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To investigate molecular biomarkers of a-synuclein and tau aggregation, autophagy, and inflammation in the saliva of de novo Parkinson's disease (PD) patients in comparison to healthy subjects (HS), and to correlate molecular data with clinical features of PD patients, in order to establish whether abnormalities of these parameters are associated with specific clusters of de novo PD patients, and their potential diagnostic power in differentiating PD patients from HS.We measured total and oligomeric a-synuclein, total-tau and phosphorylated-tau, MAP-LC3beta, and TNFalpha in the saliva of 80 de novo PD patients and 62 HS, using quantitative Enzyme-Linked Immunosorbent Assay analysis.Oligomeric a-synuclein, total-tau, MAP-LC3beta, and TNFalpha levels resulted significantly higher in patients with respect to HS, while no significant differences were detected for total a-synuclein or phosphorylated-tau. Phosphorylated-tau directly correlated with MAP-LC3beta, whereas it inversely correlated with TNFalpha in PD patients. An inverse correlation was detected between MAP-LC3beta and non-motor symptoms severity. Principal Component Analysis showed that molecular and clinical parameters were independent of each other in de novo PD patients. Receiver Operating Characteristic curve analysis reported an accurate diagnostic performance of oligomeric a-synuclein and MAP-LC3beta. The diagnostic accuracy of total a-synuclein increased when it was combined with other salivary biomarkers targeting different molecular pathways.Our study proposes a novel biomarker panel using saliva, a non-invasive biofluid, in de novo PD patients, with implications in understanding the molecular pathways involved in PD pathogenesis and the relevance of different molecular pathways in determining clinical PD subtypes. This article is protected by copyright. All rights reserved.

Cholangiocytes are the target cells in cholestatic models of ductal hyperplasia including bile duct ligation (BDL). We have shown that: (i) cholangiocytes express VEGFR-2 and VEGFR-3; (ii) VEGF-A and VEGF-C stimulate cholangiocyte proliferation via an autocrine mechanism; and (iii) chronic administration of VEGF-A prevents cholangiocyte damage induced by hepatic artery ligation. Caffeic acid phenethyl ester (CAPE) induces growth inhibition in different cells. Taurocholic acid (TC) protects cholangiocytes against injury induced by parasympathetic or sympathetic denervation. The aims of this study were to determine if: (i) CAPE induces bile duct damage; and (ii) TC prevents CAPE-induced bile duct damage by increasing cholangiocyte VEGF expression. Methods: Normal and BDL rats (immediately after surgery) were fed 1% TC or control diet in the absence/presence of daily IP injections of CAPE (10 mg/Kg BW). One week later, we evaluated: (i) cholangiocyte apoptosis, proliferation and ductal mass in liver sections; (ii) functional activity by measuring secretin-stimulated bile and bicarbonate secretion; and (iii) VEGF-A/C and VEGFR-2/R-3 expression in liver sections. In vitro, BDL cholangiocytes were exposed to CAPE (40 μM) in the absence/presence of TC (40 μM) with and without pretreatment with VEGF receptor inhibitors before evaluating cholangiocyte apoptosis and proliferation. Results: Chronic CAPE administration to BDL rats increased cholangiocyte apoptosis and decreased ductal mass. This effect was associated with reduced expression of VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3. In vivo, TC feeding partly prevented CAPE-induced changes in cholangiocyte apoptosis and growth and loss of ductal secretion. The protective effect of TC was associated with enhanced VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3. In vitro, TC partially prevented CAPE-induced increases in apoptosis and decreases in cholangiocyte proliferation. These changes were reversed by pretreatment with VEGF-receptor inhibitors. Conclusion: Manipulation of cholangiocyte VEGF expression by bile acids may be important in preventing the impairment of cholangiocyte proliferation by exogenous agents.

Reduction of biliary serotonin N-acetyltransferase (AANAT) expression and melatonin administration/secretion in cholangiocytes increases biliary proliferation and the expression of SR, CFTR and Cl(-)/HCO3 (-) AE2. The balance between biliary proliferation/damage is regulated by several autocrine neuroendocrine factors including vascular endothelial growth factor-A/C (VEGF-A/C). VEGFs are secreted by several epithelia, where they modulate cell growth by autocrine and paracrine mechanisms. No data exists regarding the effect of AANAT modulation on the expressions of VEGFs by cholangiocytes.In this study, we evaluated the effect of local modulation of biliary AANAT expression on the cholangiocytes synthesis of VEGF-A/C.The decrease in AANAT expression and subsequent lower melatonin secretion by cholangiocytes was associated with increased expression of VEGF-A/C. Overexpression of AANAT in cholangiocyte lines decreased the expression of VEGF-A/C.Modulation of melatonin synthesis may affect the expression of VEGF-A/C by cholangiocytes and may modulate the hepatic microvascularization through the regulation of VEGF-A/C expression regulating biliary functions.