Browsing by Subject "Parkinsonin tauti"

Parkinson’s disease is a progressive neurodegenerative disease, in which dopamine neurons are dying in the nigrostriatal dopaminergic pathway. This causes motor symptoms such as slowness of movement, tremor, and rigidity. In addition, various non-motor symptoms appear. All currently used medicines are symptomatic, and there are no disease modifying treatment available for Parkinson’s disease. Several neurotrophic factors have shown promise in animal models of Parkinson’s disease. One of those is cerebral dopamine neurotrophic factor (CDNF) which has been studied in different animal models, including rodents and non-human primates. CDNF is a secreted protein but it is also localized in endoplasmic reticulum (ER). CDNF has two domains, N-terminal and C-terminal, which may have distinct functions. CDNF can be retained in the ER by the ER retention sequence at the end of the C-terminal domain. The C-terminal domain also has an evolutionarily conserved disulfide bridge which is crucial for the biological activity of CDNF. The exact mechanism of CDNF is still unknown. However, it has been shown that CDNF affects the unfolded protein response (UPR) in the presence of ER stress. Neurotrophic factors do not penetrate blood-brain barrier (BBB), for this reason, they need to be injected directly to the brain. Penetration of the BBB is also a problem in the treatment of many other diseases. Various methods for enhancing the BBB penetration of drugs have been studied. For example, permeability of the BBB can be temporarily increased by focused ultrasound combined with microbubbles. Another possibility is the use of a carrier molecule, which can be transported through BBB via specific transport mechanisms. Furthermore, molecule modification offers many applications to achieve enhanced BBB penetration. In view of peripheral administration, a next generation variant of CDNF (ngCDNF) has been developed. The efficacy of this novel variant after intrastriatal injection is equal to that of CDNF in a 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease. Systemic administration could also enable treatment of non-motor symptoms of Parkinson’s disease. The aim of this experiment was to study the effects of subcutaneously injected ngCDNF on rotation behaviour, and nigrostriatal TH-positive cells in rats with 6-OHDA lesions. 6-OHDA was injected unilaterally to three different sites in the striatum. Two weeks later, the lesion size was estimated, via amphetamine- induced rotation test. ngCDNF, at two dose levels, was injected twice weekly for three weeks. Amphetamine-induced rotation test was assessed every other week, until week 12. At the end, optical density of tyrosine hydroxylase (TH) was measured from sections of the striatum, and TH positive cells in the substantia nigra were counted. In addition, the effect of ngCDNF on anxiety and depression like behaviour, learning, and locomotor activity were studied at three different levels in naïve mice. Behaviour was analyzed by open field test, forced swim test, and fear conditioning test. The ngCDNF did not seem to have clear effect on rats’ behaviour or TH positive cells and fibers compared to the control group, but positive tendency was found in the group with lower dose. The reduced efficacy of ngCDNF,via subcutaneous administration, is likely due to rapid metabolism and insufficient entry of the active form to the brain. In naïve mice, ngCDNF did not reduce anxiety-like behaviour and did not affect locomotor activity after subcutaneous injections. This result supports previous findings, which suggest that the effects of CDNF are specific to the toxin treated cells and CDNF has no effect in naïve animals.

MicroRNAs are ~22 nucleotide long RNA strands which regulate gene expression by binding to the 3’UTRs of messenger RNAs. MicroRNAs are predicted to regulate about a half of all protein-coding genes in the human genome thus affecting many cellular processes. One crucial part of microRNA biogenesis is the cleaving of pre-miRNA strands into mature microRNAs by the type III RNase enzyme, Dicer. Dicer has been shown to be downregulated due to aging and in many disease states. Particularly central nervous system disorders are linked to dysregulated microRNA processing. According to the latest studies, Dicer is crucial to the survival of dopaminergic neurons and conditional Dicer knockout mice show severe nigrostriatal dopaminergic cell loss, which is a hallmark of Parkinson’s disease. By activating Dicer with a small-molecule drug, enoxacin, the survival of dopaminergic cells exposed to stress is significantly improved. However, enoxacin, which is a fluoroquinolone antibiotic, activates Dicer only at high concentrations (10-100 μM) and is polypharmacological, which may cause detrimental side effects. Therefore, enoxacin is not a suitable drug candidate for Dicer deficiencies and better Dicer-activating drug candidates are needed. The aim of this work was to develop a cell-based fluorescent assay to screen for Dicer-activating compounds. Assays which measure Dicer activity have already been developed, but they have some pitfalls which don’t make them optimal to use for high-throughput screening of Dicer-activating compounds. Some are cell-free enzyme-based assays and thus neglect Dicer in its native context. The RNA to be processed by Dicer does not represent a common mammalian RNA type. Most assays do not have internal normalizing factors, such as a second reporter protein to account for e.g. cell death, or the analysis method is not feasible for high-throughput screening data. Considering these disadvantages, the study started by designing a reporter plasmid in silico. The plasmid expresses two fluorescent proteins, mCherry (red) and EGFP (green), and a mCherry transcripttargeting siRNA implemented into a pre-miR155 backbone which is processed by Dicer. Thus, measuring the ratios of red and green fluorescence intensities will give an indication on Dicer activity. The plasmid also has additional regulatory elements for stabilizing expression levels. The plasmid was then produced by molecular cloning methods and its functionality was tested with Dicer-modulating compounds. The assay was optimised by testing it in different cell lines and varying assay parameters, and stable cell lines were created to make large-scale screening more convenient. Finally, a small-scale screen was done with ten pharmacologically active compounds. Transiently transfected, in Chinese hamster ovarian cells, mCherry silencing was too efficient for reliable detection of improvement in silencing efficiency due to floor effect. With an inducible, Tet-On, system in FLP-IN 293 T-Rex cells, the expression could be controlled by administering doxycycline and the improvement in silencing was quantifiable. The assay seemed to be functional after 72 hours and 120 hours of incubation using enoxacin (100 μM) as a positive control. However, the screening found no compounds to significantly reduce mCherry/EGFP fluorescence ratio and, additionally, the effect of enoxacin was abolished. Therefore, a more thorough analysis on the effects of enoxacin was done and, although statistically significant, enoxacin was only marginally effective in reducing mCherry/EGFP fluorescence ratio after 72 hours of treatment. It should be noted from the small-scale screening that metformin and BDNF, compounds previously shown to elevate Dicer levels, showed similar effects to enoxacin. The quality of the assay in terms of high-throughput screening was determined by calculating Zfactors and coefficients of variations for the experiments, which showed that the variability of the assay was acceptable, but the differences between controls was not large enough for reliable screening. In conclusion, the effects of metformin and BDNF should be further studied and regarding the assay, more optimisation is needed for large-scale, high-throughput, screening to be done with minimal resources.

Parkinson's disease is a progressive neurodegenerative disease where dopaminergic neurons die in the substantia nigra pars compacta. Dopamine depletion induces typical parkinsonian motor symptoms which are treated by the golden standard medication levodopa and compounds enhancing the effect of levodopa. However in 4-6 years after the initiation of the chronic levodopa therapy abnormal involuntary movements (AIMs, also called levodopa-induced dyskinesia, LID) often develop and can notably worsen the quality of life. The most effective treatment for LID is deep brain stimulation (DBS), but as an invasive method its use is rare and not suitable for all patients. To date the only effective therapy for LID with marketing authorisation is amantadine. The disadvantage of amantadine is loss of efficacy which might appear less than a year after the initiation of medication. The pathophysiology of LID is a diverse phenomenon and includes dysfunctions in several different neurotransmitter systems both in the basal ganglia and in surrounding brain areas. The role of nicotinic acetylcholine receptors (nAChRs) in the pathophysiology of LID has been studied recently. Both nicotine and several nicotine-like agents have been shown to alleviate LID in preclinical studies and nicotine itself has been tested in a clinical phase II study as a potential LID medication. Of various different nAChR subtypes, the α7 receptor seems to be a potential option for future therapy of LID. It has been shown that α7 nAChR knock out mice display an increase in LID suggesting that this nicotinic receptor subtype has an inhibitory impact on the development of LID. Other studies have confirmed this view by showing that a selective α7 nAChR agonist (ABT-107) alleviates LID in primates and is neuroprotective for dopaminergic neurons in rats. Based on these observations, the aim of this study was to examine the effect of a novel α7 nAChR agonist (AZD0328) on LID in a 6-OHDA mouse model of Parkinson's disease. C57BL/6J female mice (n=17) were injected unilaterally 6-OHDA solution (3 µg) into the right medial forebrain bundle (MFB). Degeneration of dopaminergic neurons was detected two weeks after the 6-OHDA injection by measuring the motor performance in rotating rod with accelerated speed and with amphetamine-induced rotametry (2.5 mg/kg, i.p.). In the beginning of the chronic treatment, levodopa (4.5 mg/kg, s.c.) was administered twice daily for four days and then continued once daily (from Mon to Sun) to the end of the experiments. Levodopa treatment had been ongoing for 10 days before the first testing of drug effects. The pretreatment (AZD0328 0.06, 0.19, 1.9 mg/kg or 0.9 % saline, s.c.) was given 30 minutes before levodopa. The study was conducted using a within subject design so that each mouse received all four treatments on four test days during three weeks. Mice were videorecorded for 1 minute 20, 40, 60, 80 and 100 minutes after the levodopa injection was given. After the last recording day mice were killed under anesthesia via perfusion fixation and brains were collected for immunohistochemical staining to measure the extent of degeneration of dopaminergic neurons. 54 % of mice who survived from surgery (13/17) were dyskinetic (n=7). AZD0328 alleviated axial dyskinesia statistically significantly 40 minutes after levodopa injection but the statistical analysis did not reveal which of the doses was the most effective. The pretreatment with AZD0328 did not affect orolingual or forepaw dyskinesia. A potential mechanism of AZD0328 in alleviating LID might be the desensitization of α7 nAChRs which would happen only at very low doses. This means that LID are only attenuated when receptors are temporarily activated and then immediately gradually inactivated. The doses used in this study might have only activated the α7 nAChRs which might explain why no clear alleviation of LID was observed. On the other hand, the acute treatment may also be insufficient to develop desensitization. Additional studies are needed to investigate the effects of chronic administration of AZD0328 on LID in mice.

Parkison's disease is a progressive neurodegenerative disorder that is characterized by degeneration of dopaminergic neurons in the nigrostriatal dopamine pathway. This is responsible for the major symptoms of Parkinson's disease. The current therapies only treat symptoms without being able to slow down, or reverse, the neurodegenerative process. Therefore current research is directed toward prevention of dopaminergic neuron degeneration. Prolyl oligopeptidase (POP) is a serine peptidase which cleaves small proline-containing peptides. A number of neuropeptides are affected in Parkinson's disease and POP contributes to the degradation of many of these neuropeptides. Reduction in POP activity has observed in Parkinson's disease. However, it is not known if changes in POP activity were a cause or a consequence of Parkinson's disease. POP inhibitors are substrate-like compounds. In our study we used KYP-2047, a novel brain-penetrating POP inhibitor. Administration of KYP-2047 has previously been shown to increase slightly neurotensin levels after a single dose. Neurotensin is an endogenous neuropeptide that has antidopaminergic actions in the brain. A number of neurotensin receptors has been observed to decrease in substantia nigra and striatum after degeneration of nigrostriatal pathway in laboratory animals or Parkinson patients. When given into the brain, neurotensin and neurotensin analogs have decreased rigidity and tremor caused by 6-hydroxydopamine (6-OHDA). The aim of this study was to determine the interactions between POP and neurotensin and their connections with dopamine deficit in a rat model of Parkinson's disease (the turning model of Ungerstedt). POP activity was also studied. In this study two different kinds of lesions were used. Intracerebral injections of 6-OHDA were given either into the MFB (medial forebrain bundle) or striatum. Rotational behaviour was measured five weeks post-lesion. The MFB lesion Wistar rats were given levodopa/carbidopa -suspension with KYP-2047 or entacapone or both of them. The striatum lesion rats were given amphetamine with KYP-2047. Studies were organized in a cross-over manner once a week. Rotational behaviour did not change when a POP inhibitor was given suggesting that neurotensin levels were apparently not much changed. Differences in POP activity assay were not noticed when compared to normal and lesioned cerebral hemisphere. This indicates that there is no POP in long dopaminergic neurons.

Parkinson's disease is characterized primarily as a bradykinetic disorder with severe nigral cell loss. In addition to motor symptoms, up to 85 % of patients with Parkinson's disease experience pain and in about 60 % of cases pain is related to Parkinson's disease. Most of it is classified as musculoskeletal pain. Bradykinesia and muscle cramps lead to pain by causing malpositions of joints and trunk. Up to 40 % of parkinson patients experience pain caused by dystonia. Neuritic or radicular pain is also related to Parkinson's disease. Less than 10 % of patients have primary central pain. Pain threshold and nociceptive flexon reflex threshold are lower among patients with Parkinson's disease than in healthy subjects. Common comorbidities, namely restless legs syndrome and depression can also exacerbate pain. The pathology of pain in patients is not well understood. It is known that basal ganglia take part in pain perception and modulation. Lesions in basal ganglia can interfere pain perception and cause the exacerbation of pain. The modulation of pain in central nervous system is altered and descending inhibitory tracts are thought to work insufficiently. Levodopa alleviates the pain in about 60 % of patients with Parkinson's disease suffering from pain. Levodopa normalizes dopamine function at least partly in basal ganglia and that way alleviates the pain caused by dysfunction of dopamine tracts. Levodopa relieves motor symptoms and so alleviates the secondary pain caused by muscle cramps and stiffness. Levodopa raises the pain thresholds of patients to normal level. Levodopa may have also a direct analgesic effect via dopamine D2 receptor activation. The mutations of the gene that codes catechol-O-methyltransferase (COMT) change its activity and are related to pain perception. Low COMT activity is related to several functional differences including increased sensitivity to pain and increased response to opioids. Also COMT inhibitors sensitize mice and rats to pain. The mechanism underlying the sensitization is not well understood. We examined the effects of COMT gene disruption and COMT inhibition in acute pain models. In the first part of our study, we examined the effect of COMT inhibitor OR-486 in COMT deficient mice. We tried to clarify wether sensitization to pain is caused by COMT inhibition or some other mechanism. We also tested the effects of endogenous opioids (swim stress) and exogenous opioid (morphine) in COMT deficient mice. In the second part, we tested the effects of an atypical COMT inhibitor CGP 28014 in acute pain models. CGP 28014 does not inhibit COMT in vitro but it inhibits the Omethylation of catecholamines in vivo. The main finding of our study was the sensitization to pain caused by CGP 28014. The result gives support to hypothesis claiming that sensitization to pain is caused by O-methylation of catecholamines. The results of our study are also in line with the theory that low COMT activity is related to pain sensitization and increased response to opioids.

Parkinson's disease is a progressive neurodegenerative disease. The incidence of the disease is 1.5-2 per cent after age 60. Typical symptoms are tremor, rigidity and bradykinesia. At the late stage of the disease patients have psychic disorders, for example dementia, anxiety and depression. Motor impairment is caused by degenerative loss of dopamine cells in nigrostriatal tract. Current treatment of the disease relieves the symptoms but it cannot stop or slow the progress of the disease. Neurotrophic factors and gene therapy have been trialled to improve the treatment of Parkinson's disease and the results have been encouraging. Neurotrophic factors are proteins that regulate actions of neurons. It has been discovered that they are neuroprotective and neurorestorative. The results with glial cell line-derived neurotrophic factor (GDNF) have been encouraging in in vivo studies of Parkinson's disease. There has been variability in success of clinical trials though. GDNF degrades quickly in vivo but overexpression of GDNF in cells can be produced with viral vector adeno-associated virus. Two different forms of GDNF, pre-α-pro-GDNF (α-GDNF) and pre-β-pro-GDNF (β-GDNF), are produced as precursors and they are activated proteolytically. Based on in vitro studies, some differences in secretion of precursors have been discovered. α-GDNF is secreted constitutively and secretion of β-GDNF is dependent on physiological stimulation. Previous in vitro studies have focused on α-GDNF, but β-GDNF might be a better solution for treating Parkinson's disease based on physiological regulation system. Cerebral dopamine neurotrophic factor (CDNF) is recently discovered and less studied than GDNF. It has been discovered that CDNF also has neuroprotective and neurorestorative effects in animal models of Parkinson's disease. The aim of the first part of this study was to discover the neurorestorative effect of single injection of CDNF injected above substantia nigra for rats that received injection of 6-hydroxydopamine (6-OHDA) into medial forebrain bundle. One week later rats received PBS, GDNF or CDNF injection. The degree of the lesion was estimated with apomorphine (0.1 mg/kg s.c.) or d-amphetamine sulphate (2.5 mg/kg) induced rotation test. The rats were perfused nine weeks post-lesion and their brains were sliced. Tyrosine hydroxylase (TH) positive dopamine cells were stained by immunohistochemistry. The amount of TH positive cells in substantia nigra was counted and optical density of TH positive fibres in striatum was measured. The aim of the second part of the study was to research the neuroprotective effect of two different precursors of GDNF, dsAAV1-pre-α-pro-GDNF and dsAAV1-pre-β-pro-GDNF, given with viral vectors. The dopamine cells in nigrostriatal tract were destroyed with a 6-OHDA injection into striatum and viral vectors were injected two weeks later. Rats in control group received injection of dsAAV1-GFP. The degree of the lesion was evaluated with d-amphetamine sulphate (2.5 mg/kg) induced rotation tests and cylinder test. The rats were perfused eight weeks post-lesion and their brains were processed for immunohistochemistry. The results of the study were interesting and supporting previous studies. The success of the neurotrophic factor treatment is dependent on a successful injection of protein or viral vector, and the dose is dependent on the size of the lesion. Neurotrophic factors and gene therapy needs to be studied more before wide clinical usage.

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.

The α5 subunit of nicotinic acetylcholine receptors forms functional receptors with other subunits as a structural subunit. It affects the structural and functional properties of the nicotinic receptor by increasing calcium permeability and accelerating desensitization. In the mammalian brain, the α5 mRNA is widely expressed, mostly in substantia nigra pars compacta, ventral tegmental area and interpeduncular nucleus. Its protein has been identified in various distinct brain areas, such as striatum, cortex and medial habenula. In the dorsal striatum partaking in motor functions, the α5 subunit modulates the release of dopamine, thus it is believed to have an impact on motor function. In the experimental part of the thesis mice lacking the α5 subunit were injected unilaterally with neurotoxin 6-hydroxydopamine (6-OHDA) in the striatum. The purpose was to determine the importance of the subunit with regard to the lesion extent and motor function. The motor functions of α5-deficient and wild type control mice were assessed in amphetamine- and apomorphine-induced rotametry. After the tests the mice were euthanized and their substantia nigra and striatal brain samples were collected for further analysis. The number of dopamine cells in the medial and dorsal tier of substantia nigra were determined, so as to quantify the extent of the lesions and to explain the research group's previous finding about the α5-deficient mice spinning less ipsilaterally in amphetamine induced rotametry. The α5-deficient mice were found to turn less ipsilaterally compared to the control mice in the amphetamine-induced rotametry and in the apomorphine-induced rotametry, first less contralaterally and subsequently more contralaterally than the control mice. The results of male mice, that were less in number, were excluded from the results as the difference between genders was significant in the wild type mice in the amphetamine-induced rotametry. There was no significant difference in the number of remaining dopamine cells between the genotypes after the lesioning in either of the areas of interest. However, the wild type mice tended to have less cells remaining in the medial tier of the substantia nigra. The observed differences between the genotypes in the rotametries could be accounted by differences in the amount of dopamine released from striatal neurons or differences in striatal dopamine receptor quantities or function. The results support the hypothesis about the contribution of the α5 subunit containing acetylcholine nicotinic receptors in motor function.