Biomarkers of Catecholaminergic Neurodegeneration

(A)NINDS PD风险研究：在内部NINDS PDRisk研究（NIH临床方案09 N 0010，NCT NCT 00775853）中，我们根据遗传学、做梦行为、嗅觉下降和站立时血压下降（直立性低血压，OH），对有帕金森病（PD）风险的个体进行了随访。足够数量的参与者已经完成了5个1.5年的随访评估，开始得出关于大脑或心脏中存在儿茶酚胺缺乏生物标志物的阳性和阴性预测值的结论。因此，所有8种儿茶酚胺能生物标志物将发生PD的组与未发生PD的组区分开来。缺乏这些生物标志物的阴性预测值可能高达100%。因此，儿茶酚胺缺乏的生物标志物似乎可以有效地识别继续发展PD的风险个体。 (B)帕金森病患者皮肤、下颌下腺（SMG）和心脏交感神经中的α-突触核蛋白（AS）沉积：一项尸检观察研究：尽管广泛怀疑，但对AS沉积在儿茶酚胺能神经元中的病理生理学意义了解甚少。在一项尸检研究中，我们定量了免疫反应性AS和酪氨酸羟化酶（TH，儿茶酚胺能神经元的标志物）的数量，并同时测定了三个交感去甲肾上腺素能神经支配的结构-皮肤，SMG和myocardum-从尸检证实PD患者的去甲肾上腺素含量。在所有三种组织中，PD患者与年龄匹配的对照组相比AS沉积增加。出乎意料的是，PD患者的皮肤和SMG中的去甲肾上腺素和TH含量正常，而PD患者的心脏中存在严重的去甲肾上腺素缺乏和TH降低。这些发现表明，皮肤和SMG中的AS沉积可能是一种非致病性生物标志物，而心脏中交感神经中的AS可能是有毒的（PMID 35260194）。 (C)计算模型揭示了LBD的三阶段进展：当患者出现LBD症状时，已经有大量的儿茶酚胺能神经元损失。确定临床前LBD的生物标志物可能对于最大限度地提高疾病改善治疗的有效性至关重要。然而，临床前LBD中儿茶酚胺能神经变性的进展模式尚不清楚。我们扩展了以前发表的计算模型，该模型结合了多巴胺代谢产物3，4-二羟基苯乙醛（DOPAL）与AS的有害相互作用。建模表明，三阶段的损失，从稳态的第一阶段，以动态平衡和快速的儿茶酚胺消耗在第二阶段，以减缓进一步损失的症状第三阶段的儿茶酚胺含量。LBD患者心脏和壳核的经验性纵向神经影像学数据符合这种三相模式。将计算建模与临床实验室生物标志物相结合来检测临床前疾病，可以实现对LBD实验性治疗试验的疾病进展和靶向特异性神经元内功能障碍的个性化预测（PMID 35621196）。 (D)遗传性PD中交感神经中的α-突触核蛋白沉积：脑干神经元中AS的细胞质内含物是特发性PD的特征。PD还需要在交感神经中积累AS。在遗传形式的PD中，交感神经元内AS蓄积的相对程度尚未报道。在一项横断面观察性研究中，我们通过应用一种经过验证的方法，计算AS-酪氨酸羟化酶共定位指数，比较了常见和罕见遗传形式PD的神经元内AS沉积的幅度。神经元内AS沉积作为基因型的函数而变化，在100%的SNCA或LRRK 2突变受试者、95%的特发性PD受试者、83%的GBA突变受试者和0%的双等位基因PRKN突变受试者中高于对照范围。因此，SNCA，LRRK 2或GBA突变的个体在皮肤活检的交感去甲肾上腺素能神经中有大量的神经元内AS沉积，而双等位基因PRKN突变的个体则没有。这些发现对通过AS-TH共定位指数（PMID 34076298）诊断活体患者的LBD具有重要意义。 (E)液相色谱串联和飞行时间质谱（LC-MS/MS）测定临床生物液体和组织中的儿茶酚胺和相关化合物：通过液相色谱电化学检测测定儿茶酚胺和相关化合物在技术上受到限制。与合作者一起，我们正在探索使用市售系统的LC-MS/MS。

(A) NINDS PDRisk study: In the intramural NINDS PDRisk study (NIH Clinical Protocol 09N0010, NCT NCT00775853 ) we are following individuals at risk for developing Parkinsons disease (PD) based on genetics, dream enactment behavior, decreased sense of smell, and a fall in blood pressure during standing (orthostatic hypotension, OH). A sufficient number of participants have completed 5 1.5-year follow-up evaluations to begin to draw conclusions about the positive and negative predictive values of having biomarkers of catecholamine deficiency in the brain or heart. Preliminarily, all of 8 catecholaminergic biomarkers distinguish the group that develops PD from the group that does not. The negative predictive value of the absence of these biomarkers may be as high as 100%. Therefore, biomarkers of catecholamine deficiency seem to identify efficiently at-risk individuals who go on to develop PD. (B) Alpha-synuclein (AS) deposition in sympathetic nerves in skin, submandibular gland (SMG), and heart in PD: A post-mortem observational study: The pathophysiological significance of AS deposition in catecholaminergic neurons, although widely suspected, has been poorly understood. In a post-mortem study we quantified the amounts of immunoreactive AS and tyrosine-hydroxylase (TH, a marker of catecholaminergic neurons) and concurrently assayed norepinephrine contents in three sympathetic noradrenergically innervated structures--skin, SMG, and myocardum--from patients with autopsy-proven PD. In all three tissues PD patients had increased AS deposition compared to age-matched controls. Unexpectedly, norepinephrine and TH contents in skin and SMG were normal in PD patients, whereas in the heart PD patients had severe norepinephrine deficiency and decreased TH. These findings suggest that in skin and SMG AS deposition may be a non-pathogenic biomarker, whereas in the heart AS in sympathetic nerves may be toxic (PMID 35260194). (C) Computational modeling reveals tri-phasic progression of LBDs: By the time a patient develops symptoms of a LBD, there is already substantial loss of catecholaminergic neurons. Identifying biomarkers of preclinical LBDs may be crucial for maximizing the effectiveness of disease-modifying treatments. The pattern of progression of catecholaminergic neurodegeneration in preclinical LBDs, however, has been unknown. We extended on a previously published computational model that incorporates harmful interactions of the dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) with AS. The modeling indicated tri-phasic loss of catecholamine contents, from homeostasis in the first phase to dyshomeostasis and rapid catecholamine depletion in the second phase to slow further loss in the symptomatic third phase. Empirical longitudinal neuroimaging data in both the heart and putamen of LBD patients fit with this tri-phasic pattern. Combining computational modeling with clinical laboratory biomarkers to detect preclinical disease may enable individualized predictions about disease progression and target specific intra-neuronal dysfunctions for experimental therapeutic trials in LBDs (PMID 35621196). (D) Alpha-Synuclein deposition in sympathetic nerves in genetic PD: Cytoplasmic inclusions of AS in brainstem neurons are characteristic of idiopathic PD. PD also entails AS buildup in sympathetic nerves. Among genetic forms of PD, the relative extents of sympathetic intra-neuronal accumulation of AS have not been reported. In a cross-sectional observational study we compared magnitudes of intra-neuronal AS deposition in common and rare genetic forms of PD by applying a validated method, calculation of AS-tyrosine hydroxylase colocalization indexes. Intra-neuronal AS deposition varied as a function of genotype, above the control range in 100% of subjects with SNCA or LRRK2 mutations, 95% with idiopathic PD, 83% with GBA mutations, and 0% with biallelic PRKN mutations. Individuals with SNCA, LRRK2, or GBA mutations therefore have substantial intra-neuronal AS deposition in sympathetic noradrenergic nerves in skin biopsies, whereas those with biallelic PRKN mutations do not. These findings have important implications for diagnosing LBDs in living patients via AS-TH colocalization indexes (PMID 34076298). (E) Liquid chromatography with tandem and time of flight mass spectrometry (LC-MS/MS) to assay catecholamines and related compounds in clinical biofluids and tissues: Assays of catecholamines and related compounds by liquid chromatography with electrochemical detection are limited technologically. With collaborators we are exploring LC-MS/MS using a commercially available system.