Key Points Question Does the pathological α-synuclein (αSyn P ) detected by immunohistochemistry in the skin of individuals with Parkinson disease (PD) have aggregation seeding activity, and is skin αSyn P seeding activity a potential biomarker for diagnosis of PD and other synucleinopathies? Findings In this diagnostic study including skin samples from 160 autopsies and 41 biopsies, a statistically significant increase in αSyn P seeding activity was observed in individuals with PD and synucleinopathies compared with controls with tauopathies and nonneurodegenerative diseases. Meaning Skin αSyn P has aggregation seeding activity in patients with PD and non-PD synucleinopathies and may be a biomarker for antemortem diagnosis of PD and other synucleinopathies.

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Sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is transmissible through iatrogenic routes due to abundant infectious prions [misfolded forms of the prion protein (PrPSc)] in the central nervous system (CNS). Some epidemiological studies have associated sCJD risk with non-CNS surgeries. We explored the potential prion seeding activity and infectivity of skin from sCJD patients. Autopsy or biopsy skin samples from 38 patients [21 sCJD, 2 variant CJD (vCJD), and 15 non-CJD] were analyzed by Western blotting and real-time quaking-induced conversion (RT-QuIC) for PrPSc. Skin samples from two patients were further examined for prion infectivity by bioassay using two lines of humanized transgenic mice. Western blotting revealed dermal PrPSc in one of five deceased sCJD patients and one of two vCJD patients. However, the more sensitive RT-QuIC assay detected prion seeding activity in skin from all 23 CJD decedents but not in skin from any non-CJD control individuals (with other neurological conditions or other diseases) during blinded testing. Although sCJD patient skin contained ~103- to 105-fold lower prion seeding activity than did sCJD patient brain tissue, all 12 mice from two transgenic mouse lines inoculated with sCJD skin homogenates from two sCJD patients succumbed to prion disease within 564 days after inoculation. Our study demonstrates that the skin of sCJD patients contains both prion seeding activity and infectivity, which raises concerns about the potential for iatrogenic sCJD transmission via skin.

Definitive diagnosis of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) relies on postmortem finding of disease-associated alpha-synuclein (αSynD) as misfolded protein aggregates in the central nervous system (CNS). The recent development of the real-time quaking induced conversion (RT-QuIC) assay for ultrasensitive detection of αSynD aggregates has revitalized the diagnostic values of clinically accessible biospecimens, including cerebrospinal fluid (CSF) and peripheral tissues. However, the current αSyn RT-QuIC assay platforms vary widely and are thus challenging to implement and standardize the measurements of αSynD across a wide range of biospecimens and in different laboratories. We have streamlined αSyn RT-QuIC assay based on a second generation assay platform that was assembled entirely with commercial reagents. The streamlined RT-QuIC method consisted of a simplified protocol requiring minimal hands-on time, and allowing for a uniform analysis of αSynD in different types of biospecimens from PD and DLB. Ultrasensitive and specific RT-QuIC detection of αSynD aggregates was achieved in million-fold diluted brain homogenates and in nanoliters of CSF from PD and DLB cases but not from controls. Comparative analysis revealed higher seeding activity of αSynD in DLB than PD in both brain homogenates and CSF. Our assay was further validated with CSF samples of 214 neuropathologically confirmed cases from tissue repositories (88 PD, 58 DLB, and 68 controls), yielding a sensitivity of 98% and a specificity of 100%. Finally, a single RT-QuIC assay protocol was employed uniformly to detect seeding activity of αSynD in PD samples across different types of tissues including the brain, skin, salivary gland, and colon. We anticipate that our streamlined protocol will enable interested laboratories to easily and rapidly implement the αSyn RT-QuIC assay for various clinical specimens from PD and DLB. The utilization of commercial products for all assay components will improve the robustness and standardization of the RT-QuIC assay for diagnostic applications across different sites. Due to ultralow sample consumption, the ultrasensitive RT-QuIC assay will facilitate efficient use and sharing of scarce resources of biospecimens. Our streamlined RT-QuIC assay is suitable to track the distribution of αSynD in CNS and peripheral tissues of affected patients. The ongoing evaluation of RT-QuIC assay of αSynD as a potential biomarker for PD and DLB in clinically accessible biospecimens has broad implications for understanding disease pathogenesis, improving early and differential diagnosis, and monitoring therapeutic efficacies in clinical trials.

A definitive pre-mortem diagnosis of prion disease depends on brain biopsy for prion detection currently and no validated alternative preclinical diagnostic tests have been reported to date. To determine the feasibility of using skin for preclinical diagnosis, here we report ultrasensitive serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays of skin samples from hamsters and humanized transgenic mice (Tg40h) at different time points after intracerebral inoculation with 263K and sCJDMM1 prions, respectively. sPMCA detects skin PrPSc as early as 2 weeks post inoculation (wpi) in hamsters and 4 wpi in Tg40h mice; RT-QuIC assay reveals earliest skin prion-seeding activity at 3 wpi in hamsters and 20 wpi in Tg40h mice. Unlike 263K-inoculated animals, mock-inoculated animals show detectable skin/brain PrPSc only after long cohabitation periods with scrapie-infected animals. Our study provides the proof-of-concept evidence that skin prions could be a biomarker for preclinical diagnosis of prion disease.

Objective.To determine whether: 1) the immunofluorescence (IF) is a reproducible technique in detecting misfolded α-synuclein (α-syn) in skin nerves; and subsequently 2) IF and RT-QuIC (both in skin and CSF) show a comparable in vivo diagnostic accuracy in distinguish synucleinopathies (SOPs) from non-synucleinopathies (non-SOPs) in a large cohort of patients.Methods.We prospectively recruited 90 patients fulfilling clinical and instrumental diagnostic criteria for all SOPs variants and non-SOPs (mainly including Alzheimer disease, tauopathies, and vascular parkinsonism or dementia). Twenty-four patients with mainly peripheral neuropathies were used as controls. Patients underwent skin biopsy for IF and RT-QuIC whereas CSF was performed in patients who underwent lumbar puncture for diagnostic purposes. IF and RT-QuIC analysis were made blinded to the clinical diagnosis.Results.IF showed reproducible results between two pairs of neighbouring skin samples. Furthermore, both IF and RT-QuIC showed high sensitivity and specificity in discriminating SOPs from non-SOPs and controls but IF presented the higher diagnostic accuracy. IF presented a good level of agreement with RT-QuIC both skin and CSF in SOPs.Conclusions:1) Both IF and RT-QuIC showed a high diagnostic accuracy although IF displayed the better value as well as an optimal reproducibility; 2) they presented a good level of agreement in SOPs supporting the use of a less invasive tests such as skin IF or RT-QuIC instead of CSF RT-QuIC as diagnostic tool for synucleinopathies.Classification of evidence:This study provides Class III evidence that IF or RT-QuIC accurately distinguish SOPs from non-SOPs patients.

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