==== Front PLoS Pathog PLoS Pathog plos PLOS Pathogens 1553-7366 1553-7374 Public Library of Science San Francisco, CA USA 10.1371/journal.ppat.1011456 PPATHOGENS-D-23-00689 Research Article Medicine and Health Sciences Surgical and Invasive Medical Procedures Biopsy Medicine and Health Sciences Medical Conditions Neurodegenerative Diseases Movement Disorders Parkinson Disease Medicine and Health Sciences Neurology Neurodegenerative Diseases Movement Disorders Parkinson Disease Biology and Life Sciences Anatomy Digestive System Gastrointestinal Tract Duodenum Medicine and Health Sciences Anatomy Digestive System Gastrointestinal Tract Duodenum Biology and Life Sciences Anatomy Digestive System Gastrointestinal Tract Medicine and Health Sciences Anatomy Digestive System Gastrointestinal Tract Medicine and Health Sciences Gastroenterology and Hepatology Constipation Medicine and Health Sciences Clinical Medicine Signs and Symptoms Constipation Medicine and Health Sciences Diagnostic Medicine Medicine and Health Sciences Pharmacology Drugs Levodopa Biology and Life Sciences Biochemistry Neurochemistry Neurochemicals Levodopa Biology and Life Sciences Neuroscience Neurochemistry Neurochemicals Levodopa Research and Analysis Methods Histochemistry and Cytochemistry Techniques Immunohistochemistry Techniques Research and Analysis Methods Immunologic Techniques Immunohistochemistry Techniques α-Synuclein seeding activity in duodenum biopsies from Parkinson’s disease patients α-Synuclein seeding activity in Parkinson’s disease intestinal biopsies https://orcid.org/0000-0002-6493-8669 Vascellari Sarah Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Validation Visualization Writing – original draft Writing – review & editing 1 * Orrù Christina D. Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Validation Visualization Writing – original draft Writing – review & editing 2 Groveman Bradley R. Data curation Formal analysis Investigation Visualization Writing – review & editing 2 Parveen Sabiha Data curation Formal analysis Investigation Visualization 2 Fenu Giuseppe Data curation Investigation 3 Pisano Giada Data curation Investigation 3 Piga Giuseppe Data curation Investigation 3 Serra Giulia Data curation Investigation 3 Oppo Valentina Data curation Investigation 3 Murgia Daniela Data curation Investigation 3 Perra Andrea Investigation Visualization Writing – review & editing 1 Angius Fabrizio Visualization Writing – review & editing 1 Hughson Andrew G. Data curation Formal analysis Investigation 2 Haigh Cathryn L. Supervision 2 Manzin Aldo Supervision Writing – review & editing 1 Cossu Giovanni Conceptualization Data curation Investigation Resources Supervision Writing – review & editing 3 ‡ Caughey Byron Conceptualization Funding acquisition Resources Supervision Writing – original draft Writing – review & editing 2 ‡ 1 Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy 2 Laboratory of Neurological Infections and Immunity (LNII), Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), National Institute of Health (NIH), Hamilton, Montana, United States 3 S. C. Neurology and Stroke Unit, AOBrotzu, Cagliari, Italy Woerman Amanda L. Editor University of Massachusetts Amherst, UNITED STATES BC, CDO and AH are inventors on patent applications pertaining to aSyn RT-QuIC technology. The other authors have declared that no competing interests exist. ‡ These authors are joint senior authors on this work. * E-mail: svascellari@unica.it 30 6 2023 6 2023 19 6 e101145620 4 2023 2 6 2023 https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Abnormal deposition of α-synuclein is a key feature and biomarker of Parkinson’s disease. α-Synuclein aggregates can propagate themselves by a prion-like seeding-based mechanism within and between tissues and are hypothesized to move between the intestine and brain. α-Synuclein RT-QuIC seed amplification assays have detected Parkinson’s-associated α-synuclein in multiple biospecimens including post-mortem colon samples. Here we show intra vitam detection of seeds in duodenum biopsies from 22/23 Parkinson’s patients, but not in 6 healthy controls by RT-QuICR. In contrast, no tau seeding activity was detected in any of the biopsies. Our seed amplifications provide evidence that the upper intestine contains a form(s) of α-synuclein with self-propagating activity. The diagnostic sensitivity and specificity for PD in this biopsy panel were 95.7% and 100% respectively. End-point dilution analysis indicated up to 106 SD50 seeding units per mg of tissue with positivity in two contemporaneous biopsies from individual patients suggesting widespread distribution within the superior and descending parts of duodenum. Our detection of α-synuclein seeding activity in duodenum biopsies of Parkinson’s disease patients suggests not only that such analyses may be useful in ante-mortem diagnosis, but also that the duodenum may be a source or a destination for pathological, self-propagating α-synuclein assemblies. Author summary Misfolded α-Synuclein deposition is a hallmark of Parkinson’s disease. The gastrointestinal tract may be an initial site of α-Synuclein aggregation, and its detection might be useful in the early diagnosis of Parkinson’s disease. Here, we have used a rapid, ultrasensitive seed amplification assay (RT-QuICR) to show that pathologic α-Syn aggregates with prion-like self-propagating activity are in the upper intestine (duodenum) of Parkinson’s disease patients. Our intra vitam detection of α-synuclein seeding activity in duodenum biopsies gave high diagnostic accuracy. Quantitation revealed high levels of seeds in duodenal tissue. Thus, our findings suggest that abnormal α-Synuclein seeds in the upper intestine might be both an early accurate biomarker for Parkinson’s disease and cause of gut dysfunction. http://dx.doi.org/10.13039/100006492 Division of Intramural Research, National Institute of Allergy and Infectious Diseases Caughey Byron This work was partially supported by Intramural Research Program of the National Institute for Allergy and Infectious Diseases, National Institutes of Health (BC). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CDO, BRG, SP, AGH, CLH and BC received a salary from Intramural Research Program of the National Institute for Allergy and Infectious Diseases, National Institutes of Health. Data AvailabilityAll relevant data are within the paper and its Supporting Information files. Data Availability All relevant data are within the paper and its Supporting Information files. ==== Body pmcIntroduction Definitive diagnosis of Parkinson’s disease (PD) relies on postmortem detection of disease-associated α-Synuclein aggregates (α-SynD) in the brain [1]. According to the “dual hit hypothesis”, α-SynD may start accumulating at the site of enteric nerves in the gut or the olfactory bulb, and progressively spread in stages along neural tracts to the brain [2]. Others have suggested a reverse path of α-SynD spreading, which could originate from the brain and then reach the gastrointestinal (GI) tract [3]. Clinical and neuropathological evidence indicates that motor symptoms in PD are often preceded by GI dysfunctions [4, 5] leading some researchers to consider PD as a gut-brain disorder. One recent study showed α-SynD in the colon of pre-symptomatic mice expressing human A53T α-Syn before detecting it in the brain [6]. It is also postulated that both the gut and brain first hypotheses are possible, which would lead to a new classification of PD, depending on the different initial site of α-SynD accumulation. These include a “brain-first” subtype, in which pathology originates primarily in the brain, and a “body-first” subtype, characterized by premotor rapid eye movement behavior disorder (RBD) and GI impairment, in which pathological changes begin in the enteric or peripheral autonomic nervous systems, and follow a route that involves the brainstem [7–10]. These considerations make α-SynD detection in intestinal tissues an attractive strategy for the early diagnosis of PD. However, conventional immunological-based techniques for detection of α-SynD have produced conflicting results, likely due to the limited sensitivities of these assays [11]. One recent study suggested that immunohistochemistry (IHC) based methods are not suitable for the diagnosis of PD [12]. In contrast, an initial report by Emmi et al [13] showed IHC detection of α-SynD in 100% of duodenum samples tested from a small number of PD patients. Other studies also propose that IHC analysis of layers deeper than mucosa and submucosa, such as muscularis propria or myenteric plexuses, for enteric phosphorylated α-Syn detection could be used to predict the onset of motor symptoms in PD [14]. Recently, ultrasensitive seed amplification assays (SAAs) have been applied to help address these controversial questions. Based in principle on the RT-QuIC assay platform developed for PrP prions [15, 16], the RT-QuIC-like SAAs for synucleinopathies are techniques that exploit the ability of α-SynD to self-replicate similarly to prions, and to amplify trace amounts of pathological α-Syn in biological specimens (e.g., brain tissues, cerebrospinal fluid [CSF], submandibular glands, saliva, olfactory mucosa and skin) [17–25]. SAAs, such as α-Syn RT-QuIC [18, 19] and α-Syn PMCA [26], have been shown to detect α-SynD in intestinal tissues. However, these studies detected α-SynD seeds in only 55% of cases [27], or used postmortem samples [28]. In this study, we analyzed duodenum biopsies from PD and control patient cohorts with a relatively rapid α-Syn RT-QuIC assay (RT-QuICR), which uses a mutated version of the human α-synuclein protein and achieves faster and more specific detection of α-SynD [29] than prior SAAs [18, 26]. Given reports of tau and α-Synuclein copathologies [30–34], we also checked a subset of the biopsies for tau seeding activity using a tau RT-QuIC assay [35]. We found α-SynD, but not tau, seeding activity in the intestinal mucosa (IM) tissue in 95.7% of PD cases, suggesting both diagnostic and potential pathological implications. Results Patient demographics and clinical information Demographic and clinical information about the participants recruited for this study is given in Table 1. The mean age at the time of biopsy collection was 67 ± 8 years for PD cases and 58 ± 10 years old for healthy controls (HCs). The number of male and female patients within the PD and HC cohorts was similar. For PD patients, the mean disease duration at the time of intestinal biopsy collection was 14 ± 5 years. Thirteen of the 23 cases showed premotor RBD from 7 years before the onset of motor symptoms. At the time of biopsy collections all PD cases were on levodopa medication and the mean of Unified Parkinson’s Disease Rating Scale (UPDRS) III motor score was 25 ± 7. Several PDs reported moderate/severe constipation as a common condition of GI impairment with an average score of 11 ± 6, ranging from a minimum score of 3 to a maximum of 21. Upper GI biopsies were performed and the mucosa from proximal small intestine (duodenum) was taken from each subject and tested by RT-QuICR assay. 10.1371/journal.ppat.1011456.t001 Table 1 Demographic characteristics of Parkinson’s and healthy control patients. ID Diagnosis Age1 Sex (M/F) Disease duration1 Constipation score premotor RBD2 UPDRS III score1 Duodenal biopsies tested by RT-QuICR Parkinson’s disease 67±8 11/12 14±5 11±6 13 (57) 25±7 n = 30 IM1 73 F 6 17 yes 23 2 IM2 65 M 25 19 yes 19 1 IM3 75 M 11 3 no 24 2 IM4 65 F 15 14 yes 24 1 IM5 65 F 10 16 yes 20 1 IM6 76 F 14 19 yes 21 1 IM7 58 F 11 10 no 20 1 IM8 58 M 15 14 no 13 1 IM9 66 M 17 18 no 28 1 IM10 70 M 12 4 yes 38 1 IM11 70 F 15 6 yes 23 1 IM12 69 F 12 14 yes 27 1 IM13 68 M 19 7 no 27 1 IM14 74 F 14 5 yes 39 1 IM15 57 F 20 6 yes 28 1 IM16 69 F 12 21 no 14 1 IM17 56 M 8 18 no 19 1 IM18 77 M 6 15 yes 23 1 IM22 56 F 19 2 no 38 2 IM23 55 M 13 7 yes 26 2 IM24 77 M 20 12 no 21 2 IM26 58 F 8 4 yes 30 2 IM27 74 M 13 7 no 23 2 Healthy Controls 58 ±10 3/3 n = 12 IM19 42 M 2 IM20 59 F 2 IM28 60 M 2 IM29 72 M 2 IM30 53 F 2 IM31 62 F 2 1At time of biopsy (mean±SD) 2 Number of cases with premotor RBD and related percentages in parenthesis: n (%) Abbreviations: RBD, rapid eye movement behavior disorder; SD, Standard Deviation; UPDRS, Unified Parkinson Disease Rating Scale. Histology and immunohistochemistry of human intestinal mucosa biopsies To visualize the general morphology and cellular composition of IM tissues, selected biopsies were examined after staining with hematoxylin and eosin (H&E). Representative samples from two patients showed that IM biopsies mainly contain the mucosa layer (Fig 1A), or mucosa associated to a thin layer of submucosa (Fig 1B). These findings suggest that standard endoscopic duodenal biopsies, although variable in size, always include the mucosa and may also include small amounts of the sub-mucosal layer. In both specimens, microscopic analysis did not reveal pathological findings. We also performed IHC for thyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) but it was not informative due to the low sensitivity of the method (see S1 Fig and S1 Text). 10.1371/journal.ppat.1011456.g001 Fig 1 Tissue morphology in duodenum intestinal mucosa biopsies. (A-B): Representative photomicrographs of duodenum biopsies stained with hematoxylin and eosin. Both samples showed a normal morphology, well represented mucosa (black box) and variable amounts of submucosa (black arrow) depending on the biopsy specimen. Magnification x10. Assessment of IM tissue matrix inhibition of α-SynD RT-QuICR assay Previous studies had shown matrix inhibition of the RT-QuIC when testing tissues such as brain with higher concentrations of material [19, 36]. Therefore, to investigate the ability of the RT-QuICR to discriminate between PD and HC subjects, we initially tested a subset of IMs at 10−2 and 10−3 tissue dilutions (Fig 2). Our results showed inhibition of RT-QuICR amplification at 10−2, with most of the wells being negative for each sample. However, when seeding with a 10−3 IM a marked improvement was observed in the ability to discriminate between PD and HC samples. We therefore tested the remaining of the samples at 10−3 or further dilutions. Primary RT-QuICR fluorescence curves for representative PD (n = 3) and HC (n = 3) cases are shown in S2 Fig. 10.1371/journal.ppat.1011456.g002 Fig 2 Optimization of IM tissue dilutions used for RT-QuICR detection of α-SynD. Inverse of the time to threshold for reactions seeded with IM tissue dilutions from PD or HC cases with the designated sample codes. Each symbol indicates a single reaction well with each sample analyzed in quadruplicate at both 10−2 (black triangles) and 10−3 (green squares) dilutions. A reaction time cut off of 40 h was used. The dotted line indicates the threshold for a positive reaction. Diagnostic sensitivity of RT-QuICR detection of α-SynD in IM biopsies We next performed a blinded RT-QuICR analysis of an additional panel of IMs. A total number of 42 duodenal biopsies from 23 PD cases and 6 HCs were tested. Overall, we observed positive RT-QuICR responses in 22 out of the 23 duodenal biopsies from PD patients, and negative responses for all 6 HCs (Fig 3). The sensitivity and specificity of the RT-QuICR were 95.7% (95% CI: 78.1% to 99.9%) and 100% (95% CI: 54.1% to 100.0%), respectively (Table 2). Positive and negative predictive values (PPV and NPV) were 100.0% and 85.7% (95% CI: 46.9–97.6%), respectively. The value of diagnostic efficacy, expressed as the ratio between the sum of subjects classified as true positives and negatives, and the sum of the subjects classified as false positives and negatives, was 96.6% (95% CI: 82.2–99.9%). 10.1371/journal.ppat.1011456.g003 Fig 3 RT-QuICR detection of α-SynD in duodenum IM biopsies. Symbols represent the inverse of time to threshold for individual reaction wells seeded with a 10−3 IM tissue dilution from the designated PD or HC IM sample ID numbers. The dotted line indicates the threshold for a positive reaction. Bars show mean ±SD. Quadruplicate reaction wells are shown for each sample, with the exception of samples 6 and 7. The overall RT-QuICR status for the latter samples were inconclusive based on the initial quadruplicates. Upon repeating 6 and 7, as per the methods, both were determined to be positive overall and results from all 8 wells are shown here. 10.1371/journal.ppat.1011456.t002 Table 2 Diagnostic performance of IM α-Syn RT-QuICR. Measures Value 95% CI Sensitivity 95.7% 78.1% to 99.9% Specificity 100.0% 54.07% to 100.0% PLR NA NA NLR 0.04 0.01 to 0. 30 Disease prevalence 79.3% 60.3% to 92.0% PPV 100.0% NPV 85.7% 46.9% to 97.6% Youden’s index 0.95 Accuracy 96.6% 82.2% to 99.9% Abbreviations: PLR, Positive Likelihood Ratio; NLR, Negative Likelihood Ratio; PPV, Positive Predictive Value; NPV, Negative Predictive Value; CI, Confidence interval expressed as percentages; NA: Not available, the value cannot be calculated because the denominator is 0. Quantitation of α-SynD seeding activities in PD duodenum biopsies To measure the levels of α-SynD seeding activity in PD IM biopsies, end-point dilution analysis was performed [19]. IM samples were serially diluted from 10−3 to 10−6 for RT-QuICR analysis. Tissue concentrations of seeding units giving 50% positive replicate reactions (50% seeding doses or SD50s) were then estimated using a modified Spearman-Kärber algorithm [37]. SD50 concentrations were calculated for all the PD samples with the exception of samples 6 and 7, which did not have high enough seeding activity for the algorithm to be applied properly (Fig 4). On average, RT-QuICR-positive IMs from PD patients had 4.3 ± 0.8 log SD50 per mg of tissue (range: 2.7 to 5.7). 10.1371/journal.ppat.1011456.g004 Fig 4 RT-QuICR quantification of seeding activity in PD duodenum biopsies. Orange triangles mark log SD50/mg values from individual IM biopsies. Dark red triangles indicate values for PD IMs that did not completely reach end-point (0/4 positive wells) in our serial dilutions and therefore may be slight underestimations of the seeding activity by Spearman-Kärber analysis. However, each of those cases diluted to 2/4 or 1/4 positive wells which is at, or below, the definition of 1 SD50, respectively, in those diluted aliquots, meaning that values shown in the figure should be close to being accurate. The dotted line indicates the RT-QuICR detection limit. RT-QuICR testing of two IM biopsies from individual patients To address the question of α-SynD distribution within the duodenum intestinal mucosa, we tested two samples collected during the same procedure from the superior and descending part of duodenum from 9 individual PD patients and 6 HC patients (Fig 5A). With the exception of patient 27, who was positive in only one of the two samples, RT-QuICR detected α-synD in both biopsies from each patient. To evaluate whether α-SynD levels were consistent across the duodenum sampling areas, end-point dilution analysis was performed on each specimen. Log SD50s per mg of tissue (Fig 5B) varied by 0.5±0.4 between biopsies from the same patient. 10.1371/journal.ppat.1011456.g005 Fig 5 Comparison of seeding activities in IM samples collected from first and second duodenum segments from individual patients. (A) Inverse of time to fluorescence threshold comparisons for PD (orange) and HC (blue) cases. Each IM was analyzed in quadruplicate at 10−3 dilution, except for sample 27 which shows values from 8 reaction wells, with each symbol representing an individual reaction well from either the first (biopsy 1; triangles) or second (biopsy 2; circles) duodenum segments from the given patient. (B) Log SD50/mg comparisons from end-point dilution analysis. The dark red circle represents a PD IM that did not reach end-point, and thus is likely an underestimate of the actual value. Grey triangle indicates a positive sample for which a log SD50/mg value could not be accurately calculated due to low seeding activity. Log SD50/mg for the first samplings are the same as those reported in Fig 2. The dotted lines indicate the threshold for detection (A) and the assay’s detection limit (B). Lack of correlation between α-SynD seed concentrations and clinical parameters Spearman’s correlation analysis was performed to investigate the relationship between the concentration of α-SynD (log SD50/mg) and clinical parameters. No significant correlation between SD50 values and UPDRS III motor scores (r = – 0.23; p = 0.25; 95% CI:– 0.57% to 0.18; alpha = 0.05) (Fig 6A), constipation scores (r = 0.27; p = 0.18; 95% CI:– 0.14% to– 0.60; alpha = 0.05) (Fig 6B) and disease duration (r = 0.10; p = 0.64; 95% CI:– 0.31% to 0.47; alpha = 0.05) (Fig 6C) was observed. 10.1371/journal.ppat.1011456.g006 Fig 6 Spearman correlation between α-synD seed concentration (logSD50/mg tissue) and clinical parameters. (A) UPDRSIII score versus IM log SD50/mg while patient was on Levodopa medication. (B) Constipation score and (C) Disease duration versus seed concentration. Spearman’s r and p values are inset. Lack of 3R or 3R+4R tau seeding activity in the IM biopsies For comparative purposes, we also tested PD (n = 9) and HC (n = 1) IM biopsies for the presence of proteopathic seeds of another cytosolic protein, tau, using a tau RT-QuIC assay that ultrasensitively detects the tau seeds associated with either 3R (Pick’s disease) or 3R+4R (Alzheimer’s disease and chronic traumatic encephalopathy) tauopathies [35]. This comparison was of interest because of reported evidence of tau and synuclein co-pathologies in PD and Alzheimer’s disease, and the detection of tau in Lewy bodies [30–34]. However, no tau seeding activity was detected in any of the IM specimens (Fig 7). As an indication that the IM tissue matrix did not prevent detection of tau seeds using this K12 tau RT-QuIC assay, we found that dilutions as extreme as 10−5 of sporadic Alzheimer’s disease brain homogenates spiked into IM sample dilutions of 10−2 to 10−4 were readily detected. Simultaneous dilutions of the AD brain homogenates themselves showed positivity out to 10−8 dilutions, confirming the high sensitivity of the assay for AD tau seeds. These tau RT-QuIC assay results provided evidence that no measurable 3R or 3R+4R (Alzheimer’s-like) tau seeding activity accompanied the duodenal α-SynD seeds of PD. 10.1371/journal.ppat.1011456.g007 Fig 7 Tau RT-QuIC analysis of PD IM specimens. Colored symbols represent the inverse of the time to threshold for individual reaction wells seeded with a 10−3 (green squares) or 10−4 (blue circles) IM tissue dilution from the designated PD or HC cases. Solid downward triangles indicate control reactions seeded with sAD brain homogenate (BH) of the designated dilution with respect to solid tissue. Open downward triangles display representative data from reaction wells seeded with 1:1 mixtures 10−5 sAD BH and the designated tissue dilution of HC IM30. The dotted line indicates the threshold for a positive reaction. Error bars show mean ±SD. Quadruplicate reaction wells are shown for each sample. Discussion In this study, we have detected α-SynD, but not tau, seeding activity in enteric duodenal biopsies from patients with a clinical diagnosis of Parkinson’s disease. We estimated the average α-SynD seeding activity to be 4.3 log SD50 per mg of tissue, which was similar to that reported for RT-QuIC analysis of both post-mortem colon and skin specimens from PD patients [20, 28]. This means that the IM seeding activity was comparable to levels that we have seen in various PD brain specimens, and >3 logs higher than levels typically seen in antemortem PD CSF samples [19, 25] The diagnostic accuracy of RT-QuICR for PD in our initial duodenum biopsy panel was high, i.e., 95.7% sensitivity and 100% specificity. By comparison, a previous analysis of lower intestinal biopsies from a small cohort of PD and healthy subjects using an alternative SAA previously detected α-SynD with a diagnostic sensitivity of 55.6% and a specificity of 90.9% [27]. The reason for the increased sensitivity and specificity that we observed in the duodenum biopsies is unknown, but it could be due to the differences in the section of the intestine tested (rectum vs. duodenum) in the two patient cohorts and/or to the version of SAA used [19, 26]. α-SynD can spread from gut-to-brain and brain-to-gut via vagal pathways in animal models of body-first and brain-first PD (reviewed in [10]). Since vagal innervation is highest in the upper gastrointestinal tract, one might expect to detect more α-SynD in duodenal biopsies, compared to rectal biopsies. Emmi et al. recently detected α-Syn aggregates in duodenum biopsies from 22 patients with PD using IHC [13]. Our findings are consistent with these IHC results, and show additionally that duodenal α-SynD has self-propagating (seeding) activity. We found similar amounts of seeding activity in contemporaneous biopsies from superior and descending sections of duodenum from individual patients, suggestive of widespread α-SynD distribution within these duodenum segments. However, we assume that multiple IM collections, when possible, would improve diagnostic sensitivity. Emmi et al. [12] studied the relationship between the extent of α-SynD and the severity of extrapyramidal symptoms, but failed to find a correlation among α-SynD immunoreactive areas and the severity of motor symptoms, the global cognitive scales’ score, the quality of life, and the UPDRS scale values. Likewise, we found no significant correlation between the α-SynD seed concentrations and UPDRS III motor scores in patients on levodopa treatment. Thus, the link between the distribution of α-SynD in peripheral tissues and the stage of disease remains unclear [38]. The extent to which factors such as bilirubin content and gut inflammation might affect α-SynD seed concentrations and confound the interpretation of IM RT-QuICR results remains to be determined. Although the high diagnostic sensitivity and specificity were observed in this study, our results cannot be validated by autopsy as the patients were still alive at the time this study was completed. However, the study participants were carefully chosen based on clinical criteria. Additionally, the number of patients was limited due to the availability of patients undergoing PEG-J tube placement, patient hesitation to undergo an additional, albeit minimally invasive procedure, and restrictions placed on elective medical procedures during the Covid-19 pandemic. We caution that PEG-J for continuous L-DOPA infusion is generally used in more advanced cases of PD where oral L-DOPA is less consistently effective. Thus, the RT-QuICR results that we have obtained with our cohort of more severe PD cases may not be generalizable to patients with more moderate/mild PD that can be controlled well via oral L-DOPA. Another caveat is that the average age of the PD cases was 9 years older than the non-PD controls, and, at this time we cannot exclude the possibility that this age differential contributed in some way to our divergent RT-QuICR results for these two cohorts. In our study, 1 of 23 PD cases was RT-QuICR negative (IM16). At the time of IM homogenization, we noted that the tissue of this patient had an unusual hardened, thickened, and yellow appearance. A similar observation was made in one duplicate sample from IM27, which also gave an RT-QuICR-negative result. Because of this appearance, we speculate that these specimens might have been contaminated with bilirubin derived from the breakdown of hemoglobin in senescent red blood cells. Because blood contamination can inhibit some RT-QuIC assays [39], it is possible that residual blood components may have interfered. We also observed that in one PD patient (IM7), the RT-QuICR reaction gave a weak positive signal. This sample was collected from a patient affected by scleroderma, a condition which is known to damage peripheral nerves and mucosa [40] in the GI tract, most frequently in the duodenum [41]. Therefore, we hypothesize that the weak positive result for this subject could be due to vascular ischemia and fibrosis associated to scleroderma, which may have altered α-SynD deposition and distribution. Recent histopathological evidence indicates that enteric α-SynD deposition is more prevalent in RBD-positive than RBD-negative subjects [42]. Moreover, subjects with premotor RBD and constipation fit the criteria of “body first” phenotype [8, 43]. In our cohort about half of the patients (52.2%) presented both premotor RBD and GI symptoms of constipation. Interestingly, the RT-QuICR-negative PD patient was also RBD-negative. However, other than this case, no significant differences in RT-QuICR detection or parameters were observed between RBD positive or negative subjects. In conclusion, RT-QuICR provides a method for testing duodenal biopsies with similar sensitivity and specificity to SAAs of other diagnostically relevant samples [18, 19, 22, 25, 29] and skin [20, 23]. Paired with end-point dilution analysis, this assay can quantitatively assess levels of seeding activity among patients as well as across tissues, or in different sections of the same tissue. This could prove to be valuable for monitoring disease progression or selecting cohorts for clinical trials and monitoring their progress. While further studies are needed to elucidate the feasibility and utility of IM RT-QuICR, the potential for intra vitam monitoring and diagnosis, for example during routine esophagogastroduodenoscopy and colonoscopy screenings, holds promise for the early diagnosis of PD. Materials and methods Ethics statement This study was approved by the Institutional Ethics Committee (Prot.PG/2017/17817) of the Azienda Ospedaliera Universitaria di Cagliari, Italy. All participants signed written informed consent documents. Participants This study was approved by the Institutional Ethics Committee (Prot.PG/2017/17817) of the Azienda Ospedaliera Universitaria di Cagliari, Italy. All participants signed written informed consent documents. A total of 29 participants in this study, 23 PD patients and 6 non-neurodegenerative healthy controls (HCs) subjects, were recruited between September 2020 and September 2022 at the Neurology and Digestive Endoscopy Units of AO Brotzu Cagliari. Idiopathic PD patients were diagnosed according to the UK Brain Bank criteria, and patients with atypical parkinsonism were excluded. All PD patients were evaluated by the Movement Disorder Society UPDRS III and IV and by the Non-Motor Symptom Scale (NMSS). The presence of premotor RBD was assessed retrospectively through the REM sleep behavior disorder screening questionnaire (RBDSQ) [44]. The criteria for evaluation of constipation was based on the Constipation Scoring System [45]. A total score ranging from 0 (no constipation) to 30 with higher scores indicating more severe constipation was assigned retrospectively to each patient in according to eight variables [45], such as frequency of bowel movements, painful evacuation, incomplete evacuation, abdominal pain, length of time per attempt, assistance for defecation, unsuccessful attempts for evacuation per 24 hours, and duration of constipation. Exclusion criteria were intestinal cancer, the concomitant presence of neurological, or unstable psychiatric illness or cognitive impairment. All PD subjects included in this study were given levodopa-carbidopa intestinal gel (LCIG). HCs were matched to PD subjects for sex and age and had no neurological or cognitive disorders. Intestinal mucosa collection All PD patients underwent upper GI endoscopy for placement of an administration jejunal extension tube (PEG-J) for continuous levodopa enteral infusion, and intestinal mucosa biopsy. HCs were selected from those undergoing upper GI endoscopy biopsy for diagnostic investigations and surveillance. Two to four IM biopsy specimens from the proximal small intestine (duodenum) were taken for each participant (~20 mg of total tissue). All samples were collected in sterile containers containing 20 to 50 mL of physiological solution and immediately frozen at –80°C until analysis. Two additional IM biopsies were collected in sterile containers with 10% formalin for histological analysis and immunohistochemistry (IHC) examination. Histology and immunohistochemical analysis Histological examination was done on hematoxylin and eosin stained sections. Four μm thick sections were cut from formalin fixed-paraffin embedded biopsies. After rehydration, sections were incubated for 20 minutes with ready to use Carazzi’s Haematoxylin (Bio-Optica, Milan), soaked in tap water and counter-stained with 1% aqueous eosin Y solution for 20 seconds (Bio-Optica). Tissue examination was performed using a Leica DM6000 light microscope. Homogenization of intestinal mucosa samples Intestinal mucosa samples were homogenized following the protocol described by Bargar et al [19, 28] with some modifications. In brief, IMs were thawed and washed three times in 1× phosphate buffered saline (PBS) to remove any residual blood. Samples were weighed and homogenized at 10% (w/vol) in ice-cold homogenization buffer containing 1 x PBS, 0.1% Triton X-100, 150 mM sodium chloride (NaCl), 5 mM of ethylene-diamino-tetraacetic acid (EDTA) and Complete Protease Inhibitor without EDTA (Roche). IM homogenization was done using zirconia beads (1 mm) in a mini-Beadbeater-16 device (Bead Mill24; Thermo Fisher) at maximum speed. Samples were homogenized with 4 rounds of 1 min each at max speed, with cooling in between rounds. Next, samples were centrifuged at 500xg for 5 min and the supernatant was distributed in single use aliquots to be stored at -80°C for subsequent RT-QuICR analysis. Recombinant α-Syn (K23Q) and tau (K12CFh) protein purification Human K23Q rec α-Synuclein was purified as described by Groveman et al [19]. In brief, BL21(DE3) Escherichia coli bacteria were freshly transformed with the plasmid for recombinant K23Q α-Syn protein expression. One liter cultures were inoculated with a single colony and grown overnight using auto-induction media [46] containing 50 μg/mL kanamycin. The cells were lysed and the periplasmic fraction was isolated using an osmotic shock and acid purification protocol modified from Paslawski et al. [47]. The protein extract was filtered and loaded for purification into a 5 ml Ni-NTA column (Cytiva) on an Äkta Start chromatography system (GE). Peak fractions where then further purified using a 5 ml Q-HP column (Cytiva). The purified protein was filtered, diluted to 1 mg/ml, and dialyzed overnight at 4°C using a 3 kDa MWCO dialysis membrane. Protein concentration was determined with a UV–VIS spectrophotometer using a theoretical extinction coefficient at 280 nm of 0.36 (mg/mL) − 1 cm − 1. Lyophilized protein aliquots were stored at − 80°C. Human K12CFh rec tau substrate was purified as described by Metrick et al [35]. IM α-Syn RT-QuICR and tau RT-QuIC assays For α-Syn RT-QuICR assays 10% IM homogenates were thawed at room temperature, serially diluted in PBS containing N2 media supplement (Gibco) and tested by RT-QuICR using purified recombinant K23Q α-Syn as substrate. In brief, RT-QuIC buffer composition was: 40 mM phosphate buffer pH 8, 170 mM NaCl, 0.1 mg/ml K23Q rec-αSyn, 10 μM Thioflavin T (ThT). A volume of 98 uL of RT-QuIC buffer was loaded into wells of a black 96-wells plate with a clear bottom (Nunc). Plates were preloaded with six silica beads (0.8 mm, molecular biology grade; OPS Diagnostics) and reactions were seeded with 2 μL of IM homogenates at the specified dilutions for a final reaction volume of 100 μL. Next, plates were sealed (Nunc International sealer) and incubated in a BMG Fluostar plate reader at 42°C for 60 h with cycles of 1 min shake (400 rpm double orbital) and 1 min rest. ThT fluorescence measurements (450+/− 10 nm excitation and 480+/− 10 nm emission; bottom read) were taken every 45 min. All IMs were tested with at least 4 replicate reactions per sample dilution. Single reactions were considered positive when their fluorescence exceeded our threshold of 10% of the maximum value in the plate. IM samples were considered positive when ≥50% of replicate wells were above our ThT threshold within the established 40-h time cutoff. Samples that had 1 out of the 4 replicate reactions score as positive were deemed inconclusive and their analysis was repeated. Then, if the total percentage of positive reaction wells exceeded 25% then the sample was considered positive. The K12 tau-RT-QuIC assay was performed as described by Metrick et al [35]. Briefly, the reaction buffer composition was: 40 mM HEPES, pH 7.4, 400 mM NaF, 40 μM heparin, 10 μM ThT, 0.1 mg/mL K12 CFh substrate. A volume of 48 μL of reaction buffer was added into wells of a black 384-wells plate and seeded with 2 μL PD IM homogenate dilutions alone or spiked with 10−5 AD brain at 1:1 volumetric ratios with the designated dilution of PD IM. Plates were sealed with sealing tape and placed in an Omega FLUOStar plate reader at 42°C and subjected to rounds of 1 min shaking, 500 rpm, orbital, and 1 min rest, with ThT fluorescence reads (450 excitation, 480 emission) taken every 15 min. Reaction wells were considered positive on an individual reaction basis when their fluorescence value exceeded a threshold value of the mean fluorescence value plus 100 times the standard deviation from the first 5 h of readings. End-point dilution analysis and 50% seeding dose (SD50) calculation IMs were serially diluted 10-fold for end point analysis. Two μl of each IM dilution (10−3 to 10−6) were used to seed individual reactions. RT-QuICR testing was done as reported above. The amount of sample giving 50% positive replicate reactions (SD50) was estimated as described by Srivastava et al. [37]. Statistical analysis Student’s test and Fisher’s exact test was used to analyze differences in demographic characteristics between cases and controls. Variability of the data was presented as mean ± standard deviation or as percentages. Measures of diagnostic performance, such as sensitivity, specificity, negative predictive value, and positive predictive value, were determined for comparisons between clinical diagnosis and RT-QuICR results. Measures were expressed as percentages with 95% confidence interval (95% CI). Spearman’s rank correlation coefficient was used to determinate the correlation between SD50 values and motor UPDRSIII scores, disease duration and constipation score (95% CI; alpha = 0.05). All statistical analyses were performed and plotted using the Prism software (v.8 GraphPad). Statistical significance was set at p < 0.05. Supporting information S1 Fig Immunohistochemistry analysis of human duodenum IM biopsies. (A) Immunohistochemistry for tyrosine hydroxylase (TH). (B) Immunohistochemistry for choline acetyltransferase (ChAT). (TIF) Click here for additional data file. S2 Fig Representative ThT fluorescence curves in IM α-Syn RT-QuICR. (A) Traces from single reactions (n = 4 per case) seeded with 10−3 dilutions of duodenum IM biopsies from PDs cases 4, 5, and 15 (as colored) as a function of reaction time. (B) Traces from healthy control duodenum IM biopsies from cases 29, 30, and 31. (TIF) Click here for additional data file. S1 Data Fig 2 tab: Inverse time to threshold values (h-1) for individual quadruplicate wells seeded with 10−2 or 10−3 dilutions of IM samples with the IM sample ID numbers shown in the “Patient” column. Fig 3 tab: Inverse time to threshold values (h-1) for individual quadruplicate wells seeded with 10−3 dilutions of IM samples with the IM sample ID numbers shown in the “Patient” column. For samples 6 and 7, data from an additional 4 wells are shown. Fig 4 tab: log SD50/mg tissue values calculated for individual IM biopsies from end-point dilution data. Fig 5A tab: Inverse time to fluorescence threshold comparisons between first (biopsy 1) or second (biopsy 2) duodenum segments from a given patient for the IM sample ID numbers shown in the “Patient” column. Data are from quadruplicate wells seeded with 10−3 IM dilutions. Fig 5B tab: Log SD50/mg tissue values calculated for two IM biopsies from the same patient from end-point dilution analysis. Fig 6 tab: Individual IM log SD50/mg tissue values, UPDRSIII scores, constipation scores, and disease durations (y) associated with the designated IM sample ID in the “Patient” column. Fig 7 tab: Top 2 rows: Inverse time to threshold values (h-1) for individual quadruplicate wells seeded with 10−3 or 10−4 dilutions of IM samples with the IM sample ID numbers shown in the “Patient” row. sAD group: Analogous data from reactions seeded with 10−5 or 10−8 dilutions of sAD brain homogenate. The sAD spiked 10−5 group: data from reaction wells seeded with 1:1 mixtures 10−5 sAD BH and the designated tissue dilution of HC IM30. S2A and S2B Figs tabs: Quadruplicate ThT fluorescence readings as function of reaction time (h) for reactions seeded with 10−3 dilutions of IM sample IDs given the top row. (XLSX) Click here for additional data file. S1 Text Immunohistochemistry analysis of human intestinal mucosa biopsies. (DOCX) Click here for additional data file. The authors kindly acknowledge the Digestive Endoscopy Unit of AO Brotzu Cagliari for the assistance with subject recruitment. 10.1371/journal.ppat.1011456.r001 Decision Letter 0 Mabbott Neil A. Section Editor Woerman Amanda L. Academic Editor © 2023 Mabbott, Woerman 2023 Mabbott, Woerman https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version0 23 May 2023 Dear Dr Vascellari, Thank you very much for submitting your manuscript "α-Synuclein seeding activity in duodenum biopsies from Parkinson’s disease patients" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations. Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. When you are ready to resubmit, please upload the following: [1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out [2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments. Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments. Sincerely, Amanda L. Woerman Academic Editor PLOS Pathogens Neil Mabbott Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens ​orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens orcid.org/0000-0002-7699-2064 *********************** Reviewer Comments (if any, and for reference): Reviewer's Responses to Questions Part I - Summary Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship. Reviewer #1: In this manuscript, Vascellari and colleagues investigate αSyn seeding activity in duodenal biopsies from PD and control patients using a modified version of RT-QuIC called RT-QuICR. They find positive seeding activity in 22/23 PD patients compared to none of the 6 control samples investigated. These results suggest that RT-QuICR-mediated detection of αSyn seeding activity in intestinal biopsies may be useful as an ante mortem diagnostic test for PD. This is a well-conducted set of experiments on an important topic. The conclusions are sound and the manuscript is well written. I was left with an overall positive impression of the manuscript with only relatively minor issues for the authors to address. Reviewer #2: The authors performed asyn- (and tau) seeding assay on duodenal biopsies of PD patients and healthy controls. Despite the authors did not observe any correlation of duodenal asyn levels with clinical parameters, I find the findings highly interesting and relevant to the field, esp. in light of progress towards earlier diagnosis which is currently lacking. The manuscript is very well-written, figures are clear and the methods are executed adequate including quadruplicates which is highly recommended when using RT-QuIC. I only have a few minor comments to the discussion. Reviewer #3: In the manuscript ‘�-synuclein seeding activity in duodenum biopsies from Parkinson’s disease patients,’ the authors find that using the RT-QUIC rapid assays they are able to detect �-synuclein seeding activity. Uniquely the authors can discover �-synuclein end-point dilution seeding units per mg of tissue, allowing for possible future diagnostics. This manuscript adds to the field of understanding how misfolded proteins, like �-synuclein accumulates in the gut and should be considered for publication after the following minor concerns. ********** Part II – Major Issues: Key Experiments Required for Acceptance Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions. Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject". Reviewer #1: 1. The authors mention that their RT-QuICR results are in agreement with previous results showing αSyn pathology in the duodenum (Emmi et al., 2023). However, this does not address the previous inconsistencies in IHC data the authors note in the introduction (Corbille et al., 2016; Emmi et al., 2023; Ruffmann et al., 2018). Inclusion of IHC for αSyn in the current patient duodenal biopsy samples would be a stronger comparison and would address the important question of whether RT-QuICR is always in agreement with IHC data or does it show increased sensitivity that may explain previously noted IHC discrepancies (Corbille et al., 2016; Emmi et al., 2023; Ruffmann et al., 2018)? 2. The average age of the non-PD patients from which the healthy control biopsies were derived is nearly 10 years younger than the PD patient population used (to go along with far fewer samples in the control group). While this reviewer appreciates that there are likely challenges with obtaining sufficient numbers of perfectly aged-matched control duodenal biopsies, the authors need to discuss how the different ages could at least partially influence their findings. For instance, the 3 control samples that produced a positive response in at least one replicate reaction (IM28, IM29, and IM31) in Figures 3 and 5A are from the oldest controls, which have an average age (64.7 yr) that is much closer to the mean age of the PD sample group. Thus, IM RT-QuICR may be less discriminatory between PD and controls as age increases. References Corbille, A. G., Neunlist, M., & Derkinderen, P. (2016). Cross-linking for the analysis of alpha-synuclein in the enteric nervous system. J Neurochem, 139(5), 839-847. doi:10.1111/jnc.13845 Emmi, A., Sandre, M., Russo, F. P., Tombesi, G., Garri, F., Campagnolo, M., . . . Antonini, A. (2023). Duodenal alpha-Synuclein Pathology and Enteric Gliosis in Advanced Parkinson's Disease. Mov Disord. doi:10.1002/mds.29358 Ruffmann, C., Bengoa-Vergniory, N., Poggiolini, I., Ritchie, D., Hu, M. T., Alegre-Abarrategui, J., & Parkkinen, L. (2018). Detection of alpha-synuclein conformational variants from gastro-intestinal biopsy tissue as a potential biomarker for Parkinson's disease. Neuropathol Appl Neurobiol, 44(7), 722-736. doi:10.1111/nan.12486 Reviewer #2: (No Response) Reviewer #3: (No Response) ********** Part III – Minor Issues: Editorial and Data Presentation Modifications Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. Reviewer #1: 1. As PEG-J for continuous L-DOPA infusion is generally used in more advanced cases of PD where oral L-DOPA is ineffective, is there any concern that this may artificially select for patients with severe PD and that the results may not be generalizable to patients with more moderate/mild PD that can be controlled well via oral L-DOPA? 2. No raw traces of the ThT fluorescence curves were included. It would be useful to include some (or all) raw traces, perhaps as a supplementary figure, so that researchers conducting similar studies have examples of what such curves should look like. Were the ThT plateau fluorescence values similar across all positive samples? 3. The authors mention using “a mutated version of human α-synuclein protein to achieve faster and more specific detection of α-SynD”. While K23Q does improve specificity due to reduced propensity for spontaneous aggregation, I question if it results in “faster” detection. Previous work shows K23Q had near identical elongation kinetics to WT αSyn when seeding with WT PFF and in αSyn RT-QuIC (Groveman et al., 2018; Koo et al., 2008). 4. In calculating the SD50, several samples did not reach end-point requiring the extrapolation of the values. Why were the dilutions stopped at that point, instead of continuing them to endpoint to allow the accurate calculation of SD50? 5. In the first paragraph of the Discussion, the authors should mention how the calculated seeding activity in the duodenal biopsies compares to seeding activity calculated using PD patient brain tissue and CSF. This would provide some context to the final statement in the Abstract (“the duodenum may be a source or a destination for pathological, self-propagating α-synuclein assemblies”). References Groveman, B. R., Orru, C. D., Hughson, A. G., Raymond, L. D., Zanusso, G., Ghetti, B., . . . Caughey, B. (2018). Rapid and ultra-sensitive quantitation of disease-associated alpha-synuclein seeds in brain and cerebrospinal fluid by alphaSyn RT-QuIC. Acta Neuropathol Commun, 6(1), 7. doi:10.1186/s40478-018-0508-2 Koo, H. J., Lee, H. J., & Im, H. (2008). Sequence determinants regulating fibrillation of human alpha-synuclein. Biochem Biophys Res Commun, 368(3), 772-778. doi:10.1016/j.bbrc.2008.01.140 Reviewer #2: I only have a few minor comments to the discussion. • Line 247 – 249: The reason for the increased sensitivity and specificity that we observed in the duodenum biopsies is unknown, but it could be due to the differences in the section of the intestine tested (rectum vs. duodenum) in the two patient cohorts and/or to the version of SAA used [18, 25]- > It has been shown that alpha-synuclein can spread from gut-to-brain and brain-to-gut via vagal pathways in animals models of body-first and brain-first PD (reviewed in PMID: 34952161). Since vagal innervation is highest in the upper gastroinstestinal tract, it would make sense to detect more aggregated alpha-synuclein in duodenal biopsies, compared to rectal biopsies. This should be included in the discussion. • Line 260-261: Thus, the link between the distribution of α-SynD in peripheral tissues and the stage of disease remains unclear [37]. --> Were the biopsies tested for inflammatory markers? Inflammation could temporarily upregulate the presence of asyn deposition, masking the correlation with clinical parameters. Such potential confounder should be discussed. • Line 292: While further studies are needed to elucidate the feasibility and utility of IM RT-QuICR, the potential for intra vitam monitoring and diagnosis, for example during routine esophagogastroduodenoscopy and colonoscopy screenings, holds promise for the early diagnosis of PD. --> The use of confirmation-specific oligothiophene ligands in combination with RT-QuICR should be included as future perspective for early stratification of synucleinopathies. Several labs have shown the power of using luminescent oligothiophenes (LCO) to differentiate asyn conformation between synucleinopathies (reviewed in PMID: 35693346). The LCO’s have a much larger conformational freedom than ThT thanks to their flexible thiophene backbone, which allows conformation-specific binding to the aggregate. They yield a different spectral read out depending on the structure of the aggregate. The LCO staining could be done on a tissue section, or during the RT-QuIC procedure by replacing ThT with an LCO. Some labs have also used the LCO on the end-product of the RT-QuIC to investigate strain variability between end-product samples. Reviewer #3: Minor Concern 1: In Figure 1 the authors show tissue morphology but no evidence of �-synuclein within in the intestine. Although this may be difficult to detect it is important to show that pathologically there is �-synuclein within the tissues especially the mucosa region as this is what they claim within their studies to be important for RT-QuiCR screening. Minor Concern 2: The authors should discuss the rationale behind measuring the seeding only in the duodenum. Is there any advantages to this over the other portions of the intestine. Specifically the colon as most microbiome studies in the field are a snapshot of the intestinal tissue. Minor Concern 3: As there is no correlation between clinical signs and the RT-QuiCR, how would one have this be a proper diagnostic? How does this limitation of your assay still allow for diagnostics? Important to discuss this in the discussion. Minor Concern 4: The authors find that there are specific parameters that must be used in order to get proper results from their seeding assay, mucosa from the 1st segment and no bilirubin contamination on the sample. This is critical and should be stated clearly in the discussion and a limitation of studying all routine colonoscopy procedures. Minor Concern 5: The authors should discuss why they were unable to find tau in the intestine. Is it because its not there? The tool is not sensitive enough? ********** PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. 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Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols References: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. 10.1371/journal.ppat.1011456.r002 Author response to Decision Letter 0 Submission Version1 31 May 2023 Attachment Submitted filename: Rebuttal_Plos Pathogens.pdf Click here for additional data file. 10.1371/journal.ppat.1011456.r003 Decision Letter 1 Mabbott Neil A. Section Editor Woerman Amanda L. Academic Editor © 2023 Mabbott, Woerman 2023 Mabbott, Woerman https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version1 2 Jun 2023 Dear Vascellari, We are pleased to inform you that your manuscript 'α-Synuclein seeding activity in duodenum biopsies from Parkinson’s disease patients' has been provisionally accepted for publication in PLOS Pathogens. Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests. Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated. IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS. Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards, Amanda L. Woerman Academic Editor PLOS Pathogens Neil Mabbott Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens ​orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens orcid.org/0000-0002-7699-2064 *********************************************************** Reviewer Comments (if any, and for reference): 10.1371/journal.ppat.1011456.r004 Acceptance letter Mabbott Neil A. Section Editor Woerman Amanda L. Academic Editor © 2023 Mabbott, Woerman 2023 Mabbott, Woerman https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 16 Jun 2023 Dear Vascellari, We are delighted to inform you that your manuscript, "α-Synuclein seeding activity in duodenum biopsies from Parkinson’s disease patients," has been formally accepted for publication in PLOS Pathogens. We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication. The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. 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Best regards, Kasturi Haldar Editor-in-Chief PLOS Pathogens ​orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens orcid.org/0000-0002-7699-2064 ==== Refs References 1 Braak H. , et al ., Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging, 2003. 24 (2 ): p. 197–211. doi: 10.1016/s0197-4580(02)00065-9 12498954 2 Hawkes C.H. , Del Tredici K. , and Braak H. , Parkinson’s disease: a dual-hit hypothesis. Neuropathol Appl Neurobiol, 2007. 33 (6 ): p. 599–614. doi: 10.1111/j.1365-2990.2007.00874.x 17961138 3 Ulusoy A. , et al ., Brain-to-stomach transfer of alpha-synuclein via vagal preganglionic projections. Acta Neuropathol, 2017. 133 (3 ): p. 381–393.28012041 4 Cersosimo M.G. and Benarroch E.E. , Pathological correlates of gastrointestinal dysfunction in Parkinson’s disease. Neurobiol Dis, 2012. 46 (3 ): p. 559–64. doi: 10.1016/j.nbd.2011.10.014 22048068 5 Pfeiffer R.F. , Gastrointestinal dysfunction in Parkinson’s disease. 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