Blinded RT-QuIC Analysis of α-Synuclein Biomarker in Skin Tissue from Parkinson’s Disease Patients

Abstract

Background:

An unmet clinical need in PD is to identify biomarkers for diagnosis, preferably in peripherally accessible tissues such as skin. Immunohistochemical studies have detected pathological α-synuclein in skin biopsies from PD patients albeit sensitivity needs to be improved.

Objective:

Our study provides the ultrasensitive detection of pathological α-synuclein present in the skin of PD patients and thus pathological α-synuclein in skin could be a potential biomarker for PD.

Methods:

The real-time quaking-induced conversion assay was used to detect pathological α-synuclein present in human skin tissues. Further, we optimized this ultra-sensitive and specific assay for both frozen and formalin-fixed paraffin-embedded sections of skin tissues. We determined the seeding kinetics of the aSyn present in the skin from autopsied subjects consisting of frozen skin tissues from 25 PD and 25 controls and formalin-fixed paraffin-embedded 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 paraffin-embedded 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 α-synuclein. Moreover, this peripheral biomarker discovery study may have broader translational value in detecting misfolded proteins in skin samples as a longitudinal progression marker.

Introduction

Parkinson’s disease (PD) is a neurodegenerative disorder affecting millions of people worldwide, inflicting significant medical, social and economic burdens to the society.¹ The classic neuropathological changes of PD include the gradual onset and progressive degeneration of dopaminergic neurons in the substantia nigra and deposition of pathological α-synuclein (aSyn) aggregates widely throughout the brain.^{2, 3} Diagnosis of PD is mostly based on clinical signs and can only be definitely confirmed upon postmortem demonstration of substantia nigral aSyn.⁴ As such, misdiagnosis is common and approximately 25% of clinically diagnosed PD patients get an alternative diagnosis postmortem.^{5, 6} Moreover, recent estimates suggest that the accuracy of new clinical PD diagnoses is as low as 58% due to overlapping symptoms with other Parkinsonian movement disorders.⁷ Hence, accurate diagnosis is critical to both clinical practice and translational research, particularly for initiating early intervention or recruiting newly diagnosed patients into neuroprotection trials. Developing diagnostic tests capable of differentiating PD from similar clinical motor syndromes rests on efforts to develop minimally invasive methods to detect PD-related pathological processes, which is challenging due to individual variation in the progression and severity of the diseases. Although some biomarkers have been proposed for targeting various pathological processes of PD, such as oxidative damage⁸, mitochondrial dysfunction⁹, misfolded aSyn in the enteric nervous system tissues¹⁰, neuroinflammation¹¹, and genetic variants of aSyn¹⁰, we still lack an objective and reliable biomarker. Other biomarkers such as potential RNAs, proteins and lipids have been investigated but the results are inconclusive and confounded by large variability.² Overall, the low sensitivity and specificity associated with these preclude them as a reliable biomarkers of PD.

As a key hallmark of PD pathogenesis, the accumulation of aSyn aggregates is strongly correlated with the progression of PD.^{3, 12–16} For a confirmatory diagnosis of PD, aSyn is routinely visualized using immunohistochemistry (IHC) in autopsied central nervous system tissues. More recently, cerebrospinal fluid (CSF)^{17, 18} and several peripheral tissue biopsies from the olfactory mucosa¹⁹, gastrointestinal tract²⁰, submandibular gland (SMG)^21–24 and skin¹² are being investigated for clinical diagnosis of PD due to the availability of aSyn in the peripheral nervous system and the advances in understanding the prion-like mechanism of aSyn spreading.^{25, 26} Several IHC methods have been employed on various peripheral biopsies, but their clinical potential can only be assessed by including optimal tissue, site, and technique as well as recruiting pathologists for histological detection, sensitivity and specificity.^{27, 28} However, there are several limitations associated with these tissues such as: 1) the brain is only available post-mortem, 2) CSF collection is more invasive and involves the risk of headache and infection²⁹, 3) variabilities associated with time of collection and repeated sampling^{30, 31} and 4) SMG biopsy requires a surgeon.¹² The multi-center Systemic Synuclein Sampling Study (S4) examined various peripheral tissue biopsies for feasible diagnosis of PD and found skin punch biopsies to have no adverse effects while also being easy to collect, repeatable, and minimally invasive.¹² Accumulating evidence from skin biopsies tested using techniques with varying sensitivity, such as IHC and proximal ligation assays, confirms aSyn deposition in cutaneous autonomic nerves.^32–35 We and others recently implemented a real-time quaking-induced conversion (RT-QuIC) protein misfolding assay for detecting aSyn in various tissues including the brain, cerebrospinal fluid (CSF)^{17, 36}, and SMG²¹ for diagnostic and therapeutic applications. Furthermore, RT-QuIC has been shown to efficiently amplify prions from skin of both humans and experimental animal models^{37, 38}. Therefore, in the present study we extended the RT-QuIC assay to detect aSyn from skin tissues to address one of the major challenges in biomarker discovery for PD. The overall goal of this study was to develop and validate an ultra-sensitive RT-QuIC test to detect aSyn from autopsied skin samples from neuropathologically confirmed PD and control subjects, using frozen tissues, cryostat sections as well as slide-mounted, formaldehyde-fixed paraffin-embedded (FFPE) sections. Our results suggest that the skin RT-QuIC is highly sensitive and specific and thus may represent a most sensitive and specific peripheral tissue biomarker for the clinical diagnosis of PD.

Materials and Methods

Sample details

Frozen skin tissues of the scalp were collected from the posterior lower occipital region along the midline, about 8 cm superior to the hairline, cryostat sections and slide-mounted FFPE sections all came from Banner Sun Health Research Institute (BSHRI) in Sun City, Arizona. All subjects were neuropathologically examined at autopsy using specific diagnostic criteria for PD as a part of the IRB-approved Arizona Study of Aging and Neurodegenerative Disorders/Brain and Body Donation Program (www.brainandbodydonationprogram.org).^39–41 Controls consisted of normal elderly subjects without dementia or parkinsonism; some subjects had Alzheimer’s disease (AD) related microscopic changes that were insufficient for an AD diagnosis. In this study, we tested grossly dissected frozen skin tissues from 25 PD and 25 control subjects, skin FFPE sections from 12 PD and 12 controls, and cryostat sections from 4 PD and 4 controls. Further details of the samples, including a diagnostic summary, are in Table S1 and S2.

Processing of skin tissues, FFPE and cryostat sections

All skin samples were trimmed to ~25 mg and stored at −80 °C. Before processing, the samples were cleaned three times in Tris-buffered saline (TBS) containing 10 mM Tris-HCl and 133 mM NaCl (pH 7.4). Later, 10% (w/v) homogenates of skin samples were prepared in TBS containing 2 mM CaCl₂ and 0.25% (w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h at 350 rpm³⁸. Next, samples were homogenized with a bullet blender with 0.5-mm zirconium oxide beads at speed 10 for 5 min. After homogenization, samples were centrifuged at 500 × g for 5 min and the supernatant was used to make serial 10-fold (w/v) dilutions. For recovering slide-mounted FFPE skin sections, the slides were washed twice in 100% xylene for 10 min each. After the xylene washes, slides were submersed for 10 min each in a descending series of alcohol dilutions (100%, 70%, and 50%) to rehydrate the tissues as described previously²¹. As a final step, the slides were washed with sterile TBS before scraping skin tissue sections from the slides and then recording tissue weights to make 10% (w/v) homogenates with 1X TBS containing 2 mM CaCl₂ and 0.25% (w/v) collagenase A in a shaker at 37 °C for 4 h at 350 rpm. Cryostat sections were processed by washing 3–5 sections in 0.5 mL of 1X TBS to remove the embedding compound (OCT). The cleared tissue was pelleted at 500 × g for 5 min, weighed and resuspended to attain 10% (w/v) in 1X TBS containing 2 mM CaCl₂ and 0.25% (w/v) collagenase A. The tissue was triturated with repeated pipetting and incubated in a thermal mixer at 37 °C for 4 h at 350 rpm. Next, the samples were cup sonicated at 70% amplitude with alternate cycles of 30 sec pauses and sonicated for 5 min. The resulting homogenates were used to make serial 10-fold dilutions similar to other tissues prior to testing in RT-QuIC assay.

Purification of recombinant human aSyn protein

Purification of recombinant human WT aSyn protein was performed as described previously with minor modifications.^{17, 21} In brief, E. coli Rosetta DE2 cells were transformed with a plasmid expressing human WT aSyn inoculated in 5-mL tubes containing LB media supplemented with kanamycin. The mini-cultures were incubated overnight at 225 rpm and 37 °C and expanded to 1-L cultures the following morning. After 4.5 h of induction with 1 mM IPTG, bacterial cultures were harvested by pelleting at 4200 × g for 20 min at 4 °C. Pellets were dissolved by adding a lysis buffer composed of 50 mM Tris and 500 mM NaCl at pH 7.4. Then, bacterial lysate was heat-precipitated at 90 °C for 15 min and centrifuged to remove the unwanted precipitates at 15,000 × g for 20 min. Later, DNA was precipitated out by incubating with streptomycin (10 mg/mL) for 30 min followed by centrifugation at 23,000 × g for 30 min and supernatants were dialyzed in a 20 mM Tris HCl buffer at pH 8 overnight at 4 °C to remove the excess salt of the lysis buffer. Next, supernatants were concentrated and filtered before loading onto a Sephacryl 200 column (GE Healthcare Life Sciences) pre-equilibrated in 20 mM Tris-HCl buffer of pH 8 at 4 °C for size-exclusion FPLC. Later, fractions positive for recombinant aSyn were combined, concentrated, and 0.2-μm filtered before loading onto a HiPrep Q FF 16/10 anion-exchange column and aSyn protein was recovered between 300 and 350 mM NaCl. Fractions with recombinant aSyn were pooled and dialyzed overnight in 4 L of 20 mM Tris, pH 8, at 4 °C. The following day, the buffer was replaced with a new buffer for one hour and the protein was 0.2-μm filtered. Protein concentrations were determined using a NanoDrop spectrophotometer with an extinction coefficient of 0.5960 mg∙mL^{− 1}∙cm^{− 1} and stored at −80 °C.

RT-QuIC assay

The RT-QuIC assay was performed as described previously with some modifications^{17, 18, 21, 42} using a 96-well clear-bottom plate (Nalgene Nunc International). The reaction mixture consists of final concentrations of 40 mM phosphate buffer (pH 8.0), 170 mM NaCl, 10 μM ThT, 0.00125% sodium dodecyl sulfate (SDS) and 0.1 mg/mL of recombinant aSyn as a substrate. To seed the RT-QuIC reactions, we used a 10⁻¹ (w/v) dilution of skin tissue homogenates or FFPE tissue sections. Samples were homogenized to 10% (w/v) and diluted 10-fold with sterile TBS as described previously.^{17, 21, 43–47} Unless specified, each reaction consisted of 2 μL of test sample as a seed and 98 μL of αSyn RT-QuIC reaction mixture with six 0.8-mm silica beads (OPS Diagnostics) per each well in a 96-well plate. For samples prepared from FFPE and cryostat sections, 5 μL of test sample was added to 95 μL of RT-QuIC reaction mixture. The plates were sealed and incubated at 42 °C in a CLARIOstar (BMG) plate reader with alternating 1-min shake and rest cycles (double orbital, 400 rpm) and the gain was set to 2500 units. ThT fluorescence readings were recorded every 30 min at excitation and emission wavelengths of 450 and 480 nm, respectively. Each sample was tested in quadruplicates and most of the positive samples amplified in all 4 of their technical replicates. Threshold ThT fluorescence was calculated by averaging fluorescence of the first 10 cycles for all samples plus 10 standard deviations to determine the protein aggregation rates (PAR) for each sample. PAR was calculated by taking the inverse of the time required to cross the threshold fluorescence.

Immunoblotting

Conformation of the RT-QuIC end-products was analyzed by dot blot using a Bio-Dot Microfiltration System as described previously.^{17, 21, 44} The end-products from each well of the RT-QuIC assay plate were diluted 40-fold in 100 μL of PBS and blotted on to a nitrocellulose membrane for 1 h. Next, we incubated membranes in 1X LI-COR blocking buffer (LBB) for 30 min and then added a rabbit monoclonal aSyn filament conformation-specific antibody (MJFR-14-6-4-2; 1:2,000) and a mouse monoclonal total aSyn antibody (BD Biosciences, #610787; 1:2,000) for one hour and were triple-washed with 1X TBS for 10 min each. Later, membranes were incubated with respective secondary antibodies conjugated with IR dye (LI-COR) made in LBB (1:15,000) for 30 min followed by triple-washing with 1X TBS. Later, membranes were scanned with a LI-COR machine and densitometric quantification of dots was done using ImageJ software as described previously.^{17, 21, 43, 44, 47}

Statistical analysis

GraphPad 7.0 was used for statistical analysis. Raw data were analyzed using Student’s two-sample unpaired t-test. Asterisks were assigned as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. The number of biological replicates is expressed as “n” unless otherwise mentioned. GraphPad 8.0 was used for multivariate correlation analysis.

Results

Establishing the optimal titration for frozen skin tissues to test in the RT-QuIC assay

Since aSyn exhibits a protein-seeding phenomenon similar to prions, we examined the skin samples from PD patients for aSyn seeding activity using the ultrasensitive and specific RT-QuIC assay. We^{17, 21} and others^{18, 42, 48} have previously shown that the aSyn from α-synucleinopathy cases can be detected in various human tissues using the RT-QuIC assay with recombinant human aSyn as a substrate and ThT as a fluorescent dye to bind to the protein aggregates. Since the concentrations of tissue sample and SDS in the assay mixture are major determinants in the protein seeding activity, our initial set of experiments was conducted to optimize the concentrations of skin lysate and SDS in the RT-QuIC reaction mixture used for frozen human skin tissues.^{17, 21, 43–47, 49} For this, we tested various dilutions (w/v) of skin tissues (10⁻¹ to 10⁻⁴) from a set of unblinded subjects (3 PD and 3 controls) in the RT-QuIC assay with 0.00125% SDS in the reaction mixture (Fig 1). Among various dilutions tested, both 10⁻¹ and 10⁻² (w/v) amplified in all the biological and technical replicates at an earlier time (before 10 h) than controls (Fig 1A) but more effective amplifications were achieved with the 10⁻¹ dilution. In contrast, no specific result was seen with either the10⁻³ or 10⁻⁴ (w/v) dilution. Among the concentrations of SDS tested, 0.00125% was found to be optimal. Therefore, we used a 10⁻¹ (w/v) dilution of skin tissues with 0.00125% SDS in the RT-QuIC reaction mixture for further blinded testing of a larger set of samples.

Figure 1.

Optimization of the RT-QuIC assay for frozen skin tissue homogenates. RT-QuIC amplification profiles of the ThT fluorescence for (A) 10⁻¹, (B) 10⁻², (C) 10⁻³, and (D) 10⁻⁴ dilutions made from 10% (w/v) skin homogenates using 0.00125% SDS in the RT-QuIC reaction mixture. Each trace represents the average of 4 technical replicates for each PD (red, n=3) and control (green, n=3) subject.

RT-QuIC detection of aSyn seeding activity from skin homogenates of PD patients

After optimizing the RT-QuIC assay conditions for frozen skin tissues, we tested an independent cohort consisting of 44 frozen skin samples from 22 PD and 22 control subjects. These samples were collected from the scalp region during autopsy by the Banner Sun Health Research Institute (BSHRI) Brain and Body Donation Program (www.brainandbodydonationprogram.org) and sent to Iowa State University (ISU) after de-identifying and blinded coding of the samples. Using the optimized dilution (10⁻¹ w/v), we tested all 44 samples for aSyn seeding activity and RT-QuIC results were sent to BSHRI for unblinding. After unblinding the RT-QuIC results, we accurately identified 20 PD and 20 controls, leaving 2 false positives and 2 false negatives among 22 PD and 22 control samples. For the 2 false positive samples, all 4 technical replicates showed enhanced ThT fluorescence, whereas none of false negative sample replicates amplified. Hence, to eliminate error during sample preparation, we retested these 4 samples with new skin tissues in a blinded manner consisting of a total 8 cryostat sections. Of the cryostat sections tested, the RT-QuIC identified all samples correctly except 1 PD and 1 control, which is consistent with the frozen skin tissue results. Overall, RT-QuIC identified 21 PD and 21 control subjects among 22 in each group of human skin tissues. In all quadruplicate wells tested in the RT-QuIC assay, aSyn seeding activity occurred earlier in the skin samples from all the 21 PD cases, which crossed the threshold fluorescence within 10 h, whereas aSyn seeding activity from the 21 control skin samples did not occur during the 20-h period (Fig 2A). As such, the skin tissue homogenates from PD samples exhibited a protein aggregation rate (PAR) significantly higher than the PAR of age-matched control samples (Fig 2B). PAR was calculated as described previously^{17, 43–46} based on the inverse of time to cross the threshold fluorescence. To further confirm the amplification of aSyn in the RT-QuIC end-products, we subjected the RT-QuIC end-products from the frozen skin tissues present in the 96-well plate to a dot blot analysis with an aSyn filament conformation-specific antibody. The immunoblots were also stained for total aSyn to quantify total aSyn among these samples. Consistent with the RT-QuIC ThT fluorescence data, the RT-QuIC end-products from frozen skin tissues of PD cases contained aSyn levels that were significantly higher than control cases, whereas total αSyn levels appear to be slightly higher in controls, although the difference between control and PD dot intensities was not statistically significant (Fig. 2C and 2D).

Figure 2.

RT-QuIC detection of aSyn seeding activity from frozen skin tissues and cryostat sections. (A) Enhanced ThT fluorescence indicating more aSyn seeding activity in PD skin tissues compared to controls. (B) PAR of PD and control skin homogenates showing that PD samples had higher aSyn load compared to controls. The 22 PD (red) and 22 control (green) samples consisted of 20 frozen tissues and 2 cryostat sections, all tested in quadruplicates and expressed as the mean and standard error of 4 technical replicates. (C) A representative dot blot image consisting of 6 PD and 6 control skin RT-QuIC end-products with aSyn filament conformation-specific (top panel) and total aSyn (bottom panel) antibodies. (D) Densitometric quantification of aSyn filament and total aSyn levels, showing that the RT-QuIC end-products in PD wells had elevated levels of misfolded aSyn compared to control wells, while total aSyn levels were not significantly different between the groups. Results were analyzed using Student’s two-sample t-test. Data expressed as the mean and standard error of 4 technical replicates. ****=P<0.0001 and not significant (ns) when p>0.05.

Establishing the optimal working dilution for skin FFPE sections to test in the RT-QuIC assay

Since an earlier study of ours involving FFPE sections from SMG tissues showed excellent seeding activity for aSyn in the RT-QuIC assay²¹, we tested for aSyn seeding activity in skin FFPE sections as well. We first optimized the working dilution required for skin FFPE sections using 3 PD and 3 control skin FFPE sections. Similar to frozen skin tissues, after recovering the skin tissue from FFPE sections by serial xylene and descending grades of alcohol washes, we tested four dilutions (w/v) consisting of 10⁻¹ to 10⁻⁴ in the RT-QuIC assay using 0.00125% SDS in the reaction mixture. Among these, the 10⁻¹ (w/v) dilution amplified at a much earlier time (within 15 h) in all the biological and technical replicates of PD skin FFPE sections compared to 10⁻² and 10⁻³ dilutions (Fig 3A), whereas 10⁻⁴ dilutions and all controls failed to amplify. Hence, for testing the remaining samples, we used a 10⁻¹ (w/v) dilution for skin FFPE sections with 0.00125% SDS in the RT-QuIC reaction mixture (Fig 3).

Figure 3.

Optimization of the RT-QuIC assay for skin FFPE sections. RT-QuIC of FFPE skin tissues was tested in serial dilutions of the RT-QuIC reaction mixture that was 0.00125% SDS. ThT fluorescence profiles for (A) 10⁻¹, (B) 10⁻², (C) 10⁻³, and (D) 10⁻⁴ dilutions in the RT-QuIC assay consists of PD (red, n=3) and control (green, n=3) skin homogenates. Each trace represents the average of 4 technical replicates.

Detection of aSyn seeding activity from FFPE skin sections using the RT-QuIC assay

Next, we tested the remaining 18 skin FFPE samples (9 PD and 9 control) received from BSHRI in a blinded manner. Similarly, skin tissues from these autopsied FFPE sections were retrieved and used as a seed in the RT-QuIC assay. Interestingly, the skin FFPE homogenates from PD cases displayed enhanced ThT fluorescence within 10 h (Fig 4A) and significantly higher PAR values (Fig 4B) compared to controls. To further confirm the selective amplification of aSyn from the FFPE skin sections in the RT-QuIC assay, we did dot blot quantification of RT-QuIC end-products with aSyn filament-specific and total aSyn antibodies. Similar to frozen tissues, significantly higher levels of misfolded aSyn were seen in the RT-QuIC end-products from PD samples, whereas total aSyn levels did not differ significantly (Fig 4C & 4D). Overall, 9/12 PD and 10/12 controls were correctly identified using RT-QuIC with FFPE skin sections (75% sensitivity and 83% specificity). Taken together, these results clearly demonstrate aSyn seeding activity in skin FFPE sections, further indicating the biomarker value of skin aSyn in PD patients from archived FFPE sections.

Figure 4.

RT-QuIC detection of aSyn seeding activity from FFPE skin sections. (A) Enhanced ThT fluorescence in skin FFPE sections from PD cases compared to controls indicating more aSyn seeding activity in PD skin FFPE sections. (B) PAR of PD and control skin FFPE section homogenates showing elevated aSyn load in PD samples compared to controls. We tested 9 PD (red) and 9 control (green) samples, each in quadruplicate, with their data expressed as the mean and standard error of 4 technical replicates. (C) A representative dot blot image consisting of 5 PD and 5 control FFPE skin RT-QuIC end-products with αSyn filament conformation-specific (top panel) and total αSyn (bottom panel) antibodies. (D) Densitometric quantification of aSyn filament and total aSyn levels, showing that the RT-QuIC end-products of PD wells had elevated levels of misfolded aSyn compared to control wells, while total aSyn levels did not differ significantly between groups. Results were analyzed using Student’s two-sample t-test. Data expressed as the mean and standard error of 4 technical replicates. ***=P<0.001, ** = P<0.01, and not significant (ns) when p>0.05.

Discussion

The clinical diagnosis and treatment of PD have remained challenging despite several advances in the understanding of its pathogenesis. The definitive diagnosis of PD is only achieved at autopsy; therefore, less invasive pre-clinical diagnostic biomarkers are essential to facilitate earlier diagnosis and effective treatment. We and others have shown a highly sensitive and specific RT-QuIC assay for detecting aSyn from the brain and CSF.^{17, 18, 42} More recently, we demonstrated that RT-QuIC testing of autopsied SMG samples was able to differentiate PD from controls with 100% sensitivity and 94% specificity.²¹ Moreover, we reported the utility of both frozen tissues as well as FFPE sections as suitable material to seed the RT-QuIC reactions.^{17, 21} Although both CSF and SMG tissues also serve as suitable seeding material for the RT-QuIC, obtaining these samples is more invasive and requires specialized medical personnel, limiting their usefulness.^{12, 30}

Recently, several IHC studies of skin biopsies identified the accumulation of aSyn, thus pointing to skin as a promising tissue type.^{34, 50} Considered the largest organ in the body, the skin shares the same embryonic origin as neural tissue, and is well connected to the central nervous system^{51, 52}. Hence, it is logical to hypothesize that the disease-associated protein accumulations that precipitate in the brain may also be found in the skin. Furthermore, IHC of skin tissue proved useful for identifying the immunoreactivity of serine-129 phosphorylated aSyn, which is a major hallmark of aSyn pathology.^{53, 54} However, cutaneous IHC detection suffers from limitations related primarily to insufficient sensitivity but also due to the patchy availability of autonomic nerves in a given section and the need, for some methods, for specialized and idiosyncratic sample processing and slide interpretation.^{28, 34, 55–58} Moreover, oligomeric forms of aSyn were detected in the synaptic terminals of skin tissue using a proximal ligation assay with 82% sensitivity and 89% specificity in a recent case-control study³⁵. Overall, to address the limitations associated with the other techniques, we probed the aSyn seeding activity of skin tissues using the RT-QuIC assay. A schematic representation of skin biopsy as a potential biomarker for PD using the RT-QuIC assay is depicted in Fig 5. Our study not only demonstrates the presence of aSyn seeding activity in frozen skin samples of PD patients with high sensitivity (96%) and specificity (96%), but also that skin-based RT-QuIC determination of aSyn seeding activity and PAR may be used to effectively discriminate PD from control samples. This conclusion is further supported by the strong and concordant amplification of the ThT signal in all the technical replicates for each sample tested in the RT-QuIC assay. Along with frozen skin tissues, we also tested FFPE skin sections from the same cohort for aSyn seeding activity and we were able to detect enhanced ThT fluorescence in PD cases compared to controls with 75% sensitivity and 83% specificity. The relatively lower sensitivity and specificity for FFPE sections could be due to an insufficient quantity of aSyn present to seed the RT-QuIC reactions, possibly stemming from either the limited amount of tissue present on 5-μm thick sections or perhaps a suppressing effect of formalin fixation. Despite the relatively lower sensitivity, FFPE sections remain an alternative option in scenarios when fresh or frozen tissues are unavailable.

Figure 5.

Schematic representation of the aSyn RT-QuIC assay as a tool for monitoring PD using skin biopsy. Skin biopsies collected from the scalp were processed for testing in 96-well plates using a high-throughput RT-QuIC workflow that can be finished in 24 h. Each well consists of 2 μL of skin tissue homogenate in 98 μL of αSyn RT-QuIC reaction mixture containing recombinant human aSyn protein as a substrate. Skin samples from PD cases showed higher ThT fluorescence compared to controls due to the elevated levels of aSyn.

Moreover, the associations of the RT-QuIC PAR values with various PD patient metrics were assessed using correlation analysis. The RT-QuIC PAR values were significantly correlated with unified LB stage (p<0.0001), pathological diagnosis (p<0.0001), MMSE scores (p=0.0035), UPDRS scores (p<0.0001), disease duration (p=0.0029) and sex (p=0.0031). However, age was not significantly correlated (Table S3). Altogether, our results suggest that the aSyn present in skin can serve as a potential biomarker for PD, and if validated in longitudinal studies with larger cohorts, the RT-QuIC assay of aSyn levels could not only help in disease diagnosis but also in monitoring disease progression and identifying the efficacy of treatments. In conclusion, we report excellent sensitivities and specificities after using RT-QuIC to detect aSyn from from PD cases. Furthermore, multi-center studies of larger cohorts are warranted to better determine the diagnostic accuracy of cutaneous aSyn as a biomarker for synucleinopathies.