Pathogenesis And Treatment Of Neurodegenerative Disease

ETB研究帕金森病多巴胺神经元死亡的发病机制，发现α -突触核蛋白的分布与毒毒碱M1和M3受体的分布密切相关。为了探索该蛋白在胆碱能传递中的可能作用，我们在SH-SY5Y细胞中完成了毒毒碱受体刺激对α -突触核蛋白的影响的评估，SH-SY5Y细胞是一种表达α -突触核蛋白的人多巴胺能细胞系。在基础条件下，α -突触核蛋白在分离的所有亚细胞区室中检测到：质膜、细胞质、细胞核和两个囊泡部分。脂质部分只含有45 kda的α -突触核蛋白低聚物，而细胞质和细胞核部分同时含有α -突触核蛋白低聚物和单体，这表明α -突触核蛋白在生理上是作为脂质结合的低聚物和可溶性单体存在的。巯基苯酚的毒毒碱刺激减少了质膜上的α -突触核蛋白低聚物，同时细胞质部分的低聚物和单体都增加了。低聚物与细胞质中含有未包被的内吞囊泡的轻囊泡部分有关。α -突触核蛋白低聚物对碳醇刺激的易位与配体刺激的毒蕈碱受体内吞作用的时间过程密切相关。这些数据表明，肌碱受体刺激α -突触核蛋白寡聚物从质膜释放，随后与内吞囊泡部分的关联可能有助于肌碱受体内吞作用。可以想象，其功能可能是膜结合磷脂酶D2从α -突触核蛋白抑制中短暂释放，从而使该脂肪酶参与毒蕈碱受体内吞作用。据报道，前列腺素A(1) （PGA1）抑制NF-kappaB激活并诱导热休克蛋白的表达。由于这两种作用都可能具有神经保护作用，PGA1在神经退行性疾病（如帕金森病或亨廷顿病，其中兴奋毒性可能有助于发病）中的治疗潜力在暴露于NMDA受体激动剂喹啉酸（QA）的大鼠纹状体神经元中进行了评估。PGA1灌胃可减弱qa诱导的核小体间DNA断裂。单剂量PGA1对qa诱导的DNA断裂的抑制作用在处理后12 ~ 48 h观察。在QA暴露4小时后给予PGA1也能减弱QA诱导的DNA断裂。PGA1降低了注射QA的纹状体中D1多巴胺受体和GAD(67) mRNA的丢失，提示其减轻了QA引起的神经元丢失。PGA1通过阻断抑制性kappab - α降解，显著抑制qa诱导的NF-kappaB活化。PGA1处理显著增加纹状体70和72 kda热休克蛋白。这些结果表明，PGA1抑制NMDA受体介导的神经元凋亡的机制可能与上调神经保护性热休克蛋白和抑制NF-kappaB激活有关。鉴于其有效的神经保护活性，PGA1可能在治疗某些与兴奋性毒性相关的神经退行性疾病中被证明是有用的。先前的分支研究结果表明，所有标准疗法对纹状体多巴胺受体的间歇性刺激导致大多数晚期帕金森病患者最终失能的运动反应并发症综合征。作为我们持续尝试解决这一问题的一部分，我们进行了两项对照临床研究，以评估一种涉及脂溶性、经皮给药的D2多巴胺受体激动剂N-0923的新方法，早期的ETB研究表明，静脉给药时具有有效的抗帕金森活性。其中一项研究是一项小型住院概念验证评估，包括2周剂量递增阶段，随后是2周最高剂量剂量维持阶段。每个人的左旋多巴剂量都是可行的。中位日左旋多巴需要量下降了71%，“停药”时间显著减少。第二项研究评估了85名帕金森门诊患者的N-0923，这些患者被随机分配到安慰剂组或四剂N-0923中的一剂。在最大允许剂量下，N-0923再次被发现可以显著减少左旋多巴的剂量。在两项研究中，N-0923被认为是安全且耐受性良好的。结果表明透皮N-0923是一种持续治疗帕金森病的有效方法，允许较低的左旋多巴剂量，从而减少间歇性多巴胺能刺激的有害影响，而不会失去抗帕金森病的疗效。除了其治疗效果，N-0923将允许首次测试ETB假设，即从治疗开始持续的多巴胺能刺激将防止运动并发症的出现。根据早期的ETB研究，共表达多巴胺受体的间歇性刺激导致纹状体中棘神经元树突上NMDA受体的致敏与运动并发症综合征的发病机制有关。我们发现NMDA受体拮抗剂可以缓解帕金森病动物模型和帕金森病患者的帕金森症状以及运动并发症。在过去的一年中，完成了对非竞争性NMDA拮抗剂金刚烷胺对左旋多巴诱导的运动障碍的影响的对照评估。患有严重运动波动和峰值剂量运动障碍的帕金森患者接受第一次早晨左旋多巴剂量，随后静脉输注金刚烷胺或安慰剂2小时。静脉注射金刚烷胺可减少约50%的运动障碍，而不影响左旋多巴抗帕金森病的效果。这些结果证实了我们之前的临床前和临床观察，并加强了使用这种或其他抗谷氨酸能药物治疗帕金森病的理由。为了进一步评估这种可能性，对279例左旋多巴治疗的运动波动患者进行了另一种非竞争性谷氨酸受体拮抗剂的安全性和有效性评估。在一项多中心、对照、剂量范围研究中，雷马塞米表现出良好的耐受性，与安慰剂相比，它倾向于延长“起效”时间，提高帕金森病评分。这些发现有助于验证非人类灵长类动物的观察结果，并开发出一种关于谷氨酸拮抗剂赋予帕金森患者最大治疗益处的算法。

ETB studies of the pathogenesis of dopamine neuron death in Parkinson's disease examined the close correspondence between the distribution of alpha-synuclein and that of muscarinic M1 and M3 receptors. To explore a possible role for this protein in cholinergic transmission, we completed an evaluation of the effect of muscarinic receptor stimulation on alpha-synuclein in SH-SY5Y cells, a human dopaminergic cell line that expresses alpha-synuclein. Under basal conditions, alpha-synuclein was detected in all subcellular compartments isolated: plasma membrane, cytoplasm, nucleus, and two vesicle fractions. The lipid fractions contained only a 45-kDa alpha-synuclein oligomer, whereas the cytoplasmic and nuclear fractions contained both the oligomer and the monomer, suggesting alpha-synuclein exists physiologically as a lipid-bound oligomer and a soluble monomer. Muscarinic stimulation by carbachol reduced the alpha-synuclein oligomer in plasma membrane, with a concomitant increase of both the oligomer and the monomer in the cytoplasmic fraction. The oligomer was associated with a light vesicle fraction in cytoplasm that contains uncoated endocytotic vesicles. Translocation of the alpha-synuclein oligomer in response to carbachol stimulation corresponds closely with the time course of ligand-stimulated muscarinic receptor endocytosis. The data suggest that the muscarine receptor stimulated release of the alpha-synuclein oligomer from plasma membrane and its subsequent association with the endocytotic vesicle fraction may contribute to muscarine receptor endocytosis. Conceivably, its function may be a transient release of membrane-bound phospholipase D2 from alpha-synuclein inhibition, thus allowing this lipase to participate in muscarinic receptor endocytosis. Prostaglandin A(1) (PGA1) reportedly inhibits NF-kappaB activation and induces expression of heat shock proteins. Since both these effects could be neuroprotective, the therapeutic potential of PGA1 in neurodegenerative disorders, such as Parkinson's or Huntington's disease, where excitotoxicity may contribute to pathogenesis, was evaluated in rat striatal neurons exposed to the NMDA receptor agonist quinolinic acid (QA). Intrastriatal administration of PGA1 attenuated QA-induced internucleosomal DNA fragmentation. The inhibitory effects of a single dose of PGA1 on QA-induced DNA fragmentation were observed 12 to 48 h after treatment. PGA1 also attenuated QA-induced DNA fragmentation when administered up to 4 h after QA exposure. PGA1 decreased the loss of D1 dopamine receptors and GAD(67) mRNA in QA-injected striatum, suggesting that it reduced the neuronal loss induced by QA. PGA1 significantly inhibited QA-induced NF-kappaB activation by blocking inhibitory kappaB-alpha degradation. PGA1 treatment substantially increased striatal 70- and 72-kDa heat shock protein. These results indicate that PGA1 blunts NMDA receptor-mediated neuronal apoptosis by a mechanism possibly involving the up-regulation of neuroprotective heat shock proteins and inhibition of NF-kappaB activation. In view of its potent neuroprotective activity, PGA1 could prove useful in the treatment of certain neurodegenerative disorders related to excitotoxicity. Previous Branch findings indicated that the motor response complication syndrome that ultimately disables most patients with advanced Parkinson's disease results from the intermittent stimulation of striatal dopamine receptors by all standard therapies. As part of our continuing attempts to remedy this problem, we conducted two controlled clinical studies to evaluate a novel approach involving a lipid-soluble, transdermally administered, D2 dopamine receptor agonist, N-0923, which earlier ETB research had shown to posses potent antiparkinsonian activity when given intravenously. One study was a small, inpatient proof-of-concept evaluation consisting of a 2-week dose escalation phase followed by a 2-week dose maintenance phase at the highest dose. Each individual's levodopa dose was back-titrated as feasible. The median daily levodopa dose requirement declined by 71% in association with a significant reduction in "off" time. The second study evaluated N-0923 in 85 parkinsonian outpatients randomized to placebo or one of four doses of N-0923. At the maximum allowable dose, N-0923 co-administration again was found to permit a significant reduction in levodopa dosage. N-0923 was deemed safe and well tolerated in both studies. The results suggest that transdermal N-0923 is an effective approach to the continuous treatment of Parkinson's disease, allowing a lower levodopa dosage and thus a reduction in the harmful effects of intermittent dopaminergic stimulation without loss of antiparkinsonian efficacy. In addition to its therapeutic benefits, N-0923 will allow the first test of the ETB hypothesis that continuous dopaminergic stimulation from the onset of therapy will prevent the appearance of motor complications. Based on earlier ETB studies, sensitization of NMDA receptors on dendrites of striatal medium spiny neurons as a consequence of the intermittent stimulation of coexpressed dopamine receptors has been implicated in the pathogenesis of the motor complication syndrome. We have found that NMDA receptor antagonists can palliate parkinsonian signs as well as motor complication in animal models and patients with Parkinson's disease. During the past year controlled evaluations of the effects of the noncompetitive NMDA antagonist, amantadine, on levodopa-induced dyskinesias were completed. Parkinsonian patients with severe motor fluctuations and peak dose dyskinesias received their first morning levodopa dose, followed by a 2-hour intravenous infusion of amantadine or placebo. Intravenous amantadine reduced dyskinesias by about 50% without compromising the anti-parkinsonian benefis of levodopa. These results confirm our previous preclinical and clinical observations and strengthen the rationale for using this or other antiglutamatergic drugs in the treatment of Parkinson's disease. In a further evaluation of this possibility, an assessment of the safety and efficacy of another non-competitive glutamate receptor antagonist was completed in 279 levodopa treated patients with motor fluctuations. Remacemide, in a multicenter, controlled, dose-ranging study, appeared well tolerated and tended to prolong "on" time and improve parkinsonian scores compared with placebo. These findings help validate observations in non-human primates and develop an algorithm regarding which glutamate antagonists confer the greatest therapeutic benefit to parkinsonian patients.