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Index: Abstract Results: 108

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A rapidly changing environment requires an efficiently functioning nervous system to receive, process, and respond to stimuli. Consequently, nervous system development is tightly regulated and highly time-sensitive. Disruptions in this process may result in abnormal brain functioning and neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), ADHD, and Tourette syndrome. These disorders are associated with impaired cognition, sociability, and sensorimotor integration. While their precise etiology remains unclear, both genetic and environmental factors, including inflammation, are implicated. Matrix metalloproteinase 9 (MMP-9), a known regulator of inflammation and synaptic plasticity, is elevated in the blood and brains of NDD patients. Most studies on NDDs and inflammation focus on prenatal stages using maternal immune activation models. Since neural development continues postnatally, this dissertation investigates the effects of immune stimulation during the early postnatal period. The focus is on exuberant synaptogenesis occurring around postnatal day 7 (P7) in mice. During this period, pups received a single intraperitoneal injection of lipopolysaccharide (LPS, 0.05 mg/kg) or saline (NaCl) as control. The aim was to assess the long-term behavioral consequences of early immune activation. In the first part, behavioral alterations in adulthood were assessed using tests evaluating activity, anxiety, and sociability (cage observation, elevated plus maze, three-chamber test, and Eco-HAB™). Mice treated with LPS were less active and less anxious. Additionally, sex-specific differences in social behavior were observed. LPS-treated males showed reduced interest in unfamiliar social stimuli but greater preference for group interaction. Conversely, LPS-treated females more actively explored novel social scents and spent less time in familiar social groups. The second part examined molecular changes in P7 mice following LPS administration. Serum levels of eight inflammation-related proteins, including MMP-9, increased, with peak responses at 2 hours post-injection. Elevated MMP-9 activity was observed in the cortex of both sexes and in the hippocampus of males. Six hours post-injection, gene expression of tnf-α, il-6, and mmp-9 increased, particularly in the striatum. The third part explored the role of MMP-9 in mediating LPS-induced behavioral changes. Experiments were conducted in MMP-9 knockout (KO) and wild-type (WT) littermates. Behavioral analysis in adulthood confirmed reduced activity in LPS-treated animals. KO mice also exhibited increased anxiety. Regardless of genotype, LPS-treated males preferred social interaction, whereas females avoided each other. Notably, genotype-specific differences emerged in response to unfamiliar social stimuli: WT males from the LPS group avoided the novel social cue, while WT females showed increased interest. These effects were absent in KO mice. In conclusion, a single LPS injection during postnatal synaptogenesis induces behavioral alterations and may serve as a novel model of NDDs. Furthermore, the study highlights MMP-9 as a critical factor in social behavior, particularly in responses to unfamiliar stimuli. These findings contribute to understanding the molecular basis of NDDs and suggest a potential diagnostic or therapeutic role for MMP-9. <br>
Accumulation of toxic lipid metabolites in cardiomyocytes leads to lipotoxicity, which is associated with activation or inhibition of signaling pathways, causing stress and dysfunction of organelles, energy deficit and apoptosis. Lipotoxicity also decreases cardiac insulin sensitivity, causing a shift in cardiac metabolism toward increased fatty acid (FA) uptake, storage and oxidation. The high rate of FA oxidation increases the production of reactive oxygen species (ROS), causing oxidative stress, mitochondrial damage and disruption of Ca2+ homeostasis. Stearoyl-CoA desaturase (SCD) is a key enzyme involved in the regulation of lipid metabolism in cardiomyocytes. In addition to the formation of monounsaturated fatty acids, this enzyme regulates lipolysis and β-oxidation, thereby affecting functional parameters of the heart. Four isoforms of SCD have been identified in mice, of which SCD4 is considered the cardiac- specific isoform. The loss of SCD4 in the mouse heart has been shown to reduce AMP-activated protein kinase activation induced by myocardial infarction (MI). Lack of Scd4 expression in the heart after MI also reduced ROS formation by decreasing protein levels of the NADPH oxidase subunit. Loss of SCD4 also decreased proangiogenic factors in the heart and plasma, suggesting that SCD4 positively regulates blood vessel formation in the mouse heart after MI. However, the role of SCD4 in regulating cardiac metabolism and function remains poorly understood. The main objective of this dissertation was to determine the effects of SCD4 deficiency on cardiac structure, function and metabolism under physiological conditions and in obesity. To test this: 1) the role of SCD4 in the regulation of systemic metabolism in mice was determined; 2) the effects of SCD4 deficiency on cardiac function and structure were investigated; 3) the metabolic pathways through which SCD4 regulates lipid metabolism in the heart were identified; 4) the effects of Scd4 expression on mitochondrial structure and activity in cardiomyocytes were determined. The study was conducted on wild-type (WT) mice and SCD4-deficient (SCD4-/-) mice kept under normal conditions or fed a high-fat diet (HFD) for 8 weeks to induce obesity. In vitro studies were performed using a mouse HL-1 cardiomyocytes with silenced Scd4 expression. The study showed that loss of SCD4 had systemic effects, reducing body adiposity, hyperinsulinemia and hypercholesterolemia, and increasing insulin sensitivity in HFD-fed mice, despite SCD4 being a cardiac-specific isoform. The absence of SCD4 under normal conditions caused minor changes in heart morphology, reducing left ventricular end-diastolic diameter and volume, but prevented concentric cardiac remodeling under HFD conditions. Scd4 deficiency reduced lipid accumulation in cardiomyocytes, but did not affect the expression of other Scd isoforms in the heart. Lower lipid levels in SCD4-deficient cardiomyocytes were associated with activation of adipose triglyceride lipase and lipolysis. Simultaneous activation of lipogenesis, β-oxidation and lipolysis indicated increased lipid droplet (LD) dynamics, and downregulation of SCD4 inhibited the HFD-induced increase in LDs growth in cardiomyocytes. These results indicate that SCD4 is involved in the regulation of cardiac energy metabolism. Mitochondrial hypertrophy and ROS overproduction caused by lipid accumulation and toxicity were inhibited in cardiomyocytes with silenced Scd4 expression under lipid overload conditions.This was associated with increased mitophagy and decreased NADH dehydrogenase activity. SCD4 was also found to be involved in the regulation of Ca2+ homeostasis in the heart under HFD conditions, through increased levels of phospholamban and sarcoplasmic Ca2+-ATPase type 2.In conclusion, the results of this study showed that SCD4 not only regulates lipid metabolism in the heart,but also affects myocardial function and systemic metabolism,especially in the HFD condition. <br>
Alzheimer's disease (AD) develops silently as a pre-symptomatic stage for many years, and only after 10-20 years clinical symptoms such as memory and cognitive decline appear. There are many hypotheses regarding the causes, sequence of events and molecular mechanisms of AD development, but none of them covers the complexity of the disease etiology, what translates into the lack of effective treatments. In addition, increasing evidence indicates that modifiable environmental factors, such as an unbalanced diet or past infections, play an important role in the development of AD. In the absence of effective treatments, prevention seems to be the best way to reduce the incidence of AD. However, designing an effective preventive strategy requires a holistic understanding of the interrelationships between organs, cells, and signaling pathways linking risk factors with the AD pathology. Therefore, the aim of this study was to verify the hypothesis that an improper diet, such as the Western diet (WD), by inducing metabolic syndrome and systemic inflammation, may accelerate and/or intensify brain inflammation and neuropathological changes characteristic of AD. In the study, transgenic mice expressing the human amyloid precursor protein gene with the Swedish mutation (APPswe) were used. Mice were analyzed in 5 age groups: 4, 8, 12, 16 and 20 months, corresponding to the age ranges in humans: 25, 35, 45, 65, 75 years, respectively. The 4- to 12-month-old mice corresponded to pre-symptomatic stages where the pathological processes leading to the aggregation of Aβ peptides are taking place, while Aβ senile plaques are not yet present. In each age group, 4 experimental subgroups were tested: 1) CTR – control group, mice fed a standard diet, 2) WD – mice fed a Western diet, 3) LPS – mice fed a standard diet, which were treated with intraperitoneal administration of lipopolysaccharide (LPS) for induction of systemic inflammation, and 4) WD+LPS – WD-fed mice given LPS intraperitoneally. This study characterizes for the first time a detailed sequence of systemic and metabolic changes, followed by neuroinflammation and Aβ pathology in the hippocampus and entorhinal cortex induced by WD in APPswe mice. It has been shown that 3 weeks of feeding WD causes hypercholesterolemia and fatty changes in hepatocytes. After 5 months of WD, the induced metabolic changes were joined by obesity and non-alcoholic fatty liver disease, which indicates a developed metabolic syndrome. At the same time, an increase in the concentration of white blood cells was observed, suggesting the presence of low-grade systemic inflammation. The analysis of brain tissue showed that the hippocampus, compared to the entorhinal cortex, is a structure more sensitive to the diet composition. 3 weeks of WD accelerated by 4 months the increase in the level of APP and its C-terminal proteolytic fragments and the activation of astrocytes. After 5 months of WD, the activation of microglial cells was additionally observed to be accelerated by 4 months and its phagocytic functions disturbed, which was most likely the cause of the process of amyloid plaque deposition in the hippocampus accelerated by 8 months. Comparison of WD-induced changes with LPS-induced changes showed that WD induces stronger inflammatory response in the brain and, unlike LPS, is closely related to amyloidogenesis and not pTau(Thr231) protein phosphorylation in APPswe mice. The obtained results indicate that WD can significantly accelerate the pathology of AD, and that AD is a disorder of the whole organism, where peripheral organs may play a key role in its pathogenesis. In particular, WD-induced liver damage may affect the disturbed cholesterol metabolism and lead to impaired amyloid degradation and removal processes, which seems to be one of the main factors accelerating the development of AD. The obtained results may support the development of methods for effective prevention and early treatment of AD. <br>
The amount of information we encounter in our perceptual environment exceeds the capacities of our cognitive system, and thus efficient navigation in everyday situations requires a selective mechanism that prioritizes behaviorally relevant contents. This is the assumed role of the selective attention mechanism. While attention has been extensively studied in simplified, artificial settings, the factors that might drive the deployment of attentional resources in naturalistic settings are not fully understood. In the present thesis, I present the outcomes of research conducted in order to delineate the scope of attentional prioritization of two recognized sources of perceptual saliency – namely semantic congruency and affective relevance. In the first study, we investigated whether objects that violate the semantic structure of the real-world scene automatically engage exogenous attention for longer than semantically congruent objects. The conducted experiment involved a central presentation of a scene and a peripheral presentation of a small target letter. We found that the presentation of semantically incongruent objects did not delay responses to the target identification task, which indicates that such objects did not benefit from automatic attentional engagement. At the same time presentation of disgust-evoking scenes was related to the robust attention-hold effect. The obtained results demonstrate that the affective relevance of the scene induces automatic engagement of exogenous attention, but semantic incongruency does cause a similar effect. In the second study, we tested whether an automatic attentional response to threats can be induced at the preconscious levels of visual processing. In the present experiment, we employed event-related potentials (ERP) to compare neural activity evoked by the subliminal and supraliminal perception of fearful and neutral facial expressions. The obtained pattern of results suggests that consciously perceived fearful faces were preferentially encoded and automatically prioritized by bottom-up attention. Importantly, when perceived outside awareness fearful faces were still preferentially encoded, but we found no evidence for attentional prioritization. Therefore, our findings show that attentional prioritization of threats depends on perceptual consciousness. In the third study, we reanalyzed data collected in the second study in order to investigate the influence of attention on neural correlates of visual awareness. It has been proposed that an early ERP component called Visual Awareness Negativity (VAN) constitutes a neural marker of subjective conscious experience that is independent of attentional selection. Therefore, in the conducted analysis we investigated whether VAN is indeed not affected by exogenous attention associated with the inherent saliency of presented stimuli and endogenous attention induced by task relevance. Our findings revealed that VAN was highly dependent on attentional manipulations in both early (140–200 ms) and late time windows (200–350 ms). Thus, the obtained results challenge the view that VAN constitutes a specific, attention-independent mechanism of subjective conscious experience. Overall, the presented work contributes to a better understanding of how attention operates in naturalistic settings by elucidating the limitations of exogenous attention capture and engagement. Our findings indicate that the perception of real-world images involves the integration of bottom-up and top-down mechanisms that mutually shape the behavioral and neural response. Further, our results reveal the role of conscious evaluation and significantly add to the discussion about the relationship between awareness and attention. <br>
Appropriate response to appetitive stimuli in the environment is a crucial skill for animals, enabling their survival and reproduction. While some psychoactive substances also induce pleasurable sensations, their actions rely on the pharmacological influence on neurons. Hence, two types of appetitive stimuli are distinguished: natural and pharmacological rewards. Pharmacological rewards are associated with addiction, and at the core of substance dependence are changes in the quantity and quality of neuronal connections induced by psychoactive substances, known as plastic changes. Plastic changes also occur physiologically in the brain and are considered as the molecular bases of memorizing. It is suspected that the plastic changes induced by pharmacological rewards occur in brain regions that usually process natural rewards, encoding pathologically persistent memories of the drug. Using two mouse models of reward exposure, I investigated whether information about them is indeed processed by the same brain regions. As natural reward, mice had access to a sweet sucrose solution for two hours daily. As pharmacological reward, mice received intraperitoneal injections of a cocaine saline solution. Animals were exposed to these rewards either once or for seven consecutive days. To investigate brain structures engaged by these rewards, I examined the level of c-Fos (a protein associated with neuronal activity and synaptic plasticity) throughout the entire brain. Utilizing optical tissue clearing and light sheet microscopy, I found that both rewards engaged structures in extensive brain regions. The pattern of increased activity partially overlapped for both rewards, but in the case of many areas it was specific only to exposure to cocaine or sugar. By analyzing the increase of c-Fos level after single vs. multiple exposures of animals to sweet water, I observed that it was higher after a single administration. In the case of cocaine, these effects were opposite, and after seven days of drug administration, over half of brain structures showed altered activity. I also examined the processing of information about sugar and cocaine at the level of individual neurons within the central nucleus of the amygdala (CeA). CeA processes emotionally significant stimuli and consists of two parts: medial (CeM) and lateral (CeL). Using electrophysiological techniques, I investigated plastic changes induced by rewards in CeA. I found that both sugar and cocaine induce plastic changes in CeM. Electrophysiological analysis showed that these plastic changes resulted in enhanced synaptic strength reaching CeM. On the other hand, plastic changes in CeL were observed only in animals exposed to cocaine, and they resulted in a weakening of synaptic strength. I confirmed the involvement of CeA in processing both rewards by studying the behavior of animals with altered CeM activity. With chemogenetic tools, I blocked the motivation of animals to sweet water and delayed the onset of some behavioral effects induced by cocaine. I also discovered in CeA a population of dopamine sensitive neurons with two types of receptors: DRD1 and DRD2. I found that neurons in CeL express only DRD2, while in CeM, they have either DRD1 or DRD2 on their surface. I found that cocaine and sugar differently modulate the activity of these neurons in CeM. Cocaine increases the spontaneous activity of neurons with DRD1 and decreases those with DRD2. Sugar, on the other hand, increases the spontaneous activity of neurons with DRD2 and decreases those with DRD1. In summary, the results of the study indicate that information about sugar and cocaine is processed differently in the brain. The distinct nature of these rewards requires the characterization of individual reward system models. Information about sugar and cocaine is also variably processed by CeA neurons, but a precise understanding of these mechanisms requires consideration of the roles of different neuron populations. <br>
Associative learning involves forming associations between a neutral stimulus and a reinforcer (as in classical conditioning) or between a behavior and the outcome it produces (as in instrumental conditioning). Modern theories suggest that associative learning arises from a prediction error (PE) defined as a discrepancy between expected and actual reinforcement. A positive PE occurs when the actual reinforcement exceeds expectations, strengthening the association, whereas a negative PE happens when the reinforcement is less than anticipated, weakening the association. These prediction errors can further be categorized as either appetitive, relating to rewards, or aversive, associated with punishments. The amygdala is critical for associative learning, particularly in signaling prediction errors. Research on animals indicates that different regions of the amygdala, like the centromedial amygdala (CMA) and basolateral amygdala (BLA), serve distinct functions in associative learning. However, the precise functional roles of these areas in humans remain largely unexplored. Therefore, this study aimed to investigate the specific contributions of the CMA and BLA in the left and right hemispheres of the human brain to the process of associative learning. The fMRI study included both classical learning (experiment I) and instrumental learning (experiment II) in the appetitive and aversive contexts. In both experiments compound reinforcers comprised a gustatory (sweet, salty or tasteless liquid) and a social component (a 3-second video of a person drinking a pleasant, unpleasant, or neutral beverage). In experiment I, the participants (N = 37, 20 females) were tasked with predicting the type of reinforcement based on the cue presented on the screen. In experiment II, the participants (N = 33, 16 females) were asked to independently choose one of two simultaneously presented cues. Their responses were used to compute prediction error values according to the Rescorla- Wagner learning model, and these prediction error values were then applied as a parametric modulator of the BOLD signal. The findings revealed that the CMA in the left hemisphere is involved in signaling negative prediction errors during both appetitive and aversive classical learning, as well as negative and positive prediction errors during appetitive instrumental learning. Furthermore, the CMA in the left hemisphere was that amygdala region whose activity correlated with personality traits, such as extraversion and neuroticism, and with BMI. In the right hemisphere, CMA activity was observed in relation to negative prediction errors during appetitive classical learning. Additionally, the BLA in the left hemisphere showed activity specifically linked to positive prediction errors during appetitive instrumental learning. The findings suggest that the CMA and BLA regions in the left and right hemispheres of the brain are engaged differently in associative learning tasks involving gustatory-social reinforcements. The CMA in the left hemisphere appears to play a pivotal and universal role, likely related to adjusting the association strength between cues and reinforcers or between actions and reinforcers when the value of the reinforcement shifts. In contrast, the roles of the right CMA and the left BLA are more specific, with the right CMA primarily involved in processing negative prediction errors during aversive classical learning, and the left BLA being responsible for encoding positive prediction errors during appetitive instrumental learning. These findings not only shed light on the functional organization of the amygdala in processing prediction errors in healthy individuals but also provide valuable insights into the neural mechanisms underlying conditions like obesity and eating disorders, which are characterized by impaired learning from prediction errors in contexts involving food and social cues. <br>
Astrocytes, a type of glial cells, are essential for proper brain function, and disruptions in their homeostasis are a hallmark of numerous pathological conditions. Human astrocytes exhibit unique features compared to their mouse and primate counterparts, which are believed to be driven by alterations in the DNA sequences of regulatory regions, particularly enhancers, which increase gene expression. The precise functional contribution of individual evolutionary changes in regulatory elements active in astrocytes, however, remains unknown. The main objective of my doctoral research was to gain a deeper understanding of the role of astrocytes in shaping the characteristics of the human brain and to determine whether, and how, evolutionary changes in enhancers may have contributed to uniquely human gene expression in astrocytes and, consequently, to the emergence of traits of the human mind during evolution. In this work, I used astrocyte models generated in vitro through the differentiation of induced pluripotent stem cells (iPSCs) derived from humans, chimpanzees (Pan troglodytes), and rhesus macaques (Macaca mulatta). The resulting in vitro astrocyte model (so-called iAstrocytes) exhibited a transcriptional profile corresponding to fetal astrocytes. Based on this model, I characterized the transcriptome, regulome, and chromatin conformation using omics techniques including RNA-seq, ATAC-seq, ChIP-seq, and Intact Hi-C. I demonstrated that sequence variants associated with brain morphology are significantly enriched in genomic regions that bear the signature of active enhancers. Moreover, I identified enhancers active in human astrocytes whose sequences undergo exceptionally rapid evolutionary changes compared to other primates. I also showed that human-specific enhancers frequently contain DNA variants that distinguish modern humans from archaic hominins. These results support broader conclusions regarding the mechanisms underlying brain evolution and human cognitive abilities. In the subsequent part of the dissertation, to identify functional regulatory elements, I employed a high-throughput reporter assay (MPRA, Massively Parallel Reporter Assay) to measure the activity of 10,241 unique DNA sequences that represent potential enhancers in primate iAstrocytes. Using MPRA, I assessed the effects of mutations within sequences recognized by selected transcription factors, as well as the contributions of enhancer variants associated with human traits and diseases. In particular, I demonstrated that the presence of binding sites for USF (Universal Stripe Factors) transcription factors is essential for the activity of enhancers located near genes with higher expression in human iAstrocytes than in chimpanzee and macaque iAstrocytes. Furthermore, I showed that USF binding sites underwent recent evolutionary changes and described associations between these evolutionary modifications in the regulome and disease- linked genetic variants. In summary, the studies presented in this dissertation deepen our understanding of the role of astrocytes in the development and evolution of the human brain and provide new insights into gene regulatory mechanisms that may have contributed to the emergence of uniquely human traits. <br>
ATB0,+ is a membrane amino acid transporter, encoded by the SLC6A14 gene, with broad substrate specificity, transporting all neutral (index „0”) and basic (index „+”) amino acids in a sodium and chloride dependent way. ATB0,+ (SLC6A14) level is upregulated in many cancers, including breast cancer, in which its activity is important for growth and unlimited proliferation of transformed cells. SLC6A14 is an ideal candidate for targeting cancer therapies, as it also transports many drug precursors and amino acid-derived drugs such as valacyclovir and valgancyclovir. Previous analysis of the SLC6A14 proteome detected, among others, several heat shock proteins, including HSP70 (HSPA14) and HSP90-beta. This dissertation focuses on verifying the interaction between transporter and HSP proteins in the process of SLC6A14 exit from the endoplasmic reticulum. Interaction of the overexpressed transporter with heat shock proteins was confirmed in immunoprecipitation and immunofluorescence experiments, while direct interaction was shown in proximity ligation assay. Cell surface localization of the transporter was confirmed using biotinylation of cell surface proteins. Treatment with HSP90 and HSP70 inhibitors - radicicol or VER155008 respectively resulted in a dramatic decrease of SLC6A14 presence in the plasma membrane, an effect of the total transporter diminution. Possibility of interaction between SLC6A14 and HSP90 was tested by measuring ATPase activity of purified, recombinant HSP90-beta protein in an in vitro system. HSP activity was measured in the presence of particular peptides, representing fragments selected from the N- and C-terminus of the transporter, and it was shown that only two peptides, corresponding to C-terminal fragments located closer to the 12th transmembrane domain of SLC6A14, affected the activity although in opposite manner. The results indicate that the C- terminal fragment, which is essential for the exit of SLC6A14 from the endoplasmic reticulum via SEC24C binding, is also involved in altering the activity of HSP90-beta, suggesting that the same transporter fragment binds both HSP90-beta and SEC24C and is responsible for the proper folding of SLC6A14 and its further transport to the plasma membrane. Since high expression of both SLC6A14 and HSP90-beta has been observed in various types of cancer, we also decided to test whether HSP90-beta could act as a regulator of endogenous SLC6A14 activity in breast cancer cell lines. The study was performed on estrogen receptor-positive breast cancer cell lines and a control epithelial one. The direct interaction between SLC6A14 and HSP90-beta was confirmed by proximity ligation assay in all selected lines, but at a significantly higher level in the cancer lines, when compared to the control. The uptake of radioactive leucine, a high-affinity substrate of SLC6A14, was significantly reduced after treatment with the HSP90-beta inhibitor, in breast cancer cells with estrogen receptor (ER-α+) present. In the non-tumorigenic MCF 10A cell line, there was no effect of the HSP90 inhibitor on leucine accumulation, in the presence of chlorine ions, and leucine accumulation was much lower than in breast cancer lines. However, radicicol decreased interaction between SLC6A14 and HSP90-beta. This suggest that SLC6A14 synthesis is lower in non-tumorigenic cells, being much higher in cancer cells and this points to the involvement of HSP90-beta in regulation of SLC6A14 activity. Moreover, a cell viability assay revealed increased cell death after treatment with inhibitors: α-methyltryptophan (SLC6A14) and radicicol (HSP90), when given separately and the combination of these inhibitors significantly increased this effect. The observation that α-methyltryptophan and radicicol at low concentrations cause a synergistic cytotoxic effect in malignant breast cancer cell lines appears to be a potential strategy for combination therapy. <br>
The ATRX (alpha thalassemia mental retardation syndrome X-linked) protein is an important protein that maintains chromatin structure with helicase properties. Its presence appears to be particularly important in neural tissue, as mutations in the Atrx gene lead to alpha thalassemia mental retardation syndrome X-linked (ATRX) characterized by, among other things, mental retardation. Despite these observations, the function of the ATRX protein in neurons is still poorly understood. Literature data suggest that the main function of this protein is participation in chromatin remodeling and DNA sequence silencing. However, there are also reports indicating its role in the activation of gene expression, demonstrating its important but ambiguous role. My preliminary studies on the occurrence of ATRX protein in hippocampal neurons confirmed its localization in condensed DNA regions. Therefore, the purpose of this dissertation was to determine the localization and role of the ATRX protein in the cell nucleus of neurons in the resting state and after stimulation. I pursued this goal using primary culture of hippocampal neurons subjected to a procedure for chemically inducing long term potentiation (cLTP). Analysis of the ATRX protein localization in the nucleus of neurons showed that in the resting state ATRX localizes at chromatin, colocalizing with chromocenters, while after cLTP evoking it accompanies changes in chromatin organization by surrounding chromatin clusters or colocalizing with them as well as with euchromatin markers, indicating an ambiguous role for ATRX in neurons. In contrast, silencing of Atrx in neurons leads to changes in chromatin organization manifested by increased chromatin condensation, accompanied by changes in the pattern of post-translational modifications of histones. In addition, I identified a new nucleolar structure composed of the ATRX protein, which is required for its maintenance. I also showed that silencing of Atrx results in functional changes in neurons, as reflected by changes in the morphology of dendritic trees, which become smaller and less spread out. In conclusion, my results indicate that ATRX protein plays an important role in neurons in the organization of chromatin and nucleolar structures, and the possible relationship between chromatin structure and cell morphology requires further research. <br>
The average life expectancy of the human population continues to rise, resulting in aging societies. However, this demographic shift brings a concerning increase in the prevalence of age-related diseases, particularly cardiovascular diseases. According to the World Health Organization (WHO), cardiovascular diseases remain the primary cause of death globally. The most common include hypertension, heart attack, aortic aneurysm, and stroke. At the core of these conditions lies development of atherosclerosis – an inflammatory disease of the arteries that leads to the narrowing of vessel lumen through the formation of stable or unstable (posing greater health risk) atherosclerotic plaque (AP). Multiple studies have identified vascular smooth muscle cells (VSMCs) as one of the critical cell types essential for proper vascular function and AP stability. VSMCs derived from atherosclerotic plaques, exhibit numerous characteristics associated with cellular senescence. Cellular senescence is defined as an irreversible cell cycle arrest with preservation of full, albeit altered, metabolic functions. It can arise due to either exhaustion of replication potential (replicative senescence - RS) or exposure to various stressors (premature senescence - PS). Regardless of the trigger, both types of cellular senescence cause several morphological changes, particularly at the nuclear and gene expression level. These changes involve gradual loss of compact heterochromatin, and the formation of relaxed euchromatin. Several factors contribute to this transformation, including loss of histones, imbalances in the post-translational modifications of histones, disruptions in histone-remodeling enzymes, and alterations in proteins that stabilize chromatin structure. One such protein is HP1α (Heterochromatin Protein 1 subunit α), which, by attaching to trimethylated lysine 9 of histone 3 (H3K9me3), causes chromatin condensation, stabilization and gene silencing. Preliminary studies conducted at the Laboratory of Molecular Basis of Aging revealed a significant decrease in both H3K9me3 and HP1α protein levels during senescence and reorganization of HP1α, which forms pronounced clusters. Therefore, the main objective of this dissertation was to analyze changes in selected histone H3 modifications and their impact on chromatin structure and gene expression in senescent VSMC, fibroblasts and cells derived from atherosclerotic plaque. The second goal was to clarify the involvement of HP1α in the process. VSMCs were subjected to both replicative and premature senescence, where premature senescence was induced by doxorubicin and curcumin. This experimental framework was extended to fibroblasts, while smooth muscle cells isolated from atherosclerotic plaques were analyzed from at least six patients. The comprehensive analysis of nucleus and chromatin structure in senescent VSMCs revealed significant changes, differentiating RS from PS. One of the differentiating parameters, nuclear surface area size specific to VSMCs, helped to identify senescent cells in a population derived from atherosclerotic plaque. Furthermore, it has been demonstrated that the decline in H3K4me3, H3K9me3, H3K27me3, and H3K9Ac modifications is a universal hallmark of senescence across tested types of cells, although the level of decrease is different in PS and RS. The level of tested modifications in the ex vivo model was heavily dependent on the donor. The decrease and reorganization of HP1α in senescence prevented interaction with H3K9me3, probably due to accumulation of HP1α in PML bodies. Based on ChIP-seq data, the characteristic sites of H3K9me3 and HP1α interactions in young VSMC were selected. In addition, it was shown that VSMCs assume different distribution of H3K4me3 and H3K9me3 in the genome that depends on the type of senescence. <br>
AXL is a receptor tyrosine kinase (RTK) which together with TYRO3 and MER constitutes the TAM receptor subfamily. TAMs participate in the regulation of the immune system, phagocytic clearance of apoptotic cells and tumorigenesis. AXL and its ligand GAS6 were shown to be overexpressed in many types of human cancers, which correlated with increased tumor progression, metastasis and acquired resistance to anti-cancer therapies. In addition, AXL acts as an important receptor for the cellular entry of viruses, including ZIKA and SARS-CoV-2. Therefore, AXL is a promising therapeutic target, both for cancer treatment and anti-viral therapy, and one of its inhibitors is currently being tested in clinical trials for the treatment of cancer and COVID-19.Endocytosis facilitates uptake of fragments of the plasma membrane (PM) together with the extracellular content via endosomes. This process plays a crucial role in the regulation of RTK functions, since it may lead to degradation of RTKs in lysosomes or their recycling to the PM, which terminates or sustains RTK-mediated signaling, respectively.Despite numerous studies reporting the involvement of AXL in carcinogenesis as well as virus infections, the molecular mechanisms underlying these processes have been poorly characterized and AXL-binding proteins remained practically unknown. Additionally, none of TAM receptors have been studied so far with respect to their endocytosis. Thus, the aim of this thesis was the identification of AXL-interacting partners and the characterization of AXL endocytosis.To discover the interactome of AXL, the proximity-dependent biotin identification (BioID) was used. Its results showed that AXL interacted with proteins implicated in actin- related processes, axonogenesis, cell junction organization, signaling and endocytosis. The latter category indicated that intracellular trafficking is an important regulator of AXL function. Therefore, the mechanisms of AXL internalization have been examined in detail. It was demonstrated that, upon GAS6 stimulation, GAS6-AXL complexes were rapidly internalized into cells, and this uptake operated via multiple endocytic routes, both clathrin-mediated (CME) and clathrin-independent endocytosis (CIE). Interestingly, blocking a single endocytic route, except for clathrin-independent carriers/GPI-AP-enriched compartments (CLIC/GEEC) and ARF6-dependent endocytosis, was not sufficient to reduce endocytosis of GAS6-AXL complexes. In contrast, the inhibition of AXL kinase activity completely blocked internalization of the ligated receptor. These findings offer a mechanistic explanation for previous studies showing that AXL inhibitor treatment decreases AXL-mediated viral infections. They further provide a rationale for using pharmacological inhibition of AXL in anti-viral therapies.Subsequent analyses concerning the kinetics of AXL internalization revealed that this process operated faster than the uptake of other RTKs, such as EGFR and PDGFRβ. Moreover, in contrast to ligated EGFR, endocytosis of AXL did not lead to receptor degradation but most probably to its recycling back to the PM. The latter was associated with the prolonged phosphorylation of AXL and the sustained activation of its downstream effector AKT, which may contribute to AXL-driven cancer cell migration and invasion. Finally, the presented results revealed that depletion of AXL was sufficient to block GAS6 internalization, which supports a notion previously reported by our laboratory that AXL is a primary receptor for GAS6.Altogether, this study provides the first comprehensive analysis of the AXL interactome as well as a detailed characterization of endocytosis of AXL, the first TAM receptor studied in this respect. The results presented here shed light on the molecular mechanisms regulating AXL and AXL-mediated processes on the cellular level that significantly extends our current understanding of the role of AXL in cancer progression and viral entry
Beetles (Cassidinae) - appearance description
Beetles (Cassidinae) - location of findings - tropical America
Beetles (Cassidinae) - new specimens
The bidirectional communication between the neuronal connection network and astrocytic protrusions initiated a new approach to the previously neurocentric view of the regulation mechanisms of synaptic transmission. In the 1990s, the concept of a ‘tripartite synapse’ was defined in response to accumulated evidence of active contact between neural and glial cells, based on the idea that neurotransmitters, by activating metabotropic receptors on the surface of astrocytes, lead to an increase in the intracellular Ca2+ ion concentration in their cytoplasm and the release of gliotransmitters. To date, it is not entirely clear what the dynamics and the specific mechanism of Ca2+ ion transmission in astrocytes is and whether it leads to the release of gliotransmitters trough regulated exocytosis. Additionally, little is known about the functional diversity of astrocytes in different brain areas, particularly in the context of gliotransmission. In this study, mixed neuronal-glial cultures and total internal reflection fluorescence microscopy (TIRF) were used to investigate the mechanisms regulating exocytosis in astrocytes. It was shown that the presence of neurons in the culture significantly reduces the level of spontaneous exocytosis in astrocytes. Furthermore, neuronal activity was found to regulate the frequency of exocytosis in astrocytes, a process that largely depends on extracellular Ca2+ ions. It was also established that, unlike in hippocampal cultures, the exocytosis process induced by electrical stimulation is not inhibited in cortical cultures by the presence of TTX. This correlates with a reduced level of mRNA for the Unc13c gene in astrocytes from cortical cultures compared to those from hippocampal cultures, as investigated using fluorescent in situ hybridization (FISH). In summary, the findings presented in this dissertation describe certain mechanisms of exocytosis regulation in astrocytes, which require further investigation. <br>
Birds - Oriolus Consobrinus Rams
Both stroke and glioblastoma are brain diseases that affect enormous numbers of people around the world. The development of innovative techniques for in-vivo imaging of brain pathological changes may significantly accelerate the process of searching for new therapeutic agents. Such a technique is Optical Coherence Tomography (OCT).OCT is a non-invasive, non-contact, interferometric imaging method based on detection of backscattered light from external and internal structural elements of the examined object. OCT without the need of contrast agents allows for fast, three-dimensional imaging with high resolution of a few microns. The aim of the study was to evaluate the applicability of OCT for imaging of structural and angiographic changes in the brain of mice in models of phototoxic stroke and glioblastoma and an attempt to correlate of OCT signals with changes in the nervous tissue and vessels. Developed prototype OCT system was optimized during subsequent stages of the project and validated for quantifying disease biomarkers. First, I used an OCT system to provide in-vivo imaging of the cerebral cortex through the cranial window 24 hours after a stroke. The cerebral vascular network was visualized with high temporal and spatial resolution before, during and after the phototoxic stroke, in which a single branch of the Middle Cerebral Artery was illuminated with green light. I found that despite reperfusion of the brain's surface arteries 24 hours after the stroke, there was no blood flow in the vessels in the deeper regions of the cortex. Moreover, after 24 hours, the angiographic images in the area of the stroke showed an enhancement of the scattering signal in the area of large vessels.Subsequently, the OCT system was optimized by changing the interferometer and the scanning beam type. This modification increased the stability of the OCT system, which had a positive effect on reproducibility and quality of acquired images. The system was used for long-term (14 days) in-vivo imaging of glioblastoma tumor development in the mouse brain. The method was developed to inject Gl261 glioblastoma cells into the cerebral cortex, which finally was covered with a cranial window. Structural OCT images revealed hyporeflective (dark) tumor region, surrounded by a hyper-reflective (bright) region of normal tissue. Strong angiogenesis has been demonstrated in the area of glioblastoma growth at successive time points, with characteristic irregularly shaped newly formed vessels. Finally, an assessment of angiogenesis in the ischemic area after focal stroke was performed and an attempt was made to correlate the hypo- and hyper-reflective areas with changes in the nervous tissue and vessels visualized histologically. The region of the cortex with limited blood flow was clearly visible on angiographic images as a dark area devoid of blood vessels. During the next 14 days, blood vessels appeared in this area due to angiogenesis and reperfusion. During the first 7 days after the stroke, angiographic images revealed vessels mainly in the surface layer, and on day 14 also in the deeper layers of the cortex. On the third day after the stroke, structural OCT images showed a hyporeflective area in the ischemic core, the area of which was reduced by 70% on day 14. This area correlated with the area with microglia/macrophages presence. In some mice, the hyporeflective area was surrounded by a hyperreflective halo that correlated with the presence of activated astrocytes.This study demonstrated that the prototype multifunctional OCT system is a good tool for stroke induction and imaging of changes in the brain after stroke. The analysis of scattering signals identified in the ischemia area by OCT and their histological verification allowed for their correlation with changes at the cellular level. It has been shown that the OCT technique can be used to assess the growth of a mouse brain tumor in-vivo and to observe angiogenesis in its environment
Cellular mechanisms associated with memory consolidation, extinction, and its impairment have been the subject of scientific research for a long time. This is particularly important due to their clinical significance in patients with emotional disorders such as post-traumatic stress disorder (PTSD), anxiety, and phobias. Understanding the neurological correlates of these processes is crucial for a better understanding of the enduring nature of fear memory and the development of new therapies for anxiety disorders in humans. Most studies on fear memory extinction mechanisms focus on recent memory (from a few hours to several days after fear conditioning). At the same time, few studies have focused on late memory (e.g., several weeks after conditioning), although it is certainly more important in the context of long-term emotional disorders. Hence, the neuronal basis of remote fear memory extinction remains mostly unknown. In this dissertation, I present the results of studies on selective impairment of late context-dependent fear memory extinction in mice with impaired autophosphorylation of the alpha isoform of calcium/calmodulin-dependent protein kinase II (αCaMKII) (T286A+/-). To determine the brain regions involved in this process, the expression of the c-Fos protein, which serves as an indicator of neuroplasticity, was examined in 23 brain regions of the mice. Brain regions showing distinct differences in activation during remote fear memory extinction in T286A+/- mice compared to wild-type (WT) mice were then subjected to chemogenetic inhibition using the Designer Receptors Exclusively Activated by Designer Drugs (DREADD) system. Additionally, similar manipulations were performed at the level of neuronal projections between the nucleus reuniens (RE) and the medial septum (MS). The obtained data demonstrate that reduced autophosphorylation of αCaMKII in T286A+/- mice impairs late, but not early, fear memory extinction. The c-Fos expression pattern in the brain of these mice during extinction differs from the pattern in WT mice, suggesting differences in the processes of acquisition and consolidation of remote fear memory extinction. Specifically, after late memory extinction training, hyperactivity was observed in the RE, central-medial (CM) and medio-dorsal (MD) thalamic nuclei and the primary visual cortex (V1) in T286A+/- mice. Furthermore, I observed that remote fear memory extinction depends on the activity of the MS and RE. Chemogenetic inhibition of these structures impairs remote fear memory extinction. Interestingly, inhibiting the RE during recent memory extinction accelerates extinction, revealing the complex role of this brain region in the processes of acquisition and consolidation of fear memory extinction. In contrast, inhibiting the MS at the same time point does not affect the extinction process. Additionally, selective inhibition of glutamatergic neurons in the RE using viral vectors encoding DREADD under the αCaMKII promoter affects fear extinction during the session but has no impact on the consolidation of extinction memory, regardless of whether it is recent or remote memory. Moreover, I demonstrated that chemogenetic inhibition of the RE→MS projection impairs late, but not recent fear memory extinction. In summary, the experiments I conducted revealed the involvement of αCaMKII in the regulation of thalamic activity during long-term consolidation of fear memory. Additionally, I demonstrated the involvement of the RE and MS, as well as the RE→MS projection, in the regulation of remote fear memory extinction. <br>
Cellular response to hypoxia is regulated by hypoxia-inducible transcription factors called HIFs. Those transcription factors are heterodimers made of two HIF subunits: constitutively expressed beta subunit (HIF1B) and oxygen-dependent alpha subunits, of which there are three major isoforms: HIF1A encoded by HIF1A, HIF2A encoded by the EPAS1, and HIF3A encoded by HIF3A. HIF1A is responsible for the acute response to hypoxia, whereas HIF2A and HIF3A are responsible for the adaptation to the long-term hypoxia. During oxygen homeostasis, the concentration of the alpha subunits is low, due to their oxygen-dependent degradation. During hypoxia, this degradation process is interrupted, which leads to the accumulation of alpha subunits, their translocation to the nucleus, where they dimerize with HIF1B to form transcriptionally active complexes. Active HIF complexes bind to hypoxia-response elements (HREs) in target-gene promoters to regulate their response to hypoxia. HIF1 and HIF2 regulate the adaptation of vascular endothelial cells to low oxygen conditions, by activating signalling pathways and genes, which are responsible for endothelial cells migration, growth, differentiation and metabolism. In this dissertation, I characterised two previously described HRE motifs annotated to HIF1 and HIF2, by identifying their instances in the open chromatin regions in promoters of hypoxia-resposive genes, their association with the timepoint of gene activation under hypoxia, and their spatial distribution in the promoters of hypoxia-responsive genes. These results confirmed that the two HRE motifs do have some specificity for HIF1 and HIF2. We investigated the effects of silencing of either HIF1A or HIF2A in Human Umbilical Vein Endothelial Cells (HUVECs) on the expression of 14 pre-selected hypoxia-responsive genes. Among these genes, we identified genes that in HUVECs are regulated by HIF1 (ANKRD37, NARF, BNIP3, SLC2A1), by HIF2 (ADM, ANGPTL4, C1orf21, MAGI1, PTGIS), and by both HIF1 and HIF2 (EGLN3, LUCAT1, MIR210HG, BNIP3L), in the time-window when both HIF1 and HIF2 are active. I demonstrated a linear proportionality between the effect of HIF1 on gene activation and the count of HRE motifs annotated to HIF1 in promoter open chromatin regions. I corroborated this result by genome-wide analysis of HRE motif content in normoxic HUVECs open chromatin regions and HIF1A binding in these cells under hypoxia. This allowed us to propose a mechanism, by which higher content of HRE motifs annotated to HIF1 in open chromatin regions increases HIF1 binding, which contributes to increased gene induction due to HIF1 under hypoxia. I also report that for 232 previously identified hypoxia-responsive genes, the genes which have in their promoter regions ChIP-seq peaks for HIF1A contain more HRE motifs annotated to HIF1A, than genes which do not contain said ChIP-seq peaks in their promoter regions. I developed an ordinary differential equations (ODE) model of hypoxia signalling and transcriptional activation of hypoxia responsive genes that takes into account not only HIF1 but also HIF2. Within this model, I was able to correctly simulate the effects of a further drop of oxygen level during hypoxia on the HIF switch. These simulations results support experimentally established conclusion that residual PHD activity under hypoxia contributes to the HIF-switch. Furthermore, by simulations in the model I established that, for the simulation results to broadly agree with experiments, there is a need for a large excess of HIF1B over the two HIF alpha subunits. However, our model including both HIFs was not better than model including only HIF1 in predicting mRNA expression of hypoxia responsive genes. The results described in this dissertation illustrate the relationship between the type and number of HRE motifs in open chromatin regions in the promoters of hypoxia responsive genes and their transcriptional activation by HIF1 and HIF2. <br>
Cellular senescence is a process that significantly impacts the functioning of the whole organism. It is characterized by a stable and irreversible cell cycle arrest, while maintaining its metabolic activity. During this multi-stage process, a number of changes occur within the cell, which leads to the senescent cell achieving a specific phenotype. Along with new research on cellular senescence, awareness of the complexity and diversity of this process, which depends e.g. on the cell type or the inducing factor, is growing. One of the most important features of senescent cells that have the greatest physiological significance is senescence-associated secretory phenotype (SASP). Recently published data suggest that extracellular vesicles (EVs) may play an important and so far little-understood role in the complex and diverse functions of SASP. It has been proven that SASP may contribute to chronic inflammation, which promotes age-related diseases, such as atherosclerosis. The studies published so far have demonstrated the presence of senescent vascular smooth muscle cells (VSMC) within the atherosclerotic plaque, where they play an important role in its development. Moreover, SASP factors secreted by senescent cells can influence neighboring cells, including T lymphocytes, and modify the tissue microenvironment, thereby contributing to the promotion of inflammation. In the first part of this dissertation, I characterized and compared the premature senescence phenotype in three different types of normal cells cultured in vitro and induced to senescence by doxorubicin or hydrogen peroxide treatment. Changes in the phenotype of senescent cells were compared with the phenotype of proliferating cells and quiescent cells with temporary inhibited proliferation. I showed that the changes which correlated the most strongly with senescence were a decreased level of nuclear proteins - lamin B1, HMGB1, PARP1 and a decreased level of a protein involved in the regulation of mitosis - cyclin B1. These changes can be considered as universal markers of senescence. Next, I compared the replicative and premature senescence of VSMC at the early and late stages of this process. I have shown that the changes observed at the early stage intensify over the time after the inhibition of proliferation. I observed an increased activity of SA-β-gal, a decreased level of HMGB1 and lamin B1 proteins, an increased level of p16 protein and an increased amount of secreted cytokines. Based on the obtained results, I concluded that the phenotype of cells in the late state of senescence differs from that in the early state and is resemble the phenotype of cells undergoing replicative senescence. I also analyzed selected markers in smooth muscle cells isolated from atherosclerotic plaques as an in vivo model of senescence. I observed a decrease level of proteins that I identified, based on previous analyses, as universal markers of senescence. Moreover, cells undergoing senescence in vivo were characterized by increased secretion of SASP factors, and the secretory profile of smooth muscle cells isolated from atherosclerotic plaques was largely similar to the secretory profile of cells undergoing replicative senescence and premature senescence at a late state of this process. I characterized extracellular vesicles (EVs) secreted by VSMC cells. I have shown that VSMC undergoing replicative and premature senescence secrete significantly more EVs than control one. Based on the proteomic analysis of EVs and soluble factors, I showed differences in the composition of the secretome of control and senescent cells, as well as the secretome of cells undergoing replicative and premature senescence. I examined the influence of factors secreted by senescent VSMC on the activation, proliferation and migration of T lymphocytes and on the secretion of cytokines by these cells. <br>
Chronic and acute myeloid leukemia (CML/AML) constitute cancers that arise in the bone marrow, due to malignant transformation (by oncogenic mutations such as BCR-ABL1, FLT3-ITD and others) of myeloid progenitor cells. As myeloid leukemias develop, they expand outside the bone marrow and engraft other tissues, such as the spleen or blood. Development and expansion of myeloid leukemias has been recently shown to be significantly facilitated by immunosuppression - a state when anti-tumor immunity is attenuated and dysfunctional. Immunosuppression is largely established by suppressive cell subsets of the immune system, such as regulatory T cells (Tregs) - a type of T cells that express transcription factor Foxp3 and perform tolerogenic/suppressive function. Tregs have been shown to be upregulated in blood and bone marrow of patients with myeloid leukemias. However, as this has only recently been described, mechanisms that drive expansion and suppressive activity of Tregs in leukemias remain largely unexplored. This thesis has aimed at dissecting modulation of Tregs by leukemic extracellular vesicles (EVs) - small, lipid bilayer-enclosed structures released outside cells as mediators of intercellular communication. As EVs have been demonstrated to modulate non-immune components of the leukemic bone marrow niche and have been shown to interact with Tregs in solid tumors, they might also constitute drivers of Foxp3+ regulatory T cells in myeloid leukemias. Using ex vivo cultures of murine and human Tregs with EVs released by CML and AML cell lines, leukemic EVs were shown to upregulate suppressive phenotype and activity of Tregs, as well as level of Foxp3. Leukemic EVs also induced Foxp3 expression in non-regulatory, conventional T cells. Leukemic EVs have upregulated phosphorylation of STAT5 and downregulated mTOR-S6 signaling in T cells to promote Treg induction, activity and stability. RNA-sequencing has revealed significant remodeling of Treg transcriptome by leukemic EVs, upregulated expression of tumor Treg genes and several transcription factors engaged in this regulation. Furthermore, 23-color spectral flow cytometry and unsupervised clustering tools have revealed 2 subsets of human effector Tregs (eTreg) expanded by leukemic EVs - CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Mass spectrometric analysis of leukemic EVs' proteome revealed presence of TNF superfamily protein 4-1BBL, which was engaged in modulation of expression of effector molecules (CD30, TNFR2, LAG-3) on Tregs. Finally, in a developed immunocompetent mouse model of CML-like disease, influence of EVs on Tregs and leukemic progression was validated by development of leukemia by Rab27a deficient cells, with attenuated secretion of EVs. Rab27a deficient leukemia has exhibited reduced engraftment in animals, whereas Tregs were less abundant and exhibited a less activated, less suppressive phenotype than in wild type counterparts. Altogether, data presented in this thesis pin-point extracellular vesicles, released by chronic and acute myeloid leukemia cells, as significant modulators of regulatory T cells - their induction, suppressive phenotype, function and effector subsets. In vivo, in a mouse model of leukemia-like disease, Rab27a-mediated secretion of EVs was shown to modulate Tregs and leukemic engraftment. Therefore, EVs and EVs-Tregs interaction may be evaluated as potential therapeutic targets in myeloid neoplasms <br>
Cilia and flagella are evolutionary conserved structures which protrude from the apical surface of the eukaryotic cells in both unicellular organisms and humans. There are two major types of cilia – motile and non-motile cilia (also called primary cilia). In mammals, motile cilia are formed on the apical surface of epithelial cells lining e.g. respiratory tract. Sperm cells possess special type of cilium called flagellum. Lack or defects in motile cilia lead to primary ciliary dyskinesia that involves e.g. chronic airway diseases and defects in fertility. Motile cilia axoneme consists of nine outer microtubule doublets which are accompanied by protein complexes responsible for generation of cilia movement. In the central part of the axoneme localizes structure called central apparatus which comprises two single microtubules surrounded by multiprotein complexes called projections. Up to now, exact protein composition of these protein complexes remains unknown, therefore the function of central apparatus and its role in generation of cilia movement is only partially resolved. One of the hypotheses suggests that the signal needed for cilia beating is initiated in central apparatus. However, it cannot be excluded that functions of central apparatus are regulated by enzymatic proteins. Nevertheless, the available data are only fragmentary. This thesis focuses on so-far uncharacterized central apparatus proteins, potentially being part of ciliary MAP kinases-regulatory pathway. Experiments presented in this thesis were conducted with use of well established, unicellular model organism – Tetrahymena thermophila. Based on analysis of previously obtained ciliomes from mutants that lack C1b projection (SPEF2A-CoDel), three kinases– Mapk3, Map2k7, Nek6, two phosphatases: Pp2c and Dusp5 and kinesin Kif9 which build C2c projection were selected for further research. All of these proteins were downregulated in Tetrahymena mutants lacking C1b projection. Amino acid sequence analysis of studied proteins revealed that all of them are evolutionary conserved. Conducted research allowed to confirm that all examined proteins localize in Tetrahymena cilia and when overexpressed are present in both cilia and cell body. BioID and coimmunoprecipitation experiments showed that Dusp5 not only localizes in close proximity but also forms stable complex with C1b projection proteins in Tetrhymena - that is with Spef2A, Cfap69, Androglobin and Cfap246/Lrguk. BioID and pull-down experiments results showed also that both Dusp5 and kinesin Kif9 directly interact with Mapk3, Map2k7 and Nek6 – that is with all examined kinases. None of them interacted with Pp2c phosphatase. These results allowed to propose a model of interaction network between these proteins. Moreover, functional analysis of Dusp5 and Kif9 was also performed. For this, Tetrahymena knock-out mutants lacking Dusp5 or Kif9 were prepared. Lack of Dusp5 or Kif9 resulted in reduced cell swimming speed. Interestingly, both mutants had slower cilia regeneration rate after experimental deciliation which suggest that both of them can participate in ciliary intraflagellar transport. Additionally, no changes in cell proliferation, phagocytosis or cilia length have been observed. To sum up, conducted experiments allowed to identify and at least partly characterize novel potential regulators of the central apparatus functions. <br>
The cortical neural network consists of excitatory glutamatergic cells and inhibitory GABAergic interneurons. GABAergic cells regulate the flow of information in local neural networks, affecting the excitability of glutamatergic cells, being responsible for filtering the input signal and controlling the information output. In terms of expression of molecular markers, GABAergic neurons form three classes of cells: somatostatin (Int-SOM), parvalbumin (Int-PV) interneurons, and those expressing the 5HT3a ionotropic serotonin receptor. The last class is divided into interneurons containing vasoactive intestinal polypeptide (Int VIP) and cells not containing this protein. Many studies indicate a wide role of various classes of GABAergic interneurons in processes related to learning, memory formation, as well as its coding and expression. However, less attention has been paid to plastic changes induced by learning within different classes of interneurons. The research included in this dissertation was intended to reveal whether a simple form of learning in mice leads to plastic changes in the electrophysiological activity of three types of GABAergic cells in the layer IV of the primary somatosensory (barrel) cortex: Int SOM, Int-PV, and Int-VIP. For this purpose, one group of animals was subjected to a conditioning procedure consisting of the simultaneous application of a conditioned stimulus, tactile stimulation of the row of whiskers, and an unconditioned electric shock to the tail. A second group of mice underwent a pseudoconditioning procedure in which the electrical stimulus was not time-bound to vibrissae stimulation, but delivered randomly. The last group, naïve animals, has not been subjected to any form of manipulation. One day after the last session of procedures, electrophysiological recordings were carried out from single neurons (whole-cell patch-clamp) in brain slices in layer IV of sensory representations (barrels) corresponding to the stimulated rows of vibrissae. Experiments showed an increase in intrinsic excitability of Int-SOM in conditioned animals compared to pseudoconditioned and naïve animals. In contrast, the excitability of Int PV was reduced in pseudoconditioned mice compared to other groups of mice. Excitability of Int VIP, which were characterized by accommodation of discharges, was found to be reduced in pseudoconditioned mice compared to conditioned but not naïve mice. Analyzes of the action potentials’ shapes mainly showed that the increase in the excitability of interneurons is associated with the shortening of the duration of individual action potentials (reduced half width of the potential). On the other hand, a decrease in excitability meant an increase in the duration of the potentials. The obtained results suggest that the observed changes in the excitability of interneurons may be associated with changes in ionic conductivity responsible for the duration of the action potential. Subsequent studies using optogenetic methods showed that conditioning (but not pseudoconditioning) results in enhanced inhibition of adjacent excitatory neurons by Int SOM and Int-PV, but not Int-VIP. These results indicate that changes in the intrinsic excitability of interneurons and changes in synaptic inhibition coming from these interneurons may be divergent. In conclusion, the obtained results show that both associative learning and pseudoconditioning lead to plastic changes in the activity of all classes of GABAergic interneurons studied. In this way, changes in intrinsic excitability can be seen as a common mechanism of plasticity of GABAergic interneurons occurring as a result of various forms of learning - conditioning or pseudoconditioning. The observed modifications of intrinsic excitability may affect synaptic summation, processing of sensory information, control of the precision of excitatory cell discharges, and regulation of the output of signals transmitted to the higher layers of the barrel cortex.
CTCF is a conserved DNA-binding protein that plays a key role in regulating the three- dimensional architecture of the genome. It defines the boundaries of topologically associated domains (TADs) and controls interactions between promoters and enhancers. In mammalian cells, the positions of CTCF-binding sites (CBS) have been shown to remain essentially unchanged during embryonic stem (ES) cell to neural stem (NS) cell differentiation, despite an increase in the number and stability of chromatin loops. Changes in chromatin architecture involving CTCF may influence its role in regulating gene expression. However, it remains unclear whether, and how, this regulatory function changes during development. This doctoral thesis aimed to address this question and assess the functions of CTCF during the differentiation of ES cells into NS cells. To investigate this, a CTCF degron system enabling precise, inducible protein depletion was used, followed by transcriptome and regulome profiling in both cellular states. CTCF loss altered gene expression, but did not directly affect regulatory element activity. Genes downregulated upon CTCF depletion were highly expressed and contained CTCF binding at their promoters, whereas upregulated genes showed low basal expression and lacked promoter-bound CTCF. These findings support a model in which promoter- bound CTCF facilitates transcription by mediating contacts with distal enhancers, while intergenic CTCF acts as an insulator. The experiments outlined in this dissertation revealed that, despite stable DNA binding, CTCF regulates distinct gene sets in ES and NS cells, indicating cell-type- specific functions of this factor in development. This was further validated by genome editing at the Aldh1a3 locus. To unveil what underlies the changes in CTCF functions in development, proteomic analysis was performed independently in the lab. These data revealed enhanced interactions between CTCF and RNA-binding proteins (RBP) in the NS compared to the ES cells. The cells derived in this work help identify long non-coding RNA (lncRNA) Pantr1 as a mediator of the gain of CTCF-RBP interactions upon the ES-to-NS transition. In addition, a visualisation system was developed to monitor selected genomic loci in living cells, which has the potential, in the future, to enable real-time tracking of chromatin loop anchor dynamics. In summary, although the localisation of CTCF binding sites remains stable during differentiation, its transcriptional regulatory function is dynamic and highly context-dependent. <br>
Cytoplasmic aggregation and nuclear depletion of TAR DNA-binding protein 43 (TDP-43) represent key pathological features in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Since TDP-43 is a critical regulator of RNA processing and metabolism, its dysfunction contributes to cellular stress through both loss- and gain-of-function mechanisms. Interestingly, metabolic conditions commonly linked to poor systemic health, including type 2 diabetes mellitus (T2DM), dyslipidemia, and elevated body mass index (BMI), are associated with prolonged survival in ALS. Conversely, high levels of physical activity have been linked to increased ALS risk, suggesting a complex interplay between metabolism and TDP-43- mediated neurodegeneration. This study systematically investigates the impact of TDP-43 dysfunction—via knockdown or M337V mutation—on cellular energy metabolism and metabolic sensing, with a focus on motor neuron vulnerability. Using NSC34 motor neuron-like cells, TDP-43 knockdown induced a hypermetabolic state characterized by increased glycolysis, oxidative phosphorylation, and ATP production, accompanied by persistent activation of AMP-activated protein kinase (AMPK). In contrast, mutant TDP-43 disrupted AMPK regulation primarily under metabolic stress, leading to prolonged AMPK activation during recovery phases. Comparative analyses in BV2 microglia and N2A neuroblastoma cells highlighted distinct cell-specific metabolic responses to TDP-43 perturbation. To explore how systemic metabolic status influences TDP-43–associated metabolic changes, we treated TDP-43–deficient NSC34 motor neuron–like cells with serum from mice subjected to voluntary exercise (VE) or a high-fat diet (HFD), revealing a sex-dependent modulation of those metabolic alterations. Female-derived serum more strongly regulated glycolytic and mitochondrial responses in both motor neurons and microglia. Finally, integration of patient-derived transcriptomic datasets from ALS and FTLD postmortem tissues with NSC34 RNA-seq data identified both common and disease-specific metabolic dysregulation. ALS transcriptomes were enriched for lipid metabolism and insulin signaling pathways, while FTLD transcriptomes showed predominant alterations in RNA processing and translation. Collectively, these findings demonstrate that TDP-43 dysfunction disrupts cellular metabolism in a cell-type- and context-dependent manner, with motor neurons displaying heightened vulnerability. The data further suggest that systemic metabolic states modulate TDP-43-driven metabolic stress, providing insights into potential metabolic targets for therapeutic intervention in ALS and FTLD. <br>
The defining property of mitochondria – generation of mitochondrial membrane potential –interlinks the metabolic and signaling functions of this organelle. Mitochondrial large- conductance calcium-activated potassium channels (mitoBK) execute its fine regulation by allowing the controlled influx of potassium ions into the mitochondrial matrix. This functionendows them with unique properties, resulting in a cytoprotective phenomenon of mitoBKactivation in ischemia-reperfusion injury. The functional and structural interaction of mitoBKchannels with the electron transfer chain, and in particular, its terminal enzyme cytochrome c oxidase (COX), can be one of its molecular mechanisms.To investigate the interaction between the COX and mitoBK channels, different COX-deficientcellular models were employed. Specifically, human astrocytoma cells were depleted ofmitochondrial DNA (mtDNA) by the treatment with 2’,3’-dideoxycytidine. The comparison ofthe protein complexes formed by the mitoBK and COX in the mtDNA-depleted and WTastrocytoma cells identified the interaction of the pore-forming mitoBK subunit with the COX-containing complexes and respirasomes. Furthermore, downregulation of mitoBK-α subunits on both protein and mRNA levels occurred upon mtDNA-induced COX deficiency. Theanalysis of the retrograde signaling pathways induced by the mtDNA depletion in the mtDNA-depleted astrocytoma cells showed activation of the integrated stress response signaling.Human dermal fibroblasts with a mutation in the structural COX subunit – COX8A – were usedas another cellular model with a deficiency in COX. The organization of the electron transportchain was characterized in the COX8A-deficient fibroblasts and HEK293T cells with CRISPR/Cas9 induced mutations in COX8A and ensuing COX deficiency, identifying that theresidual COX was stabilized in the respirasomes. The decrease in the protein amount of mitoBKpore-forming subunit, as well as its protein complexes, was observed.To follow the systemic implications of this coupling, the effect of a gaseous transmitter carbonmonoxide (CO), putatively targeting both COX and mitoBK, was assessed in the patch-clampstudies. While direct application of CO-saturated solution has not exerted significantmodulation of the mitoBK channel activity, patch perfusion with CO-releasing moleculesinduced pleiotropic effects. Perfusion with heme and hemin inhibited mitoBK channels. Thesubsequent application of CO-saturated solution released this inhibition, activating mitoBKchannels in the presence of heme
The depression, according to World Health Organization is a leading cause of disability worldwide, affecting more than 270 million people. This is a multifactorial disease of still unknown pathogenesis. Unfortunately, contemporary therapies using antidepressants are not effective enough, and their therapeutic effect is usually significantly delayed. The latter fact suggests that antidepressants work by affecting long-term brain plasticity, probably associated with changes in gene activity orchestrated by reorganization of the chromatin architecture. One of the best-known pathogenic changes in depression are disturbances of the hypothalamic-pituitary-adrenal (HPA) axis regulating the release of cortisol. Cortisol, under physiological conditions, exerts a pleiotropic effect on various organs, including brain, and prepares us to "fight or flight" from danger. Nevertheless, severe stress causes disturbances in the HPA axis, which results in morphological and behavioral abnormalities. It is believed that such disruption is associated with abnormal glucocorticoid receptor (GR) function. GR regulates the activity of many genes, including the negative-feedback autoregulation of Nr3c1 gene encoding GR. Although structural changes involved in GR autoregulation on DNA level, were studies in proliferating cell, not much is known about its function in cells terminally differentiated, including neurons and astrocytes. Therefore, the presented dissertation is an attempt to investigate the architectural changes induced by GR activation in brains cells. Experiments presented in this thesis have indicated, a close relationship between activity changes of the Nr3c1 gene and its location within the cell nuclei of brain cells, under stress conditions, in three brain structures associated with the pathogenesis of depression. Application of STED super-resolution microscopy confirmed that changes in the location of the Nr3c1 gene result from its binding to active or inactive chromatin and ChIA-PET analysis clearly shown reorganization of the chromatin architecture caused by GR activation. The presented data show for a first time, that GR stimulation in brain cells leads to changes of the chromatin organization not only within this particular gene but also at the global level. And that those changes differ between neurons and astrocytes
Depression is a global medical problem frequently leading to suicides. It is assumed that depressive symptoms occur as a result of aberrant excitatory synaptic plasticity developed after chronic stress. Synaptic plasticity is an ability of neurons to modulate the strength of synaptic connections manifested by structural alterations of dendritic spines on which excitatory synapses are located. Recently discovered activation of the serotonin type 7 receptor (5-HT7R)-dependent signaling pathway leads to aberrant structural and functional synaptic plasticity in the hippocampal neurons in vitro. However, whether this signaling pathway exists in vivo or not and what its role is, it has not yet been investigated.Using a combination of behavioral, biochemical, and imaging methods, it has been demonstrated that the 5-HT7R-dependent signaling pathway is activated in the murine hippocampus and underlies the depressive-like phenotype. Additionally, the results confirmed that the activation of the 5-HT7R-dependent signaling pathway is associated with the structural remodeling of dendritic spines of anhedonic animals. The stress-resilient animals did not display the aforementioned biochemical and structural alterations indicating the specificity of the obtained results. Moreover, the implementation of the chronic unpredictable stress procedure with specific silencing of htr7 gene expression in the CA1 hippocampal subregion enabled to discover that silencing of 5-HT7R in the hippocampus is sufficient to prevent the development of anhedonia.The obtained results point out a crucial role of 5-HT7R in the pathogenesis of depressive-like behavior and the dendritic spine structure as a possible decisive molecular target for promoting stress resilience in future pharmacotherapies.
Despite the growing body of research and the inclusion of Compulsive Sexual Behavior Disorder (CSB, code 6C72) in the International Classification of Diseases (ICD-11), published in the 11th edition in recent years, many of the neural mechanisms underlying the development and persistence of symptoms of compulsive sexual behaviors (CSB) are poorly understood. The Incentive Sensitization Theory of addiction suggests that maladaptive attentional and motivational processes directed toward reward-associated cues, coupled with impaired extinction of these previously learned associations, may drive addictive behaviors through alterations of the brain reward system. The aim of this dissertation was to investigate whether individuals with CSB exhibit altered appetitive associative learning, including both appetitive conditioning and extinction of cue–reward relationships and its underlying neuronal underpinnings. Additionally, it was examined whether these alterations are specific to erotic rewards or extend to other appetitive stimuli (e.g. monetary), whether neurofunctional differences also manifest outside of any task (at rest), and whether structural changes in prefrontal regions accompany these functional abnormalities. To this end, men struggling with CSB (n=33) and healthy control subjects (n=33) underwent neuroimaging examinations whilst performing behavioral tasks, allowing to probe their self-assessed, behavioral and neuronal effects of associative learning. Obtained results show that men struggling with CSB are prone to short-term general conditioning and extinction alterations towards cues of both erotic and monetary rewards. These alterations were present in self-assessment, reaction times, and reactivity of the ventral striatum, anterior orbitofrontal cortex, and dorsal anterior cingulate cortex. Intriguingly, the notion of generalized aberrant associative learning, however, was not supported by task-based functional connectivity analyses, which provided an account of erotic-biased processing of appetitive cues in both conditioning and extinction. No group differences were observed in actual reward processing, in default brain reward system functional connectivity, nor in morphological alterations in the CSB group, suggesting CSB brain reward system alterations are functional and more context-specific. In line with Incentive Sensitization Theory, these findings contribute to the growing evidence that CSB may share certain neurobiological characteristics with addictive disorders, particularly regarding enhanced conditioning, cue-reactivity and disrupted extinction of conditioned responses. As demonstrated in the study presented in this dissertation, analytical approach towards neurobiological underpinnings of CSB drawing on functional connectivity complementing the classically used task-based activity analysis, in tandem with self-reported and behavioral indices, provide a richer view of this complex disorder. <br>
Drug craving is an intense desire or need to engage in specific behaviors related to psychoactive substance use. The phenomenon known as incubation of craving is characterized by a gradual increase in the intensity of craving symptoms over time, which increases the likelihood of relapse triggered by spatial cues associated with substance intake. Previous research has shown that re-exposure to a morphine-paired context after withdrawal elicits increased “50-kHz” ultrasonic vocalizations (USVs) in rats, which are interpreted as markers of positive affective states. This study investigated the role of serotonergic and glutamatergic co-transmission in the amygdala in modulating drug-paired context-induced behavioral responses after withdrawal. The amygdala is a brain structure involved in emotional responses to both aversive and appetitive stimuli. A classical animal model of place conditioning and a DREADD chemogenetic technique (using hM3Dq and/or hM4Di receptors) to manipulate the activity of the serotonergic and glutamatergic neurons in the amygdala, were employed. The impact of these chemogenetic manipulations on the number of ultrasonic vocalizations emitted and the distance travelled by rats during the context response was assessed. Additionally, the effects of these manipulations on levels of monoamines, their metabolites, amino acids, and neuromodulators in selected brain structures were investigated, along with the relationships between the neurochemical systems and their association with behavioral changes. This study demonstrated a significant role of serotonergic-glutamatergic co- transmission in the amygdala in shaping the drug-paired context-induced response. No strong correlation was found between the number of ultrasonic vocalizations and the distance traveled by rats, suggesting that these measures may reflect different aspects of the context response (USV – emotional, while distance – motivational). A significant effect of the chemogenetic modifications on the number of ultrasonic vocalizations in the context response test was observed: a statistically significant reduction in the number of USVs in the "5-HT-" (inhibition of serotonergic signaling) and "Glu+5-HT-" (simultaneous activation of glutamatergic signaling and inhibition of serotonergic signaling) groups and increase in the "Glu+5-HT+" (simultaneous activation of glutamatergic and serotonergic signaling) group. Moreover, the study revealed a complex network of neurochemical associations and correlations. Further analysis revealed a diverse neurochemical basis for the expression of the affective state, confirming that the same behavioral effect can result from the activity of a number of distinct neurotransmitter system networks. This experiment highlighted the complexity of the neurobiological basis of addiction, emphasizing the need for a holistic approach in studying this phenomenon. <br>
Dyslexia is a specific learning disorder characterized by reduced reading fluency, accuracy, and comprehension. It affects approximately 7-12% of the population and is more commonly diagnosed in males than females. While several cognitive and neural factors associated with dyslexia have been identified, the precise causal mechanisms underlying reading difficulties remain unclear. Since reading acquisition relies on integrating auditory and visual stimuli, deficits in low-level multisensory integration may also contribute to dyslexia. Some studies have reported such deficits, but effect sizes varied depending on whether participants were matched for sex. Despite the higher prevalence of dyslexia in males and emerging evidence of sex-based differences in its neural underpinnings, no previous studies have specifically examined sex differences in multisensory integration. Thus, the first aim of this thesis was to address this gap by directly assessing sex-specific effects in low-level multisensory integration in dyslexia. One of the latest causal theories of dyslexia, the neural noise hypothesis, proposes that reading difficulties stem from increased cortical excitability, leading to cognitive impairments in phonological awareness, rapid automatized naming, and multisensory integration in dyslexia. Non-invasive estimates of excitatory/inhibitory (E/I) balance in the brain can be obtained through various electroencephalography (EEG) power spectrum measures, including aperiodic (exponent, offset) and periodic (beta and gamma power) components. To date, no study has tested the neural noise hypothesis of dyslexia by examining EEG E/I balance biomarkers in relation to proposed cognitive deficits. Thus, the second aim of this thesis was to investigate these relationships. Regarding the first aim, a study of 88 adolescents and young adults revealed that only males with dyslexia exhibited deficits in multisensory integration of simple, non-linguistic stimuli, as assessed by a simple reaction time task. At the neural level, both males and females with dyslexia showed smaller differences in responses between multisensory and unisensory conditions in the N1 and N2 components (event-related potentials related to sensory processing) compared to controls. However, in a subsample of 80 participants matched for non-verbal IQ, only males with dyslexia exhibited a smaller difference in neural responses to multisensory versus unisensory conditions in the N1 component of the left hemisphere. These findings provide novel insights into sex-specific cognitive processes related to reading difficulties. Regarding the second aim, results from a sample of 120 participants, analyzed using Bayesian statistics, revealed no evidence of group differences in any EEG E/I balance biomarkers (exponent, offset, beta power) at rest or during a spoken language task. However, a positive indirect relationship between beta power, phonological awareness, and reading speed was found. These findings do not support the prediction that cortical hyperexcitability underlies dyslexia, underscoring the need to explore alternative neural mechanisms associated with reading difficulties. Furthermore, the observed sex-specific effects in multisensory integration highlight the potential for distinct cognitive and neural pathways in males and females with dyslexia, which should be considered in future research frameworks. <br>
Endocytosis is a process of internalizing molecules from the extracellular milieu or the cell surface and delivering them to membrane-bound organelles called endosomes, which facilitate further transport of internalized cargoes. Proteins present on endosomal membranes are recognized by the endosomal sorting complexes required for transport (ESCRT), which consist of ESCRT-0, -I, -II and -III. ESCRT mediate remodeling of the limiting membrane of endosomes and formation of intraluminal vesicles (ILVs) inside endosomes. The content of ILVs can be secreted outside the cell or transported via the endolysosomal pathway to lysosomes for degradation. In addition, lysosomes regulate Ca2+-dependent signaling and constitute platforms to sense nutrient availability.Despite a well-characterized function of ESCRT-I in regulating endosomal size and sorting, its involvement in maintaining lysosomal homeostasis remains poorly investigated. The general aim of this thesis was to characterize the role of ESCRT-I in maintaining lysosomal homeostasis and investigate the consequences of ESCRT-I depletion for lysosomal function and lysosome-related signaling.First, lysosomal morphology was characterized in colorectal cancer cell lines, RKO and DLD-1, upon siRNA-mediated depletion of ESCRT-I components, namely Tsg101 or Vps28. Quantitative microscopic analysis of lysosomal markers revealed that lack of ESCRT-I led to enlargement of lysosomes but did not impair lysosomal integrity, maintenance of acidic pH or content of degradative enzymes. The increased lysosomal size was likely due to an impaired degradation of resident membrane proteins that was observed in cells lacking ESCRT-I. This included MCOLN1, a lysosomal Ca2+ channel, whose lysosomal degradation was studied using a GFP-MCOLN1-expressing reporter cell line.To verify whether the lack of ESCRT-I induced transcriptional responses characteristic for altered lysosomal function, RNA sequencing analysis was performed. It revealed that depletion of ESCRT-I upregulated expression of genes related to autophagy and/or lysosomal biogenesis. Activation of transcription factors from the MiT-TFE family, namely TFEB and TFE3, predicted to be responsible for induced expression of these genes, was confirmed in nuclear fractions of ESCRT-I-depleted cells.Next, a mechanism involved in the activation of MiT-TFE signaling upon ESCRT-I depletion was investigated. Quantitative analysis of microscopic images revealed that in cells lacking ESCRT-I, activation of TFEB and TFE3 required Ca2+-dependent signaling and mTORC1 inhibition, but was not due to calcineurin-dependent dephosphorylation of these transcription factors. Moreover, biochemical analyses indicated that the lack of ESCRT-I inhibited mammalian target of rapamycin complex 1 (mTORC1) kinase activity specific towards TFEB and TFE3 but it did not affect canonical mTORC1 substrates. Therefore, it was verified whether the MiT-TFE activation upon ESCRT-I depletion occurred due to the reduced activity of the Rag GTPase complex, known to control the TFEB- and TFE3-specific lysosomal mTORC1 signaling. Overexpression of constitutive active RagC mutant prevented nuclear translocation of TFEB and TFE3 in Tsg101-depleted cells. Hence, the activation of MiT-TFE factors in cells lacking ESCRT-I occurred due to the inhibition of Rag GTPase–dependent mTORC1 pathway.The results presented in this thesis characterize new roles of ESCRT-I in the turnover of lysosomal membrane proteins and maintaining lysosome-related Rag GTPase-dependent, non-canonical mTORC1 signaling. Lack of ESCRT-I leads to a homeostatic response, involving inhibition of the non-canonical mTORC1 signaling and, as a consequence, activation TFEB and TFE3 factors, in an attempt to counteract lysosomal nutrient starvation
Endothelial dysfunction is the earliest symptom of the cardiovascular system dysfunction observed in obesity. Increased concentration of fatty acids associated with obesity, including the most abundant palmitic acid (PA), may contribute to the development of endothelial dysfunction. Von Willebrand factor (vWF), produced and secreted mainly by endothelial cells, plays an important role in the regulation of coagulation. Increased vWF concentration in the plasma of obese patients is associated with an increased risk of developing cardiovascular diseases caused by increased thrombosis. The aim of this thesis was to investigate the effect of PA on the vWF secretion by HUVEC cells. The experiments were carried out under mild fatty acid stress conditions that were sufficient to increase the amount of ICAM-1 and VCAM-1 adhesion molecules, which is a marker of the pro-inflammatory response of the endothelium, without affecting cell survival. The cells were incubated in the presence of PA at a concentration not exceeding 200 μM for no longer than 48 h. This approach enables the study of adaptive changes in response to a stimulus. It has been shown that under relatively mild conditions vWF secretion is increased after incubation in the presence of PA, as a result of the increased VWF gene expression and stimulated maturation of this protein. Attempts have been made to identify the mechanisms of PA's regulatory influence on vWF maturation and secretion, but no conclusive results have been obtained. The results presented in this study confirmed that PA activates NF-κB, a transcription factor involved in the regulation of the inflammatory response. Moreover, it has been shown that PA affects the content of TLR2, TLR4 and TLR6 receptor proteins. To identify the signaling pathway activated by PA, inhibitors of NF-κB and TLR2 and TLR4 receptors were used, that is cardamonin, C29 and TAK-242, respectively. It has been shown that VWF gene expression increased by PA is the result of NF-κB activation. Moreover, the involvement of the TLR4 receptor in the PA-activated endothelial cells response was confirmed. Additionally, in the presence of a TLR4 inhibitor and PA, which is its ligand, the expression of CD36, TIRAP, TLR6 genes, encoding proteins involved in TLR receptor signal transduction, was increased. In the presence of a TLR2 inhibitor, TLR4 activity was increased. Moreover, TLR2 gene silencing increased TLR4 protein level. TLR4 gene silencing increased TLR2 level, which was observed even when only mRNA level of TLR4 was decreased. This suggests a compensatory effect between TLR2 and TLR4 receptors. To conclude, this thesis shows that PA regulates the vWF secretion from endothelial cells by increased expression of the VWF gene and maturation of this protein. The involvement of the TLR4 receptor and the NF-κB signaling pathway in the cell response to PA was identified. Moreover, it has been shown that there is a compensatory effect between TLR2 and TLR4 receptors, observed at the receptor activity as well as mRNA and protein level. The obtained results suggest that a relatively small increase in the concentration of fatty acids to a level that activates the pro-inflammatory response may be sufficient to increase concentration of the extracellular vWF, potentially resulting in thrombotic complications. The involvement of PA in the regulation of the vWF secretion partially explains the increased vWF concentration observed in the plasma of obese patients. <br>
Energy homeostasis is crucial for maintenance of body weight and is coordinated through the hypothalamus, in particular the arcuate nucleus (ARC) containing the antagonistic neurons responsible for the food intake and appetite control: orexigenic AgRP/NPY, activated during fasting, and anorexigenic POMC/CART, active during satiety. Their activation depends on detection of peripheral satiety signals, as well as intracellularly on gene expression regulated molecules, such as miRNA. The Dicer cKO mice with a neurospecific and tamoxifen-inducible deletion of Dicer1 gene, the key enzyme in the miRNA biogenesis, is characterized by the development of hyperphagic obesity. A mutation in ARC is responsible for the development of the obesity phenotype. The first aim of the study was to determine the metabolic parameters of Dicer cKO mice. A series of experiments were performed to assess the metabolic changes in Dicer cKO mice including the glucose metabolism assessment, as well as the effect of physical activity on the development of obesity and changes in metabolic parameters analysed using metabolic cages. The effect of leptin infusion on food intake and body weight gain was also determined using osmotic pumps. The results indicate sex-dependent transient changes in glucose metabolism during the weight gaining phase. Studies conducted in metabolic cages showed that Dicer cKO mice consume more food and water during the hyperphagic period in both the active and inactive phases of the circadian rhythm, which is in a line with the changes in respiratory exchange ratio. Dicer cKO mice also exhibit reduced energy expenditure. It was also shown that modifications in leptin signaling slightly contribute to the development of obesity in Dicer cKO mice. Deletion of miRNAs may also affect plastic changes in ARC, thus the second aim of the study was to determine the role of the neuroplasticity marker an immediatly early gene c-fos in the development of the obesity phenomenon in Dicer cKO mice. The c-fos plays an important role in the induction of plastic changes during fasting. We performed analysis of c-fos expression in C57Bl/6J mice during fasting in ARC. It was shown that c-fos does not behave like classical IEG, as its expression is chronically elevated after 24 hours of food removal, and c-FosIR cells show co-localization with AgRP/NPY neurons. Thus, the hypothesis was advanced that constant action of peripheral hunger signals exert neuronal excitability in the ARC, preventing the silencing of the c-Fos response. To investigate this phenomenon, an optogenetic tool was used to activate AgRP neurons with the photosensitive channelrhodopsin-2 (ChR2) expression in animals with ad libitum food access. It has been shown that photostimulation of orexigenic neurons leads to immediate food intake and stimulates c-fos expression. However, without peripheral negative energy signals, the c-fos expression is silenced like canonical IEG. The c-fos expression pattern analysed in ARC of Dicer cKO mice showed the elevated levels of c-Fos protein during the hyperphagic period, implying its role in the development of obesity. To determine the role of c-fos in obesity phenotype, the CRISPR/Cas9 system was used to silence its expression. The designed guideRNA sequences were inserted into AAV viral vectors and then injected into ARC of DicerCas9 mice. However, there were no significant differences in body weight and food intake in Dicer cKO mice after administration of viral vectors carring anty-Fos guideRNAs. Instead, it was observed that mutation in orexigenic neurons in Dicer cKO mice led to lack of responsiveness to negative energy status. The obtained results demonstrate the important role of miRNAs in ARC neurons in metabolic control and maintenance of energy homeostasis. Downregulation of miRNAs lead to hyperphagia and obesity, as well as changes in the functioning of neuronal circuits involved in appetite control. <br>
The enlarged brains of homeotherms bring behavioral advantages but also incur high energy expenditures. Energy fueling evolutionary increase in brain size and enhanced cognitive abilities (CA) could come from two primary sources: according to the “expensive tissue” hypothesis postulated by (Aiello and Wheeler 1995), the evolution of a larger brain was made possible by a diet-related reduction in the size of the digestive tract and by increasing of quality (energy density) of the diet. Thereby, an evolutionary increase in brain size resulted from the brain-gut trade-off. The second hypothesis, dubbed the “expensive brain” hypothesis (Isler and van Schaik 2006), predicts that the energetic costs of an evolutionary increase in brain size were covered by increased total energy intake rather than energy savings on metabolically costly organs (such as the gut) or processes (reproduction or immunocompetence). In my thesis, I asked a question: How were the energetic costs of an enlarged brain overcome in the course of evolution? To answer this question, I used the experimental evolution animal model consisting of the line types of Swiss Webster mice artificially selected for high (H-) or low (L-) Basal Metabolic Rate (BMR), maximal (VO2max) metabolic rate (a.k.a. peak, PMR), and random bread lines (RB). The metabolism rates selected in the model are proxies of the traits implicated in the evolution of homeothermy. Thus, they are a prerequisite for the encephalization and exceptional CA of mammals, including humans. The H-BMR mice had bigger guts, but not brains, than mice of other line types. Yet, they were superior to the other line types in the cognitive tasks carried out in reward and avoidance learning contexts. Conversely, when subjected to the classical paradigm of contextual fear conditioning, the L- BMR mice lost fear response much faster than the mice of other line types (that is, their memory was inferior). Furthermore, the H-BMR mice had higher neuronal plasticity (indexed as the long-term potentiation, LTP). They also had increased numbers of neurons and dendritic spines in the hippocampus compared to their counterparts. Finally, the activity of cytochrome oxidase (CCO), a proxy of the number of neuronal mitochondria, was higher in the H-BMR mice than in other line types. The results suggest that the evolutionary increase of CA in mammals was initially associated with increased BMR and brain plasticity, rather than a direct increase in brain size. Thus, an enlarged gut was not traded off for brain size. It could be that in the course of evolution, selection for increased total energy expenditures indirectly increased BMR and the metabolic rate of better connected and more plastic individual neurons, improving CA. Thus, my study does not support the existence of the brain-gut trade-offs postulated by the ET hypothesis. Conversely, my results support the link between CA fueled by high brain metabolism reflected in H-BMR as proposed by the EB concept. <br>
Environmental factors affecting the brain during the prenatal period can disrupt the formation of neural connections, thereby increasing the risk of neurodevelopmental disorders (NDDs). A growing body of epidemiological evidence identifies infection during pregnancy as one such factor; however, the mechanisms underlying this phenomenon remain poorly understood. Studies conducted on animal models indicate that maternal immune activation (MIA) plays a pivotal role in the developmental deficits observed in offspring. Identifying the proteins involved in these processes is critical for deepening our understanding of NDDs pathogenesis and for developing potential preventive or therapeutic strategies. Lipocalin 2 (Lcn2) is a protein associated with the innate immune response. It is secreted during infection and inhibits bacterial growth by iron sequestration. Lcn2 expression is strongly upregulated in the brain during inflammatory states, and its role can vary from anti- inflammatory to promoting pathological processes. Notably, both in vivo and in vitro studies suggest that Lcn2 influences the morphology and function of neurons and glial cells. In adult mice lacking the Lcn2 gene, structural abnormalities in the dendritic tree and dendritic spines were observed, accompanied by impaired neuronal function. Moreover, these animals exhibited anxiety-like and depressive-like behaviors, as well as deficits in spatial learning. Despite these findings, the role of Lcn2 in the developing brain has yet to be investigated. This study aimed to determine the role of Lcn2 in the processes underlying developmental disorders of the mouse brain caused by maternal immune activation. A bacterial infection model was employed in which pregnant mice received intraperitoneal injections of lipopolysaccharide (LPS), a bacterial endotoxin that induces an innate immune response. First, it was demonstrated that Lcn2 is expressed in the developing brain and that MIA increases Lcn2 mRNA levels in fetal brains of both sexes. To evaluate whether these changes in Lcn2 expression influence developmental outcomes, transgenic mice were used. Pregnant Lcn2 Het females were subjected to the MIA procedure, and offspring with either wild-type or Lcn2- knockout genotypes were further analyzed. The results indicated that maternal immune activation induces sex-dependent expression of pro-inflammatory cytokines in the placenta and fetal brain, while the absence of Lcn2 modulates the inflammatory response. In wild-type mice, MIA caused changes in the density and morphology of dendritic spines and influenced the excitability of hippocampal pyramidal neurons. These neural alterations were accompanied by behavioral disturbances resembling symptoms of neurodevelopmental disorders. Interestingly, Lcn2 deletion under baseline conditions resulted in behavioral deficits similar to those observed in MIA offspring. The effects of both MIA and Lcn2 deletion were sex-dependent. However, Lcn2 silencing did not significantly affect the behavior of animals whose mothers were exposed to LPS during pregnancy. These findings cannot conclusively determine Lcn2's contribution to the development of disorders triggered by maternal immune activation, given that the absence of the Lcn2 alone produces similar behavioral impairments. However, they underscore the pivotal role of Lcn2 in proper brain development under physiological conditions. <br>
Epidemiological data indicates that the number of people suffering a stroke is increasing every year. One of the most common consequences of a stroke is aphasia, defined as an impairment of language functions. Growing attention is being paid to the occurrence of deficits in non-linguistic cognitive functions in individuals with aphasia, such as memory, attention, planning, and temporal information processing (TIP). These deficits intensify the severity of language impairments and hinder the therapy process. Given the temporal dynamics of speech production and comprehension, as well as other cognitive functions supporting language processes, TIP deficits appear to play a particularly significant role in the deficits observed in aphasia. The present thesis consists of three articles focusing on the relationship between non-linguistic cognitive functions, with a special focus on TIP efficiency, and language functions in individuals with aphasia. The first study examines the relationship between short-term and working memory, TIP, and speech comprehension. It was observed that individuals with aphasia exhibit greater short-term memory efficiency, both verbal and spatial, compared to working memory. Furthermore, higher levels of verbal and spatial short-term memory, as well as verbal and spatial working memory, were associated with better speech comprehension. Further analyses showed that the relationship between memory performance and TIP depends on the modality of the memorised material. TIP was found to be closely linked to spatial working memory. However, its significance was less pronounced for verbal short-term and working memory, as well as spatial short- term memory. In these types of memory in individuals with aphasia, the severity of speech comprehension deficits played a key role. The second study aimed to determine the relationship between the parameters of the P300 potential and the efficiency of cognitive functions in individuals with aphasia. It was demonstrated that shorter latency of the potential was associated with better TIP, psychomotor speed, spatial short-term memory, planning, word comprehension, global speech comprehension, and verbal fluency. These results suggest that in individuals with aphasia, the latency of the P300 potential may serve as an reliable indicator of cognitive function efficiency, particularly those functions for which speed of information processing is critical. The third study evaluated the effectiveness of a new training developed for individuals with aphasia, based on the Dr. Neuronowski® program. This training focused on the comprehensive improvement of various cognitive functions with an emphasis on TIP. It was shown to improve both trained non-linguistic cognitive functions and untrained language functions. After training, the improvement was observed in TIP, verbal short-term and working memory, phonemic hearing, global speech comprehension, grammar comprehension, naming, and verbal fluency. In contrast, the control training, which was based solely on language exercises, resulted in improvement only in the directly trained functions. These findings indicate that exercises that target non-linguistic cognitive functions, including TIP, provide greater benefits than training language functions alone. This presented series of studies highlights the importance of non-linguistic cognitive functions in the comprehensive understanding, diagnosis, and rehabilitation of aphasia. In particular, TIP is considered by some researchers to be a logistical basis of cognitive functions, including language functioning. Incorporating TIP exercises and other non-linguistic cognitive function training into aphasia therapy may have significant benefits for patients. <br>
Epilepsy is a widespread neurological disorder aﬀecting millions worldwide, with temporal lobe epilepsy (TLE) being its most common drug-resistant form. Current treatments primarily target neuronal excitability, neglecting the signiﬁcant contributions of glial cells, particularly astrocytes, in epilepsy progression. CD44, a cell-surface glycoprotein involved in cell adhesion, neuroinﬂammation, and synaptic plasticity, is highly upregulated in epilepsy models. This thesis investigates the role of astrocytic CD44 in epileptogenesis, aiming to elucidate its involvement in seizure development, hippocampal structural remodeling, and synaptic alterations. The central research question addresses whether astrocytic CD44 contributes to TLEʹs pathophysiological changes and whether its modulation could inﬂuence disease progression. To explore this question, an astrocyte-speciﬁc CD44 knockout mouse model was generated using the Cre-loxP system. The kainate (KA) model of temporal lobe epilepsy was employed to induce status epilepticus, replicating TLE-like pathologies. Advanced methodologies were applied, including immunohistochemistry for assessing reactive astrogliosis, mossy ﬁber sprouting and granule cell dispersion, Western blot analysis for protein expression, and serial block-face electron microscopy (SBEM) for high-resolution examination of synaptic ultrastructure. Electroencephalographic (EEG) recordings further characterized seizure patterns and severity in CD44-depleted mice compared to controls. This comprehensive approach enabled the assessment of astrocytic CD44ʹs role across molecular, cellular, and systemic levels in epileptogenesis. The thesis ﬁndings demonstrate that astrocytic CD44 deletion inﬂuences both seizure patterns and hippocampal remodeling in a KA-induced model of TLE. Speciﬁcally, CD44- deﬁcient mice showed a reduction in the frequency of behavioral seizures but an increase in electrographic non-convulsive episodes detectable only via EEG. While CD44 deletion did not signiﬁcantly aﬀect the latency to the ﬁrst spontaneous seizure or the severity of convulsive seizures, it reduced hallmark structural changes of epileptogenesis, including reactive astrogliosis, mossy ﬁber sprouting, and granule cell dispersion. SBEM analyses showed increased dendritic spine number, reduced postsynaptic density size, and decreased astrocytic ensheathment in the CD44-depleted epileptic brain compared to controls. These ﬁndings underscore the essential role of astrocytic CD44 in preserving synaptic integrity and stability during epileptic insults. These results suggest that astrocytic CD44 plays a pivotal role in hippocampal remodeling and seizure propagation. By attenuating pathological astrocyte responses, CD44 deletion may mitigate some of the structural changes associated with TLE, positioning CD44 as a potential therapeutic target. This work underscores the importance of astrocyte-driven mechanisms in epilepsy and advocates for a broader perspective in designing future treatments, integrating neuronal and glial contributions to disease progression. <br>
Fear contagion is an automatic process of aligning one animal's emotional state with another's emotional distress. It has been described in different social species, including rats and humans. Reading the emotional states of others has recently been suggested to play an essential role in detecting danger. If so, one could expect fear contagion to be a cross-species phenomenon. However, this hypothesis has yet to be tested. Both rat and human studies implicated the amygdala, a brain structure crucial for processing emotions, in fear contagion.Further, the rat studies showed that two main parts of the amygdala, which differ morphologically and functionally - the basolateral and centromedial nuclei - are involved in emotional transfer. Such a detailed analysis of the amygdala activity has yet to be performed for human-human emotional transfer. In this doctoral thesis, I aimed to test whether the cross- species (human-rat) fear transfer occurs and whether it involves the basolateral and centromedial parts of the amygdala (study 1). Their involvement was also verified during the human-human fear contagion (study 2).In study 1, the habituated rats were handled by familiar humans who underwent the fear conditioning task (or an emotionally neutral task in the control condition). Following the interaction, the rats' amygdala activations were analyzed using the expression of c-Fos, a marker of neuronal activation. I observed that the rat amygdala was activated to a greater extent in the experimental rats compared to the control rats. That was true for both the basolateral and centromedial divisions. The behavioral differences between the experimental and control rats further confirmed the successful transfer of fear from human to rat.Study 2 was performed using functional magnetic resonance imaging (fMRI). Participants (so-called observers) were placed in the fMRI scanner and watched their friends (so-called demonstrators) undergoing the classical fear conditioning paradigm. In this task, a neutral stimulus was repeatedly paired with aversive electrical stimulation applied to the forearm. I analyzed the observers' brain responses to the electric shocks administered to their friends and found enhanced activations in the amygdala. Also, here, both the basolateral and centromedial divisions were activated.The thesis provides the first neural evidence for interspecies fear contagion. The findings indicate that both main divisions of the amygdala respond when human fear is transmitted to another human and a rat. This suggests a common brain circuit involved in perceiving fear socially in humans and rats. I argue that it could have evolved to enable sharing of the emotional cues essential for survival across species
Fishing profitability
Flotillin-1 and flotillin-2 are ubiquitously expressed proteins which are N-myristoylated and/or S-palmitoylated. Thanks to these acylations, flotillins bind to the cytoplasmic leaflet of plasma membrane nanodomains, rafts. Moreover, flotillins undergo homo- and hetero-oligomerization and interact with numerous proteins. Therefore, flotillins can act as scaffolding proteins, facilitating the assembly of multiprotein submembrane complexes involved in various cellular processes.The main objective of this study was to reveal the role of flotillins and their S-palmitoylation in TLR4 signaling triggered by bacterial lipopolysaccharide (LPS). TLR4 initiates a pro-inflammatory response aiming at the eradication of bacteria which can lead to fatal sepsis, fueling interest in TLR4 signaling. The rationale for undertaking the studies was: (1) results of our mass spectrometry analysis, which showed that the amount of palmitoylated flotillin-1 increased in LPS-stimulated Raw264 macrophage-like cells, suggesting its participation in LPS-triggered signaling; (2) a line of data indicating that flotillins are involved in endocytosis and cellular trafficking of raft proteins. A typical raft protein is CD14 which assists activation of TLR4 by LPS. It was assumed that flotillins can affect LPS-induced signaling due to possible interplay with CD14.To achieve the goal, lentiviral particles were used to deliver flotillin-2-specific shRNA into Raw264 cells. Several clones of cells stably depleted of flotillin-2 were obtained, which were also found to be deficient in flotillin-1. In flotillin-depleted cells, the LPS-induced responses were diminished. The TRIF-dependent signaling pathway of TLR4 leading to activation of the IRF3 transcription factor was inhibited and the subsequent production of chemokine CCL5/RANTES was reduced. The MyD88-dependent signaling leading to the activation of the NFκB transcription factor and production of cytokine TNFα was also reduced. However, the latter effect was most pronounced in cells stimulated with low LPS concentration which requires the participation of CD14. Indeed, depletion of flotillin-1 and -2: (i) lowered CD14 mRNA level; (ii) reduced the total cellular level of CD14; (ii) decreased the amount of CD14 on the cell surface. Notably, no such changes were observed for TLR4. On the other hand, forced clustering of CD14 in the plasma membrane (the first effect of LPS binding) induced S-palmitoylation of flotillin-1 and flotillin-2, indicating mutual interactions of flotillins and CD14. Co-expression of flotillins with 23 members of the zDHHC family revealed that zDHHC5 and zDHHC8 can S-palmitoylate flotillins. After silencing of Zdhhc5 or Zdhh8, it was found that zDHHC5 participation is required for a response to LPS triggered in both TLR4 signaling pathways, with emphasis on the TRIF-dependent pathway, which may be linked with zDHHC5 involvement in S-palmitoylation of flotillins. Taken together, the data indicate that flotillins modulate the cellular level of CD14 and interact (indirectly) with CD14, thereby affecting the intensity of the LPS-induced pro-inflammatory response. Flotillins are likely to be involved in CD14 endocytosis and recycling, as well as in the transport of newly synthesized CD14 to the plasma membrane, all events may be regulated by S-palmitoylation of flotillins catalyzed among others by zDHHC5. The above results were obtained, i.a., owing to the development of a modification of a technique for detecting palmitoylated protein. It involves enrichment of 17ODYA (palmitic acid analogue)-labeled proteins and their recovery from streptavidin-coupled beads allowing simultaneous identification of several endogenous and overproduced palmitoylated proteins. The technique was used in a study conducted in collaboration with the Institute of Molecular Genetics ASCR in Prague and allowed the detection of S-palmitoylation of OPAL1, an adaptor protein of leukocytes, likely to be located in rafts.
Food is the foundation of the survival pyramid while hunger is the primary drive that motivates the search for and acquisition of nourishment. The brain is the locus of the superior centers involved in the regulation of hunger and satiety. The arcuate nucleus, located in the immediate vicinity of the median eminence in the hypothalamus, is the primary first-order center processing information about the body's energy status. Its composition includes two populations of opposing neurons: AgRP/NPY - stimulating food intake and POMC/CART - responsible for promoting satiety and appetite suppression. Any disturbances within this center and in the communication of the arcuate nucleus with second-order neurons in other centers may lead to changes in eating behavior and the development of metabolic diseases such as obesity. This dissertation chiefly aims to investigate the involvement of AgRP/NPY neurons in the development of obesity in animals with the neurospecific deletion of the Dicer1 gene. Dicer is an enzyme the endonucleolytic action of which leads to the production of mature forms of microRNA (miRNA) molecules regulating the translation process. Cells deprived of the Dicer1 gene lack functional, canonical microRNAs. The research problem required the use of several transgenic mice models. All models were based on the Cre-loxP system, where recombination was induced by Tamoxifen in some and administration of the AAV viral vector regulating the inserted transgene with the AgRP promoter or by using CRISPR/Cas9 technology in others. Analysis and modification of successive transgenic models resulted in high specificity of the introduced change, selectively in AgRP/NPY neurons. Analysis of the obtained research models evidenced that the system with the induced Dicer1 deletion in AgRP neurons (AgRPCreERT2Dicerfl/fl) does not allow for conclusions about the involvement of these neurons, due to the insufficient level of recombination in this lineage. The remaining models provided significant information. The model with the inducible loss of microRNA in mature CaMKIIα neurons and the simultaneous loss of both Npy alleles (NPY-KO/DicerCKO) excluded the involvement of NPY as a key stimulant of food intake. Meanwhile, experiments on Dicerfl/fl mice subjected to intracerebral injections of the AAV- AgRPCre vector into the arcuate nucleus, showed the significant involvement of AgRP neurons in the development of microRNA-dependent obesity. Moreover, they revealed a quantitative relationship between the number of AAV-vector particles introduced determining the number of modified neurons and the increased nutritional requirements leading to increased body weight. Mice expressing Cre and Cas9 in AgRP neurons (AgRPCreCas9) proved to be the model generating the highest degree of specificity in the targeted modification of Dicer1 in AgRP neurons. These animals, following intracerebral administration of the AAV-guide2Dicer vector, developed massive obesity associated with severe appetite while also revealing a sex- dependent effect, where only females lacking microRNA in AgRP neurons showed a significant increase in appetite and body weight. The above observations imply the important role of microRNA in regulating the functions of AgRP/NPY neurons. Selective loss of microRNA in hunger neurons leads to impairment of their functioning, which is manifested by increased appetite and development of obesity. The number of modified hunger neurons determines the magnitude of the effect, indicating there is a subtle balance between the signals of hunger and satiety. Sexual differences in the observed phenotype suggest microRNA plays a role in modulating hormone-dependent pathways. <br>
Functional Magnetic Resonance Spectroscopy (fMRS) is a non-invasive technique used to measure dynamic changes in metabolite concentrations in response to stimuli. Despite its potential for advancing our understanding of brain activation mechanisms, fMRS remains relatively novel and the temporal dynamics of glutamate (Glu), the main excitatory neurotransmitter, following stimulation have not yet been fully explored. To date, no studies have applied fMRS to the reading process, despite the potential of this approach to reveal dynamic glutamate responses that may underlie both typical reading and its impairments in dyslexia. One of the newest mechanistic account of dyslexia, the neural noise hypothesis, suggests that it could be caused by an imbalance between glutamate and gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter. In particular, an elevated concentration of glutamate in the left superior temporal sulcus (STS) was proposed to disrupt signal processing and impair reading acquisition. The aim of this thesis was to investigate glutamate concentration changes during reading-related tasks, in brain regions involved in reading: the superior temporal sulcus and the visual word form area (VWFA), as well as in one control region, the medial prefrontal cortex (mPFC). To characterize the temporal dynamics of glutamate, fMRS signals were acquired at four different delays between stimulus onset and signal acquisition. Participants with varying reading abilities, including individuals diagnosed with dyslexia and typical readers, were scanned at both 7T and 3T MR scanners. In total, 59 participants (29 with dyslexia, 13 females; 30 typical readers, 14 females) were scanned at 7T, and 40 participants (21 with dyslexia, 9 females; 19 typical readers, 11 females) at 3T. Glutamate levels were compared between groups to determine whether participants diagnosed with dyslexia exhibit higher glutamate concentrations in reading-related brain regions. While 7T scanners theoretically provide higher spectra resolution and improved metabolite separation, they also introduce technical challenges. For the VWFA, reliable analysis was not feasible due to insufficient spectral quality, highlighting the methodological difficulty of collecting data from regions susceptible to magnetic field inhomogeneities. In the STS, glutamate responses to reading-related stimulation were heterogeneous. Effects were more apparent in females, yet they were sensitive to blood oxygenated level depended (BOLD) correction and varied between 7T and 3T. No evidence of elevated glutamate in dyslexic participants within the left STS was observed, which does not support the neural noise hypothesis. Glutamate concentration changes were not limited to reading-sensitive regions, and some responses were also observed in the mPFC. A consistent glutamate response function could not be established, as glutamate changes varied across sex, group, brain region, stimulation type, and scanner. This inconsistency may reflect limited spectral quality due to a small number of averaged signals and the impact of BOLD contamination. Additionally, glutamate levels were significantly influenced by sex, age, and voxel tissue composition. While 7T improved some quality parameters, overall gains over 3T were inconsistent and region-dependent. These findings suggest that the practical advantages of ultra-high-field scanners in fMRS depend on region and are constrained by technical challenges. <br>
The functional organisation of the human brain is influenced both by innate mechanisms and individual experience. Spoken language processing, an evolutionary old skill, occurs in a neural network universal for different languages. On the other hand, reading is a skill that appeared in human evolution quite late and thus is an excellent example of neural plasticity connected to learning a new skill. Additionally, reading can be performed using not only vision but also touch. Braille alphabet is a script used by the blind population for reading using the sense of touch. Blindness enables us to see which aspects of the neuronal organisation are fixed and which change with altered experience.The current thesis focuses on the plastic changes in the organisation of the neural language network following visual deprivation. Three studies were conducted. The first focused on mapping the spoken and reading neural networks in the blind population and comparing them to the organisation of language processing in the sighted. Speech-reading convergence – a phenomenon thought to be universal in print reading was also tested for the first time in the blind population. The results of Study 1 revealed that speech-reading convergence was present in the blind subjects, but in different areas. It was found in the ventral occipitotemporal cortex (vOT), instead of the perisylvian regions. In the blind group, the vOT was active not only during reading, as in the sighted, but also during speech processing. The temporal cortex, which is involved in phonological processing in the sighted population, was disengaged during Braille reading.Thus, in Study 2, the vOT engagement in phonological processing was studied in the blind and the sighted. The blind subjects activated the left vOT during auditory phonological processing to a larger extent than the sighted subjects. However, this activation seemed not to be phonology specific. In the blind, the left vOT presented a similar activation during linguistic processing as other regions of the language network. The results of the second experiment suggest that the vOT plays a more general role in language processing in the blind population due to changed input to this structure arising from visual deprivation.Study 3 tested the differences in the cognitive correlates of print and Braille reading. Additionally, the relationship between literacy-related skills and age was studied using a cross-sectional design. The results of the third experiment indicate that the change in the modality used for reading introduces some alterations to the cognitive mechanisms of reading. Limits of the tactile modality - lower processing speed and the sequential nature of the processing augment the importance of the haptic factors for Braille reading and may cause minor deficits in some domains. On the other hand, different demands induced by the changed modality strengthen phonological skills and short-term memory. Yet, the developmental trajectory of literacy skills remains unchanged in the blind, as there were no differences in the correlations with age between the groups.Research presented in the thesis demonstrates that visual deprivation influences the functional organisation of both evolutionary old (spoken language) and newly learned skills (reading) on the neural and behavioural levels. Results underline the importance of individual experience for the organisation of specialised neural networks and are in line with the pluripotent cortex hypothesis of neural plasticity
Glioblastoma (GBM) is the most common and most aggressive brain malignancy in adults, with a median patients’ survival of only 14 months. A hallmark feature of GBM is its complex microenvironment, largely composed of microglia and macrophages: immune cells which in theory should fight with the tumor. However, factors secreted by glioma cells drive the phenotypic polarization of microglia and macrophages, so instead of limiting tumor development, they support its growth and suppress anti-tumorigenic response. As a result, these cells constitute key mediators of the immunosuppressive nature of the GBM microenvironment. Such re-programmed microglia and macrophages are collectively called glioma-associated microglia and macrophages (GAMs) and may account for up to 30% of the tumor mass. Therefore, finding molecular mechanisms responsible for shaping tumor- supportive properties of GAMs is crucial for the development of new therapeutic strategies of GBM. SorLA protein (encoded by SORL1 gene) is an intracellular sorting receptor which transports its protein cargoes between different subcellular organelles and therefore defines their final localization. While the presence of SorLA was initially thought to be limited to neurons in the brain, it was later discovered to be expressed in glial cells as well, including microglia. Interestingly, according to publicly available data from analysis of human GBM samples transcriptomes, SorLA seems to be important in shaping functional properties of GAMs. In line with this notion, the overall aim of this study was to decipher the influence of SorLA on the properties of GAMs, in particular microglia. Experiments performed in the frame of the PhD dissertation revealed that the level of Sorl1 transcript in primary mouse microglia depends on the activation mode of the cells. Furthermore, it turned out that lack of SorLA unlocks the ability of microglia to release pro- inflammatory factors, including TNF-α, when compared to wild type cells (WT). Microglia depleted of SorLA (SorLA-KO) upon co-culture with mouse glioma cells are characterized by increased level of proteins linked to interferons-related response and phagocytosis, which indicates their pro-inflammatory activation. These findings raised the hypothesis that the lack of SorLA may unlock the pro-inflammatory potential of microglia and thereby could influence the properties of glioma microenvironment. Indeed, using mouse model of glioma it was observed that SorLA-KO mice develop smaller brain tumors when compared to WT animals, which coincides with changes in microglia morphology suggesting their inflammatory activation. Simultaneously, GAMs from SorLA-KO microenvironment are characterized by increased expression of genes linked to interferons-related response, while the levels of transcripts linked to tumor-supportive functions of the cells are decreased. Finally, in glioma microenvironment of SorLA-KO mice, increased levels of markers related to necroptotic type of cell death are observed, as well as changes in the profile of infiltrating immune cells. <br> In summary, presented research indicates that SorLA is a key player in shaping the properties of GAMs and its depletion unlocks their anti-tumor response which influences the overall potential of glioma microenvironment and as a result, prevents tumor growth. <br>
Gliomas are primary tumors of the central nervous system. Diagnosis and therapy recommendations are difficult, because of the intertumoral heterogenicity of glial tumors. Current World Health Organization classification of gliomas is based on the pathomorphological and molecular characteristics of the tumor biopsy. Diagnosis is based on pathomorphological features (diffusiveness, proliferation index, a presence of necrosis) and upon specific genetic alterations detected in the tumor, which leads to specific recommendations for the therapy. Next generation sequencing (NGS) is a valuable tool to improve diagnostics of brain tumors. Traditional tissue biopsy might not present complete mutational spectrum in case of such heterogenic tumors, so alternative methods are being tested to enable more holistic view of each disease. When traditional biopsy or tumor resection are not possible, a liquid biopsy would be of great assistance to clinical practice. Liquid biopsy is a use of bodily fluids to isolate circulating cell free nucleic acids or circulating tumor cells to detect cancer markers for diagnostics, disease monitoring or prognostic. In case of primary brain tumors, cerebrospinal fluid can contain more circulating cell free DNA (cfDNA) or RNA (cfRNA) originating from the tumor, but a lumbar puncture may have side effects, so it is rarely performed on heavily symptomatic primary brain tumor patients. We sought to evaluate if improvements in cfDNA isolation, library preparation and targeted sequencing would provide reliable information regards genetic alterations in glioblastoma (GBM), most common and deadly primary brain tumor. After analysis of blood derived cfDNA potentially pathogenic variants were detected in 37/84, which based upon the current literature is an improvement from most of the studies. We employed a target gene panel encompassing 668 cancer-related genes and NGS to a set of diagnostically difficult pediatric glioma tumors. The analysis of DNA isolated from formalin fixed paraffin embedded (FFPE) sections originating from those tumors yielded the whole spectrum of potentially pathogenic mutations, some interesting variants were found, that could be further studied (MTUS, FANCA, RET). Tumor-derived cell cultures are valuable in vitro system to study tumorigenesis and screen for therapeutics, however it is not fully known if tumor cells keep their genetic alterations and cultured clones reflect molecular profile of an original tumor. Comparative analysis of somatic mutations present in tumor-derived cell lines and/or original tumors have shown some differences in variant profiles, cell cultures contained more detectable somatic mutations. This can indicate that some somatic variants can be missed in the tissue biopsy, due to its complexity as tumor contains healthy cells, microglia and macrophages that can make background noise decreasing the tumor variants detectability. On the other hand, tumor stem cells can possibly gain mutations during cell culture, as their DNA repair pathways are frequently malfunctioning, and mitosis is maximized by artificial growth factors. The current classification of gliomas is based upon tumor genotyping. Current diagnostic tests employ molecular analysis of DNA isolated from FFPE or frozen tumor samples. There are many ongoing clinical and research studies improving current diagnostic methods with the aim to create personalized therapy recommendations with use of both blood derived cfDNA and tumor derived cell cultures. Present study demonstrates how tumor derived cell lines and blood derived cfDNA can offer an insight on tumor genetic heterogeneity. <br>
Glycogen Synthase Kinase-3β (GSK-3β) was discovered for its role in the regulation of glycogen metabolism. GSK-3β is observed in all tissues and it is involved in the regulation of the activity of multiple proteins and metabolic pathways. Studies on mouse models showed that GSK-3β is important during development of central nervous system and in the adult brain. Transgenic mouse model with overexpression of the constitutively active form of GSK 3β[S9A] in the brain is characterized by behavioral changes such as memory deficits and hyperactivity. In adult transgenic mice, structural changes including decreased brain volume and increased thin spines fraction (considered as immature) in granule cells of dentate gyrus (DG) have been observed. Mechanisms underlying abnormal activity of GSK-3β in synaptic function are not fully understood. Here, we analyzed how constitutively active GSK-3β influences morphology of dendritic spines and excitatory synaptic transmission in granular cells of DG in young (3 week-old) transgenic mice. Microscopic analysis showed that increased activity of GSK-3β led to elongation of dendritic spines without changes of spine density. Next, using whole-cell patch-clamp method, we observed increased inter-event intervals of miniature excitatory postsynaptic currents (mEPSCs) while the event amplitude was not changed. Lack of changes in total spine density together with lower frequency of excitatory events suggested lower number of functional synapses. Therefore, in the next step we analyzed the presence of silent synapses. Silent synapses are non-functional (or immature) excitatory synapses, where N-methylo-D-asparate acid (NMDA) receptor is present with lack of functional α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. We observed an increase in the fraction of silent synapse in GSK-3β[S9A] mice. These results suggest that increased activity of GSK-3β decreases the stability of AMPA receptors in postsynaptic compartment and/or inhibits synapse maturation.The second aim of this study was to identify whether the abnormal activity of GSK-3β can regulate the expression level of microRNAs (miRNAs) in neurons. MiRNAs (small, non-coding RNAs), are key molecules for proper function of central nervous system. Previous studies showed that GSK-3β can regulate the expression level of microRNAs in cancer cells. Such a link has not been reported in neurons. Therefore, we analyzed miRNA expression in hippocampus of GSK-3β[S9A] mice using next generation sequencing (NGS) by Illumina MiniSeq system. Dysregulation of 24 mature and 71 precursor miRNAs in RNA samples was observed. We chose 4 miRNAs for validation by quantitative polymerase chain reaction (PCR). In transgenic mice, miR-221-5p (miR-221*) expression level was significantly downregulated. Next, to define a role of miR-221* in synaptic plasticity we used wild-type primary hippocampal cell culture. Neurons were transfected with miR-221* inhibitor, and imagining for dendritic spine analysis and mEPSCs recordings were performed. Changes of dendritic spine shape and density were not observed. We found an increase in the peak amplitude of mEPSCs, without changes of inter-event intervals after the application of miR-221* inhibitor. Our results indicate that the downregulation of miR-221* enhances excitatory synaptic transmission in hippocampal neurons. Altogether, overactivity of GSK-3β leads to a reduction of functional synapses in hippocampal granular cells of young mice. Moreover, GSK-3β can regulate miRNA expression level in neurons. In GSK-3β[S9A] mice, the expression level of miR-221* is significantly downregulated and the inhibition of miR-221* in primary hippocampal cell culture leads to changes in excitatory synaptic transmission. Structural and electrophysiological changes observed in GSK-3β[S9A] mice might in turn drive aberrant synaptic plasticity
Hemagglutinin (HA) is one of the major surface proteins of the influenza A virus. During maturation, HA0 precursor is cleaved into two subunits, HA1 and HA2 subunits. They perform different functions: HA1 is responsible for the receptor binding at the host cell surface, while HA2 mediates viral fusion in the late endosomes. The N-terminal part of the HA2 subunit acts as a fusion peptide (FP). Under low pH conditions in the late endosomes, FP is exposed and initiates fusion. The C-terminal part of the HA2 is transmembrane domain (TMD), which anchors the protein in the membrane and possibly performs an important function in the fusion process. So far, 18 subtypes of HA have been identified, classified into two phylogenetic groups based on the amino acid sequence of the protein. Currently, the most prevalent subtypes circulating in the human population are H1 (group 1) and H3 (group 2). While FP sequences are conserved between subtypes, TMD sequences diverge significantly. Although the role of FP in the fusion process has been well characterized, the function of TMD still remains poorly understood. It has been shown that the presence of TMD is essential for the complete fusion process of the viral envelope with late endosomal membranes. Some studies suggest that an interaction between FP and TMD is responsible for full fusion. However, the presence of this interaction remains a contentious issue in the literature. The main aim of the dissertation was to examine the potential interaction between FP and TMD. To examine this, series of experiments with peptides and full-length HA were carried out. Peptides with amino acids sequences corresponding to FP and TMD from H1 and H3 subtypes were used in the studies. Synthetic peptides corresponding to TMD were utilized in two versions: shorter (H1, H3) and longer (H1long, H3long). The longer versions were extended with amino acids that can interact with cholesterol. In addition, virus-like particles (VLP) were used, with full-length HA of the H1 subtype (H1-TMD-H1) or HA of the H1 subtype with the TMD fragment replaced by that from the H3 subtype (H1-TMD-H3). Liposomes and supported lipid bilayers (SLB) with different lipid compositions were utilized as artificial lipid membrane models. To examine synergic interaction between peptides, individual peptides and FP:TMD mixtures were used. The work aimed to biophysically characterize the action of the peptides in the membrane. To measure the fusion activity, a series of fluorescence experiments was performed. Moreover, membrane lipid order was investigated by fluorescence lifetime imaging microscopy (FLIM) using a dye sensitive to membrane lipid order. Finally, the fusion activity test of VLP with SLB was carried out using FLIM-FRET (fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer). The research showed a lack of interaction between FP and TMD. However, differences were observed in the membrane activity of TMDs from divergent phylogenetic groups. While H1 induced membrane ordering, H3 caused a decrease in the lipid order. Moreover, the results suggest that TMD affects the fusion activity of HA. Moreover, it was observed that TMD may influence the fusion activity of the full-length protein. The experiments with VLP revealed a tendency suggesting that H1-TMD-H3 exhibits higher fusion activity than H1-TMD-H1. <br>
High developed and developing countries are witnessing a significant increase in the percentage of elderly people, due to the progress of civilization, improvements in quality of life, and healthcare. Human aging is a complex and inevitable process of biological, psychological, and functional changes that occur over time. One aspect of this is cognitive aging, which leads to the deterioration of cognitive functions as one gets older. Hence, there is a growing demand for strategies and interventions aimed at improving the seniors life quality and supporting their cognitive abilities. Results from existing research indicate that appropriately structured cognitive training can be an effective method for enhancing cognitive abilities in the elderly, potentially delaying cognitive aging processes. The objective of present study was to assess the effectiveness of the Dr. Neuronowski® as a method designed to enhance the cognitive functions of seniors. This training program focuses on improving time perception, which also deteriorates with advanced age. The study examined whether Dr. Neuronowski® could enhance various cognitive domains such as: temporal information processing, memory, attention, and executive functions and whether there are parallel changes in the neural network 69 healthy seniors participated in this study and were randomly divided into 3 groups: A - the experimental group that participated in the Dr. Neuronowski® cognitive training (n=25); B - the active control group that underwent educational training (n=21); and C - the nonactive control group that did not participate in any training (n=23). Groups A and B underwent 24 training sessions, each session lasting 45 minutes, with a frequency of 3 meetings per week. The study design included three diagnostic measures: a pretest (before training), a posttest (immediately after the training completion to assess the effects directly), and a follow-up assessment (approximately 8 weeks after the training completion in order to evaluate maintenance of obtained effects). The use of an nonactive control group C aimed to control the repeated measure effect in the diagnostic procedures. Cognitive functions were examined using a number of neuropsychological tests. Additionally, electrophysiological procedures were used to understand the underlying neural mechanisms of the changes observed after the training. It was found that cognitive training improved time perception, short-term verbal and spatial memory, planning ability, and inhibitory control among the seniors. These behavioral changes were accompanied by changes at the electrophysiological level. A decrease in the amplitudes of mismatch negativity potential and a mental workload index was observed, indicating improvements in neural network efficiency. The behavioral outcomes were stable over time and persisted for two months after the training completion. In conclusion, the observed changes suggest that the Dr. Neuronowski® training program, based on its unique time perception component, can be an effective method for enhancing the cognitive functions of seniors. <br>
High-grade gliomas (HGGs), the most frequent and severe primary brain tumours in adults, invariably recur due to incomplete surgery or therapeutic resistance. The major checkpoint in regulation of gene expression is the initiation of transcription, which is mostly regulated by a class of DNA-binding proteins known as transcription factors (TFs). The expression of essential TFs is required by cancer cells to carry on a variety of biological processes in cancer cells such as cellular transformation, oncogenesis and progression, cell proliferation, metastasis, and chemo-resistance. In HGGs, several interconnected biological components such as somatic mutations, transcriptomic and TF dysregulations, as well as alterations in histone modifications, DNA methylation and chromatin remodelling contribute to the disease aggressiveness. Transcriptomic profiles of HGGs at recurrence have not been thoroughly investigated yet. Moreover, despite significant efforts, the specific regulation of genes overexpressed in HGGs by TFs remains largely unknown. A better understanding of events occurring in open chromatin regions in HGGs is crucial to comprehend routes of brain cancer progression. We employed targeted DNA- and RNA-sequencing to identify single nucleotide variants, small insertions and deletions, copy number aberrations (CNAs), gene expression alterations and pathway dysregulations in 16 matched pairs of primary and recurrent HGGs. The majority of somatic mutations found in primary HGGs were not found in relapsed tumours, implying a sub-clone substitution during tumour progression. A novel frame-shift insertion in the ZNF384 gene was discovered, which may play a role in extracellular matrix remodelling. The presence of focal CNAs in the EGFR and PTEN genes was found to be inversely correlated. In silico analysis of the tumour microenvironment demonstrated that tumour supportive (M2) macrophages and immature dendritic cells are enriched in recurrent HGGs indicating a prominent immunosuppressive signature in those tumours. Immunohistochemistry staining of tumour sections confirmed the accumulation of immunosuppressive cells in recurrent HGGs. We identified glioma grade-specific TFs binding sites in glioblastoma tissues as well as in human LN18 and LN229 glioma cells, using ATAC-seq data and confirmed their roles in controlling gene regulatory networks in HGGs. We explored different datasets that comprise DNA methylation profiles, histone acetylation profiles, GBM cell line RNA-seq and TCGA (the Cancer Genome Atlas) datasets (RNA-seq and lllumina 450K array DNA methylation). The comparative analyses of those profiles in gliomas of different malignancy grades revealed the importance of the c-Jun TF for the disease progression. c-Jun may play a role in the regulation of genes overexpressed in glioblastoma by binding to the gene promoters. Furthermore, we found that in the majority of c-Jun gene targets, DNA methylation plays an important role in the c-Jun dependent regulation. The bioinformatic predictions have been validated experimentally by testing c-Jun binding to various probes in the electrophoretic mobility shift assay (EMSA). Chromatin remodelling proteins SMARCA2 and SMARCA4 are frequently mutated in high-grade gliomas. To determine the role of those proteins, we performed knockdown of genes coding them in human LN18 glioma cells and tested the impact of SMARCA2 and SMARCA4 deficiencies on chromatin accessibility using the Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq). We discovered an increase in chromatin openness in SMARCA2/4 deficient cells, which affected expression of genes critical for signal transduction, including those from the transforming growth factor beta pathway: SMAD1, SMAD3, BMPR1A, and TGFBR2, implying the interdependence of chromatin remodellers and specific signalling pathways <br>
Hypoxia is a common feature of solid tumours, arising from abnormal vasculature that fails to deliver sufficient oxygen to rapidly proliferating tumour cells. This physiological stress plays a pivotal role in tumour progression by contributing to genomic instability, enhancing cellular invasiveness, metastatic potential, suppressing anti-tumour immunity, and reducing the efficacy of major treatments. In IDH-wild-type glioblastoma (GBM), the most aggressive and deadly primary brain tumour, intratumoral hypoxia is highly extensive and represents a critical determinant of poor patient survival. Moreover, the GBM microenvironment is infiltrated by diverse cell types, with glioma-associated microglia and macrophages (GAMs) constituting the predominant population. These cells can adopt immunosuppressive phenotypes and are recognised as major contributors of glioma progression. Since GAMs are commonly recruited to hypoxic niches, such stress can further enhance their tumour-promoting functions. One of key cellular responses to hypoxia is the remodelling of chromatin properties through histone modifications and DNA methylation. However, the extent to which these changes contribute to the hypoxia-driven reprogramming of the GBM transcriptome is not yet fully understood. In this study, the impact of hypoxic stress on chromatin reprograming and transcriptomic profiles within the glioma TME was investigated, particularly in GAMs. First, using the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) approach, hypoxia- dependent chromatin alterations were assessed in glioma cells. The data revealed global reduction in chromatin accessibility at promoter regions of numerous genes under hypoxic condition (<0.1% O2). Notably, specific functional pathways were affected, including those involved in mRNA processing and splicing, as well as regulators of R-loop formation. Second, hypoxia was found to alter the expression of key identity marker genes in GAMs. In glioma- co-cultured GAMs in vitro and in glioblastoma patient samples, hypoxia upregulated the expression of monocytic marker Lgals3 and downregulated the homeostatic microglial markers P2ry12 and Tmem119. In addition, hypoxic stress appeared to interfere with multiple functional markers, including genes related to lipid metabolism, phagocytosis, chemotaxis, ribosomal biogenesis, and the interferon response. Some of these hypoxia-induced changes in GAMs were fine-tuned through the changes in chromatin accessibility. Furthermore, it was found that hypoxia induced lipid droplet accumulation in myeloid cells via increased expression of lipid storage-related genes and this effect could be reversed through targeting epigenetic mechanisms with histone deacetylase inhibitors. Overall, these findings highlight hypoxic stress as a potent epigenomic and transcriptomic regulator of glioma TME which may hold significance for future basic research and clinical applications. <br>
Hypoxia/reperfusion of the heart and brain tissues leads to cell injury and necrosis and is the most common cause of death in developed countries. Mitochondrial channels are implicated in cytoprotection during reperfusion. One of them is the mitochondrial potassium channel inhibited by ATP (mitoKATP). It was proposed that the ROMK2 protein may be a pore-forming subunit of the mitoKATP channel. ROMK2 is an isoform of the ROMK1 protein that forms rectifying potassium channels in the plasma membrane. These channels interact with many proteins involved in the regulation of their activity, or in their trafficking. However, protein partners of the ROMK2 channel have not been identified so far. The main aim of the research presented in this doctoral dissertation was the identification of proteins that are part of the mitochondrial proxisome of the ROMK2 channel. For this purpose, proximity biotinylation was used. Among the biotinylated proteins, cytoplasmic proteins have been identified that participate among others in endocytosis, vesicular transport, oxidative stress, nucleotide synthesis, or lipid metabolism. In addition, subunits of inner and outer mitochondrial membrane translocases have been identified. These complexes allow the import of nuclear-encoded proteins into mitochondria. Among them, acylglycerol kinase (AGK) was found, which is involved in the transport of polytopic proteins into the inner mitochondrial membrane. AGK is a dual-function protein and participates also in the synthesis of lipids: lysophosphatidic acid (LPA) and phosphatidic acid (PA). The occurrence of a complex containing ROMK2 and AGK was supported by co-immunoprecipitation. Another goal was to investigate the pharmacological modulation of ROMK2 channel activity. To carry out these studies the ROMK2-6xHis protein was produced in bacteria Escherichia coli; bacterial membranes were solubilized with amphipathic copolymers, and the channel protein was purified by immobilized metal affinity chromatography. To study the electrical activity of ROMK2-6xHis the planar lipid bilayer technique was used. The impact of known mitoKATP channel modulators on the activity of the ROMK2 protein was tested. The ROMK2 channel was activated by the mitoKATP activator – diazoxide and blocked by mitoKATP inhibitors, i.e. ATP/Mg2+, 5-HD, and glibenclamide. These results confirmed that the ROMK2 protein may be a pore-forming subunit of mitoKATP. Additionally, it was shown that the influence of these modulators was not related to the presence of accessory proteins of ROMK channels. Finally, a functional interaction between ROMK2 and AGK was demonstrated. The activity of the ROMK2 channel was regulated by the products of the enzymatic activity of the AGK protein, i.e. LPA and PA. <br>
In addition to genetic material and molecular machinery necessary for gene expression,cell nucleus contains multiple auxiliary structures that perform diverse functions. Thesestructures include very dynamic and functionally heterogeneous PML (promyelocyticleukemia) nuclear bodies. Their major and crucial constituent is the PML protein. PML nuclearbodies often co-localize with transcriptionally active chromatin and serve as a reservoir ofmultiple other nuclear proteins. Diverse functions of PML-binding partners place PML proteinand PML nuclear bodies at the crossroads of multiple cellular processes, which may beessential for proper animal brain function. The role of PML protein in the adult mouse brainhowever remains to be elucidated. PML expression varies across mouse brain regions and alsodepends on animal age. In adult mice, a relatively high PML expression is observed inhippocampus, a brain structure implicated in learning and memory formation.The main objective of the study described in this thesis was investigation of the role ofPML protein and PML nuclear bodies in shaping the morphology of nuclei of hippocampalneurons and maintaining cognitive abilities of adult mice. To this end, two transgenic animalmodels were generated and analyzed. The first model, with hippocampal PML overexpression,was obtained by introducing an additional PML gene copy be means of injection of AAVs asa gene-carrying viral vector. The second model, with hippocampal PML ablation (knock-out),was obtained using AAVs, which deployed gRNA for PML in a Cre-dependent CRISPR/Cas9system.To characterize spatial and procedural memory formation, operational memory, andanxiety threshold, several behavioral tests were performed within the IntelliCage system. Itwas demonstrated that hippocampal PML ablation impairs operational memory and lowers theanxiety threshold. It was also shown that neither PML ablation nor its overexpression affectspatial and procedural memory formation. Confocal image analysis revealed significantmorphological alteration of neurons’ nuclei and disrupted chromatin organization in the caseof PML overexpression, but not in the case of PML ablation. Histone post-translationalmodifications indicated that in the model with PML overexpression, transcriptionally activechromatin is more densely packed. Moreover, transcriptionally inactive chromatin displays anincreased share of modifications associated with constitutive gene inactivation at the expenseof decreased share of modifications associated with transient gene inactivation. Modulation ofPML level entails commensurate reduction or increase of the nuclear presence of DAXX,a PML partner involved in transcriptional activation of neuronal immediate early genes
In alphabetic languages, learning letters and speech sounds correspondence is the first and one of the most crucial steps in reading development. Research shows that this process differs depending on how transparent the language is (how constant and repetitive the association of letters and speech sounds is, e.g., Italian is highly transparent, Polish and Dutch are moderately transparent, and English is an opaque language). According to the literature, Dutch kids learn letter-speech sound (LS) associations in their first year of formal schooling. From the neuroscience perspective, we know that the left superior temporal cortex (STC) plays an essential role in LS integration. Developmental dyslexia or family risk of dyslexia are factors that may interfere with this process. The process of LS association seems similar in alphabetic languages but has not been thoroughly examined in the blind who read the Braille alphabet using their sense of touch.The principal aim of my doctoral dissertation is to investigate how the process of LS association occurs in the typical and atypical reading development in Polish. In the first behavioral experiment, I checked how much time Polish-speaking children needed to learn the correspondence between LS pairs. As it is the case with the Dutch language, children learn this skill in the first year of schooling, but it takes them longer to automate this process (up to around the third grade of primary school). In the second experiment, I delineated the brain regions that play a role in LS integration in young readers with and without a family history of dyslexia. Children's STC activity during the LS association task varied considerably between those with and without a family history of dyslexia. The at-risk group showed more robust activation when processing congruent LS pairs than incongruent ones, while the no-risk group showed the opposite pattern – higher activation for incongruent LS pairs.In the third experiment, I found significant changes in the pattern of brain activation during the first two years of education. While the brain activity decreases in response to unimodally presented speech sounds (auditory) and letters (visually), it increases when children process multimodal LS pairs.In the last experiment, I checked what the process of LS integration looks in the blind compared to the sighted. The integration process takes place in the STC in both groups. However, the activation pattern is different. The sighted subjects showed higher activity for incongruent LS pairs in the bilateral STC, similarly to children without the family risk of dyslexia in the early stages of learning to read. In the blind, congruent pairs resulted in an increased response in the right STC. These differences may be related to lower exposure to letters in the blind or more sequential processing of Braille as compared to print reading. The experiments that comprise my doctoral dissertation lead to a conclusion that the process of letter and speech sound association in Polish takes place in the STC. Its exact course is influenced by dyslexia, family risk of dyslexia, and reading modality.
In the cortical representation of the visual field, receptive fields (RFs) form a gradient from small sizes in the center to the largest at the periphery. Center and peripheral cortical representation of the visual field differs functionally, center being engaged in sharp vision, whereas periphery in motion and attention. In this thesis I aim to analyze brain activity using functional MRI (fMRI) after visual field loss. The thesis is divided into two studies: first is devoted to the analysis of the receptive field (RF) adaptation in primary (V1), secondary (V2), and third (V3) cortical visual areas by population RF (pRF) mapping, second describes motion-based acuity by measuring individual thresholds and establishing whole-brain activations. We gathered two large groups of patients with long-term photoreceptors degeneration: Stargardt (STGD) with loss of the central retina and Retinitis Pigmentosa (RP) with loss in the peripheral retina. We also modelled peripheral vision loss in healthy participants by transiently limiting the visual field bilaterally to 10 degrees. In the first study, we found in V1, that the pRF size increased bilaterally in RP and controls in limited vision, as compared to the controls in full vision. In STDG, we found a clear separation between dorsal and ventral pRF responses, with pRF size increasing significantly only in the dorsal subdivision of V1. The response in V2 and V3 differed depending on the nature of the loss. Both controls in limited vision and RP patients showed a decrease in pRF size in V2 and V3. On the contrary, in STGD, we observed an increase in pRF size, not only in V1, but also in V2 and V3. Interestingly, in the STGD patients, this increase of pRF sizes was predominantly occurring within the dorsal subdivision of the visual cortex. In the second study, fMRI results indicated distinct functional impairments in RP patients that differed from transient loss of peripheral vision in controls in limited vision. RP patients exhibited higher thresholds for motion-acuity tasks in negative contrast and fast velocity conditions. RP patients when tested in fMRI using the same motion-acuity test, showed significantly lower activations within the cortical representation of the peripheral visual field in V1-3, in line with the behavioral response to the fast velocity in negative contrast stimuli, likely reflecting peripheral vision loss. Outside the visual cortices, we also found higher responses of putamen and dorsal anterior cingulate cortex for the RP patients, likely pointing to a faster adaptation to new stimuli for long-term loss of vision compared to transient loss of vision in controls. Results described in the thesis provides further insight into the interplay between visual field loss and cortical reorganization, emphasizing the role of dorsal subdivisions in compensatory adaptations. The findings extend the understanding of visual system plasticity and possibly direct potential therapeutic approaches for STGD and RP treatments. <br>
Kinesin-1 is a motor protein that converts energy from ATP hydrolysis into mechanical movement. This motor protein "walks" on the microtubule (MT) towards its plus end. Kinesin-1 is a heterotetramer composed of two heavy chains and two light chains. The heavy chain consists of a motor domain (amino terminus) containing a catalytic center, a neck linker, a coiled-coil domain and, at the carboxy terminus, a tail which is responsible for cargo binding. The main role of kinesin-1 in cells is to transport various cargoes from the cell body to its distal parts and to reorganize the microtubular cytoskeleton.The two kinesin-1 heavy chains contain two MT binding sites - one in the motor domain, and the other in the tail. As a result, kinesin-1 can simultaneously interact with two MTs, and cross-link them or move them against each other. In the neuron, this process is necessary during axon formation - mechanical pressure on the cell membrane initiates and directs the formation of a neurite. However, still little is known about the mechanism of MT sliding by kinesin-1, the mutual orientation of the MTs during movement, the way kinesin molecules bind between the MTs, and the regulation of these processes.In order to investigate the mechanism of MT-pair sliding driven by kinesin-1, a new in vitro motility assay was developed. Full length recombinant dimeric kinesin-1 without light chains was tested. Both kinesin-1 and MTs were marked with fluorescent dyes which enabled their individual observation and visualization using a total internal reflection microscopy (TIRFM). This technique allowed the simultaneous observation of the kinesin-1 as well as the cargo MT and stationary MT, which contained different proportions of the fluorescent dye in order to distinguish them from each other.Analysis of the movies obtained from TIRFM showed that the average velocity of MT-MT sliding by kinesin-1 (120 nm/s) is much lower than for kinesin-1 single molecules (600 nm/s) or in gliding assay (1200 nm/s). Contrary to the other two analyzes, MT-MT sliding was not smooth, there were visible breaks in movement, and there were often significant interruptions during MT transport. Experiments with polarity marked MTs showed that there were 3 ways of MT-MT sliding and stationary MT orientation: sliding of the anti-parallel MTs to the plus end, parallel MT sliding towards the minus end, and parallel sliding to the plus end of the stationary MT. Unexpectedly, it turned out that not only anti-parallel MTs are transported, as observed for other motor proteins, but also parallel MTs, which, according to the mechanism suggested in this thesis, is the result of the processive generation of movement by kinesin-1.Kinesin-1 can undergo autoinhibition – a change in conformation that prevents movement, which could make it difficult to repeatedly observe the process of MT sliding. To eliminate the flexible kinesin fragment necessary for autoinhibition (molecular hinge- 2, Δ505-610), a construct lacking hinge-2 was created using molecular biology tools. It turned out that hingeless kinesin-1 performed MT-pair sliding 6 times less frequently, with run length 5 times lower comparing to wild-type kinesin-1. Thus, the presence of an elastic fragment is necessary to compensate the lack of synchronization among the kinesin-1 molecules transporting the same MT.The effect of post-translational modifications of tubulin on MT-MT sliding by kinesin-1 was also investigated. It has been shown that for detyrosinated microtubules, the duration of the movement is longer than for the tyrosinated ones. Concerning glutamylation of microtubules, a lower percentage of movement interruptions was observed as compared to the control. On the other hand, the analysis of the velocity and run length of transport showed no significant effects of the post-translational modifications of tubulin investigated here
Lately, we can observe a steady increase in the number of autism spectrum disorder (ASD) diagnoses. The ASD population is characterised with deficits in social interactions and communication, as well as the presence of stereotyped behavior. Lack of social skills often manifests itself through empathy impairment. Until recently, empathy was thought to occur only in humans, but a growing body of research indicates that emotional contagion - the simplest form of empathy, is widely found in nature, including in primates, marine mammals, birds and rodents. Despite the importance of the phenomenon, there is still little data on the neuronal basis of sharing emotions. Aim of my PhD project was to assess the empathic abilities and the activity pattern within the amygdala and the prefrontal cortex of Fmr1KO(FVB) mice (both males and females) - commonly used mouse model of ASD. To study this phenomenon, I employed the Remote Transfer of Fear paradigm, in which mice are housed in pairs for three weeks, one labelled an Observer, and the other a Demonstrator. In the test session, the Demonstrator is subjected to aversive stimuli outside of the home cage, while the Observer remains there undisturbed. Then, the Demonstrator returns to the home cage, where it can freely interact with the Observer and the first nine minutes of interactions are recorded. The activity of the amygdala and prefrontal cortex was assessed using immunohistochemistry against c-Fos protein, a standard neuronal novelty marker. Behavior was measured by using software utilizing machine learning, for automatic pose estimation (DeepLabCut) and automatic classification and recognition of animal behavior patterns (simBA). Behavioral and c-Fos activation pattern results indicated the existence of deficits in emotional contagion in Fmr1KO(FVB) mice. Furthermore, data obtained during this study points to differences in response to stressed partner between females and males, both on behavioral and c-Fos levels. The behavior recognition model created during this study made it possible to study behavior with great accuracy, and after short re-training, can be successfully used in another study. <br>
Learning is the ability of organisms to change their behavior as a result of past experience. Learning enables adaptation to constantly changing environment, and thus proper response to positive (appetitive) and negative (aversive) stimuli. An appetitive stimulus triggers motivational responses aimed at approaching a reward, an aversive on the other hand induces withdrawal (avoidance and/or escape). Learning occurs through synaptic plasticity which is the brain's ability to alter the strength of connections between neurons. On cellular level synaptic plasticity is related to neuronal activity that drives functional and structural changes within synapses. As a consequence a subject can retain past experiences in its memory and adapt its behavior to similar situations in the future. Thus, synaptic plasticity is crucial for the development of appetitive and aversive behavior and organism survival. On the other hand, these mechanisms are also involved in the development of mental illnesses. Therefore, understanding the neuronal mechanisms involved in synaptic plasticity is crucial. In comparison to the neuronal changes underlying aversive behavior those associated with appetitive learning have been poorly characterized. Therefore, the aim of the present study was to map neuronal activity in the brain occurring during appetitive learning. For this purpose, we used a mouse model in which appetitive learning was based on place preference for a 10% sucrose. The c-Fos protein was used as a marker of neuronal activation that is related to plasticity, and its expression was imaged by light-sheet fluorescent microscopy (LSFM). This approach allowed creating a global map showing the increased c-Fos expression in nearly 170 brain structures. Next, in silico analysis of neuronal projections between the most activated brain regions were conducted. It was shown that there is a large number of connections between the central nucleus of the amygdala (CeA) and several other structures that were activated during the training. Next, the types of neurons in which c-Fos expression occurred in response to appetitive learning were identified. Since CeA mostly consists of inhibitory neurons, experiments were conducted on 3 populations of neurons: VIP+, PV+ and SST+. It was shown that during learning of appetitive events, c-Fos is expressed mainly in SST+ neurons and to a small extent in VIP+ and PV+. The last part of the study involved electrophysiological characterization of SST+ neurons in CeA. It was shown that depending on the CeA part, SST+ neurons are divided into two groups that generate a different type of discharge. Furthermore, it was shown that learning positive events results in the increased excitability of SST+ neurons in the CeA. The present study shows that appetitive learning leads to global activation of many brain structures. Moreover, it was found that CeA is crucial for the appetitive learning by the activation of SST+ neurons. <br>
Macular degeneration is the leading cause of vision loss in adults. In this condition, the photoreceptor cells within the central retina, undergo progressive degeneration, leading to a gradual loss of high-acuity vision. During the doctoral research, two series of studies were conducted in order to investigate the impact of visual training on changes occurring after retinal damage: one involving human and the other one using an animal model of central retinal damage. At first, a visual test was designed to measure visual acuity based on motion perception. The motion-acuity test involved distinguishing a circle from an ellipse made up of moving dots. The level of visual acuity was verified in a group of healthy individuals and preliminarily tested in patients suffering from retinitis pigmentosa (RP) and Stargardt disease (STGD). Both patient groups suffer from progressive degeneration of photoreceptors: RP patients in the peripheral retina and STGD patients in the central retina. To assess the feasibility of modeling peripheral retinal damage we restricted the peripheral visual field in healthy controls using welding goggles. Obtained results showed that fast motion of dots in negative contrast is the most difficult for healthy individuals (Kozak et al., 2021). In the second study, an animal model of macular degeneration was used in adult cats, which underwent bilateral photocoagulation of the central retina. Control animals and two groups of cats with retinal damage, naive and trained, were tested. 7 T-MRI scanning was performed before and after lesioning at 5 different timepoints. Cats with retinal lesions performed motion- based visual tasks with higher level of correct responses than control animals. Next, a fixel- based analysis (FBA) of diffusion data was conducted. In naive, untrained cats (RLN), the reduction in FBA metrics was 40-15% compared to the control group. Changes in the metrics affected fibers in the motion-sensitive visual area V5, the superior colliculi, hippocampus, caudate nucleus, and the optic tract. In contrast, trained cats with retinal lesions showed more spatially limited changes, and the reduction in FBA metrics was smaller compared to the RLN group. To track the temporal dynamics of changes after injury, fractional anisotropy (FA) tensor analysis was performed. FA values in naive, lesioned animals increased rapidly after the lesion, while in trained lesioned animals, the values remained stable. Our results show the stabilizing effect of training, which halts or limits the changes caused by the lesion (Kozak et al., 2024). In conclusion, we suggest that visual training focused on motion-related visual functions, specific to visual areas receiving input from the active peripheral part of the retina, may lead to a partial takeover of functions typically dependent on central vision. <br>
Malignant glioma with a wild-type IDH1/2 (glioblastoma, GBM, wt-IDH1/2) is the most common and aggressive brain tumor in adults. Current therapies for GBM are not effective and most tumors recur within months. The development of tumor is associated with deficits of the host's antitumor defense. The goal of immunotherapy is to restore the anti-tumor immune response, and immune checkpoint inhibitors offer the potential to completely eradicate cancer. However, GBM patients respond poorly to immunotherapies because the tumors are characterized by meager infiltration of functional T lymphocytes and NK cells. Myeloid cells accumulating in the GBM (including resident microglia and peripheral macrophages, collectively known as glioma associated myeloid cells - GAMs) are reprogrammed to support the tumor and exert an immunosuppressive effect in the tumor microenvironment. One of the factors responsible for these processes is the SPP1 (secreted phosphoprotein 1) protein secreted by glioma. The aim of this study was to develop new strategies to reactivate the anti-tumor response in malignant gliomas and to sensitize the tumor to the action of anti-PD-1 antibodies. These strategies involve reprogramming the phenotype of GAMs. Using a syngeneic model of glioma in mice, two therapeutic approaches were employed: i) blocking the activity of arginase 1 (Arg1) using OAT-1746 - a new, potent and selective, small- molecule inhibitor of the enzyme, and ii) preventing the action of Spp1 using a short peptide that blocks the Spp1-integrin interaction. Activation of Arg1 in GAMs leads to depletion of L-arginine, a nutrient required for T and NK cell proliferation, while activation of integrin receptors by Spp1 contributes to the induction of the pro-tumor phenotype of GAMs. The effects of immunomodulators on GL261 glioma growth in vivo were assessed using magnetic resonance imaging or the Xtreme intravital imaging. The glioma microenvironment following immunomodulator administration was characterized using multiparametric flow cytometry analysis and transcriptomic profiling (RNAseq) of isolated CD11b+ cells. The antitumor efficacy of immunomodulators administered alone or in combination with PD-1 blockade was assessed. Oral administration of OAT-1746 had no effect on the immune cell composition in the glioma microenvironment, but induced transcriptomic changes in CD11b+ cells immunosorted from the brains of inhibitor-treated mice. Administration of OAT-1746 with anti-PD-1 antibody reduced tumor growth. Intratumorally delivered RGD peptide effectively blocked the reprogramming of GAMs towards a glioma-supporting phenotype, but did not affect tumor size. The use of the RGD peptide in combination with the anti-PD-1 antibody changed the composition and functionality of immune cells and enhanced pro-inflammatory responses, which led to a reduction in tumor size. The presented results indicate that the tested immunomodulators change the glioma microenvironment, enable the influx of effector lymphocytes and support the action of immune checkpoint inhibitor immunotherapy. <br>

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