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Browsing by study line "Pharmacology"

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  • Viinamäki, Emilia (2023)
    Alpha-beta-hydrolase domain containing proteins (ABHD) are involved in lipid metabolism and its regulation in human and animal cells. Approximately 50 of these proteins have been identified and their physiological and pathophysiological functions are still further investigated. ABHD2, ABHD3, ABHD4, ABHD5, ABHD6, ABHD12 and ABHD16 are involved in the metabolism of glycerin esters and phospholipids, in particular lipid mediator 2-arachidonoylglycerol (2-AG) and its metabolites have a significant effect on neuroinflammation via the endocannabinoid system. ABHD12 and ABHD16A were at the center of focus in this thesis as enzymes regulating phosphatidylserine and pro-inflammatory lysophosphatidylserine. In this study, toxicity of five (5) abietane terpene derivatives was tested using mouse E15/16 prenatal cortical neurons, cultured in 96-well plates. There were totally 8 plates cultured in three different batches, 60 cell containing wells per each plate. Wells from each plate were divided into treatment groups of 17, three concentrations of every five compound, control and VEH groups. Those concentrations were 0,1 µM, 1 µM and 10 µM, and for the last two plates 1 µM, 10 µM and 100 µM. Treatment was also separated into three batches like the cell culturing. After treatment, number of living cortical neurons in each treatment groups were counted. For that, cells were treated with immunofluorescent NeuN and DAPI antigens and the fluorescence was imagined with automated microscope. CellProfiler was used to recognize and count the number of living cortical neurons. Immunofluorescent MAP2 antigens were also used but because the shape of MAP2-fluorescing cells, CellProfiler could not recognize them. Intensity of MAP2-fluorescence were measured from those imaged, so the work would not be wasted. One-way ordinary variance analyze ANOVA was carried out for the data to figure out if there were statistically significant results. For compound TAC174, there were several significant results with different concentrations but unfortunately, some results showed signs of toxicity and others improved cell-growth. Some significant results were also found with compounds TAC121, TAC147 and TAD40 showing sings of toxicity, but reliability of those results was questioned. Only one compound, TAC150 was not showing clear toxicity towards mouse cortical neurons, at least with lower concentrations. For conclusion, there were no clear or significant results if these compounds are toxic for cortical neurons. TAC150 showed the least sings of toxicity, therefore it could possibly be considered for further studies in medical field.
  • Peltola, Roosa (2020)
    Amyotrophic lateral sclerosis (ALS) is a rare fatal neurodegenerative disease in which both the upper and lower motor neurons degenerate. Pathological features of the disease include misfolded proteins and accumulations in the central nervous system. The molecular mechanisms of the disease include neuroinflammation, glutamate induced excitotoxicity, and endoplasmic reticulum stress (ER-stress). Numerous genetic defects have been identified in the background of ALS, the most common mutations are in the C9ORF72, SOD1, TDP43 and FUS genes. For each gene mutation, it is important to develop a reliable animal model of ALS for studying pathology and testing new therapies. The most common and most recently found gene mutation, the C9ORF72 repeat expansion mutation, does not yet have an established animal disesase model. The molecular mechanisms of the disease include neuroinflammation, glutamate induced excitotoxicity, and endoplasmic reticulum stress (ER- stress). There is no drug treatment to cure or slow ALS, so the need for new drug therapies that affect the course of the disease is significant. Cerebral dopamine neurotrophic factor (CDNF) protects and restores dopamine neurons and controls ER-stress in preclinical models of Parkinson’s disease. CDNF has also been shown to improve motor coordination as well as protect spinal cord neurons from cell destruction in ALS genetic SOD1- G93A mouse and TDP-43M337 animal models. The purpose of this master's thesis study was to characterize the changes related to neurodegeneration and neuroinflammation in the new C9ORF72-500 disease model and study ER stress of the SOD1-93A disease model and the effect of CDNF on ER stress in SOD1-model and on inflammation in C9-model. In the first sub-study, brain sections from C9ORF72 transgenic and wild-type mice at different time points were subjected to six different immunohistological stainings. The results were compared at each time point (30, 70 and 170) between the wild type and the transgenic group. In another sub-study, spinal cord sections from CDNF snd vehicle treated SOD1- G93A mice were subjected to immunofluorescence staining, after which the intensity of their ER stress marker, GRP78, was analyzed using a confocal microscope. GFAP stained brain sections from CDNF and vehicle treated C9ORF72 mice were analyzed using microscope and imaging analyses. The results of the first sub-study showed neuroinflammation at 24 weeks timepoint in the transgenic group compared to wild-type mice. Pathological features of C9-ALS, various protein accumulations, were observed only in the transgenic group, mainly at 24 weeks. No neuronal loss was observed in this study. The obtained results support the previously published research results and support the reliability of the studied disease model. In the second sub-study ER stress levels were higher in SOD1-mice compared to wild-type mice. Single intracerebroventrical CDNF injection reduced ER stress in SOD1-G93A transgenic mice almost to the same level as ER stress in wild-type mice. CDNF treatment also showed a tendency for reducing inflammation in hippocampus and motor cortex of C9ORF72 mice. The results confirm the pathological role of ER stress in ALS and show that CDNF reduces ER stress when administered as early in the disease as possible, when neuronal damage begins to occur but does not yet lead to neuronal destruction. CDNF appears to be a promising drug candidate for the treatment of ALS and should therefore be further investigated.
  • Seppälä, Katariina (2024)
    General anaesthetics are pharmaceutical agents used to induce general anaesthesia, a reversible state of unconsciousness. Caenorhabditis elegans, a nematode species, has been successfully used as a model organism in the study of gaseous anaesthetics due to its amenability to genetic modification, fully mapped nervous system connectome and high evolutionary conservation. However, C. elegans is less well characterised as a model organism in the study of non-gaseous anaesthetics. The primary aim of the study was to study the potential of ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, and urethane, a nonselective modulator of various neurotransmitter-gated ion channels, to immobilise C. elegans without activating antioxidant response signalling by transcription factor SKN-1, the nematode orthologue of the mammalian Nrf/CNC proteins. The transparent body of C. elegans enables microscopic imaging of cellular processes, but high-quality imaging requires the immobilisation of the worm. A commonly used chemical immobilising agent, sodium azide, causes SKN-1 activation in C. elegans, which may limit the use of sodium azide in studies on SKN-1 promoters. A secondary aim of this study was to study the impact of ketamine, an activator of mTOR (mechanistic target of rapamycin) signalling, on the lifespan of C. elegans. The lifespan of C. elegans has been found to increase with inhibition of the mTOR homologue pathway in previous studies. Ketamine and urethane were administered to wild-type C. elegans in aqueous media in 96-well plates. Behavioural endpoints of immobility and uncoordination were assessed manually via microscopic observation and video recordings. SKN-1 activation was studied by measuring drug-induced fluorescence in mutant strain CL2166, which carries a green fluorescent protein reporter of SKN-1 downstream target GST-4 (glutathione-S-transferase). A lifespan assay was performed with sterile C. elegans strain SS104 by incorporating ketamine in the worm maintenance agar. In this study, urethane did not appear to be a potent immobilisation agent. Ketamine was found to cause reversible weak immobilisation at a similar concentration at which sodium azide fully paralyses wild-type C. elegans. At lower doses ketamine caused uncoordinated locomotion. Short-term exposure to an immobility-inducing dose of ketamine was not found to significantly activate SKN-1. In the lifespan assay, ketamine unexpectedly appeared to significantly increase the nematode lifespan compared to control treatment.
  • Pykälämäki, Matias (2023)
    Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by the death of nigrostriatal dopaminergic neurons and formation of intraneuronal protein aggregates called Lewy bodies and Lewy neurites. These inclusions consist of a protein called α-synuclein (aSyn) but also of other proteins, lipids and cell organelles. Progressive cell death leads to nonmotor and motor symptoms. Current therapies for PD are symptomatic and do not modify the disease progression. Therefore, there is a need for the development of therapies attenuating the neurodegeneration. The pre-formed fibrils (PFF) model enables studying of aSyn aggregation and mechanisms behind inclusion formation. The PFF model is based on the exogenous aSyn fibrils’ tendency to result in formation of Lewy body -like inclusions when added in cell culture or in animals. Primary neuronal cultures of mice and rats have typically been used to model aSyn aggregation in vitro with the PFF model. Primary neuronal cultures provide practicality and are able to depict relevant features of dopaminergic neurons. To gain insight about the composition of E13.5 primary embryonic mouse midbrain culture and to enable adaptation of an existing protocol to study other cell types, this study identified and quantified several relevant cellular phenotypes in the micro island culture. The cells were fixed on day in vitro (DIV) 8 or DIV 22 and analysis was conducted using fluorescent immunocytochemistry combined with automated image analysis software, CellProfiler. On DIV 8, tyrosine hydroxylase -positive dopaminergic neurons represented 5 % of the total cells in the culture. Neuronal nuclear antigen -positive neurons resulted representing 30 % of the total cells. Gabaergic neurons were identified to be abundant in the culture and certain dopaminergic neurons were identified as immunoreactive for GABA. Choline acetyltransferase -positive cholinergic neurons were also identified to be present in the culture. The number of oligodendrocyte precursors (OPCs) was observed to be significantly smaller than the number of dopaminergic neurons. OPCs represented around 1 % of the culture on DIV 8. Glutaminergic neurons, parvalbumin-positive interneurons, microglia or astrocytes were not identified in the culture on DIV 8. The number of astrocytes was observed to increase as the incubation time was prolonged to DIV 22. Overall these findings provide valuable insights of the composition of cell phenotypes in E13.5 mouse midbrain culture. The results also provide additional validation for suitability of the original protocol to robustly produce midbrain dopaminergic cultures with minimal number of glial cells. Understanding more about the relevance and interplay of different cell phenotypes in PD pathophysiology can provide valuable insight for the development of potential therapeutic strategies.
  • Eronen, Sini-Tuulia (2022)
    Introduction: When people age, the composition of sleep changes and sleep becomes more sensitive to external disturbances, making insomnia also more common. Medication is not the first-line treatment option for insomnia. Benzodiazepines or benzodiazepine receptor agonists for the treatment of insomnia have been in the focus of past studies. The content of the dosing instructions for the supplied medicines has not been studied. The dosing instructions should provide clear instructions on how to dose the medicine prescribed to the patient. The aim of this study was to investigate the content and quality of dosing instructions prescribed for the treatment of insomnia for Finns aged ≥75 years in 2020 based on the prospective reimbursement register data by the Social Insurance Institution of Finland (Kela). Materials and methods: The reimbursed purchases of all medicines by persons aged ≥75 years from 1.1.2020 to 31.12.2020 were selected by ATC code from the medicines data according to the Insomnia: Current Care guidelines. The data was gathered from Kela’s dispensations reimbursed under the National Health Insurance scheme. The dataset consisted of 1,080,843 delivery lines, which were screened, and 328,285 lines were included in the analyses. Dosage instructions were reviewed according to the following predetermined five categories: frequency of use, dose, timing, warnings or remarks, and inappropriate instructions. In addition, 1000 dosing instructions were randomly derived to study the phrasing and appropriateness of the dosing instructions in more detail. Results: In 2020, an average of 3.8 reimbursed hypnotic drug deliveries were made per elderly person. Of the deliveries, 68% were for women. 52% of drug deliveries were partially made later and not by original prescription. In the hypnotic dataset, the three most administered drugs were zopiclone (41%), mirtazapine (34%) and zolpidem (12%). The dosage was prescribed in 98–99% of the dosage instructions. Dosing schedule was reported in 83% of dosing instructions and regularity of use was reported in 57% of them. Only 3-6% of the dosing instructions had comments or warnings. 1–2% of the dosing instructions were vague. The duration or regularity of use was clearly indicated in 5% of the dosing instructions. Only 0.1% of dosage instructions contained instructions for discontinuation or reduction. Discussion: The dose and timing of administration were well reported, but the frequency of use was reported in only about half of the dosing instructions. Only few dosing instructions contained remarks or warnings even though hypnotics are at risk for the elderly. Among the three most administered drugs for the treatment of insomnia were two benzodiazepine receptor agonists, zopiclone and zolpidem. However, they may not be suitable for the elderly according to Beers criteria and their use should be avoided. During 2020, an average of four drug deliveries were made per elderly person for the treatment of insomnia, which may indicate prolonged hypnotics use. In addition, more than half of the deliveries were partially made later and not by original prescription. Thus, several drug packages are prescribed for prescriptions, although the drug-based treatment of insomnia should only be short-lived. Conclusions: There are significant deficiencies in the contents and quality in the dosing instructions for drugs delivered to the elderly for insomnia. Minimum information on dose, timing and duration of use was not found in all dosing instructions in this study. Understandable dosing instructions and the reduction in the amount of medication in the prescription could have a further effect on reducing the long-term use of hypnotics, also increasing the safety of medicine use in the elderly.
  • Hämäläinen, Klaus (2022)
    Multiple Sclerosis (MS) is an incurable autoimmune demyelinating disease affecting the central nervous system (CNS). Although the detailed pathogenesis remains unclear, recent research has highlighted the involvement of B cells. For decades, however, MS research was based on T cell-focused animal models of autoimmune encephalomyelitis (EAE), which do not reflect the involvement of B cells in the pathogenesis. Therefore, B cell-dependent EAE models are hypothesized to allow a better understanding of MS immunohistopathology and may therefore lead to the development of efficient treatments. In our spontaneous relapsing-remitting (RR) EAE model, B cells are recruited from the endogenous repertoire by transgenic myelin oligodendrocyte glycoprotein (MOG) -reactive T cells, causing the development of EAE in 3–4-month-old mice. Interestingly, MOG-specific antibodies are present long before actual onset of clinical disease and can be detected already in 5-week-old RR mice and disease development in RR mice is dependent both on the presence of (presumably MOG-specific) B cells as well as on stimuli provided by intestinal microbiota. Firstly, we evaluated the broader usability of induced germinal center cell (iGB) culture as a model for B cell repertoire studies. Then, by using iGB culture, we studied whether MOG-specific B cells are present in secondary lymphoid organs of younger than 4-week-old and germ-free RR mice. Finally, this study aimed to investigate whether the repertoire of MOG-specific B cells undergoes significant qualitative changes from young healthy mice to older acutely sick RR mice, and whether at the time of disease onset the recruited MOG-specific B cells expand and mature in the cervical nodes (cLN) or in the CNS. To do so, following the hosting-lab’s previous single-cell RNA sequencing (scRNA-seq) of B cells derived from cLN of 5-week-old RR mice, we performed the scRNA-seq of B cells from CNS, spleen, and cLN of acutely sick RR EAE mice. We demonstrated that iGB culture is an unsuitable tool to expand pre-activated B cells, and hence, in our hands it was inappropriate for repertoire studies. However, iGB culture proved to be useful for screening different organs for MOG-specific B lymphocytes, and we found that anti-MOG antibodies were firstly detected in 3-4-week-old RR mice, and MOG-specific B cells were present also in germ-free RR mice. Our scRNA-seq results revealed many highly expanded MOG-specific B cell clonotypes in acutely sick RR mice. Moreover, the B cell repertoire of sick RR mice was more diverse, including IgG1, IgM, IgG2b, IgG2c, and IgG3 isotypes, compared to healthy 5-week-old RR mice that had only IgG1 or IgM isotypes. Two-thirds of the expanded clonotypes were primarily detected in the CNS in sick RR mice, indicating that clonotypes develop further and continue isotype switching within the CNS. We also detected more somatic mutation in the variable region of expanded clones of sick RR mice compared to 5-week-old RR mice. The results of this study clearly show an antigen-driven evolution of the MOG-specific B cell repertoire from healthy young to acutely sick RR mice, which seems to occur mainly in CNS itself. In contrast, cLN are the major initial priming site of MOG-specific B cells in healthy RR mice, even under germ-free conditions. This suggests that commensal microbiota is not required for initial recruitment of MOG-specific B cells, but for the development of EAE. To further validate our encouraging scRNA-Seq results, it is necessary, in future experiments, to confirm the MOG-specificity of expanded clonotypes.
  • Heiskanen, Vilma (2021)
    Binge eating disorder (BED) is the most common eating disorder characterized by compulsive recurrent binge eating episodes with the sense of a lack of control. During a binge eating episode, one eats a larger amount of food, typically high in fat and/or sugar, than would normally be eaten in a discrete period of time. After the episode, negative emotions, such as shame and self-disgust, are present. However, BED does not include compensatory behavior, such as vomiting or excessive exercise. Due to compulsive and uncontrollable eating behavior, it has been suggested that BED represents a food addiction. Eating energy-dense food activates the dopamine, opioid, and endocannabinoid systems in the brain. This elicits the activation of the reward process. Some drugs and medications affect the same neurotransmitter systems, which may produce neuronal alterations in the reward process, leading to an addiction. Several studies have found that cannabidiol (CBD) reduces the self- administration of cocaine, morphine, alcohol, and sucrose in rodents, suggesting an effect on the reward-response. Some of these effects have been shown to be mediated by cannabinoid receptor 2 and TRPV1 receptor. However, the effects of CBD on bingeing behavior have not been studied up to date. The aim of the study was to investigate the effect of CBD on homeostatic feeding and binge eating behavior in C57BL/6 mice. Five separate experiments were conducted. The first experiment investigated the effect of CBD (15, 50, and 150 mg/kg, i.p.) on locomotor activity in a modified open field test over a 2-hour period. In the second test, the effect of CBD (15, 50, and 150 mg/kg, i.p.) on homeostatic feeding was monitored in non-bingeing mice. Next, a limited intermittent access binge eating model without food deprivation or stressors was inducted. Mice had access to laboratory chow ad libitum, but a high energy diet (high in fat, HED) was presented in 24-hour periods every 5-8 days. Then the effect of CBD (15, 50, and 150 mg/kg, i.p.) on HED and chow intake in bingeing mice was investigated. In the fourth experiment, seven days following the administration, the after effect of CBD was studied by monitoring food intake without CBD treatment. Finally, it was investigated whether the effect of CBD can be inhibited by TRPV1 receptor antagonist AMG9810 (1 mg/kg, i.p). In each test, the food intake was monitored at the time point 0,5, 2,5, and 24 h after CBD treatment. Also, water consumption was measured in each experiment. The results revealed that CBD does not affect locomotor activity or homeostatic feeding at a dose of 15, 50, or 150 mg/kg (i.p). However, the results showed that CBD reduces the intake of HED in a dose-dependent manner (15, 50, or 150 mg/kg; i.p.) and, possibly, increases chow intake. No after effect was observed seven days following the administration. Most likely, TRPV1 does not mediate the effect of CBD on HED intake. Furthermore, no significant effects on water intake were observed. In this study, the core aims were to evaluate whether CBD affects homeostatic feeding or binge eating behavior in mice. The results provided a novel insight into the effects of CBD. The findings indicate that the acute systemic administration of CBD reduces HED intake, and possibly, simultaneously increases chow intake, suggesting a balancing effect on feeding in bingeing mice. However, the role of TRPV1 in this effect remains unclear, and further studies are needed.
  • Silmu, Veera (2021)
    Parkinsonin tauti on hitaasti etenevä hermorappeumasairaus, jossa mustatumakkeen dopamiinihermosolut tuhoutuvat. Taudille on tyypillistä dopamiinihermosoluissa esiintyvät Lewyn kappaleet, jotka koostuvat pääasiassa väärin laskostuneesta ja kasautuneesta alfasynukleiiniproteiinista. Myös neuroinflammaation uskotaan olevan osa Parkinsonin taudin patofysiologiaa. Nykyiset lääkkeet vaikuttavat ainoastaan taudin oireisiin, joten tarve uusille lääkkeille on suuri. Pilottikokeen tarkoituksena oli selvittää aiheuttaako adenoassosioidun virus- (AAV) vektorin alfasynukleiinin ja alfasynukleiinifibrillien yhdistelmämalli rotilla liikehäiriöitä ja tyrosiinihydroksylaasi- (TH) positiivisten dopamiinihermosolujen tuhoutumista mustatumakkeessa ja hermopäätteiden tuhoutumista aivojuoviossa sekä saadaanko mallilla aikaan neuroinflammatorinen vaste. Varsinaisen pitkän kokeen tarkoituksena oli selvittää aivojen dopamiinihermokasvutekijän (CDNF) mahdollinen neurorestoratiivinen vaikutus tässä mallissa. Alfasynukleiinin kasautumispatologian tasoa ja CDNF:n neurorestoratiivista vaikutusta selvitettiin käyttäytymiskokeilla sekä mustatumakkeen ja aivojuovion TH-vasta-ainevärjäyksillä. Yhdistelmämallista aiheutuvaa neuroinflammatorista vastetta selvitettiin ionisoidun kalsiumia sitovan adapterimolekyylin 1 (Iba1) ja gliaalisen fibrillaarisen happaman proteiinin (GFAP) vasta-ainevärjäyksillä. Pilottikokeen sylinterikokeessa yhdistelmämalli ei indusoinut liikehäiriötä, mutta pitkän kokeen askel- ja sylinterikokeessa mallin osoitettiin aiheuttavan unilateraalille leesiolle tyypillinen liikehäiriö. Pilottikokeen ja pitkän kokeen TH-vasta-ainevärjäyksissä mallin osoitettiin aiheuttavan TH-positiivisten dopamiinihermosolujen tuhoutumista mustatumakkeessa ja hermopäätteiden tuhoutumista aivojuoviossa. Nämä tulokset osoittavat, että yhdistelmämallilla saadaan aikaan alfasynukleiinin kasautumispatologiaa. Pilottikokeessa osoitettiin myös, että yhdistelmämallilla saadaan aikaan neuroinflammatorinen vaste, mikä osoittaa, että malli soveltuu hyvin uusien lääkkeiden vaikutuksen tutkimiseen Parkinsonin tautiin liittyvässä neuroinflammaatiossa. Pitkän kokeen sylinterikokeessa AAV-CDNF:llä ei ollut vaikutusta mallista aiheutuvaan liikehäiriöön. Sen sijaan askeltestissä kämmenen suunnan mittauksessa AAV-CDNF korjasi liikehäiriötä. AAV-CDNF ei kuitenkaan suojannut TH-positiivisia hermosoluja mustatumakkeessa tai hermopäätteitä aivojuoviossa, minkä perusteella johtopäätöstä CDNF:n neurorestoratiivisesta vaikutuksesta ei voida tehdä.
  • Lähteenmäki, Ida (2024)
    Alzheimer's disease is a progressive neurodegenerative disease of the central nervous system, in which accumulation of amyloid β plaques and hyperphosphorylated tau proteins cause neuronal death, loss of synapses, and impaired neurotransmission. Alzheimer's disease's main symptoms are memory dysfunction (dementia) and decline in cognition. One of the most significant factors believed to cause dementia in Alzheimer's disease is the destruction of dendritic spines. Dendritic spines are small protrusions of dendrites where most of the glutamatergic synapses are located and where mainly excitatory synaptic functions occur. Loss of dendritic spines is directly correlated to the loss of synaptic function, which then causes memory dysfunction and impaired cognition. Also, the morphology of dendritic spines is important for their stability and strength. The spine head's size is correlated to the number of postsynaptic receptors. Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are unconventional neurotrophic factors, which have neuroprotective and -restorative effects via regulating endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Accumulation of unfolded and/or misfolded proteins in the lumen of ER causes ER stress, which then activates UPR, which again maintains protein homeostasis by reducing the amount of unfolded/misfolded proteins. In this study, we injected CDNF, MANF or PBS into the hippocampus of C57BL/6 mice to analyze whether they increase dendritic spine density and improve their morphology, especially by increasing the density of mature spines. This study shows that MANF increases dendritic spine density and CDNF does not. MANF increases the density of most of the mature and immature spines but interestingly decreases the density of mature stubby spines. These results are very promising and MANF's effect on dendritic spine formation should be studied further. In another separate study, we investigated whether CDNF and MANF activate the ERK1/2 pathway on organotypic hippocampal slices. This pathway is important for memory formation, and another neurotrophic factor, brain-derived neurotrophic factor (BDNF), has been shown to increase dendritic spine density by stimulating the ERK1/2 pathway. CDNF and MANF activate UPR, but there is some evidence that UPR might also activate ERK1/2, which could then explain CDNF's and MANF's mechanism of action. CDNF and MANF-treated hippocampal slices had visually thicker and darker bands in ERK1/2 antibody staining but any conclusion cannot be made because of the sample sizes being too small.
  • Lahtinen, Katja (2024)
    Cardiac fibrosis (CF) is a physiological response to various stress factors encountered by the heart, with the aim of maintaining proper functioning of this vital pump in an altered situation such as increased mechanical stress or sudden injury in heart muscle. CF is characterized by excessive production of extracellular matrix (ECM) components and stress fibers in cardiac tissue, accompanied by morphological changes of the heart muscle. The responsible cells behind these changes are fibroblasts (FBs) that undergo phenotypic change by transdifferentiating into myofibroblasts (myoFBs). Although being initially a supportive response, CF can lead to deterioration of the heart performance and even heart failure, if prolonged. Given the lack of effective enough therapies against CF, and the strong involvement of CF in cardiovascular diseases (CVDs) that are associated with high mortality rate, the need for new effective therapies is urgent. Indeed, a diversity of approaches to fight CF have been proposed, among them protein kinase C (PKC) and its signaling cascades. PKC has been shown to play a role in fibrosis and many studies suggest antifibrotic properties of PKC, yet the results are challenged by the opposite findings. Despite the dichotomous results, new small molecules that function as partial agonist of PKC seemed to be a promising strategy for the treatment of fibrosis. To further explore the role of PKC activation in CF, the aim of this study was to first develop and characterize a human cardiac fibroblast (HCF)-based CF model, in which the effects of seven new PKC modulators on HCFs could then be evaluated. To create the CF model and provoke a fibrotic response, HCFs were treated with either transforming growth factor β1 (TGF-β1), Angiotensin II (Ang-II), endothelin-1 (ET-1), or combination of treatments, followed by determination of HCF proliferation activity and α-smooth muscle actin expression (α-SMA), a marker of myoFBs. After the treatments, the original goal was to continue in compound testing phase by exposing the HCFs to the PKC-modulators to see whether any differences could be determined in α-SMA expression or proliferation activity. However, no considerable effects of fibrosis-inducing treatments on the activation of HCFs were observed, thus preventing this progression. Nevertheless, toxicity tests were performed on the compounds and the results indicated relatively low overall toxicity for the lower concentration: six out of seven compounds yielded over 70% HCF viability at 3 μM concentration with three of them reaching even over 80% viability, while the corresponding value for the previously published PKC agonist HMI-1a3 was 54%. Although these results are promising for the lower concentrations of PKC-modulators, it is obvious that more in-depth studies are required prior to drawing any unambiguous conclusions.
  • Sipola, Kirsi (2021)
    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder caused by degeneration of motor neurons in brain and spinal cord. The degeneration of motor neurons leads to muscle atrophy and paralysis. Currently there is no cure for ALS. Available drugs for ALS can lengthen the survival time by a couple of months. Several factors involve the pathophysiology of ALS, such as endoplasmic reticulum stress and neuroinflammation. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a protein which has shown neuroprotective effects on animal models of Parkinson disease and brain ischemia. C-terminal fragment of MANF can cross the blood-brain barrier, allowing it to be administered subcutaneously instead of injected directly into the brain. The experimental part consists of two parts. The aim of the first part was to study the pharmacokinetic properties of next generation MANF (C-MANF). The aim of the second part was to elucidate the effect of twice a week administered subcutaneous injection of C-MANF in genetic SOD1-G93A mouse model and its neuroprotective effects by assessing protection of lumbar motor neurons. Pharmacokinetic properties of C-MANF were determined in wild type mice after a single subcutaneous injection of C-MANF at different time points by using indirect ELISA assay. The effects of C-MANF in SOD1-G93A mouse model were assessed by subcutaneous injection of either C-MANF or PBS twice a week and by monitoring clinical score and motor behavior of mice from 10 weeks of age to clinical endpoint. Hematoxylin eosin staining was used to study neuroprotective effects of C-MANF. C-MANF administered subcutaneously is absorbed into the blood circulation and the highest serum concentration of C-MANF is after 60 minutes of dosing. Subcutaneously injected C-MANF also crosses the blood-brain barrier and reach the brain in 120 minutes. C-MANF did not preserve motor function or ameliorated ALS symptoms in SOD1-G93A mouse model. In this study C-MANF did not increase the survival of SOD1-G93A mice. C-MANF did not significantly protect motor neurons from degeneration even though there was a slight trend between the groups. No beneficial effects were observed with C-MANF in SOD1-G93A mouse model and therefore the dose and frequency of administration of C-MANF were not optimal. Subcutaneously injected C-MANF provides a safer dosing option for neurodegenerative disorders.
  • Cavonius, Karin (2021)
    Huntington’s disease (HD) is a rare but devastating neurodegenerative disease, progressively culminating in severe brain atrophy and death. The disease is caused by an inherited mutation resulting in a CAG trinucleotide repeat expansion in the huntingtin gene, leading to the production of a neurotoxic protein, known as mutant huntingtin, with an abnormally long polyglutamine stretch. Even though the genetic background of HD is known, the cellular pathways affected in the disease are complex and not completely understood. Increasing evidence indicates that endoplasmic reticulum (ER) stress – a condition of disturbances in normal ER activity, leading to accumulation and aggregation of misfolded proteins in the ER lumen – is a central factor in the pathogenesis of HD and other neurodegenerative diseases. In the literature review of this thesis, known pathogenic cellular mechanisms of HD and how these cellular mechanisms are connected to ER stress, are discussed. Unpublished data from previous studies in our laboratory have indicated that the ER luminal protein canopy homolog 2 (CNPY2) could play a role in the regulation of neuronal survival, including the viability of mutant huntingtin expressing neurons. The aim of the experimental part of this study was to gain insight into a possible function of CNPY2 in HD, by examining the levels of the protein in neuronal models of HD under various conditions, such as ER stress, and by searching for potential interacting partners of CNPY2 amongst known ER stress regulators. The obtained results show that the levels of CNPY2 are increased in striatal neurons expressing mutant huntingtin, and that the secretion of CNPY2 is increased by these neurons, compared to control neurons expressing normal huntingtin. Further, we show that CNPY2 interacts with the major ER stress regulator binding immunoglobulin protein (BiP) in human neuroblastoma cells treated with the ER stress inducer tunicamycin, and that the intracellular levels of CNPY2 are altered by tunicamycin treatment. Together, these findings indicate that CNPY2 could be involved in the pathogenesis of HD. However, further research on the functions of CNPY2 and its role in ER stress regulation is required to understand the nature of this involvement.
  • Lähdesmäki, Emmi (2023)
    The most typical symptoms of dementia include impairment of cognitive brain functions, such as memory and thinking. Most common forms of dementia include Alzheimer's disease, vascular dementia, Lewy body dementia, and frontotemporal dementia, which is caused by degeneration of the frontotemporal lobe. Alzheimer's disease is the most common form of dementia, covering about 75% of all the cases. The pathophysiology of Alzheimer's disease includes beta-amyloid plaques and tau proteins, which accumulate in the brain, and which have been linked to damage to nerve pathways and the appearance of the typical symptoms of the disease. The disorder is progressive, but the exact cause remains unknown. However, old age (>65 years), the APOE-4 gene, lifestyle, and some comorbidities, such as cardiovascular diseases, are considered risk factors. Even though extensive research has been conducted, there is currently no curative treatment for Alzheimer's disease. Sleep disorders can be both a symptom of Alzheimer's disease and a risk factor for the onset of the disorder. Therefore, the mechanisms of sleep and circadian rhythm are connected to the pathophysiology of Alzheimer's disease, for example through the glymphatic system that cleans the brain mainly during deep sleep. Many drugs for Alzheimer's disease have a recommended time of administration. The dosing time can be very important issue in terms of the effectiveness of the drug. According to a recent study, sleep and circadian rhythm have not been considered in most studies on new rapid-acting antidepressants. Therefore, we carried out an analogous systematic literature review for Alzheimer's disease and dementia. The aim of this study was to find out whether sleep and circadian rhythm have been considered in the most cited preclinical and clinical drug research articles for Alzheimer's disease and dementia during the last decade (2010–2020). In addition, it was examined which drug groups the studied compounds belonged to, and what was the sex distribution of the test subjects in the studies. The number of subjects was also determined from clinical studies, and the animal species from preclinical studies. The research articles analysed in the study were collected with a systematic literature review of Scopus database. The study found that most studies did not include any consideration of sleep or circadian rhythm. Most of the investigated compounds were small molecules, followed by supplements and herbs, and rest classified as biological drugs. Most of the clinical trials were relatively small studies with less than a hundred subjects or hundreds of subjects. Among the 100 most cited clinical research articles, there were 14 reanalyses and observational studies that were not included in this analysis of subject numbers. In clinical studies, most of the test subjects were usually female, while preclinical studies used commonly male animals. To conduct more open and reliable science in the future, drug research should pay more attention to the subjects’ sleep patterns, the time of drug administration, and reporting on these issues in the articles, which is usually part of the requirements of scientific journals. This could potentially narrow the translational gap between preclinical and clinical research.
  • Granqvist, Riikka (2021)
    Parkinson´s disease (PD) is the second most common neurodegenerative disease in the world after Alzheimer´s disease. There is still no drug that alters the state of the disease. It has been found that Endoplasmic reticulum (ER) stress is one mechanism in PD. ER stress occurs due to accumulation of unfolded proteins. ER stress triggers Unfolded protein response (UPR) that protects against ER stress by decreasing unfolding of proteins. In the beginning, UPR has protective effect, but in prolonged ER stress UPR triggers apoptotic cell death. There are several key mediators in the UPR pathway. Characterisation of ER stress in PD models may be important for the current and future drug development of PD. If ER stress is a significant factor that affects the disease development, it would be important to find a drug that alters these mechanisms and UPR. This may be a way to halt the disease development. Different animal models of PD, like 6-OHDA (6-hydroxydopamine) and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model, have similarities in their mechanisms. It has been found that ER stress occurs both in the brain of PD patients and animal models of PD. That is why studying and further characterisation in animal models is relevant. The aim of this study was to characterize ER stress in 6-OHDA rat model. The expression of some key mediators of the UPR were determined in this study. There were male and female Spraque Dawley rats in this experiment. 6-OHDA or saline was injected intrastriatally in 3 spots by stereotaxic surgery. Two weeks after 6-OHDA lesions, amphetamine-induced rotation test was conducted to the rats. The rats were divided into groups based on lesion size according to the results. For this study, the rats were euthanised at week 2 or week 4 post lesion. The rats were euthanised by carbondioxide, and the death was confirmed by decapitation. The brains were collected and stored in -80°C. Striatum and substantia nigra were collected later. Total RNA was isolated from these samples. Part of the RNA sample was used to conduct cDNA synthesis. Finally, the gene expression of Atf4, Ire1α, Xbp1s, Xbp1t, Grp78 and Chop was measured from these cDNA samples by qPCR (quantitative polymerase chain reaction). The qPCR data describes the expression of exact gene. The data was processed prior to statistical analysis. By statistical analysis, it was possible to compare the expression of these genes between 6-OHDA group and vehicle group. In addition, comparison was made between 6-OHDA treated groups at week 2 and 4. According to the results, only Chop expression had increased in 6-OHDA lesioned rats at week 2 compared to the vehicle group. In other genes there were no statistical differences, unlike in several other studies where the expression was found to be increased. Thus, the characterisation of this model requires further studying, possibly by increasing the sample size and studying later time points as well.
  • Reunanen, Saku (2020)
    Parkinson’s disease (PD) is a neurodegenerative disease in which dopaminergic neurons that form the nigrostriatal pathway gradually die. This causes the main motor symptoms of Parkinson’s disease: tremor, rigidity and bradykinesia. While PD affects 1-2% of total population, all currently used medicines are symptomatic, and there is no disease modifying therapy available at present. Although several different animal models for Parkinson’s disease exist, the lack of adequate animal models is often cited as a major obstacle for predicting the clinical success of potential drug candidates. Lewy bodies (LBs) are abnormal aggregates that develop and spread inside nerve cells of human PD patients, their main structural component being α-synuclein. Because α-synuclein is thought to play a major role in the pathology of PD, much research has been focused on it. Different α-synuclein-based animal models of PD exist today, of which the most recent are based on using direct injections of preformed α-synuclein fibrils (PFFs). These new α-synuclein based disease models have helped to understand the disease process in PD better, but cell death in these models takes longer to achieve and is often less pronounced compared to traditional neurotoxin based animal models of PD. The aim of this study was to participate in the development and characterization of a novel mouse model of PD. This new model combines PFF-injections with the commonly used neurotoxin 6-OHDA, which should result in more robust dopamine pathway degeneration than what is seen with the current PFF-based models. The main hypothesis of this study was that the combination of intrastriatal injections of PFFs and a low dose of 6-OHDA would cause gradual spreading of the α-synuclein aggregation pathology in the nigrostriatal dopamine pathway and progressive dopamine neuron loss leading to motor deficits. C57BL/6 mice were stereotactically injected unilaterally with both PFF and 6-OHDA, and their performance was assessed every other week with different behavioral tests until week 12. At the end, brains were collected and optical density of tyrosine hydroxylase (TH) and dopamine transporter (DAT) was measured from striatal sections, and TH and DAT positive cells in the substantia nigra were counted. The amount of Lewy bodies present in the brain slices was also counted from the cortex and substantia nigra areas of the brain. In the histological assays, statistically significant reductions of both TH and DAT were found in the brain sections of the PFF + 6-OHDA combination group and the amount of TH and DAT positive cells were lower in this group compared to the group receiving vehicle treatment only. However, the results of behavioral tests were non-significant, although a non-statistical positive trend in the amphethamine-induced rotations test was observed where mice receiving PFF + 6-OHDA rotated the most. Taken together, combination model that utilizes both PFF and 6-OHDA injections seems like a promising candidate in modelling PD in mice, but much more research and further development of the model is required before this combination model is ready and robust for use in drug development.
  • Anttila, Emmi (2021)
    Mild traumatic brain injury (TBI) is defined as an injury that disrupts the normal functioning of the brain and is the result of external force to the head. It is the most common type of traumatic head injury, and it is common especially in contact sports and within military personnel. Mild TBI typically causes no clear structural changes to the head, but it can induce persistent clinical symptoms, as well as microscopic pathological changes to the brain that may eventually lead to neurodegeneration and increase the risk for several diseases. Mild TBI is a risk factor for several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and chronic traumatic encephalopathy. The primary objective of this study was to develop a repetitive mild TBI mouse model for future research purposes in the field of head trauma and neurodegeneration. The injury was induced as a closed head injury with an electromagnetic impactor. Literature and pilot experiments were used to define the parameters of the impactor required to induce a brain injury of desired severity. The characterization criteria of the mild TBI model considered the criteria used to define human mild TBI, as well as long term effects often reported after repetitive mild TBI: neurodegeneration as tau protein related pathology, neuroinflammation, and memory deficits. The secondary objective of this study was to tentatively test a prolyl oligopeptidase (PREP) inhibitor on the behavioral and histological effects of mild TBI. The functioning of the mild TBI model was studied by histopathological and behavioral assessments. After baseline behavioral assessment and repetitive (1 injury every 24 hours altogether 5 times) mild TBI inductions, the mice were monitored for approximately 3 months, during which several rounds of behavioral tests were performed. Barnes maze and novel object recognition tests were used to assess memory functions, and locomotor activity test was used to assess general locomotor activity. After euthanasia, brain histopathology was performed to study the amount of tau protein and the level of neuroinflammation. Due to the low number of animals in the study, the results are directional and need to be confirmed in subsequent studies. The histopathology showed greater amount of neuroinflammation and tau protein in the brains of injured mice, but statistical evaluations could not be made. Memory functions were slightly worse in the injured mice compared to controls, but significance of the results is unclear. Locomotor activity was not influenced by the mild TBIs. PREP inhibition treatment increased the locomotor activity of the mice, but the significance is unclear. The mild TBI model seems promising and the characterization criteria were partially met. The results of the study need to be verified in subsequent studies with a greater amount of animals. The model developed here can be used to study the involvement of head trauma in neurodegeneration, as well as treatment alternatives to changes caused by mild TBIs. As there currently are no curative treatments to neurodegenerative diseases, research regarding neurodegeneration and its risk factors is highly important.
  • Katajamäki, Jani (2021)
    Cytochrome P450 (CYP) enzyme inhibition is one of the most common reasons for adverse drug-drug interactions. An especially harmful form of inhibition is time-dependent inhibition (TDI) in which the inhibition potency increases over time and persists even after discontinuation of the drug. Both direct and time-dependent inhibition can be efficiently screened with the so-called cocktail method containing several CYP-selective probe substrates in a single reaction mixture. This method is practical especially in ADME studies of drug development, as it offers lower costs, consumption of fewer reagents and faster implementation in comparison to conventional methods. In addition, the cocktail method can be used to establish new diagnostic CYP inhibitors in vitro. The aim of this Master’s thesis was to participate in the development and optimization of a new cocktail assay method. The method was developed for screening of major drug-metabolizing CYP enzymes in vitro both in a direct and time-dependent manner using pooled human liver microsomes. Based on preliminary testing, included probe substrates were divided into two cocktails to avoid significant inter-substrate interactions: cocktail I containing tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4, and cocktail II containing coumarin/CYP2A6, (S)-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2. First, cocktail incubation conditions were optimized, followed by the determination of probe reaction kinetics, kinetic parameters (Km, Vmax) and inter-substrate interactions with single- or dual-substrate incubations. Finally, suitable probe substrate concentrations and the composition of cocktails was evaluated based on the obtained results. As a result of assay optimization, optimal incubation conditions for yet unoptimized cocktail II were established. In optimized incubation conditions, all probe reactions exhibited saturable Michaelis-Menten kinetics except for tacrine 1-hydroxylation (CYP1A2), which exhibited biphasic kinetics instead (Km1: 7.36, Km2: 517). The selected probe substrate concentrations were all below or near their respective Km values except for (S)-mephenytoin 4’-hydroxylation (40 µM vs. Km of 12.5 µM); however, its concentration could not be reduced in order to maintain sufficient metabolite formation for UHPLC-MS/MS-analysis. Dual-substrate incubation assays demonstrated a need for the reduction of bupropion concentration below 100 µM due to its inhibitory effects on CYP2C8 and CYP3A4. In addition, chlorzoxazone/CYP2E1 and testosterone/CYP3A4 were tested as complementary probe substrates for the cocktails; however, they proved to be unsuitable for both cocktails due to significant interactions (>40% inhibition). Prior to the deployment of the method, some adjustments of probe substrate concentrations are still required in addition to consideration of the suitability of less commonly used CYP3A4 and CYP2E1 probe reactions to improve cocktail coverage. Lastly, validation of the method with known time-dependent model inhibitors should also be conducted. Besides to improvement of the cocktails, new information was generated on inter-cocktail probe-probe interactions and enzyme kinetics of probe reactions, especially for the less-studied astemizole O-demethylation (CYP2J2) and tacrine 1-hydroxylation (CYP1A2). Generated information can be used, for example, in the development of new cocktails.
  • Tuominen, Elsi (2021)
    Neurodegenerative diseases and neuronal injury after trauma are common causes of neuronal loss. Adult brain has only a limited regenerative capability to replace the lost neurons caused by several distinct brain diseases. Direct reprogramming of brain resident cells into neurons could provide a promising strategy for efficiently replacing non-functional neurons. To date, the focus has been put largely on astrocyte-to-neuron reprogramming despite the relatively low yield of newly generated neurons reported in vivo. According to our hypothesis oligodendrocytes possess a more diverge transcriptomic profile when compared to neurons and astrocytes thus allowing better cell-specific targeting of reprogramming. Here, we establish the molecular tools for direct neuronal reprogramming of human oligodendrocytes to neurons. We investigate whether the expression of a known neural fate specification factor under selected oligodendrocyte-specific promoters is sufficient to induce oligodendrocyte-to-neuron transformation. Furthermore, we test the established tools in vitro using an immortalized human oligodendrocyte cell line. Our preliminary data shows that the human ERBB3 promoter and a single transcription factor transfected cells express doublecortin (DCX), an early marker of neuronal identity. Only recently, the direct in vitro reprogramming of human oligodendrocyte precursor cells into functional neurons has been reported. The direct reprogramming of oligodendrocytes into neurons provides an exciting alternative of neuronal replacement for astrocyte-to-neuron reprogramming. Overall, the field of direct reprogramming offers interesting possibilities for regenerative medicine providing a method for the production of newly generated disease and patient-specific cells.
  • Pihl, Enni-Eveliina (2024)
    Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the nigrostriatal pathway, resulting primarily in motor dysfunctions. The key neuropathological features in PD are intraneuronal inclusions, Lewy bodies (LB), resulting from aggregation and accumulation of misfolded α-synuclein (αSyn). Although it has not yet been undisputedly proven whether the accumulation of αSyn and formation of LBs is the initial cause for the loss of dopaminergic neurons or simply an epiphenomenon, several PD models have been successfully developed by inducing αSyn aggregation. One of the approaches utilizes pre-formed fibrils (PFFs) generated from recombinant αSyn monomers in vitro. The model is based on the discovery that exogenous PFFs induce αSyn aggregation in the affected cells, which eventually leads to formation of LB-like inclusions. In previous studies, neurotrophic factors (NTFs), such as brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and mesencephalic astrocyte-derived neurotrophic factor (MANF) have been shown to reduce the formation of PFF-induced LB-like aggregates. Although the effects of exogenous NTFs have been studied in PFF-based models, the effects of PFFs on the endogenous levels of NTFs has not received much attention. Therefore, the aim of this study was to assess the effects of PFFs as well as cell maturation on the endogenous levels of BDNF, GDNF, and MANF in primary mouse embryonic midbrain dopaminergic cultures. The midbrain dopaminergic neurons isolated from E13.5 mice were cultured for 15 days, during which αSyn aggregation was induced with PFFs on day 8 in vitro (DIV8). Cell lysate samples were collected at three time points: on DIV0 1 hour after plating, DIV8 1 hour after addition of PFFs, and DIV15. The expression levels of BDNF, GDNF, and MANF mRNA were measured with quantitative PCR (qPCR). An unsuccessful attempt to optimize a TRI Reagent™-based RNA extraction protocol was also conducted during the project. The data suggested that the expression of BDNF and MANF is affected by cell maturation regardless of the absence or presence of PFFs. However, further research is needed to confirm the results. Additionally, GDNF was shown to be expressed at extremely low levels in mouse dopaminergic neurons. Lastly, the study revealed that the reference genes Gapdh and Hprt1 commonly used in qPCR are affected by cell maturation and PFFs, thus not suitable for studying PFF-treated mouse midbrain dopaminergic neurons.
  • Kullbäck, Jonas (2024)
    Congenital heart diseases develop during heart development and encompass structural abnormalities in the heart present at birth, with hypoplastic left heart syndrome (HLHS) representing a rare but life-threatening subtype. HLHS is characterised by the underdevelopment of left-sided heart structures, resulting in a major blood flow obstruction of the heart, impairing systemic circulation. Current knowledge of HLHS aetiology is scarce, which makes the development of effective treatments challenging. Therefore, identifying the disease mechanisms causing HLHS is essential. Notably, HLHS is linked with mutations in the NKX2-5 gene, which encodes for a cardiac transcription factor and has a pivotal role in heart development together with the transcription factor GATA4. This makes these genes intriguing research targets in HLHS. This study aims to enlighten how HLHS patient-derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) differ from those derived from healthy donors in terms of stress response by subjecting hiPSC-CMs to pro-hypertrophic stimuli, namely endothelin-1 (ET-1) and cyclic mechanical stretching. Additionally, the effects of GATA4-targeted compounds on these hypertrophy models were also studied, which included two inhibitors (3i-1262 and 3i-1000) and one activator (3i-0777) of GATA4-NKX2-5 interaction. Differentiation of CMs was performed using a small-molecule induction protocol based on sequential Wnt pathway activation and inhibition. The effects of ET-1 and cyclic mechanical stretching were analysed by High-content analysis for pro-B-type natriuretic peptide (proBNP) expression, and quantitative PCR for hypertrophic gene expression, respectively. Both ET-1 and cyclic mechanical stretching effectively induced hypertrophy in their respective models. This was observed in all cell lines as a higher hypertrophic response of proBNP in ET-1 exposed hiPSC-CMs and upregulation of hypertrophic genes NPPA and NPPB in stretched hiPSC-CMs. GATA4-targeted compounds did not show statistically significant effects on ET-1-induced hypertrophy or stretching-induced hypertrophic gene expression in any cell line, but various trends could be distinguished. As expected, both inhibitor compounds, 3i-1262 and 3i-1000, showed a tendency for antihypertrophic effects since they decreased the percentage of proBNP+ cells in all cell lines. Unexpectedly, the activator compound 3i-0777 also decreased the percentage of proBNP+ cells. We also observed that HLHS-disease cell line HEL 149 seemed to differ from the three other cell lines showing a phenotype that exhibits similar gene expression patterns as seen in heart failure patients. This was mainly observed as a statistically significantly lower basal MYH6 gene expression. However, the limited experimental setup of this study requires further experiments to detect significant differences and draw definitive conclusions regarding the effects of GATA4-targeted compounds on hypertrophic stimuli.