Development of a novel tear-based biomarker assay for diagnosis of Parkinson's disease using RT-QuIC

使用 RT-QuIC 开发一种新型基于泪液的生物标志物检测方法来诊断帕金森病

• 批准号: 10227242
• 负责人: Sarah F Hamm-Alvarez
• 金额: $ 24.75万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227242
• 关键词: Address; Adoption; Affect; Affinity; Age; Alzheimer' s Disease; Antibodies; Appearance; Area; Biochemistry; Biological Assay; Biological Markers; Bradykinesia; Brain; Cerebrospinal Fluid; Clinical; Clinical Research; Collection; Corpus striatum structure; Data; Detection; Development; Diagnosis; Disease; Disease Progression; Early Diagnosis; Enzyme-Linked Immunosorbent Assay; Eye; Eye diseases; FYN gene; Film; Goals; Human; Impaired cognition; Incidence; Individual; Knowledge; Lacrimal gland structure; Lewy Bodies; Link; Measurement; Measures; Methods; Movement Disorders; Nerve Degeneration; Neural Pathways; Neurites; Neurodegenerative Disorders; Parkinson Disease; Pathologic; Pathology; Patients; Phase; Population; Primary Care Physician; Protein Isoforms; Proteins; Reaction; Receiver Operating Characteristics; Recombinants; Reflex action; Rest Tremor; Saliva; Science; Secure; Seeds; Sensitivity and Specificity; Serum; Severity of illness; Signal Transduction; Sjogren' s Syndrome; Societies; Source; Specificity; Techniques; Testing; Thyroid Gland; Time; Tissues; Translations; Visit; alpha synuclein; amyloid fibril formation; base; clinical development; clinical diagnostics; cognitive testing; cohort; detection assay; detection limit; detection platform; detection sensitivity; diagnostic biomarker; disease diagnosis; disorder control; dopaminergic neuron; exosome; improved; interest; light scattering; motor impairment; motor symptom; mutant; nanoparticle; nervous system disorder; neurotoxicity; non-motor symptom; novel; novel diagnostics; novel therapeutics; pre-clinical; prevent; prion-like; prospective; protein aggregation; protein oligomer; sex; therapeutic development; tool

One percent of individuals over the age of 60 suffers from Parkinson's disease (PD), with this number increasing to five percent by age 80. A diagnosis of PD is typically made after manifestation of defining motor symptoms including resting tremor, rigidity and bradykinesia; however by the time that patients present with clinically- established PD, they may have lost from 30-70% of dopaminergic neurons in the striatum. As such, there is great interest in the availability of definitive biomarkers enabling earlier diagnosis prior to the development of the debilitating motor symptoms signaling irreversible neurodegeneration. Biofluids explored as sources have traditionally included serum, saliva and cerebrospinal fluid (CSF). Tears represent an optimal new biofluid for PD biomarkers since they are acquired non-invasively (unlike CSF), are more concentrated than saliva, and are produced by the lacrimal gland which is highly responsive to neural pathways affected by PD. In fact, PD can manifest with initial changes in proteins in the tissues of the eye in parallel with changes in primary neurites that often occur prior to the development of clinical signs. We have found, in PD patient cohorts, that the oligomeric α-synuclein composition of tears is increased compared to tears from healthy controls. Due to limitations in the sensitivity of conventional ELISA used for these measurements, PD patients (8-18%) have tear oligomeric α- synuclein values below the threshold of the assay's sensitivity. ELISA also lacks the ability to measure the actual capacity of oligomeric α-synuclein to promote protein aggregation, a feature linked to disease pathology. Here we apply and optimize a real-time quaking-induced conversion (RT-QuIC) assay to detect pathological oligomeric α-synuclein in tears. We propose two Aims. Aim 1: To develop an RT-QuIC assay capable of detection of α-synuclein aggregates in human tears. We will utilize recombinant wild type α-synuclein to optimize the detection of oligomeric α-synuclein utilizing RT-QuIC, determine whether bulk tears or isolated tear exosomes provide a more effective seed, and optimize the collection matrix. We will also decrease the lag phase of α-synuclein aggregation without compromising reaction specificity by utilizing aggregation-prone α-synuclein mutants, enhancing the feasibility of adoption of this assay to a clinical diagnostic. Aim 2: To test the ability of RT-QuIC to distinguish pathological α-synuclein aggregates in tears of diagnosed PD patients versus healthy controls and neurological disease controls. Using optimized conditions, we will compare RT-QuIC to ELISA in the ability to detect oligomeric α-synuclein in tears of diagnosed PD patients versus age- and sex- matched healthy and neurological disease controls, determining its sensitivity and specificity in discriminating those with PD. Our goal at study conclusion is to secure data sufficient to advance this assay to a clinical study to test its ability to detect undiagnosed PD patients in a mixed population. If tears can be used for early detection of PD, this may assist with the development of new therapies which offer true neuroprotective qualities.

60岁以上的人中有1%患有帕金森病（PD），而且这一数字还在增加 到80岁时下降到5%。PD的诊断通常是在表现出明确的运动症状后做出的 包括静止性震颤、僵硬和运动迟缓;然而，当患者出现临床- 在已经建立的PD中，他们可能已经失去了纹状体中30-70%的多巴胺能神经元。因此， 人们对能够在疾病发展之前进行早期诊断的确定性生物标志物的可用性非常感兴趣， 使人衰弱的运动症状是不可逆的神经退化的信号。作为来源的生物流体已经被探索 传统上包括血清、唾液和脑脊液（CSF）。眼泪代表了一种最佳的新生物流体， PD生物标志物，因为它们是非侵入性获得的（与CSF不同），比唾液更浓缩， 由泪腺产生，泪腺对受PD影响的神经通路高度敏感。事实上，PD可以 表现为眼睛组织中蛋白质的初始变化，与初级神经突的变化平行， 通常发生在临床症状出现之前。我们发现，在PD患者队列中， 与健康对照组的泪液相比，泪液中的α-突触核蛋白成分增加。由于限制， 为了确定用于这些测量的常规ELISA的灵敏度，PD患者（8-18%）具有泪液寡聚体α- 突触核蛋白值低于测定灵敏度的阈值。ELISA也缺乏测量实际的 寡聚体α-突触核蛋白促进蛋白质聚集的能力，这是与疾病病理学相关的特征。这里 我们应用并优化了实时振荡诱导转换（RT-QuIC）测定来检测病理性 低聚α-突触核蛋白。我们提出两个目标。目的1：开发能够检测和分析蛋白质的RT-QuIC测定法。 人泪液中α-突触核蛋白聚集体的检测。我们将利用重组野生型α-突触核蛋白， 利用RT-QuIC优化寡聚体α-突触核蛋白的检测， 外泌体提供了更有效的种子，并优化了收集基质。我们还将减少滞后期 通过利用易于聚集的α-突触核蛋白， 突变体，增强了采用该测定进行临床诊断的可行性。目标2：测试能力 RT-QuIC用于区分诊断PD患者泪液中的病理性α-突触核蛋白聚集体与 健康对照和神经疾病对照。使用优化的条件，我们将比较RT-QuIC ELISA检测诊断PD患者泪液中寡聚体α-突触核蛋白的能力与年龄和性别的关系， 匹配的健康和神经系统疾病对照，确定其灵敏度和特异性， PD的人。我们在研究结束时的目标是获得足够的数据，以将该检测方法推向临床 一项旨在测试其在混合人群中检测未确诊PD患者的能力的研究。如果眼泪可以用来 早期发现PD，这可能有助于开发新的治疗方法，提供真正的神经保护 的天性.