Alpha-synuclein seeds in olfactory mucosa of patients with isolated rapid-eye-movement sleep behaviour disorder

Isolated REM sleep behaviour disorder is an early-stage α-synucleinopathy in most, if not all, affected subjects. Detection of pathological α-synuclein in peripheral tissues of isolated REM sleep behaviour disorder patients may identify those progressing to Parkinson’s disease, dementia with Lewy bodies or multiple system atrophy, with the ultimate goal of testing preventive therapies. Real-Time Quaking-Induced Conversion (RT-QuIC) provided evidence of α-synuclein seeding activity in cerebrospinal fluid and olfactory mucosa of patients with α-synucleinopathies. Aim of this study was to explore RT-QuIC detection of α-synuclein aggregates in olfactory mucosa of large cohort of subjects with isolated REM sleep behavior disorder compared to Parkinson’s disease and controls. This cross-sectional case-control study was performed at the Medical University of Innsbruck, Austria, the Hospital Clinic de Barcelona, Spain, and the University of Verona, Italy. Olfactory mucosa samples obtained by nasal swab in 63 patients with isolated REM sleep behavior disorder, 41 matched Parkinson’s disease patients and 59 matched controls were analysed by α-synuclein RT-QuIC in a blinded fashion at the University of Verona, Italy. Median age of isolated REM sleep behavior disorder patients was 70 years, 85.7% were male. All participants were tested for smell, autonomic, cognitive and motor functions. Olfactory mucosa was α-synuclein RT-QuIC positive in 44.4% isolated REM sleep behavior disorder patients, 46.3% Parkinson’s disease and 10.2% controls. While the sensitivity for isolated REM sleep behavior disorder plus Parkinson’s disease versus controls was 45.2%, specificity was high (89.8%). Among isolated REM sleep behavior disorder patients with positive α-synuclein RT-QuIC, 78.6% had olfactory dysfunction compared to 21.4% with negative α-synuclein RT-QuIC, p<0.001. The extent of olfactory dysfunction was more severe in positive than negative α-synuclein RT-QuIC olfactory mucosa isolated REM sleep behavior disorder patients (p<0.001). We provide evidence that α-synuclein RT-QuIC assay enables the molecular detection of neuronal α-synuclein aggregates in olfactory mucosa of patients with isolated REM sleep behavior disorder and Parkinson’s disease. Although the overall sensitivity was moderate in this study, nasal swabbing is attractive as simple, non-invasive test and might be useful as part of a screening battery to identify subjects in the prodromal stages of α-synucleinopathies. Further studies are needed to enhance sensitivity, and better understand the temporal dynamics of α-synuclein seeding in the olfactory mucosa and spreading to other brain areas during the progression from isolated REM sleep behavior disorder to overt α-synucleinopathy, as well the impact of timing, disease subgroups and sampling technique on the overall sensitivity. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported in part by the Ministero della Salute GR201302355724 to MB and GZ, Cariverona: Development and validation of a novel molecular assay for alpha-synuclein in patients with Parkinson's disease and other alpha-synucleinopathies to GZ and Brain Research Foundation Verona. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: EK Nr: 1131/2018 All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Data available upon request to the corresponding author * α-syn : α-synuclein IQR : Interquartile range MDS : Movement disorders society MDS-UPDRS : Movement disorders society Unified Parkinson’s Disease Rating Scale MoCA : Montreal cognitive assessment p-α-syn : Phosphorylated α-synuclein OM : Olfactory mucosa REM : Rapid eye movement RBD1Q : REM sleep behaviour disorder single question RT-QuIC : Real-Time Quaking Induced Conversion SCOPA-AUT : SCales for Outcomes in PArkinson’s disease - AUTonomic dysfunction ThT : Thioflavin T UPSIT : University of Pennsylvania Smell Identification Test