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 ?-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 ?- synuclein values below the threshold of the assay's sensitivity. ELISA also lacks the ability to measure the actual capacity of oligomeric ?-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 ?-synuclein in tears. We propose two Aims.
Aim 1 : To develop an RT-QuIC assay capable of detection of ?-synuclein aggregates in human tears. We will utilize recombinant wild type ?-synuclein to optimize the detection of oligomeric ?-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 ?-synuclein aggregation without compromising reaction specificity by utilizing aggregation-prone ?-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 ?-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 ?-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.
Parkinson's disease is diagnosed largely based on the appearance of defining motor symptoms but by this time, patients may have lost the majority of their dopaminergic neurons. Based on our findings that oligomeric ?-synuclein is increased in tears of patients with Parkinson's disease, we will develop a more sensitive assay, real-time quaking induced conversion, for accessible use as a clinical diagnostic. Knowledge gained may address the urgent need for new diagnostic biomarkers for Parkinson's disease to identify patients at earlier disease stages before irreversible damage has occurred, and provide a new tool to aid in the development of therapeutics with true neuroprotective qualities.
