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Background: Rituximab is used in the treatment of CD20+ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas.Methods and Findings: Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a 51Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-beta-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells.Conclusions: Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab. [ABSTRACT FROM AUTHOR]

Aims: Epilepsy is one of the most common chronic neurological disorders, affecting around 50 million people worldwide, but its underlying cellular and molecular events are not fully understood. The Golgi is a highly dynamic cellular organelle and can be fragmented into ministacks under both physiological and pathological conditions. This phenomenon has also been observed in several neurodegenerative disorders; however, the structure of the Golgi apparatus (GA) in human patients suffering from epilepsy has not been described so far. The aim of this study was to assess the changes in GA architecture in epilepsy., Methods: Golgi visualisation with immunohistochemical staining in the neocortex of adult patients who underwent epilepsy surgery; 3D reconstruction and quantitative morphometric analysis of GA structure in the rat hippocampi upon kainic acid (KA) induced seizures, as well as in vitro studies with the use of Ca 2+ chelator BAPTA-AM in primary hippocampal neurons upon activation were performed., Results: We observed GA dispersion in neurons of the human neocortex of patients with epilepsy and hippocampal neurons in rats upon KA-induced seizures. The structural changes of GA were reversible, as GA morphology returned to normal within 24 h of KA treatment. KA-induced Golgi fragmentation observed in primary hippocampal neurons cultured in vitro was largely abolished by the addition of BAPTA-AM., Conclusions: In our study, we have shown for the first time that the neuronal GA is fragmented in the human brain of patients with epilepsy and rat brain upon seizures. We have shown that seizure-induced GA dispersion can be reversible, suggesting that enhanced neuronal activity induces Golgi reorganisation that is involved in aberrant neuronal plasticity processes that underlie epilepsy. Moreover, our results revealed that elevated cytosolic Ca 2+ is indispensable for these KA-induced morphological alterations of GA in vitro., (© 2023 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Background: Epilepsy affects millions of people worldwide, yet we still lack a successful treatment for all epileptic patients. Most of the available drugs modulate neuronal activity. Astrocytes, the most abundant cells in the brain, may constitute alternative drug targets. A robust expansion of astrocytic cell bodies and processes occurs after seizures. Highly expressed in astrocytes, CD44 adhesion protein is upregulated during injury and is suggested to be one of the most important proteins associated with epilepsy. It connects the astrocytic cytoskeleton to hyaluronan in the extracellular matrix, influencing both structural and functional aspects of brain plasticity., Methods: Herein, we used transgenic mice with an astrocyte CD44 knockout to evaluate the impact of the hippocampal CD44 absence on the development of epileptogenesis and ultrastructural changes at the tripartite synapse., Results: We demonstrated that local, virally-induced CD44 deficiency in hippocampal astrocytes reduces reactive astrogliosis and decreases the progression of kainic acid-induced epileptogenesis. We also observed that CD44 deficiency resulted in structural changes evident in a higher dendritic spine number along with a lower percentage of astrocyte-synapse contacts, and decreased post-synaptic density size in the hippocampal molecular layer of the dentate gyrus., Conclusions: Overall, our study indicates that CD44 signaling may be important for astrocytic coverage of synapses in the hippocampus and that alterations of astrocytes translate to functional changes in the pathology of epilepsy.

Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Brain-Derived Neurotrophic Factor is one of the most important trophic proteins in the brain. The role of this growth factor in neuronal plasticity, in health and disease, has been extensively studied. However, mechanisms of epigenetic regulation of Bdnf gene expression in epilepsy are still elusive. In our previous work, using a rat model of neuronal activation upon kainate-induced seizures, we observed a repositioning of Bdnf alleles from the nuclear periphery towards the nuclear center. This change of Bdnf intranuclear position was associated with transcriptional gene activity. In the present study, using the same neuronal activation model, we analyzed the relation between the percentage of the Bdnf allele at the nuclear periphery and clinical and morphological traits of epilepsy. We observed that the decrease of the percentage of the Bdnf allele at the nuclear periphery correlates with stronger mossy fiber sprouting-an aberrant form of excitatory circuits formation. Moreover, using in vitro hippocampal cultures we showed that Bdnf repositioning is a consequence of transcriptional activity. Inhibition of RNA polymerase II activity in primary cultured neurons with Actinomycin D completely blocked Bdnf gene transcription and repositioning occurring after neuronal excitation. Interestingly, we observed that histone deacetylases inhibition with Trichostatin A induced a slight increase of Bdnf gene transcription and its repositioning even in the absence of neuronal excitation. Presented results provide novel insight into the role of BDNF in epileptogenesis. Moreover, they strengthen the statement that this particular gene is a good candidate to search for a new generation of antiepileptic therapies., Competing Interests: The authors have declared that no competing interests exist.

The overall approach to the treatment of multiple myeloma (MM) has undergone several changes during the past decade. and proteasome inhibitors (PIs) including bortezomib, carfilzomib, and ixazomib have considerably improved the outcomes in affected patients. The first-in-class selective PI bortezomib has been initially approved for the refractory forms of the disease but has now become, in combination with other drugs, the backbone of the frontline therapy for newly diagnosed MM patients, as well as in the maintenance therapy and relapsed/refractory setting. Despite being among the most widely used and highly effective agents for MM, bortezomib can induce adverse events that potentially lead to early discontinuation of the therapy with negative effects on the quality of life and outcome of the patients. Although peripheral neuropathy and myelosuppression have been recognized as the most relevant bortezomib-related adverse effects, cardiac and skeletal muscle toxicities are relatively common in MM treated patients, but they have received much less attention. Here we review the neuromuscular and cardiovascular side effects of bortezomib. focusing on the molecular mechanisms underlying its toxicity. We also discuss our preliminary data on the effects of bortezomib on skeletal muscle tissue in mice receiving the drug.

Entosis is a phenomenon, in which one cell enters a second one. New clinico-histopathological studies of entosis prompted us to summarize its significance in cancer. It appears that entosis might be a novel, independent prognostic predictor factor in cancer histopathology. We briefly discuss the biological basis of entosis, followed by a summary of published clinico-histopathological studies on entosis significance in cancer prognosis. The correlation of entosis with cancer prognosis in head and neck squamous cell carcinoma, anal carcinoma, lung adenocarcinoma, pancreatic ductal carcinoma and breast ductal carcinoma, is shown. Numerous entotic figures are associated with a more malignant cancer phenotype and poor prognosis in many cancers. We also showed that some anticancer drugs could induce entosis in cell culture, even as an escape mechanism. Thus, entosis is likely beneficial for survival of malignant cells, i.e., an entotic cell can hide from unfavourable factors in another cell and subsequently leave the host cell remaining intact, leading to failure in therapy or cancer recurrence. Finally, we highlight the potential relationship of cell adhesion with entosis in vitro, based on the model of the BxPc3 cells cultured in full adhesive conditions, comparing them to a commonly used MCF7 semiadhesive model of entosis.

Introduction: The main component of extralysosomal proteolysis is the ubiquitin-proteasome system (UPS), which is supplemented by tripeptidyl peptidase II (TPPII). That system is a target for anticancer strategies by using proteasome inhibitors. Data from several studies on leukemic cells share evidence for the beneficial and potential role of TPPII in cell survivability. Therefore, the aim of this work was to analyze the effect of AAF-cmk, a membrane permeable semi-specific TPPII inhibitor, on human monocytic leukemic cells U937 for translational research., Material and Methods: We studied the viability of U937 cells incubated with AAF-cmk using tetrazolium salt reduction assay (MTT) and apoptosis induction by assessing caspase activation by Western blotting and Annexin V binding assays. Transmission electron microscopy (TEM), a gold standard for apoptosis and autophagy detection, was used to assess the ultrastructure of U937 cells., Results: Incubation of cells with AAF-cmk reduced their viability and induced apoptosis by intrinsic pathway. In groups treated with AAF-cmk, activation of caspases 9 and 3 was observed and caspase inhibition by zVDA restored cell viability. TEM revealed the presence of ultrastructural features of apoptosis and authophagy. Moreover, we identified two types of protein aggregates. The first one was found in close proximity to the endoplasmic reticulum (ER) and corresponds to Aggresome-Like Structure (ALIS); however, the second novel type of aggregate was not related to ER elements, but rather to free cytosolic ribosomes. This type did not correspond to the aggresome neither in localization nor the structure, thus we referred these aggregates as ALiSNER (Aggresome-Like Structure Not Associated With the ER)., Conclusions: Our results provide novel and important findings about the role of TPPII in protein homeostasis and cell survival. Since semispecific TPPII inhibitor AAF-cmk displays cytotoxic activity against leukemic U937 cells in vitro it can be considered as a potential anticancer agent.

Multiple myeloma (MM) accounts for ∼13% of all hematologic malignancies. Bortezomib treatment is effective in MM, but can be complicated with neurological side effects. We describe a patient with symptomatic MM who had a reversible metabolic myopathy associated with bortezomib administration and pathologically characterized by excessive storage of lipid droplets together with mitochondrial abnormalities. In a single-center prospective study, 14 out of 24 patients with symptomatic MM were treated with bortezomib and, among these, 7 developed muscular signs and/or symptoms. The myopathy was characterized by a proximal muscle weakness involving lower limbs and was an early complication. Complete resolution of muscle weakness occurred after treatment discontinuation. Conversely, none of the patients who received a treatment without bortezomib developed muscular symptoms. Experimental studies demonstrate that in primary human myoblasts bortezomib at low concentrations leads to excessive storage of lipid droplets together with structural mitochondrial abnormalities, recapitulating the pathologic findings observed in patient's muscle. Our data suggest that patients treated with bortezomib should be monitored for muscular signs and/or symptoms and muscle weakness should alert the clinician to the possibility of myopathy. Bortezomib-induced metabolic myopathy is a potentially reversible entity with important implications for management and treatment of patients with MM., (© 2017 American Association of Neuropathologists, Inc. All rights reserved.)

The importance of the extracellular matrix (ECM) glycoprotein tenascin-C (TnC) and the ECM degrading enzymes, matrix metalloproteinases (MMPs) -2 and -9, in cerebellar histogenesis is well established. This study aimed to examine whether there is a functional relationship between these molecules in regulating structural plasticity of the lateral deep cerebellar nucleus. To this end, starting from postnatal day 21, TnC- or MMP-9-deficient mice were exposed to an enriched environment (EE). We show that 8 weeks of exposure to EE leads to reduced lectin-based staining of perineuronal nets (PNNs), reduction in the size of GABAergic and increase in the number and size of glutamatergic synaptic terminals in wild-type mice. Conversely, TnC-deficient mice showed reduced staining of PNNs compared to wild-type mice maintained under standard conditions, and exposure to EE did not further reduce, but even slightly increased PNN staining. EE did not affect the densities of the two types of synaptic terminals in TnC-deficient mice, while the size of inhibitory, but not excitatory synaptic terminals was increased. In the time frame of 4-8 weeks, MMP-9, but not MMP-2, was observed to influence PNN remodeling and cerebellar synaptic plasticity as revealed by measurement of MMP-9 activity and colocalization with PNNs and synaptic markers. These findings were supported by observations on MMP-9-deficient mice. The present study suggests that TnC contributes to the regulation of structural plasticity in the cerebellum and that interactions between TnC and MMP-9 are likely to be important for these processes to occur.

Synaptic cell adhesion molecules regulate signal transduction, synaptic function, and plasticity. However, their role in neuronal interactions with the extracellular matrix (ECM) is not well understood. Here we report that the CD44, a transmembrane receptor for hyaluronan, modulates synaptic plasticity. High-resolution ultrastructural analysis showed that CD44 was localized at mature synapses in the adult brain. The reduced expression of CD44 affected the synaptic excitatory transmission of primary hippocampal neurons, simultaneously modifying dendritic spine shape. The frequency of miniature excitatory postsynaptic currents decreased, accompanied by dendritic spine elongation and thinning. These structural and functional alterations went along with a decrease in the number of presynaptic Bassoon puncta, together with a reduction of PSD-95 levels at dendritic spines, suggesting a reduced number of functional synapses. Lack of CD44 also abrogated spine head enlargement upon neuronal stimulation. Moreover, our results indicate that CD44 contributes to proper dendritic spine shape and function by modulating the activity of actin cytoskeleton regulators, that is, Rho GTPases (RhoA, Rac1, and Cdc42). Thus CD44 appears to be a novel molecular player regulating functional and structural plasticity of dendritic spines., (© 2016 Roszkowska et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Enhanced levels of Matrix Metalloproteinase-9 (MMP-9) have been implicated in the pathogenesis of epilepsy in humans and rodents. Lack of Mmp-9 impoverishes, whereas excess of Mmp-9 facilitates epileptogenesis. Epigenetic mechanisms driving the epileptogenesis-related upregulation of MMP-9 expression are virtually unknown. The aim of this study was to reveal these mechanisms. We analyzed hippocampi extracted from adult and pediatric patients with temporal lobe epilepsy as well as from partially and fully pentylenetetrazole kindled rats. We used a unique approach to the analysis of the kindling model results (inclusion in the analysis of rats being during kindling, and not only a group of fully kindled animals), which allowed us to separate the molecular effects exerted by the epileptogenesis from those related to epilepsy and epileptic activity. Consequently, it allowed for a disclosure of molecular mechanisms underlying causes, and not consequences, of epilepsy. Our data show that the epileptogenesis-evoked upregulation of Mmp-9 expression is regulated by removal from Mmp-9 gene proximal promoter of the two, interweaved potent silencing mechanisms-DNA methylation and Polycomb Repressive Complex 2 (PRC2)-related repression. Demethylation depends on a gradual dissociation of the DNA methyltransferases, Dnmt3a and Dnmt3b, and on progressive association of the DNA demethylation promoting protein Gadd45β to Mmp-9 proximal gene promoter in vivo. The PRC2-related mechanism relies on dissociation of the repressive transcription factor YY1 and the dissipation of the PRC2-evoked trimethylation on Lys27 of the histone H3 from the proximal Mmp-9 promoter chromatin in vivo. Moreover, we show that the DNA hydroxymethylation, a new epigenetic DNA modification, which is localized predominantly in the gene promoters and is particularly abundant in the brain, is not involved in a regulation of MMP-9 expression during the epileptogenesis in the rat hippocampus as well as in the hippocampi of pediatric and adult epileptic patients. Additionally, we have also found that despite of its transient nature, the histone modification H3S10ph is strongly and gradually accumulated during epileptogenesis in the cell nuclei and in the proximal Mmp-9 gene promoter in the hippocampus, which suggests that H3S10ph can be involved in DNA demethylation in mammals, and not only in Neurospora. The study identifies MMP-9 as the first protein coding gene which expression is regulated by DNA methylation in human epilepsy. We present a detailed epigenetic model of the epileptogenesis-evoked upregulation of MMP-9 expression in the hippocampus. To our knowledge, it is the most complex and most detailed mechanism of epigenetic regulation of gene expression ever revealed for a particular gene in epileptogenesis. Our results also suggest for the first time that dysregulation of DNA methylation found in epilepsy is a cause rather than a consequence of this condition.

Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a "stellate"-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of astrocytes.

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The expression level of complement regulators in ovarian and corpus uteri tumors was not fully established so far. In current manuscript we performed gene expression analysis by the real-time PCR approach to investigate both membrane bound - CD55 and CD59 and fluid phase - factor H and factor H-like 1 complement regulators. We found increased CD55 expression in corpus uteri tumors when compared to control tissues, whereas in ovarian cancer CD55 expression was lower than in control sections. Additionally we found CD59 expression to be more prominent in ovarian cancer than in corpus uteri tumor samples. We observed also the strong positive correlation between the level of expression of the whole group of regulators, which was particularly significant between the expression of factor H and factor H- like 1. In conclusion we present novel results which implicates different role of particular complement inhibitors in the regulation of the complement system in two cancer types examined. Strong positive correlation between examined proteins implicates similar pattern of the regulation which should be taken into consideration with regards to the possible immunotherapy applied as adjuvant therapeutic approach in these two indications. The inhibition of complement regulation may serve as a strategy to potentiate the efficacy of such treatment., (Copyright © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

CD44 is the major surface hyaluronan (HA) receptor implicated in intercellular and cell-matrix adhesion, cell migration and signaling. It is a transmembrane, highly glycosylated protein with several isoforms resulting from alternative gene splicing. The CD44 molecule consists of several domains serving different functions: the N-terminal extracellular domain, the stem region, the transmembrane domain and the C-terminal tail. In the nervous system, CD44 expression occurs in both glial and neuronal cells. The role of CD44 in the physiology and pathology of the nervous system is not entirely understood, however, there exists evidence suggesting it might be involved in the axon guidance, cytoplasmic Ca(2+) clearance, dendritic arborization, synaptic transmission, epileptogenesis, oligodendrocyte and astrocyte differentiation, post-traumatic brain repair and brain tumour development.

The acquisition of proper dendrite morphology is a crucial aspect of neuronal development towards the formation of a functional network. The role of the extracellular matrix and its cellular receptors in this process has remained enigmatic. We report that the CD44 adhesion molecule, the main hyaluronan receptor, is localized in dendrites and plays a crucial inhibitory role in dendritic tree arborization in vitro and in vivo. This novel function is exerted by the activation of Src tyrosine kinase, leading to the alteration of Golgi morphology. The mechanism operates during normal brain development, but its inhibition might have a protective influence on dendritic trees under toxic conditions, during which the silencing of CD44 expression prevents dendritic shortening induced by glutamate exposure. Overall, our results indicate a novel role for CD44 as an essential regulator of dendritic arbor complexity in both health and disease., (© 2014. Published by The Company of Biologists Ltd.)

Inhibition of the proteasome offers many therapeutic possibilities in inflammation as well as in neoplastic diseases. However, clinical use of proteasome inhibitors is limited by the development of resistance or severe side effects. In our study we characterized the anti-tumor properties of the novel proteasome inhibitor BSc2118. The sensitivity of tumor lines to BSc2118 was analyzed in comparison to bortezomib using crystal violet staining in order to assess cell viability. The In Vivo distribution of BSc2118 in mouse tissues was tracked by a fluorescent-modified form of BSc2118 (BSc2118-FL) and visualized by confocal microscopy. Inhibition of the 20S proteasome was monitored both in cultured cell lines and in mice, respectively. Finally, safety and efficacy of BSc2118 was evaluated in a mouse melanoma model. BSc2118 inhibits proliferation of different tumor cell lines with a similar potency as compared with bortezomib. Systemic administration of BSc2118 in mice is well tolerated, even when given in a dose of 60 mg/kg body weight. After systemic injection of BSc2118 or bortezomib similar proteasome inhibition patterns are observed within the murine organs. Detection of BSc2118-FL revealed correlation of distribution pattern of BSc2118 with inhibition of proteasomal activity in cells or mouse tissues. Finally, administration of BSc2118 in a mouse melanoma model shows significant local anti-tumor effects. Concluding, BSc2118 represents a novel low-toxic agent that might be alternatively used for known proteasome inhibitors in anti-cancer treatment.

The deleted in liver cancer (DLC) protein family is composed of proteins that are hypothesized to function predominantly by regulating the activity of the small GTPases. The aim of the present study was to determine the expression and exact localization of DLC1 in hepatocellular carcinoma (HCC) tissue sections. In two types of HCC tissues, typical and fibrolamellar, immunohistochemical and immunofluorescent analysis were performed to assess DLC1 immunoreactivity. Additionally, the DLC1 gene status was determined by the fluorescence in situ hybridization. According to the observations, DLC1 is often lost in cancer cells; however, it can remain within the stromal component of tumor sections. The DLC1 immunoreactivity was particularly noticeable within the capsules surrounding the tumor masses. It was found that the DLC1 gene was deleted in 52% of HCC cases. In addition, the hemizygous deletion was observed to be independent of the HCC subtype. The results indicate that although the loss of DLC1 is a common step during hepatocarcinogenesis, this protein may be present in the tumor microenvironment.

The interior of the neuronal cell nucleus is a highly organized three-dimensional (3D) structure where regions of the genome that are linearly millions of bases apart establish sub-structures with specialized functions. To investigate neuronal chromatin organization and dynamics in vivo, we generated bitransgenic mice expressing GFP-tagged histone H2B in principal neurons of the forebrain. Surprisingly, the expression of this chimeric histone in mature neurons caused chromocenter declustering and disrupted the association of heterochromatin with the nuclear lamina. The loss of these structures did not affect neuronal viability but was associated with specific transcriptional and behavioural deficits related to serotonergic dysfunction. Overall, our results demonstrate that the 3D organization of chromatin within neuronal cells provides an additional level of epigenetic regulation of gene expression that critically impacts neuronal function. This in turn suggests that some loci associated with neuropsychiatric disorders may be particularly sensitive to changes in chromatin architecture.

Recent studies in neurons indicate that the large-scale chromatin architectural framework, including chromosome territories or lamina-associated chromatin, undergoes dynamic changes that represent an emergent level of regulation of neuronal gene-expression. This phenomenon has been implicated in neuronal differentiation, long-term potentiation, seizures, and disorders of neural plasticity such as Rett syndrome and epilepsy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Currently, there are about 20 antiepileptic drugs on market. Still, seizures in about 30% of patients with epilepsy are not adequately controlled, or the drugs cause quality-of-life-compromising adverse events. Importantly, there are no treatments to combat epileptogenesis, a process that leads to the development of epilepsy and its progression. To fill the gaps in the treatment of epilepsy, there is an urgent need for identification of novel treatment targets. Data emerging over the recent years have shown that different components of the extracellular matrix (ECM) contribute to many components of tissue reorganization during epileptogenesis and the ECM is also a major regulator of synaptic excitability. Here, we review the role of urokinase-type plasminogen activator receptor interactome, matrix metalloproteinases, tenascin-R, and LGI1 in epileptogenesis and ictogenesis. Moreover, the role of the ECM in epilepsy-related comorbidities is reviewed. As there is active development of new imaging methods, we also summarize the data available on imaging of the ECM in epilepsy.

Learning how to avoid danger and pursue reward depends on negative emotions motivating aversive learning and positive emotions motivating appetitive learning. The amygdala is a key component of the brain emotional system; however, an understanding of how various emotions are differentially processed in the amygdala has yet to be achieved. We report that matrix metalloproteinase-9 (MMP-9, extracellularly operating enzyme) in the central nucleus of the amygdala (CeA) is crucial for appetitive, but not for aversive, learning in mice. The knock-out of MMP-9 impairs appetitively motivated conditioning, but not an aversive one. MMP-9 is present at the excitatory synapses in the CeA with its activity greatly enhanced after the appetitive training. Finally, blocking extracellular MMP-9 activity with its inhibitor TIMP-1 provides evidence that local MMP-9 activity in the CeA is crucial for the appetitive, but not for aversive, learning.

Studies in cultured cells have demonstrated the existence of higher-order epigenetic mechanisms, determining the relationship between expression of the gene and its position within the cell nucleus. It is unknown, whether such mechanisms operate in postmitotic, highly differentiated cell types, such as neurons in vivo. Accordingly, we examined whether the intranuclear positions of Bdnf and Trkb genes, encoding the major neurotrophin and its receptor respectively, change as a result of neuronal activity, and what functional consequences such movements may have. In a rat model of massive neuronal activation upon kainate-induced seizures we found that elevated neuronal expression of Bdnf is associated with its detachment from the nuclear lamina, and translocation toward the nucleus center. In contrast, the position of stably expressed Trkb remains unchanged after seizures. Our study demonstrates that activation-dependent architectural remodeling of the neuronal cell nucleus in vivo contributes to activity-dependent changes in gene expression in the brain.

Matrix metalloproteinases (MMPs) are involved in tissue repair, cell death and morphogenesis. We investigated the role of the gelatinases MMP-2 and MMP-9 in the pathogenesis of neuronal death induced by prolonged seizures in the developing brain. Seven-day-old rats, MMP-9 knockout mice and transgenic rats overexpressing MMP-9 received intraperitoneal injections of pilocarpine, 250 mg/kg, to induce seizures. After 6-72 h pups were sacrificed, tissue from different brain regions was isolated and expression of MMP-9 mRNA and protein was analyzed by real-time PCR or Western blot. Additionally, brains were fixed and processed for TUNEL-staining, immunohistochemistry and in situ zymography. We found increased numbers of TUNEL-positive cells 24 h after pilocarpine-induced seizures, most pronounced in cortical areas and the dentate gyrus, and less pronounced in thalamus. At 6-24 h, MMP-9 mRNA levels showed significant elevation compared to sham-treated controls; this effect resolved by 48 h, whereas MMP-2 mRNA levels remained stable. Cortical gelatinolytic activity, monitored by in situ zymography, was enhanced following pilocarpine-induced seizures. The MMP inhibitor GM 6001 ameliorated cell death following pilocarpine-induced seizures in infant rats. MMP-9 knockout mice were less susceptible to seizure-induced brain injury. Transgenic rats overexpressing MMP-9 were equally susceptible to seizure-induced brain injury as wild type rats. Our results suggest a significant contribution of MMP-9 to cell death after pilocarpine-induced seizures in the developing brain. As indicated by Western blot analysis, MMP-9 activation may be linked to activation of the Erk/CREB-pathway. The findings implicate involvement of MMP-9 in the pathophysiology of brain injury following seizures in the developing brain., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Quantitative analysis of changes in dendritic spine morphology has become an interesting issue in contemporary neuroscience. However, the diversity in dendritic spine population might seriously influence the result of measurements in which their morphology is studied. The detection of differences in spine morphology between control and test group is often compromised by the number of dendritic spines taken for analysis. In order to estimate the impact of dendritic spine diversity we performed Monte Carlo simulations examining various experimental setups and statistical approaches. The confocal images of dendritic spines from hippocampal dissociated cultures have been used to create a set of variables exploited as the simulation resources., Results: The tabulated results of simulations given in this article, provide the number of dendritic spines required for the detection of hidden morphological differences between control and test groups in terms of spine head-width, length and area. It turns out that this is the head-width among these three variables, where the changes are most easily detected. Simulation of changes occurring in a subpopulation of spines reveal the strong dependence of detectability on the statistical approach applied. The analysis based on comparison of percentage of spines in subclasses is less sensitive than the direct comparison of relevant variables describing spines morphology., Conclusions: We evaluated the sampling aspect and effect of systematic morphological variation on detecting the differences in spine morphology. The results provided here may serve as a guideline in selecting the number of samples to be studied in a planned experiment. Our simulations might be a step towards the development of a standardized method of quantitative comparison of dendritic spines morphology, in which different sources of errors are considered.

Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, is a potent anticancer agent, which restricts tumor cell growth both in vitro and in vivo. Thus far curcumin was shown to induce death of cancer cells. This study reports the induction of cellular senescence of human colon cancer cells HCT116 upon curcumin treatment. The SA-β-galactosidase activation was observed both in p53+/+ and p53-/- cells, however the latter ones were less sensitive to the prosenescent activity of curcumin. Upregulation of p53 and p21 proteins was observed in p53+/+ HCT116, while p53-independent induction of p21 was noticed in p53-/- HCT116. Moreover, the senescence of HCT116 cells was accompanied by autophagy, that was confirmed by electron microscopy observations of autophagosomes in the curcumin-treated cells as well as LC3-II expression, punctue staining of LC3 and increased content of acidic vacuoles. Inhibition of autophagy, due to the diminished expression of ATG5 by RNAi decreased the number of senescent cells induced by curcumin, but did not lead to increased cell death. Altogether, we demonstrated a new antitumor activity of curcumin leading to cancer cell senescence and revealed the presence of a functional link between senescence and autophagy in curcumin-treated cells., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Hepatocellular carcinoma (HCC) remains a major problem in oncology. The molecular mechanisms which underlie its pathogenesis are poorly understood. Recently the Small Heterodimer Partner (SHP), an orphan nuclear receptor, was suggested to be involved as a tumor suppressor in hepatocellular carcinoma development. To date, there are no such studies regarding fibrolamellar carcinoma, a less common variant of HCC, which usually affects young people and displays distinct morphological features. The aim of our project was to evaluate the SHP levels in typical and fibrolamellar hepatocellular carcinoma with respect to the levels of one of the cell cycle regulators, cyclin D1. We assessed the immunoreactivity levels of SHP and cyclin D1 in 48 typical hepatocellular carcinomas, 9 tumors representing the fibrolamellar variant, 29 non malignant liver tissues and 7 macroregenerative nodules. We detected significantly lower SHP immunoreactivity in hepatocellular carcinoma when compared to non malignant liver tissue. Moreover, we found that SHP immunoreactivity is reduced in fibrolamellar carcinoma when compared to typical hepatocellular carcinoma. We also found that SHP is more commonly lost in HCC which arises in the liver with steatosis. The comparison between the cyclin D1 and SHP expression revealed the negative correlation between these proteins in the high grade HCC. Our results indicate that the impact of loss of SHP protein may be even more pronounced in fibrolamellar carcinoma than in a typical form of HCC. Further investigation of mechanisms through which the loss of SHP function may influence HCC formation may provide important information in order to design more effective HCC therapy.

During the last decade, multiple data have been obtained, pointing to an involvement of extracellular, including extrasynaptic, proteolysis in epilepsy pathogenesis. The most productive avenues of investigations have been analyses of seizure-evoked gene and protein expression patterns, both hypothesis-driven and unbiased (e.g., DNA microarrays), complemented by functional analyses in animal models, as well as expression and gene polymorphism studies carried out on human tissue In result, serine proteases (e.g., tPA, thrombin, trypsin-like proteases, etc.), metalloproteinases, natural protease inhibitors, as well as complement components, and reelin have been identified as a novel molecular system, emerging as a key factor in the development of epilepsy, in addition to well known contribution of ion channels and signal transduction pathways. The extracellular location of the enzymes makes them particularly attractive potential targets for future pharmacological therapeutic interventions., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An increasing body of data has shown that matrix metalloproteinase-9 (MMP-9), an extracellularly acting, Zn(2+)-dependent endopeptidase, is important not only for pathologies of the central nervous system but also for neuronal plasticity. Here, we use three independent experimental models to show that enzymatic activity of MMP-9 causes elongation and thinning of dendritic spines in the hippocampal neurons. These models are: a recently developed transgenic rat overexpressing autoactivating MMP-9, dissociated neuronal cultures, and organotypic neuronal cultures treated with recombinant autoactivating MMP-9. This dendritic effect is mediated by integrin β1 signalling. MMP-9 treatment also produces a change in the decay time of miniature synaptic currents; however, it does not change the abundance and localization of synaptic markers in dendritic protrusions. Our results, considered together with several recent studies, strongly imply that MMP-9 is functionally involved in synaptic remodelling., (@ 2011. Published by The Company of Biologists Ltd)

Matrix metalloproteinases (MMPs) play an essential role in tissue repair, cell death, and morphogenesis. The aim of the present study was to investigate potential involvement of selected MMPs in the pathogenesis of neuronal apoptosis induced by the NMDA antagonist MK-801 (dizocilpine) or the GABA(A) agonist phenobarbital in infant rats, transgenic rats overexpressing MMP-9 and MMP-9 knockout mice. Seven-day-old rats or knockout mice received intraperitoneal injections of MK-801, 1 mg/kg, or phenobarbital, 50 mg/kg. At different survival intervals following administration of the compounds (1-72 h), pups were sacrificed, tissue from different brain regions was isolated, and the expression and activity of MMP-2 and MMP-9 were analyzed by real-time PCR, western blot, and zymography. In addition, brains were fixed and processed for TUNEL staining. In all the brain regions analyzed, we found an increased number of TUNEL-positive cells 24 h after administration of MK-801. After treatment, we detected no significant increase in MMP-2 or MMP-9 mRNA expression in cortical areas. No changes in the MMP-9 protein expression or gelatinolytic activity of MMP-2 were observed in conjunction with MK-801 or phenobarbital-induced neuroapoptosis in any brain region analyzed. The extent of neurodegeneration induced by MK-801 or phenobarbital was not altered in MMP-9 transgenic rats and was increased in MMP-9 knockout mice compared to wild-type rats and mice. Treatment with the panmetalloproteinase inhibitor GM6001 did not confer protection against MK-801-induced apoptotic cell death in the developing rat brain. Our results suggest that activation of MMP-9 and MMP-2 does not contribute to pathogenesis of neuronal apoptosis caused by NMDA antagonists or GABA(A) agonists in the developing rat and mouse brain.

Structural remodeling or repair of neural circuits depends on the balance between intrinsic neuronal properties and regulatory cues present in the surrounding microenvironment. These processes are also influenced by experience, but it is still unclear how external stimuli modulate growth-regulatory mechanisms in the central nervous system. We asked whether environmental stimulation promotes neuronal plasticity by modifying the expression of growth-inhibitory molecules, specifically those of the extracellular matrix. We examined the effects of an enriched environment on neuritic remodeling and modulation of perineuronal nets in the deep cerebellar nuclei of adult mice. Perineuronal nets are meshworks of extracellular matrix that enwrap the neuronal perikaryon and restrict plasticity in the adult CNS. We found that exposure to an enriched environment induces significant morphological changes of Purkinje and precerebellar axon terminals in the cerebellar nuclei, accompanied by a conspicuous reduction of perineuronal nets. In the animals reared in an enriched environment, cerebellar nuclear neurons show decreased expression of mRNAs coding for key matrix components (as shown by real time PCR experiments), and enhanced activity of matrix degrading enzymes (matrix metalloproteinases 2 and 9), which was assessed by in situ zymography. Accordingly, we found that in mutant mice lacking a crucial perineuronal net component, cartilage link protein 1, perineuronal nets around cerebellar neurons are disrupted and plasticity of Purkinje cell terminal is enhanced. Moreover, all the effects of environmental stimulation are amplified if the afferent Purkinje axons are endowed with enhanced intrinsic growth capabilities, induced by overexpression of GAP-43. Our observations show that the maintenance and growth-inhibitory function of perineuronal nets are regulated by a dynamic interplay between pre- and postsynaptic neurons. External stimuli act on this interaction and shift the balance between synthesis and removal of matrix components in order to facilitate neuritic growth by locally dampening the activity of inhibitory cues.

The role of adult brain neurogenesis (generating new neurons) in learning and memory appears to be quite firmly established in spite of some criticism and lack of understanding of what the new neurons serve the brain for. Also, the few experiments showing that blocking adult neurogenesis causes learning deficits used irradiation and various drugs known for their side effects and the results obtained vary greatly. We used a novel approach, cyclin D2 knockout mice (D2 KO mice), specifically lacking adult brain neurogenesis to verify its importance in learning and memory. D2 KO mice and their wild-type siblings were tested in several behavioral paradigms, including those in which the role of adult neurogenesis has been postulated. D2 KO mice showed no impairment in sensorimotor tests, with only sensory impairment in an olfaction-dependent task. However, D2 KO mice showed proper procedural learning as well as learning in context (including remote memory), cue, and trace fear conditioning, Morris water maze, novel object recognition test, and in a multifunctional behavioral system-IntelliCages. D2 KO mice also demonstrated correct reversal learning. Our results suggest that adult brain neurogenesis is not obligatory in learning, including the kinds of learning where the role of adult neurogenesis has previously been strongly suggested.

Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by inducing production of reactive oxygen species such as singlet oxygen. PDT leads to oxidative damage of cellular macromolecules, including proteins that undergo multiple modifications such as fragmentation, cross-linking, and carbonylation that result in protein unfolding and aggregation. Because the major mechanism for elimination of carbonylated proteins is their degradation by proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to accumulation of carbonylated proteins in endoplasmic reticulum (ER), aggravated ER stress, and potentiated cytotoxicity toward tumor cells. We observed that Photofrin-mediated PDT leads to robust carbonylation of cellular proteins and induction of unfolded protein response. Pretreatment of tumor cells with three different proteasome inhibitors, including bortezomib, MG132, and PSI, gave increased accumulation of carbonylated and ubiquitinated proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells of murine (EMT6 and C-26) as well as human (HeLa) origin to PDT-mediated cytotoxicity. Significant retardation of tumor growth with 60% to 100% complete responses was observed in vivo in two different murine tumor models (EMT6 and C-26) when PDT was combined with either bortezomib or PSI. Altogether, these observations indicate that combination of PDT with proteasome inhibitors leads to potentiated antitumor effects. The results of these studies are of immediate clinical application because bortezomib is a clinically approved drug that undergoes extensive clinical evaluations for the treatment of solid tumors.

CD44 is a multifunctional cell surface glycoprotein which regulates cell-cell and cell-matrix interactions in a variety of tissues. In particular, the protein was found to be expressed in glial cells of developing, but not adult, peripheral nerves, where it takes part in signaling mediated by ErbB class of receptors for neuregulins. Here, we demonstrate, using high resolution morphological methods, tissue fractionation and RT-PCR, that CD44 is strongly expressed in terminal Schwann cell (TSC) at the neuromuscular junction (NMJ) of the adult rat skeletal muscle. As CD44 is also expressed by Schwann cells of the non-myelinated Remak bundles of the proximal peripheral nerves, it appears to be a marker of non-myelinating Schwann cell subpopulation. The analysis of transgenic rats bearing a mutated superoxide-dismutase gene (SOD1(G93A)) causing familial amyotrophic lateral sclerosis (ALS) revealed that TSC activation and morphological plasticity at the NMJ, caused by ongoing denervation-reinnervation is associated with a strong increase in CD44 expression therein. Notably, CD44 immunoreactivity is present in fine axon-escheating processes of the glial cells that guide reinnervation. In addition, we found that both in normal and SOD1(G93A) muscle, CD44 expressed in TSC partially colocalizes with immunoreactivities of neuregulin receptors ErbB2 and ErbB3. The colocalization appears to reflect a physical interaction, as evidenced by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) analysis between CD44 and ErbB3. Importantly, TSC activation upon ALS-like neurodegeneration results in significant increase in molecular proximity of CD44 and ErbB3, which may have an impact on glial plasticity at the NMJ.

Synaptic plasticity involves remodeling of extracellular matrix. This is mediated, in part, by enzymes of the matrix metalloproteinase (MMP) family, in particular by gelatinase MMP-9. Accordingly, there is a need of developing methods to visualize gelatinolytic activity at the level of individual synapses, especially in the context of neurotransmitters receptors. Here we present a high-resolution fluorescent in situ zymography (ISZ), performed in thin sections of the alcohol-fixed and polyester wax-embedded brain tissue of the rat (Rattus norvegicus), which is superior to the current ISZ protocols. The method allows visualization of structural details up to the resolution-limit of light microscopy, in conjunction with immunofluorescent labeling. We used this technique to visualize and quantify gelatinolytic activity at the synapses in control and seizure-affected rat brain. In particular, we demonstrated, for the first time, frequent colocalization of gelatinase(s) with synaptic N-methyl-D-aspartic acid (NMDA)- and AMPA-type glutamate receptors. We believe that our method represents a valuable tool to study extracellular proteolytic processes at the synapses, it could be used, as well, to investigate proteinase involvement in a range of physiological and pathological phenomena in the nervous system.

Matrix Metalloproteinase-9 (MMP-9) is an extracellularly operating enzyme involved in the synaptic plasticity, hippocampal-dependent long term memory and neurodegeneration. Previous studies have shown its upregulation following seizure-evoking stimuli. Herein, we show that in the rat brain, MMP-9 mRNA expression in response to pentylenetetrazole-evoked neuronal depolarization is transient. Furthermore, we demonstrate that in the rat hippocampus neuronal activation strongly induces JunB expression, simultaneously leading to an accumulation of JunB/FosB complexes onto the -88/-80 bp site of the rat MMP-9 gene promoter in vivo. Surprisingly, manipulations with JunB expression levels in activated neurons revealed its moderate repressive action onto MMP-9 gene expression. Therefore, our study documents the active repressive influence of AP-1 onto MMP-9 transcriptional regulation by the engagement of JunB.

Yin Yang 1 (YY1) is a ubiquitous transcription factor belonging to Polycomb group proteins. Its expression patterns in the adult brain have not been before clearly elucidated. Using immunohistochemical stainings, we show a distribution of YY1 protein throughout the adult rodent brain. Furthermore, we characterize a cellular localization of YY1 protein and mRNA in the adult rat hippocampus. We have found that YY1 is expressed in all major brain regions, although not ubiquitously in all cells, and its expression levels vary significantly depending on the brain structure. In most of the regions YY1 is not very abundant, but in the olfactory bulb, cerebellar cortex, hippocampus, cerebral cortex, wall of the lateral ventricle and rostral migratory stream intense YY1 staining is observed. In the rat hippocampus, YY1 protein and mRNA are very strongly expressed in neurons, and to a lesser extent in oligodendroglia and microglia. In contrast, we have not detected YY1 protein in astrocytes, which are the most abundant component of hippocampal glia. Moreover, we show that in the adult rodent brain, YY1 is expressed exclusively in the cell nuclei, except of a molecular layer of cerebellar cortex, where it is also present in the cytoplasm. Interestingly, YY1 staining is accumulated in a form of granules in cell nuclei of different types of brain cells. Thus, our data demonstrate that in the adult rodent brain YY1 is predominantly localized to neurons.

In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.

Large conductance Ca(2+)-activated potassium channels (BK(Ca) channels) are expressed in the plasma membrane of various cell types. Interestingly, recent studies provided evidence for the existence of BK(Ca) channels also in mitochondria. However, the molecular composition of these channels as well as their cellular and tissue distribution is still unknown. The goal of the present study was to find a candidate for the regulatory component of the mitochondrial large conductance calcium activated potassium (mitoBK(Ca)) channel in neurons. A combined approach of Western blot analysis, high-resolution immunofluorescence and immunoelectron microscopy with the use of antibodies directed against four distinct beta subunits demonstrated the presence of the BK(Ca) channel beta4 subunit (KCNMB4) in the inner membrane of neuronal mitochondria in the rat brain and cultured neurons. Within the cell, the expression of beta4 subunit was restricted to a subpopulation of mitochondria. The analysis of beta4 subunit distribution throughout the brain revealed that the highest expression levels occur in the thalamus and the brainstem. Our results suggest that beta4 subunit is a regulatory component of mitochondrial BK(Ca) channels in neurons. These findings may support the perspectives for the neuroprotective role of mitochondrial BK(Ca) channel in specific brain structures.

A soluble complement inhibitor factor H (FH) and its splice variant factor H-like protein (FHL) have been recently discovered to play a major role in malignant cell escape from complement-mediated cytotoxicity in lung-, ovarian- and glia-derived neoplasms. The role of FH in colon cancer has not yet been examined. Here, we studied immunocytochemically FH/FHL expression in tumor samples derived from 40 patients, with both primary colon adenocarcinoma and metastatic foci in the liver. FH/FHL immunoreactivity was present in stroma of both primary and metastatic tumors, in virtually all patients. The cellular immunoreactivity was observed infrequently. Importantly, when analyzed quantitatively, FH/FHL immunoreactivity was significantly increased in liver metastases when compared with the primary sites. In addition, we have analyzed FH and FHL expression in 5 colon cancer cell lines: SW480, SW620, HCT116, HT-29 and Lovo. FH mRNA and FH secretion were observed in SW620 and HT-29 cells, whereas FHL was produced only by HT-29 cell-line. By confocal and electron microscopy, FH immunoreactivity was associated with the plasma membrane and intracellular vesicular structures. Finally, we have analyzed the role of FH in the susceptibility of SW620 colon cancer cells to complement-mediated damage. When FH function was blocked, using specific antibody, the cells became more susceptible to lysis. Taken together, our results suggest an important role of FH/FHL in colon cancer cells defense against complement-mediated cytotoxicity, and in metastatic process., ((c) 2008 Wiley-Liss, Inc.)

Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling-induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.

GLT1 is one of the major transporters responsible for maintenance of glutamate homeostasis in the brain. In the present study, glutamate transporter 1-deficient GLT1 homozygous (-/-) and heterozygous (+/-) mice were investigated with the intention that they may provide a model of hyperglutamatergic state resulting in various behavioral alterations. The GLT1 (-/-) mice had lower body and brain weight, mild neuronal loss in CA1 hippocampal region as well as focal gliosis and severe focal neuronal paucity in layer II of the neocortex. The short life-span of GLT1 (-/-) precluded us from systematic behavioral studies in these mice. In contrast, GLT1 (+/-) mice exhibiting a 59% decrease in GLT1 immunoreactivity in their brain tissue, showed no apparent morphological brain abnormalities, and their life-span was not markedly different from controls. Behaviorally, GLT1 (+/-) presented moderate behavioral alterations compared to their wildtype littermates, such as: mild sensorimotor impairment, hyperlocomotion (at 3 month of age only), lower anxiety (at 6 months), better learning of cue-based fear conditioning but worse context-based fear conditioning. Our results suggest that GLT1 (+/-) mice may serve as a potentially useful model to study neurodegenerative disease conditions with mild hyperglutamatergic activity.

The phenomenon of dendritic transport and local translation of mRNA is considered to be one of the most fundamental mechanisms underlying long-term synaptic plasticity. Matrix metalloproteinase 9 (gelatinase B) (MMP-9) is a matrix metalloproteinase implicated in synaptic long-term potentiation and hippocampus-dependent memory. It was recently shown to be prominently up-regulated in the hippocampal dentate gyrus (DG) upon kainate-mediated seizures. Here, using a high resolution nonradioactive in situ hybridization at the light- and electron-microscopic levels, as well as subcellular fractionation, we provide evidence that in the rat hippocampus, MMP-9 mRNA is associated with dendrites and dendritic spines bearing asymmetric (excitatory) synapses. Moreover we observe that after kainate treatment the number of dendrites and synapses containing MMP-9 mRNA increases markedly. Our results indicate that we are observing the phenomenon of dendritic transport of seizure-induced MMP-9 mRNA.

Background: Understanding of the molecular mechanisms of prefrontal cortex (PFC) plasticity is important for developing new treatment strategies for mental disorders such as depression and schizophrenia. Long-term potentiation (LTP) is a valid model for synaptic plasticity. The extracellular proteolytic system composed of matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) has recently been shown to play major role in the hippocampal plasticity., Methods: We tested whether induction of hippocampal-prefrontal LTP results in accumulation of tissue inhibitor of MMP-1, TIMP-1 mRNA, in the PFC of rats and whether adenovirally driven overexpression of TIMP-1 affects LTP. Additional study of slices was done with a specific MMP-9 inhibitor., Results: The TIMP-1 is induced in the rat medial PFC by stimuli evoking late LTP; its overexpression blocks the gelatinolytic activity of the MMP family; its overexpression before tetanization blocks late LTP in vivo; and MMP-9 inhibitor prevents late LTP in vitro., Conclusions: We suggest a novel extracellular mechanism of late LTP in the PFC, engaging TIMP-1-controlled proteolysis as an element of information integration. Our results may also be meaningful to an understanding of mental diseases and development of new treatment strategies that are based on extracellular mechanisms of synaptic plasticity.

Matrix metalloproteinase-9 has recently emerged as an important molecule in control of extracellular proteolysis in the synaptic plasticity. However, no synaptic targets for its enzymatic activity had been identified before. In this report, we show that beta-dystroglycan comprises such a neuronal activity-driven target for matrix metalloproteinase-9. This notion is based on the following observations. (i) Recombinant, autoactivating matrix metalloproteinase-9 produces limited proteolytic cleavage of beta-dystroglycan. (ii) In neuronal cultures, beta-dystroglycan proteolysis occurs in response to stimulation with either glutamate or bicuculline and is blocked by tissue inhibitor of metalloproteinases-1, a metalloproteinase inhibitor. (iii) Beta-dystroglycan degradation is also observed in the hippocampus in vivo in response to seizures but not in the matrix metalloproteinase-9 knock-out mice. (iv) Beta-dystroglycan cleavage correlates in time with increased matrix metalloproteinase-9 activity. (v) Finally, beta-dystroglycan and matrix metalloproteinase-9 colocalize in postsynaptic elements in the hippocampus. In conclusion, our data identify the beta-dystroglycan as a first matrix metalloproteinase-9 substrate digested in response to enhanced synaptic activity. This demonstration may help to understand the possible role of both proteins in neuronal functions, especially in synaptic plasticity, learning, and memory.

Photodynamic therapy is a promising antitumor treatment modality approved for the management of both early and advanced tumors. The mechanisms of its antitumor action include generation of singlet oxygen and reactive oxygen species that directly damage tumor cells and tumor vasculature. A number of mechanisms seem to be involved in the protective responses to PDT that include activation of transcription factors, heat shock proteins, antioxidant enzymes and antiapoptotic pathways. Elucidation of these mechanisms might result in the design of more effective combination strategies to improve the antitumor efficacy of PDT. Using DNA microarray analysis to identify stress-related genes induced by Photofrin-mediated PDT in colon adenocarcinoma C-26 cells, we observed a marked induction of heme oxygenase-1 (HO-1). Induction of HO-1 with hemin or stable transfection of C-26 with a plasmid vector encoding HO-1 increased resistance of tumor cells to PDT-mediated cytotoxicity. On the other hand, zinc (II) protoporphyrin IX, an HO-1 inhibitor, markedly augmented PDT-mediated cytotoxicity towards C-26 and human ovarian carcinoma MDAH2774 cells. Neither bilirubin, biliverdin nor carbon monoxide, direct products of HO-1 catalysed heme degradation, was responsible for cytoprotection. Importantly, desferrioxamine, a potent iron chelator significantly potentiated cytotoxic effects of PDT. Altogether our results indicate that HO-1 is involved in an important protective mechanism against PDT-mediated phototoxicity and administration of HO-1 inhibitors might be an effective way to potentiate antitumor effectiveness of PDT.

Background/aims: To analyze, by means of immunocytochemistry, the cases of fibrolamellar variant of hepatocellular carcinoma (FLC), diagnosed in our Department., Methodology: The material comprised 4 FLC cases of tumors resected surgically. Besides the routine morphological assessment, we used a panel of immunohistochemical stainings including: hepatocellular cytokeratin, CK7, CK19, Ki67, PCNA, chromogranin A, synaptophysin, NSE, insulin, calcitonin, parathormon, CD34, EBV (LMP), Bcl2, cyclin D1., Results: In 3 out of 4 cases, we observed co-expression of CK7 with hepatocellular CK. In addition, there was positive staining with some endocrine markers in the majority of patients. In one case, we found strong cyclin D1 immunoreactivity which correlated with EBV (LMP) immunoreactivity, in the same patient. The score of PCNA positivity varied between 15 and 90%. In all cases Ki67 was negative., Conclusions: The incidence of FLC, among all hepatocellular carcinomas diagnosed in our Department was 5.1%. In accordance with other reports, all our FLC cases were young patients without underlying liver disease. We were unable to find a correlation between FLC cellular immunophenotype, and histological and clinical markers of malignancy. In addition, it appears that PCNA is a better marker of cell-proliferation in FLC than Ki67. The significance of EBV infection in FLC requires further study.

Histological, clinical and immunohistochemical analysis of 6 cases of primary liver lymphomas (PLL) are presented. PLL represents 4.3% of primary malignant liver tumors diagnosed in our department. The patients were relatively young people, who despite the presence of a large tumor, were in good general health status. There were no signs of scirrhosis, and cancer markers were normal. All lymphomas were CD20, CD79a, BAX positive, CD3, CD30, EMA, CD10, CD5, CD59, c-myc, Bcl2, EBV(LMP), CK negative. The proliferation index (Ki67) was high, ranging from 50-100%. In two cases positive staining for Bcl6 and in another one for cyclin D1 was obtained. The major histological type of the tumor was diffuse large B-cell lymphoma. Positive immunohistochemical results with BAX and the lack of Bcl2, c-myc and CD59 are associated with better prognosis. We have not confirmed the value of Bcl6 and CD10 stains as a predictor of poor outcome. Despite clinically advanced stage at the time of diagnosis, if treated appropriately, the primary lymphoma of the liver has relatively good prognosis (five of our patients are alive).