Neuroprotection of Active Principles from the Leaves of Laurus nobilis and Mori Cortex Radicis in Neuronal Cell Culture and Rodent Models of Parkinsons Disease: Regulatory Effect on the Ubiquitin-Proteasome

Abstract

PD is characterized by selective loss of dopaminergic (DAergic) neurons in the substantia nigra (SN) and the presence of Lewy bodies, the pathological hallmarks of PD composed of aggregated a-synuclein (ASYN) occurring inside neurons. The modification of ASYN may initiate the accumulation of abnormal proteins by impairment of the ubiquitin-proteasome proteolytic (UPP) pathway. A failure of the UPP pathway, which degrades intracellular proteins, has also been thought to play an important role in the pathogenesis of PD. The aims of this study were to assess the neuroprotective effect of the active principles from the Leaves of Laurus nobilis, Mori Cortex radicis, and Cudrania tricuspidata against dopamine (DA)-induced neuronal toxicity by regulation of UPP and ASYN-mediated pathways in both in vitro and in vivo models of PD. We observed a marked increase in apoptosis and the generation of ROS and decreased cell viability by DA. Pretreatment of the cells for 24 h with each of cstunolide, dehydrocostuslactone, and spirafolide from Laurus nobilis before DA exposure notably increased the cell survival and lowered the intracellular ROS level. Moreover costunolide had inductive effects on the expression of Nurr1 (nuclear receptor related-1), DA transporter (DAT), and vesicular monoamine transporter type 2 (VMAT2) in DA treated human DAergic SH-SY5Y cells, and a reciprocal reduction in ASYN was observed at both the transcriptional and translational levels in vitro. E6-associated protein (E6-AP) has been known to promote the degradation of ASYN. Flow cytometric and western blotting analyses revealed that reynosin significantly protected against DA-induced cell death in both SH-SY5Y cells and 6-hydroxydopamine (6-OHDA)-induced tyrosine hydroxylase (TH)-positive cell loss in the SN region of rat brain. In addition, reynosin concomitantly up-regulated E6-AP protein expression and down-regulated over-expression of the ASYN protein in both DA-treated cells and a rodent PD model system. And the treatment with moracenin D isolated from Mori Cortex radicis resulted in an up-regulation of Nurr1 mRNA levels and a down-regulation of ASYN mRNA levels. Additionally, the ASYN protein expression was decreased in accordance with an increase in Nurr1 protein expression. The protective effect of moracenin D was presumably due to the correlative effects on the up-regulation of nurr1 and down-regulation of ASYN expressions against DA induction. Iisozaluzanin C, magnolialide, santamarine, lucentolide, 1β-hydroxyarbusculin A, and methyl 1β, 2β, 6-tryhydroxy-5, 7H-eudesma-4(15), 11(13)-dien-12-oate (METO) were isolated from the chloroform extract of Laurus nobilis. Incubation of DA alone increased in fluorescence, as the protein aggregation. However co-incubation of magnolialide or 1β-hydroxyarbusculin A with DA significantly inhibited protein aggregation, as determined by thioflavin S fluorescence. Further magnolialide, isozaluzanin C, santamarine, and lucentolide co-treated with DA increased proteasome activity significantly in the DA-induced markedly decreased proteasome activity. Parkin has E3 ubiquitin ligase activity and is mediated UPP pathway. Therefore the mutants were designed point mutation of E3 ligase activity associated RING region. After cell lysates of the over-expressed Parkin were prepared and conducted E3 ligase activity by autoubiquitination assay. The C289G missense mutation is in functional domains in the Parkin protein; altered decreasing the E3 ligase activity in both cell lysates of immunoprecipitated C289G mutant and C289G mutant with 6-OHDA. 5, 7-Dihydroxychromone significantly increased the decreasing E3 ubiquitin ligase activity of Parkin by 6-OHDA-induced in C289G mutant cells. Taken together, the present results indicated that the neuroprotective effects of isolated compounds from natural plants on DA-induced cytotoxicity and mutation of Parkin in models of PD may be, at least in part, attributable to the its potent regulation of UPP or ASYN-mediated pathways. Therefore these active principles could be considered as a candidate therapy for the treatment of PD.