Proteomic Identification Of Proteins Associated With Methamphetamine-induced Neurotoxicity

BACKGROUNDMethamphetamine(MA) is a new kind of substance of abuse. Its use has increased considerably in recent years. With the large population of abuse only secondary to cannabis, MA has become one of the most devastating druges in the world. MA belongs to the incitant family of amphetamine, and has multiple pharmalogical and toxicological effects. Recent reports revealed that brain is the main target of MA-toxicity. MA-induced histological changes of the brain, which include dopamine (DA) depletion, dopaminergic nerve terminals degeneration, decreasing of the activity of tyrosine hydroxylase and the number of DA uptake sites, as well as neuron apoptosis, are simillar to that of neurodegenerative disorders such as Alzimer's disease and Parkinson's disease. MA-induced neurotoxicity is a complex process, which may include oxidative stress, disregulation of temperature homeostasis, ion dysregulation and activation of apoptosis pathways. Yet a number of mechanisms appear to contribute to MA-induced neurotoxicity, the exact is still unknown.OBJECTIVEAnimal models were used to survey the toxicity of MA by histopathological, behavioral and neurochemical methods. On the bases of these results, proteomic method was used to screen brain proteins associated with MA neurotoxicity. The pathogenesis of MA neurotoxicity was concluded according to the functions of differantly expressed proteins and further researches were conducted according to these pathways.METHODS1. Animal protocols and toxicity observationWistar rats, weighting from 200g to 220g (the experimental animal center of Southern Medical University, China) were divided randomly into two groups (n=20 each group). The methamphetamine hydrocloride was dissolved in saline. Rats received i.p. injections of saline or MA (eight injections, 15 mg/kg/injection, at 12 h intervals).The body weight, food and wafter-intake changes and behavioral changes of rats were recorded. The histological changes of heart, liver, spleen, lung and kindy were observed by HE stain. The counts of dopamine and its metabolites in striatum, hippocampus, and frontal cortex were detected by HPLC. The histological changes of brain were observed by HE stain and Glees silver stain. Imunohistochemical detection of GFAP was used to evaluate the proliferation ofglia cells.2. Proteomic profiling of proteins associated with MA-induced neurotoxicityProteins of striatum, hippocampus, and frontal cortex were separated respectively by two-dimensional gel electrophoresis. The differently expressed proteins were identified by tandem mass spectrum and data base search. The pathogenesis of MA-induced neurotoxicity was speculated according to the functions of identitied proteins.3. The relationship between NOS activity and MA neurotoxicity in the process of MA-indueed oxidative stress.According to the research results above, the enzyme activity of NOS may be increased in the oxidative stress after MA administration. The resulting overproducing of NO may cause neurotoxicity. In the present study, 7-nitroindazole (7-NI), a specific inhibitor of nNOS, was used to evaluate the potential role of NOS in the pathogenesis of MA neurotoxicity. Wistar rats were divided randemly into 4 groups. Rats of group 1-3 recieved i.p. injections of saline, 7-NI or MA respectively. Rats of group 4 recieved i.p. injections of 7-NI 30 min before each MA injection. The NOS activity in striatum of MA group and saline group were analysised. The rental temperature and dopamine countent in striatum were mesured in four groups.4. Identification of nitrated proteins after MA administrationProteins of rat stfiatum were seperated by two-dimensional electrophoresis and gels were transferred to PVDF membrane. Nitrated proteins on the membrane were detected by immunoreactivity with 3-nitrotyrosine (3-NT). Nitrated proteins were identified by tandem mass spectrum and data base search.RESULTS1. MA has obvious appetite inhibition effects. The food-intake volume of rats decreased after MA injection (P＜0.01). The body weight of rats also decreased (P＜0.01). Rats showed obvious behavior changes approximately 10-20 min after MA injection, such as easy to be irritated, moving ahead constantly, and rapid revolving movements. The score of stereotyped behavior increased significantly in MA-treated group (P＜0.01). Dopamine contents in striatum, hippocampus and frontal cortex decreased after MA administration (P＜0.01). Injuries of neurons and axons were observed after MA treatment, such as swelling of neurons, proliferation of glial cells, neuronophagy, glial cell tubercles, snaky like and segmental enlargement of axons and increased expression of GFAP. Hydropic degeneration and fatty degeneration of hepatic cells, as well as interstitial pneumonia were found in a proportion of animals.2. 14 protein spots in striatum, 12 spots in hippocampus and 4 spots in frontal cortex were found differently expressed after MA injection by proteomic method. These proteins are related to oxidative stress, proteosome function, neurodegeneration/apoptpsis and mitochondrial dysfunction. Although the patterns of protein expression change in three brain regions are different, some proteins increased or decreased simultaneously in two or three brain regions.3. The enzyme activity of NOS in striatum increased after MA injection (P＜0.01). 7-NI has no effect on animal body temperature, nor did it has any effects on MA-induced hyperthermia (P＞0.05). Dopamine content in striatum of 7-NI+MA group is higher than that of MA group (P＜0.01).4. 6 nitrated protein spots in striatum of rats were found by two-dimensional 3-NT blot. Among them, the intensity of 3-NT immunoreactivity of 5 spots increased after MA injection. Three proteins were identified by tandem mass spectrum as tropomodulin 1, tropomodulin 2, and mu-crystallin. Tropomodulin 1 and tropomodulin 2 are two members of tropomodulin family that have simillar functions. They cap the pointed ends of an actin filament and stabilize the filament and regulate its length. The function of mu-crystallin is not fully understood by now. It may be involved in the process of neural proliferation and oxidative stress.CONCLUSION1. There is obvious neurotoxicity after MA administration, which can cause injury on brain regions such as striatum, hippocampus and frontal cotex as well as behavior changes of rats. MA also has some extent of toxic effects on liver and lung.2. Oxidative stress, abnormal of ubiquitin-mediated protein degragation, neurodegeneration/apoptosis, and motochondrial dysfunction are the main mechanisms involved in MA-induced neurotoxicity. There are some same pathways in the pathogenesis of MA neurotoxicity in different brain regions.3. The increased enzyme activity of NOS and the resulting NO overproducing in the process of oxidative stress induced by MA administration play an important role in MA-induced neurotoxicity.4. Nitration of tropomodulin may disturbance the normal cytoskeletal framework of neurons and may cause axons injury and activating cellular responses that lead to apoptosis. Lack of normal function of tropomodulin may also relate to behavior changes of rats. The nitration of vital structure proteins in neurons_may be a key pathway in MA-induced neurotoxicity.