PDF(Pubmed)
Abstract：
UNASSIGNED: Loss of proteasome function, proteinopathy, and proteotoxicity may cause neurodegeneration across the human lifespan in several forms of brain injury and disease. Drugs that activate brain proteasomes in vivo could thus have a broad therapeutic impact in neurology.
UNASSIGNED: Using pigs, a clinically relevant large animal with a functionally compartmental gyrencephalic cerebral cortex, we evaluated the localization and biochemical activity of brain proteasomes and tested the ability of small molecules to activate brain proteasomes.
UNASSIGNED: By Western blotting, proteasome protein subunit PSMB5 and PSMA3 levels were similar in different pig brain regions. Immunohistochemistry for PSMB5 showed localization in the cytoplasm (diffuse and particulate) and nucleus (cytoplasm < nucleus). Some PSMB5 immunoreactivity was colocalized with mitochondrial (voltage-gated anion channel and cyclophilin D) and cell death (Aven) proteins in the neuronal soma and neuropil in the neocortex of pig and human brains. In the nucleus, PSMB5 immunoreactivity was diffuse, particulate, and clustered, including perinucleolar decorations. By fluorogenic assay, proteasome chymotrypsin-like activities (CTL) in crude tissue soluble fractions were generally similar within eight different pig brain regions. Proteasome CTL activity in the hippocampus was correlated with activity in nasal mucosa biopsies. In pilot analyses of subcellular fractions of pig cerebral cortex, proteasome CTL activity was highest in the cytosol and then ~50% lower in nuclear fractions; ~15-20% of total CTL activity was in pure mitochondrial fractions. With in-gel activity assay, 26S-singly and -doubly capped proteasomes were the dominant forms in the pig cerebral cortex. With a novel in situ histochemical activity assay, MG132-inhibitable proteasome CTL activity was localized to the neuropil, as a mosaic, and to cell bodies, nuclei, and centrosome-like perinuclear satellites. In piglets treated intravenously with pyrazolone derivative and chlorpromazine over 24 h, brain proteasome CTL activity was modestly increased.
UNASSIGNED: This study shows that the proteasome in the pig brain has relative regional uniformity, prominent nuclear and perinuclear presence with catalytic activity, a mitochondrial association with activity, 26S-single cap dominance, and indications from small molecule systemic administration of pyrazolone derivative and chlorpromazine that brain proteasome function appears safely activable.
UNASSIGNED: Using pigs, a clinically relevant large animal with a functionally compartmental gyrencephalic cerebral cortex, we evaluated the localization and biochemical activity of brain proteasomes and tested the ability of small molecules to activate brain proteasomes.
UNASSIGNED: By Western blotting, proteasome protein subunit PSMB5 and PSMA3 levels were similar in different pig brain regions. Immunohistochemistry for PSMB5 showed localization in the cytoplasm (diffuse and particulate) and nucleus (cytoplasm < nucleus). Some PSMB5 immunoreactivity was colocalized with mitochondrial (voltage-gated anion channel and cyclophilin D) and cell death (Aven) proteins in the neuronal soma and neuropil in the neocortex of pig and human brains. In the nucleus, PSMB5 immunoreactivity was diffuse, particulate, and clustered, including perinucleolar decorations. By fluorogenic assay, proteasome chymotrypsin-like activities (CTL) in crude tissue soluble fractions were generally similar within eight different pig brain regions. Proteasome CTL activity in the hippocampus was correlated with activity in nasal mucosa biopsies. In pilot analyses of subcellular fractions of pig cerebral cortex, proteasome CTL activity was highest in the cytosol and then ~50% lower in nuclear fractions; ~15-20% of total CTL activity was in pure mitochondrial fractions. With in-gel activity assay, 26S-singly and -doubly capped proteasomes were the dominant forms in the pig cerebral cortex. With a novel in situ histochemical activity assay, MG132-inhibitable proteasome CTL activity was localized to the neuropil, as a mosaic, and to cell bodies, nuclei, and centrosome-like perinuclear satellites. In piglets treated intravenously with pyrazolone derivative and chlorpromazine over 24 h, brain proteasome CTL activity was modestly increased.
UNASSIGNED: This study shows that the proteasome in the pig brain has relative regional uniformity, prominent nuclear and perinuclear presence with catalytic activity, a mitochondrial association with activity, 26S-single cap dominance, and indications from small molecule systemic administration of pyrazolone derivative and chlorpromazine that brain proteasome function appears safely activable.
摘要：
■蛋白酶体功能丧失，蛋白质病，和蛋白毒性可能导致神经变性在人类的整个生命周期中的几种形式的脑损伤和疾病。因此，体内激活脑蛋白酶体的药物可能在神经病学中具有广泛的治疗作用。
■使用猪，一种临床相关的大型动物，具有功能房室性脑皮层，我们评估了脑蛋白酶体的定位和生化活性，并测试了小分子激活脑蛋白酶体的能力。
■通过蛋白质印迹，蛋白酶体蛋白亚基PSMB5和PSMA3水平在不同猪脑区域相似。PSMB5的免疫组织化学显示位于细胞质(弥散和颗粒)和细胞核(细胞质<细胞核)中。某些PSMB5免疫反应性与线粒体（电压门控阴离子通道和亲环素D）和细胞死亡（Aven）蛋白共定位在猪和人脑新皮层的神经元体细胞和神经纤维中。在细胞核中，PSMB5免疫反应性弥漫性，颗粒，并聚集，包括核仁周围的装饰。通过荧光分析，在八个不同的猪脑区域中，粗组织可溶性部分中的蛋白酶体胰凝乳蛋白酶样活性（CTL）通常相似。海马中的蛋白酶体CTL活性与鼻粘膜活检中的活性相关。在猪大脑皮层亚细胞部分的初步分析中，蛋白酶体CTL活性在细胞质中最高，然后在核部分中降低约50％；约15-20％的总CTL活性在纯线粒体部分中。用凝胶内活性测定，26S-单端和双端蛋白酶体是猪大脑皮层中的主要形式。通过一种新颖的原位组织化学活性测定，MG132抑制蛋白酶体CTL活性定位于神经纤维，作为马赛克，和细胞体，原子核,和类似中心体的核周卫星。在24小时内用吡唑啉酮衍生物和氯丙嗪静脉注射的仔猪中，脑蛋白酶体CTL活性适度增加。
■这项研究表明，猪脑中的蛋白酶体具有相对的区域均匀性，具有催化活性的突出的核和核周存在，线粒体与活动的联系，26S-单上限优势，和适应症从小分子全身给药吡唑啉酮衍生物和氯丙嗪脑蛋白酶体功能似乎安全激活。
■使用猪，一种临床相关的大型动物，具有功能房室性脑皮层，我们评估了脑蛋白酶体的定位和生化活性，并测试了小分子激活脑蛋白酶体的能力。
■通过蛋白质印迹，蛋白酶体蛋白亚基PSMB5和PSMA3水平在不同猪脑区域相似。PSMB5的免疫组织化学显示位于细胞质(弥散和颗粒)和细胞核(细胞质<细胞核)中。某些PSMB5免疫反应性与线粒体（电压门控阴离子通道和亲环素D）和细胞死亡（Aven）蛋白共定位在猪和人脑新皮层的神经元体细胞和神经纤维中。在细胞核中，PSMB5免疫反应性弥漫性，颗粒，并聚集，包括核仁周围的装饰。通过荧光分析，在八个不同的猪脑区域中，粗组织可溶性部分中的蛋白酶体胰凝乳蛋白酶样活性（CTL）通常相似。海马中的蛋白酶体CTL活性与鼻粘膜活检中的活性相关。在猪大脑皮层亚细胞部分的初步分析中，蛋白酶体CTL活性在细胞质中最高，然后在核部分中降低约50％；约15-20％的总CTL活性在纯线粒体部分中。用凝胶内活性测定，26S-单端和双端蛋白酶体是猪大脑皮层中的主要形式。通过一种新颖的原位组织化学活性测定，MG132抑制蛋白酶体CTL活性定位于神经纤维，作为马赛克，和细胞体，原子核,和类似中心体的核周卫星。在24小时内用吡唑啉酮衍生物和氯丙嗪静脉注射的仔猪中，脑蛋白酶体CTL活性适度增加。
■这项研究表明，猪脑中的蛋白酶体具有相对的区域均匀性，具有催化活性的突出的核和核周存在，线粒体与活动的联系，26S-单上限优势，和适应症从小分子全身给药吡唑啉酮衍生物和氯丙嗪脑蛋白酶体功能似乎安全激活。
