Fluorescent Peptide-based Probe for the Detection of Alpha-synuclein Aggregates in the Gut

Background Parkinson’s disease (PD) is diagnosed clinically by motor symptoms, with no molecular diagnostic test currently available. By the time motor symptoms manifest, significant irreversible neurodegeneration has already occurred, limiting the effectiveness of neuroprotective therapies and drug interventions. Recent identification of pathological alpha-synuclein (α-syn) aggregates in the gastrointestinal (GI) tract of prodromal PD patients offer a potential avenue for early disease diagnosis. This study aims to explore specific fluorescence labelling of α-syn aggregates in the GI tract using a peptide-based probe for early diagnosis of PD. Methods We used primary hippocampal neuronal cells and wild-type mouse tissues with the addition of pre-formed α-syn fibrils to identify the most suitable peptide fluorescent probe ( P1 ) for staining α-syn aggregates in cells and tissues. We validated the probe labelling in GI tract tissues from three mouse models, including PFF-injected mice and two transgenic PD mouse strains. We quantified labelling accuracy by confocal imaging and protein analysis. Results We found that P1 labelled α-syn aggregates with high accuracy (87% in comparison to Serine129-phosphorylated α-syn antibody) and high specificity for labelling their aggregated forms over monomeric forms. In GI tract tissues, P1 labelled α-syn aggregates across tissue layers (mucosa, sub-mucosa, muscularis externa) and achieved comparable performance to antibody staining. Higher degree of probe labelling was found in older mice due to increased accumulation of α-syn aggregates with ageing. Notably, α-syn aggregates were readily detectable in the colonic mucosae using P1 , indicating the potential use of this probe for early PD diagnosis during colonic examinations like colonoscopy. Conclusion We have developed a peptide-based fluorescent probe and demonstrated its rapid and specific labelling of α-syn aggregates. We highlight the probe’s ability to label these aggregates rapidly over α-syn monomers and survey the abundance of α-syn aggregates throughout the entire length of the GI tract. These support the further development of P1 as a specific fluorescent imaging biomarker for colonic α-syn aggregates for the early detection of PD. ### Competing Interest Statement The authors have declared no competing interest. * α-syn : Alpha-synuclein BCA : Bicinchoninic acid BSA : Bovine serum albumin ELISA : Enzyme-linked immunosorbent assay ENS : Enteric nervous system FBS : Fetal bovine serum FRET : Förster Resonance Energy Transfer FCSS : Fluorescence cross-correlation spectroscopy GI : Gastrointestinal H&E : Hematoxylin and eosin IF : Immunofluorescence LC-MS : Liquid chromatography–mass spectrometry min : minutes PAGE : Polyacrylamide gel electrophoresis PBS : Phosphate buffered saline PD : Parkinson’s disease PFF : Pre-formed fibrils PPV : Positive predictive value s : seconds SAA : Seed amplification assay pS129 : Phosphorylated α-syn at residue serine 129 SDS : Sodium dodecyl sulfate TEM : Transmission electron microscopy TBS : Tris buffered saline TBS-T : Tris buffered saline with 0.1% tween-20 WB : Western blot