Neuroprotective and neuroregenerative effects of Bone morphogenetic proteins

骨形态发生蛋白的神经保护和神经再生作用

• 批准号: 7966793
• 负责人: Yun Wang
• 金额: $ 56.59万
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7966793
• 关键词: Adult; Alleles; Animals; Attenuated; Binding; Biological; Biological Assay; Blood Vessels; Bone Morphogenetic Proteins; Brain; Cells; Cerebral Infarction; Cerebral Ischemia; Cerebral cortex; Chloride Ion; Chlorides; Corpus striatum structure; Data; Dominant-Negative Mutation; Dose; Embryo; Event; Fiber; Galactosidase; Glucose; Histocytochemistry; Hour; Human; Hydrogen Peroxide; Impaired cognition; In Situ Nick-End Labeling; Injection of therapeutic agent; Injury; Intraventricular; Ischemia; Ischemic Brain Injury; Label; LacZ Genes; Lesion; Ligation; Measures; Mediating; Messenger RNA; Methamphetamine; Monitor; Motor; Motor Activity; Mus; Nerve Degeneration; Neuraxis; Neurologic; Neurons; Neurotoxins; Oxidopamine; Oxygen; PC12 Cells; Parkinson Disease; Polymerase Chain Reaction; Rattus; Receptor Serine/Threonine Kinase; Reperfusion Therapy; Reporter; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Rodent Model; Role; Saline; Signal Transduction; Staining method; Stains; Stroke; Subgroup; Testing; Tetrazolium; Time; Tissues; Toxic effect; Treatment Factor; Tretinoin; Tyrosine 3-Monooxygenase; Vitamin A; alitretinoin; bone morphogenetic protein 7; bone morphogenetic protein receptor type II; bone morphogenetic protein receptors; caspase-3; density; deprivation; dopaminergic neuron; drug of abuse; immunoreactivity; in vivo; mRNA Expression; male; middle cerebral artery; nerve supply; neurotoxicity; nigrostriatal pathway; osteosarcoma; protective effect; response

1. Bone morphogenetic protein-7 reduces toxicity induced by high doses of methamphetamine: Methamphetamine (MA) is a drug of abuse as well as a dopaminergic neurotoxin. We previously demonstrated that pretreatment with bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine Vmediated neurodegeneration in a rodent model of Parkinsons disease. In this study, we examined the neuroprotective effects of BMP7 against MA-mediated toxicity in dopaminergic neurons. Primary dopaminergic neurons, prepared from rat embryonic ventral mesencephalic tissue, were treated with MA. High doses of MA decreased tyrosine hydroxylase immunoreactivity (THir) while increasing TUNEL labeling. These toxicities were significantly antagonized by BMP7. Interaction of BMP7 and MA in vivo was first examined in CD1 mice. High doses of MA (10 mg/kg x 4 s.c.) significantly reduced locomotor activity and THir in striatum. Intra-cerebroventricular administration of BMP7 antagonized these changes. In BMP7 +/- mice, MA suppressed locomotor activity and reduced TH immunoreactivity in nigra reticulata to a greater degree than in wild type BMP7 +/+ mice, suggesting that deficiency in BMP7 expression increases vulnerability to MA insults. Since BMP7 +/- mice also carry a LacZ-expressing reporter allele at the BMP7 locus, the expression of BMP7 was indirectly measured through the enzymatic activity of -galactosidase (-gal) in BMP7 +/- mice. High doses of MA significantly suppressed -gal activity in striatum, suggesting that MA may inhibit BMP7 expression at the terminals of nigrostriatal pathway. In conclusion, our data indicate that MA can cause lesioning in the nigrostriatal dopaminergic terminals and that BMP7 is protective against MA Vmediated neurotoxicity in central dopaminergic neurons. 2. Nigrostriatal Alterations in Bone morphogenetic protein Receptor II dominant negative mice: BMPs exert their biological effects by binding to type I and type II serine-threonine kinase receptors. The purpose of this study is to examine the neurotrophic and protective roles mediated through BMP Receptor II (BMPRII) using the BMPRII dominant negative (BMPRIIDN) mice. Adult male BMPRIIDN mice and their wild type controls (WT) were placed in the activity chambers for 3 days to monitor locomotor activity. Animals were sacrificed for tyrosine hydroxylase (TH) immunostaining. To examine the sensitivity to dopaminergic neurotoxin, a subgroup of BMPIIDN and WT mice were injected with high doses of methamphetamine (10 mg/kg s.c., X4) or saline injection and were sacrificed for TUNEL histochemistry at 4 days after injection. We found that BMPRIIDN mice had lower locomotor activity than the WT. There is a significant decrease in TH neuronal number in substantial nigra compacta, TH fiber density in the substantial nigra reticulata, and TH immunoreactivity in striatum in the BMPRIIDN mice, suggesting that deficiency in endogenous BMP signaling reduces dopaminergic innervation and motor function in the nigrostriatal pathway. Administration of methamphetamine increased TUNEL labeling in nigra in the BMPRIIDN mice. In conclusion, our data suggest that endogenous BMPs have trophic effects on nigrostriatal dopaminergic neurons. Deficiency in BMP signaling increases vulnerability to insults induced by high doses of methamphetamine. 3. BMP7 reduces synergistic injury induced by methamphetamine and ischemia in mouse brain: Previous studies have indicated that methamphetamine (MA) potentiates neurodegeneration induced by ischemia in brain. We, and others, have reported that BMP7 is protective against MA and ischemic brain injury. The purpose of this study is to examine whether BMP7 reduces synergistic injury induced by both MA and cerebral ischemia. Adult CD-1 mice were treated with MA (10 mg/kg x 4, each dose two hours apart) or saline. Using the quantitative real time polymerase chain reaction, we found that MA suppressed the expression of BMP7 mRNA in the cerebral cortex one day after injection. Ischemic and reperfusional injuries were introduced by ligation of the right middle cerebral artery for 90 min after MA injection. Animals were sacrificed for caspase 3/7 activity assay and tri-phenyl-tetrazolium chloride staining at 1 hour and 2 days after reperfusion, respectively. Cerebral infarction and caspase-3/7 activity were enhanced in the stroke animals pretreated with MA; both responses were attenuated by pretreatment with BMP7. In conclusion, our data suggest that MA facilitates cerebral infarction after ischemia possibly mediated, in part, through the suppression of BMP7. 4. 9-Cis-Retinoic acid reduces ischemic brain injury in rodents via bone morphogenetic protein Retinoic acid (RA), a biologically active derivative of vitamin A, has protective effects against damage caused by H2O2 or oxygen-glucose deprivation in mesangial and PC12 cells. In cultured human osteosarcoma cells, RA enhances the expression of BMP7, a trophic factor that reduces ischemia- or neurotoxin Vmediated neurodegeneration in vivo. The purpose of this study is to examine whether RA reduces ischemic brain injury through a BMP7 mechanism. We found that intracerebroventricular administration of 9-cis-retinoic acid (9cRA) enhanced BMP7 mRNA expression, detected by RTPCR, in rat cerebral cortex at 24 hours after injection. Rats were also subjected to transient focal ischemia induced by ligation of the middle cerebral artery (MCA) at one day after 9cRA injection. Pretreatment with 9cRA increased locomotor activity and attenuated neurological deficits 2 days after MCA ligation. 9cRA also reduced cerebral infarction and TUNEL labeling. These protective responses were antagonized by BMP antagonist noggin given at one day after 9cRA injection. Taken together, our data suggest that 9cRA has protective effects against ischemia -induced injury and these effects involve BMPs.

1.骨形态发生蛋白-7减少高剂量甲基苯丙胺引起的毒性： 甲基苯丙胺（MA）是一种滥用药物，也是一种多巴胺能神经毒素。我们先前证明，在帕金森病啮齿动物模型中，骨形态发生蛋白7（BMP 7）预处理可减少6-羟基多巴胺V介导的神经变性。在这项研究中，我们研究了BMP 7对MA介导的多巴胺能神经元毒性的神经保护作用。原代多巴胺能神经元，从大鼠胚胎腹侧中脑组织制备，用MA处理。高剂量的MA降低酪氨酸羟化酶免疫反应性（THir），同时增加TUNEL标记。这些毒性被BMP 7显著拮抗。首先在CD 1小鼠中检查BMP 7和MA的体内相互作用。高剂量MA（10 mg/kg x 4 s.c.）显著降低纹状体中的运动活性和THir。脑室内注射BMP 7可拮抗这些变化。在BMP 7 +/-小鼠中，MA抑制运动活性，并降低网状黑质中TH免疫反应性的程度大于野生型BMP 7 +/+小鼠，表明BMP 7表达的缺陷增加了对MA损伤的脆弱性。由于BMP 7 +/-小鼠在BMP 7基因座处也携带LacZ表达报告基因等位基因，因此通过BMP 7 +/-小鼠中β-半乳糖苷酶（β-gal）的酶活性间接测量BMP 7的表达。 高剂量的MA显著抑制纹状体的-gal活性，表明MA可能抑制黑质纹状体通路末端的BMP 7表达。 总之，我们的数据表明，MA可以引起黑质纹状体多巴胺能末梢的病变，BMP 7对MA V介导的中枢多巴胺能神经元的神经毒性具有保护作用。 2.骨形态发生蛋白受体II显性阴性小鼠的黑质纹状体改变： BMP通过与I型和II型丝氨酸-苏氨酸激酶受体结合发挥其生物学效应。 本研究的目的是使用BMPRII显性阴性（BMPRIIDN）小鼠来检查通过BMP受体II（BMPRII）介导的神经营养和保护作用。将成年雄性BMPRIIDN小鼠及其野生型对照（WT）置于活动室中3天以监测运动活动。处死动物进行酪氨酸羟化酶（TH）免疫染色。 为了检查对多巴胺能神经毒素的敏感性，给BMPIIDN和WT小鼠的亚组注射高剂量的甲基苯丙胺（10 mg/kg s.c.，X4）或盐水注射，并在注射后4天处死用于TUNEL组织化学。我们发现BMPRIIDN小鼠的运动活性低于WT。在BMPRIIDN小鼠中，黑质中TH神经元数量、黑质网状中TH纤维密度和纹状体中TH免疫反应性显著减少，表明内源性BMP信号传导的缺乏减少了黑质纹状体通路中的多巴胺能神经支配和运动功能。 在BMPRIIDN小鼠中，甲基苯丙胺的给药增加了黑质中的TUNEL标记。总之，我们的数据表明，内源性骨形成蛋白对黑质纹状体多巴胺能神经元有营养作用。 BMP信号传导的缺陷增加了对高剂量甲基苯丙胺诱导的损伤的脆弱性。 3. BMP 7减少小鼠脑中甲基苯丙胺和缺血诱导的协同损伤： 以往的研究表明，甲基苯丙胺（MA）增强脑缺血引起的神经退行性变。我们和其他人已经报道了BMP 7对MA和缺血性脑损伤具有保护作用。本研究的目的是检查BMP 7是否减少MA和脑缺血诱导的协同损伤。成年CD-1小鼠用MA（10 mg/kg x 4，每次给药间隔2小时）或盐水处理。应用定量真实的时间聚合酶链反应（RT-PCR）技术，发现MA在注射后第1天抑制了大脑皮层BMP 7 mRNA的表达。MA注射后，通过结扎右侧大脑中动脉90 min造成缺血和再灌注损伤。分别于再灌注后1h和2d处死动物进行caspase 3/7活性测定和氯化三苯基四氮唑染色。脑梗死和caspase-3/7的活性增强中风动物预处理MA，这两个反应减弱预处理BMP 7。总之，我们的数据表明，MA促进脑梗死缺血后可能介导的，部分通过抑制BMP 7。 4. 9-顺式维甲酸通过骨形态发生蛋白减轻啮齿动物缺血性脑损伤 维甲酸（RA）是维生素A的生物活性衍生物，对H2 O2或氧糖剥夺引起的系膜细胞和PC 12细胞损伤具有保护作用。在培养的人骨肉瘤细胞中，RA增强BMP 7的表达，BMP 7是一种营养因子，可减少体内缺血或神经毒素V介导的神经变性。本研究的目的是研究RA是否通过BMP 7机制减少缺血性脑损伤。我们发现，侧脑室注射9-顺式维甲酸（9 cRA）后24小时，通过RTPCR检测到大鼠大脑皮质中BMP 7 mRNA的表达增加。 在注射9 cRA后1天，大鼠还经受通过结扎大脑中动脉（MCA）诱导的短暂局灶性缺血。 用9 cRA预处理增加MCA结扎后2天的自发活动和减弱的神经功能缺损。9 cRA还可减少脑梗死和TUNEL标记。在注射9 cRA后1天给予BMP拮抗剂noggin可拮抗这些保护作用。综上所述，我们的数据表明，9 cRA对缺血诱导的损伤具有保护作用，并且这些作用涉及BMP。