Chronic lorcaserin treatment reverses the nicotine withdrawal-induced disruptions to behavior and maturation in developing neurons in the hippocampus of rats

Preclinical data have shown that treatment with serotonin (5-HT)2C receptor agonists inhibits the behavioral effects of nicotine, including self-administration, reinstatement, and locomotor responses to nicotine. Since the data on the effects of 5-HT2C receptor agonism on nicotine withdrawal signs are limited, we aimed to investigate whether 5-HT2C receptor agonism alleviated the behavioral and neurobiochemical (hippocampal neurogenesis) consequences of nicotine withdrawal in Sprague-Dawley rats. Our data indicate that withdrawal from nicotine self-administration induced locomotor hyperactivity, lengthened immobility time (the forced swim test), induced ‘drug-seeking’ behavior and deficits in cognition-like behavior (the novel object recognition task). A two-week exposure to the 5-HT2C receptor agonist lorcaserin attenuated locomotor hyperactivity and induced recovery from depression-like behavior. Analyses of brain slices from nicotine-withdrawn animals revealed that lorcaserin treatment recovered the reduced number of doublecortin (DCX)-positive cells, but it did not affect the number of Ki-67- or 5-bromo-2’-deoxyuridine (BrdU)-positive cells or the maturation of proliferating neurons in drug-weaned rats. To summarize, we show that lorcaserin alleviated locomotor responses and depression-like state during nicotine withdrawal. We propose that the modulatory effect of lorcaserin on the ‘affective’ aspects of nicotine cessation may be linked to the positive changes caused by the compound in hippocampal neurogenesis during nicotine withdrawal

Oxygen-glucose deprivation in organotypic hippocampal cultures leads to cytoskeleton rearrangement and immune activation : link to the potential pathomechanism of ischaemic stroke

Ischaemic stroke is characterized by a sudden loss of blood circulation to an area of the brain, resulting in a corresponding loss of neurologic function. As a result of this process, neurons in the ischaemic core are deprived of oxygen and trophic substances and are consequently destroyed. Tissue damage in brain ischaemia results from a complex pathophysiological cascade comprising various distinct pathological events. Ischaemia leads to brain damage by stimulating many processes, such as excitotoxicity, oxidative stress, inflammation, acidotoxicity, and apoptosis. Nevertheless, less attention has been given to biophysical factors, including the organization of the cytoskeleton and the mechanical properties of cells. Therefore, in the present study, we sought to evaluate whether the oxygen-glucose deprivation (OGD) procedure, which is a commonly accepted experimental model of ischaemia, could affect cytoskeleton organization and the paracrine immune response. The abovementioned aspects were examined ex vivo in organotypic hippocampal cultures (OHCs) subjected to the OGD procedure. We measured cell death/viability, nitric oxide (NO) release, and hypoxia-inducible factor $1\alpha$ (HIF-$1\alpha$) levels. Next, the impact of the OGD procedure on cytoskeletal organization was evaluated using combined confocal fluorescence microscopy (CFM) and atomic force microscopy (AFM). Concurrently, to find whether there is a correlation between biophysical properties and the immune response, we examined the impact of OGD on the levels of crucial ischaemia cytokines (IL-$1\beta$, IL-6, IL-18, TNF-$\alpha$, IL-10, IL-4) and chemokines (CCL3, CCL5, CXCL10) in OHCs and calculated Pearsons’ and Spearman’s rank correlation coefficients. The results of the current study demonstrated that the OGD procedure intensified cell death and nitric oxide release and led to the potentiation of HIF-$1\beta$ release in OHCs. Moreover, we presented significant disturbances in the organization of the cytoskeleton (actin fibers, microtubular network) and cytoskeleton-associated protein 2 (MAP-2), which is a neuronal marker. Simultaneously, our study provided new evidence that the OGD procedure leads to the stiffening of OHCs and a malfunction in immune homeostasis. A negative linear correlation between tissue stiffness and branched IBA1 positive cells after the OGD procedure suggests the pro-inflammatory polarization of microglia. Moreover, the negative correlation of pro- and positive anti-inflammatory factors with actin fibers density indicates an opposing effect of the immune mediators on the rearrangement of cytoskeleton induced by OGD procedure in OHCs. Our study constitutes a basis for further research and provides a rationale for integrating biomechanical and biochemical methods in studying the pathomechanism of stroke-related brain damage. Furthermore, presented data pointed out the interesting direction of proof-of-concept studies, in which follow-up may establish new targets for brain ischemia therapy

Study of 53BP1 foci formation dynamics after chromatin damage induced by visible light: dependence on wavelength and dose of light

Uszkodzenia DNA, w szczególności pęknięcia dwuniciowe, są wysoce niebezpieczne dla zachowania ciągłości genomu. Mechanizm odpowiedzi komórkowej na dwuniciowe pęknięcia DNA jest wciąż badany, wiadomo jednak, że niesie on ze sobą szereg potranslacyjnych modyfikacji histonów takich jak metylacja reszty lizyny 20 histonu H4 (H4K20me2), ubikwitynacje lizyny 15 histonu H2A (H2AK15ub) czy fosforylacje. Takie modyfikacje rozpoznawane są przez domeny czynnika naprawczego 53BP1: domena Tudor oddziałuje z H4K20me2 a UDR z H2AK15ub. 53BP1 bierze udział w wyborze drogi naprawczej dwuniciowego uszkodzenia DNA. Naprawa uszkodzeń DNA komórek, będących w fazie G1/G0, odbywa się głównie na drodze łączenia niehomologicznych zakończeń (NHEJ), promowana przez 53BP1, natomiast komórek będących w fazie S i G2 cyklu komórkowego – rekombinacji homologicznej (HR), związanej z antagonistą 53BP1, białkiem BRCA1. Białka naprawcze, w eksperymentach stosujących do wywołania uszkodzenia DNA promieniowanie jonizujące, UV bądź światło widzialne, tworzą wyraźne ogniska (foci) w miejscu uszkodzenia.Celem niniejszej pracy magisterskiej było zbadanie dynamiki powstawania ognisk białka fuzyjnego EGFP-53BP1 po wywołaniu lokalnego uszkodzenia chromatyny za pomocą światła widzialnego, w zależności od długości fali światła oraz od jego dawki.Doświadczenia przeprowadzano na komórkach żywych, po wprowadzeniu do nich wektora plazmidowego, w celu wywołania przejściowej ekspresji białka fuzyjnego EGFP-53BP1. Po ekspozycji chromatyny na światło widzialne (488 nm – 594 nm), techniką parkowania wiązki lasera, zaobserwowano powstawanie ogniska białka fuzyjnego w miejscu uszkodzenia. Nie zaobserwowano gromadzenia białka EGFP-53BP1, po ekspozycji na światło o długości fali 633 nm. Odnotowano, że maksymalne wartości powierzchni focus białka nie zależą od dawki światła lecz istnieje dawka minimalna wywołująca uszkodzenie. Zauważono również, że EGFP-53BP1 wykazuje co najmniej dwa typy tworzenia ogniska. Ognisko EGFP-53BP1 może nie zanikać do mitozy, a nawet trwać w komórkach potomnych. Pokazano również, że po wywołaniu uszkodzenia, za pomocą światła o długościach fali krótszych niż 561 nm, doszło do znacznego fotoblaknięcia fluorescencji EGFP, przez co fototoksyczność białka fluorescencyjnego mogła wywołać zaburzenia układu. Wobec tego postanowiono podjąć próbę skonstruowania nowego wektora plazmidowego niosącego gen mRFP-53BP1, za pomocą klasycznego klonowania molekularnego.DNA damages especially double-strand breaks are highly dangerous for maintenance genome integrity. Mechanism of double-strand DNA damage response is still being prospected, however, it is known that it promotes a number of histone post-tranlational modifications, such as dimethylation of Lys20 of histone 4 (H4k20me2), ubiquitilation of Lys15 of histone H2A (H2AK15ub) or phosphorylations. Such modifications are recognized by 53BP1 (repair factor) domains: Tudor interacts with H4K20me2 and UDR motif binds to H2AK15ub. 53BP1 takes part in choosing of repair pathway in response to DSBs of DNA. DNA damages repair for G1/G0 phase cells are mostly performed by nonhomologous end joining that 53BP1 promotes, by contrast, for S/G2 phase cells it is followed by homologous recombination that the 53BP1 antagonist – BRCA1 promotes. Repair proteins create noticeable foci in experiments that use ionising radiation, UV or visible light to induced DNA damages.The purpose of this master's thesis was to investigate of EGFP-53BP1 foci formation dynamics after local chromatin damage induced by visible light, in particular on wavelenght and dose of light dependence.Experiments were carried on living cells after imposition of plasmid vector to elicit transient expression of EGFP-53BP1 fusion protein. EGFP-53BP1 focus formation in site of damage was observed after chromatin exposure to visible light (488 nm – 594 nm) by laser beam park technique. Focus formation was not observed after usage of 633 nm light wavelenght. 53BP1 exhibits at least two types of foci formation. Maximum values of foci surfaces do not depend on dose of light but the minimal dose of light exist. EGFP-53BP1 focus can be continued untill mitosis and go on after cell division. Photobleaching effect appeared after induction of local chromatin damage by shorter than 561 nm wavelenghs. It could cause activation of fototoxicity of EGFP and could disturb studied system. Because of that side effect, the trial to get plasmid vector with mRFP-53BP1 protein was made

Changes in nanomechanical properties of single neuroblastoma cells as a model for oxygen and glucose deprivation (OGD)

Although complex, the biological processes underlying ischemic stroke are better known than those related to biomechanical alterations of single cells. Mechanisms of biomechanical changes and their relations to the molecular processes are crucial for understanding the function and dysfunction of the brain. In our study, we applied atomic force microscopy (AFM) to quantify the alterations in biomechanical properties in neuroblastoma SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) and reoxygenation (RO). Obtained results reveal several characteristics. Cell viability remained at the same level, regardless of the OGD and RO conditions, but, in parallel, the metabolic activity of cells decreased with OGD duration. 24 h RO did not recover the metabolic activity fully. Cells subjected to OGD appeared softer than control cells. Cell softening was strongly present in cells after 1 h of OGD and with longer OGD duration, and in RO conditions, cells recovered their mechanical properties. Changes in the nanomechanical properties of cells were attributed to the remodelling of actin filaments, which was related to cofilin-based regulation and impaired metabolic activity of cells. The presented study shows the importance of nanomechanics in research on ischemic-related pathological processes such as stroke

Translocation of chromatin proteins to nucleoli—The influence of protein dynamics on post‐fixation localization

International audienceIt is expected that the subnuclear localization of a protein in a fixed cell, detected by microscopy, reflects its position in the living cell. We demonstrate, however, that some dynamic nuclear proteins can change their localization upon fixation by either crosslinking or non-crosslinking methods. We examined the subnuclear localization of the chromatin architectural protein HMGB1, linker histone H1, and core histone H2B in cells fixed by formaldehyde, glutaraldehyde, glyoxal, ethanol, or zinc salts. We demonstrate that some dynamic, weakly binding nuclear proteins, like HMGB1 and H1, may not only be unexpectedly lost from their original binding sites during the fixation process, but they can also diffuse through the nucleus and eventually bind in nucleoli. Such translocation to nucleoli does not occur in the case of core histone H2B, which is more stably bound to DNA and other histones. We suggest that the diminished binding of some dynamic proteins to DNA during fixation, and their subsequent translocation to nucleoli, is induced by changes of DNA structure, arising from interaction with a fixative. Detachment of dynamic proteins from chromatin can also be induced in cells already fixed by non-crosslinking methods when DNA structure is distorted by intercalating molecules. The proteins translocated during fixation from chromatin to nucleoli bind there to RNA-containing structures