Innovative approaches for the diagnosis of Parkinson’s disease and multiple system atrophy based on the analysis of the olfactory mucosa

Parkinson's disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases whose diagnosis is particularly complex, especially in the early stages, because the symptoms are similar to each other and to those of other diseases, including dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). All these disorders share a similar pathological process: the change in the structure of some proteins normally present in the brain which thus lose their function, begin to aggregate and deposit in specific brain areas, causing irreparable damage. In particular, PD, MSA and DLB are called α-synucleinopathies because they present aggregates of the α-synuclein protein (αSynD), which however are localized in different brain structures. PSP and CBD are instead called tauopathies because they are characterized by the presence of aggregates of the tau protein. These protein aggregates are considered disease-specific biomarkers because their detection and distribution (which can only be determined post-mortem on the patient's brain tissue) are used to formulate a definite diagnosis. As long as the patient is alive the diagnosis is only probable and does not have absolute accuracy. Consequently, some diagnoses made in life may change after the neuropathological assessments. Several evidence suggests that misfolded proteins can also appear in peripheral tissues such as the olfactory mucosa (OM, easily and periodically collectible with a nasal swab), but in such small quantities as not to be detectable with common diagnostic techniques. The recent advances in molecular and structural biology have provided insights into the processes involved in the pathogenesis of neurodegenerative diseases and have made it possible to recapitulate the protein misfolding process in vitro in a limited period of time through the development of innovative techniques, called seed amplification assays (SAAs), among which the real-time quaking-induced conversion (RT-QuIC). This new methodology exploits the ability of misfolded proteins to transmit their abnormal conformation to normal monomers, which are used as substrate of the reaction. Abnormally folded proteins are able to interact with these substrates and induce monomers to change conformation and subsequently aggregate. Therefore, the addition of misfolded proteins (considered “seeds”) to the substrate is able to trigger an aggregation phenomenon, known as “seeding effect” that might be exploited for diagnostic and therapeutic purposes. In my Ph.D. project I have firstly optimized the RT-QuIC assay, with the aim of analyzing OM samples collected from patients with PD, MSA, CBD and PSP, and evaluating the efficacy of the test in detecting traces of misfolded αSynD in α-synucleinopathy derived samples. The results of our study showed that most OM samples from patients with PD and MSA induced aggregation of the recombinant substrate protein, suggesting the presence of traces of αSynD. In contrast, the PSP and CBD samples had no effect on the substrate (since they do not contain abnormal αSyn). Interestingly, the RT-QuIC reaction products acquired biochemical and biophysical characteristics useful to discriminate, with a good degree of accuracy, patients with PD from patients with MSA. Moreover, by exposing neuronal-like differentiated SH-SY5Y cells to these products, we observed the induction of different inflammatory pathways. These findings suggested the existence of a link between the morphology of the aggregates and their inflammatory properties. To deepen this aspect, we have produced three different recombinant aggregates of αSyn, in order to generate, in a controlled environment, artificial αSyn seeds resembling to some extent the αSynD strains present in OM, and test their behavior by RT-QuIC without the presence of other tissue factors. Although capable to efficiently seed the aggregation of the substrate, αSv1, αSv2, and αSv3 did not transmit their seed-specific properties to the reaction products which showed comparable biochemical properties, instead. Probably, our experimental setting was too artificial to properly recapitulate the phenomenon of the seeding effect exerted by αSynD in RT-QuIC. However, when used to stimulate SH-SY5Y cells, αSv1, αSv2, and αSv3 acted on different activators of inflammatory pathways, thus strengthening the existence of a correlation between morphological and inflammatory properties of αSyn fibrils. In the last part of my project, we decided to evaluate how much the RT-QuIC assay could be used for diagnostic purposes in the field of α-synucleinopathies, by studying its reproducibility in other laboratories. Together with an American lab we have so analyzed a group of OM samples with the same experimental protocol and we obtained a 96% concordance of results. Furthermore, we observed that the OM of MSA behaved differently according to the pathological subtype. In fact, we know that this disease can manifest itself in a cerebellar form (MSA-C) or associated with parkinsonism (MSA-P). In our test, only MSA-P samples induced a seeding effect, allowing us to discriminate between the two pathological subtypes. These preliminary studies provide evidence that RT-QuIC of OM samples represents a reliable assay for supporting the diagnosis of α-synucleinopathy and may limit the negative effects that misdiagnosis produces in terms of costs for the healthcare system and improve overall patient care, treatment, and possible enrollment in future clinical trials.