α-Synuclein seeding activity in duodenum biopsies from Parkinson's disease patients

Abstract

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.

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.

Fig 2. Optimization of IM tissue dilutions used for RT-QuICR detection of α-Syn^D.

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.

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.

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.

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).

Fig 6. Spearman correlation between α-syn^D 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.

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.