Mechanisms of Parkinson Disease and Related Disorders

通过应用一种独特和新颖的神经成像-神经化学方法，我们获得了路易体疾病交感神经元儿茶酚胺囊泡摄取减少的第一个证据(Goldstein等人，J Clin Invest 2011；121：3320-3330)。 我们发现，终末期帕金森病患者的壳核组织在调整了减少的多巴胺能神经支配后，含有增加的DOPAL浓度，以DOPAC/DOPAL比率衡量的ALDH活性降低。这些发现与帕金森病患者DOPAL积聚和壳核ALDH活性降低是一致的(Goldstein等人，EUR J Neurol 2011；18：703-710)。 在含有内源性多巴胺和DOPAL的大鼠嗜铬细胞瘤PC12细胞中，我们测试了囊泡摄取阻断是否增加了内源性DOPAL的产生。我们还检查了细胞内DOPAL是否有助于细胞凋亡，并且由于α-突触核蛋白寡聚体可能是帕金森病的致病因素，因此寡聚化α-突触核蛋白。在利血平阻断囊泡摄取的情况下，PC12细胞中儿茶酚的代谢被抑制或不被抑制，代谢酶DOPALdaidzein的乙醛脱氢酶和AL1576的乙醛还原酶。以6F-多巴胺或LC-MS方法为基础，通过13C-多巴胺孵育来定量囊泡摄取；在有或没有大豆苷元+AL1576的情况下，通过外源性多巴胺的细胞凋亡反应来定量DOPAL的毒性；以及在有或没有L-芳香族氨基酸脱羧酶或单胺氧化酶的抑制下，通过DOPA孵育过程中产生的突触核蛋白二聚体来定量DOPAL诱导的突触核蛋白寡聚。利血平可抑制95-97%的囊泡摄取，并迅速增加细胞内多巴胺的含量(p=0.0008)，大豆苷元+AL1576可增强多巴胺对利血平的反应。细胞内DOPAL促进多巴胺诱导的细胞凋亡和DOPA诱导的突触核蛋白二聚化。这一发现符合儿茶酚醛假说，根据该假说，胞液中儿茶酚胺的囊泡隔离减少和儿茶酚醛解毒作用受损导致了以帕金森病为特征的儿茶酚胺能去神经(Goldstein等人，J Neurochem(正在出版))。 儿茶酚醛假说预测，遗传性ALDH活性低的小鼠应该有与衰老相关的黑质纹状体神经生成，表现为帕金森氏症。在与R.Strong(大学)的合作研究中我们直接在ALDH1A1和ALDH2基因敲除的小鼠身上测试了这一预测，这两种已知的异构体在黑质多巴胺神经元中表达。双基因敲除小鼠先天性地增加了纹状体DOPAL和年龄相关的运动能力缺陷，黑质酪氨酸羟基酶免疫反应神经元的丢失，以及纹状体多巴胺的减少(Wey等，PLoS One 2012；7：E31522)。 儿茶醛假说还预测，遗传性2型囊泡单胺转运体(VMAT2-LO)活性低的小鼠应该有纹状体多巴胺耗竭和衰老相关的帕金森综合症。在与G.Miller(Emory)正在进行的一项合作研究中，我们证实了VMAT2-LO小鼠的纹状体多巴胺和心脏去甲肾上腺素含量在给定的儿茶酚胺合成率下减少。这些小鼠现在已知会出现模仿帕金森病的运动和非运动异常。我们正在进行涉及静脉注射的研究。注射6F-多巴胺和高效液相电化学法或13C-多巴胺和LC MS法在体内证实VMAT2-LO小鼠的囊泡对儿茶酚胺的摄取减少。 利用这些数据和儿茶酚胺资源倡议下的许多其他合作研究数据，我们正在研究DOPAL在大脑各区域的浓度以及DOPAL、多巴胺、DOPAC、二羟基苯乙醇(DOPET，多巴胺的替代脱氨代谢物)和DOPA(多巴胺的前体)之间的关系。来自小鼠模型的数据已经被用来验证多巴胺：多巴作为给定数量的多巴胺合成量的囊泡摄取的指标，以及DOPAC：DOPAL来指示ALDH的活性。我们正在将这些指标应用于帕金森病患者的死后纹状体组织。初步来说，老鼠和人类纹状体的DOPAL水平是其他任何大脑区域的10倍以上。在两个物种的区域和个体中，DOPAL与多巴胺、DOPAC和DOPET相关，但与DOPA无关。重要的是，与对照组相比，帕金森病患者被发现有低壳核多巴胺：多巴和低DOPAC：DOPAL。这些发现提供了新的神经化学证据，证明帕金森病患者纹状体对多巴胺的囊泡摄取减少和ALDH活性降低，正如儿茶酚醛假说所预测的那样。 体外研究表明，DOPAL能有效地寡聚α-突触核蛋白。我们发现金属离子(尤其是Cu2+)增强了DOPAL诱导的α-突触核蛋白的寡聚，提示儿茶酚醛、金属离子和α-突触核蛋白的有害相互作用可能是路易体疾病中儿茶酚胺能神经元相对选择性丧失的原因。 路易小体病发病机制的Braak分期概念是基于死后证据，即α-突触核蛋白沉积的时空序列，在黑质神经元突触核病之前自主神经系统受累。1例原发性慢性自主神经衰竭患者在四年内接受了两年一次的脑部18F-DOPA和心肌18F-多巴胺扫描。心肌放射性低表明心脏去甲肾上腺素能去神经持续存在。纹状体18F-DOPA来源的放射性起初正常，两年后略有下降，四年后明显下降，并伴有痴呆和帕金森病。在这种情况下，心脏去甲肾上腺素能去神经和随后的进行性纹状体多巴胺能去神经的神经成像证据在体内支持Braak分期(Goldstein等人，Clin Auton res 2012；22：57-61)。

By applying a unique and novel neuroimaging-neurochemical approach, we obtained the first evidence for decreased vesicular uptake of neuronal catecholamines in sympathetic neurons in Lewy body diseases (Goldstein et al., J Clin Invest 2011;121:3320-3330). We found that putamen tissue from patients with end-stage PD contains an increased concentration of DOPAL after adjustment for decreased dopaminergic innervation and that ALDH activity measured by the DOPAC:DOPAL ratio is decreased. These findings are consistent with DOPAL buildup and decreased putamen ALDH activity in PD (Goldstein et al., Eur J Neurol 2011;18:703-710). In rat pheochromocytoma PC12 cells, which contain endogenous dopamine and DOPAL, we tested whether vesicular uptake blockade augments endogenous DOPAL production. We also examined whether intracellular DOPAL contributes to apoptosis and, since alpha-synuclein oligomers may be pathogenetic in PD, oligomerizes alpha-synuclein. Catechols were assayed in PC12 cells after reserpine to block vesicular uptake, with or without inhibition of enzymes metabolizing DOPALdaidzein for aldehyde dehydrogenase and AL1576 for aldehyde reductase. Vesicular uptake was quantified by novel HPLC methodology based on 6F-dopamine or LCMS methodology using 13C-dopamine incubation; DOPAL toxicity by apoptosis responses to exogenous dopamine, with or without daidzein+AL1576; and DOPAL-induced synuclein oligomerization by synuclein dimer production during DOPA incubation, with or without inhibition of L-aromatic-amino-acid decarboxylase or monoamine oxidase. Reserpine inhibited vesicular uptake by 95-97% and rapidly increased cell DOPAL content (p=0.0008), and daidzein+AL1576 augmented DOPAL responses to reserpine. Intracellular DOPAL contributed to dopamine-evoked apoptosis and DOPA-evoked synuclein dimerization. The findings fit with the catecholaldehyde hypothesis, according to which decreased vesicular sequestration of cytosolic catecholamines and impaired catecholaldehyde detoxification contribute to the catecholaminergic denervation that characterizes PD (Goldstein et al., J Neurochem (in press)). The catecholaldehyde hypothesis predicts that mice with inherited low ALDH activity should have aging-related nigrostriatal neurogeneration manifesting as Parkinsonism. In a collaborative study with R. Strong (Univ. of Texas, San Antonio) we tested this prediction directly in mice with knockout of ALDH1A1 and ALDH2, the two isoforms known to be expressed in substantia nigra dopamine neurons. Double knockout mice had congenitally increased striatal DOPAL and age-dependent deficits in motor performance, loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra, and reduced striatal dopamine (Wey et al., PLoS ONE 2012;7:e31522). The catecholaldehyde hypothesis also predicts that mice with inherited low activity of the type 2 vesicular monoamine transporter (VMAT2-Lo) should have striatal dopamine depletion and aging-related Parkinsonism. In an ongoing collaborative study with G. Miller (Emory) we have confirmed decreased striatal dopamine and cardiac norepinephrine contents for given rates of catecholamine synthesis as indicated by tissue DOPA in VMAT2-Lo mice. These mice are now known to develop both motor and non-motor abnormalities mimicking PD. We are conducting studies involving i.v. injection of 6F-dopamine and HPLC-electrochemical assays or 13C-dopamine and LCMS assays to confirm in vivo that VMAT2-Lo mice have decreased vesicular uptake of intra-neuronal catecholamines. Using these and numerous other data from collaborative studies under the Catecholamine Resource Initiative, we are surveying DOPAL concentrations across brain areas and relationships among DOPAL, dopamine, DOPAC, dihydroxyphenylethanol (DOPET, an alternative deaminated metabolite of dopamine), and DOPA (the precursor of dopamine). Data from mouse models have been used to validate dopamine:DOPA as an index of vesicular uptake for a given amount of dopamine synthesis and DOPAC:DOPAL to indicate ALDH activity. We are applying these indices in post-mortem striatal tissues from PD patients. Preliminarily, mouse and human striatum contain more than 10-fold greater DOPAL levels than found in any other brain region. Across regions and individuals of both species, DOPAL correlates with dopamine, DOPAC, and DOPET but not with DOPA. Importantly, PD patients have been found to have low putamen dopamine:DOPA and low DOPAC:DOPAL, compared to controls. These findings provide novel neurochemical evidence for decreased vesicular uptake of dopamine and decreased ALDH activity in striatum in PD, as predicted by the catecholaldehyde hypothesis. In vitro studies have shown that DOPAL potently oligomerizes alpha-synuclein. We have found that metal ions (especially Cu+2) augment DOPAL-induced oligomerization of alpha-synuclein, suggesting that deleterious interactions of catecholaldehydes, metal ions, and alpha-synuclein might explain relatively selective loss of catecholaminergic neurons in Lewy body diseases. The Braak staging concept of Lewy body disease pathogenesis is based on post-mortem evidence for a spatiotemporal sequence of alpha-synuclein deposition, with autonomic nervous system involvement before synucleinopathy in substantia nigra neurons. A patient with primary chronic autonomic failure underwent biennial brain 18F-DOPA and myocardial 18F-dopamine scanning over four years. Low myocardial radioactivity indicated cardiac noradrenergic denervation that persisted. Striatal 18F-DOPA-derived radioactivity initially was normal, two years later was decreased subtly, and by four years was clearly decreased and accompanied by dementia and Parkinsonism. In this case, neuroimaging evidence of cardiac noradrenergic denervation and subsequent progressive striatal dopaminergic denervation provided in vivo support for Braak staging (Goldstein et al., Clin Auton Res 2012;22:57-61).