Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease (AD) and is characterized by bradykinesia with rigidity, tremor and postural instability [
1,
2]. Neurodegenerative parkinsonisms resemble PD in some of its clinical feature and include Multiple System Atrophy (MSA), Progressive Supranuclear Palsy (PSP) and Corticobasal Degeneration (CBD) [
3,
4]. Although highly heterogeneous from a clinical and neuropathological point of view, all these diseases share a common pathological mechanism that involves the accumulation of abnormally folded proteins in the Central Nervous System (CNS) [
5,
6]. In particular, PD is characterized by the presence of α-synuclein aggregates in neuronal perikarya and neuronal process (referred to as Lewy bodies and Lewy neurites) mainly located in the brainstem, while neurodegenerative parkinsonisms can be divided in two molecular classes [
7,
8]. The first molecular class corresponds to MSA that is characterized by accumulation of abnormal conformers of α-synuclein (also referred to as α-synucleinopathies) in oligodendrocytes of cerebellum, pons and basal ganglia [
3,
9‐
11]. The second molecular class includes disorders characterized by brain accumulation of abnormal forms of tau protein which cause PSP or CBD (hence referred to as tauopathies). PSP shows aggregates of 4-repeat tau (4R) in neurons (neurofibrillary tangles), astrocytes (tufted astrocytres) and oligodendrocytes (coiled bodies) prevalently located in basal ganglia and brainstem, while CBD is characterized by the presence of 4R tau in oligodendrocytes (coiled bodies), astrocytes (astrocytic plaques), and neurons located in neocortex and basal ganglia [
3]. One of the most intriguing aspects of these diseases is therefore the fact that the same protein might account for different pathologies. Probably this is due to conformation-dependent changes in the protein affected. Indeed, either α-synuclein or tau can acquire different abnormal conformations, that can be considered analogous to the “strains” of prion diseases [
3,
12‐
17]. It can be hypothesized that different strains of α-synuclein are responsible for PD or MSA while different strains of tau cause PSP or CBD. These structural differences might dictate specific tropism of these proteins for defined neuroanatomical regions or even cell types. Currently, both α-synuclein and tau are considered disease-specific biomarkers and definite diagnosis of PD and neurodegenerative parkinsonisms relies on their identification and anatomical distribution in brains collected at autopsy. Conversely, the in vivo diagnoses based on clinical criteria and neuroimaging are characterized by unsatisfactory sensitivity and specificity, and several cases might be misdiagnosed, especially in the early stages when clinical symptoms overlap [
18‐
20]. Moreover, in some instances, the co-occurrence of different protein aggregates may take place in the brain. For instance, cases of PSP, CBD and AD confirmed at neuropathological level, were found to concurrently contain aggregates of α-synuclein in the form of Lewy bodies or, in other cases, aggregates of α-synuclein in isolation may phenotypically present as PSP or CBD [
21‐
25]. Similarly, cases of MSA or PD were found to also contain aggregated species of tau [
26‐
29]. The in vivo diagnostic accuracy for PD is at about 80 and 20% of patients classified as probable PD are indeed misdiagnosed [
19]. Even worse, only 26% of patients with a diagnosis of possible PD are confirmed at autopsy [
30]. The diagnostic accuracy of MSA is even lower and the clinical diagnosis is confirmed at autopsy in only 62% of patients [
31‐
34]. Diagnostic accuracy of PSP has a specificity of about 95% for probable and 80–93% for possible cases [
35‐
39] while only the 68% of patients diagnosed in vivo as CBD are confirmed at autopsy [
40]. Therefore, a defined discrimination of these diseases is difficult with the current clinical diagnostic criteria alone. Compelling evidence suggests that trace amount of these abnormally folded proteins are present in cerebrospinal fluid (CSF) and peripheral tissues of diseased patients. Unfortunately, their concentration is well below the limits of detection of the conventional diagnostic techniques. By exploiting an innovative technique named Real Time Quaking Induced Conversion assay (RT-QuIC), developed in the field of prion diseases [
41,
42], it was recently shown the presence of minute amount of abnormal α-synuclein in the CSF of PD patients with 95% sensitivity and 100% specificity [
43]. Similarly, Soto and colleagues successfully identified α-synuclein in the CSF of patients with PD with 89% sensitivity and 97% specificity [
44]. More recently, Groveman improved the assay and allowed quantitation of α-synuclein present in the CSF of patients with PD and Dementia with Lewy bodies (DLB) [
45]. Finally, Saijo and colleagues showed the presence of abnormal 3R-tau in the CSF of patients with Frontotemporal dementia (FTLD) [
46] with 100% sensitivity and 94% of specificity. Therefore, RT-QuIC represents a fundamental tool that might significantly increase the diagnostic accuracy of these diseases. The technique exploits the prion-like properties of abnormally folded α-synuclein and tau proteins, which become capable of interacting with their normal counterparts forcing them to acquire similar pathological structures. While in vivo this feature is considered to be responsible of the progression of α-synuclein or tau pathology within the brain (by transmission of protein misfolding), in vitro this property has been exploited in RT-QuIC for detecting minute amount of abnormally folded proteins in a known biological samples (CSF or blood or urine) [
47‐
56]. In RT-QuIC, samples are incubated with a recombinant protein used as substrate of reaction. The presence of pathological proteins in the samples triggers the aggregation of the substrate, generating amyloid fibrils whose formation is monitored in real time with the use of the Thioflavin-T (ThT) fluorescent dye [
57,
58]. Recent evidence has demonstrated that abnormal α-synuclein or tau proteins can accumulate in the olfactory epithelium collected post-mortem from patients with AD [
59]. To the best of our knowledge, there are no reports that have evaluated the ability of RT-QuIC to detect trace amount of abnormally folded proteins in OM samples collected from patients with a clinical diagnosis of PD or neurodegenerative parkinsonisms. RT-QuIC has been successfully optimized for the analysis of OM samples collected from patients with prion diseases, so far [
60]. We therefore decided to perform RT-QuIC experiments, using human recombinant α-synuclein (rec-αS) as reaction’s substrate, aimed at evaluating the presence of abnormal α-synuclein in OM samples collected from a consecutive series of patients affected by different neurodegenerative parkinsonian syndromes, to evaluate if seeding activity for α-synuclein can differentiate between synucleinopathies and tauopathies. Moreover, we evaluated if different strains of α-synuclein (MSA or PD) might have imprinted their aberrant structure to the same rec-αS used as RT-QuIC substrate. In contrast to the widely used CSF, OM samples can be periodically collected with a non-invasive procedure, thus representing optimal tissues for RT-QuIC analysis.