Browsing by Subject "Parkinson's disease"

Parkinson's disease is a neurodegenerative disease where the nigrostriatal dopaminergic cells die gradually causing severe motor symptoms. Current treatment of the disease relieves the symptoms but does not affect the progression of the disease, nor does it have a neuroprotective effect. The most important drug for the treatment of Parkinson's disease is L-dopa, the precursor of dopamine. With long-term use, L-dopa loses its efficacy and patients start to get adverse effects. The most significant adverse effects are abnormal involuntary movements called dyskinesias. In the literature review of this thesis Parkinson's disease and its treatment is briefly described. Review focuses on the description of the brain cholinergic and histaminergic systems and their receptors along with the available studies about cholinergic and histaminergic neurotransmission in Parkinson's disease 6-hydroxydopamine (6-OHDA) rodent model. The experimental part of this thesis consisted of two different set of experiments and in both of these the dopamine neurons were destroyed unilaterally by injecting 6-OHDA into the striatum. The aim of the first experiment was to examine histamine H3-receptor antagonist JNJ-39220675 and α7-nicotinic receptor agonist PHA-543613, and their combination therapy effects on motor function and the concentrations of striatal neurotransmitters in hemiparkinsonian mice. Effects on motor function were studied two and four weeks after the 6-OHDA injection with cylinder test, the D-amphetamine-induced rotations, and the inverted grid test. After behavioral tests, mice were sacrificed and striatal neurotransmitter concentrations were determinated by HPLC. The aim of the second experiment was to examine if nicotine can relieve L-dopa-induced dyskinesias. In this experiment 6-OHDA was injected at two sites into the striatum, which was intended to produce more extensive destruction of dopaminergic neurons than in the first experiment. The extent of the lesion by 6-OHDA was verified before starting chronic L-dopa treatments with cylinder test. One month after the 6-OHDA injection, five mice were sacrificed and their striatum and substantia nigra sections were measured for destruction of dopaminergic neurons by immunohistochemical TH-staining. Chronic L-dopa treatment with benserazide was started 49‚àí63 days after the 6-OHDA injection. At the same time, mice were divided into two groups. Half of them got normal drinking water and half got nicotine water. During the chronic L-dopa treatment, development of dyskinesias was observed once a week by video tracking. The cylinder test was also done once again after starting the L-dopa treatment. In the first experiment, H3-receptor antagonist JNJ-39220675 showed promising results in improving motor function. Mice used the impaired (contralateral) paw more in the cylinder test and rotated less to the ipsilateral side in the D-amphetamine-induced rotation test than control animals two weeks after the 6-OHDA injection. Combination therapy also reduced the ipsilateral rotations but in the cylinder test it had no effect two weeks after 6-OHDA injection. Because the asymmetry in behavioural tests were caused by destroying dopaminergic neurons, balancing of the motor skills can result from decreased levels of dopamine in the intact side or from increased dopamine levels or stronger dopaminergic postsynaptic transmission in the lesion side. The results four weeks after 6-OHDA injection are not reliable because the striatal samples showed that dopamine concentrations in the lesion side were very close to that of the intact side indicating recovery from the lesion. In the second experiment, mice developed dyskinesias which were decreased with nicotine treatment. Mice also used the contralateral side paw less indicative of loss of dopamine neurons. In agreement, TH-immunostaining confirmed significant loss of TH-positive neurons. Based on these findings, the 6-OHDA injection site, the selected drug doses, and the experimental design seem to fit the evaluation of dyskinesias. The occurrence of dyskinesias and nicotine's effect on them was seen strongest in the body movements. Dyskinesias in forelimbs were minor, but the nicotine treatment decreased them also.

Laminins are a family of heterotrimeric glycoproteins found mainly in basement membranes. They interact with numerous other extracellular matrix components and cell surface receptors, including integrins and α-dystroglycan. Laminins play roles in myriad of functions including tissue morphogenesis, organogenesis, maintenance of tissue integrity and compartmentalization. In central nervous system laminins are involved in every major developmental stage from neural tube closure to synaptogenesis. Laminin expression in central nervous system decreases after maturation but has been found inducible by injury after trauma or disease. Since laminins are known to promote neurite outgrowth and neuronal survival, this has been proposed as a regenerative response to injury. Although the effects of endogenous laminin are clearly inadequate for repair, laminin based compounds could be powerful therapeutic agents. In previous in vivo studies KDI-tripeptide, a neurite outgrowth promoting fragment from γ1-laminin, has proved effective neuroprotective and regeneration promoting compound. Encouraged by these results I set out to test whether KDI would rescue midbrain dopaminergic neurons in unilateral 6-hydroxydopamine-induced rat model of Parkinson's disease. KDI (1-30µg) was injected to the striatum six hours prior to 6-hydroxydopamine. The severity of the lesion was then evaluated by measuring D-amphetamine induced rotation 2, 4 and 6 weeks postlesion and by assessing the number of neurons in substantia nigra pars compacta and optical density of striatum after tyrosine hydroxylase immunostaining at week seven. The only effective KDI dose studied was 3 µg. Compared to control it decreased Damphetamine induced rotational behaviour significantly at week four. KDI, however, failed to save tyrosine hydroxylase positive dopaminergic neurons in substantia nigra pars compacta or their axons in striatum. KDI might be usable in treating Parkinson's disease but it's mode of action doesn't appear to rely on protecting dopaminergic neurons or promoting the branching of their axons. KDI is known to inhibit ionotropic glutamate receptors and could therefore improve motor function by opposing striatal denervation induced overactivity of glutamatergic subthalamic nucleus neurons.

Parkinson's disease is a neurodegenerative disorder where dopaminergic neurons in substantia nigra are gradually destroyed. Less than 10% of Parkinson's disease cases are genetic. For example mutations in α-synuclein, LRRK2-, parkin-, PINK1- and DJ-1 are known to cause Parkinson's disease. There is still no curative treatment for Parkinson's disease. Alpha-synuclein is linked to Parkinson's disease through Lewy bodies. Three point mutations causing Parkinson's disease have been found in a gene coding α-synuclein. Alpha-synuclein has been expressed in Drosophila melanogaster, C. elegans and mouse. Main function of LRRK2-protein is thought to be kinase activity. Mutations in LRRK2-gene are the most common known cause of Parkinson's disease. LRRK has been expressed in Drosophila melanogaster, C. elegans and mouse. LRRK2 knock-out Drosophila melanogaster and mouse have also been studied. Parkin is a neuroprotective protein and its deficiency results in a loss of neurons in substantia nigra. Mutations in Parkin cause 50% of recessive Parkinson's disease. Parkin knock-out Drosophila melanogaster and mouse and Drosophila melanogaster and mouse expressing human Parkin are Parkin animal models. PINK1 is a mitochondrial protein coded by nucleus. DJ-1 is thought to have a part in mitochondria maintenance and protection. Both PINK1- and DJ-1 knock-out Drosophila melanogaster and mouse have been studied. None of the genetic animal models of Parkinson's disease is identical to symptoms and pathology of human Parkinson's disease. The purpose of the experimental part of this thesis was to examine non-drug induced behavioural test and Cerebral dopamine neurotrophic factor (CDNF) in 6-OHDA lesioned rats. CDNF protects and restores dopaminergic neurons. The non-drug induced behavioural tests included in this study were stepping test, cylinder test and staircase test. An old and widely used drug induced test for Parkinson's disease, amphetamine-induced rotation test, has problems that have led to a seek for replacing and complementary test methods. In amphetamine-induced rotation test dopamine agonist is given to a unilaterally 6-OHDA lesioned animal. The agonist causes rotational behaviour that can be measured with designed equipment. The stepping test measures forelimb akinesia in rats. In the experimental setting the rat is moved sideways when it is held only one front paw on a table and adjusting steps are counted. In the cylinder test front paw preference is measured. In the experimental setting the rat is placed in a transparent cylinder and the front paw preference is counted on rears and on ground contacts after a rear. The staircase test measures front paw coordination and function. In the experimental setting the number of sucrose pellets picked up from a double staircase is counted. There were no significant differences between lesioned groups in stepping test, cylinder test or in staircase test. It is possible that the 6-OHDA lesion used in the experiment was not extensive enough. Different non-drug induced behavioural tests supplement each other and they should be combined for the best result. Combining different behavioural tests enables more reliable results and versatile information than the amphetamine-induced rotation test alone.

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 progressive degenerative brain disease that causes degeneration of dopaminergic neurons in the substantia nigra. Characteristic motor symptoms in Parkinson's disease are caused by dopamine deficiency in striatum. Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the dopamine biosynthesis. Because of this TH has a significant role in the function of the dopaminergic system. TH activity is regulated by several mechanisms. The most important regulatory mechanism is phosphorylation of TH protein by spesific protein kinases. Alterations in the function of TH have been associated with Parkinson's disease. The most prominent findings are decreased TH protein and TH mRNA content in the nigrostriatal dopaminergic neurons. A possible pathogenic role of TH in Parkinson's disease has also been suggested. In addition TH might be a potential therapeutic protein for gene therapy. One possible approach is viral vector-mediated gene transfer of TH gene directly into the brain. Simultaneous gene transfer of TH gene and neurotrophic factor gene could both enhance dopamine synthesis and prevent remaining dopaminergic neurons from dying. None of the current treatments of Parkinson's disease can halt or retard dopaminergic neuron degeneration. Novel treatments are being developed and amongst other strategies neurotrophic factors have proven promising candidates for the treatment of Parkinson's disease. Member of CDNF/MANF family of neurotrophic factors, cerebral dopamine neurotrophic factor (CDNF), is currently being studied. Previous studies have demonstrated the neuroprotective and neurorestorative effects of CDNF but more research is needed for optimal administration technique and dose. The aim of this work was to study the neuroprotective effect of AAV vector-mediated delivery of CDNF (AAV-CDNF) in a rat model of Parkinson's disease. Rats' brains were unilaterally lesioned with intrastriatal injection of 6-OHDA two weeks after viral vector injections and amphetamine-induced rotational behavior was monitored for ten weeks. The CDNF protein expression after intrastriatal AAV vector-mediated gene transfer was analyzed with immunohistochemical staining of brain sections. We confirmed that CDNF protein is expressed in rat brain after intrastriatal injection of AAV-CDNF. AAV-CDNF treatment also reduced the amphetamineinduced ipsilateral rotations nearly as much as AAV-GDNF treatment. AAV-CDNF treatment also had an effect on the amount of remaining TH-immunoreactive cells in the substantia nigra pars compacta and the optical density of striatal TH-immunoreactive fibers but these results did not reach statistical significance. The immunohistochemical measures did not correlate completely with the behavioral data and further studies are needed to confirm the results obtained here. The results of this research support the conclusion that AAV-CDNF treatment has a neuroprotective effect on nigrostriatal dopaminergic neurons.