Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

Parkinson's disease (PD) is diagnosed clinically by motor symptoms, with no clinical molecular diagnostic test currently available. By the time motor symptoms manifest, significant irreversible neurodegeneration would have already occurred, limiting the effectiveness of the therapies. Recent identification of pathological α-synuclein (α-syn) aggregates in the gastrointestinal tract of prodromal PD patients offers a potential avenue for early diagnosis. This study aims to explore specific fluorescence labeling of α-syn aggregates in the gastrointestinal tract using a peptide-based probe for early indication of PD risk. We used primary hippocampal neuronal cells and wild-type mouse tissues with the addition of preformed α-syn fibrils (PFFs) to identify the most suitable probe (<b>P1</b>) for staining α-syn aggregates. We found that <b>P1</b> labeled α-syn aggregates with high accuracy (87% in comparison to Serine129-phosphorylated α-syn antibody) and exhibited high labeling specificity for aggregated α-syn forms over monomeric forms. In a wide range of gastrointestinal tissues from PFF-injected mice and transgenic mice, <b>P1</b> labeled α-syn aggregates across tissue layers (mucosa, submucosa, muscularis externa) and achieved comparable performance to antibody staining. A higher degree of probe labeling was found in older mice due to increased accumulation of α-syn aggregates with aging. α-Syn aggregates were readily detectable in the colonic mucosae using <b>P1</b>, even in colon samples that were briefly flushed with <b>P1</b>, indicating the potential use of this probe in colonic biopsy samples and intact colon imaging. These support further development of <b>P1</b> as a specific fluorescent imaging biomarker for colonic α-syn aggregates for the early indication of PD risk.