Browsing by Subject "pharmacology"

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  • Restitutti, Flavia (Helsingin yliopisto, 2015)
    This series of investigations aimed to evaluate in dogs the interaction between MK-467, a peripheral α2-adrenoceptor antagonist with poor penetration into the central nervous system, and dexmedetomidine, a selective α2-adrenoceptor agonist commonly employed in small animal clinical practice due its potent sedative effects. The objective of this study was to find an optimal dose-ratio of dexmedetomidine and the antagonist that could attenuate or prevent major cardiovascular changes without any significant effect on the sedation induced by the agonist. The effects on blood flow in abdominal organs and on plasma concentrations of glucose, insulin, non-esterified free fatty acids, lactate and cortisol of this optimal dose were then evaluated. The sedative effects were assessed subjectively by means of a composite sedation score. Simultaneously, hypnosis was evaluated through the bispectral index. Haemodynamic parameters that were evaluated comprised cardiac output, arterial blood pressure, heart rate, central venous pressure and systemic vascular resistance. Time-intensity parameters derived from contrast-enhanced ultrasound imaging were used to assess blood flow in selected abdominal organs. Three doses of MK-467 (250, 500 and 750 µg/kg) were tested against dexmedetomidine alone (10 µg/kg). All treatments were administered IV. Sedation was significantly lower and BIS significantly higher with the medium and highest doses of MK-467 than with dexmedetomidine. However, bioequivalence between dexmedetomidine and the combination was reached with all treatments for the two parameters analysed. Early cardiovascular effects of dexmedetomidine were not completely prevented with the lowest dose of MK-467, and the highest dose reduced mean arterial pressure. The middle dose of MK-467 (500 µg/kg) provided the best cardiovascular stability. Addition of the peripheral antagonist attenuated dexmedetomidine-induced changes in organ blood flow evaluated by the CEUS. An increase in plasma glucose was observed in dexmedetomidine-treated dogs, but not when MK-467 was added. Inversely, plasma insulin concentration was reduced with dexmedetomidine, but not when dexmedetomidine was combined with MK-467. Plasma non-esterified free fatty acids concentration decreased transiently with the combination, while with dexmedetomidine alone the reduction persisted throughout the observation period. Plasma lactate concentration increased with dexmedetomidine, but not with the combination. In conclusion, the addition of MK-467 attenuated or prevented the early cardiovascular effects of dexmedetomidine, not having clinically relevant effects on the sedation induced by the latter. Some metabolic changes induced by dexmedetomidine were halted by MK-467.
  • Lepola, P.; Wang, Siri; Tötterman, A.M.; Gullberg, Ninna; Harboe, Kristine Moll; Kimland, Elin E. (2020)
    Objective The aim of this study was to assess the marketing status of the new paediatric medicinal products listed in the 10-year report as initially authorised between 2007 and 2016, reflecting the product availability in four Nordic countries. Design This is a cross-sectional study. Setting Analysis of the national medicine agency's databases in Denmark, Finland, Norway and Sweden. Data source New medicinal products with paediatric indications and new paediatric formulations listed in the Annex of European Medicines Agency's EU Paediatric Regulation 10-year report. Data analysis The products were classified according to national marketing status between January 2019 and March 2019, whether a product was authorised and whether the product was marketed. Main outcome measures The percentages of the new medicinal products with paediatric indications and new paediatric formulations having a valid marketing authorisation and being marketed, both in terms of the sums of all countries and separately for each country. Results Across the four countries, 21%-32% (16/76-24/76) of the new medicinal products were not marketed. Of the new formulations relevant to children, 29%-50% (16/56-28/56) were not marketed, and a significant proportion of these products had never been marketed. Conclusions This study reflects the reality of the implementation of the Paediatric Regulation. The results show that several new paediatric medicines and new formulations are not marketed. This affects the product availability. Similar data from other countries are needed to evaluate the overall European status to find remedies to current situation and increase the availability of the medicines for children. ©
  • Kolberg, Matthias; Bruun, Jarle; Murumagi, Astrid; Mpindi, John P.; Bergsland, Christian H.; Holand, Maren; Eilertsen, Ina A.; Danielsen, Stine A.; Kallioniemi, Olli; Lothe, Ragnhild A. (2017)
    Patients with malignant peripheral nerve sheath tumor (MPNST), a rare soft tissue cancer associated with loss of the tumor suppressor neurofibromin (NF1), have poor prognosis and typically respond poorly to adjuvant therapy. We evaluated the effect of 299 clinical and investigational compounds on seven MPNST cell lines, two primary cultures of human Schwann cells, and five normal bone marrow aspirates, to identify potent drugs for MPNST treatment with few side effects. Top hits included Polo-like kinase 1 (PLK1) inhibitors (volasertib and BI2536) and the fluoronucleoside gemcitabine, which were validated in orthogonal assays measuring viability, cytotoxicity, and apoptosis. DNA copy number, gene expression, and protein expression were determined for the cell lines to assess pharmacogenomic relationships. MPNST cells were more sensitive to BI2536 and gemcitabine compared to a reference set of 94 cancer cell lines. PLK1, RRM1, and RRM2 mRNA levels were increased in MPNST compared to benign neurofibroma tissue, and the protein level of PLK1 was increased in the MPNST cell lines compared to normal Schwann cells, indicating an increased dependence on these drug targets in malignant cells. Furthermore, we observed an association between increased mRNA expression of PLK1, RRM1, and RRM2 in patient samples and worse disease outcome, suggesting a selective benefit from inhibition of these genes in the most aggressive tumors.
  • Kopra, Jaakko (Helsingin yliopisto, 2016)
    Midbrain dopamine neurons exert a powerful influence on behavior and their dysfunction is associated with many neurological and neuropsychiatric diseases, including Parkinson s disease (PD). Dopamine neurons are large, complex and sensitive cells. Hence, their survival and correct function requires coordinated action of various transcription and regulatory factors both during development and aging. Potentially, one such factor is glial cell line-derived neurotrophic factor (GDNF). Ectopically applied GDNF is best known for its potent ability to protect and restore damaged dopaminergic neurons both in vitro and in vivo. GDNF-based therapies have been tested in clinical trials with PD patients with variable success. However, the function of endogenous GDNF in brain dopamine system development, aging and disease is poorly understood. Improvement in GDNF-based therapies requires better understanding of the physiological functions of GDNF in the brain. The current knowledge of endogenous GDNF function remains obscure, mainly due to the lack of proper animal models. The present study investigated the regulatory role of endogenous GDNF in the development, maintenance and function of midbrain dopamine neurons utilizing novel mouse models: GDNF conditional knock-out (cKO) mice and GDNF hypermorphic (GDNFh) mice over-expressing GDNF from the endogenous locus. GDNF cKO mice enable GDNF deletion solely from the central nervous system during embryonic development or later in adulthood, preserving its vital role in kidney development. Midbrain dopamine systems of these new mouse strains were studied with immunohistochemical, neurochemical, pharmacological, behavioral and molecular biology methods. We found more substantia nigra dopaminergic cells and elevated striatal dopamine levels in immature and adult GDNFh mice. In cKO mice, dopamine levels and cell numbers were unaltered, even upon aging, and regardless of the timing of GDNF deletion. Both mouse strains exhibited enhanced dopamine uptake, while responses to amphetamine were augmented in GDNFh mice and reduced in cKO mice. GDNFh mice also released more dopamine and GDNF elevation protected them in a lactacystin-based model of PD. Overall, dopamine neurons were more sensitive to moderate elevation than complete absence of endogenous GDNF, which suggests that they can adaptively compensate for GDNF loss. This highlights the limitation of broadly utilized gene deletion approaches in analyzing gene function. Our results indicate a clear role for endogenous GDNF in midbrain dopamine neuron development and function, but also demonstrate that GDNF is not required for their maintenance during aging. Furthermore, the ability of endogenous GDNF to protect animals in a PD model without the side effects associated with ectopic GDNF application suggests that elevation in endogenous GDNF levels may be an important future route for PD therapy.
  • Renko, Juho-Matti (Helsingin yliopisto, 2021)
    Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of nigrostriatal dopamine neurons and propagating Lewy body pathology. Dopamine depletion in the striatum gives rise to the cardinal motor symptoms of PD. Current PD medications are based on replenishing striatal dopamine and provide symptomatic relief to the motor deficits. However, troublesome adverse effects and diminished efficacy complicate their long-term use. There is a great unmet medical need for a therapy that could slow or halt the progression of the disease. Neurotrophic factors (NTFs) are secreted proteins that promote neuronal growth, differentiation and survival. They are able to prevent the progression of neurodegeneration and restore aberrant neuronal function in a variety of preclinical models of PD. Nonetheless, outcomes from clinical trials have been disappointing. The purpose of this work was to characterize the effects of cerebral dopamine neurotrophic factor (CDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF) and novel small molecule receptor tyrosine kinase RET agonists (BT13 and BT44) on nigrostriatal dopamine system and support their preclinical development as potential neurotrophic therapies of PD. To further clarify the functional effects of glial cell line-derived neurotrophic factor (GDNF), CDNF and MANF in the normal rat brain, microdialysis measurements were performed after a bolus injection of NTFs into the striatum. We saw augmented stimulus-evoked dopamine release and elevated dopamine turnover in the striatum of MANF-injected rats. GDNF injection increased in vivo tyrosine hydroxylase (TH) and catechol-O-methyltransferase activity and decreased monoamine oxidase A activity. These data are relevant when considering exogenously administered NTFs as a potential therapeutic approach for PD, since they have to be compatible with the existing dopaminergic medications of the patients. We also investigated the distribution properties of 125I-labeled and unlabeled CDNF after a nigral injection to intact rats. CDNF readily diffused into the brain areas surrounding the injection site and colocalized with TH-immunoreactive neurons in the substantia nigra. We did not detect active transportation of CDNF to distal brain areas. This characterization provides valuable insights into the selection of optimal delivery site and protocol for CDNF therapy. Our in vitro assays showed that RET agonists BT13 and BT44 were able to induce RET phosphorylation and activate downstream pro-survival signaling cascades Akt and ERK. They also supported the survival of cultured midbrain dopamine neurons from wild-type, but not from RET knockout, mice. The functional effects of BT13 and BT44 were evaluated in a unilateral 6-hydroxydopamine rat model of PD, where both compounds alleviated amphetamine-induced turning behavior. BT44 also showed potential to restore striatal TH-immunoreactive fibers. As blood-brain barrier penetrating compounds, BT13 and BT44 serve as promising leads that can be further developed into a disease-modifying therapy for PD.
  • Peled, Nitai (Helsingfors universitet, 2014)
    Nitai Peled1, Miao Zefeng1, Tuija Tapaninen1,2, Pertti J. Neuvonen1,2 and Mikko Niemi1,2 1Department of Clinical Pharmacology, University of Helsinki, Finland 2HUSLAB, Helsinki University Central Hospital, Helsinki, Finland Rifampicin is a broad spectrum antibiotic used in the treatment of tuberculosis and staphylococcal infections. Through activation of pregnane X receptor (PXR), rifampicin induces the expression of several drug metabolizing enzymes and drug transporters. Previous studies suggest that rifampicin can induce the expression of certain drug transporters (e.g., ABCB1) in blood. Our aim was to investigate possible effects of rifampicin on drug transporter gene expression in whole blood. In a randomized crossover study, 12 healthy volunteers took 600 mg rifampicin or placebo once daily for 5 days (Tapaninen et al 2010). On the morning of day 6, 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 16 ABC transporters, 18 SLC transporters and 4 SLCO transporters were expressed above the quantification limit in most samples. Rifampicin had no significant effect on the expression of any transporter. However, SLC5A6 (sodium-dependent multivitamin transporter, SMVT) and ABCB4 (multidrug resistance protein 3, MDR3) expression tended to be increased by rifampicin (by 19% and 18%; P=0.066 and P=0.096, respectively). In conclusion, multiple drug transporter genes are expressed in whole blood, but rifampicin has limited effects on their expression. References: Tapaninen T, Neuvonen PJ, Niemi M. Rifampicin reduces the plasma concentrations and the renin-inhibiting effect of aliskiren. Eur J Clin Pharmacol 2010;66:497-502.
  • Yoo, Bo Ram (Helsingfors universitet, 2017)
    Cardiovascular disease is one of the leading causes of mortality worldwide. Upon myocardial infarction, billions of cardiomyocytes are lost, a fibrotic scar forms, and the heart's contractile function is compromised. Mammalian cardiomyocytes lose most of their proliferative capacity shortly after birth. This decline in proliferative capacity is associated with a switch from glycolysis to oxidative phosphorylation, yielding more ATP, but also inevitably forming reactive oxygen species (ROS). Therefore, finding a way to extend the proliferative window seems crucial to cardiac repair. microRNAs (miRNAs) are short, single-stranded noncoding RNAs that repress gene expression after transcription by binding to their target mRNAs. SIRT1-7, mammalian homologs of the Sirt2 protein in yeast, have been implicated in the regulation of metabolic homeostasis, cell proliferation, cardiac hypertrophy, and aging. The objective of our research was to investigate the differential expression of SIRT1-7 between day 1 and day 7 neonatal mice. Since cells continue to divide until day 7, we wanted to compare the differences in sirtuin expression during the two time points. By doing so, we hoped to gain insight into ways we could regulate sirtuin protein expression by manipulating miRNA and sirtuin gene expression in diseased hearts, thereby promoting the fetal gene program and inducing cells to reenter the cell cycle. Proteins were isolated from whole cell lysates of cardiac tissue of day 1 and day 7 neonatal mice, and western blotting technique was used to analyze SIRT1-7 expression. Expression of SIRT3 and 7 was significantly higher in day 7 as opposed to day 1 in at least two of the three runs, with SIRT7 levels being higher in day 7 in all three runs. Our study provides a basis for carrying out more quantitative analysis to validate gene and protein expression and protein activity, since expression is different at the gene and protein levels and does not necessarily translate into activity.
  • Shi, Jin (Helsingin yliopisto, 2014)
    Milk proteins which make up 3.5% of the bovine milk are classified into casein and whey proteins. A high intake of milk proteins, especially whey proteins, has been shown to exert the beneficial effects on obesity and obesity related diseases in both humans and animals via unknown mechanisms. The aim of the present study was to investigate the potential of different whey proteins, i.e. whey protein isolate (WPI), alpha-lactalbumin (α-lac), lactoferrin (LF) and microfiltered native whey (MFNW), and their mechanisms of actions to prevent and treat diet-induced obesity and its consequences in C57Bl/6J mice. In the present study, all of the tested whey proteins were given as the only protein source in high-fat diets with a constant protein (18 % of the energy): carbohydrate (21 % of the energy): fat (61 % of the energy) ratio. We used weekly body weight measurements, daily food intake monitoring, apparent fat digestibility, dual-energy X-ray absorptiometry, oral glucose tolerance test, monitoring of fat pad weights, as well as biochemical measurements in order to assess the metabolic effects of whey proteins. Compared to casein, WPI (rich in lactoperoxidase, LF, growth factors and immunoglobulins) and LF accelerated weight and fat loss under energy restriction, and inhibited weight and fat regain during the ad libitum feeding after energy restriction without interfering with energy intake or apparent fat digestibility in C57Bl/6J mice. Both WPI and LF ameliorated fatty liver formation, and exerted beneficial effects on glucose tolerance under high-fat-feeding. The beneficial effects of WPI occurred in a dose-dependent manner. In addition, LF reduced the adipose tissue inflammation after weight regain, a property not shared with WPI. The further biochemical analysis indicated that these effects of both WPI and LF are mediated, at least partly, via the inhibition of mTOR nutrient sensing pathway and the activation of SIRT3 in the liver. Alpha-lac has been reported as one of the most effective whey protein fractions for accelerating weight and fat loss during energy restriction in the same mouse model. It was observed that the effects of α-lac on body weight and fat under energy restriction could be reproduced by supplying an amino acid mixture with an identical amino acid profile, which indicates that the anti-obesity effects of α-lac were mainly mediated by its individual amino acid composition. The MFNW produced by polymeric membranes using novel microfiltration technology, prevented weight gain and fat accumulation without interfering with energy intake or glucose homeostasis during ad libitum high-fat-feeding. The findings also suggest that the beneficial effects of MFNW are largely due to its rich α-lac content. In summary, the intake of whey proteins exerts anti-obesity effects in C57Bl/6J mice during high-fat-feeding. WPI and LF enhance weight loss, prevent weight regain and ameliorate obesity induced fatty liver formation. The anti-obesity effects of WPI are attributable, to a large extent, to its LF content. The anti-obesity effects of α-lac are mainly due to its amino acid composition. The observed beneficial effects of MFNW point to a possible method to generate whey proteins with high bioactive value on a large scale.
  • Elmadani, Manar; Khan, Suleiman; Tenhunen, Olli; Magga, Johanna; Aittokallio, Tero; Wennerberg, Krister; Kerkelä, Risto (2019)
    Background-Small molecule kinase inhibitors (KIs) are a class of agents currently used for treatment of various cancers. Unfortunately, treatment of cancer patients with some of the KIs is associated with cardiotoxicity, and there is an unmet need for methods to predict their cardiotoxicity. Here, we utilized a novel computational method to identify protein kinases crucial for cardiomyocyte viability. Methods and Results-One hundred forty KIs were screened for their toxicity in cultured neonatal cardiomyocytes. The kinase targets of KIs were determined based on integrated data from binding assays. The key kinases mediating the toxicity of KIs to cardiomyocytes were identified by using a novel machine learning method for target deconvolution that combines the information from the toxicity screen and from the kinase profiling assays. The top kinases identified by the model were phosphoinositide 3-kinase catalytic subunit alpha, mammalian target of rapamycin, and insulin-like growth factor 1 receptor. Knockdown of the individual kinases in cardiomyocytes confirmed their role in regulating cardiomyocyte viability. Conclusions-Combining the data from analysis of KI toxicity on cardiomyocytes and KI target profiling provides a novel method to predict cardiomyocyte toxicity of KIs.
  • Liu, Li; Rippe, Catarina; Hansson, Ola; Kryvokhyzha, Dmytro; Fisher, Steven; Ekman, Mari; Sward, Karl (2021)
    Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) are co-factors of serum response factor (SRF) that activate the smooth muscle cell (SMC) gene program and that play roles in cardiovascular development and mechanobiology. Gain and loss of function experiments have defined the SMC gene program under control of MRTFs, yet full understanding of their impact is lacking. In the present study, we tested the hypothesis that the muscarinic M-3 receptor (CHRM3) is regulated by MRTFs together with SRF. Forced expression of MYOCD (8d) in human coronary artery (SMC) followed by RNA-sequencing showed increased levels of M-2, M-3, and M-5 receptors (CHRM2: 2-fold, CHRM3: 16-fold, and CHRM5: 2-fold). The effect of MYOCD on M-3 was confirmed by RT-qPCR using both coronary artery and urinary bladder SMCs, and correlation analyses using human transcriptomic datasets suggested that M-3 may also be regulated by MRTF-B. Head-to-head comparisons of MYOCD, MRTF-A and MRTF-B, argued that while all MRTFs are effective, MRTF-B is the most powerful transactivator of CHRM3, causing a 600-fold increase at 120h. Accordingly, MRTF-B conferred responsiveness to the muscarinic agonist carbachol in Ca2+ imaging experiments. M-3 was suppressed on treatment with the MRTF-SRF inhibitor CCG-1423 using SMCs transduced with either MRTF-A or MRTF-B and using intact mouse esophagus in culture (by 92 +/- 2%). Moreover, silencing of SRF with a short hairpin reduced CHRM3 (by >60%) in parallel with alpha-actin (ACTA2). Tamoxifen inducible knockout of Srf in smooth muscle reduced Srf (by 54 +/- 4%) and Chrm3 (by 41 +/- 6%) in the urinary bladder at 10days, but Srf was much less reduced or unchanged in aorta, ileum, colon, trachea, and esophagus. Longer induction (21d) further accentuated the reduction of Chrm3 in the bladder and ileum, but no change was seen in the aorta. Single cell RNA-sequencing revealed that Mrtfb dominates in ECs, while Myocd dominates in SMCs, raising the possibility that Chrm3 may be driven by Mrtfb-Srf in the endothelium and by Myocd-Srf in SMCs. These findings define a novel transcriptional control mechanism for muscarinic M-3 receptors in human cells, and in mice, that could be targeted for therapy.
  • Hakala, Elina (Helsingfors universitet, 2011)
    The aim of this study was to explore the functions of T-type calcium channels, and their possible role in neuronal stem cells migration. The role of T-type calcium channel in mature brain is known to be in producing electroencephalographic oscillations. This action in turn is the key factor in some neuronal physiological and pathophysiological functions, like non-REM sleep, memory, learning and absence epilepsy. In addition, T-type calcium channels have peripheral actions, but this study concerns on its neuronal functions. This low-voltage activated channels functions in neurogenesis is less known than its role in mature brain. It is known to promote neuronal proliferation and differentiation, but what comes to its possible actions in neuronal migration, is poorly studied. This study shows some evidence of T-type calcium channel taking part in neuronal migration in mice embryonic subventricular zones progenitor cells. Selective T-type antagonists, ethosuximide, nickelchloride and a scorpion peptide toxin kurtoxin, decreased the rate of migration in differentiating progenitor cells. This study consists of a literature review and an experimental part. Another aim of this study is to consider an alternative approach to stem cell therapies based on invasive transplantation of the cells. This other attempt is non-invasive manipulating of endogenous stem cells to proliferate and migrate to the injured or depleted area in the brain, differentiate into a desired phenotype and stop their division after they have completed their mission. Non-invasive altering of the stem cells is awaiting pharmacological solutions to resolve the problems being faced in this effort. There are some non-invasive therapies already being used successfully to cure pathological conditions such as spinal cord injury. These methods could be used as well in stem cell based therapies in the treatment of neurodegenerative diseases and brain injuries. These methods are still in the beginning of their way and lacking the full understanding of the key factors that affect neuronal development. These factors include some important endogenous inducing and inhibiting substances. One of the most important inducing substances is calcium ion regulating a variety of events in neurogenesis. T-type calcium channel, as being widely expressed during early brain development, and decaying by neuronal maturation, might have a pivotal role in conducting progenitor cells.
  • Es-safi, Imane; Mechchate, Hamza; Amaghnouje, Amal; Kamaly, Omkulthom Mohamed Al; Jawhari, Fatima Zahra; Imtara, Hamada; Grafov, Andriy; Bousta, Dalila (2021)
    Depression and anxiety are major mental health problems in all parts of the world. These illnesses are associated with a number of risk factors, including oxidative stress. Psychotropic drugs of a chemical nature have demonstrated several side effects that elevated the impact of those illnesses. Faced with this situation, natural products appear to be a promising alternative. The aim of this study was to evaluate the anxiolytic and antidepressant effects of the Petroselinum sativum polyphenols in vivo, as well as its correlated antioxidant properties in vitro. Anxiolytic activity of the extract (50 and 100 mg/kg) was evaluated using the open field and the light-dark chamber tests, while the antidepressant activity was evaluated using the forced swimming test. The antioxidant activity of the extract was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical test and the FRAP (iron-reducing capacity) test. The phenolic extract showed very powerful anxiolytic and antidepressant-like effects, especially at a dose of 100 mg/kg, decreasing the depressive behavior in mice (decreased immobility time) and also the anxiolytic behavior (tendency for discovery in the center and illuminated areas) better even than those of paroxetine and bromazepam (classic drugs) concomitant with those results the extract also showed an important antioxidant capacity. These preliminary results suggest that Petroselinum sativum exhibits anxiolytic and antidepressant potential for use as a complement or independent phytomedicine to treat depression and anxiety.
  • De Lorenzo, Francesca (2020)
    Neurodegenerative diseases are characterized by the dysfunction and death of specific neuronal populations. Parkinson’s disease (PD) is caused by the progressive loss of dopamine neurons in the substantia nigra, whereas motor neurons (MNs) in the motor cortex, brain stem, and spinal cord degenerate and die in amyotrophic lateral sclerosis (ALS). Accumulation of misfolded proteins and endoplasmic reticulum (ER) stress are some common hallmarks in the pathophysiology of neurodegenerative diseases. ER stress triggers the unfolded protein response (UPR), a physiological response that aims at restoring the ER homeostasis by degrading misfolded proteins, attenuating protein translation, and increasing the expression of ER chaperones important for protein folding. Initially the UPR is protective, but, upon prolonged ER stress, the UPR switches from an adaptive to a pro-apoptotic response. Cerebral dopamine neurotrophic factor (CDNF) is an ER resident protein with neurotrophic properties that is protective and restorative in preclinical models of PD. The mechanism underlying CDNF’s action is still unclear, but experimental data suggest a possible involvement of CDNF in the ER homeostasis. The aim of this thesis work was to study the therapeutic potential of CDNF in PD and ALS rodent models and investigate CDNF mode of action, with a special focus on the ER stress response. Herein, we report that co-administration of CDNF and glial cell line-derived neurotrophic factor (GDNF) showed an additive neurorestorative effect in the unilateral 6-hydroxydopamine rat model of PD, suggesting a different mechanism of action for these two proteins. We found that GDNF activated the pro-survival MAPK/ERK and PI3K/AKT pathways in the striatal dopamine neurons within 1 hour from protein administration. In contrast, CDNF activated only the PI3K/AKT pathway and at 4 hours upon treatment. Furthermore, CDNF, but not GDNF, reduced the expression of UPR markers ATF6, p-eIF2α, and GRP78. Therefore, the ability of CDNF to regulate ER stress was thoroughly investigated in three rodent models of ALS with different genetic etiology and disease progression. We showed that CDNF decreased the ER stress response specifically in MNs, by attenuating all three branches of the UPR, initiated by transducers inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6). CDNF treatment was effective in all three models, indicating that CDNF’s therapeutic effect was independent of disease etiology. CDNF rescued MNs from ER-stressed induce cell death, halting the progression of the disease and ameliorating the motor deficit in the SOD1-G93A mouse model and in the TDP43-M337V rat model. Finally, we identified that depleting endogenous CDNF from the SOD1-G93A model worsened the motor symptoms in the mice, but did not affect their lifespan. The ER stress response in the Cdnf -/- SOD1-G93A mice was especially exacerbated in the skeletal muscle, where CDNF is normally highly expressed, and an overexpression of homologous protein mesencephalic astrocyte-derived neurotrophic factor (MANF) was detected in the same tissue. We observed a reduction in the number of lumbar MNs in Cdnf -/- SOD1-G93A compared to classical SOD1-G93A mice, which would explain the aggravated motor impairment. At this point, however, we could not determine whether the increase in MNs loss was caused by CDNF depletion in MNs, or rather a consequence of CDNF-deficiency in the degenerating muscle cells, targets of MNs. It was previously reported that, in mice, endogenous CDNF is important for the development and maintenance of enteric submucosal neurons, as well as for the regulation of gastrointestinal transit. Remarkably, we found that Cdnf -/- mice had less lumbar MNs at 4 months, compared to WT littermates, although this decrease did not result in any motor deficit. These findings suggest that CDNF may also have a role in the development and/or survival of MNs. Altogether, these studies indicate that ER stress is an important therapeutic target for neurodegenerative diseases, such as PD and ALS, and that CDNF is a promising drug candidate, due to its ability to attenuate all three pathways of UPR.
  • Vashchinkina, Elena (Helsingin yliopisto, 2013)
    Dopamine (DA) neurons of the ventral tegmental area (VTA) are critical for decision-making and motivation and have also been implicated in the development of addictive behaviors. The activity of these neurons and the subsequent changes in DA concentrations in the target regions of the VTA are strictly regulated by both excitatory and inhibitory inputs. Among those inhibitory inputs, GABAergic transmission is mediated by phasic and tonic currents generated through different GABAA receptor subtypes. Although the phasic currents arising through the activation of synaptic GABAA receptors have been well described, much less is known about extrasynaptic GABAA receptors mediating tonic currents and modulating neuronal activity in the VTA. Here, pharmacologically selective receptor modulators, transgenic mouse models and brain slice electrophysiology were all exploited to probe the role of extrasynaptic GABAA receptors in mediating neuroplasticity in VTA DA neurons. Even though they possess distinct molecular sites of action, gaboxadol (THIP) and ganaxalone (GAN) enhanced tonic inhibition by selective targeting of the extrasynaptic δ subunit-containing GABAA receptors located on VTA GABA neurons. The tonic inhibition induced in these neurons appeared to be sufficient to disinhibit DA neurons and induce persistent neuroplasticity in the glutamate synapses on VTA DA neurons, which resulted from insertion of new GluA2 subunit-lacking AMPA receptors into the synapses. Screening of reward-related behaviors associated with VTA DA activity revealed that THIP failed to induce any reinforcement during self-administration either in mice or baboons. Moreover, both THIP and GAN produced conditioned place aversion in mice. The study performed in δ subunit-knockout mice further supported the proposal that tonic inhibition of the VTA GABA neurons contributes to conditioned aversive behavior and THIP- and GAN-induced neuroplasticity. The c-Fos mapping of brain regions, which could take part in THIP-induced aversive behavioral effects and/or neuroplasticity on VTA DA neurons, revealed the bed nucleus of stria terminalis (BNST), a part of the so-called extended amygdala circuitry, as a possible participant in mediating the aforementioned THIP-induced aversive effects. In summary, these studies demonstrate that tonic inhibition mediated by δ subunit-containing GABAA receptors appears to be a significant component of the inhibition in the VTA, and thus important for the control of motivated behavior.
  • Svarcbahs, Reinis (Helsingin yliopisto, 2019)
    Neurodegenerative disorders are characterized by accumulation of toxic protein species that are followed by a gradual loss of neurons in certain brain regions and person’s loss of movement and dementia. The cause of the protein accumulation is not fully understood but is partially influenced by the disturbances in the protein degradation pathways, post-translational protein modifications that facilitate either gain-of-toxicity or loss-of-function of these proteins. In Parkinson’s disease, the best-known aggregation prone protein is alpha-synuclein (aSyn) that is the main component of Lewy bodies, the histopathological hallmarks of Parkinson’s disease and other synucleinopathies. Several studies suggest that aSyn aggregates can damage neurons by various mechanisms, and propagate toxicity by cell-to-cell transfer thus making it a tempting target for drug therapy. Current drug therapies can only relieve symptoms of neurodegenerative diseases but do not address, for example, protein aggregate clearance or pharmacological deceleration of the inclusion formation. In previous studies, prolyl oligopeptidase (PREP) has been shown to enhance the aggregation of aSyn. PREP inhibitors have been shown to reduce the aggregation and increase the clearance of aggregates via enhanced autophagy. However, the mechanisms of how PREP affects aSyn aggregation and regulates autophagy, and if this has a long-term impact on aSyn toxicity, have not been studied. The aim of this study was to investigate the role of PREP deletion, restoration, overexpression, and catalytical inhibition on the cellular signaling pathways and aSyn aggregation. The first part of the work was done in PREP knockout cells and knockout mice where the aSyn protein was overexpressed alone or together with PREP. We showed that absence of PREP decreases aSyn-overexpression mediated behavioral and cellular toxicity in mouse brain. Additionally, we found that PREP knockout cells exhibit reduced stress response and toxicity in the presence of protein overload, have increased autophagic activity, and remove excess aSyn into the cell media. The second part studied effects of chronic PREP inhibition by KYP-2047 on aSyn aggregation and on motor disturbances in the aSyn viral vector overexpression PD mouse model. The main finding showed that after chronic PREP inhibition, animals lost pathological unilateral motor behavior due to reduction in aSyn oligomer species in the nigrostriatal pathway. The third part concentrated on mapping the role of PREP in the pathways responsible for the autophagy initiation. The main finding was discovery of PREP’s role in negatively regulating one of the most important protein phosphatase complexes, protein phosphatase 2A (PP2A), via direct protein-protein interaction. Besides, this interaction could be altered with PREP inhibitor treatment that resulted in upregulation of PP2A activity and explained the functional results of autophagy induction after PREP inhibition. In summary, the findings of this study underline mechanisms through which PREP might be mediating aSyn related pathology and underlines the potential of PREP inhibition as an attractive drug target in reducing aSyn aggregate formation and boosting clearance from the affected brains. PREP involvement in the PP2A network regulation and additional functional data warrants further PREP investigation in the context of other neurodegenerative disorders and PP2A-related ailments.