Colonic mucosal α-synuclein lacks specificity as a biomarker for Parkinson disease

Abstract

Objective: To determine the utility of detecting a-synuclein (aSyn) in colonic mucosal biopsy tissue as a potential diagnostic biomarker for Parkinson disease (PD). Methods:We used the paraffin-embedded tissue (PET) blot, which degrades physiologic nonaggregated aSyn using proteinase K and enhances antigen retrieval allowing sensitive and selective detection of remaining protein aggregates, to detect aSyn in colonic mucosal biopsies from 15 patients with early PD (,3 years), 7 patients with later PD (.5 years), and 11 individuals without PD. aSyn and serine 129-phosphorylated aSyn (Ser129p-aSyn) were assessed by PET blot and conventional immunohistochemistry.Results: PET blot-resistant aggregated aSyn and Ser129p-aSyn was present in 12 of 15 individuals with early PD, 7 of 7 individuals with later PD, and 11 of 11 control subjects. The number of biopsies positive by PET blot relative to conventional immunohistochemistry was significantly lower in both PD groups compared with the control group for both aSyn and Ser129p-aSyn, whereas routine immunohistochemistry was positive more often in PD, but was positive in as many as 9 of 11 control individuals.Conclusion: Strong evidence of the presence of aggregated hyperphosphorylated aSyn in individuals with and without PD, using such a sensitive and specific method as the PET blot, suggests that colonic deposition of aSyn is not a useful diagnostic test for PD. The utility of detecting aSyn in the colon as a biomarker in combination with other assessments remains to be determined. An accurate diagnostic test for early or prodromal Parkinson disease (PD) is a significant unmet need. Studies suggesting that abnormal deposition of a-synuclein (aSyn) may originate in the peripheral autonomic nervous system, 1,2 coupled with evidence of a prion-like spread of synucleinopathy, 3 have generated much interest in evaluating enteric nervous system (ENS) aSyn to assess early PD neuropathology before disease spread and long before markers reliant on advanced disease are affected. 4 Several recent studies of aSyn staining in colonic tissue from living patients with PD report sensitivities ranging from 72% to a remarkable 100%. [5][6][7] However, the description of some degree of aSyn labeling in controls in these studies and reports of aSyn in colonic tissue from individuals without PD have raised concerns regarding specificity. 8,9 The aim of the present study was to optimize the immunohistochemical detection of aSyn in colonic biopsies by adapting the paraffin-embedded tissue (PET) blot, a method developed to increase sensitivity and specificity of detecting aggregated proteins in a related proteinopathy, Creutzfeldt-Jakob disease. The PET blot degrades nonaggregated proteins allowing the selective detection of remaining, presumably pathologic, protein aggregates.

Detection of alpha‐synuclein conformational variants from gastro‐intestinal biopsy tissue as a potential biomarker for Parkinson's disease

“…Each technique is specific for a particular variant or conformation of aSyn. For IHC, antibodies reactive for total (T‐aSyn‐Ab) and phosphorylated (P‐aSyn‐Ab) aSyn were selected as they have been most widely used for the detection of aSyn in the GI tract making our results comparable with these previous studies 4, 14, 15, 17, 19, 20. We also used recently developed antibodies specific for the oligomeric forms of aSyn (O‐ASN‐Abs) 26, as transformation of monomeric aSyn to oligomeric conformations is increasingly recognized as the early, key pathological event in the fibrillization process of aSyn 27.…”

“…neuritic and ganglionic ) were considered to be specifically neuronal and putatively pathological. Different antibodies showed drastically different sensitivities (and specificities) for the neuronal pattern of staining, with the highest sensitivity being achieved with the O‐aSyn‐Ab, with a positive signal in 39% of PD vs. 24% HC, followed by the P‐aSyn‐Ab with 14% of PD vs. 24% HC, and finally T‐aSyn‐Ab that surprisingly, and in contrast with other studies 4, 15, 19, 20, 33 showed no neuronal staining in any of the examined cases. We found no difference in specificity whether we used O/P‐aSyn‐Ab.…”

“…17 and Visanji et al . 19 detected neuronal aSyn pathology in all of their HC. In another study, colonic biopsies from PD and HC showed no difference in phosphorylated aSyn expression levels measured by Western blotting 36.…”

“…It has, however, become increasingly apparent that the high sensitivity and specificity of GI aSyn detection to identify PD patients reported in the earlier studies 12, 13, 14, 15, 16 has not been sustained in subsequent studies repeatedly detecting aSyn accumulation also in the GI tract of neurologically healthy individuals 17, 18, 19, 20, 21. This was also confirmed by a recent multicentre study, which showed limited diagnostic value for detecting aSyn deposition in the GI biopsies by immunohistochemistry (IHC) 22.…”

Rotenone and elevated extracellular potassium concentration induce cell‐specific fibrillation of α‐synuclein in axons of cholinergic enteric neurons in the guinea‐pig ileum

“…However, "pathological" α-synuclein species have been detected in the gut of healthy asymptomatic ageing adults (39,40,41,42). Early studies of the GI tract in Parkinson's disease reported Lewy bodies and neurites comparable to those observed in brain (43,44,45).…”