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INSTYTUT ARCHEOLOGII I ETNOLOGII POLSKIEJ AKADEMII NAUK
INSTYTUT BADAŃ LITERACKICH POLSKIEJ AKADEMII NAUK
INSTYTUT BADAWCZY LEŚNICTWA
INSTYTUT BIOLOGII DOŚWIADCZALNEJ IM. MARCELEGO NENCKIEGO POLSKIEJ AKADEMII NAUK
INSTYTUT BIOLOGII SSAKÓW POLSKIEJ AKADEMII NAUK
INSTYTUT CHEMII FIZYCZNEJ PAN
INSTYTUT CHEMII ORGANICZNEJ PAN
INSTYTUT FILOZOFII I SOCJOLOGII PAN
INSTYTUT GEOGRAFII I PRZESTRZENNEGO ZAGOSPODAROWANIA PAN
INSTYTUT HISTORII im. TADEUSZA MANTEUFFLA POLSKIEJ AKADEMII NAUK
INSTYTUT JĘZYKA POLSKIEGO POLSKIEJ AKADEMII NAUK
INSTYTUT MATEMATYCZNY PAN
INSTYTUT MEDYCYNY DOŚWIADCZALNEJ I KLINICZNEJ IM.MIROSŁAWA MOSSAKOWSKIEGO POLSKIEJ AKADEMII NAUK
INSTYTUT PODSTAWOWYCH PROBLEMÓW TECHNIKI PAN
INSTYTUT SLAWISTYKI PAN
SIEĆ BADAWCZA ŁUKASIEWICZ - INSTYTUT TECHNOLOGII MATERIAŁÓW ELEKTRONICZNYCH
MUZEUM I INSTYTUT ZOOLOGII POLSKIEJ AKADEMII NAUK
INSTYTUT BADAŃ SYSTEMOWYCH PAN
INSTYTUT BOTANIKI IM. WŁADYSŁAWA SZAFERA POLSKIEJ AKADEMII NAUK
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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>
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
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>
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.
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.
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
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>
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
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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>
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
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
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/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.
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
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>
Malignant gliomas are primary tumours of the central nervous system. They remain one of the hardest to treat brain tumours due to their invasive phenotype, the immunosuppressive microenvironment and anatomical localisation in vital areas of the brain. The intratumoural heterogeneity and overexpression of enzymes involved in DNA replication and repair, impair the effectiveness of commonly used therapies, leading to an inevitable relapse of the tumour.The RecQ helicases are considered ‘guardians of the genome’, as they play the fundamental roles in DNA replication, repair and maintaining genome stability. The RecQ family is composed of several structurally related helicases, including BLM (Bloom Syndrome) helicase. The involvement of RecQ helicases in tumorigenesis and responses to therapies in malignant brain tumours is not fully understood, thus we aimed to elucidate the role of BLM in these processes.The Cancer Genome Atlas (TCGA) dataset analyses and immunostaining of numerous tumour sections demonstrated that BLM is overexpressed in high grade gliomas (WHO grade 3 and 4). In malignant gliomas BLM localisation was detected in the cytoplasm whereas in benign tumours BLM was present mostly in the nuclear compartment. High BLM levels were detected in several glioma cell lines and primary patient derived cultures. To decipher the role of BLM in gliomas, BLM deficient LN18 and LN229 cells were generated using CRISPR/Cas9 genome editing technology. BLM deficiency (KO) had minor effects on basal cell viability and proliferation. However, the cell responses to chemotherapeutics, in particular to the combination of temozolomide (TMZ, a DNA methylating agent common used in glioblastoma therapy) and olaparib (OLA, a PARP inhibitor) were changed. Unexpectedly, BLM KO cells were more resistant to the combined treatment than wild type (WT) cells that underwent apoptosis. Moreover, this effect was exclusive for TMZ combined with PARP inhibitors. The BLM KO cells were not sensitive to PARP inhibitors, in contrast to expected the synthetic lethality. BLM KO cells displayed the therapy induced cellular senescence (LN229) or polyploidy (LN18). The polyploidy in p53-deficient LN18 cells was reversed by forced p53 expression. Interestingly, RecQL4-depleted LN18 and LN229 cells responded to TMZ+OLA similarly to WT cells which indicates specialised and non-overlapping functions of RecQ family helicases.To obtain more insights into the functions of distinct RecQ helicases we generated blm and recql4 knock-out in Xenopus frogs to evaluate roles of the helicases in the embryonic development. Considerable mortality of tadpoles was noted and helicase-depleted tadpoles displayed morphological and functional developmental abnormalities.Altogether, the present study demonstrates important and specific functions of BLM and RecQL4 helicases in glioma cell responses to chemotherapy. As BLM expression was highly elevated and inversely correlated with survival of patients with malignant gliomas, we postulate that BLM could be a therapeutic target. The assessment of BLM levels, p53 and MGMT, a DNA repair enzyme, status might be helpful in choosing the proper therapy against malignant gliomas
Malignant gliomas are the most common CNS tumors and the most aggressive one is glioblastoma multiforme (GBM). The treatment of GBM includes tumor resection combined with radio- and chemotherapy, however due to diffuse growth surgical resection is difficult and the blood-brain barrier limits the penetration of many drugs. Thus, the effectiveness of therapy is very low and the average survival of GBM patients is 15 months. Large-scale analysis of gene expression based on data from TCGA database showed significant differences in transcriptional profiles in GBM. This led to identification of three molecular subtypes of GBM: proneural, mesenchymal and classical. Mes-GBM is characterized by the highest aggressiveness and the strongest infiltration of myeloid cells: microglia and monocytes/macrophages (so called GAMs), which in the tumor microenvironment (TME) undergo reprograming into tumor supportive cells. In Laboratory of Molecular Neurobiology IBD PAS, osteopontin (Spp1) and lactadherin (Mfg-E8) were identified as proteins potentially involved in this process. Both Spp1 and Mfg-E8 are integrin ligands and their expression is increased in many cancers. The aim of this study was to assess their role in the pathogenesis of malignant gliomas, in particular in modulation of TME. For this purpose, a rat C6 glioma model was used, in which intracranial tumors bear the histopathological similarities to human GBMs. To further determine a degree of similarity between the rat C6 gliomas and human GBMs, global changes in gene expression were analyzed and compared to signatures specific to human GBM subtypes, showing the greatest similarity to Mes-GBM. Analysis of genes expressed in microglia isolated from C6 gliomas confirmed the tumor-supportive properties of these cells. To verify the hypothesis about participation of Spp1 and Mfg-E8 in the pro-tumoral activation of GAMs and TME modulation, C6 glioma cells with stably silenced expression of Spp1 and Mfg-E8 were used. Significant inhibition of the growth of Spp1 and Mfg-E8 depleted gliomas in vivo was observed. In Mfg-E8 depleted tumors, also the migration of glioma cells into brain parenchyma was restricted. The analysis of the GAMs phenotype showed that both proteins are involved in the induction of the pro-tumoral, immunosuppressive activation. In Mfg-E8 depleted tumors, the percentage of CD3+ cells increased. In Spp1 depleted tumors, an increase in the percentage of cells expressing the pro-inflammatory interleukin 1β was observed, which may indicate the initiation of an efficient antitumor response. The analysis of Arg-1+ and Trem2+ cells in control tumors showed that the proteins are expressed in distinct populations of myeloid cells found in different tumor regions, confirming the heterogeneity of GAMs in glioblastoma. The RGD motif of Spp1 was identified as crucial for a tumor growth. A rescue experiment was performed, in which constructs encoding different variants of Spp1 were used. Reestablishing expression of the wild type Spp1 in glioma cells depleted of Spp1 (shSpp1), Spp1 with a mutation at the thrombin cleavage site or Spp1 with a deletion of the C-terminal region restored tumor growth. Expression of the Spp1 variant with a point mutation in the RGD motif did not restore tumor growth. However, the application of a short peptide containing the RGD motif, which could potentially act as a competitive inhibitor for Spp1 and Mfg-E8, did not affect tumor size and microglial activation in vivo compared to the control peptide (SCR). Altogether, this study demonstrated that Spp1 and Mfg-E8 are crucial for in vivo tumor growth and reprograming of the TME, especially for the activation of GAMs. Although Spp1 in cancer shows a pleiotropic effect and different regions of the protein are important in many aspects of tumor development (such as adhesion, migration or interaction with GSCs), the RGD motif and its interactions with target integrins play the key role in tumor growth in vivo.
MBD3 (methyl CpG binding domain 3) protein belongs to the MBD family of proteins, responsible for reading the DNA methylation pattern. MBD family proteins bind the methyl-CpG domain and are also involved in heterochromatin formation. Interestingly, MBD3 protein does not have the ability to selectively recognize methyl-CpG islands, however, its characteristic feature is the ability to bind to 5-hydroxymethylcytosine and unmethylated DNA. A study by Bednarczyk and colleagues (Bednarczyk et al. 2016) using a rat model of temporal epilepsy induced by electrical stimulation of the amygdala showed an increase in NuRD complex proteins, including Mbd3 protein, in the brain of epileptic animals. A greater number of regions of DNA to which the NuRD complex, containing the Mbd3 protein, attaches was also observed in stimulated animals, compared to a group of control animals. The aim of the experiments conducted in this dissertation was to investigate whether the Mbd3 protein participates in the processes leading to changes in the seizure threshold. The effects of pentylenetetrazole (PTZ)-induced convulsions on Mbd3 protein levels and Mbd3 mRNA expression in vivo were investigated. An increase in Mbd3 protein levels was demonstrated in the entorhinal cortex and amygdala of rats 4 hours after the induced convulsion.The effects of decreasing and increasing Mbd3 protein levels on seizure threshold in vivo were further evaluated. Commercially designed AAV viral vectors were used to modify Mbd3 levels. It was shown that lowering the level of Mbd3 prolongs the latency time to the onset of a convulsion induced by PTZ injection.Behavioral experiments have shown that downregulation of MBD3 protein increases anxiety in animals. In contrast, overexpression of Mbd3 decreases anxiety responses in animals and increases their excitability and activity in the open field test. The effects of decreasing and increasing Mbd3 protein levels on epileptogenesis were also investigated in a kindling model using PTZ. Elevation of Mbd3 protein levels was shown to accelerate epileptogenesis and the development of tonic-clonic seizures. In order to identify the role of Mbd3 protein in the regulation of gene expression, in vitro experiments were conducted using a model of magnesium deficiency-induced epileptic-like discharges. Overexpression of Mbd3 in vitro was shown to induce changes in gene expression in a time- and state-specific manner in neurons.The data obtained from this project indicate the involvement of the Mbd3 protein in epilepsy pathology.
Mitochondria are specialized, multifunctional and dynamic organelles involved in many processes in the cell. They are the main place for the generation of chemical energy (ATP) as well as the formation of reactive oxygen species (ROS). Due to the wide range of performed functions, mitochondria play a key role in signal transduction in the cell. For instance, they are first to react to stress conditions, and one of the signalling pathways that enables them to adapt to those changes is the mitochondrial retrograde signalling cascade, i.e. the mitochondria− nucleus−mitochondria signalling. Such feedback signaling and mitochondrial adaptation allow to keep the cell in good condition. Mitochondrial dysfunction has been observed in many diseases and in the process of cellular ageing. However, research on mitochondrial metabolism in early ageing is still lacking. In addition, natural compounds are sought that, through their properties, can contribute to delaying the aging process. The study aims to investigate how mitochondria adapt in the early stage of phase of induced (which was caused by short-term oxidative stress by tert-butyl hydroperoxide) and replicative (depending on telomere shortening) ageing in the primary line of fibroblasts and to investigate the influence of the phytoestrogen - daidzein on these processes. Therefore, the elements leading to mitochondrial adaptation through regulation of the retrograde signalling cascade were characterized and mitochondrial dynamics processes such as network reorganization, mitochondrial biogenesis and autophagy/mitophagy were investigated. In the early stages of ageing, both accelerated and replicative, a reverse signalling cascade is induced by elevated levels of ROS in the cell. In response to stress caused by ageing, mitochondrial adaptation is disparate for the model of induced and replicative ageing. Studies on the effect of phytoestrogen daidzein on the ageing process have shown that it has a positive influence on the functioning of mitochondria in both types of ageing. A complete and thorough analysis of mitochondrial dysfunction may become an attractive strategy to delay ageing and age-related diseases. <br>
Mitochondria carry out a variety of important cellular functions including ATP synthesis as well as reactive oxygen species production. They are also implicated in many crucial cellular processes, in regulation of the levels of several substantial for the cell metabolites and e.g., in the initiation of the apoptosis process. Therefore, it is not surprising that mitochondria of tumor cells could be a potential target in chemotherapy. Substantial number of evidence indicate that both, apoptosis and reactive oxygen species production involve a small p66Shc adaptor protein, which demonstrates the unique prooxidative properties comparing to other ShcA family members (p46Shc and p52Shc). Taking into account the fact that p66Shc can play a dual role as a negative regulator of proliferation and as oxidative stress sensor, p66Shc seems to be a promising target concerning cancer proliferation, tumor progression and chemotherapeutic treatment.My doctoral dissertation presents a comprehensive evaluation of the role of p66Shc protein in mitochondrial physiology of breast cancer cells. Moreover, describes response of these cells to chemotherapeutic treatment with the use of doxorubicin agent. Furthermore, the use of human breast cancer cell lines (MDA-MB-231 and MCF-7) and their genetically modified clones presenting different level of p66Shc protein allowed me to demonstrate how the p66Shc protein can affect the mitochondrial metabolism of human breast cancer cells.The comparative analysis of two breast cancer cell lines characterized with relatively different level of p66Shc and their noncancerous equivalents (MCF-10) have revealed that both tumor cell lines: MDA-MB-231 and MCF-7 being two various subtypes of breast cancer and characterized with different metastasis abilities significantly differ in most of studied cellular parameters. Furthermore, changes in the level of p66Shc protein (in individual breast cancer cell lines) exert different effects in the same clones respectively: MDA-MB-231 and MCF-7 tumor cell lines (overexpressing p66Shc protein, overexpressing of Ser36-mutated p66Shc as well as knockout of p66Shc). Knocking out p66Shc in both breast cancer cell lines caused significant changes observed mostly in mitochondrial physiological parameters. Interesting, in both of examined breast cancer cell lines, I did not found positive correlation between overexpression of p66Shc (containing serine 36 residue responsible for the prooxidative properties of the p66Shc protein) and the level of reactive oxygen species. I have shown that clone of MDA-MB-231 (which is more metastatic type of breast cancer comparing to MCF-7 cell line) lacking p66Shc protein represents the most glycolytic phenotype comparing to other
Myosin VI (MVI) is a unique motor protein that, unlike other myosins, moves toward the "minus" end of actin filaments. MVI is involved in migration, adhesion, exo- and endocytosis, stabilization of the Golgi apparatus structure, cytokinesis and gene expression. Its involvement in these processes involves cargo transport and/or cargo anchoring with actin filaments. The presence of MVI in the nucleus has been known since 2006, when it was shown that in transcriptionally active HeLa cells, MVI colocalizes with the RNA polymerase II complex (Pol II) and with newly formed mRNA transcripts. Subsequent studies conducted in the Laboratory of Molecular Basis of Cell Motility, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, on PC12 neurosecretory cells derived from rat adrenal medulla tumor confirmed these observations. Moreover, it was shown that after stimulation of PC12 cells with KCl the level of MVI in the nucleus increases, and this is accompanied by an increase in its colocalization with the active form of Pol II. In addition, mass spectrometry analysis allowed for identification of a number of new potential nuclear partners of MVI, which may determine its role in the cell nucleus. Among them was nucleolin, a nucleolar protein involved in ribosome biogenesis as well as S6, a structural protein of the small ribosomal subunit (40S). The lack of literature data on the function of MVI in the nucleolus encouraged me to investigate the potential role of this motor protein in this region of the nucleus.In my study, I confirmed the interaction of MVI with nucleolin [a protein characteristic of the granular component (GC) of the nucleus, where the assembly of ribosome subunits takes place] and the ribosomal protein S6 (involved in the export of the small subunit of the ribosome from the nucleus to the cytoplasm). I also showed that other nucleolar proteins also interact with MVI. These included upstream binding factor (UBF) [characteristic of the fibrillar center (FC) of the nucleolus, where rDNA transcription takes place], fibrillarin [characteristic of the dense fibrillar component (DFC) of the nucleolus, where pre-rRNA maturation takes place] and B23 protein [characteristic of the GC]. Thus, the interaction of MVI with nucleolar proteins and S6 suggests the involvement of MVI in various steps of ribosome biogenesis. Using electron microscopy, I determined the subnuclear localization of MVI; MVI is mainly found in the DFC and the GC region. Further analysis using electron microscopy showed that under actinomycin D (ActD)-induced nucleolar stress, the nucleolus disintegrates. In addition, I observed that in MVI-knockdown cells the ultrastructure of the nucleolus was less compact compared to control cells, which might indicate that MVI is involved in the maintaining the normal organization of the nucleolus. Moreover, confocal microscopy analysis showed that MVI is involved in the localization of the nucleolar protein B23 under control and nucleolar stress conditions, which may suggest the functionality of these interactions. In addition, I observed that reduced level of MVI is accompanied by disruption of the organization of the endoplasmic reticulum (ER). Also, I observed that in cells with reduced levels of MVI, staining against the ribosomal protein S6 was significantly less intense compared to control cells, which may suggest the involvement of MVI in the organization and function of ribosomes. In contrast, I did not show an effect of reduced MVI levels on 45S pre-rRNA level.In summary, my results indicate that MVI is involved in the organization of the nucleolus and ER but does not play a significant role in rDNA transcription. Since the maintenance of proper nucleolar organization is important for ribosome biogenesis (the disruption of which leads to many serious diseases), the results presented here open the possibility for further research into the mechanisms of MVI function in the nucleolus
The neuromuscular junction (NMJ) is a chemical synapse of the peripheral nervous system that enables skeletal muscle contraction through acetylcholine-mediated neurotransmission. This synaptic structure is composed of three main elements, motor neuron, myofiber, and Schwann cells, that tightly regulate one another throughout ontogenesis via multiple signaling pathways. Disruption of these pathways can result in neuromuscular disorders that reduce the efficiency of muscle contraction and lead to muscle fatigue, wasting, and, in some cases, premature death. The cause of many neuromuscular disorders remains unknown to this date and any cure or treatment is often missing. Studying the molecular mechanisms underlying NMJ function in physiological and pathological contexts could shed light on these unmet clinical needs and even lay the path to discovering therapeutic targets. Impairments in cellular processes regulating the presence of acetylcholine receptors (AChRs) on the myofiber surface are one of the most devastating for proper NMJ function. Most importantly, many signaling pathways and developmental processes involved in receptor clustering and maintenance remain poorly understood, and researchers continue to unveil new roles for proteins whose relevance at the NMJ had been unknown before. The actin cytoskeleton and actin-remodeling proteins are at the core of many postsynaptic-regulating processes, including local delivery and recycling of synaptic components, stabilization of postsynaptic complexes, and recruitment of other cytoskeletal filaments. Through my PhD research, I identified and characterized for the first time some of the functions of the cytoskeletal regulators drebrin and myosin VI in the formation and maintenance of the postsynaptic machinery. Specifically, I found that drebrin plays an important role in AChR clustering and maturation through its ability to rearrange actin filaments. Moreover, its mechanisms of action at the postsynaptic machinery seem to involve microtubule organization and interaction with rapsyn, a key AChR clustering mediator and stabilizer. Myosin VI, on the other hand, does not seem to be crucial neither for NMJ formation nor maturation, however its absence in mice significantly impairs NMJ structure and leads to reduced muscle strength, particularly in females. Altogether, my results provide an insight into the mechanisms through which drebrin mediates AChR cluster formation, maturation, and maintenance, as well as the functional consequences of myosin VI loss in the skeletal muscle of mice
Non-alcoholic fatty liver disease (NAFLD) affecting approximately 24% of the worldwide population is the main leading cause of chronic liver disease. The increased number of fatty liver cases considered as the hepatic manifestation of the metabolic syndrome, is rising in parallel with obesity and type 2 diabetes. This is mainly associated with the sedentary habits and overconsumption of hypercaloric diets. The initial stage of NAFLD - non-alcoholic fatty liver (NAFL) is characterized by a metabolic remodelling of the liver to compensate the overload of fat accumulation. Although, this compensatory event seems to be abolished during disease progression. Along this process, mitochondrial function impairment and an exacerbation of oxidative stress have been described to trigger hepatic signalling pathways associated with the initiation of inflammation, fibrosis and cirrhosis (transition to non-alcoholic steatohepatitis (NASH)). Despite the advances in the field, the primary mechanisms underlying the development of NAFL and its progression into NASH are complex and still incomplete. In this context, my first aim was to study the hepatic and mitochondrial redox-associated alterations in a NAFL stage. I have characterized hepatic proteome, mitochondrial structure and function, reactive oxygen species (ROS) production and antioxidant defences in a mouse model of early NAFLD stage. Induction of NAFL resulted from a chronic feeding (16 weeks) of mice with different diets: high-fat, high-sucrose or high-fat plus high-sucrose diets. I have shown (see Chapter 1), that no excess of mitochondrial ROS took place in NAFL. Therefore, I have suggested that other organelles as peroxisomes rather than mitochondria contribute to hepatic oxidative stress. Moreover, I established that fat and sucrose (components of Western diet) differentially impair autophagy. In the second aim, I investigated the specific end-points for mitochondrial dysfunction that represent “a point of no return” and which drive disease progression along time. NAFL development has been studied with the use Western diet (WD) in an early NAFLD stage mice model. The combination of high-fat and high-sucrose representing Western diet good resemble human NAFLD features of the disease development. In this part of my thesis (see Chapter 2), I demonstrated for the first time the sequential events of mitochondrial alterations during NAFL development and progression. I showed that in a more progressive NAFL stage previously observed mitochondrial adaptation in NAFL was followed by a progressive decrease of mitochondrial respiration concomitant with a higher susceptibility to mitochondrial permeability transition pore (mPTP) opening. Importantly, it was proven that mitochondrial ROS are not the first hit causing disease progression. Instead, my findings continue to support the role of peroxisomes as possible contributors to the hepatic oxidative damage in the origin of hepatic injury and progression of the disease. <br>
Opis syntezy hemopyrrolu
The opossum, Monodelphis domestica is a small, omnivorous marsupial species from Brazil. Like other marsupials, opossums are born at an earlier stage of development than eutherians. Newborn opossums weighing only 100-120 mg, are at a developmental stage comparable to that of mice at day 12 after conception, and at 6 weeks of embryonic development in humans. Almost all brain structures, including the neocortex, develop within three weeks after birth, which is slower than in mice or rats. The slow and protracted postnatal development of the opossum brain allows to study early stages of the development in the mammalian brain structures.Neurotrophins and their receptors play an important role in processes involved in shaping the nervous system. They have been shown to be essential for division, migration, survival and differentiation of progenitor cells, as well as in synaptic plasticity which is crucial for learning and memory.The aim of the current study was to investigate the role of neurotrophin receptors, mainly TrkB, a specific receptor for brain-derived neurotrophic factor (BDNF) and TrkC, a specific receptor for neurotrophin 3 (NT-3) in the development of two brain structures, the neocortex and the cerebellum. The in vivo electroporation technique was used. The shRNA construct for the trkB or trkC gene was injected into the lateral ventricles of the opossum brain at postnatal day 7 (P), when deep layers of the cerebral cortex begin to form. The effect of injections on the development of neocortex in opossums was analyzed 2 or 5 days after electroporation. The inhibition of TrkB or TrkC receptors activity in the neocortex of opossums at P7, resulted in a reduction of the number of proliferating progenitor cells in vivo, which was not due to an increase in apoptosis. Moreover, the lack of TrkB or TrkC receptors activity stopped the migration and caused the arrest of newborn neurons in the intermediate zone, therefore, they could not penetrate the subplate zone.In order to study the role of neurotrophin receptors in development of the opossum cerebellum, first, developmental sequences and timing of different types of cerebellar cells generation were examined using BrdU injections into opossums at different ages (from newborn to adult). After a month or three, the immunohistochemical staining was performed to label BrdU cells of the cerebellum and detect specific cellular markers in various cell types. The presented study showed that Purkinje neurons and deep cerebellar nuclei cells are formed between P1-P5. Approximately three weeks later, the proliferation of granule cells started. Cerebellar cell cultures from P3 or P22 opossums were used to study the influence of the TrkC receptor on the development of Purkinje or granule cells, respectively. The plasmid shRNA targeting trkC was used to reduce TrkC receptors activity in cells that were cultured for the next 8 days after electroporation. Analysis of dendritic arbor structures showed that TrkC downregulation resulted in an increase in the number of dendrites and branching of Purkinje cells but had no effect on cerebellar granule cells. The presented results show that the selected neurotrophin receptors have a significant impact on the proliferation and migration of neocortical progenitor cells and development of the dendritic tree in Purkinje cells of the opossum cerebellum, demonstrating region-specific and cell-specific effects
P2X7 is an ionotropic nucleotide receptor that acts as a cation permeable channel upon ATP stimulation. This receptor can also form a large transmembrane pore or transmit an ATPdependent signal without creating a channel at all. P2X7 receptors control many physiological and pathological cellular processes, and their increased expression is often associated with tumor progression. Since nucleotides are important signaling molecules in the central nervous system, P2X7 also plays an important but ambiguous role in glioblastoma biology. Therefore, our research aimed to investigate the expression and function of the P2X7 receptor in three human glioblastoma cell lines (U-138, U-251, LN-229) and in one rat glioma cell line (C6). Although the receptor mRNA and protein were detected in all the studied cells, we found profound differences in their level. In U-138 human cell line, the receptor seemed to be inactive, while in U-251 human and C6 rat cell line its activation resulted in calcium influx and large pore formation. The viability of studied cells upon the administration of specific P2X7 agonist – BzATP – was not affected for U-138 and U-251, whereas for C6 cells a stimulatory effect was observed. This process is accompanied by an increase of prosurvival proteins expression (CD133, HSPA1, HSPA5) as well as an increase in phosphorylation of kinases influencing the progress of the cell cycle (Akt and p38 MAPK). It was also shown that P2X7 activation promoted cell adhesion, mitochondria depolarization, and overproduction of reactive oxygen species in C6 cells in vitro. The effect of the P2X7 receptor on the growth of C6 glioma tumors in vivo was also investigated. These results are in the line with the majority of the data obtained in vitro. The administration of BBG, a P2X7 inhibitor, effectively inhibited growth of the tumor mass and tumor development, reduced the amount of ATP with a simultaneous decrease of cancerassociated pro-survival protein expression. A decreased level of negative prognostic cancer markers (CD133, HSPA1, HSPA5, Akt, p38 MAPK, NOS-2) and proteins related to the epithelial-mesenchymal transition (N-cadherin, vimentin, β-catenin) were noted. It has also been shown that the P2X7 receptor may be involved in shaping the glioblastoma tumor microenvironment by modulating the immune response and regulating the level of inflammatory markers. These data bring some new insight into P2X7 influence on the biology of glioma. For the first time, the results showing the receptor-promoting effect on the proliferation of glioma cells in vitro were shown in correlation with the growth of neoplastic tumors in vivo. Moreover, the cell signaling pathways were investigated to elucidate the molecular mechanisms activated by P2X7 receptor in glioblastoma cells as well as the receptor engagement in shaping of glioma tumor microenvironment through modulation of inflammation marker profile. <br>
Patients with major depressive disorder (MDD) and borderline personality disorder (BPD) face difficulties in autobiographical memory (AM) recall and emotion regulation (ER). Despite frequent co-occurrence of MDD and BPD, they are rarely studied together and compared to each other, especially using a neuroimaging methodology. The main goal of the dissertation was to compare MDD and BPD in autobiographical memory and emotion regulation processes at behavioral and neural levels.The functional magnetic resonance imaging (fMRI) study comprised two tasks, carried out with three groups of women: diagnosed with MDD, diagnosed with BPD, and healthy control (HC). The AM task regarded the recall of sad and happy memories. In the ER task participants were instructed to use one of two ER strategies: cognitive reappraisal or mindful acceptance while looking at sad pictures.In the AM task, the MDD group experienced more sadness than the HC after the sad recall, while BPD participants experienced less happiness than HC after the happy recall. No significant differences were found between the MDD and BPD groups. The emotional autobiographical memory recall in all participants taken together led to the engagement of brain regions previously reported as crucial for this process However, there were no significant differences between the groups. The functional connectivity analysis of the main effect of recall revealed significant connections between all the above-mentioned regions involved in autobiographical memory recall for all participants. The only group difference was found between the MDD and BPD groups taken together, and the HC group. During recall of sad and happy memories, the clinical groups had a significantly stronger connection between the left precuneus and the right occipital cortex, as compared to the HC group.In case of the emotion regulation task, the behavioral results showed that each group rated their emotional state as less sad after using either of the strategies than after passively viewing sad pictures. Moreover, ratings of emotional state were less sad after the CR regulation than after MA, even though participants rated themselves as more successful in following MA’s instructions. There were no significant between-group differences in ratings of the emotional state after the strategies. Analysis of the neuroimaging data for both emotion regulation strategies taken together showed broad activations within brain regions previously associated with emotion regulation, such as the thalamus, middle cingulate, prefrontal, occipital, temporal, and insular cortices. No significant between-group differences were found. The functional connectivity analyses did not reveal any significant results.Although the between-group results were mostly statistically insignificant, results of the autobiographical memory task indicate several group differences. The neuroimaging result differentiating the groups showed stronger functional connectivity between the left precuneus and the right occipital cortex during emotional recall in the clinical groups than in the HC group. One possible explanation of this result is that in these disorders vivid autobiographical memory recall requires stronger cooperation of regions engaged in visual imagery (occipital cortex) and in recollection of contextual details (precuneus).
Potassium channels present in the inner mitochondrial membrane are involved in the regulation of many cellular processes. Activation of the mitochondrial large conductance calcium regulated potassium channel (mitoBKCa) protects the cardiomyocytes and brain cells during ischemia/reperfusion. On the other hand, inhibition of the mitochondrial Kv1.3 channel activity increases the cancer cells death. The experiments described in this dissertation focused on electrophysiological and biochemical studies on identification and characterization of the mitochondrial BKCa channels in selected model cell lines and the search for new low molecular weight modulators (activators and inhibitors) of this channel activity. Characterization of new mitoBKCa channel modulators could contribute to a better understanding of the mechanisms leading to both cytoprotection and cell death.The aim of this dissertation was to identify the mitochondrial BKCa channel in human bronchial epithelial cells and to compare the activity of this channel with the exogenous mitochondrial VEDEC isoform of BKCa channel.The first part of the dissertation presents the results of electrophysiological studies using the patch-clamp technique, which allowed to characterize the mitoBKCa channel in bronchial epithelial cells. It is worth noting that this is the first description of a mitochondrial potassium channel in this type of cell in the literature. Then, experiments on the properties of the mitochondrial VEDEC isoform of the BKCa channel were described in the new developed research model HEK293-BK_DEC cells.In the next part of this dissertation, the influence of low molecular weight synthetic compounds and of plant origin on the activity of the described mitoBKCa channels was examined. One of the investigated naringenin derivatives, 7-O-prenyl-naringenin, has been shown to activate the mitochondrial channel that occurs endogenously in bronchial epithelial cells and exogenously in HEK293-BK_DEC cells. It was also found that another derivative of flavonoids - chalcone 4 ', 5, 7-tri-O-methyl-naringenin completely inhibits the activity of both tested channels. Experiments were also carried out to identify new potassium channel inhibitors with a structure similar to the channel inhibitor - paxilline. Selected compounds reduced the open probability of the mitoBK_DEC channel but not as effectively as the commonly used paxilline.In conclusion, this dissertation presents the new localization of the mitochondrial BKCa channel in bronchial epithelial cells and investigates the properties of the VEDEC isoform of this channel in the newly developed HEK293-BK_DEC cell model. The effect of potential new modulators, synthetic and natural compounds on the activity of mitoBKCa channels are also presented
The process of aging is a complex biological phenomenon that results in a decline in cellular function and tissue degeneration. Late-onset Alzheimer's disease (LOAD), the most prevalent cause of dementia, is a neurodegenerative disease that is often diagnosed in advanced stages. The etiology of LOAD is multifaceted and includes lifestyle, environmental, and genetic factors. Detecting neurodegenerative diseases early is crucial for global healthcare and for affected individuals, as it enables the potential for early prevention and treatment. Therefore, understanding how Alzheimer's disease risk genes impact the brain function of healthy individuals is crucial in advancing this process. This dissertation describes a study on the relationship of LOAD risk genes and brain function/structure and basic health indicators in middle-aged individuals without symptoms of dementia. A genetic screening involving 200 participants was conducted to assess two LOAD risk genes: APOE (encoding apolipoprotein E) and PICALM (encoding phosphatidylinositol binding clathrin assembly protein). A comprehensive demographic data was collected, along with a battery of psychometric tests. Based on the screening results, distinct groups were defined including individuals with no risk (N), carriers of risk variant exclusively in the APOE gene (A+P-), and carriers of risk variants in both the APOE and PICALM genes (A+P+). The groups differences were studied with neuroimaging techniques, involving both structural methods (magnetic resonance imaging, MRI) and functional approaches (electroencephalography, EEG; and functional MRI, fMRI). Extended blood tests were also performed, including microRNA panel associated with Alzheimer's disease. The groups were similar in demographic characteristics, and most psychometric tests yielded comparable results. Importantly, no differences between control and risk groups were found in memory abilities as assessed by The California Verbal Learning Test (CVLT). In terms of health indicators, the at-risk groups differed from control group in standard blood test parameters, showing slightly elevated levels of eosinophils and hemoglobin content in red blood cells. Analysis of circulating miRNAs in plasma revealed downregulation of miR-29b- 3p, a trait reported in scientific literature as characteristic of Alzheimer's disease (AD) patients. The findings indicated a reduction in the complexity of the EEG signal and a phenomenon termed “slowing” of the EEG. Analysis of brain responses during MSIT showed differences between the study groups in the components related to the attention and cognitive control (N2 event-related potential component) and during response execution phase (late sustained potential, LSP). In terms of brain structure, a reduced thickness of the cerebral cortex is one of the symptoms of Alzheimer's disease; our study showed similar changes in the right temporal pole for individuals with risky gene variants. fMRI connectivity showed significant alterations in small clusters within some areas (e.g., posterior cingulate cortex) linked to the default mode network (DMN). Furthermore, task-related fMRI revealed differences in brain activation between the groups in areas partially associated with this network. Disruption of the DMN is frequently observed in the neurodegenerative diseases. Moreover, alterations in regions linked to the so-called signature of Alzheimer's Disease, such as the angular gyrus, inferior temporal gyrus, and supramarginal gyrus, were also found. An important finding is the absence of a linear accumulation of effects along the risk level axis. APOE and PICALM seem to influence the organism in a complex way. Longitudinal studies are essential to identify individuals from our experimental groups, who will eventually develop symptoms of the disease. <br>
Relationships between individuals that make up a group develop through a series of social behaviors, such as establishing hierarchies and forming bonds. Social learning, or the ability to gain information from the behavior of other individuals, is one of the most essential elements of the social behavioral repertoire of mammals. The presented dissertation describes the development of experimental protocols for evaluating social learning in groups of mice tested under semi-natural conditions. For this purpose, the Eco-HAB system was used. The Eco-HAB reflects the most important features of the mice's natural environment, while allowing fully automated evaluation of social behavior. The developed protocols were then used to study how information about the location of rewards in the environment spreads among individuals in the group, and how social relationships in the group, including the social network, affects responses to social information about food. It was discovered that mice have the ability to effectively learn about rewards found in the environment by other familiar individuals based on the scent traces the conspecifics leave behind, without the need for direct contact with group members. In addition, it was discovered that the effectiveness of social learning depends on the social hierarchy and structure of the social network. Namely, individuals that are the centers of the network, show the most intense response to social stimuli associated with reward. What is more, a pioneering way to study the above-described parameters in a new habitat (inter- environmental transfer of social information) previously populated only by "scout" mice was also developed. The creation of the aforementioned experimental protocols made it possible to conduct research on the neural mechanisms of social learning. The second part of this dissertation describes studies on the importance of neuronal plasticity in the prelimbic part of the prefrontal cortex for the ability to learn socially from group members. For this purpose, specially designed nanoparticles containing TIMP1 (Tissue Inhibitor of Metalloproteinases 1), an inhibitor of the enzyme MMP9 (Matrix Metallopeptidase 9) which is key for synaptic plasticity, were used. MMP9 in the brain is involved in the formation of neural connections by regulating the maturation of dendritic spines. In addition, numerous studies show that, at the behavioral level, manipulation of MMP9 activity affects learning and memory consolidation. The nanoparticles used in this study, when injected into the brain, gradually release their contents, which made it possible to manipulate neuronal plasticity over a long period of time, and thus observe behavioral effects over many days. Over the course of the study, it was shown that the ability of mice to transmit information and learn the location of a potential reward based on the odor traces left in the environment depends on the undisturbed activity of MMP9 protein in the prelimbic part of the prefrontal cortex. It was discovered that reducing the level of MMP9 protein activity with its inhibitor TIMP1 decreases reward-seeking motivation in response to socially transmitted reward information. Moreover, the described manipulation of neuronal plasticity has been shown to interfere with the animals' ability to use social information in novel environments. It also resulted in significant remodeling of the social networks and, consequently, the in-group social interactions. The findings of the presented study are a significant contribution to the development of knowledge about social learning and the underlying neuronal mechanisms. In particular, the development of new experimental protocols contributes to the versatility of the Eco-HAB, the automated system for tracking social behavior developed at the Nencki Institute. <br>
Schizophrenia still remains a significant burden on patients, societies and healthcare systems. Current therapies focus mainly on alleviating only the positive symptoms, and, at the same time, cause numerous side effects. Furthermore, a large number of patients doesn’t respond to the treatment. Due to that there is a strong medical need to develop innovative drugs with a new mechanism of action. Cyclic nucleotides are an important secondary messenger dependent on the activation of G proteins that are hydrolysed by phosphodiesterases. Phosphodiesterase 10A (PDE10A) is selectively expressed in medium spiny neurons in the striatum where it affects the intensity and duration of signalling through the regulation of cAMP and cGMP concentration. As schizophrenia and many other psychiatric and neurological disorders are linked to striatum and disturbances of its circuits, the inhibition of PDE10A activity is considered as an potential therapeutic approach. The main aim of this study was to identify an innovative, low-molecular-weight PDE10A inhibitor and to characterize its pharmacokinetic and pharmacodynamic properties. The lead molecule CPL500036 was selected based on the results of screening assays. The identified compound was by highly active towards PDE10A and selective against another member of the phosphodiesterase family as determined by in vitro enzymatic assays. CPL500036 was shown to be metabolically stable and further studies have confirmed that it is characterized by high oral bioavailability and good penetration of the blood-brain barrier in rats. Additionally, the administration of CPL500036 led to increased phosphorylation of GluR1 in the rats’ striatum. In the second part of this thesis, a set of in vivo pharmacokinetic and pharmacodynamic experiments was performed in rats aiming at further characterisation of CPL500036. Intravenous and intragastric administrations allowed to determine the pharmacokinetic parameters confirming high bioavailability, dose-dependent exposure and good penetration into the brain. CPL500036 administration in rats resulted in an increase in the concentration of cyclic nucleotides and in the induction of expression of early response genes. Both effects were observed selectively in the striatum. In conclusion, a novel, innovative PDE10A inhibitor with promising pharmacological properties has been identified. Characteristics of CPL500036 allows for the continuation of preclinical and clinical development of the molecule as a new potential therapy in diseases related to the basal ganglia, including schizophrenia. <br>
Serum Response Factor (SRF) is a key transcription factor that regulates gene expression in the adult brain in response to neuronal stimulation. Recent findings reveal the role of SRF and MKL in prenatal neuronal cell development. Despite this knowledge, the role of SRF and MKL in postnatal neuronal development remains poorly understood. Studies indicate a link between single nucleotide changes (SNP) in MKL1/2 an elevated risk of neurodevelopmental diseases associated with abnormalities in the process of neuronal maturation. The influence of SRF and MKL on development of dendritic trees and spines in neurons is still poorly studied. The results presented in this thesis show that deletion of the SRF during early postnatal development in vitro decreases the number and total length of dendrites and reduces dendritic tree complexity. Neurons lacking SRF also exhibit a lower density of dendritic spines and an increased number of immature spines. Additionally, the study investigated the influence of SNP in MKL, identified in humans, on their protein function in neurons in vitro. The subcellular localization of the proteins, their ability to activate transcription, and their effect on dendritic tree maturation were analyzed. Results demonstrate that overexpression of MKLs, influenced by BDNF, alters their neuronal localization, activates SRF-dependent transcription, and increases the complexity of the dendritic tree in vitro. Moreover, we identified SNP in MKL1/2 that disrupted their function. The study was extended with the analysis of the development of human neuronal cells obtained by differentiation from induced pluripotent stem cells. Neurons arising from neural stem cells where SRF was silenced using shRNA exhibited an increase in the number of basal dendrites. In conclusion, the downregulation of the SRF and SNP in MKL contributes to abnormalities in the neuronal structure during development in vitro. These morphological changes are often observed in animal models of neurodevelopmental diseases. <br>
Spinal cord injury leads to the disruption and degeneration of neural pathways originating in supraspinal centers. Consequently, patients experience partial or complete loss of motor function, the extent of which depends on the location and severity of spinal damage. Such injuries often result in permanent disability due to limited effectiveness of current treatment methods. One promising strategy involves enriching the neuronal network below the injury site with neurotrophins – low molecular weight proteins that promote neuron survival and synaptic plasticity processes. In this dissertation, adult rats subjected to complete spinal cord transection (SCT) at the lower thoracic segments were administered an intraspinal AAV vector carrying the brain-derived neurotrophic factor gene (AAV-BDNF). Animals overexpressing BDNF demonstrated rapid and significant improvement in motor function; by the third week post-injury, they regained the ability for alternating limb movements, weight-bearing, and plantar stepping during treadmill training. These results, obtained in a group of 14 animals, strongly confirmed the therapeutic potential of the AAV-BDNF vector previously observed in a chronic experiment conducted by our Team. In the 6-week experiment, the best functional improvement occurred in the third week post-SCT and AAV-BDNF injection, but side effects in the form of clonic movements emerged in later postoperative periods, likely related to overstimulation of the network. This study aimed to elucidate the molecular basis of BDNF's therapeutic action. To this end, neurochemical and structural changes in the spinal cord and neuromuscular junction were characterized during the period of greatest functional improvement. Given indications of different SCT sensitivities in the neuronal circuits of two antagonistic muscles of the ankle joint: the flexor (Tibialis anterior, TA) and extensor (Soleus, Sol), motoneurons and neuromuscular junctions of these two muscles were examined. Combining cellular and subcellular mRNA distribution analysis, assessed via fluorescent in situ hybridization, and protein distribution analysis using immunofluorescence in tissue sections, provided a comprehensive response to questions about the impact of injury and BDNF overexpression on cellular responses in the spinal cord and muscles. Transmission electron microscopy was employed to examine changes in the ultrastructure of the neuromuscular junction. It was demonstrated that, in the third week post-vector administration, BDNF overexpression in the spinal cord concentrated in neurons of the L1-L2 lumbar segments, close to the injection site, while in the area of the hind limb motoneurons, located in the L3-L6 segments, the transgenic protein was observed only in fibers of transduced cells. Based on this characterized distribution of recombinant BDNF, it was concluded that locally introduced vector leads to the transgenic protein's action on neurons located throughout the entire lumbar segment. The therapy was proven to have several positive effects: it maintained reduced SCT post-injury receptivity of motoneurons to BDNF, caused increased transcriptional activity of subsynaptic myocyte nuclei, and contributed to the normalization of neuronal signaling markers in the muscle. Moreover, AAV-BDNF injection preserved the structural integrity of neuromuscular junctions, with the Soleus motor unit responding more favorably to therapy. These results contribute to understanding the mechanism of action of the neurotrophin BDNF and point to a potentially useful research direction: targeted therapy for selected types of motor units. <br>
Stress-related disorders are highly prevalent diseases all over the world. Accumulating data indicate that the serotonergic system is strongly linked with the pathogenesis of depression. Numerous studies based on molecular biology, genetic, histological, and behavioral approaches have shown that serotonin receptors, in particular 5-HT1A and 5-HT7, might mediate the stress response in both rodents and humans. However, mechanisms explaining an involvement of 5-HT1A and 5-HT7 receptors in stress-related diseases are not completely understood. Previous studies of our research group have shown that 5-HT1A and 5-HT7 receptors form heterodimers in the recombinant system, in neuronal cultures and in the mouse brain, which in turn leads to changes in the receptor-mediated signaling. In this regard, the aim of this project was to investigate functional implication of 5-HT1AR/5-HT7R heterodimerization by modeling depression in animals.First, a theoretical study based on meta-analysis was performed to verify the applicability of the chronic unpredictable stress protocol for modeling depression in different strains of rodents. Using this approach, we have demonstrated that both rats and mice showed anhedonic behavior after implementation of the chronic unpredictable stress protocol. In this study, C57BL/6J mice were chosen as the best model due to their higher susceptibility to stress protocol upon shorter stress duration in comparison to other rodent strains, availability of transgenic lines bred on C57BL/6J genetic background, and lower cost of depression modeling compared to rats. The depressive phenotype was assessed based on anhedonic and despair parameters as well as body weight fluctuation. Second, the investigation of 5-HT1A and 5-HT7 receptors expression profiles in different mouse brain regions during postnatal development was performed. Thus, our data have shown that the 5-HT1AR protein level was upregulated in the prefrontal cortex and hippocampus compared to the raphe nuclei, whereas the level of the 5-HT7R did not differ. Additionally, applying qRT-PCR it has been demonstrated that the 5-HT1ARs mRNA is the dominant subpopulation in comparison to the 5-HT7Rs mRNA in the prefrontal cortex and hippocampus during brain development. Third, the interaction between 5-HT1A and 5-HT7 receptors using depression-like model in C57BL/6J mice was investigated. Noteworthy, the most prominent changes in heterodimerization profile of 5-HT1A and 5-HT7 receptors were observed in the medial prefrontal cortex and hippocampal dentate gyrus of C57BL/6J mice. We have obtained a decrease in the number of 5-HT1AR/5-HT7R heterodimeric complexes in the stressed anhedonic mice in comparison to stressed control and stressed resilient animals. In contrast, no significant changes in the 5-HT1AR and 5-HT7R heterodimerization profile were detected in the dorsal raphe nuclei. In conclusion, our data revealed that the chronic unpredictable stress paradigm represents a robust and reproducible model for the depression-like behavior in rodents. Moreover, the number of 5-HT1AR/5-HT7R heterodimers was decreased in the prefrontal cortex of C57BL/6J mice upon chronic unpredictable stress, suggesting functional role of 5-HT1AR and 5-HT7R interaction in development of depressive-like behavior.
Studies using spinal cord injury (SCI) models investigated molecular changes in neurotransmission-related molecules in motoneurons (MNs) mostly at the late postlesion phase, when hyperexcitability considered to be the reason of muscle spasticity is well established. However, in experimental SCI in rodents the onset of spasticity is seen as early as one week postinjury. Patterns and relations of expression level of genes coding for membrane proteins instrumental for excitatory vs inhibitory neurotransmission in the subacute phase of SCI when excitability starts to restore, are not clear.The aim of my work was to clarify the direction and extent of transcriptional regulation of receptors mediating excitatory and inhibitory neurotransmission and of functionally associated channels in hindlimb MNs of adult rats, at the second week postinjury. I hypothesized that fast molecular changes in lumbar MNs develop in response to the loss of inputs. These responses may disturb the balance of excitatory and inhibitory receptors and related ion channels in MNs. Because after SCI the extent of impairment of inputs to MNs innervating extensor and flexor muscles operating at the ankle joint is different, I examined separately MN pools innervating ankle extensor (Gastrocnemius lateralis; GL) and flexor (Tibialis anterior; TA) muscles.A promising way to treat SCI is by spinal cord enrichment with brain derived neurotrophic factor (BDNF). Previous studies showed that BDNF overexpression induced with AAV-BDNF injection caudal to the lesion site improves locomotor abilities and upregulates transcript levels of glutamatergic and GABAergic markers in the interneurons, presynaptic to MNs. While the study showed beneficial role of BDNF in adapting the network to increased activity, undesirable behavioral effects suggesting overexcitability were observed in time, which set my second aim: to characterize the effect of spinal AAV-BDNF administration on gene expression studied in the first part of my project, and identify target molecules of pro-excitogenic potential.Prior to complete spinal cord transection (SCT) at the thoracic Th11 level, retrograde tracers were injected to the respective muscles to identify MNs. After SCT, PBS or AAV-BDNF was injected bilaterally to the lumbar L1/2 segment. Non-lesioned rats with injected tracers served as controls. At two weeks postlesion, locomotor performance of spinal rats was evaluated on a running treadmill. After animal perfusion, GL and TA MNs were isolated from longitudinal spinal sections by laser-assisted microdissection, mRNA was isolated and reverse- transcribed into cDNA. Transcript levels of selected neurotransmitter receptors, ion channels and Cl- transporters were assayed using quantitative PCR.
Successful social interaction involves reciprocal contact with other individuals and requires well-orchestrated responses from interaction partners. In social species, specialized brain areas and neural networks ('social brain') mediate social interaction and allow individuals to survive and thrive. Dysfunctions of these brain networks result in decreased motivation to initiate social interaction and/or incapacity to communicate and understand social information, which causes problems with maintaining social interaction. Different mental disorders affect various aspects of social interactions. However, the neuronal circuits underlying the initiation and maintenance of social contact have yet to be discovered. As one of the primary hypotheses explaining social dysfunctions is a deficiency in reward processing, one of the promising targets to treat social impairments are neuronal circuits known to process rewards. Here, I investigated the neuronal circuit comprising the anterior cingulate cortex (ACC), orbitofrontal cortex (OFC), central amygdala (CeA), and ventral tegmental area (VTA) to verify their role in the initiation and maintenance of social interaction. Further, I tested the specificity of these circuits in social interaction by comparing their role in social interaction and food motivation. I found that the CeA cells activated by social interaction or food rewards receive projections from the ACC and OFC. Next, I discovered that chemogenetic inhibition of the ACC-CeA projection modulates the maintenance of social interaction but not the initiation of social interaction. On the other hand, inhibition of the OFC-CeA projection diminished both the social approach and the maintenance of social contact. Inhibition of either projections decreases food motivation. Further, using a c-fosdependent construct containing opsins that targets behaviorally activated neurons, I labeled the CeA cells involved in social and food reward. Optogenetic manipulations revealed that the functional overlap between these circuits is limited. To identify the CeA outputs that can be specific to social interaction, I used chemogenetic manipulations. I found that the CeA-VTA projection and the dopaminergic VTA-ACC and VTA-OFC pathways are involved in social interaction but not food motivation. Moreover, I identified the CeA-VTA and VTA-ACC projections as critical for initiating social contact and the CeA-VTA for maintaining it. Unlike most published studies, I define the circuits regulating appetitive social behaviors by their functional connectivity with other structures rather than the markers they express. Such defined neuronal circuits could serve as therapeutic targets for rescuing social deficits. <br>
The transmission of information between neurons in the central nervous system (CNS) occurs mainly at synapses. The postsynaptic part of most excitatory synapses in the brain is located at dendritic spines, which are small membranous protrusions of nerve cells. Dendritic spines are highly dynamic structures, undergoing structural and functional plasticity. Changes in the dendritic spine shape correlate with the degree of maturation of synapses and the strength of neuronal connections. The proper synapse function is crucial for effective memory and learning. Furthermore, abnormalities in the maturation process or in the number of dendritic spines are frequently observed in the clinical picture of different neurodevelopmental diseases such as autism spectrum disorders, fragile X syndrome or Rett syndrome. Formation and maturation of dendritic spines is a very complex phenomenon, and despite numerous studies, the molecular mechanisms underlying the processes of synapse formation, and development are still not fully understood. Recent analyses have shown a correlation between the occurrence of certain neurodevelopmental disorders in humans and point mutations within genes whose expression is regulated by Serum Response Factor (SRF) or its cofactors. SRF protein is a transcription factor significantly involved in both physiological and pathological processes occurring in neurons. SRF regulates long-term potentiation (LTP), long-term depression (LTD), neuronal activity-dependent gene expression, neuronal network formation or epileptogenesis. SRF activity is controlled by its coactivators including myocardin-related transcription factors, MRTF. Moreover, there is also a relationship between single nucleotide mutations within mrtf genes, and the occurrence of autism spectrum disorders or schizophrenia. Until now, the role of SRF protein and its coactivators on the process of dendritic spine formation and maturation was unknown. The results presented herein showed that SRF or MRTF regulates dendritic spike maturation and synapse function in developing neurons. Silencing SRF expression in vitro and in vivo impaired dendritic spine structure and increased the number of immature spines with no apparent changes in their overall density. Moreover, downregulation of MRTF protein had a similar effect on dendritic spike morphology as SRF depletion. Additionally, the observed structural changes were
Tuberous sclerosis complex (TSC) is a rare genetic disease characterized by a highly variable clinical picture. Numerous cellular and tissue dysplasias are observed in this disease in multiple organs, including the central nervous system. Some of these characteristic neuropathological changes can already be detected during fetal development. Patients with TSC also exhibit neurological symptoms such as epilepsy which often develop during the first two years of life. Equally important but often overlooked manifestations of this disease are TSC-associated neuropsychiatric disorders (TANDs). The TSC pathogenesis results mainly from hyperactivity of the mTORC1 pathway caused by loss-of-function mutations in TSC1 or TSC2 genes. This work aims to characterize neuroanatomical, behavioral, and molecular changes in the zebrafish model of TSC tsc2vu242/vu242, and investigate the effects of selected drugs on the observed phenotypes. In the first part of this thesis, I showed that homozygous mutants exhibit alterations in locomotor activity, associated with hyperactivity of the mTORC1 pathway and seizures. Furthermore, based on in-depth behavioral analysis, it was found that tsc2vu242/vu242 larvae exhibit phenotypes similar to TANDs, such as increased anxiety and cognitive impairment. The quantitative analysis of cortisol levels further supported the hypothesis of increased anxiety in this model. The second part of the study examined pathologies of the selected neuronal circuits in the brain of tsc2vu242/vu242 mutants identifying disturbances in the morphology of the anterior commissure and the directed axonal growth leading to impaired axon fasciculation. These abnormalities coexisted with altered mRNA levels of genes of the Dock-Elmo-Rac1 pathway, which is involved e.g., in axon elongation, indicating a molecular basis for the observed neuroanatomical changes. Molecular analysis also revealed the probable disturbance of inhibitory neurotransmission, which is one of the reasons for the initiation of epileptic seizures and the development of intellectual disability in the TSC patients. Furthermore, the effects of the TSC clinical drugs, rapamycin and vigabatrin, on selected neurological and behavioral changes were examined. Rapamycin was effective in rescuing both behavioral phenotypes and neuroanatomical abnormalities in the anterior commissure of the brain in the tsc2vu242/vu242 mutants. Vigabatrin reversed phenotypes reflecting seizures but did not improve the parameters associated with increased anxiety in the tsc2vu242/vu242 mutants. In addition, a novel compound, ANA-12, was identified as a potential treatment for TSC-related symptoms, as it had a positive effect on some aspects of anxiety-related behavior and improved anterior commissure morphology. <br>
The volume of information flowing in from the world is enormous and overwhelming. Yet, individuals may not be constantly aware of this, as they do not permanently experience the consequences of this immense influx of information. This is primarily attributed to the selection process, which is not always contingent on conscious choices. One possible criterion for the selection of information is its association with the self, leading to more efficient processing. This effect is called the self-prioritisation effect (SPE). Although SPE is observed in many different contexts, the factors driving this phenomenon are still ambiguous. Scientists propound two factors, familiarity and emotionality, focusing mainly on the former. In this thesis, using electrophysiological techniques, I aimed to investigate the role of these two factors independently. The first two studies were devoted to the emotionality factor. As emotions might be perceived objectively or subjectively, this distinction was transferred to the emotionality factor. In the first study, a plausible role of objective emotionality was investigated by comparing the processing of one’s own face and emotional unknown faces. ERPs analysis (with P3 and LPP in the focus of attention) and cluster-based permutation tests revealed that the processing of the self-face is unique and does not resemble the processing of the objectively emotional faces. In the follow-up study, subjective emotionality was in the spotlight. To assess its impact on the SPE, a face of a close person was introduced into the study. Such a person presents a similar combination of familiarity and emotionality factors as is possessed by the self; thus, the face of a close-other seems to be the best comparison to the self-face. Moreover, as the COVID-19 pandemic significantly impacted human lives in the last few years, the study's goals were expanded, and the SPE was tested for partial facial information. Source analysis indicated that the processing of partially covered faces is associated with the brain area typically linked to the face processing, fusiform gyrus. Amplitudes of early (P1) and late (P3, LPP) ERP components consistently indicated that all covered faces require more attentional resources to be processed, and SPE is not impoverished by the surgical-like masks, as the self-face in both conditions (with and without mask) evoked significantly higher P3 and LPP amplitudes. Furthermore, a significant difference between the processing of the self-face and the close-other’s face was depicted. This pattern of results undermines the plausible role of subjective emotionality, and in combination with findings from the first study, it deflates the role of emotionality in general. The last study was dedicated to the familiarity factor. The familiarity of the presented stimuli was equalised to disentangle the mutual impact of both factors. Apart from the highly familiar stimuli as one’s own and close-other’s faces, we used unknown abstract shapes assigned to the participant and freely chosen close-other. Our findings revealed no differences in the processing of newly acquired information (similar P3 and LPP amplitudes in both cases). As the typical pattern of face processing was manifested (larger P3 and LPP for the self-face), the lack of differences between the self-assigned shape and the shape assigned to the close-other might be interpreted as a further substantial argument in favour of familiarity as a driving factor of self-prioritisation. The findings presented in this thesis indicate that familiarity is a driving factor in the SPE. Through various paradigms and diverse analytical techniques, we have demonstrated that high familiarity of self-related information is crucial for the self-prioritisation effect. By shedding light on the intricate interplay between familiarity and emotionality, my work contributes to a deeper understanding of how individuals process information and make decisions based on SPE.
While the relationship between epilepsy and circadian dysregulation is known, we know very little about circadian oscillations of the transcription factors which are responsible from modulating the gene expression in health and disease pathologies. This study aims to characterise circadian dynamics of one of the identified and prominent transcription factors in epilepsy – the Zbtb14. In pursuit of this objective, protein rhythmicity of the Zbtb14 is observed in the ventral and dorsal hippocampus and the somatosensory cortex using immunochemistry, and in cytoplasmic and nuclear protein extracts of the hippocampus using western blot. The downregulated genes with ZF5 motif in their promoters identified in an in vitro model of epileptiform discharges is characterised. Zbtb14 protein expression is investigated in two time points in an in vivo model of epilepsy model. The study showed that Zbtb14 protein only has a rhythmic expression in the ventral hippocampus but not in the dorsal hippocampus and the somatosensory cortex. Additionally, the cytoplasmic and nuclear dynamics of the Zbtb14 protein are different. I identified the downregulated genes in in vitro model of epileptiform discharges are mainly responsible from synaptic plasticity and transmission. Furthermore, the epilepsy pathology affected the Zbtb14 transcription factor expression in a time-dependent manner. My research shows that the studies on circadian regulations of the transcription factors can be beneficial target to unravel the disease pathologies and potential therapeutics. <br>
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