A novel super-resolution microscopy platform for cutaneous alpha-synuclein detection in Parkinson's disease.

Alpha-synuclein (aSyn) aggregates in the central nervous system are the main pathological hallmark of Parkinson's disease (PD). ASyn aggregates have also been detected in many peripheral tissues, including the skin, thus providing a novel and accessible target tissue for the detection of PD pathology. Still, a well-established validated quantitative biomarker for early diagnosis of PD that also allows for tracking of disease progression remains lacking. The main goal of this research was to characterize aSyn aggregates in skin biopsies as a comparative and quantitative measure for PD pathology. Using direct stochastic optical reconstruction microscopy (STORM) and computational tools, we imaged total and phosphorylated-aSyn at the single molecule level in sweat glands and nerve bundles of skin biopsies from healthy controls (HCs) and PD patients. We developed a user-friendly analysis platform that offers a comprehensive toolkit for researchers that combines analysis algorithms and applies a series of cluster analysis algorithms (i.e., DBSCAN and FOCAL) onto STORM images. Using this platform, we found a significant decrease in the ratio of the numbers of neuronal marker molecules to phosphorylated-aSyn molecules, suggesting the existence of damaged nerve cells in fibers highly enriched with phosphorylated-aSyn molecules. Furthermore, our analysis found a higher number of aSyn aggregates in PD subjects than in HC subjects, with differences in aggregate size, density, and number of molecules per aggregate. On average, aSyn aggregate radii ranged between 40 and 200 nm and presented an average density of 0.001-0.1 molecules/nm. Our STORM analysis thus highlights the potential of our platform for identifying quantitative characteristics of aSyn distribution in skin biopsies not previously described for PD patients while offering valuable insight into PD pathology by elucidating patient aSyn aggregation status.