Identification And Characterization Of Differentially Regulated Proteins In Rat Model Of Methamphetamine-induced Neurotoxicity In Response Toα-synuclein RNA Interference

Backgroud:Methamphetamine (METH) is an emerging addictive drug, belonging to Amphetamine-Typed Stimulants (ATS). METH can elicit a wide spectrum of pharmacological and toxicological responses, such as central excitability, hallucination, appetite repression and sympathomimetic effects. Due to its fast-acting and sustained effects, low cost, ease of production through chemical synthesis, METH has become one of the two heavily abused drugs, which poses a tremendous burden to the succumbed individuals, their family and the whole society as well. Therefore, it is imperative to elucidate the mechanisms underlying METH’s neurotoxicity and addiction.It has been shown by many clinical studies that METH intoxication causes serious damage of a number of vital organs such as the heart, brain, lung, live and kidney. However, the brain is the most affected organ. METH abuse causes overt psychobehaviral changes, affording lesions in multiple areas such as substantia nigra, striatum and hippocampus. It is demonstrated that dopaminergic and5-HT-ergic termini in the brain were damaged by METH abuse, resulting in decreased level of dopamine (DA), dopamine transporter (DAT) and tyrosine hydroxylase (TH) activity, exhaustion of5-HT and its metabolites, depletion of dopamine binding sites on vesicular monoamine transporter2(VMAT-2), resulting in axonal damage and neuronal demise.The mechanisms underlying the neurotoxicity of METH are still not well understood, however, it has been shown that the following cascades are involved:1) oxidative stress damage by excessive dopamine oxidation,2) calcium overload and disrupted cellular homeostasis resulted from glutamate-induced excitotoxcity,3) mitochondrial damage and malfunction4) activation of apoptotic pathways, oxidative stress is considered one of the most important mechanisms underpinning METH-induced neurotoxicity.In our previous study, we have investigated the differentially expressed genes in the rat striatum, cortex and hippocampus after METH injection. The results showed that oxidative stress, perturbed energy metabolism, proteasome dysfunction and apoptosis are responsible for the neurotoxicity of METH. In addition, in striatum of METH-injected rat, we found that ROS, NOS, ONOO-、NO, MDA and DDAH were significantly increased, whereas SOD level was decreased. These findings suggest that NO may afford damage to the CNS through DDAH/ADMA/NOS pathway. Of notice, we found that the expression of a-synuclein (a-sys) was remarkably increased in the striatum, cortex and hippocampus of METH-injected rat.Physiologically, a-sys is an acidic soluble protein localized in neuronal nuclear membrane and presynaptic terminus, consisting of140amino acid residues.α-sys plays pivotal role in neural transmission and plasticity, capable of binding to a number of signaling molecules, scaffold proteins, enzymes, irons and chaperones. a-sys knock-out mouse shows no overt phenotype, suggesting potential redundancy may exist in vivo. High concentration or overexpression of a-sys is toxic to neurons. At high concentration, a-sys forms oligomers of P-sheet, which are more neurotoxic, hinting that abnormal aggregation and fibrillogenesis may underline neurodegenerative disorders such as Parkinson’s disease (PD), and Alzheimer’s disease. METH-injected brains show neuopathological changes similar to what was observed in the aforementioned neurodegenerative diseases, moreover, the incidence of PD is increased in METH abusers. Recently, striatal Lewys body-like inclusions have been observed in METH-injected mouse. Reduced dopamine release, oxidative stress, calcium overload, mitochondrial damage and apoptosis are contributing together to the pathogenesis of PD, for which a-sys plays an important role. When a-sys is of high concentration, it engages in the initiating the process of neuronal damage, in return, a-sys itself becomes an effector protein to drive neurodegeneration and neuronal loss. Nitration of a-sys makes its oligomer more stable and neurotoxic. Therefore, a-sys is a potential target of METH-induced neurotoxicity.We found that in dopaminergic cell line SH-SY5Y, silencing α-sys via RNAi inhibited METH-induced cell viability reduction, downregulation of TH,DAT, VMAT-2, dopamine exhaustion, elevation of ROS,NOS and NO, reduction of mitochondrial ΔΨm, calcium influx and cytochrome C release to cytosol, preventing METH-induced cell death. However, it is unclear as to how a-sys is involved in METH-induced neurotoxicity and what the target proteins are. Therefore, we aim to sort out these questions.RNAi has become an instrumental tool to delineate gene function. Compared with antisense-mediated gene suppression, RNAi is more potent, sustained in terms of silencing off target genes (100to10,000times more potent), providing high sequence-specificity and avoidance of immunogenicity. iTRAQ (isobaric tags for relative and absolute quantitation) is an in vitro polypeptide labeling technique that is based on the covalent labeling of the N-terminus and side chain amines of peptides from protein digestions with tags of varying mass of4to8different istotopes, enabling simultaneous quantification and comparison of proteins from4or8different samples when coupled with tandem mass spectrometer. Due to its higher sensitivity, iTRAQ can identify more differentially regulated proteins.Therefore we aim to knock down a-sys expression using RNAi, which may serve as a good platform to study the role played by a-sys in METH-induced neurotoxicity. Furthermore, iTRAQ technique will be utilized to identify differentially expressed proteins, whose roles in METH-induced neurotoxicity will be investigated. Aims:To establish rat model of a-sys-RNAi, in which the effect of knockdown of a-sys gene expression on METH-induced dopaminergic toxicity and oxidative stress damage will be investigated using behavioral and enzymology methods. In addition, proteins differentially regulated in the striatum in response to a-sys RNAi in METH-induced neurotoxicity rat model will be identified using iTRAQ technique and their roles with respect to a-sys in the pathogenesis of METH-induced neurotoxicity will be characterized.Methods:1. The establishment of rat model of a-sys-RNAiAdult male Wistar rats were randomized into four groups,①Control (CON);②Model;③Empty vector;④RNAi:a-sys-ShRNA lentiviruses were produced and characterized in our previous work. The intended substances were delivered into rat striatum via stereotaxic injection as following:saline for CON and Model groups,) for Empty vector group, a-sys-ShRNA lentivirus for RNAi group。Striatum was collected2weeks postinjection and a-sys expression was examined using real-time quantitative PCR and Western blot.2. The effect of a-sys-RNAi on METH-induced neurotoxicity in vivoSaline was intraperitoneally administered to rats in CON group, while METH (15mg/kg, one injection at8am and6pm respectively,4consecutive days,8injection in total) was intraperitoneally injected to rats in Model, Empty vector and RNAi groups. Animal behavior, weight, food intake and water consumption were closely monitored. Striatal DA and TH levels were assayed with ELISA; activity of ROS, NOS was examined with fluorescence enzymology assay kits.3. The identification and characterization of differentially expressed proteins in METH-induced neurotoxicity in response to α-sys-RNAiStriatal tissues from various groups (n=6/group) were homogenized using SDT lysis buffer, sonicated and spun, protein concentration of the supernatants was measured using BCA method. Subsequently, the quality of the protein was checked by subjecting20ug protein sample to SDS-PAGE and Coomassie blue staining.400microgram protein was subjected to trypsin digestion and peptide quantification.64microgram of tryptic peptides were labeled using iTRAQ Reagent-4plex Multiplex Kit and separated through nanoflow liquid chromatography system (Easy nLC), followed by peptide identification and validation through Q-Exactive mass spectrometer.Results:1. Establishment of a-sys-RNAi rat model:a-sys mRNA level was reduced80%in a-sys-shRNA lentivirus transduced rat striatum, striatal a-sys protein level was also significantly decreased as evidenced by Western blot.2. Food intake was significantly (p<0.001) after METH injection, confirming METH’s suppression of appetite; animal weight loss was significant (p<0.001). Behavioral changes observed in METH-injected rat manifested as hyperactivity and irritability, with stereotypical behavior score was higher than control group (p<0.001). Water consumption was also increased in METH-injected rats, presumably due to increased activity. These abnormalities were all lessened in RNAi group.3. Striatal DA level and TH activity were significantly decreased in METH-injected rats, while DA level and TH activity in RNAi-treated rats were significantly higher than METH-injcted rats (p<0.001)4. Striatal ROS level was significantly increased ater METH injection, while ROS in RNAi treated rats were significantly decreased compared with METH-injected rats (p<0.001)5. Striatal NOS activity and NO level were significantly lowered in METH-injected rat, while NOS activity and NO level were increased in RNAi rats compared with METH-injected rats(p<0.001).6Using iTRAQ techniques, a total of2327proteins in striatum were identified,65of which were associated with a-syn. Among these65proteins,9proteins are cytoskeleton proteins,5proteins are involved in synaptic transmission,5proteins are involved in cell proliferation and apoptosis,3proteins are concerned with ubiquitin,2 proteins are ribosomal proteins.Conclusions1. Rat model a-sys-RNAi was established and a-sys knockdown were confirmed at transcriptional and translational level.2. METH-induced behavioral phenotypes were suppressed by RNAi-mediated silencing of a-sys. This was accompanied with increased TH activity and DA level, decreased ROS, reduced NOS activity and NO level.3. a-syn knockdown led to downregulation of protein arginine methyltransferase5(PRMT5), resulting in NO production; a-syn regulates ubiquitin-proteasome system through regulating ubiquitin carboxyl-terminal hydrolase24and VCIP1354. a-syn plays a pivotal role in METH-induced neurotoxicity, contributing to oxidative stress, apoptosis, cytoskeletal damage and synaptic transmission dysfunction.