2. IRCSS, Institute of Neurological Sciences, Bologna, Italy Skin nerve phosphorylated alpha-synuclein deposits in idiopathic REM sleep behavior disorder Elena Antelmi,1,2 V. Donadio,2 A. Incensi,2 G. Plazzi,1,2 R. Liguori1,2   1. Department of Biomedical and Neuromotor Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy. 2. IRCSS, Institute of Neurological Sciences, Bologna, Italy

iRBD 15 yrs 90% α-Syn mediated neurodegenerative disease - Autonomic symptoms Several yrs before pheno-conversion Postuma et al, 2015 - Olfaction, color vision 5 yrs before pheno-conversin Postuma et al, 20112011 Neuroimaging findings: SPECT DAT-Scan Transcranial sonography Structural neuroimaging2011 15 yrs 90% α-Syn mediated neurodegenerative disease iRBD Time-window to observe pre-clinical synucleinopaties and to test potential markers of impending neurodegeneration Time-window to apply neuroprotective treatments/approches

In iRBD: from Tolosa and Vilas, Brain 2015 p-α-synuclein deposits 8/9 (89%) patients with iRBD vs 8/12 (67%) patients with PD Vilas et al, Lancet Neurol 2016 p-α-synuclein deposits in 4/17 patients with iRBD, 1/19 patients with PD and none of the HC Sprenger et al, Neurology 2015 from Tolosa and Vilas, Brain 2015

In PD patients, at skin biopsy, p-α-syn in vivo has been found In 20% (# 2/10) of iPD patients at the chest vs none at the leg site (Miki et al, 2010); In 51% (# 16/31) of iPD patients vs none of of the HC (11/31 at the back, 6/31 proximal leg, 4/31 distal leg, 5/31 finger) (Doppler et al, 2014); In none (# 0/6) of iPD patients (samples from distal legs) (Navarro-Otano et al, 2014); In 100% (# 21) of iPD patients at cervical site and in 52 and 24% of iPD at thigh and leg sites, respectively and in none of the patients with other form of parkinsonism (# 20) and none of the HC (# 30)(Donadio et al, 2014); In 67% (# 20/30) of iPD and of MSA vs none of the HC and of patients with tauopathy, proximal-distal gradient (Doppler et al, 2015); In 100% (# 10) of iPD patients vs none of MSA patients at the ventral forearm (Zange et al, 2015) In 100% (# 16) of iPD patients at cervical site and 75 and 31% at the tight and leg sites, respectively vs 100% (# 14) of PAF patients in all sites and vs none of the HC (# 15) (Donadio et al, 2016).

Easy to obtain/perform/not invasive Aim of the study To search for misfolded p-α-synuclein deposits in skin biopsy samples as a precocious bio-marker of impending neurodegeneration in patients with iRBD Ideal Biomarker 100% sensitivity 100% specificity Not expensive Easy to obtain/perform/not invasive Long lead-time

Aim of the study Methods To search for misfolded p-α-synuclein deposits in skin biopsy samples as a precocious bio-marker of impending neurodegeneration in patients with iRBD Methods Inclusion criteria Patients with iRBD (vs HC and iPD) Positive chronic history of «dream-enacted behaviors» plus PSG-detection of RSWA and of at list one clear RBD episode Absence of motor impairement at the clinical examination Absence of cognitive signs at the neuropsychological investigation Normal Brain MRI No assumption of drugs that have been related with RBD (i.e. anti-depressant, beta-blockers)

Methods investigations History-tacking; neurological examination and smell test (sniffing stick test kit) Questionnaires: ESS, PSQI, BDI, STAI, BIS-11 Optical coherence tomography, pupillometry, color vision assessment SCOPA-AUT questionnaire Full-night video-PSG (conventional EEG, bilateral EOG, submentalis, bilateral flexor digitorum superficialis of the arms, bilateral anterior tibialis EMG, respiratory parameters, electrocardiogram, infra-red video) Brain MRI SPECT DatScan Nerve conduction velocities study MMSE and neuropsychological tests Blood test; CSF analysis for neurodegenerative biomarkers 3 mm punch biopsy at the proximal sites (bilateral paravertebral C8 level) and distally (bilateral distal leg; 10 cm above the lateral malleolus), two different samples (3 to 4 cm apart) were taken

minimally invasive requiring no suture 3 mm punch biopsies minimally invasive requiring no suture minor discomfort for the patient 1. Ten µm skin sections double-immunostained overnight with rabbit monoclonal phosphorylated α-synuclein at Ser129 (p-syn; 1:500, Abcam, ab-51253) and mouse pan-neuronal marker protein gene product (PGP) (1:750; Abcam, ab72911). 2. Sections were then washed and incubated for one hour after adding secondary antibodies (i.e. mouse or rabbit Alexa Fluor 488 and rabbit or mouse cyanine dye fluorophores 3.18; 1:200 or 1:400, Jackson ImmunoResearch, 711-545-152 for r-AlexaFluor488 and 715-165-150 for m-cyanine 3). 3. Sections were finally analyzed under a Nikon confocal microscopy (Eclipse Ti A1). Intraneural p-α-syn staining was demonstrated by a colocalization with PGP. Two authors experienced in immunofluorescence analysis carried out the analysis, blind to the clinical diagnosis.

Antelmi et al, Neurology 2017 12 iRBD vs 50 age and sex-matched HC Antelmi et al, Neurology 2017

p-α-syn @ skin biopsy: # 10/18 iRBD (sensitivity 55.6%) # 20/25 early PD (sensitivity 80%) none of the HC (specificity 100%) Percentage of samples positive for p-α-syn correlated negatively with the DAT binding at the FP-CIT-PET, olfactory function and positively with total LR for RBD to present prodromal PD

* San Raffaele Hospital, Milan yes * San Raffaele Hospital, Milan

Thank you