Neurodegeneration associated-proteins in human olfactory epithelium: immunocytochemical and biomolecular study in healthy subjects and patients with synucleinopathies

Olfactory impairment is considered an initial disturbance of several neurodegenerative diseases (NDs), including Parkinson’s disease (PD) and Alzheimer’s disease (AD). In addition, smell impairment precedes a decade, or even longer, the onset of motor or cognitive symptoms. Olfactory signals are detected by olfactory receptor proteins (ORPs) expressed in the cilia of olfactory receptor neurons (ONs). ONs are the distinctive cellular components of the peripheral olfactory epithelium (OE) and lie in the nasal vault. ONs axons pass the cribriform plate and reach the olfactory bulb (OB) where the olfactory stimuli are processed and sent to the superior nuclei of the CNS. Previous studies in AD and other neurodegenerative disorders have shown the presence of β-amyloid deposits in the OB, neurofibrillary tangles, as well as Lewy body pathology. OB represents the brain area earlier involved in the neuropathological process, decades before the development of clinical symptoms. Therefore, OB can be considered a target in the study of neurodegenerative diseases in their early molecular processes. Moreover, the OB of healthy subjects presents deposits of aggregated proteins confirming that these aggregates are deposited in a prodromal disease stage. Since the OB is an early accumulation site of aggregated proteins and the synapses derive from the ONs, it is possible that the first event of protein aggregation occurs in OE. ONs are directly exposed to the external environment including chemical/physical toxic injuries and such micro-environment predisposes to abnormal protein processing and folding (Sammeta and McClintock 2010). In addition, ONs and all other mature cell components have a half-life of three months and programmed apoptosis. The neural activity is maintained by a constant cellular turn-over, which is sustained by the basal stem cells. This regeneration process is persistent during the whole life of an individual, albeit with a decreasing rate with aging. Extensive scientific literature indicates the neuronal damage as the consequence of exposure to toxic injuries leading to neurodegeneration and ONs are a natural model of this noxious process (Lema Tomé, Tyson et al. 2013). The hypothesis of this pathological pathway is supported by several studies, in which aggregated forms of α-synuclein, tau and β-amyloid are detected in olfactory mucosa (OM) biopsies as well as in autoptic samples of patients with Parkinson’s disease (PD), Lewy body dementia (LBD), Frontotemporal dementia (FTD) and Alzheimer disease (AD) (Funabe, Takao et al. 2013) (Saito, Shioya et al. 2016) (Tabaton, Cammarata et al. 1991) (Talamo, Rudel et al. 1989) (Crino, Greenberg et al. 1995) (Arnold, Lee et al. 2010). In this study, we investigated for the first-time primary ONs sampled ex vivo using olfactory brushing (OBg) in normal subjects and patients with different neurodegenerative disorders. Because of its convenient location, OE is easily accessible and can be sampled to obtain the ONs in the tissue outer layer. This sampling method is harmless and non-invasive, bypassing potential artifacts due to post mortem specimens as well as avoiding the invasiveness of biopsy procedures. Recently, we showed that OBg procedure in Creutzfeldt-Jakob Disease (CJD) patients allows efficient OM sampling for the Real-Time Quaking-Induced Conversion (RT-QuIC) assay. We specifically amplified pathological prion protein (PrPSc) providing a diagnostic intra vitam test with sensitivity and specificity nearly to 100% (Orrú, Bongianni et al. 2014). For the purpose of our study, we firstly defined the phenotypic characterization of the human olfactory cells sampled with OBg from healthy subjects. Distinct antibodies were selected to analyze the olfactory epithelium cells: olfactory marker protein (OMP), neuron-specific class III β-tubulin (TUJ-1), protein gene product 9.5 (PGP 9.5), Pan-Cytokeratin (PCK). Secondly, we aimed to determine the expression patterns of the major misfolded proteins involved in the main neurodegenerative diseases. In particular, the selected proteins were: α-synuclein, APP/beta-amyloid, tau, and TDP-43. The identification of the expression patterns of these proteins in the ONs might provide information to understand the abnormal molecular mechanisms in the initial misfolding species involved in the pathological process. Moreover, in this study, we speculated on the subcellular locale where the protein aggregation may occur. Furthermore, by demonstrating the constitutive expression of the native NDs-associated proteins in the OE, we could assume that they may represent a potential template for triggering the aggregation process. Based on the immunocytochemistry analysis, we investigated the α-synuclein expression in patients affected by different synucleinopathies. In fact, α-synuclein misfolding and aggregation mechanisms are involved in the pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (LBD) and multiple system atrophy (MSA), which are all characterized by α-synuclein fibrils deposition (Spillantini, Schmidt et al. 1997). Finally, we analyzed the immunocytochemistry results in OM samples tested by α-synuclein RT-QuIC (α-syn RT-QuIC).