Browsing by discipline "Farmakologi"

Nucleus accumbens, located in ventral striatum, is an important part of the brain reward system. Accumbens integrates information coming from various brain areas, and it’s important for feeling pleasure, reward learning and reward seeking, including drugs of abuse. Cholinergic interneurons represent a few percent of accumbal cells. Earlier research suggests that accumbal cholinergic activity decreases drug seeking and eating. The aim of this study was to examine the role of cholinergic interneuron activity in alcohol drinking and alcohol related locomotor activity. Cholinergic interneurons (ChI) were manipulated using DREADDs (Designer Receptors Exclusively Activated by Designer Drugs), which can be selectively activated with clozapine-n-oxide (CNO). To express DREADDs selectively in ChIs, a cre-dependent viral vector that contained a gene coding for a cell-activating hM3D(Gq)-mCherry (n=9), cell-inhibiting hM4D(Gi)-mCherry (n=9), or control mCherry(n=8), was injected to nucleus accumbens of ChAT-cre- mice. Alcohol drinking was measured using Drinking In the Dark (DID)- model. Three hours after lights-out, the water bottles were replaced with 20% alcohol for two hours, for three days. On the fourth day, mice were injected with CNO or vehicle and alcohol was given for four hours. These cycles were repeated six times. In the locomotor assay, mice were injected with CNO or vehicle, followed by injection of alcohol or saline. Locomotor activity of the mice was observed for 30 minutes. In the DID- assay, the DREADD ligand CNO did not have effects on alcohol drinking within any of the three groups. However, Gi- mice drank more alcohol than Gq-mice even without the presence of CNO. These results are not reliable enough to draw conclusions, as they were confounded by unusually low drinking volumes. In the locomotor assay, CNO alone did not affect locomotion in any group. Together with alcohol, however, CNO decreased locomotion in all three groups, compared to alcohol alone. This is consistent with recent reports suggesting that CNO may have nonspecific behavioral effects.

This review focuses on neurotrophic factors, especially CDNF, and Amyotropic lateral sclerosis (ALS). This review finds out which neurotrophic factors have been studied in clinical trials of ALS and what kind of results have been got. Neurotrophic factors are important for development and function of neurons because they prevent apoptosis of neurons. They also play role in differentiation, development and migration of neurons. It is also known that many of the neurotrophic factors have protective and restorative properties. ALS is a rare neurodegenerative disease which causes the destruction of motor neurons and leads to death in three years. The disease degenerate the upper and lower motor neurons. Symptoms are muscle weakness, muscle atrophy, cramps and problems with swallowing. At the moment there is no cure for ALS so it is important to study neurotrophic factors that could prevent the progression of the disease and perhaps to protect or repair destroyed motor neurons. This is why it is important to study potential of CDNF in ALS. The experimental part consists of three different parts. The purpose of the first part study was to determine the distribution of CDNF after intraventricular delivery at different time points. CDNF was labeled with 125I (125I-CDNF). The distribution was determined by gammacounter and autoradiography. To determine the stability of the injected 125-I CDNF we performed SDS-PAGE. The second part studied the diffusion volume of CDNF after intraventricular injection with seven wild type mice. After stereotaxic surgery CDNF-immunohistochemistry staining from coronal sections was done. The last experimental part studied the effect of single intracerebral injection of CDNF on motivation, locomotor activity, anxiety and depression with male and female mice. Light-dark box, open field, rotarod, forced swim test (FST), elevated plus maze and fear conditioning were carried out with male mice. After behavioural tests mice were sacrified for HPLC-analysis. Light-dark box and IntelliCage were carried out with female mice before c-fos staining. Gammacounter and autoradiography shows that 125I-CDNF distributes widely after intracerebroventricular injection. It spread throughout to the brain and also all the way to the spinal cord after one and three hours from injection. After 24 hours 125I-CDNF was cleared so the CDNF signal was very weak. SDS-PAGE showed the stability of radioactive CDNF. CDNF increased locomotor activity and decreased anxiety in male mice. But a statistically significant difference appeared in forced swim test and fear conditioning test. HPLC-analysis supported these results partly. CDNF also increased motivation of female mice in IntelliCage experiment. C-fos staining was observed in CDNF group and PBS group so quantitative analysis should be done from these sections so that reliable conclusions could be done. However, because CDNF distributed to spinal cord and it showed some effect on locomotor activity, motivation and depression it might be potential for ALS disease.

Literature review: There is a need for new disease modifying therapies for Alzheimer disease. In order to develop these, animal models with better Alzheimer disease pathology are required. Old rat models like giving scopolamine or MK-801 or using aged rats don't have many of the characteristics of Alzheimer disease although they diminish cognitive functions in different models. Newer models like transgenic and Aβ-injected or -infused rats have much more analogy to the pathology of Alzheimer disease - at least when Aβ-pathophysiology is concerned. Taupathophysiology however doesn't occur in either of the models. On the other hand Aβ has a bigger role in the pathophysiology of Alzheimer disease so it's more important to have that in both models. These two models seem to be quite similar in modelling the disease. Injecting or infusing Aβ to the brain of the rat may be easier to conduct in practice than to create a transgenic rat line. However in transgenic rats Aβ-pathophysiology is developed intracellularly like in Alzheimer disease instead of giving aggregated Aβ outside of the brain. Still both of the models can be used as well to study new therapies especially affecting Aβ-pathophysiology. Experimental part: The purpose of the study was to validate elevated plus-maze (EPM) as a cognition model with a Trial1/Trial2-protocol (T1/T2) in mice. In this experiment a-five-minute-trial was used and thus different parameters related to cognition were measured. The memory of the mice was tried to be disrupted with time delay (1-18 d) between trials or with muscarinic receptor antagonist scopolamine (0.1-0.8 mg/kg i.p.) 30 minutes pre-T1. These experiments were conducted in C57BL/6J- and ICR:(CD-1)-mice. The only group in the time interval experiment that had a trend of forgetting was the 18 d group of ICR:(CD-1)-mice. Thus 21 d interval was also studied, but clear signs of forgetting couldn't be seen. Scopolamine didn't disrupt memory in ICR:(CD-1)-mice but in C57BL/6J-mice it did significantly with doses 0.2-0.8 mg/kg. Based on this 0.2 mg/kg was selected to be used in further studies in C57BL/6J-mice. In this model the nootropic effects of donepezile (0.3, 0.8 and 1.5 mg/kg s.c.), memantine (5.0 and 10.0 mg/kg s.c.) and an experimental 5-HT6-antagonist SB742457 (1.5 and 6.0 mg/kg s.c.) were studied. These compounds were allocated 40 minutes pre-T1 and scopolamine 30 minutes pre-T1. Memantine (5.0 mg/kg) clearly and donepezile (1.5 mg/kg) with a strong trend enhanced cognition disrupted by scopolamine. These results suggest that EPM can be used when testing nootropic effects.

Nicotinic acetylcholine receptors are ion channel receptors that consist of five subunits and have an important role in modulating neurotransmitter release in the central nervous system. The literature review part of this thesis presents an overview of the structure, function and diverse subunit composition of nicotinic receptors and reviews the scientific literature on their function as modulators of neurotransmitter release. Relevant literature on the role of the nicotinic receptors of the striatum, the hippocampus and the prefrontal cortex in the modulation of the release of dopamine, glutamate, GABA, acetylcholine, noradrenalin and serotonin is reviewed. Finally, a summary for each of the brain areas and some conclusions are presented. The experimental part of this thesis consists of a series of experiments, where the ability of morphine to activate the presynaptic nicotinic receptors modulating dopamine release in the mouse striatum was investigated based on opioid-nicotine-interactions reported earlier. The possible effect of morphine was studied by measuring the release of radiolabeled dopamine from perfused synaptosomes prepared from mouse striatum. In addition, the effect of nicotine was studied to confirm the correct functioning of the method and to obtain data for comparison with the morphine results. Both nicotine and morphine elicited the release of [3H]dopamine from striatal synaptosomes. The release of [3H]dopamine elicited by morphine was blocked by nicotinic antagonists, suggesting that the effect of morphine was mediated by nicotinic receptors. Use of the selective antagonist α-conotoxin MII revealed that the effect of morphine, similar to nicotine, was mediated in part by α6β2* receptors and in part by other receptors, possibly α4β2*. In addition, the opioid antagonist naloxone blocked the effects of both nicotine and morphine, likely via direct antagonism of nicotinic receptors. However, the concentrations of morphine and naloxone needed for affecting [3H]dopamine release were very high, which suggests that the clinical relevance of the effects described here is likely to be small. The involvement of opioid receptors was deemed to be unlikely but, along with possible non-specific effects by high concentrations, could not be completely ruled out.

Endoplasmic reticulum stress (ER-stress) is the result of accumulation of unfolded and misfolded proteins in the ER. The unfolded proteins activate the unfolded protein response (UPR), which seeks to reduce the protein load in the ER and reduces ER-stress. When ER-stress is prolonged, the UPR will activate apoptosis. Amyotrophic lateral sclerosis (ALS) is a rare, progressive neurodegenerative disease that affects lower and higher motorneurons. The cause of ALS is unknown but ER-stress is known to play a role in the disease progression. CDNF is a new neurotrophic factor, which is known to play a role in protein folding in the ER. CDNF is neuroprotective and neurorestorative in animal models of Parkinson's disease. Thus, CDNF is a potential new drug candidate for treating ALS. The aim of this work was to examine the effect of CDNF on disease state and life span in transgenic SOD1(G93A)-mice. CDNF or PBS was injected into the mouse's ventricle in stereotaxic surgery when the mice were about 90 days old. Clinical status and motor coordination was monitored twice a week throughout the study. The mice were dissected when they reached the end point that was set for the study. Deepfrozen gastrocnemius muscles were stained with antibodies, to examine the integrity of the neuromuscular junctions (NMJ). Quantitative PCR (qPCR) was executed on deepfrozen spinal cord and motor cortex samples to measure the expression of ER-stress genes. The results showed that CDNF improves motor coordination and delays disease progression in SOD1 female mice. The NMJs were notably more damaged in SOD1 mice than in wild type mice, but CDNF did not have any significant effect on NMJ integrity. ER-stress could be observed in the spinal cord and motor cortex of SOD1 mice and CDNF decreased ER-stress in the motor cortex. CDNF did not decrease ER-stress in the spinal cord where the expression of apoptosis related genes was increased. Thus, CDNF is a potential new drug candidate for treating ALS and it should be studied further.

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease in which both upper and lower motor neurons degenerate gradually. The disease leads to a total paralysis of almost all skeletal muscles and to death within 3-5 years after onset. At the moment there are two disease modifying medicines available, riluzole and edaravone. Neither is able to cure the disease or even to stop or remarkably slow down its progression. Endoplasmic reticulum (ER) stress has been proposed as one of the pathophysiological mechanisms underlying ALS. During ER stress misfolded of unfolded proteins accumulate in ER lumen. As a defense mechanism, the cell launches unfolded protein response (UPR). UPR response aims to reduce the protein load in ER and restore cell’s normal functions. If the damage is already beyond repair, UPR signal cascades lead to programmed cell death. Neurotrophic factors (NTFs) regulate the growth of nervous tissue and participate in repairing processed. Many of the known NTFs have first seemed promising in the preclinical models of ALS but however failed in clinical trials. Cerebral dopamine neurotrophic factor (CDNF) differs drastically both in structure and function from conventional NTFs. CDNF has seen to relieve ER stress and improve motor behavior in the animal models of Parkinsons’s disease. Recently CDNF entered clinical trials in Parkinson’s patients. Since ER stress is believed to be present not only in ALS but also in Parkinson’s disease and other neurodegenerative diseases, it might have an effect in treating ALS patients. SOD1-G93A is a well-established animal model of ALS in which the animals show typical motor impairments comparable to human disease. In this study we used a novel mouse line obtained from crossing traditional SOD1-G93A model and CDNF knock out models. The study aimed to evaluate the effect of endogenic CDNF loss in survival, onset of symptoms, motor behavioral and spinal motor neuron degeneration in the new line. ER-stress and autophagy marker levels were studied with quantitative polymerase chain reaction (CNDF) and western blotting techniques. Spinal motor neuron loss was examined by anti-choline acetyltransferase antibody (ChAT) stainings. SOD1-G93A CDNF knock out animals were observed to have more severe motor impairments in the early stages of the disease compared to the traditional SOD1-G93A mice. In addition, the degeneration of spinal motor neurons appeared to be more severe in the new line. There were no statistically significant differences in ER stress between the genotypes although a trend of increased ER stress was observed. Endogenous CDNF loss had no effect on the healthy animals. The results suggest that CNDF is a potential treatment for ALS and it might have only little side effect since it does not seen to affect healthy tissue. In medical usage, CDNF might be most effective when administered immediately after disease onset. However, this might be difficult because of the challenges in ALS diagnosis.

Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine's effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.

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.

Literature review: The plasma membrane DA transporter (DAT) belongs to the family of Na+/ClÙÄÉ≠ dependent neurotransmitter transporters. DAT is the primary mechanism for clearance of dopamine from the extracellular space and transporting it back to the presynaptic nerve terminals. There's a great interest in the DAT and its regulation as its substrate, dopamine, mediates a wide array of physiological functions e.g. locomotor activity, cognition and the control of motivated behaviors. With selective transport DAT limits the intensity and the duration of dopaminergic signal. Its function is regulated by several kinases, phosphatase and protein-protein interactions. The altered expression of DAT may be related to several neurological diseases such as Parkinson's disease, addiction and ADHD. To study DAT's function, several genetically modified mouse lines including DAT knockout mice, DAT knockdown mice and DAT knock in mice with elevated DAT levels have been generated. Experimental part: Glial cell line-derived neurotrophic factor (GDNF) plays important role in the survival and function of dopaminergic neurons, learning, memory and synaptic plasticity. More recently, several studies have shown that GDNF can also negatively regulate the actions of abused drugs. The aim of this study was to investigate GDNF's role and mechanism of action in plasticity and function of the dopaminergic neurons projecting to striatum. For that purpose, we used in vivo microdialysis in freely moving mice. We chose two different mouse lines: MEN2B mice with constitutive active Ret-signaling and elevated striatal dopamine concentrations, and GDND-cKO mice that lack GDND in the central nervous system. Microdialysis guide cannula was implanted in the dorsal striatum in the stereotaxic surgery and the mice were allowed to recover for 5-7 days. The concentrations of dopamine and its metabolites DOPAC and HVA and also 5-HIAA were determined from the samples by highperformance liquid chromatography. Microdialysis was performed twice for every mouse on days 1 and 4. Between microdialysis days, the mice were given amphetamine 1 mg/kg i.p. on days 2 and 3. In the microdialysis experiment, the mice received amphetamine stimulation (100 µM/60 min) via microdialysis probe. The placements of microdialysis probes were verified from fixed brain sections after the experiments. Amphetamine increased the dopamine output in both mouse lines, but there were no statistically significant differences in striatal dopamine concentrations between genotypes neither after acute nor chronic administration. However, there was a difference between the dopamine outputs in days 1 and 4 in both MEN2B and GDNF-cKO mice: The striatal dopamine concentrations were significantly lower on the second microdialysis day. This may be a sing from tolerance to the drug. However, without more research, it is not possible, by this experiment, to draw direct conclusions of GDNF's role in addiction and in plasticity in striatum. It is possible that the differences between genotypes are too small to be seen with microdialysis. Development of compensatory mechanisms in mice cannot be ruled out either. Effects may also vary between different brain areas.

Gemfibrozil is a fibric acid derivative used in the treatment of dyslipidemia. It activates peroxisome proliferator activated receptor α (PPARα) and alters lipoprotein metabolism gene expression. PPARα may also regulate the expression of drug disposition genes (e.g., CYP3A4). The aim of this study was to investigate possible effects of gemfibrozil on drug transporter gene expression in human whole blood. In a randomized crossover study, 10 healthy volunteers took 600 mg gemfibrozil or placebo twice daily for 6 days (Filppula et al. 2013). On the morning of day 3, a venous blood RNA sample was collected from each participant into a PaxGene® tube. The expression of 18 ABC, 24 SLC and 10 SLCO transporters was investigated using reverse transcription quantitative real-time PCR (RT-qPCR) with OpenArray® technology on a QuantStudio™ 12 K Flex Real-Time PCR system (Life Technologies, Paisley, UK). FPGS, TRAP1, DECR1 and PPIB served as reference genes. A total of 31 transporters, including 15 ABC transporters, 13 SLC transporters and 3 SLCO transporters were significantly expressed in whole blood during the placebo phase. Gemfibrozil appeared to alter the expression of three transporters. The expression of SLCO3A1 was increased by 34% by gemfibrozil (P=0.004), but gemfibrozil reduced the expression of ABCG2 and ABCC3 by 31% (P=0.018) and 50% (P=0.045), respectively. However, none of the differences remained statistically significant after correction for multiple testing. In conclusion, these data suggest that gemfibrozil might alter the gene expression of certain drug transporters in human blood.