FWHM, matrix size 200×200). For each patient, right & left nontumorous liver (NL), lung and all HCC lesions were contoured by automatic algorithms on PET/CT with mean activity concentration (Bq/ml) and volume measured for each VOI. They are then corrected for the 90Y positron branching ratio (3.186×10−5) and time decay factors to obtain the actual PET-measured activities (PA) in tumor, NL and lung. The TD, NTD and lung dose were calculated by D(Gy)=PA(GBq)×49.67/W(kg). Partial volume correction was not considered due to voluminous lesions (>6cm) in all patients. The accuracy of 90Y PET/CT for dosimetry measurement was checked by comparing the summation of PA in tumor, NL and lung with the injected activity. Results 33/34 patients received reduced IA with ILD ranging from 40 to 100Gy (median: 60Gy). The IA was 2.45±1.11GBq. 90Y PET/CT post radioembolization showed high TD (>120Gy) in 30/34 patients (mean: 215±72Gy, range: 139~363Gy) and 73~117Gy in the other 4 patients. The NTD in all patients was less than the limiting dose of 70Gy recommended by the literature (mean: 37±11Gy, range: 16~55Gy). The lung shunting on PET/CT was minimal in 33/34 patients (mean: 1.8±1.2%, range: 0.4~4.6%) and mild in 1 patient (14.6%), lower than that on 99mTc-MAA planar (mean: 6.8±3.8%, range: 2.9~15.4%) in all patients. The summation of PA in tumor, NL and lung was comparable to the IA (91.2~99.3% with a median of 96.4% of IA), indicating that quantification by 90Y PET/CT for dosimetry measurement is feasible and accurate. The TNR on pretreatmet 99mTc-MAA SPECT/CT strongly correlated with 90Y PET/CT post radioembolization (mean=5.4±3.0 vs 5.6±3.6, linear curve: TNRY90=1.044×TNRMAA-0.033, P Conclusions PET/CT post 90Y-radioembolization could provide accurate dosimetry measurement and confirmation of biodistribution in HCC patients, thus obviating the conventional use of Bremsstrahlung-SPECT. The TNR predicted by 99mTc-MAA SPECT/CT was accurate but lung shunting in 90Y treatment was overestimated by 99mTc-MAA planar imaging, therefore, the threshold limit might possibly be given greater allowance. Quantitative optimization of IA of 90Y glass microspheres could be individualized and guided by target TD or limiting NTD instead of a routinely fixed ILD.

PM487. Presynaptic protein Piccolo knockdown in the prefrontal cortex induces cognitive and emotional impairment in mice.

Abstract: s | 77 1Chonnam National University, Republic of Korea, 2Orygen Youth Health, Australia, 3Deakin University, Australia, 4Medical University of Vienna, Austria, 5University Hospital Jena, Germany Abstract Object: This study investigated the relationship between erythrocyte membrane fatty acid (FA) levels and the severity of symptoms of individuals at ultra-high risk(UHR) for psychosis. Methods: The study sample consisted of 80 neuroleptic-naïve UHR patients. Associations between baseline erythrocyte membrane FA levels, measured by gas chromatography, and scores on the Positive and Negative Syndrome Scale (PANSS), the Global Assessment of Functioning Scale, and the Montgomery–Asberg Depression Rating Scale (MADRS) were investigated. After correlation analysis in all participants, subjects were divided into three groups according to the predominance of positive or negative symptoms based on PANSS subscale scores; membrane FA levels in the three groups were then compared. Results: PANSS negative symptom scores were negatively correlated with two saturated FAs (myristic and margaric acids), one ω-9 monounsaturated FA (MUFA;nervonic acid), and oneω-3 polyunsaturated FA(PUFA; docosapentaenoic acid). Negative symptom scores were positively correlated with twoω-9 MUFAs(eicosenoic and erucic acids) and two ω-6 PUFAs (γ-linoleic and docosadienoic acids). PANSS positive symptom scores were correlated only with nervonic acid. No associations were observed between FAs and MADRS scores. In subjects with dominant negative symptoms, the sum of the ω-9 MUFAs and the ω-6:ω-3 FA ratio were both significantly higher than in those with dominant positive symptoms, whereas the sum of ω-3 PUFAs was significantly lower. Conclusions: Abnormalities in FA metabolism may contribute to the neurobiology of psychopathology in UHR individuals. In particular, membrane FA alterations may play a role in negative symptoms, which are primary psychopathological manifestations of schizophrenia-related disability.Object: This study investigated the relationship between erythrocyte membrane fatty acid (FA) levels and the severity of symptoms of individuals at ultra-high risk(UHR) for psychosis. Methods: The study sample consisted of 80 neuroleptic-naïve UHR patients. Associations between baseline erythrocyte membrane FA levels, measured by gas chromatography, and scores on the Positive and Negative Syndrome Scale (PANSS), the Global Assessment of Functioning Scale, and the Montgomery–Asberg Depression Rating Scale (MADRS) were investigated. After correlation analysis in all participants, subjects were divided into three groups according to the predominance of positive or negative symptoms based on PANSS subscale scores; membrane FA levels in the three groups were then compared. Results: PANSS negative symptom scores were negatively correlated with two saturated FAs (myristic and margaric acids), one ω-9 monounsaturated FA (MUFA;nervonic acid), and oneω-3 polyunsaturated FA(PUFA; docosapentaenoic acid). Negative symptom scores were positively correlated with twoω-9 MUFAs(eicosenoic and erucic acids) and two ω-6 PUFAs (γ-linoleic and docosadienoic acids). PANSS positive symptom scores were correlated only with nervonic acid. No associations were observed between FAs and MADRS scores. In subjects with dominant negative symptoms, the sum of the ω-9 MUFAs and the ω-6:ω-3 FA ratio were both significantly higher than in those with dominant positive symptoms, whereas the sum of ω-3 PUFAs was significantly lower. Conclusions: Abnormalities in FA metabolism may contribute to the neurobiology of psychopathology in UHR individuals. In particular, membrane FA alterations may play a role in negative symptoms, which are primary psychopathological manifestations of schizophrenia-related disability. PM487 Presynaptic protein Piccolo knockdown in the prefrontal cortex induces cognitive and emotional impairment in mice. Yoshiaki Miyamoto1, Ryo Inagaki1, Keiji Sato1, Shin-ichi Muramatsu2, Toshitaka Nabeshima3, Kyosuke Uno1, Atsumi Nitta1 1Department of Pharmaceutical Therapy and Neuropharmacology, Faculty of Pharmaceutical Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, JAPAN. 2Division of Neurology, Department of Medicine, Jichi Medial University, Shimotsuke 329–0498, JAPAN. 3Nabeshima Laboratory, Meijo University, Nagoya 468–8503, JAPAN. Abstract Piccolo, a protein of the cytomatrix at the active zone, is involved in the exocytosis and endocytosis of the synaptic vesicles at the presynaptic bouton. It is reported recently that the expression level of Piccolo-encoded PCLO mRNA and Piccolo itself changed in the brain of the bipolar disorder and schizophrenia patient. In the present study, therefore, to clarify the neurological function of Piccolo in the development of mental disorders, we generated Piccolo deficient animal by bilaterally microinjecting the adeno-associated virus vector containing PCLO miRNA into the prefrontal cortex of the mouse. In the electrophysiological analysis, the Piccolo knockdown mice were attenuated the paired-pulse facilitation and long-term potentiation in the prefrontal cortex. In the same prefrontal cortex, the phosphorylation level of synaptic vesicle binding protein Synapsin significantly decreased in the Piccolo knockdown mice, but the remarkable abnormalities were not observed on the shape of dendrite and spine and on the expression level of Synaptophysin, NMDAR1 and PSD-95 in the mice. In the behavioral analysis, the Piccolo knockdown mice showed decreased cognitive dysfunction including object recognition, spatial learning and working memory. Furthermore, the locomotor activity in the novel environment increased in the Piccolo knockdown mice, and its hyperlocomotion was improved by the treatment with atypical antipsychotic drug risperidone. Similarly, the impaired prepulse inhibition of the acoustic startle responses in the Piccolo knockdown mice was ameliorated by risperidone. These results suggest that Piccolo in the prefrontal cortex regulates the release of the neurotransmitter from the presynaptic membrane, and it consequently plays a functional role in the synaptic plasticity, cognitive memory and emotional activity. Furthermore, our observations indicate that the mouse with Piccolo deficient in the prefrontal cortex is useful animal model for the mental disorders such as the manic symptom of bipolar disorder or the positive symptom of schizophrenia.Piccolo, a protein of the cytomatrix at the active zone, is involved in the exocytosis and endocytosis of the synaptic vesicles at the presynaptic bouton. It is reported recently that the expression level of Piccolo-encoded PCLO mRNA and Piccolo itself changed in the brain of the bipolar disorder and schizophrenia patient. In the present study, therefore, to clarify the neurological function of Piccolo in the development of mental disorders, we generated Piccolo deficient animal by bilaterally microinjecting the adeno-associated virus vector containing PCLO miRNA into the prefrontal cortex of the mouse. In the electrophysiological analysis, the Piccolo knockdown mice were attenuated the paired-pulse facilitation and long-term potentiation in the prefrontal cortex. In the same prefrontal cortex, the phosphorylation level of synaptic vesicle binding protein Synapsin significantly decreased in the Piccolo knockdown mice, but the remarkable abnormalities were not observed on the shape of dendrite and spine and on the expression level of Synaptophysin, NMDAR1 and PSD-95 in the mice. In the behavioral analysis, the Piccolo knockdown mice showed decreased cognitive dysfunction including object recognition, spatial learning and working memory. Furthermore, the locomotor activity in the novel environment increased in the Piccolo knockdown mice, and its hyperlocomotion was improved by the treatment with atypical antipsychotic drug risperidone. Similarly, the impaired prepulse inhibition of the acoustic startle responses in the Piccolo knockdown mice was ameliorated by risperidone. These results suggest that Piccolo in the prefrontal cortex regulates the release of the neurotransmitter from the presynaptic membrane, and it consequently plays a functional role in the synaptic plasticity, cognitive memory and emotional activity. Furthermore, our observations indicate that the mouse with Piccolo deficient in the prefrontal cortex is useful animal model for the mental disorders such as the manic symptom of bipolar disorder or the positive symptom of schizophrenia. PM488 Activation of Galphaq proteins coupled with dopamine D1-like receptor and alpha1-adrenoceptor in rat brain membranes Yuji Odagaki, Masakazu Kinoshita, Toshio Ota Saitama Medical University, Japan Abstract Objectives: Previously, we reported 5-HT2A receptorand M1 muscarinic acetylcholine receptor (mAChR)-mediated Gαq activation in rat brain membranes (Eur J Pharmacol 726: 109–115, 2014). In this paper, it was noticed that Gαq proteins were also activated by dopamine and (-)-epinephrine, although these effects were much lower than that elicited by 5-HT or carbachol. In the present study, activation of Gαq coupled to dopaminergic and adrenergic receptor was pharmacologically characterized in rat brain membranes. Methods: Receptor-mediated activation of Gαq in rat brain membranes was assessed by guanosine-5’-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation assay (Eur J Pharmacol 726: 109–115, 2014), with minor modifications. Results: In cerebral cortical membranes, dopamine and (-)-epinephrine stimulated specific [35S]GTPγS binding to Gαq in a concentration-dependent manner, with EC50 values of 65 and 0.46 μM, to the maximal percent increase over basal binding of 99 and 70 %, respectively. The responses in hippocampus and striatum were lower than those in cerebral cortex for either compound. Pharmacological characterization using a series of dopaminergic and