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: 10057848
• 负责人: Sarah F Hamm-Alvarez
• 金额: $ 19.12万
• 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 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057848
• 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; System; Techniques; Testing; Thyroid Gland; Time; Tissues; Translations; Visit; alpha synuclein; amyloid fibril formation; base; clinical development; clinical diagnostics; cognitive testing; cohort; 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岁以上的个人中有1％患有帕金森氏病（PD），这一数字增加 到80岁到5％。通常在定义运动症状的表现后进行PD的诊断 包括静止的树，刚性和胸肌；但是，到患者在临床上出现的时候 建立的PD，它们可能从纹状体中的多巴胺能神经元中的30-70％损失。因此，有 对确定生物标志物的可用性极大的兴趣，可以在开发之前实现早期诊断 衰弱的运动症状信号传达不可逆的神经变性。探索的生物流体作为来源 传统上包括血清，唾液和脑脊液（CSF）。眼泪代表最佳的新生物流体 PD生物标志物由于非侵入性（与CSF不同），比唾液更集中，并且是 由泪腺产生，对受PD影响的神经途径高度响应。实际上，PD可以 与眼睛组织中蛋白质的初始变化与原发性神经运动的变化并联 通常发生在开发临床体征之前。我们发现，在PD患者队列中，低聚 与健康对照的泪液相比，泪液的α-突触核蛋白组成增加了。由于限制 用于这些测量的常规ELISA的敏感性，PD患者（8-18％）的泪液寡聚α- 综合素值低于测定敏感性的阈值。 Elisa也缺乏测量实际的能力 寡聚α-突触核蛋白促进蛋白质聚集的能力，该特征与疾病病理有关。这里 我们应用并优化实时Quaking诱导的转化率（RT-QUIC）测定方法来检测病理 泪液中的寡聚α-突触核蛋白。我们提出了两个目标。目标1：开发能够 在人眼泪中检测α-核蛋白聚集体。我们将利用重组野生型α-突触核蛋白 使用RT-Quic优化寡聚α-突触核蛋白的检测 外泌体提供了更有效的种子，并优化了收集矩阵。我们还将减少滞后阶段 α-突触核蛋白聚集，而无需损害反应特异性，通过利用聚集 突变体，增强了对临床诊断的采用可行性。目标2：测试能力 RT Quic以区分病理α-突触核蛋白聚集体，诊断PD患者的眼泪 健康控制和神经疾病控制。使用优化的条件，我们将比较RT Quic 在诊断性PD患者的眼泪与年龄和性别的眼泪中检测到寡聚α-突触核蛋白的能力 匹配健康和神经系统疾病控制，确定其在区分方面的敏感性和特异性 那些带有PD的人。我们在研究结论中的目标是确保足以将此测定推向临床的数据 研究以测试其在混合人群中检测未诊断未诊断的PD患者的能力。如果眼泪可以用于 早期检测PD，这可能有助于开发新疗法，这些疗法提供真正的神经保护作用 品质。