Neuroprotective and neuroregenerative effects of Bone morphogenetic proteins

1. 骨形态发生蛋白-7可降低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。我们之前证明，在帕金森病啮齿动物模型中，用骨形态发生蛋白 7 (BMP7) 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，我们检查了 BMP7 对多巴胺能神经元中 MA 介导的毒性的神经保护作用。用MA处理从大鼠胚胎腹侧中脑组织制备的原代多巴胺能神经元。高剂量的 MA 降低了酪氨酸羟化酶免疫反应性 (THir)，同时增加了 TUNEL 标记。 BMP7 可显着拮抗这些毒性。首先在 CD1 小鼠中检查了 BMP7 和 MA 体内的相互作用。高剂量的 MA（10 mg/kg x 4 s.c.）显着降低纹状体的运动活性和 THir。脑室内注射 BMP7 可以拮抗这些变化。在BMP7/-小鼠中，与野生型BMP7/小鼠相比，MA在更大程度上抑制了黑质网状组织的运动活性并降低了TH免疫反应性，这表明BMP7表达的缺陷增加了对MA损伤的脆弱性。由于BMP7/-小鼠在BMP7基因座上也携带表达LacZ的报告等位基因，因此通过BMP7/-小鼠中β-半乳糖苷酶(-gal)的酶活性间接测量BMP7的表达。 高剂量的MA显着抑制纹状体中的β-gal活性，表明MA可能抑制黑质纹状体通路末端的BMP7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损伤，并且BMP7可以保护中枢多巴胺能神经元免受MA V介导的神经毒性。 2. 骨形态发生蛋白受体 II 显性失活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来发挥其生物学作用。 本研究的目的是使用 BMPRIIDN 小鼠检查 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，对 BMPIIDN 和 WT 小鼠亚组注射高剂量甲基苯丙胺（皮下注射 10 mg/kg，X4）或注射生理盐水，并在注射后 4 天处死进行 TUNEL 组织化学分析。我们发现 BMPRIIDN 小鼠的运动活性低于 WT 小鼠。 BMPRIIDN 小鼠中黑质致密区的 TH 神经元数量、黑质网状区的 TH 纤维密度以及纹状体中的 TH 免疫反应性显着减少，表明内源性 BMP 信号传导的缺陷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。 施用甲基苯丙胺增加了 BMPRIIDN 小鼠黑质中的 TUNEL 标记。总之，我们的数据表明内源性 BMP 对黑质纹状体多巴胺能神经元具有营养作用。 BMP 信号传导缺陷会增加对高剂量甲基苯丙胺引起的损伤的脆弱性。 3. BMP7减少甲基苯丙胺和小鼠脑缺血引起的协同损伤： 先前的研究表明，甲基苯丙胺（MA）会增强脑缺血引起的神经退行性变。我们和其他人已经报道，BMP7 可以预防 MA 和缺血性脑损伤。本研究的目的是检测BMP7是否可以减轻MA和脑缺血引起的协同损伤。成年 CD-1 小鼠用 MA（10 mg/kg x 4，每次剂量间隔两小时）或盐水治疗。利用实时定量聚合酶链反应，我们发现MA在注射一天后抑制了大脑皮层中BMP7 mRNA的表达。 MA 注射后结扎右侧大脑中动脉 90 分钟，造成缺血性和再灌注损伤。分别在再灌注后1小时和2天处死动物进行半胱天冬酶3/7活性测定和氯化三苯基四唑染色。在用MA预处理的中风动物中，脑梗死和caspase-3/7活性增强；这两种反应均因 BMP7 预处理而减弱。总之，我们的数据表明，MA 可能部分通过抑制 BMP7 介导缺血后促进脑梗塞。 4. 9-顺式视黄酸通过骨形态发生蛋白减少啮齿类动物的缺血性脑损伤 视黄酸 (RA) 是维生素 A 的一种生物活性衍生物，对系膜细胞和 PC12 细胞中的 H2O2 或氧糖剥夺引起的损伤具有保护作用。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，通过 RTPCR 检测，脑室内注射 9-顺式视黄酸 (9cRA) 可以增强注射后 24 小时大鼠大脑皮层中 BMP7 mRNA 的表达。 注射9cRA一天后，大鼠还通过结扎大脑中动脉（MCA）诱导短暂性局灶性缺血。 MCA 结扎后 2 天，9cRA 预处理可增加运动活动并减轻神经功能缺损。 9cRA还减少了脑梗塞和TUNEL标记。这些保护性反应被注射 9cRA 一天后给予的 BMP 拮抗剂头蛋白拮抗。综上所述，我们的数据表明 9cRA 对缺血引起的损伤具有保护作用，并且这些作用涉及 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.