Neurodegenerative diseases(NDs)are fatal and incurable conditionscharacterized bythe progressive accumulation in specific brain regions of abnormally folded(misfolded)proteins, which are considered disease-specific biomarkers (DSB).These misfoldedproteins are able to spread through neuroanatomical connected regions and to accelerate the conformational conversion of native monomers (seeding), thus progressively amplifying the pathological process. Primary tauopathies are NDs associated withthe accumulation of misfolded tau and include Corticobasal degeneration (CBD), Progressive supranuclear palsy (PSP), Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and other cases of Frontotemporal dementia (FTD). Alzheimer\u2019s disease (AD) can be considered a secondary tauopathy as it is characterized by tau misfolding in addition to amyloid-\u3b2 (A\u3b2) protein deposition. Synucleinopathies comprise a group of NDsassociated with the accumulation of misfolded \u3b1-synuclein (\u3b1S), including Parkinson\u2019s disease (PD) and other atypical parkinsonisms known as Multiple system atrophy (MSA) and Dementia with Lewy bodies (DLB).Given the overlap between clinical symptoms amongNDsand the lackofsensitive and specificdiagnostic tests, the definite diagnosis of NDs lay on neuropathological detectionof these misfolded proteinsin post-mortembrain tissues. However, recent findings have raised the possibility that trace-amount of DSBmight circulate in peripheral tissues and body fluids of affected individuals, thus constituting easily accessiblebiomarkers. For this reason, in my PhD projectweevaluated the ability of an extremely sensitivetechnique, named Real-Time Quaking Induced Conversion (RT-QuIC), to detect seeding activity of misfolded tau eventually present in peripheral tissues, such as olfactory mucosa (OM), and body fluids(urine and cerebrospinal fluid)collected from patients with clinical diagnosis of primary (FTDP-17, FTD, PSP, CBD) and secondary (AD) tauopathies.RT-QuIC assaywas optimized using a recombinant tau protein fragment named tauK18 (4R-tau)as substrate, whose aggregation was efficiently triggered(seeded) by the addition of minute amount (attograms) of tauK18 pre-formed fibrils (PFFs)previouslygenerated in vitro.We demonstrated that tauK18 RT-QuIC assay wasable to detect seeding activity of misfolded tau contained in brain samples of neuropathologically confirmed cases of FTDP-17, PSP,and AD. Thus, we performed RT-QuIC analysis of (i) OM, (ii) CSF and (iii) exosomes extracted from urine samples collected from patients with different primary and secondary tauopathies. As a comparison,we included in theanalysis samples belonging topatients with different synucleinopathies (PD,MSA,and DLB), Multiple sclerosis (MS), Non-demented patients (NDP) and healthy controls (HC). Results showed that tauK18 RT-QuIC assay was able to detect tau seeding activity in CBD and PSP OM samples, but also in some PD, MSA, DLB and MS cases. Similarly, RT-QuIC analysis of CSF samples displayed smalldifferences in tauseeding activity between AD and NDP cases. On the other hand, RT-QuIC analysis of urinary exosomes revealed that AD, FTD and CBD samples triggered tauK18 aggregation with higher efficiency if compared to HC, thus potentially discriminating between tauopathies and healthy subjects. We investigated the ability of PFFs generated in vitrofrom other NDs-associated proteins (3R-tau fragment named tauK19, \u3b1S, A\u3b21-40,and A\u3b21-42) to influence tauK18 aggregation (cross-seeding) and we found that some conformational variants of \u3b1S PFFs were able to cross-seed tauK18 aggregation, thus representing a potential issue for our assayand possibly explaining results obtained with theanalysis of OM samples. Moreover, preliminary structural analysis showed that final reaction productswere characterized by different morphologies when seeded by different (i) OM samples or by (ii) PFFs generated in vitrofrom tauK19, \u3b1S, A\u3b21-40,and A\u3b21-42, suggesting that biophysical assessments might help in discriminating between different seeding-competent samples. Although further retrospective analysis isrequired to confirm results obtained with ourtauK18 RT-QuIC assay, this preliminary study might lay the basis for the development of a new diagnostic approachwhich combines RT-QuIC and biophysical techniques to detect tau seeding activity in peripheral tissues and body fluids of patients with tauopathiesand to discriminatebetween different pathological conditions
