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

基本信息

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
项目类别：
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&apos 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&apos 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

项目摘要

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，这一比例下降至百分之五。帕金森病的诊断通常是在出现明确的运动症状后做出的包括静息性震颤、强直和运动迟缓；然而，当患者出现临床症状时建立 PD 后，他们可能失去了纹状体中 30-70% 的多巴胺能神经元。人们对确定的生物标记物的可用性非常感兴趣，这些标记物能够在开发出早期诊断之前进行早期诊断令人衰弱的运动症状标志着不可逆的神经变性，正如来源所探索的那样。传统上包括血清、唾液和脑脊液 (CSF)，泪液是一种最佳的新型生物流体。 PD 生物标志物因为是非侵入性获得的（与脑脊液不同），比唾液浓度更高，并且由泪腺产生，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同时发生我们发现，在 PD 患者群体中，寡聚体通常发生在出现临床症状之前。由于泪液的限制，与健康对照的泪液相比，眼泪的 α-突触核蛋白成分有所增加。与用于这些测量的传统 ELISA 的灵敏度相比，PD 患者 (8-18%) 具有泪液寡聚 α- 突触核蛋白值低于测定灵敏度的阈值，ELISA 也缺乏测量实际的能力。寡聚 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。我们应用并优化实时振动诱导转换 (RT-QuIC) 测定来检测病理性我们提出了两个目标 1：开发一种能够检测泪液中寡聚 α-突触核蛋白的 RT-QuIC 检测方法。检测人类眼泪中的 α-突触核蛋白聚集体我们将利用重组野生型 α-突触核蛋白来检测。利用 RT-QuIC 优化寡聚 α-突触核蛋白的检测，确定是大量眼泪还是孤立眼泪外泌体提供了更有效的种子，并优化了收集矩阵，我们还将减少滞后期。利用易于聚集的 α-突触核蛋白，在不影响反应特异性的情况下进行 α-突触核蛋白聚集目标 2：测试能力。 RT-QuIC 区分诊断 PD 患者泪液中病理性 α-突触核蛋白聚集体与使用优化条件，我们将比较 RT-QuIC。 ELISA 检测诊断 PD 患者泪液中寡聚 α-突触核蛋白的能力与年龄和性别的关系匹配健康和神经疾病对照，确定其区分的敏感性和特异性我们得出研究结论的目标是获得足够的数据以将这种检测方法推向临床。一项研究测试其在混合人群中检测未确诊帕金森病患者的能力是否可以使用眼泪。早期检测帕金森病，这可能有助于开发提供真正神经保护作用的新疗法品质。