AN EXPLORATIVE ASSESSMENT OF POTENTIAL NOVEL DIAGNOSTIC AND PROGNOSTIC BIOMARKERS FOR IDENTIFICATION OF PRODROMAL PARKINSON¿S DISEASE

ABSTRACT Background - Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer, primarily affecting about 6 million people worldwide. An early identification of PD is one of the main challenges in neurological research because to date, its diagnosis is still largely based on the clinical assessment of cardinal motor signs (bradykinesia, rigidity, resting tremor and postural instability) resulting by a progressive degeneration of dopaminergic neurons of the substantia nigra and locus coeruleus. However, impaired motor function appears when over 60% of the dopaminergic neurons are degenerated in the brain. In recent years, several evidence indicates that the onset of PD happens years to decades before the occurrence of classic motor symptoms. Pathological and imaging studies, for example, suggest that signs of nigrostriatal lesion can be detected 5–10 years before this clinical stage, and various observational prospective studies reveal that several non-motor symptoms (NMS) occur in this pre-diagnostic phase. Actually NMS such as olfactory impairment, cardiovascular dysautonomia such as orthostatic hypotension (OH) and rapid eye movement (REM) behaviour disorder (RBD) are currently being studied as features of prodromal PD and seem to be correlated to the early neuropathological process of disease. Beside these clinical manifestations, other biological alterations such as elevated oxidative stress and pro-inflammatory response have been involved in the cascade of events leading to degeneration of dopaminergic neurons. Recently, microRNAs (miRNAs) have been recognized as potent post-transcriptional regulators of PD-related gene expression. Consequently, the characterization of several NMS together with the assessment of molecular biomarkers linked to inflammation and oxidative damage, could be a potential methodological approach for the early identification of PD patients. Objectives - The main objective of my study was to explore potential novel diagnostic and prognostic biomarkers of PD. Specific study aims were, in patients with prodromal and established PD: a) to evaluate clinical markers such as olfactory and cardiovascular autonomic functions; b) to measure circulating mediators of oxidative stress and inflammatory response as early biomarkers of organ failure; c) to correlate biological findings with clinical functional alterations; d) to characterize specific circulating miRNA profiles in plasma samples. Methods - For this purpose, we recruited 15 patients with overt PD (Hoehn and Yahr stage I-III, on L-DOPA and dopamine agonists combination therapy), 11 subjects diagnosed with idiopathic RBD (iRBD) confirmed by lack of atonia during the REM sleep phase on polysomnography and 12 age- and gender-matched controls (CTRL). All enrolled subjects underwent the following assessments: total olfactory score (TOS) using Sniffin' Sticks Extended Test; autonomic function by measuring heart rate variability during deep breathing (DB) test, which expresses parasympathetic function, lying to standing (LS) test and the Valsalva manoeuvre (VM), that gives information about both sympathetic and parasympathetic function; antioxidant/oxidative stress mediators [glutathione (GSH), the most important endogenous scavenger, assessed in total and reduced form and in plasma and blood samples according to a high performance liquid chromatographic (HPLC) method; plasma malondialdehyde (MDA), a marker of lipid peroxidation, assayed by HPLC with fluorescence detection; 8-hydroxy-2-deoxyguanosine (8-OHdG), index of oxidative DNA damage, and 3-nitrotyrosine (3-NT), a stable end product of peroxynitrite oxidation, analyzed by commercial ELISA kits]; inflammatory response [plasma concentrations of tumor necrosis factor alpha (TNF) and interleukin 1-beta (IL1), the most important inflammatory cytokines, by ELISA commercial kits, while urine neopterin levels, a sensitive marker of cellular-mediated inflammation, were measured by an isocratic HPLC method]. Biochemical parameters were than correlated with clinical functional results. The miRNA profiling was performed in a subpopulation of the enrolled subjects (4 PD, 4 iRBD and 4 CTRL) by small RNA Sequencing, using Miseq sequencer (Illumina). The differentially expressed (DE) miRNAs analysis, based on the negative binomial distribution, was performed with DE Seq2 by performing three comparisons:1) iRBD versus CTRL; 2) PD versus iRBD; 3) PD versus CTRL. Subsequently, the relative expressions of specific miRNAs were validated in all study population by quantitative real-time (qRT) PCR using miScript PCR System kit (Qiagen). Results - A significant worsening trend was observed in total olfactory score, blood reduced GSH, LS and VM ratio and neopterin from the reference controls to iRBD and PD groups. In the multivariable ordinal logistic regression model, only low blood reduced GSH levels (p=0.037, OR=0.994; 95% CI 0.988 – 1.000), adjusted by history of hypertension, total olfactory score, LS ratio and VM ratio, were associated to PD status. Functional anosmia was similarly prevalent in iRBD (36%) and PD (33%) patients, but was absent in CTRL (p= 0.097). OH was more common among iRBD (73%) and PD (60%) than in controls (25%) (p=0.055), independently of antihypertensive treatment. A direct correlation was observed between total olfactory score and blood reduced GSH concentrations (R=0.034, p=0.037) and with VM ratio (R=0.43 p=0.015). Conversely, an inverse relation was found between total olfactory score and urine neopterin levels (R=-0.39 p=0.016). The results on circulating miRNA profiles found about 889 thousand sequenced reads mapped to mature miRNA sequences annotated in miRBase v21, by small RNA sequencing analysis. After data processing, no statistically significant DE miRNA was observed in the PD versus CTRL, whereas we found 33 DE miRNAs (18 downregulated, 15 upregulated, p-value <0.005) in the comparison between PD and iRBD and 6 (3 downregulated, 3 upregulated, p-value <0.005) in iRBD versus CTRL. Four common DE miRNAs (miR-101, miR-1260a, miR-142, miR15a) were dysregulated between the two different comparisons. In the PD patients, three miRNAs (miR-101, mir-142 and miR15a) were downregulated (Fold Change < -0.5) and only mir-1260a was upregulated (Fold Change > 0.5) with respect to iRBD. Conversely, miR-101, miR-142 and miR15a were upregulated and miR-1260a downregulated in iRBD compared to CTRL. The NGS results have not been validated by RT-PCR analysis till now because these miRNAs are poorly expressed in plasma. This condition makes very difficult, from a methodological point of view, their extraction. Discussion - The main findings of the present study are that reduced systemic antioxidant capacity is independently associated to overt PD and iRBD, a condition now established as prodromal PD, and correlates with olfactory and sympathetic dysfunction. Moreover, progressive cardiovascular autonomic dysfunction, expressed as altered sympathetic (VM ratio, OH) or parasympathetic (LS ratio) response to testing, is found from prodromal state to overt disease and correlates with olfactory dysfunction. Increased concentrations of neopterin, an inflammatory biomarker, are associated with worse olfactory dysfunction. The NGS analysis highlights a miRNA profiling in PD and iRBD subjects that needs to be verify, by changing and modifying the methodological approach for miRNA quantification. Conclusions - Reduced systemic antioxidant capacity is found in prodromal and overt PD and may represent, in association with olfactory loss and cardiovascular autonomic dysfunction, a useful additive biomarker of disease. Our pilot findings need to be confirmed in a larger population to establish their actual clinical value for an early diagnosis of PD.