Uri Ashery ^1* Ofir Sade ¹ Daphna Fischel ¹ Noa Barak-Broner ^1,2 Shir Halevi ¹ Irit Gottfried ¹ Dana Bar- On ^1,2 Stefan Sachs ³ Anat Mirelman ^4,5,6 Avner Thaler ^5,6 Aviv Gour ^6,7 Meir Kestenbaum ^6,7 Mali Gana Weisz ⁵ Saar Anis ^6,8 Claudio Soto ⁹ Melanie S. Roitman ⁸ Nir Giladi ^4,5,6 Nirit Lev ^4,6,7 Roy Alcalay ^5,6 Sharon Hassin-Baer ^6,8 Kathrin Doppler ¹⁰ Markus Sauer ³

• ¹ School of Neurobiology, Biochemistry, Biophysics, Life Sciences Faculty, and the Sagol School of Neuroscience,Tel Aviv University, Tel Aviv, Israel, Tel Aviv University, Tel Aviv, Israel
• ² Specialty Research and Development, Teva Pharmaceutical Industries, Ltd., Netanya, Israel
• ³ Department of Biotechnology and Biophysics, Biocenter, University of Wuerzburg, Würzburg, Bavaria, Germany
• ⁴ Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
• ⁵ Movement Disorders Division, Neurological Institute, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
• ⁶ Faculty of Medical and Health Studies, Tel Aviv University, Tel Aviv, Israel
• ⁷ Department of Neurology, Meir Medical Center, Kfar Saba, Central District, Israel
• ⁸ Movement Disorders Institute, Department of Neurology, Chaim Sheba Medical Center, Ramat Gan, Jerusalem, Israel
• ⁹ Mitchell Center for Alzheimer's Disease and Related Brain Disorders, Department of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas, United States
• ¹⁰ Department of Neurology, University Hospital Würzburg, Würzburg, Bavaria, Germany

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 (dSTORM) 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 (HC) 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 dSTORM 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 than in HC subjects, with differences in aggregate size, density, and number of molecules per aggregate. On average, aSyn aggregate radii ranged between 40-200 nm and presented an average density of 0.001-0.1 molecules/nm 2 . Our dSTORM 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.