Browsing by Subject "virusvektori"

Part I: Parkinson's disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.

Parkinson's disease is a neurodegenerative disease which is characterized by progressive loss of dopaminergic neurons in the substantia nigra and formation of intracellular Lewy bodies. α-synuclein is an essential part of Lewy bodies. In addition, mutations in the α-synuclein gene have been found to cause rare familial forms of Parkinson's disease. Animal models of Parkinson's disease are created by neurotoxins, transgenic animals and viral vectors. Transgenic animal models and viral vector models seem to reflect the pathology of Parkinson's disease better than the traditional neurotoxin models. In the transgenic animal models, the transgene and the promoter used in the expression of the transgene guide the pathology and motor dysfunctions that the animal model exhibits. In the viral vector models, it is important to use a suitable animal strain and a correct viral serotype in order to express the transgene sufficiently enough in the laboratory animals. The aim of the study was to investigate the ability of adeno-associated viral vector (AAV1-vector) to transfect WT- or A53T-α-synuclein gene into the striatum or the substantia nigra, and the effects of their overexpression on motor functions and concentrations of striatal dopamine and its metabolites in mice. In addition, the effect of a prolyl endopeptidase (PREP) inhibitor on the overexpression of A53T-α-synuclein in the mouse nigrostriatal pathway was studied, as PREP has been found to stimulate the aggregation of α-synuclein and therefore perhaps to increase neurotoxicity of α-synuclein. There were no statistically significant differences between the groups in the motor function tests (locomotor activity, rotarod and balance beam walk). Green fluorescent protein immunostaining showed that the GFP gene was weakly transfected into the striatum by the AAV1-vector, and no overexpression was observed. There were only minor differences in the striatal concentrations of dopamine and its metabolites. Finally, PREP-activity measurements showed that PREP-inhibitor (KYP-2047) treatment had poorly reduced PREP-activity. In this study, the viral vectors did not induce the overexpression of α-synuclein, although previously AAV2- and AAV6-vectors have been efficient in mice and rats. High PREP-activities that were found in most of the samples probably resulted from failed installations of mini-pumps that delivered the PREP-inhibitor. While in this study the viral vectors were not a successful attempt in the creation of an animal model of Parkinson's disease, they are an important method to model Parkinson's disease in the future.