Blinded RT‐QuIC Analysis of α‐Synuclein Biomarker in Skin Tissue From Parkinson's Disease Patients

Abstract

A BS TRACT: Background: An unmet clinical need in Parkinson's disease (PD) is to identify biomarkers for diagnosis, preferably in peripherally accessible tissues such as skin. Immunohistochemical studies have detected pathological α-synuclein (αSyn) in skin biopsies from PD patients albeit sensitivity needs to be improved. Objective: Our study provides the ultrasensitive detection of pathological αSyn present in the skin of PD patients, and thus, pathological αSyn in skin could be a potential biomarker for PD. Methods: The real-time quaking-induced conversion assay was used to detect pathological αSyn present in human skin tissues. Further, we optimized this ultra-sensitive and specific assay for both frozen and formalin-fixed paraffinembedded sections of skin tissues. We determined the seeding kinetics of the αSyn present in the skin from autopsied subjects consisting of frozen skin tissues from 25 PD and 25 controls and formalin-fixed paraffinembedded skin sections from 12 PD and 12 controls. Results: In a blinded study of skin tissues from autopsied subjects, we correctly identified 24/25 PD and 24/25 controls using frozen skin tissues (96% sensitivity and 96% specificity) compared to 9/12 PD and 10/12 controls using formalin-fixed paraffinembedded skin sections (75% sensitivity and 83% specificity). Conclusions: Our blinded study results clearly demonstrate the feasibility of using skin tissues for clinical diagnosis of PD by detecting pathological αSyn. Moreover, this peripheral biomarker discovery study may have broader translational value in detecting misfolded proteins in skin samples as a longitudinal progression marker.

Streamlined alpha-synuclein RT-QuIC assay for various biospecimens in Parkinson’s disease and dementia with Lewy bodies

“…However, technical challenges of the IHC-based method may have limited its clinical utility [ 39 ]. Most recently, several tissue-specific αSyn RT-QuIC platforms have been reported with nasal brushing [ 18 ], SMG [ 19 ] and skin [ 20 , 21 ] samples from PD patients, but procedures for tissue extraction and reagent compositions for RT-QuIC assay differed for individual tissues and among different studies. Therefore, a simplified and standardized protocol is desired to uniformly perform RT-QuIC assay and compare distribution of αSyn D among multiple tissue types.…”

“…Recent studies have utilized αSyn RT-QuIC assay to detect seeding activity of αSyn D aggregates in brain homogenate (BH) and CSF samples from PD and DLB, with a diagnostic sensitivity great than 90% and specificity of 82–100% for CSF samples [ 14 – 17 ]. Moreover, the αSyn RT-QuIC method has been extended to evaluate the presence of αSyn D from non-CNS sources, such as nasal brushing [ 18 ], salivary gland [ 19 ] and skin [ 20 , 21 ]. The RT-QuIC platform has the advantage of high throughput, fast turnaround, and automated recording of aggregation kinetics in real time.…”

The Latest Research on RT-QuIC Assays—A Literature Review

“…Some studies using the RT–QuIC assays to detect α-syn on peripheral tissues [ 48 , 50 , 56 , 57 ]. With the α-synuclein in brushes of OM, α-synuclein RT–QuIC showed 55.5% and 81.8%, positive in PD and MSA, respectively.…”

“…Finally, the assay showed 84.4% specificity. The most recent research has been successful in detecting phosphorylated α-synuclein (PαSyn) seeding activity in the skin by RT–QuIC [ 56 , 57 ]. RT–QuIC analysis of the αSynP seeding activity in autopsy abdominal skin samples revealed a 93–94% sensitivity and a 93–98% specificity in synucleinopathies (PD, DLB, and MSA) [ 56 ].…”

Vagus Nerve and Stomach Synucleinopathy in Parkinson’s Disease, Incidental Lewy Body Disease and Normal Elderly Subjects: Evidence Against the “Body-First” Hypothesis

“…It is possible that the initial “seeding” of aSyn, whether from the environment or by spontaneous internal generation, is accomplished by transiently-existing forms that may not locally incite the more familiar forms of aSyn but do so in the CNS once transmitted there. Assessment of PNS with α-synuclein seeding assays such as RT-QuIC or protein misfolding cyclic amplification (PMCA) may be more sensitive than IHC and may be more effective at uncovering PNS aSyn [96, 97]. Recent studies have suggested that bidirectional spread of α-synuclein aggregates may not necessarily pass through the vagus nerve but rather via a hematogenous route [61].…”