Objective: To validate detection of α-synuclein pathology in the gastrointestinal (GI) tract as a diagnostic marker of Parkinson’s disease (PD).

Background: α-synuclein (ASN) deposition in the GI tract may be an early event in PD pathogenesis and is thus a potential [pre-symptomatic] diagnostic biomarker. Recent studies however suggest that using conventional immunohistochemistry (IHC) for ASN does not distinguish between PD patients and controls, thus more sensitive and specific techniques are needed.

Methods: The Discovery cohort from the Oxford Parkinson’s disease Centre is an ongoing, prospective, longitudinal study investigating clinical and molecular characteristics of early PD. We screened all 1278 participants [879 PD, 259 healthy controls, 140 ‘at risk’ individuals (relatives, RBD or gene carriers] to identify those having previously undergone a GI biopsy (17.8%). We obtained tissue biopsies from 51 PD patients and 20 controls which were stained for two antibodies (Ab) for phosphorylated (WAKO, 1:10,000) and total (clone KM51, 1:1000) ASN and for calretinin (neuronal marker; clone 5A5, 1:200). In addition, we have adapted two tissue based-techniques, i.e. paraffin embedded tissue (PET) blot and proximity ligation assay (PLA), that detect synaptic and oligomeric species of ASN, respectively.

Results: Using phosphorylated ASN-Ab, the pathological deposition of misfolded ASN in nerve cell processes was encountered in 12 PD cases (24%), of which 5 were post-diagnosis and 7 pre-diagnosis (range 1 – 13 years), and in 2 controls (10%). Using total ASN-Ab, no staining was seen in controls but ASN pathology was only seen in 6 PD patients (12%). PET blot, which degrades physiologic non-aggregated ASN using proteinase K and enhances antigen retrieval allowing the detection of synaptic ASN aggregation, improved the sensitivity to 44%. Using fluorescent double-labelling of ASN-PLA technique together with calretinin, we detected two distinct types of oligomeric ASN signal in the colonic biopsy samples, i.e. diffuse and cellular.

Conclusions: ASN-IHC in colonic biopsy samples as a diagnostic biomarker of PD has low sensitivity and specificity. The paraffin embedded tissue (PET) blot and the proximity ligation assay (PLA) measure synaptic and oligomeric species of ASN, respectively, which allows a selective detection of earliest, pathological, neuronal accumulation of ASN. ASN-PLA provides a more easily quantifiable signal than ASN-PET.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/colonic-synuclein-a-potential-diagnostic-biomarker-in-parkinsons-disease/