Cutaneous Autonomic Pilomotor Testing to Unveil the Role of Neuropathy Progression in Early Parkinson's Disease

PURPOSE OF THE PROTOCOL With the goal of developing novel endpoints for early stage trials of disease modifying therapies in Parkinson's disease (PD) the objective of this study is to a) assess reproducibility b) external validity and reliability c) to perform a longitudinal assessment of sudomotor (QDIRT) and pilomotor (QPART) responses in early PD patients.

Moreover, this study aims to assess the hypothesis that functional impairment in pilomotor and sudomotor nerve fibers in early PD correlates with a-synuclein deposition mediated structural fiber damage and may therefore constitute a potential non- invasive biomarker for autonomic involvement in PD.

BACKGROUND In skin biopsy studies of PD, alpha-synuclein accumulation in cutaneous autonomic nerve fibers has been linked to autonomic nervous system disturbances even in the early stages of the disease. (1) The investigators recently introduced a non-invasive technique to assess autonomic adrenergic fiber function using the quantitative pilomotor axon-reflex test (QPART). (2)

OBJECTIVES Demonstrating generalizability and reliability of the QPART technique, the anticipated positive results of this protocol might lend support to the utility of QPART as a diagnostic tool. This might be of particular interest in clinical practice as the QPART technique has been shown to assess small fiber function with low variability and comparatively low technical effort whereas vasomotor assessment is limited by high variability and sudomotor assessment requires technical demanding settings. (3) Moreover, the anticipated positive correlation between functional and structural pilomotor nerve fiber impairment might support the capacity of the QPART technique to assess specific fiber damage and thus provide a non-invasive tool to 1.) study pilomotor nerve damage in research studies and 2.) allow for longitudinal clinical assessment in PD patients to monitor disease progression.

ETHICAL STANDARD The study protocol has been approved by the institutional review board of Technische Universität Dresden (IRB study reference: EK349082015) as well as by the institutional review board of each participating site (Berlin accepted the protocol based on Technische Universität Dresden IRB approval; Budapest IRB number: 004513-004/2016/OTIG; Boston IRB number: 2016P000141)

This study will be conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent will be obtained from each study participant. The consent will be reviewed with the subject by one of the study physicians. The subject will sign the informed consent before any procedures pertaining to the study are initiated. A copy of the signed informed consent will be given to the participant and another copy kept on file. Conduction of this study will comply with the GCP (Good Clinical Practice) guidelines.

STUDY PROCEDURES Quantitative Pilomotor Axon-Reflex Test (QPART): Iontophoresis of phenylephrine is used to induce axon-reflex mediated piloerection. A drug delivery capsule electrode (LI-611; Perimed®, Järfälla, Sweden) is affixed on the testing area on the dorsal forearm. The inner chamber of this capsule, open to the skin surface, is filled with 0.4ml of 1 % phenylephrine solution. The drug delivery electrode is then connected to the iontophoresis stimulation box (Phoresor II-Auto-PM850; IOMED Inc., Salt Lake City, USA). Iontophoresis is performed over a 1cm diameter skin region with 0.5mA over 5 minutes. Silicone impressions of piloerection are obtained to create a local topographic map of piloerection. A silicone based two-phase material with high liquidity (Honigum Light, DMG, Hamburg, Germany) is placed over the skin for 10 seconds. The silicone cures for 5 minutes, has toner applied to mark the pilomotor impressions, excess toner is wiped free and the silicone is scanned to capture the image digitally. Silicone impressions of erect hair follicles are quantified by number and area and spatial spread. A high resolution scanner (Fuel 3D® Technologies Limited, Chinnor, United Kingdom) connected to a personal computer equipped with image analyzing software (Fuel3D® Studio Starter; Fuel 3D® Technologies Limited, Chinnor, United Kingdom) is used to quantify additionally the goosebump reaction.

Sympathetic Skin Response (SSR): The skin conductance level (SCL) is measured in µSievert (µS) from two medial phalanges (index and third finger) with a Powerlab® polygraph (AD Instruments, Bella Vista, Australia). The maximum increase in amplitude following sudden deep respiration (Sympathetic Skin Response; SSR) is calculated to quantify the functional reactivity of sweat glands as previously described. (4)

Quantitative Direct and Indirect Test of Sudomotor Function (QDIRT): Axon-reflex mediated sweating is measured after iontophoresis of 10% acetylcholine by drying the stimulated region on the volar aspect of the forearm and dusting with indicator dye (povidone-iodine and cornstarch) followed by repeated digital photographs taken every 15 seconds for 7 minutes with a 18 Megapixel camera (EOS 60D; Canon Inc., Tokyo, Japan) with 100-mm macro lens. Images are digitally analyzed for droplet number, size, and axon-reflex spread as previously described. (5)

Evaluation of perceived motor and autonomic symptoms: Progression of motor and autonomic symptoms are assessed through validated symptom scores MDS-UPDRS motor part and SCOPA AUT.

Skin biopsies: a-synuclein/PGP Ratio; Immunhistochemical staining for a-synuclein and intraepidermal nerve fibers will be performed in 3 skin punch biopsies obtained from the QPART testing sites and two control sites (lower legs). Three-millimeter biopsies will be obtained following local anesthesia with 2% lidocaine. Biopsy specimens will be fixed in Zamboni solution for 18 hours and cryoprotected overnight (20% glycerol and 20% 0.4 M Sorensen buffer). Skin blocks are cut using freezing microtome and 20 tissue sections are analyzed. 20 tissue sections will analyzed. Alpha synuclein/intraepidermal nerve fiber density ratios will be calculated.

TIMELINE Subjects will be assessed for eligibility by a listed study physician. At baseline all subjects will undergo medical history, physical examination, QPART, SSR, QDIRT, MDS-UPDRS and SCOPA AUT assessment as described above. These testing procedures will be repeated, after 1-2 weeks, 1 year, 2 years, and 3 years post-baseline. All testing will be performed in a temperature and humidity controlled environment. Only at Dresden-Site subjects will additionally undergo the skin punch biopsy procedure as described above at baseline, after 1 year and after 3 years.