Neuroprotective and neuroregenerative effects of Bone morphogenetic proteins

1.骨形态发生蛋白-7减轻大剂量甲基苯丙胺的毒性： 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。我们先前已经证明，骨形态发生蛋白7(BMP7)可以减少帕金森病啮齿动物模型中6-羟基多巴胺V介导的神经退行性变。在这项研究中，我们研究了BMP7对MA介导的多巴胺能神经元毒性的神经保护作用。从大鼠胚胎腹侧中脑组织中分离出原代多巴胺能神经元，用MA处理。大剂量MA可降低酪氨酸羟化酶免疫反应性(THIR)，增加TUNEL标记。BMP7可显著拮抗这些毒性。首次在CD1小鼠体内研究了BMP7与MA的相互作用。高剂量MA(10 mg/kg x 4 s.c.)显著降低纹状体的运动活性和THIR。脑室内注射BMP7可拮抗上述变化。在BMP7+/-小鼠中，MA比野生型BMP7+/+小鼠更大程度地抑制黑质网状体内的运动活性和降低TH免疫反应性，这表明BMP7表达缺失增加了对MA侮辱的易感性。由于BMP7+/-小鼠在BMP7基因座上也携带LacZ表达的报告等位基因，因此通过-半乳糖苷酶(-GAL)的酶活性间接检测BMP7+/-小鼠BMP7的表达。大剂量MA可显著抑制纹状体GAL活性，提示MA可能抑制黑质纹状体通路终末BMP7的表达。综上所述，我们的数据表明，MA可以引起黑质纹状体多巴胺能终末的损害，BMP7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。本研究的目的是利用BMPRII显性阴性(BMPRIIDN)小鼠，研究BMP受体II(BMPRII)介导的神经营养和保护作用。成年雄性BMPRIIDN小鼠及其野生型对照组(WT)在活动室中连续3天监测运动活动。处死动物进行酪氨酸羟化酶(TH)免疫组织化学染色。为检测BMPIIDN和WT小鼠对多巴胺能神经毒素的敏感性，BMPIIDN和WT小鼠分别注射大剂量甲基苯丙胺(10 mg/kg，sc，X4)或生理盐水，于注射后4d处死动物，进行TUNEL组织化学染色。我们发现BMPRIIDN小鼠的运动能力低于WT小鼠。BMPRIIDN小鼠黑质致密带内TH神经元数量、黑质网状体内TH纤维密度和纹状体TH免疫反应性均显著降低，提示内源性BMP信号转导通路的缺失降低了黑质纹状体通路中的多巴胺能神经支配和运动功能。给予甲基苯丙胺可增加BMPRIIDN小鼠黑质的TUNEL标记。综上所述，我们的数据提示内源性BMPs对黑质纹状体多巴胺能神经元有营养作用。BMP信号的缺失增加了对高剂量甲基苯丙胺造成的侮辱的易感性。 3.Bmp7可减轻甲基苯丙胺与脑缺血的协同损伤作用： 已有研究表明，甲基苯丙胺(MA)可加重脑缺血所致的神经变性。我们和其他人已经报道，BMP7对MA和缺血性脑损伤具有保护作用。本研究的目的是探讨BMP7是否能减轻MA和脑缺血所致的协同损伤。成年CD-1小鼠分别给予MA(10 mg/kg×4，每次间隔2小时)或生理盐水。应用实时定量聚合酶链式反应发现，MA在注射1d后抑制大脑皮层BMP7mRNA的表达。在MA注射后，结扎右侧大脑中动脉90min，造成缺血和再灌注损伤。分别于再灌注1h和2d处死动物，进行caspase3/7活性测定和氯化三苯四氮唑染色。脑梗塞和caspase-3/7活性在MA预处理的卒中动物中增强；BMP7的预处理减弱了这两种反应。综上所述，我们的数据提示MA促进脑缺血后的脑梗塞，可能部分是通过抑制BMP7来实现的。 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，对过氧化氢或缺氧缺糖引起的肾小球系膜细胞和PC12细胞损伤具有保护作用。在培养的人骨肉瘤细胞中，RA增强BMP7的表达，BMP7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。本研究的目的是探讨RA是否通过BMP7机制减轻缺血性脑损伤。我们发现，侧脑室注射9-顺式维甲酸(9cRA)后24小时，大鼠大脑皮层BMP7基因表达增强。9cRA注射后1d，结扎大脑中动脉(MCA)造成大鼠短暂性局灶性脑缺血模型。在大脑中动脉结扎后2天，9cRA可增加大鼠的运动能力，减轻神经功能缺失。9cRA还可减少脑梗塞和TUNEL标记。注射9cRA后1d给予BMP拮抗剂noggin可拮抗上述保护反应。综上所述，我们的数据提示，9cRA对缺血诱导的损伤具有保护作用，这些作用涉及BMP。

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.